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Fever of unknown origin (FUO) poses a diagnostic challenge to the clinicians, with a differential diagnosis as varied as neoplastic and infectious diseases. In developing countries, the infectious causes are responsible for more cases of FUO, with tuberculosis as one of the main causes of classic FUO. Disseminated tuberculosis with negative pulmonary findings is a diagnostic problem. This study examines the diagnostic utility of the polymerase chain reaction (PCR) in samples of bone marrow aspirate in 85 patients presenting with diverse clinical symptoms. Using primers specific for Mycobacterium tuberculosis, tubercular etiology was detected in 33% of patients clinically suspecte do f tuberculosis while culture on Lowenstein-Jensen medium grew M. tuberculosis in only one patient (2.5%). None of these patients had been diagnosed by microscopy. Clinical improvement with ATT was observed in 85% of the patients with positive PCR. PCR demonstrated much higher sensitivity and specificity, thereby facilitating early therapeutic decisions for suspected extrapulmonary tuberculosis.

Histopathological and mycobacteriological examinations have limited utility in the diagnosis of genital tuberculosis. In this double-blind study, 61 samples, consisting of endometrial aspirates (EAs), endometrial biopsies (EBs) and fluid from the pouch of Douglas (POD), from 25 women suffering from infertility were investigated for the presence of the mpt64 gene of Mycobacterium tuberculosis by PCR and correlated with laparoscopic findings. PCR demonstrated M. tuberculosis DNA in 14 out of 25 patients (56.0 %), compared to one smear with acid-fast bacilli (1.6 %) and two culture-positive samples (3.2 %). The presence of M. tuberculosis DNA was observed in 53.3 % of EBs, 47.6 % of EAs and 16.0 % of POD fluid samples. All patients with laparoscopy suggestive of tuberculosis, 60 % of those with a probable diagnosis and 33 % of those with incidental findings were positive by PCR. However, one EA sample from an infertile patient with normal laparoscopy was also positive. Multiple sampling from different sites and amplification of the mpt64 gene segment by PCR offered increased sensitivity in determining tuberculous aetiology in female infertility.

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.

Genotypes of Mycobacterium tuberculosis causing disease were investigated in pulmonary tuberculosis patients admitted to two adjacent wards of a tuberculosis hospital in Delhi, India. Genetic markers, the insertion sequence IS6110, a direct repeat sequence, and a polymorphic GC-rich sequence supported the circumstantial epidemiologic link between eight strains of M. tuberculosis, suggesting their possible involvement in small-scale, interpersonal transmission of both drug-sensitive and drug-resistant tuberculosis. This is the first report of a suspected acquisition of M. tuberculosis among hospitalized patients in India. The use of multiple molecular typing markers and techniques unequivocally identified the exact clonality of strains isolated from the hospital. The result of this study emphasizes the need for more comprehensive investigation of high-risk situations for tuberculosis transmission and long-term follow-up analysis for identifying such instances of unsuspected transmission.

We report 2 isolates of Mycobacterium fortuitum from patients with pulmonary tuberculosis lesions hybridizing to IS6110 probe in restriction fragment length polymorphism (RFLP) typing. Results of polymerase chain reaction-hybridization formats using the non-specific region of IS6110 for the molecular detection of mycobacteria in clinical material should be interpreted with caution.

Setting: Since conventional bacteriological methods rarely detect Mycobacterium tuberculosis in cerebrospinal fluid (CSF) and are of limited use in the diagnosis of tuberculous meningitis (TBM), clinical features suggestive of TBM supported by indirect evidence such as CSF examination and computerized tomography (CT) of the head have been used for the early diagnosis of TBM. Objective: We evaluated the efficacy of polymerase chain reaction (PCR) in the diagnosis of TBM. Methods: Coded CSF samples from 40 patients with TBM and from 49 patients with other neurological disorders were processed. In the absence of a reliable sensitive and specific test for M. tuberculosis in CSF, we used a set of established clinical criteria as the gold standard. Accordingly, the patients were divided into definite, highly probable, probable and possible TBM. The samples were decoded only after completion of the laboratory tests. Results: PCR was positive in 24, 1920, 1316 patients with highly probable, probable and possible TBM respectively. None of the samples were positive by conventional bacteriological methods. However, 349 CSF samples from non-TBM patients were also found positive by PCR. PCR detected M. tuberculosis genomic DNA in the CSF of 85% of clinically suspected TBM cases and 6.1% of non-tuberculous controls. Conclusion: PCR, along with the suggested clinical criteria, offers a rapid and fairly accurate diagnosis of TBM.