Eight monoclonal antibodies (MAbs) directed against the 14,000-molecular-weight (14K) antigen of Mycobacterium tuberculosis reacted specifically with mycobacteria of the M. tuberculosis complex. The nucleotide sequence of the gene encoding the 14K antigen was determined by using recombinant DNA clones isolated from lambda gt11 and cosmid libraries of the M. tuberculosis genome. The DNA sequence of the 14K protein gene coded for a polypeptide of 144 amino acids with a calculated molecular mass of 16,277 Da. The 14K antigen has a marked homology with proteins belonging to the alpha-crystallin family of low-molecular-weight heat shock proteins, which includes the 18K antigen of M. leprae. The eight MAbs recognized at least four distinct epitopes localized within the following three regions of the 14K protein: amino acids 10 to 92 (MAbs F67-8 and F67-16), amino acids 41 to 92 (F159-1 and F159-11), and amino acids 41 to 144 (F23-41, F24-2, F23-49, and TB68).

A test based on the polymerase chain reaction (PCR) was developed for the detection of the Mycobacterium tuberculosis complex in clinical samples. In this test, a 245-bp sequence of the insertion element IS986 was amplified and detected by agarose gel electrophoresis in the presence of ethidium bromide and by Southern blot and dot blot hybridization by using a 188-bp digoxigenin-labeled probe. We tested clinical specimens from 227 patients suspected of having tuberculosis. These included 102 cerebrospinal fluid, 48 sputum, 18 pleural fluid, 5 bronchoalveolar lavage, 18 blood, 7 pus, 8 bone marrow, and 6 urine samples and 15 tissue biopsy specimens. We also tested sputum samples from 75 patients with diseases other than tuberculosis. Sputum samples were first decontaminated, and all samples were treated with proteinase K-detergent solution to extract the DNA. Part of each sample was spiked with M. tuberculosis to provide a semiquantitative assay and to control for the loss of mycobacteria or interference with the PCR which may cause false-negative results. One femtogram of M. tuberculosis DNA could be detected. PCR was positive for all 32 culture-positive (for M. tuberculosis) and Ziehl-Neelsen staining (ZN)-positive samples, 10 of 12 culture-positive and ZN-negative samples, and all 4 culture-negative and ZN-positive samples. PCR detected M. tuberculosis complex bacteria in 35 of 178 cultureand ZN-negative samples. Clinical data supported the diagnosis of tuberculosis in the majority of the 35 patients from whom those samples were obtained.

ObjectivesThe high prevalence of isoniazid-resistant Mycobacterium tuberculosis is often explained by a high mutation rate for this trait, although detailed information to support this theory is absent. We studied the development of isoniazid resistance in vitro, making use of a laboratory strain of M. tuberculosis.MethodsSpontaneous isoniazid-resistant mutants were characterized by molecular methods allowing identification of the most commonly encountered resistance-conferring mutations. Additionally, we determined the in vitro mutation rates for isoniazid and rifampicin resistance, and characterized the genome of a triple-resistant strain.ResultsResults confirm that the in vitro mutation rate for isoniazid resistance (3.2 × 10−7 mutations/cell division) is much higher than the rate for rifampicin resistance (9.8 × 10−9 mutations/cell division). However, in the majority of the in vitro mutants katG was partially or completely deleted and neither of the two most common in vivo mutations, katG-S315T or inhA-C(-)15T, were found in 120 isogenic mutants. This implies that clinically prevalent resistance mutations were present in <0.8% of isoniazid-resistant strains selected in vitro (95% CI 0%–2.5%). The triple-resistant strain had acquired isoniazid resistance via a 49 kbp deletion, which included katG. Apart from previously identified resistance-conferring mutations, three additional point mutations were acquired during sequential selection steps.ConclusionsThese outcomes demonstrate that the in vivo mechanism of isoniazid resistance is not reflected by in vitro experiments. We therefore conclude that the high in vitro mutation rate for isoniazid resistance is not a satisfactory explanation for the fact that isoniazid monoresistance is significantly more widespread than monoresistance to rifampicin.

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

Direct smear examination with Ziehl-Neelsen (ZN) staining for the diagnosis of tuberculosis (TB) as employed in most low-income countries is cheap and easy to use, but its low sensitivity is a major drawback. The low specificity of chest X-rays, used for the diagnosis of smear-negative TB, risks high levels of overdiagnosis. Major advances in molecular techniques, which rapidly identify mycobacterial DNA in sputa, may overcome these obstacles. In this study, the AMPLICOR PCR system was used to diagnose pulmonary TB in a developing country with high prevalences of both TB and human immunodeficiency virus (HIV). The sensitivity and specificity of this technique were compared to those of the usual diagnostic techniques. Sputum specimens were collected from 1,396 TB suspects attending the Rhodes Chest Clinic, Nairobi, Kenya. The specimens were analyzed for the presence of Mycobacterium tuberculosis by PCR; culture on Löwenstein-Jensen medium was used as the "gold standard." All culture-positive samples were genotyped to identify the mycobacterial species. The sensitivity and specificity of PCR were 93 and 84%, respectively. HIV status did not affect the sensitivity of PCR. A total of 99.7% of the true smear-positive and 82.1% of the true smear-negative TB patients were correctly identified by PCR. PCR detected M. tuberculosis in 11.7% of the culture-negative suspects, 60% of which had one or two PCR-positive sputum specimens. Of the 490 positive cultures, 486 were identified as M. tuberculosis. The high sensitivity of Amplicor PCR merits usage in a clinical setting with high TB and HIV burdens. Thus, PCR can be considered as an alternative to ZN staining in combination with chest X-ray for diagnosis of TB; however, cost-effectiveness studies and operational studies are required to support an evidence-based decision of introducing PCR for TB control in high-burden environments.