Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.

Histopathological and Molecular Investigation of Natural Cases of Bovine Tuberculosis Infection in Cattle

Bovine tuberculosis (bTB), a chronic infection in cattle caused by Mycobacterium tuberculosis/bovis, that impacts productivity and represents a major public health threat. Although the considerable economic costs and zoonotic risk consequences associated with the disease, accurate estimates of bTB prevalence are lacking in many countries, including India. Therefore, in the current study for collection of tubercular lesions the postmortem examination of 100 cattle was conducted. All major viscera and regional lymph nodes were examined and incised. Histopathology was performed in the cases where gross lesions were suggestive of tuberculosis. PCR was performed on the tissue and faecal samples by using IS6110 insertion sequence, Mycobacterium tuberculosis/bovis complex PCR kit. In 12 animals, nodular lesions with casseating mass suggestive of tuberculosis were observed in the lung tissue. All the 12 lung impression smear and only five faecal smear showed acid fast bacilli stained by Ziehl-Neelsen staining. Histologic features comprised a classic granuloma as a characteristic lesion of tuberculosis composed of a central caseous necrosis with mantle of macrophages, lymphocytes, plasma cells, epithelioid macrophages and Langhan’s giant cells and were observed in all 12 cases. All the tissue samples and 11 faecal samples were positive for the Mycobacterium tuberculosis complex using IS6110 sequence. 8 tissue samples and 4 faecal samples were positive by using Mycobacterium tuberculosis/bovis complex PCR kit. It can be concluded that there was good agreement between histopathology, acid fast staining and PCR. It can also be concluded that faecal samples which are easier to collect should be preferred for diagnosis of TB by PCR in cattle

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples

Abstract INTRODUCTION: This study evaluated the performance of the IS6110-TaqMan® assay in different types of biological samples and tissues for laboratory diagnosis of extrapulmonary tuberculosis. METHODS: 143 biological samples and tissues from patients with suspected extrapulmonary tuberculosis from the health services of Recife/Pernambuco/Brazil were evaluated with the IS6110-TaqMan® assay. RESULTS: The sensitivities of the IS6110-TaqMan® assay calculated for blood, urine, both blood and urine samples, tissue biopsies, extrapulmonary body fluid samples, and all samples from patients calculated together were 55.9%, 33.3%, 68.8%, 43.8%, 29.6%, and 73.7%, respectively, and the specificities were 80%, 100%, 78.6%, 100%, 100%, and 84.2%, respectively. CONCLUSIONS The accuracy of qPCR was high in various clinical sample types. The analysis of more than one type of clinical sample collected from the same patient with extrapulmonary tuberculosis enhances the diagnostic power of the IS6110-TaqMan® assay when compared with the use of only one clinical sample.

Performance of a molecular assay to detect Mycobacterium tuberculosis complex DNA in clinical specimens: multicenter study in Brazil

BACKGROUND In high tuberculosis (TB) burden countries, there are few data on the performance of new molecular commercialised assays developed locally. OBJECTIVE To evaluate the performance of a new molecular commercialised assay for TB diagnosis (Detect-TB) in three laboratories. METHODS A total of 302 sputum samples from an equal number of patients with presumptive diagnosis of pulmonary tuberculosis (PTB) were submitted for routine smear microscopy, culture, and Detect-TB assay at three different sites in Brazil (the cities of Caxias do Sul, São Paulo and Canoas). FINDINGS Seventy four (24.7%) TB cases were diagnosed (65 bacteriologically confirmed). When compared to smear microscopy/culture results, the overall sensitivity and specificity of Detect-TB assay was 84.6% (CI 95%; 73.7-91.6) and 93.1% (CI 95%; 89.1-95.8), respectively. When compared to bacteriological and clinical diagnostic criteria, the sensitivity and specificity of Detect-TB assay was 74.3% (CI 95%; 63.3-82.9) and 92.9% (CI 95%; 88.7-95.6), respectively. Among the three sites - Caxias do Sul, São Paulo and Canoas - the sensitivity and specificity were respectively 94.7% and 97.8%; 71.4% and 93.9%, 82.1% and 88.9%. MAIN CONCLUSIONS These findings suggest that the Detect-TB assay could be applied routinely in reference laboratories across different regions in Brazil.

Utility of multiplex real-time PCR in the diagnosis of extrapulmonary tuberculosis

Abstract Objective The diagnosis of extrapulmonary tuberculosis is still a challenge because of its pauci-bacillary nature. The aim of the study was to evaluate the role of a multiplex PCR assay in the diagnosis of extrapulmonary tuberculosis and to compare the efficiency of two targets, IS6110 and MPB64 to detect Mycobacterium tuberculosis. Methods 150 extrapulmonary samples (61 pus/aspirate, 46 tissue, 32 body fluids, and 11 urine) from clinically suspected cases of tuberculosis were included in the study. All the samples were subjected to direct fluorescent microscopy, TB culture (BacT/ALERT 3D, biomerieux, Durham, North Carolina, USA) and a Multiplexed Tandem PCR targeting two mycobacterial DNA sequences, IS6110 and MPB64. Master-Mix reagents and primers were prepared by AusDiagnostics Pvt. Ltd (Alexandria, New South Wales, Australia). The performance of the assay was assessed using a composite gold standard, which included clinical characteristics, microbiology smear as well as culture, histopathology, cytology, radiology, and response to antitubercular therapy. Results 20.3%, 23.6%, and 45.3% of specimens were positive by smear, culture, and PCR, respectively. The sensitivity and specificity of the multiplex PCR was 91.9% and 88.4%, respectively, using the composite gold standard. Positive and negative predictive values of the PCR were estimated as 85.1% and 93.8%, respectively. Higher positivity was observed with target IS6110 (44.6%) as compared to target MPB64 (18.9%). The sensitivities of IS6110 and MPB64 individual targets were 90.3% and 64.5%, respectively, and specificities were 88.4% and 97.7%, respectively. Conclusion PCR can play an important role in rapid and accurate diagnosis of extrapulmonary tuberculosis. IS6110 alone is an effective target in our part of the country.

Molecular characterization of mycobacterium tuberculosis isolates from Tehran, Iran by restriction fragment length polymorphism analysis and spoligotyping

Abstract: INTRODUCTION Characterization of Mycobacterium tuberculosis (MTB) isolates by DNA fingerprinting has contributed to tuberculosis (TB) control. The aim of this study was to determine the genetic diversity of MTB isolates from Tehran province in Iran. METHODS MTB isolates from 60 Iranian and 10 Afghan TB patients were fingerprinted by standard IS6110-restriction fragment length polymorphism (RFLP) analysis and spoligotyping. RESULTS The copy number of IS6110 ranged from 10-24 per isolate. The isolates were classified into 22 clusters showing ≥ 80% similarity by RFLP analysis. Fourteen multidrug-resistant (MDR) isolates were grouped into 4 IS6110-RFLP clusters, with 10 isolates [71% (95% CI: 45-89%)] in 1 cluster, suggesting a possible epidemiological linkage. Eighteen Iranian isolates showed ≥ 80% similarity with Afghan isolates. There were no strains with identical fingerprints. Spoligotyping of 70 isolates produced 23 distinct patterns. Sixty (85.7%) isolates were grouped into 13 clusters, while the remaining 10 isolates (14.2%) were not clustered. Ural (formerly Haarlem4) (n = 22, 31.4%) was the most common family followed by Central Asian strain (CAS) (n = 18, 25.7%) and T (n = 9, 12.8%) families. Only 1strain was characterized as having the Beijing genotype. Among 60 Iranian and 10 Afghan MTB isolates, 25% (95% CI: 16-37) and 70% (95% CI: 39-89) were categorized as Ural lineage, respectively. CONCLUSIONS A higher prevalence of Ural family MTB isolates among Afghan patients than among Iranian patients suggests the possible transmission of this lineage following the immigration of Afghans to Iran.

PCR interna con ADN extraído directamente de portaobjetos positivos para confirmar o excluir el diagnóstico de tuberculosis: enfoque en la bioseguridad / In-house PCR with DNA extracted directly from positive slides to confirm or exclude the diagnosis of tuberculosis: focus on biosafety

The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3%, 100%, 100% and 89.7%, respectively whereas accuracy was 90.7%. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method

La posibilidad de obtener ADN a partir de frotis es una valiosa alternativa para remediar la falta de muestras cuando estas son totalmente utilizadas para la baciloscopia; esta opción soluciona, además, el problema de bioseguridad asociado a la posibilidad de accidente al transportar frascos que contienen muestras clínicas potencialmente infectivas. Por lo tanto, el propósito de este estudio fue utilizar para el diagnóstico de la tuberculosis la secuencia de inserción IS6110 para amplificación del ADN a partir de frotis que resultaron positivos por baciloscopia. Del análisis de 52 baciloscopias positivas surge que la sensibilidad, la especificidad, el valor predictivo positivo y el valor predictivo negativo de esta técnica fueron, respectivamente, del 52,3%, del 100%, del 100% y del 89,7%; y la precisión fue del 90,7%. La PCR IS6110 para el diagnóstico de tuberculosis, desarrollada con ADN extraído de frotis positivos, es un método rápido, simple, específico y seguro

of multiplex polymerase chain reaction using IS6110 and Protein b for the diagnosis of extra-pulmonary tuberculosis: North India.

Background: Prompt and accurate diagnosis of extra-pulmonary tuberculosis (TB) is highly challenging. Current conventional techniques lack sensitivity and are time-consuming. Here, we report our experience with multiplex polymerase chain reaction (MPCR) using two targets namely IS6110 and protein antigen b in the diagnosis of extra-pulmonary TB. Materials and Methods: A total of 150 patients of extra-pulmonary TB visiting tertiary care center in north India between September 2008 and December 2009 were included in the study. Sixty-six biopsy samples and 84 were body fluids from these patients were subjected for microscopy (Ziehl-Neelsen), culture on LJ medium and for Multiplex PCR using IS6110 and Protein b antigen. Results: Smear positivity was noted in 11 samples (7.33%), and LJ culture yielded Mycobacterium tuberculosis in 8 biopsies and 9 body fluids with overall positivity of 11.3%. The multi-targeted PCR could detect M. tuberculosis in a total of 112 samples. Of 112 positive samples, only Protein b band was detected in 7 samples and only IS6110 was detected in 5 samples. Overall Protein b, PCR could detect 71.33% of the cases, whereas IS6110 was positive in 66.6% of the cases. Overall the sensitivities of microscopy, culture, IS6110 PCR, Protein b PCR and MPCR were 7.33%, 11.3%, 66.67%, 71.3% and 74.6%, respectively. Thus by using more than two targets the sensitivity increased from 66.67% of IS6110 to 74.6% in MPCR. Conclusion: Multiplex polymerase chain reaction using IS6110 and Protein b antigen is a highly sensitive and specific tool in the diagnosis of pauci-bacillary conditions like extra-pulmonary TB.

Evaluation of Nested Polymerase Chain Reaction for the Diagnosis of Pulmonary Tuberculosis.

Utility of Polymerase Chain Reaction (PCR) test for diagnosis of pulmonary tuberculosis was evaluated in 50 clinically diagnosed cases. All samples were also processed for microscopy by Ziehl-Neelsen (ZN) staining and culture on Lowenstein-Jensen medium (LJ). PCR was positive in all microscopy and culture positive specimens. There were 11 samples positive by PCR but failed to grow in culture. With respect to culture the sensitivity (SN), specificity (SP), Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of PCR was 100%, 57%, 68% and 100% respectively. The values for PCR on final evaluation taking into consideration clinical, radiological, microbiological evidence and response to antitubercular treatment were SN- 100%, SP-88%, PPV-94% & NPV-100% respectively. PCR detects M. tuberculosis complex with greater sensitivity and should be useful for rapid diagnosis of tuberculosis.

Polymerase chain reaction targeting insertion sequence for the diagnosis of extrapulmonary tuberculosis.

Background & objectives: Diagnosis of extrapulmonary tuberculosis (EPTB) is difficult using conventional diagnostic methods. This study was conducted to evaluate the use of polymerase chain reaction (PCR) in diagnosis of definitive and probable extrapulmonary tuberculosis patients, and to assess the performance of insertion sequence (IS) 6110 based PCR assay as compared to conventional culture by Lowenstein-Jensen (LJ) method for the diagnosis of EPTB. Methods: A total of 178 non repeated clinical specimens were collected from clinically suspected extrapulmonary tuberculosis patients. The specimens included 59 ascitic fluid, 54 pleural fluid, 25 cerebrospinal fluid (CSF), 12 fine needle aspiration (FNA), 8 urine, 7 pus, 6 synovial fluid, 2 skin tissue, one pericardial fluid, one liver abscess, one pancreatic cyst fluid, one omental biopsy and one semen sample. All these clinical samples were subjected to Ziehl-Neelsen staining (ZN) for acid fast bacilli (AFB) and culture on LJ medium. PCR was performed by targeting 123bp fragment of insertion sequence IS6110 of Mycobacterium tuberculosis (MTB). Results: Of the 178 specimens, 10 (5.61%) were ZN smear positive for AFB, six (3.37%) were L-J culture positive from 10 AFB smear positive cases and 48 (26.96%) were PCR IS 6110 positive for M. tuberculosis. Interpretation & conclusions: PCR using IS6110 primer was able to pick up more EPTB patients compared to conventional L-J culture method for detection of M. tuberculosis. False positive PCR IS6110 in three CSF samples may be due to latent TB infection which was limitation in this study.

Diagnostic appraisal of simultaneous application of two nested PCRs targeting MPB64 gene and IS6110 region for rapid detection of M. tuberculosis genome in culture proven clinical specimens.

Background: Early diagnosis of tuberculosis is critical for its effective management and prevention. Several gene amplifi cation methods are used in the detection of tubercle bacilli from clinical specimens. MPB64 gene and IS6110 region have been identifi ed as potential gene targets for the specifi c detection of Mycobacterium tuberculosis from direct clinical specimens. Objective: The present study was conducted to evaluate the diagnostic utility of simultaneous application of two nested polymerase chain reaction (nPCRs) targeting MPB64 and IS6110 region for the detection of M. tuberculosis genome. Materials and Methods: A total of 100 and 354 clinical specimens from the control group and clinically suspected tuberculosis patients, respectively, were included in the study. nPCRs targeting MPB64 and IS6110 region were performed. Results and Conclusion: All of the 100 clinical specimens from the control group were negative for both nPCRs. Out of the 354 clinical specimens, 339 were positive for both culture and nPCRs, 10 and 5 were positive for culture, and nPCR targeting IS6110 and MPB64 regions, respectively. To conclude, nPCRs targeting MPB64 and IS6110 region are reliable and specifi c targets when applied simultaneously on clinical specimens to attain 100% sensitivity for the detection of M. tuberculosis genome.

IS6110 Restriction Fragment Length Polymorphism Typing of Drug-resistant Mycobacterium tuberculosis Strains from Northeast South Africa.

Tuberculosis (TB) remains a deadly infectious disease affecting millions of people worldwide; 95% of TB cases, with 98% of death occur in developing countries. The situation in South Africa merits special attention. A total of 21,913 sputum specimens of suspected TB patients from three provinces of South Africa routinely submitted to the TB laboratory of Dr. George Mukhari (DGM) Hospital were assayed for Mycobacterium tuberculosis (MTB) growth and antibiotic susceptibility. The genetic diversity of 338 resistant strains were also studied. DNA isolated from the strains were restricted with Pvu II, transferred on to a nylon membrane and hybridized with a PCR-amplified horseradish peroxidase 245 bp IS6110 probe. Of the 338 resistant strains, 2.09% had less than 5 bands of IS6110, and 98% had 5 or more bands. Unique restriction fragment length polymorphism (RFLP) patterns were observed in 84.3% of the strains, showing their epidemiological independence, and 15.7% were grouped into 22 clusters. Thirty-two strains (61.5%) from the 52 that clustered were from Mpumalanga, 16/52 (30.8%) from Gauteng, and 4/52 (9.6%) from Limpopo province. Clustering was not associated with age. However, strains from male patients in Mpumalanga were more likely to be clustered than strains from male patients in Limpopo and/or Gauteng province. The minimum estimate for the proportion of resistant TB that was due to transmission is 9.06% (52-22=30/331). Our results indicate that transmission of drug-resistant strains may contribute substantially to the emergence of drug-resistant tuberculosis in South Africa.

An in-house multiplex PCR test for the detection of Mycobacterium tuberculosis, its validation & comparison with a single target TB-PCR kit.

Background & objectives: The conventional techniques used in TB diagnosis like AFB (acid fast bacilli) smear microscopy lack sensitivity and the gold standard, culture test takes time. A test based on multiplex polymerase chain reaction (PCR) targeting the 38 kDa gene and IS6110 insertion sequence, specific to Mycobacterium tuberculosis was developed to further increase the sensitivity of a TB-PCR kit targeting only 38 kDa gene developed earlier in the same laboratory. The multiplex test was validated using sputum samples from pulmonary TB (PTB) cases. The sensitivity and specificity were compared with AFB smear examination and Lowenstein-Jensen (LJ) culture test. Methods: Multiplex PCR amplifying 340 and 245 bp sequence of 38 kDa gene and IS6110, respectively was standardized and analytical sensitivity was verified. Sputum samples (n=120) obtained from PTB cases were subjected to AFB smear examination, LJ culture and a multiplex as well as single target PCR test. Additionally, 72 non-TB respiratory samples were included in the study as negative controls. Results: Analytical sensitivity of multiplex PCR was found to be 100 fg for 38 kDa gene and 1 fg for IS6110. Multiplex PCR, using both the targets, showed highest sensitivity of 81.7 per cent, followed by 69.2 per cent for L-J culture test and 53.3 per cent for AFB smear when clinical diagnosis was considered as a gold standard. The sensitivity of detection of M. tuberculosis in AFB smear positive and negative samples by multiplex PCR was 93.7 and 67.9 per cent, respectively. Sensitivity of 77.1 per cent observed for the detection of M. tuberculosis with single target PCR increased to 89.2 per cent with multiplex PCR in culture positive samples. Four samples showed positive PCR results only with primers for 38 kDa gene. Interpretation & conclusions: Multiplex PCR increased the sensitivity of single target PCR and will be useful in diagnosing paucibacillary smear negative samples. Further, it can also be used to detect samples with M. tuberculosis strains lacking IS6110.

Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis.

Purpose: Extrapulmonary tuberculosis (EPTB) is emerging problem in developing and developed countries. The diagnosis of EPTB in its different clinical presentations remains a true challenge. IS6110-based polymerase chain reaction (PCR) is used for rapid identification and positivity rate of the Mycobacterium tuberculosis complex in clinical isolates of different sites of EPTB. The present study was carried out to study the prevalence of M. tuberculosis complex in clinical isolates of EPTB at tertiary care centres in Lucknow. Materials and Methods: Seven hundred fifty-six specimens were collected from the suspected cases of EPTB which were processed for Mycobacteria by Ziehl Neelson (ZN) staining and BACTEC culture. All the specimens were also processed for IS6110-based PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of the M. tuberculosis complex. Results: Of these 756 specimens, 71(9.3%) were positive for acid fast bacilli (AFB) by ZN staining, 227(30.1%) were positive for mycobacteria by BACTEC culture and IS6110 PCR were positive for M. tuberculosis complex in 165 (20.7%) isolates. We found a significant difference in sensitivities of different tests (P<0.05). Conclusions: This study reveals the positivity of M. tuberculosis complex in clinical isolates of EPTB case in tertiary care hospitals in Northern India. 72.7% of M. tuberculosis complex was confirmed by IS6110-PCR in culture isolates from different sites of EPTB. The high prevalence of the M. tuberculosis complex was seen in lymph node aspirate and synovial fluid. However, utility of PCR may play a potentially significant role in strengthening the diagnosis of EPTB especially targeting IS6110.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75 percent) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Evaluation of a nested-pcr for Mycobacterium tuberculosis detection in blood and urine samples

The polymerase chain reaction (PCR) and its variations, such as the nested-PCR, have been described as promising techniques for rapid diagnosis of tuberculosis (TB). With the aim of evaluating the usefulness of a nested-PCR method on samples of blood and urine of patients suspected of tuberculosis we analyzed 192 clinical samples, using as a molecular target the insertion element IS6110 specific of M. tuberculosis genome. Nested-PCR method showed higher sensitivity in patients with extrapulmonary tuberculosis (47.8 percent and 52 percent in blood and urine) when compared to patients with the pulmonary form of the disease (sensitivity of 29 percent and 26.9 percent in blood and urine), regardless of the type of biological sample used. The nested-PCR is a rapid technique that, even if not showing a good sensitivity, should be considered as a helpful tool especially in the extrapulmonary cases or in cases where confirmatory diagnosis is quite difficult to be achieved by routine methods. The performance of PCR-based techniques should be considered and tested in future works on other types of biological specimens besides sputum, like blood and urine, readily obtainable in most cases. The improving of M. tuberculosis nested-PCR detection in TB affected patients will give the possibility of an earlier detection of bacilli thus interrupting the transmission chain of the disease.

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2 percent (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85 percent and 98 percent, and 94 percent and 100 percent, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.

Detección de microbacterias del complejo tuberculosis en tejido periodontal: aplicado biología molecular / Detection of mycobacteria tuberculosis complex in periodontal tissue: molecular biology applied

El objetivo de esta investigación fue la detección molecular de micobacterias del complejo tuberculosis en el tejido periodontal afectado de un grupo de pacientes VIH+ y VIH- con tuberculosis pulmonar. Se incluyeron 20 pacientes, 10 VIH+ y 10 VIH- con diagnóstico microbiológico convencional de tuberculosis, provenientes de la consulta de Tisiología, y de Sala de VIH, del Hospital Simón Bolívar, Caracas. De cada uno de los pacientes fue obtenido el consentimiento informado previo al inicio de la investigación, se le realizó una minuciosa historia clínica y un examen clínico bucal, con el fin de establecer la condición periodontal de cada uno de los sujetos participantes en este estudio. Se tomaron muestras de tejido periodontal afectado de cada uno de los pacientes con indicación de cirugía periodontal. Resultados: 50% (10/20) de los pacientes con TBC VIH- presentaron gingivitis crónica localizada y el 30% (3/20) de los pacientes con TBC VIH+ presentaron gingivitis crónica generalizada. El grupo restante mostró gingivitis localizada y cuadros sugerentes de periodontitis. Por medio de la reacción en cadena de la polimerasa (PCR) en 60% (12/20) de las muestras de tejido periodontal se detectó la secuencia IS6110, que está presente en micobacterias del complejo tuberculosis. La amplificación de blancos moleculares es una metodología sensible para la detección de este grupo de microorganismos en enfermedad periodontal y pudiera ser utilizada en el diagnóstico diferencial de lesión de la cavidad bucal

Efficacy of an in-house polymerase chain reaction assay for rapid diagnosis of Mycobacterium tuberculosis in patients with tubercular lymphadenitis: Comparison with fine needle aspiration cytology and conventional techniques.

Introduction: Tubercular lymphadenitis (TB-L) is the most common manifestation of extrapulmonary tuberculosis. Excisional biopsy with histopathological examination, Ziehl-Neelsen staining (ZNS) and culture and fine needle aspiration (FNA) cytology, although useful in the diagnosis of TB-L, cannot diagnose a substantial proportion of cases. We investigated the role of an in-house polymerase chain reaction (PCR) assay targeting the IS6110 gene from the FNA material in the diagnosis of the disease. Materials and Methods: The clinical profile of 150 patients with lymphadenopathy was noted and the fine needle aspirate was collected. After cytological processing, ZNS and culture on Lowenstein-Jensen media, mycobacterial DNA was isolated from the residual aspirate material and IS6110 gene PCR was performed. Results of cytology, ZNS, culture and IS6110 gene PCR were compared. Results: There were 49 confirmed patients of TB-L based on laboratory parameters (either culture isolation of Mycobacterium tuberculosis or any two of cytology, ZNS, PCR positive) and clinical response to therapy. Sensitivity and specificity of FNA was 89.8% and 96%, of ZNS was 40.8% and 99%, of culture was 40.8% and 100% and of IS6110 gene PCR test was 100% and 92.1%. Conclusion: IS6110 PCR can be considered a valuable adjunct to cytology, ZNS and culture techniques in the diagnosis of TB-L.

Evaluation of a nested PCR targeting IS6110 of Mycobacterium tuberculosis for detection of the organism in the leukocyte fraction of blood samples.

Purpose: Tuberculosis poses a serious health problem in resource-poor settings such as India. Polymerase chain reaction (PCR) is presently seen as a promising alternative to conventional smear microscopy and culture techniques. Undiagnosed fever is a condition where the aetiology could include tuberculosis in a significant percentage. This paper evaluates a nested PCR (nPCR) using Hotstar Taq for the detection of M. tuberculosis in patients with febrile illness using insertion element, IS6110 as a target. Material and Methods: A total of 355 samples (301 HIV status unknown and 54 HIV seropositives) from patients primarily with febrile illness were tested for the presence of M. tuberculosis. Blood culture was done in a commercial automated blood culture system and nPCR in DNA extracts from buffy coat samples. Hotstar Taq polymerase was used to enhance the sensitivity of nPCR and the lower limit of detection was determined by using cloned plasmid. Results: Among the patients tested, 2% were positive by automated culture system and 6.8% of patients were positive by nPCR. Majority of the positives were from HIV seropositive individuals. The sensitivity of the nPCR was 100% and the specificity was 95.1%. The lower limit of detection was less than 1 genome copy per microlitre. Among the nPCR positives, patients from rural community were significantly higher than from the peri-urban community. Conclusions: The nPCR had a high sensitivity and specificity on buffy coat samples using Hotstar Taq polymerase in the reaction mix. Thus the technique is a valuable tool in the diagnosis of tuberculosis.