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December 2011

Mycobacterium tilburgii” Infection in Two Immunocompromised Children: Importance of Molecular Tools in Culture-Negative Mycobacterial Disease Diagnosis

ABSTRACT

Mycobacterium tilburgii” is a nontuberculous mycobacterium that cannot be cultured by current techniques. It is described as causing disseminated disease in adults. We present the first cases of disseminated disease in 2 immunocompromised children. This paper stresses the importance of molecular techniques for correct mycobacterial identification and guidance to immunological diagnosis.

CASE REPORTS

Case A was a 3.5-year-old boy of nonconsanguineous Dutch parents with an unremarkable medical history. He was admitted to the hospital with complaints of fatigue, chronic cough, 4 kg of weight loss, and an intermittent elevated temperature (ranging from 37.5 to 38.8°C) occurring over several weeks. Two courses of treatment with antibiotics (erythromycin and amoxicillin-clavulanate) had been given by a general physician, but his clinical condition did not improve. He had been vaccinated in accordance with the Dutch Vaccination Program and did not receive Mycobacterium bovis bacillus Calmette-Guérin (BCG). He had not traveled abroad, and there were no documented tuberculosis contacts.
At physical examination, a slightly dyspnoeic boy was seen. His weight was 18.6 kg (73rd percentile), and his height was 105.6 cm (63rd percentile). Breathing frequency was 24/min, and retractions were noted in his supraclavicular and flank regions. On auscultation, pulmonary sounds were almost absent in the left posterobasal region. There was generalized lymphadenopathy, especially in the cervical and axillar regions. One lymph node (1.5 by 1.0 cm) in the right supraclavicular region was palpable. Liver and spleen were enlarged (liver, 2 cm; spleen, 4 cm below costal margin).
Blood analysis showed leucocytosis of 40 × 109/liter, with 18% eosinophils, 45% neutrophils, 23% lymphocytes, and 4% monocytes. A chest radiograph showed a retrocardial atelectasis and a pronounced right hilar region.
Based on this clinical description, the differential diagnosis included mycobacterial disease and malignancy (lymphoma). A tuberculin skin test (TST) initially showed an induration of 14 mm after 24 h but was read as 4 mm after 48 h. A gamma interferon release assay (IGRA; Quantiferon Gold In-Tube, Cellestis GmbH, Darmstadt, Germany) had an indeterminate result. A histological examination of the supraclavicular lymph node showed a mixed cellular infiltrate without the presence of granuloma or giant cells. Auramine staining revealed multiple positive rods. PCR for M. tuberculosis targeting IS6110 was negative. Malignancy was excluded; a bone marrow biopsy specimen showed no abnormalities.
In the period before cultures were grown, antituberculosis therapy was started with administration of isoniazid, rifampin, pyrazinamide, and ethambutol, resulting in clinical improvement within 2 weeks.
Two months after the start of therapy, the boy presented with generalized exanthema suggestive of an allergic reaction. Mycobacterial cultures of the lymph node tissue were still negative. Because an infection by nontuberculous mycobacteria (NTM) was suspected, therapy was changed to clarithromycin and ciprofloxacin. Formalin-fixed paraffin wax-embedded histopathology specimens of lymph node tissue were subjected to 16S rRNA sequencing, which identified “Mycobacterium tilburgii” as the causative agent (see below). Additional analyses of the gamma interferon (IFN-γ)-interleukin-12 (IL-12) pathway revealed an IFN-γ receptor 1 deficiency. Treatment with clarithromycin and ciprofloxacin was continued for 8 months. At follow-up 11 months after treatment, the child was in good clinical condition, with normal inflammatory response parameters, and therapy was discontinued.
Case B was a 4-year-old boy of consanguineous Turkish descent with, again, an unremarkable medical history. He was admitted to the hospital with complaints of malaise, fever, abdominal pain, diarrhea, weight loss, and night sweats occurring over the previous 2 weeks. Since week 1, he had had a swollen and painful cervical lymph node that grew gradually. There was no history of traveling abroad or documented tuberculosis contacts. He was vaccinated without BCG according to Dutch National Vaccination Program guidelines.
At physical examination, a moderately ill boy was seen, with weight of 15.7 kg (5th percentile) and height of 108 cm (50th percentile). His temperature was 38.6°C. A tender swollen lymph node with a diameter of 5 cm was palpable in the right lower cervical region. Furthermore, there was generalized lymphadenopathy in the axillary and inguinal regions in combination with hepatosplenomegaly. Auscultation of the thorax revealed no abnormalities.
Blood analysis showed a normal leukocyte number and differential counts. His C-reactive protein level was elevated (62 mg/liter). Ultrasound analysis of the abdomen revealed intra-abdominal lymphadenopathy and hepatosplenomegaly without focal abnormalities.
Based on this clinical description, the differential diagnosis included lymphoma and infection, with NTM especially suspected. The TST result was negative; an IGRA was not performed. Histology of the cervical lymph node showed granulomatous inflammation. Ziehl-Neelsen staining revealed numerous acid-fast bacilli. Cultures remained negative.
A PCR analysis of the lymph node sample targeting the IS6110 element of M. tuberculosis was negative. Subsequent analysis using an Inno-LiPA Mycobacteria v. 2 assay (Innogenetics, Ghent, Belgium) identified the acid-fast bacilli as M. simiae. At that time, no material was left to perform additional tests.
Initial treatment consisted of administration of rifampin, clarithromycin, and ciprofloxacin. Due to the progression of the hepatomegaly, persistent lymphadenopathy, and suspicion of a defect in the IFN-γ-IL-12 pathway, subcutaneous IFN-γ was added to the antimicrobial therapy. Analysis of the IFN-γ-IL-12 pathway function revealed an IL-12 receptor deficiency. After 3 months, cotrimoxazole was added due to insufficient clinical response and the suspicion of M. simiae as the causative agent. Because of a diagnosis of progressive abdominal lymphadenopathy 6 months after the start of therapy, an abdominal lymphadenectomy was performed to decrease the mycobacterial load.
A 16S rRNA gene sequence analysis was performed using paraffin wax-embedded tissue obtained during an abdominal lymphadenectomy; M. tilburgii was identified as the causative agent (see below). Therapy was changed to administration of amikacin, clarithromycin, ethambutol, moxifloxacin, and prothionamide, with continuation of IFN-γ administration. Currently, 20 months after presentation, the boy is in good clinical condition and continues to receive suppressive treatment with moxifloxacin, clarithromycin, and IFN-γ.
DNA extracted from paraffin wax-embedded tissues was used to perform 16S rRNA gene sequence analysis as previously described (12). Briefly, amplification was performed using one general 16S and one mycobacterium-specific primer, yielding a 500-bp PCR product of the 16S rRNA DNA. This product included hypervariable regions A and B. The obtained sequences are compared to those stored in GenBank (National College of Biotechnology Information [NCBI]; http://www.ncbi.nih.gov), using the Basic Local Alignment Search Tool. Quality control included saline solution and paraffin-wax-embedded human lymph node tissue from the pathology department as negative controls.
By the techniques described above, sequences from samples of both patients were shown to be identical to the M. tilburgii 16S rRNA gene sequence deposited under accession number Z50172. For additional identification, extracted DNA was used to perform 16S-23S internal transcribed spacer (ITS) sequencing and rpoB and hsp65 gene sequencing, using previously published primers (1, 8, 10). The ITS sequences of samples from both patients proved identical to the M. tilburgii ITS sequence (GenBank accession number AJ580827); no matches to the hsp65 and rpoB sequences in the GenBank database were found, as no hsp65 or rpoB sequences had been previously stored for this species. The GenBank accession numbers of the sequences used are as follows: AY353699, DQ185132, AY365190, and GQ166762 (M. sherrisii), GQ153280, Y14186, AF547875, and GQ153313 (M. simiae), U57632, Y14189, AF547882, and GQ153311 (M. triplex), X60070, Y14183, and AF547837 (M. genavense), AF480583, AF317658, AF547851, and EU109300 (M. lentiflavum), and AJ580826, AJ580827, HM588695, and HM069330 (M. tilburgii).
Figure 1 illustrates the phylogenetic relationships of M. tilburgii to most closely related NTM species, as inferred from the obtained sequences.
Fig. 1.
Fig. 1. Phylogenetic positioning of Mycobacterium tilburgii and its closest related species based on concatenated 16S rRNA, ITS, hsp65, and rpoB gene sequences.
Nontuberculous mycobacteria (NTM) are recognized as infectious agents in humans. Diseases caused by NTM include pulmonary infections of patients with preexisting pulmonary disease (3), cervicofacial lymphadenitis in immunocompetent but young children (4), and disseminated disease in immunocompromised patients (11). Detection of NTM in samples from sterile sites (obtained, e.g., by tissue biopsy) is usually indicative of infection (11). When mycobacteria are found upon microscopy, the bacterial load is generally high enough to provide positive cultures.
Mycobacterial disease is suspected in clinical cases based on symptoms or within an epidemiological context. A dilemma arises when cultures remain negative but acid-fast bacilli have been seen on histology. In these situations, the clinician should be aware of the possibility that the disease was caused by an NTM species that is difficult or impossible to culture on standard media.
Advanced molecular techniques have improved the identification and taxonomic classification of NTM (3). Amplification and sequencing of the 16S rRNA gene by PCR have become important diagnostic tools for use when acid-fast organisms are found in clinical samples. Although PCR is preferably performed using fresh and/or liquid specimens, it is possible to extract DNA coding for 16S rRNA from formalin-fixed paraffin wax-embedded tissue and to use this as a source for microorganism identification (12).
In both of our cases, PCR for identification of the IS6110 element of M. tuberculosis was performed on fresh biopsy material. The remainding tissue was used for culture and pathology analyses. For case B, an additional INNO-LiPA assay was performed, using the 16S-23S internal transcribed spacer (ITS) as its target. The assay misidentified the DNA samples as M. simiae, since the ITS sequences of M. tilburgii closely resemble those of M. simiae. Misidentification has been previously noted for other NTM species as well (9, 14). Since M. simiae can be easily grown on routine culture media, the diagnosis in case B was reconsidered and paraffin wax-embedded tissue was send to the appropriate laboratory for additional PCR procedures, resulting in identification of M. tilburgii.
M. tilburgii was first identified in a sample from an immunocompetent adult patient in the city of Tilburg, The Netherlands (2); the species presented in the form of a disseminated infection and was later described as a causative pathogen in infections of three patients with HIV-AIDS and 1 patient with prolonged prednisolone treatment (5, 6) In both of our pediatric cases, M. tilburgii was associated with defects in the IFN-γ-IL-12 pathway. Defects in that pathway are known to increase susceptibility to mycobacterial disease (7, 13). IFN-γ stimulates macrophage activation and increases superoxide production through its receptors; such production is necessary for bacterial killing. Enhancement of superoxide formation is mediated by the IFN-γ-IL-12 loop (13). In retrospect, it would have been interesting to learn whether the first adult patient described in the literature was immunocompetent or whether she had defects in the IFN-γ-IL-12 pathway as well.
We have no good explanation to account for the fact that M. tilburgii has mostly been described as the cause of infections of patients living in The Netherlands. We assume that the use of paraffin wax-embedded tissues as samples for 16S rRNA gene amplification and sequencing is not common. In both of our cases, the additional tests were performed in the Dutch reference laboratory for mycobacterial disease, since the laboratories at both academic hospitals did not perform PCR on DNA extracted from paraffin wax-embedded material.
Since M. tilburgii cannot be cultured, no information on drug susceptibility is available. Clinical experiences described in the literature show the use of various therapy durations and antibiotics combinations, some of which were initially intended for treatment of infections by M. tuberculosis complex or Mycobacterium avium and all of which included clarithromycin. In 3 out of 5 cases, a quinolone was included in the treatment regimen (2, 5, 6). In case A, the patient responded well to standard antituberculous medication and no relapse occurred when a combination of clarithromycin and ciprofloxacin was administered. The case B patient did not respond well to the initial regimen that included clarithromycin and ciprofloxacin. Clinical improvement occurred only after debulking and intensifying antimicrobial therapy.
In conclusion, 3 lessons can be learned from the presented cases: (i) nonculturable NTM can cause disease in humans and the disease is not restricted to adults; (ii) formalin-fixed paraffin wax-embedded tissue is suitable for mycobacterial recognition when mycobacteria have been seen on microscopy; and (iii) establishing the exact nature of the causative agent is helpful to guide the search for a specific underlying immunodeficiency and to establish optimal therapy.

Nucleotide sequence accession numbers.

The hsp65 and rpoB sequences obtained in this work are available in GenBank under accession numbers HM588695 (hsp65) and HM069330 (rpoB).

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Information & Contributors

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Published In

cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 49Number 12December 2011
Pages: 4409 - 4411
PubMed: 22012013

History

Received: 12 August 2011
Returned for modification: 27 August 2011
Accepted: 11 October 2011
Published online: 21 December 2020

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Authors

Nico G. Hartwig [email protected]
Department of Pediatrics, Subdivision Pediatric Infectious Diseases and Immunology, Erasmus MC—Sophia Children's Hospital, Rotterdam
Adilia Warris
Department of Pediatric Infectious Diseases & Immunology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Esther van de Vosse
Department of Infectious Diseases, Leiden University Medical Center, Leiden
Adri G. M. van der Zanden
Laboratory for Medical Microbiology and Public Health, Enschede
Tanja Schülin-Casonato
Department of Clinical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen
Jakko van Ingen
Department of Clinical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen
Rob van Hest
Tuberculosis Control Section, Department of Infectious Disease Control, Regional Public Health Service Rotterdam-Rijnmond, Rotterdam, The Netherlands

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