Carrion's Disease: the Sound of Silence
SUMMARY
Carrion's disease (CD) is a neglected biphasic vector-borne illness related to Bartonella bacilliformis. It is found in the Andean valleys and is transmitted mainly by members of the Lutzomyia genus but also by blood transfusions and from mother to child. The acute phase, Oroya fever, presents severe anemia and fever. The lethality is high in the absence of adequate treatment, despite the organism being susceptible to most antibiotics. Partial immunity is developed after infection by B. bacilliformis, resulting in high numbers of asymptomatic carriers. Following infection there is the chronic phase, Peruvian warts, involving abnormal proliferation of the endothelial cells. Despite potentially being eradicable, CD has been expanded due to human migration and geographical expansion of the vector. Moreover, in vitro studies have demonstrated the risk of the development of antimicrobial resistance. These findings, together with the description of new Bartonella species producing CD-like infections, the presence of undescribed potential vectors in new areas, the lack of adequate diagnostic tools and knowledge of the immunology and bacterial pathogenesis of CD, and poor international visibility, have led to the risk of increasing the potential expansion of resistant strains which will challenge current treatment schemes as well as the possible appearance of CD in areas where it is not endemic.
INTRODUCTION
The Pre-Inca and Inca Periods
Although no written documents from the pre-Inca and Inca cultures are available, there is evidence that Carrion's disease was known to the pre-Columbian cultures (1). Among the most ancient suggestive findings is the description of a pre-Incan mummy from the Nazca area (southern Peru) which had granulomatous lesions containing Giemsa-staining microorganisms which were also present in blood vessels and in internal organ granulomas. These lesions were compatible with the typical chronic-phase warty lesions (see “Peruvian Warts” within Clinical Presentation of Carrion's Disease below), and the microorganisms were compatible with Bartonella bacilliformis, the etiological agent of Carrion's disease (2). Some pottery artwork pieces made by the Mochica and Chimú cultures in the pre-Columbian period (huacos) show images of people with warty lesions, as do some stone figures from the Huaylas culture. Moreover, Quechua, the language of the ancient Peruvians, has words to describe fever (“rupha”), anemia (“sirki”), and eruption (“kcepo” and “ticti”) (1).
In 1525, during the Inca period, the imperial city of Cusco suffered an epidemic during which more than 200,000 lives were lost. At that time the Incan emperor was in Tumibamba (present-day Cuenca, Ecuador), and he and his heirs and other family members were also victims of this epidemic. Some authors have suggested that he was infected by the messenger who delivered the reports of the epidemic (1, 3), but others believe that it was from the transfer of his mummy to Cusco after his death that the epidemic later arose (4). Although some authors attributed this epidemic to Carrion's disease, it is not completely clear because the warty phase was never described (1, 3). In fact, the presence of the first American outbreak of smallpox or measles (also supported by the occurrence of rash) is highly probable, being imported either through the Spanish settlements in Panama (4) or from the Plate river settlements by the expedition of the Portuguese explorer Aleixo Garcia to the Inca emporium in 1524 (5).
The Spanish Conquest
In Coaque (current Manabi Province, Ecuador) in 1531, Pizarro lost a quarter of his men to an outbreak of a warty epidemic involving muscle and skeletal pain similar to what is described in Carrion's disease (1, 6). Later, Juan Calvete described that during the invasion in 1547 to end the Gonzalo Pizarro rebellion, many soldiers became sick with skin eruptions (7). For some authors, these epidemics were associated with bartonellosis (6, 8) (see “Carrion's disease in other countries where it is endemic” below), but for others, they were not. Some of the reasons to the contrary are that Coaque is a coastal area almost at sea level and that the Carrion's disease in Ecuador was not firmly described until the 1920s (9).
The 17th and 18th Centuries
In 1630, Gago de Vadillo described the first report of the presence of a warty disease in an area in Huaylas, in the Ancash department, and raised the hypothesis that the warts were the consequence of the drinking water (10).
The next landmark in the history of bartonellosis was in 1764, when Cosme Bueno mentioned two important new facts: (i) the disease is very bothersome and dangerous in the absence of warts, which are related to the development of the chronic phase, which causes low mortality and therefore is a good prognostic indicating benignity, and (ii) the origin of the illness seems to be a small insect named uta (6, 11).
The 19th Century: the Tragic Era
The event that first sparked medical attention to this disease was the appearance of a febrile disease that killed thousands of Chinese and Chilean men in 1870 while they were working on the railroad in Cocachacra, a village between Lima and Oroya city. The unexplained epidemic appeared suddenly. Fever and anemia were rampant, as was the number of deaths, which, according to some reports, was more than 7,000. Indeed, it was said that “every railroad tie has cost a life.” The new disease became known as Oroya fever despite Oroya city being quite far away and the absence of cases there (6).
The most remarkable contribution was made by Daniel Alcides Carrion, a young Peruvian medical student who inoculated himself with wart blood from a hospitalized child on 27 August 1885. The young researcher wanted to describe and understand the preeruption symptoms of the Peruvian warts because of the difficulty in diagnosing the disease before the appearance of the wart (11–13). The results of this experiment were a huge surprise for everybody, including himself. Some expected the eruption of a normal wart with no associated dangers, while others considered that the inoculation would not lead to anything. However, 21 days after inoculation, the first symptoms appeared, followed by rapid disease progression, which was completely compatible with the clinical features of Oroya fever (1, 6, 11). Despite his rapidly failing health, Carrion had the wisdom to clearly understand what was happening and said to his medical student friends:
“Up to today, I thought I was only in the invasive stage of the verruga as a consequence of my inoculation, that is, in the period of anemia that precedes the eruption. But now I am deeply convinced that I am suffering from the fever that killed our friend, Orihuela. Therefore, this is the evident proof that Oroya fever and the verruga have the same origin, as Dr. Alarco once said” (12).
Unfortunately, this experiment ended tragically. Daniel Carrion died on 5 October, 39 days after inoculation. In his honor, 5 October was declared National Medicine Day in Peru, and the disease was named after him. His experiment of self-infection proved that Oroya fever and the Peruvian warts are 2 different phases of the same illness (1, 6, 8).
The 20th Century and Modern Times
In 1909, Alberto Barton announced the discovery of the causal agent of Carrion's disease. He also described that the bacillus multiplies in patients with Oroya fever and decreases until its practical disappearance in the Peruvian wart phase of the illness (14).
Nonetheless, some researchers were not convinced. Richard Strong led the first Harvard Expedition to South America in 1913 and was able to find the bacillus in erythrocytes and other body tissues only in patients in the acute phase of the disease, and not in Peruvian warts, thereby contradicting previous findings determining that Oroya fever and Peruvian warts were 2 different illnesses (3).
In the same period, Charles Townsend attempted to discover the vector of the disease, spending several months unsuccessfully studying the possible role of ticks, acarus, and diurnal bugs. Thereafter, he decided to investigate the traditional native belief which considered a nocturnal sand fly called “titira” the vector. After several studies, he confirmed the traditional knowledge proposing that the vector was the “titira,” a member of the Phlebotomus genus named Phlebotomus verrucarum (currently known as Lutzomyia verrucarum) (15).
Further doubts finally vanished in 1927, when Hideyo Noguchi isolated the etiological agent from blood from patients with both Oroya fever and Peruvian warts (16, 17). Furthermore, the microorganisms isolated from the acute phase of the disease were inoculated into monkeys, producing both anemia and the characteristic eruptions on the skin (18). Noguchi had decisively proven the work of Daniel Carrion, and his results were later confirmed in 1937 by the second Harvard Expedition to Peru. The etiological agent of Carrion's disease was then named Bartonella bacilliformis in recognition of the discovery by Alberto Barton years previously (3).
Meanwhile, several other human inoculations were carried out with B. bacilliformis. In 1913, Strong inoculated a volunteer. Fortunately, the outcome was only the appearance of a Peruvian wart at the site of the inoculation. In 1928, Garcia Rosell suffered accidental inoculation when making a blood transfusion in a patient with Oroya fever, developing moderate fever and some time later a Peruvian wart. This was the inverse of Carrion's experiment; the formation of a Peruvian wart was also possible from inoculation with blood from a patient with Oroya fever. Nevertheless, Kuczynski-Godard and Garzón went further and inoculated themselves with B. bacilliformis in 1937 and 1942, respectively. While Kuczynski-Godard presented the Oroya fever phase 19 days after inoculation, which progressed to the Peruvian wart phase (6, 19), Garzón died on 12 September (20).
Until now, only the chapter on human infectious diseases related to smallpox has been definitively closed. Poliomyelitis is being actively eliminated through intensive vaccination, and yaws, measles, and several other infectious diseases have been included in eradication agendas. However, a series of chapters on the history of Carrion's disease have been lost, and several issues have apparently been forgotten, with a large amount of information being accessible only in local publications or unpublished theses. This illness continues to be present in the poorest regions and affects the most disadvantaged populations, far from the developed world, and is consequently completely outside international focus. Indeed, the study of Carrion's disease shows that this is a truly neglected disease, which only a few have attempted to describe and understand (Fig. 1).
FIG 1
EPIDEMIOLOGY
Peru
Carrion's disease is considered to be of special relevance in Peru, with the reporting of new cases being mandatory. Information about the situation outside Peru is scarce and mostly outdated. Thus, the main data regarding the ecology and sociology of the disease are limited to Peru, although they can likely be extrapolated to the neighboring countries affected.
Peru has an area of 1,285,216 km2. It is the 19th largest country in the world and the third largest in South America (21). Moreover, its orography favors its great diversity of climates and habitats. The south and center Pacific coastal areas are desert, which transform to humid areas of mangrove woodlands on the northern coasts. Parallel to the Pacific Ocean, the center of the country is fully occupied by the Andes mountain range, with the Huascaran peak towering 6,768 m above sea level (masl). Meanwhile, at the center and north of the eastern side of the mountains, there are humid woodlands that give way to a deep jungle in the border areas of the country (Fig. 2).
FIG 2
According to the World Bank classification, Peru is an upper-middle-income country (http://www.worldbank.org/en/country/peru), with a population of about 31 million, ranking 84th in the 188 positions of the human development index (22). The high percentage of poverty in this country is of note, accounting for 21.7% of the population in 2015 (23) and being extremely high in the rural mountain areas (49.0% of inhabitants).
The internal migratory phenomenon during the 20th and 21st centuries resulted in an enormous population concentration in both the capital, the metropolitan area of Lima (9,886,647 inhabitants in 2015; 31.4% of total country inhabitants), and the coastal areas, which are home to approximately 55% of the total country inhabitants (21, 23).
Illiteracy has markedly decreased and now affects only 5.7% of the population, although it continues to affect up to 15.5% of the population in some rural areas (23). Moreover, the rate of infant mortality has decreased from 56.6/1,000 live births in 1990 to 12.9/1,000 live births in 2013 (24). Despite the high inequity between rural and urban areas, malnutrition has also decreased in the last 10 years. Overall, the level of chronic malnutrition in children under five is 18.1%, being 10.1% in urban areas and reaching 31.9% in rural areas (http://www.minsa.gob.pe/portada/Especiales/2015/Nutriwawa/directivas/005_Plan_Reduccion.pdf). Access to health care facilities in rural areas remains one of the unsolved challenges for this country. The availability of hospitals and an effective national health system to the poorest populations is very low in Peru. Access or proximity to the closest health care center is often lacking, leading to a lack of appropriate medical assistance in some areas. Another important issue is the uncontrolled and illegal sale of medications (25).
Characteristics of the Affected Population
The persistence of Carrion's disease in regions where it is endemic is associated mainly with poverty, warm weather, living conditions, low levels of education, and the characteristics of the region that define the presence of the vector. Andean areas have been largely neglected during the 19th and 20th centuries, resulting in small communities which are difficult to reach, and access to higher education requires migration to urban areas.
Carrion's disease usually affects men, with a slightly higher prevalence than for women (26–29). Furthermore, Carrion's disease has been considered an occupational disease in light of vector exposure of seasonal workers, mainly men, in most of the affected areas (e.g., coffee plantation workers living outside and traveling to areas of endemicity) (30). Moreover, the relationship between coffee plantations and the presence of Lutzomyia spp. has also been described (31).
Both children and pregnant women are especially affected. Fetal deaths, miscarriages, and premature births rank among the most serious complications affecting pregnant women (32). Children are the most affected by the acute phase of the disease (28, 33). Additionally, high levels of malnutrition enhance the severity of this disease (34).
Age and the presence of family members with bartonellosis are the best predictors of the possible development of B. bacilliformis infection (35). Patients are clustered in households. Eighteen percent of cohort households account for 70% of the cases. This pattern of bartonellosis implies that 80% of cases take place in 20% of households (“20/80 rule”). Thus, people living in a patient's household have a risk to become infected that is 2.6 times greater that that for the remaining area inhabitants (35). This finding has been related to the lack of intermediate hosts and person-to-sand fly to person transmission (36). It has also been shown that cases were more likely to report bites than controls in houses near or far away (37).
Carrion's Disease in Peru
Carrion's disease is an endemic illness found mostly in the inter-Andean valleys located between 500 and 3,200 masl (38). Peru is the country most affected by this disease, with the department of Ancash having the greatest endemicity. It is estimated that >1.6 million inhabitants live in areas of endemicity, which account for around 145,000 km2 (39), with the seroprevalence among the general population of these areas being >60% (6). Although there are exceptions, the warty phase of the disease seems to prevail in regions of endemicity, whereas severe Oroya fever appears to be more common in areas of nonendemicity (40).
Despite a trend toward a decrease in the number of cases over the last decade, there was a peak in the diagnosis of Carrion's disease in 2004, with 11,130 cases being reported and a cumulative incidence of 40.4 per 100,000 inhabitants (Fig. 3). From 2004 to 2010, about 134,000 sprayings with insecticides were done in order to control the disease (41). Nonetheless, the number of cases increased in the provinces of Cutervo (Cajamarca, 2010 and 2011), Pataz (La Libertad, 2011) and Huancabamba (Piura, 2011 and 2013) (41) after discontinuation of this activity from 2010 onwards. Overall, 247 Carrion's disease-related deaths were officially reported in Peru from 2002 to 2016, with most of these deaths belonging to 4 departments (Cajamarca, Ancash, Amazonas, and Piura) (Fig. 2 and 3) (http://www.dge.gob.pe/portal/index.php?option=com_content&view=article&id=347&Itemid=249). These data provide a general picture of the situation of Carrion's disease in Peru. Nonetheless, we believe that not all cases are reported or even diagnosed and thus that these numbers are likely underestimated; therefore, the real extent of the problem remains largely unknown.
FIG 3
Human bartonellosis is considered an emerging disease which is expanding to new areas (42), influenced by climate changes such as El Niño (15, 43, 44). El Niño causes a warming in sea temperature every 5 to 7 years, which favorably affects the vector ecology, increasing the number of cases in regions of endemicity as well as outbreaks in areas of nonendemicity (38, 45). Elevation of the sea surface temperature seems to happen just before the appearance of an increase in the number of cases of bartonellosis (44). Indeed, Chinga-Alayo et al. reported an almost 4-fold increase in monthly cases during an El Niño cycle (45). Additionally, geographic expansion and the development of outbreaks should be taken into account in the epidemiology of the disease (40). In the last years, several outbreaks have been described in the literature (26, 28, 37, 46–49). Thus, Gray et al. (26) described a febrile outbreak in 1987 with a mortality rate of up to 88% in untreated cases. Maguiña reported the first outbreak in indigenous communities in the jungle in 1992, with 10 to 11% lethality (48). Kosek et al. described an outbreak in the Amazonas department in 1996 with both acute and chronic cases, with a low case-mortality rate of 0.7% (49). Ellis et al. reported a case-control study during an outbreak of acute bartonellosis in the Urubamba Valley (Cusco department) in 1998 (37), and more recently, Sánchez Clemente et al. (28) described a 2003 outbreak in Caraz (an area of endemicity in the Ancash department). In 2013 to 2014, 428 cases were reported in the Piura department, with an outbreak in the Lalaquiz district (44, 50, 51). Finally, in 2016, 33% of the total number of cases were reported in Huanuco (52).
Masuoka et al. (36), showed that Carrion's disease in the Caraz area was not more frequent near rivers but was related to agrarian activities; these agrarian areas, not necessarily next to rivers, are where sand flies encountered the most adequate living conditions, including the presence of vegetal sugars and humans.
Carrion's Disease outside Peru
Carrion's disease in other countries where it is endemic.
Some neighboring countries, such as Ecuador and Colombia, are also affected by Carrion's disease. There has been a cutaneous form of the disease in Ecuador possibly since pre-Columbian times (9). Despite being reported in Zaruma in the 1880s, it has only been since the 1930s that more reports of bartonellosis started to reappear in Ecuador (9, 53, 54). The distribution of the disease is widespread throughout the country (9), and a less aggressive form seems to be present, with an unrecognized acute phase and a milder warty phase (54). Although no further data are available, in 1997 Amano and coworkers (55) showed the presence of patients with verrucous lesions as well as a high seropositivity proportion of 21% in contacts of index cases in the coastal northern Ecuadorian province of Manabi (Fig. 2); as mentioned above, in 1531 Pizarro reported an outbreak of a warty epidemic in this area (1, 6). Nonetheless, molecular studies showed that the amplified fragment of the 16S rRNA gene had only 96.5% of identity with that of B. bacilliformis (55). In this regard, the recent description of other Bartonella spp. able to produce Carrion's disease-like symptoms should be taken into account (56, 57). Nonetheless, the illness seems to be underreported. In fact, one of the most relevant Peruvian areas of endemicity is on the Ecuadorian border (54), where only sporadic cases have been reported (D. Larreategui, “Caso de bartonelosis,” presented at the Primer Encuentro Andino de Infectología, Quito, Ecuador, 24 to 28 November 2010) since an outbreak in 1997 (58). In Colombia, the disease was absent until 1936, when a devastating outbreak leading to more than 6,000 deaths started (9, 59). It was proposed that the disease had been imported from Peru by soldiers after the war between Colombia and Peru in 1932 to 1934 (9). Thereafter, only sporadic cases, and none during the present century, have been reported (9).
Carrion's disease in areas where it is not endemic.
In addition, sporadic cases have been reported in Chile and Bolivia (26). In distant geographical areas, such as Southeast Asia or Guatemala, compatible clinical symptoms have also been described (38). Once again, the possible role of other Bartonella species cannot be ruled out, and the cosmopolitan distribution of Bartonella rochalimae should particularly be considered (60) (see Bartonellaceae below).
Peru receives more than 700,000 tourists yearly (37). Although the illness seems to be outside the most relevant tourist destinations, several imported Bartonella-like cases have been described in travelers to and immigrants from areas of endemicity (57, 61–63). Thus, in 1992, a chronic travel-related case of Carrion's disease was diagnosed in Italy 4 months after return from a lengthy journey (7 months) to Ancash, Peru. A retrospective analysis of the clinical history showed the presence of an empirically treated febrile illness immediately after the patient's return from Peru (63). In this regard, it should be taken into account that >35% of traveler's febrile illnesses remain without a definitive diagnostic, mostly receiving empirical treatment with antimicrobial agents (64). Other cases described involved immigrants from areas of endemicity, such as a chronic case reported in an Ecuadorian immigrant to Spain (61). Moreover, relevant periods of migration to other countries have taken place since the end of the 20th century. Thus, it was estimated that the negative net migration rate of Peruvian inhabitants to other countries in the period 1990 to 2012 was >2.5 million, with the number of Ecuadorian people living in other countries in 2008 also being around 2.5 million (http://www.migracionoea.org/index.php/es/parte-iii-informes-por-pais.html) (65). This and the high prevalence of carriers in areas of endemicity (50), together with the periodic visits of emigrants to related areas of endemicity and the possible long duration of asymptomatic infections (50, 62), underline the undoubtable presence of asymptomatic carriers in countries where the disease is not endemic.
Control and Prevention
In the last third of the 19th century and the beginning of the 20th century, the first actions to prevent this infection in foreign workers (e.g., railway or road construction workers) involved attempts to remain outside areas of endemicity during the evening (66). This was followed by the use of different insect repellents. Among these, the use of petroleum-based insecticides was tested after the observation of the apparent lack of cases among workers on night trains traveling though areas of endemicity. A reduction in the presence of Phlebotomus was achieved by spraying over approximately 1 week (67).
Thereafter, the main actions were aimed at vector control, considering that the vector is the primary target for the control and prevention of Carrion's disease. The use of dichlorodiphenyltrichloroethane (DDT) sprayings was efficiently used to control sand flies after World War II, and it has continued to be applied in areas of endemicity (1). This use of DDT in Peru was successful in reducing the sand fly population to negligible numbers as well as virtually eliminating the appearance of new cases of bartonellosis, and this remnant effect persisted for at least 1.5 years (68). In a study done in Peru between 1945 and 1947, DDT spraying of stone walls and houses reduced the sand fly population to an extremely low level (68, 69), as did DDT spraying of school buildings in a zone with Peruvian warts (70) and the spraying of village dwellings during an Oroya fever epidemic (26). However, it is not clear if DDT sprayings continue at present and, if so, which areas are covered.
Other preventive measures have been taken by health authorities. Although they are not always directed against Carrion's disease, these measures also play a role in the control of the sand fly population and are useful in the fight to stop this illness. These measures include regular maintenance of irrigation canals or gullies together with campaigns aimed at making the population aware of the risks of stagnant water in the transmission of other vector-borne diseases such as dengue. Moreover, albeit not frequent, the use of mosquito nets in windows or beds in areas of endemicity has also been described (71). However, a great deal of work is still necessary in order to make the population truly aware of a problem which can be prevented and treated (71).
While vaccination approaches will be the best option, only few attempts along this line have been made line (see Immunology below). Recent studies on antigenic candidates open the door to better diagnostic tools (see “Serological Techniques” below) but also to the future development of vaccines.
Indeed, the described association between climate phenomena and illness outbreaks (45) open the door to better program of preventive actions which may be developed for hot areas prior to predictable outbreaks.
BARTONELLACEAE
Although Bartonella spp. were first described in 1905 by Graham-Smith (thus the former name Grahamella for the genus) as a parasite present in mole blood (72), the genus Bartonella was proposed after the description of B. bacilliformis. Afterwards, phylogenetic studies demonstrated a close phylogenetic and phenotypic relationship between B. bacilliformis and Rochalimaea quintana, (73, 74). Following these observations, the genera Bartonella and Rochalimaea were combined to become only Bartonella (75), and later on the genus Grahamella was also added to the genus Bartonella (76). Therefore, the genus Bartonella belongs to the Bartonellaceae family, a member of the alpha-2 subgroup of the alphaproteobacteria (77), along with Rickettsia and Brucella (38). Bartonella organisms are widely dispersed throughout nature (78). Most of these bacteria are pleomorphic Gram-negative coccobacillary or bacillary rods (0.6 μm by 1.0 μm) and are considered facultative intracellular pathogens. Among other characteristics, they are erythrocyte adherent, fastidious, and aerobic. When cultured, usually from 5 to 15 days, and up to 45 days, in primary culture is needed to form colonies on blood-containing media (77, 78).
Until 1993, the genus Bartonella contained only 1 species, B. bacilliformis (79). Currently, 35 species of Bartonella have been identified (http://www.bacterio.net/index.html) and have international standing in nomenclature, and other species have been proposed, several of which are classified as “Candidatus” species (80). New species of Bartonella continue to be discovered, but frequently only partial genetic data obtained by molecular analysis are available (81–83). B. rochalimae was reported in 2007 to have caused an Oroya fever-like disease in a traveler returning from Peru (57), and a retrospective study also identified this species in the blood of a Peruvian patient with Carrion's disease (84). B. ancashensis was first reported in 2013 isolated from the blood of 2 patients with Peruvian warts (56, 85). Moreover, preliminary data have shown several new Bartonella species to have a close phylogenetic relationship with B. bacilliformis, such as in the case of the recently described “Candidatus Bartonella rondoniensis,” which shares between 84 and 91% identity with B. bacilliformis (82). Although there are few studies on these species, B. rochalimae has been described to be present in fleas (86) and to be widely disseminated (87–90). On the other hand, B. ancashensis has been described only in the area of Ancash, and no information about its vectors or reservoirs is currently known. “Candidatus Bartonella rondoniensis” has been described in Chagas illness vector kissing bugs (Eratyrus mucronatus) in French Guiana (82).
The members of this genus have a small genome, which is especially of note in B. bacilliformis (ca. 1.4 Mb). This finding is common in different microorganisms living as obligate host-associated bacteria, in which genome shrinkage has been proposed (91). The different environmental pressures with respect to free-living microorganisms might underlie this finding. With regard to Bartonellaceae, it has been estimated that the current species have lost around 1,500 genes compared to their original ancestor (92).
Different approaches for the study of the Bartonella genus phylogeny have been carried out in the last years. Thus, the use of the 16S rRNA gene sequence was first investigated, albeit with unsatisfactory results, because of the high degree of conservation of this gene. Thereafter, phylogenic approaches based first on alignments of single genes or noncoding DNA regions, such as the gltA, nuoG, or groEL genes or the 16S-23S intergenic spacer (ITS) (92–96) and then on the use of concatenated gene sequences (97) have been proposed.
Different authors have proposed to separate the genus Bartonella into different phylogenetic lineages based on type IV secretion systems (T4SS) acquired by the different species (97–99), which favor distinct host adaptability. Thus, lineage 1 includes B. bacilliformis, whereas lineage 2 contains ruminant-specific species. Lineage 3 is composed of species infecting diverse mammals; lineage 4 includes, for example, Bartonella henselae or Bartonella quintana. Regarding B. ancashensis, while some authors classify it as the first member of a new lineage (100), others include this species within lineage 1 together with B. bacilliformis (101) (Table 1).
TABLE 1
| Microorganismc | Human diseased | Reservoir(s)e | Vector(s)f | Yrg | Fla | T4SSb | Lin | ||
|---|---|---|---|---|---|---|---|---|---|
| VirB | Vbh | Trw | |||||||
| CD-related Bartonella spp. and closely related “Candidatus” species | |||||||||
| B. ancashensish | CD-like | ND | ND | 2015 | + | + | − | − | 5i |
| B. bacilliformisj | CD | Humansk | Sand flies | 1913 | + | − | − | − | 1 |
| B. rochalimae | CD-like, Bact | Canids | Fleas | 2012 | + | + | − | − | 3 |
| “Candidatus Bartonella rondoniensis”l | ND | Kissing bugs | —m | ND | ND | ND | ND | ND | |
| Bartonella spp. causing other human illness | |||||||||
| B. alsatica | End | Rabbits | Rabbit fleas | 1999 | − | + | − | + | 4 |
| B. clarridgeiae | CSD, End | Cats | Cat fleas | 1996 | + | + | − | − | 3 |
| B. doshiae | Bact | Rodents | Fleas | 1995 | − | + | + | + | 4 |
| B. elizabethaen | End, Nrt | Rats | Rat fleas | 1993 | − | + | + | + | 4 |
| B. grahamii | Nrt | Wild mice | Rodent fleas | 1995 | − | + | + | + | 4 |
| B. henselaen | BA, Bact, CSD, End, Nrt | Cats | Cat fleas | 1992 | − | + | + | + | 4 |
| B. koehlerae | Bact, End | Cats | Cat fleas | 2000 | − | + | + | + | 4 |
| B. mayotimonensis | End | Bats | Fleas, flies | — | ND | ND | ND | ND | ND |
| B. melophagi | CF, Per | Sheep | Sheep ked | — | + | − | + | − | ND |
| B. quintanan,o | TF, BA, End | Humans | Body lice | 1917 | − | + | + | + | 4 |
| B. schoenbuchensis | Bact | Cervids | Deer flies | 2001 | + | − | + | − | 2 |
| B. tamiaep | Fever | Rodents? | Ticks, flies | — | − | − | − | − | ND |
| B. tribocorum | Bact | Rats | Rat fleas | 1998 | − | + | + | + | 4 |
| B. vinsonii arupensis | End, Bact | Mice | Rodent fleas | 2000 | − | + | − | + | 4 |
| B. vinsonii berkhoffi | End | Dogs | Fleas | 1996 | − | + | −/+q | + | 4 |
| B. washoensis | End, Myo, Men | Rodents | Rodent fleas | — | − | + | + | + | ND |
| Other Bartonella spp. | |||||||||
| B. apis | Honey beesr | Honey beesr | 2016 | + | − | − | − | ND | |
| B. acomydis | Rodents | ND | 2013 | − | ND | ND | ND | ND | |
| B. birtlesii | Rodents, shrews | Ticks | 2000 | − | + | + | + | ND | |
| B. boviss | Cattle | Ticks | 2002 | + | − | −/+t | − | 2 | |
| B. callosciuri | Rodents | ND | 2013 | − | ND | ND | ND | ND | |
| B. capreoli | Cervids | Deer flies | 2002 | + | − | + | − | 2 | |
| B. chomelii | Cattle | Ticks | 2004 | + | − | + | − | 2 | |
| B. coopersplainsensis | Rodents | Fleas | 2009 | − | ND | ND | ND | ND | |
| B. florencae | Shrews | ND | 2014 | − | + | + | + | ND | |
| B. fuyuanensis | Rodents | ND | 2016 | − | ND | ND | ND | ND | |
| B. heixiaziensis | Rodents | ND | 2016 | − | ND | ND | ND | ND | |
| B. jaculi | Rodents | ND | 2013 | − | ND | ND | ND | ND | |
| B. japonica | Rodents | ND | 2010 | − | ND | ND | ND | ND | |
| B. pachyuromydis | Rodents | ND | 2013 | − | ND | ND | ND | ND | |
| B. peromysciw | Rodents | ND | 1942 | − | ND | ND | ND | ND | |
| B. queenslandensis | Rats | ND | 2009 | − | + | + | + | ND | |
| B. rattaustraliani | Rodents | Ticks | 2009 | − | + | + | + | ND | |
| B. senegalensis | Rodents? | Rodent ticks | 2014 | − | + | − | + | ND | |
| B. silvatica | Rodents | ND | 2010 | − | ND | ND | ND | ND | |
| B. talpaew,x | Rodents | ND | 1911 | − | ND | ND | ND | ND | |
| B. taylorii | Rodents | ND | 1995 | − | + | −/+y | + | 4 | |
| B. vinsonii vinsoniin | Mice | ND | 1982 | − | + | − | + | 4 | |
a
Abbreviations: T4SS, type IV secretion system-coupling protein (in some cases data have not been reported but were determined from analysis of GenBank sequences); Fla, flagellum; VirB, VirB/VirD4 system; Vbh, VirB homolog (sometimes referred to in the literature as Vbh/TraD); Lin, Lineage or clade (only those microorganisms previously classified in the literature as belonging to any of the described clades [92, 97–101] are indicated); BA, bacillary angiomatosis; Bact, bacteremia; CD, Carrion's disease; CF, chronic fatigue; CSD, cat scratch disease; End, endocarditis; Men, meningitis; Myo, myocarditis; Nrt, neuroretinitis; Per, pericarditis; TF, trench fever; ND, no data.
b
The presence of T4SS is presented according to different reports (92, 97–101). Note that in some cases the presence (+) or absence (−) of T4SS has been reported in species and strains which are not present in GenBank. In addition, these systems have also been reported as being encoded on plasmids and chromosomes (373), which may lead to differences in their presence/absence within members of the same species. This should be taken into account, especially in light of the low number of available Bartonella genomes present in GenBank (see footnote c) or Bartonella strains in which the presence of T4SS has been explored. In addition to examining previously published data, a search for the presence of these systems in GenBank was made using the B. tribocorum (GenBank accession number AM260525.1) VirB/VirD4, Vbh, and Trw operon sequences was made. Some “Candidatus” species not included in this table (see criteria in footnote c), such as “Candidatus Bartonella australis” or “Candidatus Bartonella rattamassiliensis,” possess one or more of these T4SS.
c
Species listed in alphabetical order within groups. Only those names with standing in nomenclature (80), as well as those causing human infection or closely related to B. bacilliformis, are presented (see more in-depth information at http://www.bacterio.net/-foreword.html). Species with at least one genome sequence present in GenBank (https://www.ncbi.nlm.nih.gov/genome/?term=Bartonella) are highlighted in bold. Note that at the time of writing this review, only 27 Bartonella species (either with or without standing nomenclature) and an additional 12 unnamed Bartonella-related microorganisms fulfilled this criterion. Of these, B. henselae has 23 genomes, B. bacilliformis has 15 genomes, B. quintana has 14 genomes, B. apis and B. birtlesii have 6 genomes each, B. vinsonii has 5 genomes (2 belonging to B. vinsonii subsp. arupensis and 3 belonging to B. vinsonii subsp. berkhoffii), and B. elizabethae has 4 genomes; no more than 3 genomes are present for the remaining Bartonella spp. sequenced.
d
Some of these species have been only sporadically reported as a cause of human illness.
e
In most cases, the Bartonella spp. have also been reported in other reservoirs; e.g., macaques act as a B. quintana reservoir (374).
f
In most cases, other vectors have been described.
g
Year of official recognition as a new species (previously published reports may be found in the literature) (http://www.bacterio.net/-foreword.html). Note that despite having been reported in the literature some time ago, some species described earlier, such as B. talpae, have only recently been correctly defined from bacterial cultures.
h
In some articles reported as Bartonella ancashi.
i
j
Also present in early literature as “Bartonia bacilliformis.”
k
Humans are the only known reservoir of B. bacilliformis.
l
To date, only a single description of “Candidatus Bartonella rondoniensis” in kissing bugs has been reported, with the use of molecular tools. No isolate is available to perform further studies. This Bartonella species is closely related to B. bacilliformis and B. ancashensis (82).
m
—, not yet officially recognized as a new species (80).
n
Prior to 1993 classified within the Rochalimaea genus.
o
This species may also be found as Rickettsia quintana, Rickettsia pediculi, Rickettsia wolhynica, Rickettsia weigli, Burnetia (Rocha-limae) wolhynica, or Wolhynia qintanae.
p
The in silico analysis does not show the presence of any characterized T4SS or a flagellin-encoding gene.
q
Although some B. vinsonii subsp. berkhoffi strains have been reported to possess only the VirB4/VirD system (97), the Winnie strain (GenBank accession number CP003124.1) has both the Virb4/VirD and Vbh systems (92).
r
Considered a honey bee symbiont.
s
t
Although reported to be Vbh operon positive (97), B. bovis shows no identity when genomes present in GenBank (https://www.ncbi.nlm.nih.gov/GenBank/) are analyzed using the Blast tool. Note that the positive B. bovis strain reported (strain A96) is neither totally nor partially present in GenBank.
w
Prior to 1995 was classified within the genus Grahamella.
x
This species may also be found in previous reports as Grahamia talpae.
y
B. taylorii strain 8TBB (GenBank accession number AIMD01000055.1) possesses 2 T4SS.
The T4SS are involved in adherence to erythrocytes, facilitating host-specific adhesion (102). The exception is lineage 1, in which the T4SS is considered to be absent, thereby implicating other virulence proteins in erythrocyte adherence and invasion (see Pathogenesis and Virulence Factors below). The presence of an independently acquired VirB4/D system in B. ancashensis (101) may lead to a modification of this concept even if its classification in lineage 1 becomes definitive.
All these proposals usually classify B. bacilliformis as an outlying species, but a study analyzing 428 concatenated gene sequences described this species as being closely related to Bartonella bovis and Bartonella schoenbuchensis, both of which are found in ruminants, with Bartonella tamiae being an outlier species (92). A similar outlying classification is also given to Bartonella apis when included in the classifications (101). It is of note that an analysis of the B. tamiae and B. apis genomes showed the lack of T4SS (identical to the case for lineage 1) as well as the absence of flagella in B. tamiae (Table 1).
Several arthropod vectors, such as fleas, ticks, flies, and lice, have been described for Bartonella spp. (86–90, 103–105). The recent identification of B. quintana in bedbugs such as Cimex lectularius and Cimex hemypterus also makes these insects potential new vectors of Bartonella (106, 107). Bartonella coinfections are frequent, with a rate of coinfection in fleas from rats of 90% (104).
In animals and humans, the diversity of clinical and pathological manifestations associated with Bartonella infections may be related to the wide variety of cells which may be infected, including erythrocytes, pericytes, endothelial cells, dendritic cells, CD34+ progenitor cells, and various macrophage-type cells (108). This finding might have facilitated the presence of coevolution phenomena. In fact, almost all Bartonella spp. have a mammalian reservoir host (109). Thus, Bartonella vinsonii subsp. berkhoffii, “Candidatus Bartonella melophagi,” and “Candidatus Bartonella australis” have coevolved with canids, sheep, and kangaroos, respectively (108). B. rochalimae has been described in raccoons, coyotes, and red foxes (60), and in addition, different reports have described the presence of closely related B. rochalimae microorganisms in rats and rat fleas (110, 111). Nonetheless, the newly described B. apis is considered a honey bee symbiont (112). In fact, the evolution from insect-associated gut symbiont to a mammalian pathogen through the loss of genetic material and the acquisition of horizontal virulence factors has been proposed, with B. tamiae being representative of a transition stage (113, 114). Bartonella spp. have a close phylogenetic relationship with nitrogen-fixing soil microorganisms such as Rhizobium spp. This finding, together the symbiont role of B. apis, which seems to maintain the nitrogen fixation capacity, as well as the recent detection of Mesorhizobium spp. and Bradyrhizobium spp. in sand flies and other insects (suggested to be beneficial in female insects in the absence of blood) (115), suggests the possible evolution of these soil microorganisms to different Bartonella spp. in insects.
Several species have been associated with different diseases and syndromes in humans, with B. bacilliformis, B. henselae, B. quintana, Bartonella elizabethae, and Bartonella clarridgeiae being the most relevant. In addition B. rochalimae and B. ancashensis have recently been related to Carrion's disease-like syndromes (56, 57, 78, 85, 116). Moreover, the role of several Bartonella spp. in the development of endocarditis in immunocompetent persons has been shown (108). Furthermore, other Bartonella spp. have sporadically been described as a cause of different diseases (117–121) (Table 1). The term “bartonellosis,” historically attributed to infections with B. bacilliformis, at present has a wide definition and includes infections caused by any Bartonella spp. However, if not expressly indicated, in the present article “bartonellosis” always means infection caused by B. bacilliformis.
Bartonella bacilliformis
Classically B. bacilliformis has been considered a common “ancestor” of the Bartonella genus, but this evolutionary scenario has recently been questioned, and B. bacilliformis has been related to B. schoenbuchensis and B. bovis, as mentioned above. Indeed, it has been proposed that B. bacilliformis might have originated from a small bacterial population after successful accidental transfer from local camelids (Lama spp. and Vicugna spp.) to humans (92). Nonetheless, despite the presence of Bartonellaceae in Old World camelids (122), at present no Bartonella sp. has been recovered from any of the current 4 local camelid species.
B. bacilliformis is a Gram-negative, nonfermentative, aerobic, pleomorphic coccobacillus, with dimensions of 0.2 to 0.5 μm by 1 to 2 μm and 2 to 16 flagella that confer high mobility. Biochemical tests are not useful for presumptive identification of Bartonella species, but peptidase activity in l-proline and l-lysine is useful for that of B. bacilliformis (39). The growth of this microorganism is slow in blood-containing media such as Leptospira medium with rabbit, sheep, horse, or human blood (16) or Columbia blood agar with 5% sheep blood. The optimum temperature is 28 to 30°C, and although the organism is aerobic, microaerophilic conditions provide better results (15, 16). The colonies are typically small, round, and lenticular, ranging from translucent to opaque (39). Occasionally, the colonies may adopt other forms, such as the so-called T1 morphology (colonies having regular edge, a small halo, and a “bubble” centered on the colony) (123, 124) (Fig. 4). B. bacilliformis can remain viable at 4°C for long periods of time (125, 126), similar to what has been described for other Bartonella spp. (127). As mentioned above, the B. bacilliformis genome consists of a single circular DNA molecule ranging from 1.39 Mbp (strain Peru-18; GenBank accession no. KK097689 to KK097708) to 1.44 Mbp (strain ATCC 35685D-5; GenBank accession no. NZ_CP014012.1), which has a GC content of 38.2 to 38.3% (https://www.ncbi.nlm.nih.gov/genome/genomes/524). The high percentage of AT content is also observed in other Bartonellaceae, being a distinctive trait of host-dependent microorganisms (39).
FIG 4
Genetic Diversity
The phylogenetic relationships and genetic diversity among B. bacilliformis isolates have not been extensively studied, although several studies have addressed this aspect. Interestingly, in a study assessing the differentiation of B. bacilliformis isolates by the evaluation of gltA and 16S-23S ribosomal DNA intergenic spacer regions as well as an amplified fragment length polymorphism (AFLP), B. bacilliformis strains causing acute disease were also found to cause asymptomatic infection (40). This finding may be related to different compatible scenarios, including alterations in the expression of different genes related to host-pathogen interactions but also possible horizontal acquisition/loss of genetic material or previous exposure to the pathogen leading to acquisition of partial immunity. Moreover, the same study revealed that 3 different outbreaks in new illness expansion areas were caused by 3 different minority genotypes of B. bacilliformis and not by those genotypes most often encountered in regions of endemicity (40). The use of infrequent restriction site PCR has shown a relative high degree of genotypic variation within populations of B. bacilliformis (128).
More recently, a multilocus sequence typing (MLST) approach consisting of the amplification of 7 different genetic loci (ftsZ, flaA, ribC, rnpB, rpoB, bvrR, and groEL) was developed (129), and in 2017 a webpage devoted to bringing order to and information about B. bacilliformis MLST was created (https://pubmlst.org/bbacilliformis/). To date, phylogenetic analyses have identified up to 14 distinct sequence types among B. bacilliformis strains (129–131). A comparative genomic approach revealed that the evolution of B. bacilliformis is shaped predominantly by mutations. Mutational divergence leads to subspeciation, but mutational convergence between clones of a subspecies shows evidence of common adaptive evolution (131).
Currently up to 15 B. bacilliformis genomes are present in GenBank. Of these, although present in GenBank as a “representative genome” together with strain KC583 (NC_008783), the genome of strain Ver097 (NZ_KL503802 to NZ_KL503807) differs greatly from the remaining B. bacilliformis genomes, suggesting that it may, in fact, be a nonrecognized new Bartonella species or a B. bacilliformis subspecies, similar to what has been also proposed for other strains (Ramirez, Vega, and Peru53) which are currently not included in GenBank (131, 132). Confirmation of the Ver097 strain as a new species will make it a new Bartonella species recovered from Carrion's disease-like syndromes.
CLINICAL PRESENTATION OF CARRION′S DISEASE
The clinical presentation of Carrion's disease involves 2 syndromes that occur independently or sequentially (39): an acute phase called Oroya fever and a chronic phase named Peruvian warts. However, the presence of Peruvian warts has also been described in the clinical care of acute or recovery phases of Oroya fever (133, 134).
Once a person is bitten by an infected sand fly, an asymptomatic infection or a mild to severe disease is presented. While the average incubation time of Carrion's disease is 61 days (between 10 and 210 days) (78), the severity of the disease is probably determined by individual predisposition and the specific virulence of the strain causing infection. According to Noguchi, strains isolated from people with very mild anemia or Peruvian warts are presumably less virulent (135). Similarly, Kosek et al. proposed that a strain of B. bacilliformis with diminished virulence had caused the infection when disease manifestations were milder than the typical manifestations of Oroya fever, (49). In light of the description of B. rochalimae and, especially, B. ancashensis and the possible misidentification of presumptive new Carrion's disease-causing Bartonella species as B. bacilliformis, studies are needed to evaluate whether these species have different virulence potentials to cause severe Oroya fever, mild infections, or Peruvian warts.
Nomenclature and International Classifications
Carrion's disease is referred to by a great variety of names (Table 2). This is related to the history of the disease, including the lack of consensus as to a single or dual nature of differently related syndromes (present until the first third of the 20th century) (see History above), as well as the particularities of the areas and inhabitants mainly affected (see Epidemiology above).
TABLE 2
| Names for Carrion's diseasea in: | |
|---|---|
| Acute phase | Chronic phaseb |
| Oroya fever (fiebre de Oroya) | Peruvian wart (verruga Peruana) |
| Anemia de Carrión | Cutaneous bartonellosisc |
| Anemia perniciosa de las quebradas | Mucocutaneous bartonellosisc |
| Bartonellosisd | Botón de los Andes |
| Fiebre aguda verrucosa | Verruga Andícola |
| Fiebre de Castilla | Verruga blanda |
| Fiebre del Guáitarae | Verruga de Ancash |
| Fiebre grave de Carrión | Verruga de Castilla |
| Fiebre maligna de las quebradas | Verruga de los conquistadores |
| Fiebre maligna verrucosa | Verruga de crapaudf |
| Fiebre verrucosa de Guáitarae | Verruga de los libertadores |
| Tifus palúdico de la Oroya | Verruga de quinua |
| Verruga de sangre | |
| Verruga de zapo/sapof | |
| Verruga Ecuatorianag | |
| Verruga hemorrágica Peruana | |
| Verruga mular | |
| Verruga nodular | |
| Verruca Peruviana | |
a
Most of these names are present in Spanish scientific literature.
b
It should be highlighted that in several articles, especially early articles, the use of the term Peruvian wart and other names including the word “verruga” (wart) as synonyms for Carrion's disease is usual, irrespective of the exact illness phase.
c
From 1993 onwards, this name was extended to infections by other Bartonella spp.
d
Until 1993 this name was exclusively used to designate infections by B. bacilliformis. Thereafter, it may refer to human infections related to any Bartonella sp. Additionally, note that bartonellosis may also not be used exclusively to designate systemic infections.
e
Term used mainly in Colombia.
f
Note that “crapaud” (used in some early French literature) and “sapo” mean “toad” in English. Similarly, “zapo” is phonetically closely similar to “sapo.”
g
Proposed in Ecuador to explain the apparent lesser severity of local clinical cases.
Carrion's disease is a notifiable disease in Peru (http://www.minsa.gob.pe/renhice/documentos/normativa/RM506-2012-MINSA%20-%20DS%20046%20DGE%20Notificacion%20Enfermedades%20Eventos%20Vigilancia%20Epidemiologica%20Salud%20Publica.pdf), and according to the guidelines of the World Health Organization, it is internationally reported under the codes A44.0 (Systemic bartonellosis—Oroya Fever) and A44.1 (Cutaneous and mucocutaneous bartonellosis—Verruga Peruana) or the code A44.9 (Bartonellosis, unspecified) (http://apps.who.int/classifications/icd10/browse/2016/en).
In the Medical Subject Headings (MeSH) of the National Center for Biotechnology Information, B. bacilliformis infections are classified together with those caused by other Bartonellaceae under the code D001474, while in the Universal Medical Language System (UMLS), Carrion's disease is codified under the codes C0029307 (including other Bartonella infections) and C0348974 (systemic bartonellosis). The illness is also codified in other disease-normalizing international coding systems, such as Disease Ontology developed by Northwestern University, Institute for Genome Sciences and Center for Genetic Medicine, and the University of Maryland School of Medicine under the code DOID:0050398. In addition, it is of interest that this illness is considered to be an orphan disease and is included in the ORPHANET list under the code ORPHA64692.
Oroya Fever
The major clinical consequences of infection with B. bacilliformis result from invasion of the erythrocytes by the bacteria in the acute phase of Carrion's disease. B. bacilliformis infects the erythrocytes, which are subsequently destroyed in the spleen and liver, leading to severe hemolytic anemia and transient immunosuppression. Although not all the infected erythrocytes are removed from the circulation, hemolytic anemia involves the destruction of the infected erythrocytes by the reticuloendothelial system, with the erythrocytes remaining within the circulation for a shorter time than normal, thereby leading to the development of anemia (136). Although increased production of erythrocytes in response to the great destruction of these cells may be up to five times greater than normal, in Oroya fever there is a substantial reduction in the number of erythrocytes with a compensatory increase in plasma, so that the total blood volume is not severely affected (136). On the other hand, a deregulation of the immune system causes a deterioration of cellular immunity leading to immunosuppression, predisposing the patients to opportunistic infections (137, 138). The amount of microorganisms in the blood is higher in patients with severe anemia than in patients with warts (135). In fact, in a study by Maguiña et al., the mean percentage of infected erythrocytes in this phase of the disease was 61% (range, 2 to 100%), and in 25% of patients, >90% of the erythrocytes were infected at the time of admission (139).
The symptoms of Oroya fever are indistinguishable from the initial symptoms of other infectious diseases such as malaria, typhoid fever, dengue, tuberculosis, or even viral hepatitis. The onset is usually gradual, with malaise, fever, headache, and mild chills, and can include pallor, hepatomegaly, abdominal pain, and other unspecific symptoms (29, 33, 78, 140). The acute phase seems to have a greater effect on children and teenagers up to the age of 15 years (6, 29, 35, 43, 49). Overall, patients in the acute phase are younger than those in the eruptive phase; thus, in a study by Maguiña et al. (139), it was observed that patients in the acute phase have a mean age of 14.6 years while those in the eruptive phase has a mean age of 18.4 years, suggesting the development of acquired immunity following exposure (35). The mortality rate in the acute phase is 40 to 85% in untreated patients; however, in reference centers (where severe cases are treated) with appropriate and timely treatment, this rate can be reduced to values of around 10% (6, 15, 78, 139, 141, 142). Complications or secondary opportunistic infections can dramatically worsen the clinical outcome, and unfortunately, they are quite common (29, 33, 78, 142–144). There are several reports on noninfectious hematological, cardiovascular, and neurological complications (29, 33, 140). In regard to opportunistic infections, the most common pathogens involved are Salmonella spp., which are responsible for 90% of the deaths in Oroya fever (1). Moreover, mortality is increased during outbreaks, mainly in new transmission areas because health personnel are unaware of the disease and laboratory staff members are not trained to diagnose the disease (6). For example, in the late 1990s in La Convencion, a district of the Cusco department, acute cases of Carrion's disease were systematically reported as viral hepatitis, with mortality rates of 39% in some hospitals (27). As indicated above, the risk of mortality is high in pregnant women during acute bartonellosis, with the possible presentation of severe complications, including fetal death (32, 78, 141).
Peruvian Warts
The chronic phase of Carrion's disease, called Peruvian warts, is characterized by the development of dermal eruptions, occurring weeks or months after Oroya fever, and persists from 1 month to 1 year (1, 78). Nonetheless, the chronic phase may occur in the absence of previous reported acute illness (145). Lesions vary in size and number, mainly affecting the arms and legs, although other body areas can also be affected (145). The microorganisms are observed within the warts, both intracellularly and within the extracellular matrix (145). The lesions are classified as follows (141, 145): (i) miliary, i.e., small (diameter, <3 mm), often numerous, reddish papules situated at the papillary and medial dermis, may be pruriginous; (ii) mular, i.e., erythematous nodules (diameter, >5 mm) which tend to extend deeper into the hypodermis with the possible involvement of subcutaneous tissue and muscle; and (iii) subdermic, i.e., diffuse subdermic nodules showing no changes in the overlying skin, larger and more prominent than the miliary and mular lesions.
The eruptions are most often miliary (145), although different types of warts may commonly be found in the same patient (6). Miliary lesions are normally painless, while the mular lesions may be painful, and two-thirds of patients complain of bleeding warts.
The eruptive phase is a more common manifestation in inhabitants of regions of endemicity, and the risk of developing Peruvian warts increases with age (49). This phase tends to heal spontaneously, and the mortality rate is insignificant. These eruptions are frequently accompanied by mild systemic manifestations, including fever, malaise, osteoarticular pain, lymphadenopathy, and headache (78, 145). Notwithstanding, in more severe cases, complications, including bleeding, may be observed in 66% and secondary infection in 12% of the patients, occasionally leading to fatal outcomes in the absence of timely blood transfusions (6, 139). In fact, in the period from 2005 to 2016, only 1 death related to the chronic phase of Carrion's disease was reported (146).
Around 50% of patients with Peruvian warts are bacteremic (35, 147), with bacteremia being significantly correlated with the onset of the lesions as well as low hemoglobin levels. On the other hand, the number and distribution of the lesions and the clinical symptoms are not associated with bacteremia (147).
Although scarcely described, some reports have also indicated the presence of internal granulomas, which may be present in different organs, including the lungs, brain, or spleen (62, 133). By far the most ancient description of the presence of these granulomas was in a Tiahuanaco mummy, being suggestive of Carrion's disease (2). Although the relationship of these lesions with B. bacilliformis has not been definitively established, confirmation of the extension and the clinical parameters of the disease should be described and their true clinical relevance evaluated.
In a recent study on a patient with Peruvian warts, the presence of a concomitant infection by B. ancashensis (detected by molecular tools) and B. bacilliformis (detected by molecular and culture methods) was observed. After treatment with rifampin, a blood culture of B. ancashensis was obtained in one patient (100). This report highlighted the possible coinfection by different strains or closely related microorganisms, as well as the need for in-depth analysis of the real role of B. ancashensis in the development of chronic Carrion's disease. Moreover, this study lead to the question as to whether the two microorganisms were transferred together from the same vector or whether the patient had two different infectious processes.
Asymptomatic Carriers
It has long been known that these microorganisms can persist in blood for many years after the infection (70). Indeed, one study described the isolation of B. bacilliformis from an individual who had visited Ecuador 3 years previously (62). Moreover, asymptomatic carriers have also been described in areas of endemicity (35, 50, 148, 149). It is believed that these carriers perpetuate the disease and can introduce it into new areas, either within a stable setting with a vector in the area (as described in Colombia in the mid-1930s) or within an unstable setting in which no vector is present. Indeed, Chamberlin and coworkers analyzed the prevalence of asymptomatic B. bacilliformis carriers in an area of endemicity by PCR and found that 0.5% were positive (35). More recently, a study including an area of endemicity (Huancabamba) and an postoutbreak area (Lalaquiz), both in the northern Peruvian department of Piura, showed that approximately half of the volunteers in the area of endemicity and about 40% of the individuals in the postoutbreak area had a positive result by quantitative real time-PCR (qPCR), demonstrating the high number of asymptomatic carriers in these zones (50). It is of concern that the positivity observed was near the qPCR detection limit, showing that the presence of bacteria in the blood may have been very low. This suggests that the real number of B. bacilliformis carriers may be underestimated when less-sensitive techniques are used. Although reinfections cannot be ruled out, the presence of microbiological failures seems evident, as all volunteers from the postoutbreak area were treated with ciprofloxacin during the outbreak (50).
PATHOGENESIS AND VIRULENCE FACTORS
Successful infection of a mammalian host by a bacterial pathogen typically involves a series of intimate host-pathogen interactions. B. bacilliformis can invade a variety of different human cell types in vitro (150), but it is known that B. bacilliformis infects the erythrocytes and endothelial cells in Oroya fever and Peruvian warts, respectively. Erythrocyte infection has several advantages, such as being hidden from the humoral immune system, the absence of lysosomes, and a reasonably long life (141). Mature erythrocytes are nonendocytic, and thus, erythrocyte involvement in the invasion process is necessarily passive (150), with several determinants and virulence factors being involved in the pathogenesis of B. bacilliformis, including motility, erythrocyte deformation, and invasive factors (151–155).
In contrast, the endothelial cells actively participate in the uptake of B. bacilliformis by a process similar to phagocytosis (150). The objectives of this complex adaptation to the human host are perhaps to achieve the persistence and maintenance of a reservoir state for vector-based transmission and immune evasion.
Erythrocytes and Oroya Fever
The first study reporting the ability of B. bacilliformis to penetrate erythrocytes was performed in 1969 (156). Erythrocyte invasion is probably the most important step in the pathogenesis of Carrion's disease, since it is related to the worst outcome of the illness. It is possible that competence for erythrocyte invasion must be acquired by an adaptation process in the primary niche (157). Although it is controversial, some authors have described colonization of a primary niche after intravenous inoculation and before erythrocyte infection (38, 151, 157, 158). Moreover, the incubation period (60 days on average) of Oroya fever after infection is long (158). Erythrocytes lack the normal actin-based structures employed by most cells during endocytosis, and therefore, as mentioned above, B. bacilliformis plays an active role during erythrocyte invasion (159, 160). Infection of erythrocytes by B. bacilliformis involves at least 2 important steps: (i) the binding of the bacteria to the surface of the erythrocytes and (ii) deformation of the erythrocyte membrane, including deep invaginations and membrane fusion leading to the formation of intracellular vacuoles containing bacteria (161).
Polar flagella, composed of multiple 42-kDa flagellin subunits, provide the bacterium with a high degree of mobility during its search for erythrocytes (160, 162), which is essential for erythrocyte binding and deformation by potentially providing mechanical force (158, 161). A mutant lacking flagella has a nonmotile phenotype (163), and a significant decrease in the efficiency of invasion has been described after preincubating B. bacilliformis with antiflagellum antibodies (162). Nonetheless, spontaneous mutants (e.g., with T1 morphology) which despite being flagellated have reduced motility or nonmotility it has been described (124). These flagellated nonmotile variants can be bound to erythrocytes, promoting surface contact interaction. The optimal binding of bacteria to the surface of erythrocytes requires B. bacilliformis energy, most likely proton motive force-dependent motility (123, 140), with the whole in vitro process taking approximately 6 h (161).
Beyond flagella, the extracellular protein deformin plays a fundamental role in erythrocyte invasion. This protein is able to deform human erythrocytes by producing deep invaginations in their membranes (155, 164). Studies with purified deformin have shown the capacity of this protein to achieve these invaginations even in the absence of bacteria (155). It is likely that erythrocyte invasion is related to the concomitant action of flagellum-based motility and deformin (Fig. 5).
FIG 5
In addition, the ialA and ialB genes seem to facilitate erythrocyte invasion. The proteins encoded by these genes have shown the ability to invade human erythrocytes in the absence of the other Bartonella virulence factors (165).
Studies cloning the ialAB locus in Escherichia coli have shown an invasion increase of 6- to 39-fold when both genes were present, highlighting that both genes are needed during the invasion processes. The ialA gene reduces stress-induced dinucleotide levels during invasion, thereby enhancing pathogen survival, and ialB has been shown to probably encode a secreted protein with a direct role in human erythrocyte parasitism (153, 155, 165, 166). It has also been shown that anti-IalB antibodies can block the interaction between IalB and erythrocytes (166). A study by Coleman and Minnick (167) showed that higher ialB mRNA levels occur under acidic conditions (pH 5.0) or in bacteria grown at low temperatures (20°C). Therefore, the erythrocyte invasion would be diminished at 37°C and near neutral pH by downregulation of ialB expression, emphasizing the chronic nature of Carrion's disease. Since the insect midgut becomes acidified at around 3 days postingestion and that female sand flies need to ingest a blood meal every 4 to 5 days, these data suggest that ialB expression is upregulated, leading to maximization of the B. bacilliformis erythrocytic invasion capacity when the sand fly feeds again. Those authors also suggested that if sand fly feeding is interrupted, the ialB expression of ingested B. bacilliformis would be upregulated, then being under optimal conditions to adhere to and invade erythrocytes when transferred to another person during the insect's next feed (167).
Erythrocyte adherence and invasion undoubtedly involve other proteins and factors, some of which have already been identified. For example, actin, α- and β-spectrin, band 3 protein, glycophorin A, and glycophorin B are erythrocyte proteins implicated in the specific binding of B. bacilliformis to the human erythrocyte membrane (152, 164). Moreover, the B. bacilliformis interaction with actin and spectrin suggests that erythrocyte internalization may be related to alterations in cytoskeletal structure (152, 164). B. bacilliformis repeat proteins also seem to play a role in the adherence of the bacteria to host cells by sharing common domains and structural characteristics with the trimeric autotransporter adhesion proteins of B. henselae and B. quintana; these proteins are involved in adhesion to host cells and extracellular matrix proteins (168).
The severity of the hemolytic anemia characteristic of Oroya fever seems to be unique among Bartonella spp. and could be explained by massive infection of the erythrocytes (79). Hemolytic activity is contact dependent and is due to a Bartonella protein, not requiring direct involvement of the erythrocyte proteins.
B. bacilliformis has 3 hemin-binding proteins (Hbps) (168). These proteins are located on the bacterial surfaces and act as hemin receptors. Moreover, data obtained from the analysis of other Bartonella spp. also suggest their possible role as adhesins (169). Other studies have suggested that these proteins may favor the development of the most adequate microaerophilic conditions (170). Two interesting aspects have also been observed in B. quintana and may probably be extrapolated to B. bacilliformis. HbpA (also known as Pap31) is the most frequently expressed of the 5 B. quintana Hbps irrespective of the culture or hemin temperature used. This is of special concern since the equivalent HbpA is an immunologically dominant protein of B. bacilliformis and is considered a good candidate for use in enzyme-linked immunosorbent assay (ELISA) (171) (see “Serological Techniques” within Diagnosis of Carrion's Disease below). Moreover, it has been observed that while the levels of HbpA, HbpD, and HbpE expression increased under simulated human conditions, those of HbpB and HpbC rose when insect conditions were simulated.
In addition, Bartonella spp. also possess another heme acquisition system, the so-called hemin utilization (Hut) system (172); however, to our knowledge, no study has been done for B. bacilliformis.
Lysis of erythrocytes results in intact ghost cells that may contain numerous highly motile B. bacilliformis organisms. B. bacilliformis might use these ghosts to allow host immune system evasion, facilitating the invasion of the microvasculature endothelial cells (173).
In vivo experiments in rats have shown a prolonged period of intraerythrocytic colonization, demonstrating for the first time the persistence of B. tribocorum in erythrocytes (157). The persistence of intracellular erythrocyte parasitism is central in the pathogenesis of all Bartonella spp., allowing them to persist within an immunological environment and increasing the possibility of transmission by blood-sucking arthropods (38, 157, 158) (Fig. 6).
FIG 6
The role of other proteins, such as the 3 Brp proteins belonging to the trimeric autotransporter adhesion (TAA) family, remains to be elucidated, but TAA-related proteins in other Bartonella spp. have been involved in adhesion processes in addition to autoagglutination, inhibition of phagocytosis, and induction of a proangiogenic response (168).
Endothelial Cells and Peruvian Warts
The Peruvian wart appears in the chronic phase of Carrion's disease, with infection of the endothelial cells and their pronounced proliferation resulting in the characteristic skin eruptions. Epithelial or endothelial cells participate more actively in bacterial uptake than erythrocytes, which is facilitated, in part, by the microfilament-dependent activity of these host cells (140, 150). Definite morphological evidence of the presence of B. bacilliformis in Peruvian wart lesions is achieved by the finding of Rocha-Lima's inclusions by light microscopy. Moreover, the bacteria are also found in abundance in the extracellular spaces when there are florid lesions, while they are absent in the resolving nodules (174).
The existence of numerous capillaries within Peruvian warts suggests that infection of endothelial cells induces a local angiogenic response, that is, the formation of new blood vessels. This fact was experimentally demonstrated with a report that B. bacilliformis stimulates endothelial cell proliferation by up to 3 times, as well as the production of the tissue type plasminogen activator (t-PA), in human vascular endothelial cells (HUVECs). B. bacilliformis extracts also stimulated the formation of new blood vessels in an in vivo model for angiogenesis (175). t-PA is involved in different physiological and pathogenic processes, including transformation of plasminogen in plasmin and subsequent fibrinolysis, which, although controversially (because of lack of full in vitro/in vivo correlation), has been reported to be involved in angiogenic processes (176). Further studies confirmed and expanded these results, identifying a heat shock and highly immunogenic protein of B. bacilliformis, GroEL, which plays a key role in the induction of vascular cell proliferation (177). GroEL is actively secreted by the supernatants of B. bacilliformis cultures and acts in a dose-dependent manner by increasing HUVEC numbers by 6- to 20-fold. Cell proliferation is inhibited in the presence of anti-GroEL antibodies (177). The levels of expression of this protein are regulated by different factors, such as temperature or DNA supercoiling relaxation (178), which may be produced at different levels during the different infection phases. Furthermore, the increase in cell numbers could be caused by either increased cell division or reduced cell death. An antiapoptotic mechanism has been reported in Bartonella, accounting in part for its ability to induce vascular proliferation in vivo and enhancing the survival of the host cells and, therefore, itself (179) (Fig. 7).
FIG 7
In situ hybridization has shown that high levels of expression of angiogenesis factors such as vascular endothelial growth factor (VEGF) receptors and angiopoietin-2 are observed in the endothelium of Peruvian warts. Cerimele and colleagues showed that VEGF was produced in the overlying epidermis, demonstrating cooperation between infected endothelium and the overlying epidermis to induce angiogenesis (180). In vitro infection of human endothelial cells by B. bacilliformis results in activation of Rho family GTPases (Rho, Rac, and Cdc42, which are key signaling proteins in pathways involving actin organization), and subsequently the endothelial cell cytoskeleton is remodeled to form filopodia and lamellipodia, which lead to the bacterial invasion of cell. This demonstrates that B. bacilliformis stimulates its own entry into endothelial cells (181–183). B. bacilliformis is a paradigmatic example of strong and specific host adaptation, having the ability to manipulate the host cell with a refined elegance for the purpose of bacterial entry. Indeed, despite its lethality in the acute phase, it has developed the capacity to modify its virulence characteristics during infection, allowing it to survive for a long time in intimate interplay with the host immune system.
IMMUNOLOGY
Little is known about the immunology of Carrion's disease, and more studies in this field are definitely warranted in the future. In fact, most of the present knowledge is inferred from what has been obtained by analyzing other members of the genus. Interleukin-10 (IL-10) secretion is involved in immune modulation by the Bartonella genus (158). IL-10 is an anti-inflammatory cytokine involved in the downregulation of the inflammatory response induced by bacterial products (158, 184). It interferes with the innate immunity by suppressing inflammatory mediators such as T helper cells, monocytes/macrophages, and dendritic cells, among others, interfering with the adaptive immune response (158, 185). Accordingly, the few studies on Carrion's disease have shown a significant elevation of IL-10 and gamma interferon (IFN-γ) in patients in the acute phase, especially in the systemic inflammatory response, which would explain the severe disease course developed by some patients (186). Elevation of IL-10 levels was also described in 2 patients in the acute phase of Carrion's disease during pregnancy, being more prominent in the patient with a more serious clinical outcome (187). Finally, a case of neurobartonellosis with 98% parasitemia showed the highest IL-10 levels among 13 patients diagnosed with acute disease. The patients showed an initial elevation of IFN-γ and significantly low CD4+ and CD8+ T lymphocyte counts, which resolved after appropriate treatment (188). The elevated IL-10 secretion leads to an immune peripheral tolerance which greatly favors asymptomatic infection and persistent infection (158, 188, 189). An analysis of up to 30 different cytokines and chemokines from inhabitants of both postoutbreak areas and areas of endemicity showed the presence of significant differences in some cytokine levels related to the presence or absence of asymptomatic bacteremia and immunoglobulin M (IgM) and IgG levels (190). Thus, in asymptomatic carriers, lower levels of hepatocyte growth factor (HGF) (P = 0.005), IL-15 (P = 0.002), IL-6 (P = 0.05), IFN-γ-inducible protein 10 (IP-10) (P = 0.008), MIG (P = 0.03), and MIP-1α (P = 0.03) were observed, showing a trend to higher IL-1RA levels (P = 0.059). When age was considered, all these cytokines except IL-6 remained significantly underexpressed. In addition, IL-12 was also negatively correlated with the presence of asymptomatic B. bacilliformis bacteremia Moreover, EGF and eotaxin levels were positively, albeit moderately, correlated with bacteremia. HGF, IL-15, IP-10, MIG, and MIP-1α have been shown to be involved in inflammatory processes and T cell and NK cell activation (191–193). Thus, this cytokine profile leads to attenuation of inflammatory processes as well as a reduction in the immune response (190). In the same study, a multimarker analysis developed to show an association with the presence of markers found that HGF, IL-6, IL-15, and IP-10, as well as granulocyte colony-stimulating factor (G-CSF), IFN-γ, MIG, MIP-1α, and RANTES, were negatively associated with the presence of asymptomatic bacteremia, while IL-2, IL-8, IL-10, MCP-1, MIP-1β, and tumor necrosis factor (TNF) had a positive association (190).
Similarities have been observed between Carrion's disease and infections with other Bartonella spp. Circulating IL-10 levels have been described to be significantly higher in patients with cat scratch disease (caused by B. henselae) and in asymptomatic homeless people with B. quintana bacteremia. Moreover, this specific immune profile and an attenuation of the inflammatory response may also account for the chronic persistence of B. quintana (194, 195). In vivo studies showed IL-10 production in culture supernatants of spleen cells from mice inoculated with B. henselae (196). The lack of bacteremia in IL-10 knockout mice infected with B. birtlesii supports the key role of IL-10 in the establishment of Bartonella infection (197).
High rates of seroprevalence are observed in regions of endemicity, and native populations seem to be less susceptible to infection and advanced hemolytic disease than foreigners. It is believed that antibodies may provide long-term protective immunity (198). In addition, an opposite correlation between patient age and disease incidence or severity in regions of endemicity suggests that humoral immunity confers partial immunological protection related to the patient's immune status (35, 50, 198). In the first stages of the acute phase of bartonellosis, total IgM antibody levels rise, while IgG and IgA levels remain normal. Total IgG levels increase after the second to fourth week of treatment. In the eruptive phase, there is a significant, albeit not very marked, increase of total IgA, IgG, and IgM levels (43, 199).
The transient depression of the cellular immunity that occurs during the acute hematic phase is of note (200), with patients presenting lymphopenia with a decrease in CD4+ and a slight increase of CD8+ T lymphocytes. Recent data support the possibility that this immunosuppression remains long after the end of the acute phase (190). These findings may reflect an immune tolerance (lack of a specific immune response to an antigen) which might be triggered by B. bacilliformis, leading to an immune evasion and installation of persistent bacteremia. In fact, a resemblance between this immunosuppression and AIDS has been established (138). Immunosuppression develops in both diseases, causing a deterioration of cellular immunity, which could thereby explain the predisposition to opportunistic infections (6, 138, 140). Treatment may induce immune reconstitution and favor the manifestation of atypical symptoms. The intensity of this inflammatory response probably depends on previous exposure to Bartonella and on individual genetic predisposition (138).
Data on the immune response in the eruptive phase are very scarce. Leukocyte levels remain normal, with a slight tendency to present lymphocytosis (6, 140). Significantly elevated IFN-γ and IL-4 levels were reported in 21 patients in the chronic phase of the disease (187). Interestingly, for healthy preexposed and asymptomatic carriers it was observed that those having elevated IgG levels were more prone to have high eotaxin levels (P = 0.006), while IgG seropositivity was correlated with VEGF levels (P = 0.047), both of which are involved in angiogenic processes (190).
The development of a vaccine inducing both humoral and cellular immune responses is perhaps the most valuable approach to advance toward disease eradication and, overall, to improve the lives of people living along with Carrion's disease as well as to improve public health strategies to manage this disease (39, 166, 168). Several proteins involved in the interactions of B. bacilliformis with the human host, such as flagellin, FtsZ, or IalB, have been proposed as good candidates for the development of a vaccine (166, 168).
During the Colombian outbreak of the 1930s to 1940s, glycerinated and phenolizated B. bacilliformis-rich blood was studied as a possible vaccine, but unsuccessfully (201). The only in-field study regarding vaccines for Carrion's disease was carried out in 1943 by Howe and Hertig (202) using a suspension of 4 inactivated strains of B. bacilliformis and consisted of the active immunization of 22 military guards posted to zones with warty disease. During 1 month of exposure, 75% of nonimmune individuals developed Peruvian warts, and in the other group with 4 months of exposure, approximately 90% of the guards became infected, with two-thirds becoming incapacitated by severe illness (202). Eighty-six percent of the guards developed high titers of agglutinins for B. bacilliformis as a result of one or more inoculations. Although active immunization did not prevent the development of infection and positive cultures in 55% of the guards, only one guard required hospitalization. These results showed improvement in the course of potentially severe Carrion's disease on comparison with the controls. On the other hand, the sensitization of rabbits with B. bacilliformis increased the lethality when they were subsequently administered Bartonella metabolites, indicating that hyperreactive states are dependent on the degree of sensitization (203).
TRANSMISSION
Different routes of transmission of Bartonella have been described or proposed. Nonetheless, the most common and relevant route of Carrion's disease transmission is vector-borne transmission.
Vectors
Townsend identified P. verrucarum as the vector of Carrion's disease by observing its habits and characteristics, the conditions of zones of endemicity, and the distributions of the disease and the insect (15, 204). Nevertheless, the role of L. verrucarum as the vector of Carrion's disease was not firmly supported until the studies of Noguchi et al. in the late 1920s (205), being corroborated by Hertig in 1942 (206) and definitively established in 2004 (207) (see below). Currently, Lutzomyia and Phlebotomus are two different genera within the Phlebotominae subfamily, belonging to the Psychodidae family. While Phlebotomus members live in temperate regions of the Old World, the Lutzomyia genus is present in tropical and subtropical areas of the Americas, from Argentina and Chile to the United States (208).
According to the Theodor classification, the Lutzomyia genus has at least 26 subgenera, accounting overall for approximately 400 species (209). According to both morphological and molecular criteria, a subclassification has been proposed in which L. verrucarum and at least 40 related species, including Lutzomyia maranonensis and Lutzomyia columbiana, have been placed in the so-called verrucarum group (210, 211). The Psychodidae family has a complex taxonomy which is under constant revision; the most recently proposed taxonomy by Galati has resulted in new genera and in the reclassification of different species, including L. verrucarum (classified within the Pintomyia genus, Pifanomyia subgenus, and verrucarum series) (212) (Fig. 8). The Theodor classification (genus Lutzomyia and species L. verrucarum) is used in the present article.
FIG 8
Although L. verrucarum lives in relatively highly arid zones, the presence of humidity and organic material facilitates the establishment of Lutzomyia (31). These findings, together with the presence of sugar sources, have been related to the association between traditional coffee plantations and the presence of Lutzomyia spp. (31). Both male and female Lutzomyia organisms feed on honey dew from aphids or vegetable sugars, but females (except for some autogenous species) also have blood requirements for egg development (208). Thus, L. verrucarum females are responsible for the transmission of B. bacilliformis to humans. The mechanism of this transmission is still unclear (39).
L. verrucarum has classically been distributed at altitudes of 500 to 3,200 masl in inter-Andean valleys and slopes of the center and northern Peruvian Andes (39, 78). L. verrucarum and other Lutzomyia species have a unimodal annual distribution pattern, reaching the population maximum prior to the onset of the summer rainy season (39). Sand fly population densities are directly correlated with the average minimum environmental temperatures and relative humidity. Sand flies are usually found inside houses (35, 37, 213). They have nocturnal habits, feeding from dusk, taking advantage of the decrease of temperature and relative increase of humidity (15, 39, 140). In fact, unusually high numbers of sand flies were collected after El Niño affected Peru in 1997 to 1998 (35, 44).
In the brilliant study by Noguchi et al. (205), several insect species were collected, including three species of Lutzomyia (L. verrucarum, Lutzomyia noguchii, and Lutzomyia peruensis), from districts of Peru in which the disease prevails. The presence of B. bacilliformis in the insects was established by infecting Macaca mulatta (rhesus macaque) with the extract of the crushed insects. To corroborate the results, the blood from the animals was cultured in vitro, yielding cultures of B. bacilliformis which when inoculated in other monkeys, produced verrucous lesions. After recovering, the monkeys showed resistance to a human strain of B. bacilliformis that was later inoculated. The results showed that L. noguchii very likely carried B. bacilliformis and that L. verrucarum was a probable vector. However, there continue to be reservations with respect to L. peruensis (205). Studies in which monkeys were directly bitten by wild L. verrucarum sand flies were later performed by Hertig, resulting in infection of 5 out of 8 monkeys (206). The presence of B. bacilliformis in L. verrucarum was later confirmed by PCR and qPCR (207). In a later study done in Cusco using PCR, 1% of the L. peruensis organisms collected were infected with B. bacilliformis, and this vector was implicated in the transmission of bartonellosis to humans (37).
These data, together with the geographical distribution of L. peruensis, highlight the risk of introducing the illness in Bolivia, from which sporadic cases have been reported (38).
The distribution of Carrion's disease and the presence of L. verrucarum and L. peruensis do not always seem to match, raising the possibility of the involvement of other Lutzomyia species in disease transmission (213, 214). In Peru, in zones of endemicity in the Cajamarca and Amazonas departments, L. maranonensis and Lutzomyia robusta were considered the probable vectors (213, 215). On the other hand, in the Peruvian jungle of the Huanuco department, Lutzomyia serrana was found to be the most probable vector (216). Along this line, the presence of L. verrucarum in Ecuador and Colombia has never been reported (9). As indicated, Colombia was free from Carrion's disease prior to 1936. Indeed, in the first outbreak more than 6,000 deaths were reported (59), highlighting the possible adaptation of another arthropod to the vector role in Carrion's disease; it was postulated that the closely related L. verrucarum species L. columbiana was involved in disease transmission. The results were based on the collection of insects in areas of endemicity using traps followed by the identification of the species collected. However, B. bacilliformis was not identified in any of these studies.
More recently the vector role of L. maranonensis has been confirmed in northern Peru (Cajamarca). Thus, Ulloa-Urizar et al. (217), analyzing 97 pools containing 5 females of L. maranonensis each by PCR, found 2 positive pools, which were confirmed by sequencing as B. bacilliformis. These results expand the area of known established vectors, describing the vector presence in different areas of northern Peru and Ecuador (Table 3).
TABLE 3
| Species | Group | Series | Status | Distributiona |
|---|---|---|---|---|
| L. verrucarum | Verrucarum | Verrucarum | Confirmedb | PE |
| L. peruensis | Vexator | Peruensis | Confirmed | BO, PE |
| L. maranonensis | Verrucarum | Verrucarum | Confirmed | EC, PE |
| L. noguchii | Vexator | Peruensis | Highly probablec | PE |
| L. columbiana | Verrucarum | Verrucarum | Potentiald | CO |
| L. pescei | Vexator | Peruensis | Potential | PE |
| L. robusta | Verrucarum | Serrana | Potential | EC, PE |
| L. serrana | Verrucarum | Serrana | Potential | BO, BR, BZ, CO, CR, EC, GF, GT, HN, MX, NI, PA, PE, VE |
a
BO, Bolivia; BR, Brazil; BZ, Belize; CO, Colombia; CR, Costa Rica; EC, Ecuador; GF, French Guyana; GT, Guatemala; HN, Honduras; MX, Mexico; NI, Nicaragua; PA, Panama; PE, Peru; VE, Venezuela.
b
The presence of Bartonella bacilliformis has been confirmed by use of molecular tools.
c
There were early studies on infecting Macaca mulatta with the extract of the crushed insects, but in the absence of molecular confirmation, there is a risk of insect misidentification.
d
Present in areas of endemicity without established vectors; distribution and vector ecology support the proposal.
Vectors may play a role in the maintenance of vertical transmission by vector-borne microorganisms. This has been described for some viruses, such as dengue virus or chikungunya virus, among others (218, 219), as well as in bacteria, including Bartonella spp., such as B. schoenbuchensis (220) or B. quintana (107). Two compatible pathways have been proposed to explain this phenomenon: transovarial transmission and the contact of eggs and larvae with contaminated insect feces or diuretic fluids (221).
The role of L. verrucarum in the maintenance of the microorganism by vertical transmission was assessed by Ponce and Solorzano (222). L. verrucarum from an area with a high prevalence of insects and illness were fed blood from two patients diagnosed with Oroya fever, with a positive blood smear with 3 and 80% of infected erythrocytes, respectively. After oviposition and the death of the insects, PCR was performed to detect the microorganism. The results showed that insects fed blood with a higher bacteremia had a shorter life, and most of the infected females (35 out of 36) were unable to perform oviposition, while no Bartonella-positive descendant of the remaining infected female was obtained, supporting the hypothesis of a higher mortality of L. verrucarum in the presence of higher bacteremia and the absence of vertical transmission of B. bacilliformis in L. verrucarum (222). A recent report compared B. bacilliformis colonization in a competent (L. verrucarum) and noncompetent (L. longipalpis) vectors. Initially, no differences in the colonization of the two fly species were observed. However, at day 3 the bacteria remained in the abdominal midgut of L. longipalpis, being progressively digested and disappearing at day 7. In L. verrucarum, B. bacilliformis colonizes the digestive tract lumen, persisting for more than 14 days. Thus, L. longipalpis eliminates B. bacilliformis, while bacteria in L. verrucarum survive on blood meal digestion, colonizing the entire digestive tract of the sand fly (223). Although sporadic transmission of Carrion's disease related to L. longipalpis cannot be ruled out because the microorganism remains viable within L. longipalpis for up to 11 days, no mechanical transmission or isolation of viable B. bacilliformis has been obtained from feces or diuretic body fluids (223). B. bacilliformis was not observed in feces or diuretic body fluids of L. longipalpis or in eggs of L. verrucarum and L. longipalpis (223). Unfortunately, analysis of feces or diuretic body fluids was not carried out in the L. verrucarum group.
Other types of vectors cannot be excluded. One report described the transmission of B. bacilliformis from 2 experimentally infected to 2 healthy rhesus monkeys by the bite of the tick Dermacentor andersoni. The infection was mild, and the bacteria were recovered from the lymph nodes and blood of the animals (224). Members of the Eratyrus genus, E. mucronatus and E. cuspidatus, are present in some coastal Ecuadorian areas, and E. mucronatus can also be found in jungle areas of Peru (225). Taking into account the currently expanding distribution of Carrion's disease, this finding, together with the previously mentioned description of “Candidatus Bartonella rondoniensis,” which is closely related to B. bacilliformis, in E. mucronatus make it necessary to evaluate the role of these or other kissing bugs as potential vectors of Carrion's disease.
While nothing is known about the vectors of B. ancashensis, B. rochalimae is widely distributed worldwide through fleas and also ticks. In areas where Carrion's disease is endemic, B. rochalimae has been recovered in ticks and fleas from cats and dogs in both Cajamarca and Lima (86, 90). Moreover, although uncultured, B. rochalimae was detected before to its first full description (57) in a flea from a human from the Cuzco department (226).
Vertical Transmission
To the best of our knowledge, the first proposal of the vertical transmission route was made by Tomas de Salazar as far back as 1858 (142, 227), with the report of a case leading to the death of both the mother following labor and the newborn several days after birth (227). Afterwards, Campodonico and Odriozola described similar findings in 1895 and 1898, respectively (228, 229). Thereafter, in the first third of the 20th century, Monge and Strong in 1912 and 1913, respectively, and both Malpartida and Colaretta in 1937 also reported different cases of vertical transmission (3, 48, 227, 230). More recently, several authors have also reported different cases (15, 48, 231–233), with the most evident description being of a B. bacilliformis-positive culture obtained from the blood of a preterm child collected 90 min after birth (231) (Table 4).
TABLE 4
| Yrb | Mother | Children | Reference(s)c | ||
|---|---|---|---|---|---|
| Diagnosis | Outcome | Diagnosis | Outcome | ||
| 1858 | OF | Death | OF | Death | 227 |
| 1894 | NRd | NR | PWe | Death | 228, 229 |
| 1898 | PW | NR | PW | NR | 228 |
| 1937 | OF | Recovery | OF | Death | 230 |
| 1937 | OF | NR | OF | Death | 51 |
| 1993 | OF | Recovery | OF | Recovery | 232f |
| 2003g | PW | NR | AB? | NR | 231 |
| 2015 | PW | NR | OF | Recovery | 233 |
a
Abbreviations: OF, Oroya fever; PW, Peruvian warts; AB, asymptomatic bacteremia; NR, not reported.
b
If the year of the case was not explicitly reported, the publication year is indicated.
c
Other reports, such as that by Monge (376), highlight the presence of cases in newborns but do not present any specific case. In addition, note that in most of the cases a possible natural sand fly bite transmission cannot be ruled out. In addition, the existence of unreported cases or reports in local congresses or journals is highly probable.
d
In the report it is stated that the parents were healthy, but it is not indicated whether this was stated after physical examination; thus, the presence of Peruvian warts cannot be ruled out.
e
The patient presented a high number of verrucous lesions, but the remaining symptoms may be related to the acute phase of Carrion's disease.
f
A later publication of this case in Laborat-Acta (380) has been reported in different revisions, but despite intensive efforts, we have been unable to find this article.
g
The clearest and an undoubtable case. A B. bacilliformis culture-positive blood sample was collected from a newborn 90 min after birth.
Although the phase of infection is not stated in some of the reports and involves the acute phase in others (48, 227), it seems that in several cases the mother was asymptomatic or presented Peruvian warts (142). This finding may also be related, in part, to the serious adverse events associated with mother or fetal deaths, miscarriages, or preterm births related to acute phase of the disease during pregnancy (15, 32, 142, 234). In fact, in 1898 Odriozola explicitly described the extreme severity of the illness in newborns (229).
Blood Transfusion
Although direct blood contact with contaminated blood is a direct pathway of infection, the number of reports on B. bacilliformis transfusion transmission is scarce and limited to countries where it is endemic. Thus, in 1972 a Carrion's disease-related death of a newborn after a blood transfusion was reported (48), while in 2015 the development of Oroya fever in a polytransfused immunosuppressed patient was described (235). Perhaps the frequent associated use of antimicrobial agents in transfused patients diminishes the risk of disease development.
Blood donors are usually apparently healthy people, and as such those who are infected are actually asymptomatic carriers who probably have a low bacterial load in blood, thereby hindering the detection of this microorganism. Moreover, there are no studies on how long a person can be an asymptomatic carrier, with as long as 3 years having been described in a specific case of an Ecuadorian expatriate (62). This, together with the ability of the microorganism to survive for long periods of up to 30 months in blood stored at 4°C (124), leads to a true risk when transfusions are made in areas of endemicity. In fact, in a PCR analysis of 42 samples from blood donors in an area of nonendemicity in the north of Peru, 2.4% of the samples were found to be positive for B. bacilliformis (236). To avoid this risk, blood banks from Peruvian areas of endemicity include Carrion's disease among infectious diseases to be considered, while no data about other countries are available (237, 238). Although not considered, B. bacilliformis testing in blood banks outside countries where it is endemic would be extremely valuable due to the natural movement of people who may provide unexpected infected donations in far-away areas. In fact, as indicated above, it has been estimated that more than 2.5 million Peruvians live outside the country, with 22.1% of these individuals being from mountain areas (65).
Organ transplantation.
To the best of our knowledge no B. bacilliformis infection related to organ transplantation has ever been described. Nonetheless, it should be noted that strong evidence of other posttransplant Bartonella sp. infections has been reported (239).
RESERVOIRS OF B. BACILLIFORMIS
Various candidates have been suggested as the reservoir of Carrion's disease. Euphorb plants were initially proposed as reservoirs because of their geographical correlation with warty zones as well as the seasonal incidence of the disease and the period of greatest plant growth. Herrer tried to recover B. bacilliformis from euphorb plants in zones of endemicity and to experimentally infect these plants with cultures of this microorganism (240). Thereafter, he determined whether latex from plants allowed the development of B. bacilliformis in culture. The results of all of the above-described experiments were negative: B. bacilliformis was not recovered from the Euphorbiaceous plants, no infection was produced in the plants, and the conditions for the in vitro development of the bacteria in latex were adverse. Altogether, these results demonstrated that euphorb plants do not act as a reservoir of Carrion's disease (240).
Several studies have been performed in animals in the search for other potential reservoirs. Noguchi successfully demonstrated that M. mulatta is susceptible to infection by B. bacilliformis. The microorganisms were found in the red blood cells of the animals and recovered in in vitro cultures. The clinical manifestations in monkeys are less severe than those of human disease, and the nodules which developed resembled those of the Peruvian wart (18). Similarly, in 1942, Hertig (206) was able to isolate B. bacilliformis from blood of rhesus macaques experimentally exposed to the bite of wild L. verrucarum. More recently, it has been shown that owl monkeys (Aotus nancymaae), which are present in the South American jungles, including those of northern Peru, may be experimentally infected. Six monkeys were inoculated, 3 intradermally (in order to simulate the bites of the sand fly) and 3 intravenously. In 4 out of the 6 monkeys (2 from each group) the presence of the microorganism was observed within the erythrocytes at 21 days after inoculation. Moreover, a more-than-4-fold increase in IgM levels was observed in the 3 intradermally and 1 intravenously inoculated animals. Similar to the results of the previous study, the symptoms were less evident, and no positive PCR or successful culture was obtained (241).
These data warn about a latent risk of the expansion of Carrion's disease, with the possible arrival of infected sand flies to areas inhabited by owl monkeys (present in different areas of northern Peru) which might be infected and act as natural dispersing agents toward neighboring areas in Brazil.
Cooper et al. proposed that human bartonellosis is a zoonosis in which wild animals, most probably rodents, are the natural reservoir (58, 242). However, this proposal was based on case-control studies using questionnaires. No animal was tested for B. bacilliformis, and the results were never confirmed in later studies. The isolation of B. bacilliformis from the blood of a single rodent belonging to the genus Phyllotis from an area of endemicity in central Peru (Rimac valley) was reported, but no attempts to infect other rodents were successful (68). To our knowledge no other isolation of B. bacilliformis from blood samples of an animal has been reported, and possible misidentification cannot be ruled out (6). It is known that inhabitants of zones of endemicity usually have pets which are susceptible to infection with Bartonella spp., and these animals can present warts similar to those of patients with Peruvian warts. However, the presence of B. bacilliformis in warts from these animals has never been microbiologically demonstrated (6). A survey in which the blood of 50 animals from 11 homes of families with children who had recently had bartonellosis was collected was unable to identify an animal reservoir for B. bacilliformis (243). Moreover, in a study done by Solano and colleagues (244), blood smears and blood cultures were obtained from dogs, guinea pigs, and wild mice as well as healthy individuals from areas of endemicity in Peru. No positive results were obtained for blood smears, and no positive cultures were obtained from the wild animals. On the other hand, 12% of human samples were found to be positive for B. bacilliformis (244). Different studies showed that between 47% and 54% of patients with warty disease were bacteremic (35, 147), while Gomes et al. (50) observed 40 to 50% of bacteremic asymptomatic carriers, suggesting that patients with chronic disease and asymptomatic carriers are the most likely reservoirs.
Despite the proposal involving camelids as the original source of primitive B. bacilliformis (92), humans are currently the only established reservoir for Carrion's disease, although the absence of other vegetal or animal reservoirs for B. bacilliformis has never been definitively demonstrated. Most studies have been carried out with techniques with a low sensitivity, and thus, studies using techniques able to detect low bacteremia, such as serological techniques and/or qPCR, are needed.
DIAGNOSIS OF CARRION′S DISEASE
Carrion's disease affects the poorest populations from remote isolated rural areas, which have poor communications and poorly equipped laboratories (38). Currently, the diagnosis of Carrion's disease presents important limitations. The initial febrile stage is often misdiagnosed because its symptomatology is similar to that of other illnesses (27, 37–39, 139, 250). Although the warty phase is easier to diagnose, incorrect diagnosis can be made, especially in regions where the disease is not present and in cases imported from areas where it is endemic. One example was reported by Maguiña et al., in which erroneous initial diagnoses were made in 19% of patients, including skin tumor in 7 patients, hemangioma in 5 patients, polyarthralgias in 2 patients, and systemic lupus erythematosis in 1 patient (139). Moreover, without the diagnosis of asymptomatic carriers, perpetuation of the disease will never end.
Routine Diagnostic Techniques
In rural areas of endemicity, the diagnosis of Oroya fever is based mainly on clinical symptoms and Giemsa-stained peripheral blood smears, a low-cost and easy-to-use technique. Nonetheless, this method is requires expertise, and despite the high specificity of 96%, a very low sensitivity (24 to 36%) has been reported, especially in mild cases of disease and in the subclinical and chronic phases of illness (37, 38, 251). Moreover, the unspecific initial symptoms of Carrion's disease should be taken into account, since they are common to several pathologies present in these areas, such as different arboviral diseases, malaria, or tuberculosis, thereby making diagnosis based only on clinical symptoms difficult (15). In fact, outbreaks of Carrion's disease have been reported using microscopy tools and clinical diagnostic techniques (http://www.promedmail.org/direct.php?id=20130304.1569888) although analysis by molecular tools revealed another etiological cause (252). In the chronic stage of the disease, clinical diagnosis is made by the presence of cutaneous angiomatous skin lesions. While microscopic techniques have 36% sensitivity in the acute phase, a decrease to less than 10% is observed in Peruvian warts (140), related to lower blood bacterial carriage. Histopathological diagnosis is possible, albeit difficult, with staining of sections with Warthin-Starry silver or Giemsa stain demonstrating the presence of bacteria in the skin eruptions (39).
B. bacilliformis culture does not have clinical utility in diagnosis due to the culture requirements and especially because of the low bacterial growth rate (1 to 6 weeks). Moreover, it is cumbersome and time-consuming, and contaminations have been described in 7 to 20% of the cultures (15, 139). Additionally, the sensitivity of this method is extremely low. In one study a considerable portion of Peruvian wart cases yielded negative blood cultures (70), and another study showed that only 13% of patients with Peruvian warts had a positive culture or blood film (139). Moreover, a study in which cultures from persons known to be infected with B. bacilliformis were performed over a 6-month period supported the suggestion that the proportion of positive cultures is much higher in the first months of infection than later (70). Finally, different researchers have evaluated the use of different insect-based liquid culture media (253–255) to isolate and grow Bartonella spp., but to our knowledge, no study on the utility of these media in the isolation and growth of B. bacilliformis has been developed.
Molecular Techniques
Several PCR approaches have been described. However, these studies usually involve a small number of samples, and additionally, as occurs with the remaining diagnostic tools, they are hampered by the lack of a standard case definition. Moreover, it is necessary to identify genetic targets able to differentiate human-pathogenic strains from those that are less virulent (256). PCR approaches are more effective than optical microscopic and culture techniques, being able to diagnose Carrion's disease patients in the acute phase previously classified as negative by thin blood smear (250). Nonetheless, a critical issue is the detection limit of these techniques, raising doubts about their usefulness in the detection of low bacteremia or asymptomatic carriers. Moreover, it should be kept in mind that routine implementation of molecular techniques in remote endemic rural areas is difficult because of the lack of technical resources and trained personnel (38). In fact, molecular techniques are used only in reference centers, located mainly in Lima (Peru). This fact also leads to another problem, which is the need for rapid, consistent transfer of samples and the return of diagnostic results from reference centers to local health facilities.
PCR can be done directly in affected tissues and/or blood or in enrichment cultures (39). With enrichment, the conventional PCR positivity increases by 55% compared with that for original blood samples (257). This enrichment technique needs 14 days for development, which leads to an unaffordable delay in the processing of samples, making it impracticable in clinical diagnosis. The use of dried blood spots (DBS) may make transportation of samples from areas of endemicity to reference laboratories easier. Moreover, the small blood volumes make the use of DBS especially interesting in the pediatric setting.
Several PCR assays for the detection and identification of Bartonella species have been reported in the literature. A nested PCR using the cell division gene ftsZ to differentiate among B. bacilliformis, B. quintana, and B. henselae was described by Kelly et al. (258). Additional approaches include several PCR restriction fragment length polymorphism (RFLP) methods, including use of RNA polymerase beta subunit (rpoB) gene analysis (259), the citrate synthase (gltA) gene (260, 261), the ftsZ gene (262), and the 16S rRNA gene (263). Molecular analyses based on only one-step PCRs have also been described. The riboflavin synthesis genes, such as ribC, ribD, and ribE, are absent in humans and present in bacteria and therefore are useful either for bacterial DNA detection in human samples or for the differentiation of Bartonella species (264). The ialB gene has been proposed for the diagnosis of B. bacilliformis on demonstrating high sensitivity and specificity in 10 blood samples from acute-phase patients with confirmed thin blood smears (265). Nonetheless, further studies showed its presence in other members of the genus, such as B. henselae and B. birtlesii, thus making it a useful tool for Bartonella sp. infection (166) but not discriminatory for B. bacilliformis. The amplification of the 16S rRNA gene has also been proposed for the diagnosis of Carrion's disease in Peru (250). However, several studies have warned about the utility of the 16S rRNA gene as a means of differentiating Bartonella species due to the high conservation of this gene (266, 267). Hypervariable intergenic transcribed spacer (ITS) 16S-23S rRNA amplification allows differentiation between B. bacilliformis and B. ancashensis from the main pathogenic Bartonella spp. (268). In fact, the ITS region has been used in different studies of Bartonella spp. (89, 269). Another PCR approach is multiplex PCR based on amplification of the ITS gene combined with reverse line blotting, which allows distinguishing among 20 different Bartonella species (270).
The detection limits of 3 different PCR approaches have recently been determined in blood and DBS artificially infected with Bartonella. Two of these approaches, targeting the 16S rRNA and fla genes, had a detection limit of 5 CFU/μl when infected blood was tested. This sensitivity was maintained in the case of 16S rRNA gene amplification when applied to DBS but decreased to 500 CFU/ml in the case of fla amplification. The third approach targeted the ITS region and showed a detection limit of 500 CFU/μl in both artificially infected blood and DBS. The sensitivity of the 16S rRNA PCR approaches seems to be high enough to diagnose acute cases. Nonetheless, there continue to be doubts regarding the usefulness of PCR in the detection of asymptomatic carriers (271).
Finally, recent studies have demonstrated the utility of more sensitive molecular techniques such as qPCR. Using this technique, Smit et al. showed that 24.6% of 65 children were positive whereas only 3% were blood culture positive (272), and Gomes et al. detected 38% of asymptomatic carriers in a postoutbreak zone and 50% in an area of endemicity (50). In another study with animal blood samples, the sequences of a region of the ssrA gene amplified by qPCR were able to discriminate Bartonella species (256). Along the same line, a study on the detection of the ITS region using qPCR, which is able to detect a minimum of 13 Bartonella spp., including B. bacilliformis, in silico, showed a sensitivity of 50 genomes per reaction (273). A two-step protocol consisting of a genus-specific qPCR with the gltA gene as the target followed by pyrosequencing of qPCR-positive specimens directed against a segment of the rpoB gene was also described for differentiation of at least 11 medically relevant Bartonella spp. Despite being a two-step protocol, it can be done in 5 h, making it an alternative to the time-consuming sequencing of a PCR product (274). On the other hand, a qPCR targeting the NADH dehydrogenase gamma subunit gene (nuoG) was found to be sensitive and specific enough to detect diverse Bartonella species, maintaining minimal cross-reactivity to mammalian host and arthropod vector organisms (94). Angkasekwinai et al. (275) described a loop-mediated isothermal amplification with pap31 as the target gene. This is a simple method to detect the presence of B. bacilliformis DNA within 1 h and requires less specialized equipment. This method has a detection limit of between 1 and 10 copies/μl, depending on whether bacterial genomic DNA is used alone or in the presence of human DNA, respectively. Nonetheless, its usefulness remains to be validated in human clinical samples.
Serological Techniques
Serological tests are useful diagnostic tools for Carrion's disease, especially when combined with other techniques such as PCR and blood culture. However, immune diagnostic methods for this disease are relatively underdeveloped (39), and as mentioned above, the most-studied approaches, such as indirect fluorescence antibody assay (IFA) or enzyme-linked immunosorbent assay (ELISA), have the disadvantage of limited technical resources in areas of endemicity. In this context, the characterization of the antigens expressed during B. bacilliformis infection, which is essential in the elucidation of Carrion's disease pathogenesis, may also lead toward the development of rapid diagnostic tools (38, 276). A rapid diagnostic test which does not need experienced personnel or high-technology machinery could be easily introduced in low-income areas after basic training. Moreover, results can be obtained quickly, thereby facilitating early initiation of antibiotic treatment.
In 1988 the first study identifying B. bacilliformis antigens was published (277), which described 24 proteins, including two main antigens of 65 and 75 kDa. BB65 is a heat shock protein later identified as GroEL, and the 75-kDa antigen corresponds to the cell division protein FtsZ (277–280). Later studies have described the identification of GroEL, leading to the identification of antigenic candidates using anti-human IgG as a secondary antibody (50). Although Gomes et al. (50) detected anti-GroEL IgM antibodies, other authors have found that GroEL does not bind to Bartonella IgM, binding to IgG antibodies 2 weeks after infection and remaining detectable at least until the third year after B. bacilliformis infection. However, no anti-GroEL antibodies are detected in the 40% of Peruvian wart patients (278). GroEL has mitogenic activity against HUVECs, resulting in the development of warts. The partial inhibition of this mitogenic activity when anti-GroEL antibodies are present suggests their protective role in asymptomatic carriers (177). Recently, it has been reported that this protein has been tested in a rapid immunochromatographic assay, using rabbit IgG polyclonal anti-GroEL antibodies with a sensitivity equal to or lower than 102 CFU/ml (281).
The first study which attempted to identify the outer membrane proteins (OMPs) of B. bacilliformis led to the identification of 14 OMPs ranging from 11.2 to 75.3 kDa. The most prominent immunoprecipitants with rabbit anti-Bartonella hyperimmune serum were proteins of 31.5, 42, and 45 kDa, which may represent the immunodominant OMPs of the pathogen (159).
An antigenic lipoprotein of 43 kDa was found by Padmalayam et al. by performing a screening of a genomic DNA lambda library with serum from a patient in the chronic phase of the disease (276). An ELISA using the 43-kDa lipoprotein in its recombinant form was described. The sensitivities of IgG and IgM ELISAs were 70.4 and 85.2%, respectively, and the specificity for both IgG and IgM was 90%. Despite the good sensitivity and specificity, 3 sera from chronic-phase patients had high titers of IgM, indicating that the IgM ELISA does not discriminate between the two phases of the disease (282).
Another ELISA using the recombinant flagellin gene (fla) of B. bacilliformis was assessed against sera from patients with bartonellosis and healthy controls. The sensitivity of this method was 58%, and when sera from people with other pathologies were tested, a specificity of 69% was obtained. Moreover, 15% of sera from healthy people also gave a positive result (283). One possible explanation for the worse results on comparison with the 43-kDa lipoprotein ELISA may be that the flagellin of B. bacilliformis is very similar to other flagellins and may share epitopes with other bacteria (282). More recently the immunological dominance and high expression of Pap31 (HbpA) in B. bacilliformis cultures was described, and this was considered a good candidate for use in ELISA (171). Furthermore, Hbps have been proposed as serodiagnostic tools for B. quintana infections (284), and this usefulness probably could be extrapolated to other Bartonella infections. More recently, anti-Pap31 IgM has been detected in serum samples from healthy inhabitants of both postoutbreak areas and areas of endemicity irrespective of their asymptomatic carrier status (50). That study also described 2 new antigenic candidates: the two subunits of succinyl coenzyme A (succinyl-CoA) synthetase (SCS-α and SCS-β) (50). These new antigenic candidates are implicated in the tricarboxylic acid cycle. They are described as immunogenic protein of Brucella melitensis (SCS-α) (285) and as being involved in the pathogenesis of B. henselae infection (SCS-β) (286). Finally, through in silico analysis, the antigenic potential of 16 outer membrane or secreted proteins has been described (287).
Although further studies are needed, the use of more than one antigenic candidate may be the best option for a rapid diagnostic test. Indeed, other authors have suggested the need to evaluate other proteins of B. bacilliformis in order to improve the sensitivity or specificity of the serological test, and in order to achieve this goal, a cocktail of recombinant proteins or synthetic peptides would be necessary (283).
B. bacilliformis immunoblot sonication of whole organisms is remarkably sensitive to B. bacilliformis antibodies from sera of patients with chronic disease (94%) and also yields reasonable results in acute disease (70%). However, some cross-reactions with sera with antigenically similar bacteria such as Brucella spp. and Chlamydia psittaci were reported in 34% and 5% of the cases, respectively (251). Indeed, an intense cross-reactivity between B. bacilliformis and C. psittaci due to common surface epitopes has also been reported previously (288).
Chamberlin et al. (148) described an IFA with irradiated B. bacilliformis whole-cell antigen preparation cocultivated with Vero cells in which the 81% of acute confirmed cases were positive. Interestingly, 74% of the volunteers having a positive IFA reported bartonellosis within the last year, which decreased to 39% when a more distant or nonbartonellosis episode was reported. Overall, 45% of the volunteers from an area where Carrion's disease is endemic were seropositive for B. bacilliformis by this technique (148).
TREATMENT OF CARRION′S DISEASE
The treatment of Carrion's disease differs depending on which phase of the disease is presented and includes blood transfusions and antimicrobial agents. With regard to antimicrobial therapy, different antibacterial agents have been tested since the beginning of the antibiotic era (289). Nonetheless, the current clinical practice relies mostly on personal experience and expert opinion (290). In any case, no clinical assays have been developed to define the real usefulness of the different antibiotic schedules proposed to treat Oroya fever, and only one clinical assay (www.isrctn.com/ISRCTN16597283?q=&filters=recruitmentCountry:Peru&sort=&offset=2&totalResults=24&page=1&pageSize=10&searchType=basic-search) has been developed for the chronic phase (291).
Treatment of Oroya Fever
It is often considered that B. bacilliformis was the most lethal bacterium in the preantibiotic era. Left untreated, Oroya fever is frequently referred to as having one of the highest death rates of all infectious diseases, being from 40 to 85% (141) and achieving 88% in a study by Gray and colleagues (26). Fortunately, effective treatment is now available, and the mortality rates have decreased.
The first treatments were based on traditional folklore, including herbal extracts such as “uña de gato” (Uncaria tormentosa) or “agua de mote con vino o chancaca” (water in which maize has been cooked and then mixed with wine or chancaca) (292), as well as dubious procedures based on the use of oral or endovenous glycerin, tripaflavin, chaulmogra oil, or para-aminobenzoic acid, among other substances (293, 294). Repeated injections of citrate blood from the same patient were also made (295). It is of note that in several areas of endemicity, some of these traditional treatments were reported to remain in use as late as 1978 (6). In order to “provoke the sprout” of warts, herbal extracts such as those mentioned above or others such as “quisuar” (Buddleja incana) infusion were used. Moreover, for the treatment of symptomatic pain and discomfort, thermal baths were made or methods to “absorb negative energies” such as the so-called “sobada de cuy” (rubbing a black guinea pig all over the patient's body) were applied.
Salvarsan and quinine were also tested for use during acute infections, with unsatisfactory results (289, 294). Immunotherapies were also tested, with immune rabbit serum with high antibodies titers being used in the early 1940s without conclusive results (134).
Blood transfusions are perhaps the most classical treatment and are recommended to treat the severe anemia typically found in the acute phase of the illness (33, 62, 145, 296). Thus, although the life of transfused erythrocytes is shortened, the survival is normal in at least 50% of cases (297).
Although several studies were developed in the 19th century, it is largely considered that the first antimicrobial discovered was penicillin by Alexander Fleming in 1929, which was introduced in clinical practice coinciding with World War II (298–300). Regarding Carrion's disease, the first treatments with penicillin were reported in 1944 (289, 379). Subsequently, several antibiotics were tested. A series of case reports in the literature described the use of antimicrobial agents such as streptomycin, chloromycetin (chloramphenicol), aureomycin (chlortetracycline), ilocitin (erythromycin), furadantin (nitrofurantoin), or acromycin (tetracycline) with satisfactory results, while terramicin (oxytetracycline), neomycin, or sulfamides showed poor or unsatisfactory results (294, 301–303).
Since 2003 and according to the Ministry of Health of Peru recommendations and guidelines, ciprofloxacin is the drug of choice for adults in the acute phase of Carrion's disease, while in severe cases, ceftriaxone should be added to the treatment (304) (Table 5). However, as mentioned above, B. bacilliformis is intrinsically resistant to nalidixic acid (305), and it has been suggested that ciprofloxacin is not adequate for treatment of the acute phase and should be removed from the current guidelines (38, 306). Nonetheless, the issue of the most adequate antimicrobial remains controversial. On one hand, successful treatment has been reported, as in the case of a patient with a massive erythrocyte infection (more than 95%) who received a 10-day course of ciprofloxacin and ceftriaxone with full recovery (307), while on the other hand, therapeutic failure and persistent bacteremia of up to 22.6% have been described in patients who have completed treatment schedules (308).
TABLE 5
| Demographic group | Phase | Illness | First-line treatment | Second-line treatment | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Antibiotic(s) | Dosec> | Interval | Duration | Route | Antibiotic(s) | Dosec | Interval (h) | Duration (days) | Route | ||||
| Childrenb | OF | Uncomplicated | AMC | 20 | 12 h | 14 days | p.o. | CIP | 5 | 12 | 14 | p.o. | |
| CHLd | 16.6/10 | 8 | 3/11 | p.o. | |||||||||
| SXT | 5 | 12 | 14 | p.o. | |||||||||
| Severe | CIP + CRO | CIP, 5–7.5 | 12 h | 14 days | i.v./p.o. | CIP + CAZ | CIP, 5–7.5 | 12 | 14 | i.v./p.o. | |||
| CRO, 70 | 24 he | 7–10 days | i.v. | CAZ, 16.6–33.3 | 8 | 7–10 | i.v. | ||||||
| CIP + AK | CIP, 5–7.5 | 12 | 14 | i.v./p.o. | |||||||||
| AK, 7.5 | 12 | 7–10 | i.v./i.m. | ||||||||||
| PW | AZM | 10 | 24 h | 7 days | p.o. | RFP | 10 | 24 | 21–28 | p.o. | |||
| ERY | 7.5–12.5 | 6 | 14 | p.o. | |||||||||
| CIP | 5 | 12 | 14 | p.o. | |||||||||
| Teenagers and adults | OF | Uncomplicated | CIP | 500 mg | 12 h | 14 days | p.o. | AMC | 1 g | 12 | 14 | p.o. | |
| SXT | 800 mg | 12 | 14 | p.o. | |||||||||
| CHLd | 16.6/10 | 8 | 3/11 | p.o. | |||||||||
| Severe | CIP + CRO | CIPf, 400/200 mg | 12 h | 3 days/11 days | i.v./p.o. | CIP + CAZ | CIPf, 400/200 mg | 12 | 3/11 | i.v./p.o. | |||
| CRO, 2 g | 24 h | 7–10 days | i.v. | CAZ, 1 g | 8 | 7 | i.v. | ||||||
| CIP+AK | CIPf, 400/200 mg | 12 | 3/11 | i.v./p.o. | |||||||||
| AKg, 7.5 | 12 | 7–10 | i.v./i.m. | ||||||||||
| PW | AZM | 500 mg | 24 h | 7 days | p.o. | RIF | 600 mg | 24 | 21–28 | p.o. | |||
| ERY | 500 mg | 6 | 14 | p.o. | |||||||||
| CIP | 500 mg | 12 | 14 | p.o. | |||||||||
| Pregnant or breast-feeding women | OF | Uncomplicated | AMC | 1 g | 12 h | 14 days | p.o. | CHLd | 16.6/10 | 8 | 3/11 | p.o. | |
| SXT | 800 mg | 12 | 14 | p.o. | |||||||||
| AMX | 1 g | 8 | 14 | p.o. | |||||||||
| Severe | CRO + CHL | CRO | 1 g | 12 h | 10 days | i.v. | CRO + AK | CRO, 1 g | 12 | 10 | i.v. | ||
| AK, 500 mg | 12 | 7–10 | i.v./i.m. | ||||||||||
| CHLh | 1 g/500 mg | 8 h/6 h | 5 days/11 days | i.v./p.o. | CAZ + AK | CAZ, 1 g | 12 | 10 | i.v. | ||||
| AK, 500 mg | 12 | 7–10 | i.v./i.m. | ||||||||||
| PW | AZM | 1 g | 1 day/1 wki | 3 w | RFP | 600 mg | 24 | 21–28 | p.o. | ||||
| ERY | 500 mg | 6 | 14 | p.o. | |||||||||
a
Based on current official guidelines (34, 377). Abbreviations: OF, Oroya fever; PW, Peruvian warts; p.o., oral; i.v., intravenous; i.m., intramuscular; AK, amikacin; AMC, amoxicillin plus clavulanic acid; AMX, amoxicillin; AZM, azithromycin; CAZ, ceftazidime; CIP, ciprofloxacin; CRO, ceftriaxone; CHL, chloramphenicol; ERY, erythromycin; RFP, rifampin; SXT, co-trimoxazole.
b
Individuals up to 14 years old or weighing <45 kg (if the weight is higher the teenager/adult guidelines will be followed). Doses for children are reported in milligrams per kilogram of body weight. Note that specific situations or complications may require schedule modifications (34, 377). In addition, for adults/teenagers and for pregnant women when no units are indicated, the doses are reported in milligrams per kilogram of body weight.
c
For amoxicillin plus clavulanic acid or co-trimoxazole, the dose is in reference to the first antibiotic compound of each combination.
d
Fifty milligrams per kilogram per day for 3 days; thereafter, 30 mg/kg/day for 11 days.
e
In the guidelines 70 mg/kg/day is indicated, but no dosage information is provided.
f
Four hundred milligrams every 12 h for 3 days; thereafter, 200 mg every 12 h.
g
The maximum dose is 1.5 g/day.
h
One gram every 8 h for 5 days (i.v.) followed by 500 mg every 6 h for 11 days (p.o., if possible).
i
One gram per day once a week for 3 weeks.
Prior to the inclusion of ciprofloxacin in the guidelines, some studies reported the use of chloramphenicol with good results (6, 139, 294, 309–311). In fact, although chloramphenicol had been extensively used since 1956 (48), a medical consensus considered this drug to be the treatment of choice for bartonelosis in acute cases from 1998 to 2003. It was then replaced by ciprofloxacin and relegated to rescue therapy. The reason for the change was the lack of clinical response in some patients (312–314), which had been sporadically reported early in the 1950s (310). Moreover, similar to the case for chloramphenicol (15, 145) ciprofloxacin provided good coverage against Salmonella infections (315). In the study by Gray and colleagues mentioned above, none of 10 patients treated with chloramphenicol died, compared with 88% mortality among nontreated patients (26). In another study, 19 patients received chloramphenicol for 5 days and similar results were obtained; the temperature returned to normal within 24 h, and the erythrocyte count and size were reestablished. Relapse was infrequent, and when it occurred, further administration of chloramphenicol resulted in recovery (316). Moreover, in a retrospective report on 215 patients, clinical cure was obtained with chloramphenicol in 89% of the cases (313). A good response to therapy was also achieved in 23 patients with the use of chloramphenicol and another antibiotic (139).
The acute illness is often complicated by other infections, usually with Salmonella spp., which greatly intensifies the clinical condition of the patient (33, 143). In fact, the lack of improvement after 72 h of treatment may allow suspicion of a coinfection. A treatment scheme based on chloramphenicol has the advantage that it is a low-cost, broad-spectrum antibiotic which also covers potential coinfections (39). However, despite the effectiveness of chloramphenicol in some patients, therapeutic failures and persistent bacteremia, leading to asymptomatic carrier status, have been reported in other patients receiving this drug (37, 139, 308). In a study by Maguiña et al., 3 out of 42 patients did not respond well to chloramphenicol therapy, and it is notable that these 3 patients had an initial microorganism burden of >80% (139). Another study reported 50% persistent bacteremia after chloramphenicol treatment in 66 patients; 28.8% presented posterior Peruvian warts, and the remaining presented positive blood cultures or PCR results (308). Moreover, in Europe as well as other locations, the use of chloramphenicol in humans is restricted, and its use in livestock production is strictly forbidden because of its potential to produce side effects in the bone marrow (317).
Regarding Salmonella coinfections and ciprofloxacin, in the last years the percentage of clinical isolates of Salmonella spp. resistant to nalidixic acid has been on the rise worldwide (318–321), and it is extremely high in Peru (322, 323). This may lead to therapeutic failure during ciprofloxacin treatment of bloodstream Salmonella infections (324), being an established risk for the development of ciprofloxacin resistance (325). Moreover, although has been postulated that the development of ciprofloxacin-resistant Salmonella is difficult because of the severe effects on bacterial fitness, with impaired growth and decreased virulence capacity (326, 327), reports on ciprofloxacin-resistant isolates and the emergence of successful ciprofloxacin-resistant Salmonella clones have been described worldwide in the last years (319, 320).
These findings, together with the relevant presence of extended-spectrum β-lactamase (ESBL)-carrying Salmonella spp. in the area (323), indicate a scenario in which correct and early diagnosis and analysis of resistance levels of both B. bacilliformis and concomitant opportunistic pathogens are needed.
Treatment of Peruvian Wart
From 1969 to 1975 streptomycin was the drug of choice for the treatment of the chronic phase of Carrion's disease, and in the mid-1970s rifampin was introduced and became the first-line drug, showing better results than streptomycin (139, 328). In a study of 260 chronic-phase patients receiving rifampin, clinical cure was observed in 93.1% (313). Nonetheless, treatment failure with rifampin for Peruvian wart has also been reported (309, 329, 330), and alternatives have been used in the treatment of chronic-phase patients. A good response was obtained in an eruptive case treated with chloramphenicol (63), and in another case, a 12-year-old child was treated with sultamicillin and deflazacort for 10 days and showed rapid improvement of the overall symptoms and complete remission of skin lesions at 21 days (331). More recently, after the successful use of azithromycin (309), the Ministry of Health of Peru proposed the use of this antimicrobial (304) for the eruptive phase of the disease (Table 5).
MECHANISMS OF ANTIMICROBIAL RESISTANCE
In intracellular bacteria, such as B. bacilliformis, antibiotic resistance is related mainly to wild-type-specific target-encoded gene sequences or those acquired by inactivation or structure modification of antibiotic targets related to encoding DNA events such as insertions, deletions, or punctual mutations.
Although no plasmid has yet been reported in B. bacilliformis, their presence has been determined in other members of the genus Bartonella, with the first being described in 2003 in Bartonella grahamii (332). Thereafter, the presence of plasmids has been reported in other Bartonella spp., such as B. tribocorum, B. schoenbuchensis, and Bartonella rattaustraliani; interestingly, most of these plasmids encode proteins belong to T4SS (85, 92, 333). Moreover, it has been shown that other species of Bartonella may exchange plasmids with other microorganisms within amoebae (333). This finding suggests that the gut eukaryotome of Lutzomyia spp. might potentially act to favor the horizontal exchange of genetic material between B. bacilliformis and other microorganisms.
Fortunately, Bartonella spp. continues to be highly susceptible to antibiotics (334). Antimicrobial resistance levels have been established in a few clinical or collection isolates of B. bacilliformis (123, 305, 314, 335–337), and it should be taken into account that in vitro susceptibility does not consistently correlate with the in vivo outcomes for patients (330). In addition to the constitutive nalidixic acid resistance and related diminished fluoroquinolone susceptibility described previously (305, 338), in a recent report 26% of isolates presented resistance to ciprofloxacin (314). The current scenario is completed with the relatively high MICs of clindamycin and colistin (335), the sporadic low resistance to chloramphenicol, and the trend toward diminished susceptibility to some aminoglycosides (314, 336).
Despite the description of antibiotic treatment failure as early as 1948 (339), information about the mechanisms of antimicrobial resistance in B. bacilliformis is scarce and is mostly focused on constitutive quinolone resistance. Indeed, studies analyzing the quinolone resistance-determining regions (QRDRs) of B. bacilliformis clinical isolates, including several recovered prior to the introduction of quinolones, showed an alanine at both position 91 of GyrA and position 85 of ParC, which are responsible for the intrinsic resistance of B. bacilliformis to quinolones (305, 337). This characteristic has also been reported in other members of the Bartonella genus from which the DNA sequence encoding GyrA has been determined (338), as well as in other microorganisms having constitutive resistance to quinolones, such as Brevundimonas diminuta (340).
To our knowledge only four in vitro studies have been developed with B. bacilliformis, selecting in vitro resistance to different antimicrobial agents, including chloramphenicol, ciprofloxacin, coumermycin A1, erythromycin, and rifampin (123, 330, 341, 342). In these studies, ciprofloxacin resistance ranked among the most easily selected, while chloramphenicol resistance ranked among the most difficult to obtain (123). Additionally, 3 reports have also analyzed the presence of antibiotic resistance-related mutations in clinical isolates of B. bacilliformis (337, 343, 344), but 1 of these studies did not include the MICs of the antibiotic (344) (Table 6).
TABLE 6
| Study type | Mutation(s) causing resistance toa: | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Cip | Azm | Chl | Rif | Cou A1 | Strep | Trim | ||||
| GyrA | GyrB | L4 | L22 | 23S rRNA | 23S rRNA | RpoB | GyrB | S7 | DHFR | |
| In vitro | D90→G, A91→V, D95→G, D95→N | E475→K | Δ62-65, Q66→K, G70→R, H74→Yb | 83::VSEAHVGKS | A1983→G | G2372→A | Q527→R, H540→Y, S545→F, S588→Y | G124→S, R184→Q, T214→A, T214→I | ||
| In vivoc | G89→C, A91→V, S92→P, D95→N, D95→Y | S472→F ?d | A39→Te, D48→Ee | H115→Re | ||||||
a
Abbreviations: Cip, ciprofloxacin; Azm, azithromycin; Chl, chloramphenicol; Rif, rifampin; Cou A1, coumermycin A1; Strep, streptomycin; Trim, trimethoprim; DHFR, dihydrofolate reductase. Data are reported in the following format: wild-type amino acid (or DNA base for the 23S rRNA gene), protein (or gene for the 23S rRNA gene) position→mutant amino acid (or DNA base for the 23S rRNA gene) (e.g., for D90→G, D is the wild-type amino acid, 90 is the position in the GyrA protein, and G is the amino acid found in the resistant strain). Bold indicates that alterations (the same or other) involved in antibiotic resistance development have been found in an equivalent position in other microorganisms (either in vitro or in vivo). In addition, the presence of polymorphisms has been observed in some of these genes (e.g., T13→A in L4 or R9→C in L22) (123).
b
c
The MIC has been reported for only one ciprofloxacin-resistant clinical isolate with an Ala91→Val substitution (343). In the remaining cases, the presence of mutations has been reported without data on antibiotic resistance.
d
The authors indicated the change Ser472→Phe. B. bacilliformis possesses a Leu at amino acid codon position 472. A probable scenario is a reading mistake and the presence of the change in position 473 or 474 (both presenting Ser as the wild-type amino acid).
e
The role in the development of antibiotic resistance remains to be determined.
In both in vivo (343, 344) and in vitro (123, 329, 342) studies, the most frequently described amino acid substitutions involved in the development of ciprofloxacin resistance are in the classical QRDR of GyrA, affecting positions 91 and 95. This is in accordance with what has been widely described in the equivalent amino acid positions (e.g., positions 83 and 87 in E. coli numeration) in other microorganisms and related to the mechanisms of quinolone target-DNA interactions (345). In addition, unusual mutations, such as those affecting codon 89 (Gly89→Cys) and 90 (Asp90→Gly), have been described in vivo and in vitro, respectively (40, 344). The same and other amino acid substitutions have been reported, albeit rarely, at equivalent positions in other microorganisms, such as E. coli (345, 346). Interestingly, the equivalent Asp90→Gly substitution has previously been described in E. coli (Asp82→Gly) having the atypical nalidixic acid-susceptible, ciprofloxacin-resistant pattern (346). Unfortunately, in the study describing this mutation in B. bacilliformis, only an increase in the MIC of ciprofloxacin from 0.3 to 0.9 μg/ml was reported, with no determinations of the MIC of nalidixic acid (342). Regarding other quinolone targets, the Glu475→Lys substitution in GyrB causes a slight increase in the MICs of ciprofloxacin (123). The region surrounding the equivalent amino acid position is largely involved in the development of decreased susceptibility or resistance to fluoroquinolones. Thus, amino acid substitutions have been observed in the equivalent Glu475 position of other microorganisms, such as Salmonella spp. (Glu465→Leu), Streptococcus pneumoniae (Glu474→Lys), Bacteroides fragilis (Glu478→Lys), and Helicobacter pylori (Glu463→Lys) (347–350). Although there are no data on quinolone susceptibility, the Ser472→Phe substitution in GyrB has also been described (344). Nonetheless, careful review of all the gyrB genes of B. bacilliformis recorded in GenBank showed the presence of Leu as a wild-type amino acid 472, while Ser is present at both positions 473 and 474, suggesting that mutations are located at one of the two latter points. Accordingly, an analysis of what has been described in other microorganisms showed the presence of the same and other amino acid substitutions in the equivalent Ser474 position of Mycobacterium spp., Proteus mirabilis, Pseudomonas spp., and Salmonella spp., among others (350–354). Furthermore, the GyrB amino acid changes Gly124→Ser, Arg184→Gln, and Thr214→Ala/Ile have been involved in the development of coumermycin A1 resistance (341).
Regarding rifampin resistance, three “hot” regions have been established in the rpoB gene in other microorganisms. These regions are the so-called cluster I, cluster II, and cluster III (following E. coli numeration, amino acid 505 to 537, 563 to 575, and 684 to 690, respectively), in which mutations leading to rifampin resistance have been described (355, 356). Regarding B. bacilliformis, 4 different mutations in the rpoB gene were observed in the 2 in vitro studies carried out to date (123, 329). Accordingly, three of these mutations are found in the equivalent cluster I region, i.e., Glu527→Arg, His540→Tyr, and Ser545→Phe, with the remaining Ser588→Tyr being located in the equivalent cluster II region. Amino acid substitutions leading to rifampin resistance have been described in equivalent positions in other Bartonellaceae as well as microorganisms such as E. coli or Mycobacterium tuberculosis (357–360). In accordance with what has been widely described in other microorganisms (361, 362), including other members of the Bartonella genus (363, 364), macrolide resistance has been related to the 23S rRNA gene mutation A1983G (equivalent to A2058G following E. coli numeration) (329). This finding is frequently found in microorganisms with a small number of rrn loci (361, 362, 365). Nonetheless, in a larger study selecting independent azithromycin-resistant mutants, only alterations affecting L4 and/or L22, including insertions and deletions, were found (123). These alterations were located within the regions involved in macrolide resistance development in other microorganism and were at the same positions in several cases (123). In one study, the fitness cost of antibiotic resistance was clearly highlighted on analyzing 2 chloramphenicol-resistant B. bacilliformis mutants with the G2372→A 23S rRNA gene mutation (123). Thus, in both mutants the 23S rRNA substitution reverted, and the resistance levels substantially fell after 5 passages without antibiotic pressure (123). It has been suggested that structural changes at the peptidyl transferase center affecting chloramphenicol binding impair ribosome function (366). This involvement at the fitness level also agrees with the enhanced difficulty in selecting chloramphenicol resistance and with the high persistence of bacteremia of up to 50% following chloramphenicol treatment (308) even if transitory chloramphenicol-resistant low-fitness B. bacilliformis is selected in vivo during treatment. Thereafter, immune system pressure may lead to the control of the disease but not necessarily to its clearance. In fact, on assessing the stability of the resistance acquired to chloramphenicol, ciprofloxacin, azithromycin, and rifampin, only resistance to rifampin, which was related to different mutations in the rpoB gene, remained constant. This emphasizes the serious risk of selection of stable rifampin resistance during treatment (123).
Additionally, mutations in aminoglycosides and folate inhibitor targets have also been observed in B. bacilliformis clinical isolates. However, their association with resistance remains to be established (344).
Efflux Pumps
Efflux pumps are involved in the expulsion of toxic substances from the bacteria, including antimicrobials, contributing to the intrinsic resistance to antibiotics. Efflux pumps can be specific, extruding a single substrate, or generalist, affecting a variety of compounds, including antibiotics from different families. This is important for the development of multiresistance, since only one type of pump can confer simultaneous resistance to a wide range of antimicrobial agents (367). Although not specifically studied, different efflux systems have been identified by sequence homology during the genomic sequencing of different B. bacilliformis strains (131).
To our knowledge, only one study has analyzed the role of 2 efflux pump inhibitors, phenylalanine-arginine-β-naphthylamide (PAβN) and artesunate, in resistant mutants obtained in vitro (123). This study showed the first evidence of the role of efflux pumps in the levels of resistance to ciprofloxacin, azithromycin, and rifampin, being especially of note for chloramphenicol; thus, one chloramphenicol-resistant mutant presenting a decrease in the MIC from 4 μg/ml to 1 μg/ml and 1.5 μg/ml (with PAβN and artesunate, respectively) was described.
ERADICATION OF CARRION′S DISEASE
In 1997, the Dahlem criteria on the feasibility of eradicating an illness were described (368). These criteria consider that the effectiveness of an intervention, the sensitivity of the diagnostic tools, and the fact that humans are the only vertebrate reservoir are the 3 main factors which should be taken into account when determining the actions to be implemented.
Based on these criteria, Carrion's disease is a potentially eradicable illness, mainly due to its restricted geographic location, the lack of nonhuman reservoirs, and other factors such as the advances toward obtaining better, inexpensive, and sensitive diagnostic tools. Some of the tools proposed are based on antigenic candidates, which may also lead to the design of preventive strategies and enhance the current good activity of antibacterial agents (50, 271, 272, 369).
Illness eradication may be approached from different flanks, including the use of vaccines, as in the successful history of worldwide smallpox eradication in 1980 (370), or by massive antibiotic treatments, as has been proposed to eradicate yaws (371).
Regarding Carrion's disease, the characterization of several antigenic candidates, which have been explored in the field of development of new diagnostic tools (50, 159, 276, 277), might also be useful for the development of vaccines. Meanwhile, the current high levels of antibiotic susceptibility presented by B. bacilliformis can support the mass treatment approach, but the possibility of resistance development, which might underlie the high number of microbiological failures in clinical treatments and lead to the emergence of resistant strains, precludes the exclusive use of mass treatments for Carrion's disease eradication purposes. Probably the most efficient approach will be a mixed formula: the use of a protective vaccine combined with massive antibiotic treatment.
Nonetheless, the absolute lack of visibility of this illness, related to the nature of the affected populations, is the Achilles heel which prevents strong social and political commitments to efforts to eradicate Carrion's disease. Therefore, it is necessary to construct a narrative involving groups from policymakers to field-based physicians and to obtain the cooperation of community leaders and local educators, the support of international health authorities and developed world-based public and private fundraisers, and the collaboration of local and foreigner researchers and of journalists. It is necessary to act but also to listen: to listen to local inhabitants, because they are the target population of any action which will be designed, and to eliminate their doubts, their suspicions, and their fears.
EPILOGUE
While the hands of the research clock point to mark the future, and exciting new scientific and technical developments allow the development of direct methods to prevent, diagnose, and treat infectious diseases, Carrion's disease remains lost in time, broken and stranded, submerged in darkness and oblivion. Carrion's disease is an illness which is restricted to small isolated rural areas of middle-income countries far from the tourist routes and the developed world and consequently completely outside international focus and forgotten by the great majority of funding sources. It affects mainly the poorest populations and thus may not be perceived as relevant or generate sufficient interest to receive funding or encourage scientific interest. This lack of visibility is clearly the biggest obstacle to achieve the eradication of Carrion's disease. Notwithstanding this, although the number of people affected is low compared with those affected by other infectious diseases and the clinical management of Carrion's disease is relatively easy with the administration of antibiotics, this disease is currently expanding to new areas, and albeit few for now, people are dying from this disease, especially during outbreaks. Indeed, any effort to improve the diagnosis and treatment of Carrion's disease would have a great impact on the communities living side by side with this disease, and in the end, we should ask the question, “Will only those who are affected benefit, or will we all?”
ACKNOWLEDGMENTS
We are grateful to Donna Pringle for language correction. Figures 1, 2, 3, 6, 7, and 8 were prepared/modified with the artistic support of Patrick Lane, ScEYEnce Studios. Figures 4 and 5 were adapted to journal format by Nuno Santos.
J.R. was supported by a fellowship from program I3SNS of the ISCIII (grant number CES11/012). C.G. was supported by a Ph.D. fellowship of the ISCIII (FI12/00561).
ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya.
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Author Bios
Cláudia Gomes
Institute for Global Health, Barcelona Centre for International Health Research, Hospital Clínic, Universitat de Barcelona, Barcelona, Spain
Cláudia Gomes obtained an Ms.C. in microbiology at the Universidade Nova de Lisboa, Portugal, and a Ph.D. at the Barcelona Institute for Global Health (ISGlobal), University of Barcelona, Spain. She has been a research fellow at ISGlobal with a grant from the Instituto de Salud Carlos III and at the National Institute of Infectious Diseases in Tokyo, Japan, with a grant from the Canon Foundation in Europe. Her research interests have been focused on infectious diseases from low- and middle-income countries, and during the last 4 years she had been working on Carrion's disease, with an emphasis on diagnosis as well as the mechanisms of antimicrobial resistance.
Joaquim Ruiz
Institute for Global Health, Barcelona Centre for International Health Research, Hospital Clínic, Universitat de Barcelona, Barcelona, Spain
Joaquim Ruiz, Ms.C., Ph.D., is a microbiologist who graduated from the Science Faculty of the Universitat Autònoma de Barcelona. He has developed his scientific career at the Hospital Clinic of Barcelona, being Associate Research Professor at the Barcelona Institute for Global Health and Visiting Lecturer at the Universidad Peruana Cayetano Heredia in Lima. During recent years he has focused his research on the knowledge of the dynamics of the development of antibiotic resistance and dissemination through different environments and on the study of infectious diseases in low- and middle-income countries, mainly diarrheagenic pathogens affecting children. Since 2008 he has been leading a research line devoted to the understanding and knowledge of Carrion's disease.
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Clinical Microbiology Reviews
Volume 31 • Number 1 • January 2018
eLocator: 10.1128/cmr.00056-17
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© 2017 American Society for Microbiology. All Rights Reserved.
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Published online: 29 November 2017
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2018.
Carrion's Disease: the Sound of Silence. Clin Microbiol Rev
31:10.1128/cmr.00056-17.
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