Mycobacterium Tuberculosis In Clinical Samples Research Articles

Metagenomic next-generation sequencing of osteoarticular tissue for the diagnosis of suspected osteoarticular tuberculosis.

In the detection of unknown infectious disease pathogens, the overall efficacy of traditional detection methods, such as culture, is low, and traditional PCR testing is also limited to the gene sequences of known pathogenic microorganisms. Metagenomic next-generation sequencing (mNGS) performs DNA sequencing by studying the entire microbial community genome in a given sample, without the need for isolation and culture. Previous studies have shown that mNGS performs better on pulmonary and extrapulmonary samples when compared with Xpert, traditional pathogenetic tests, and even parallel diagnostics. However, it should be emphasized that only a few studies have explored the performance of mNGS in detecting Mycobacterium tuberculosis in clinical samples associated with bone and joint infections. We conducted this retrospective study to provide additional data to support the use of mNGS in the clinical setting to identify pathogens within abscesses or tissue samples associated with bone and joint infections.

Comparison of Xpert MTB RIF (G4) With Xpert MTB (Ultra) in Diagnosis of Mycobacterium Tuberculosis in Clinical Samples

Abstract Background: Tuberculosis (TB) continues to be a major cause of morbidity and mortality worldwide. The main strategy to fight the pandemic is the development of rapid, accurate diagnostic tests, which helps to reduce the time for initiation of therapy. In 2010, WHO recommended the use of Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) (G4), which is a cartridge-based semi-automated nucleic acid amplification test (NAAT) as a primary test with an aim to improve the detection of MTB. Later on, to improve the diagnostic efficacy, the new and improved cartridges called Xpert Ultra were introduced in 2017. Materials and Methods: One thousand and sixty-nine clinical samples were utilized for the study for microscopy, culture, and NAATs (Xpert MTB/RIF G4 and Ultra). The samples were randomly allocated. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated while assuming trace results as positive and negative in two separate scenarios. Results: The sensitivity of Xpert MTB/RIF Ultra cartridge was higher (100%) than Xpert MTB/RIF G4 cartridge (95.23%) and the specificity of Ultra cartridge was lower (71.79%) when compared to G4 cartridge (91.63%). However, the specificity of Xpert Ultra cartridge improved by reclassifying the “trace” interpretation as negative (89.1%). The NPV was 100% and 99.09% for Xpert Ultra cartridge and the Xpert G4 cartridge, respectively, and did not change by reclassifying the “trace” interpretation as negative. In contrast, the PPV varied greatly between the two tests, 26.66% for Xpert Ultra cartridge and 66.66% for Xpert G4 cartridge. The PPV of Xpert Ultra cartridge was improved to 48.48% by including “trace” samples in the negative pool. Conclusion: Xpert Ultra and G4 shows good performance as a first line diagnostic test. Caution should be exercised when interpreting ‘trace’ results in a country like India without parallel confirmatory tests like microscopy and culture. Data is limited on this newly incorporated value assigned in the automated report. Conducting further studies on results interpreted as ‘trace’ will help in improving the guidelines and as well as utilisation of such tests.

Heteroresistance to rifampicin & isoniazid in clinical samples of patients with presumptive drug-resistant tuberculosis in Central India.

A combination of resistant and susceptible Mycobacterium tuberculosis (MTB) isolated from clinical specimens is referred to as heteroresistance. Heteroresistance leads to difficulties in drug resistance testing and may adversely affect treatment outcomes. The present study estimated the proportion of heteroresistance among MTB in clinical samples of presumptive drug-resistant tuberculosis (TB) patients in Central India. A retrospective analysis of data generated from line probe assay (LPA) at a tertiary care hospital in Central India between January 2013 and December 2018 was carried out. A heteroresistant MTB in a sample was indicated by the presence of both wild-type and mutant-type patterns on an LPA strip. Data analysis was carried out on interpretable 11,788 LPA results. Heteroresistance in MTB was detected in 637 (5.4%) samples. Of these, heteroresistance in MTB was detected in 413 (64.8%), 163 (25.5%) and 61 (9.5%) samples with respect to rpoB, katG and inhA genes, respectively. Heteroresistance is considered a preliminary step in the development of drug resistance. Delayed or suboptimal anti-tubercular therapy in patients with heteroresistance of MTB may elicit full clinical resistance and negatively impact the National TB Elimination Programme. Further studies are, however, needed to determine the impact of heteroresistance on treatment outcomes in individual patients.

Comparison between the PaxView TB/NTM MPCR-ULFA Kit and Xpert MTB/RIF for Mycobacterium Tuberculosis Detection in Indonesian Clinical Isolates

The PaxView TB/NTM MPCR-ULFA Kit, which targets the IS6110 and mtp40 genes for Mycobacteriumtuberculosis (MTB) detection, is a novel tool that substitutes gel electrophoresis for universal lateral flowassays. The sensitivity and specificity of this method were compared with those of established methodologiesusing Indonesian clinical isolates. In this study, 317 sputum specimens isolated from tuberculosis (TB)suspects were examined to evaluate the performance of the PaxView TB/NTM MPCR-ULFA Kit comparedto that of smear microscopy and the Xpert MTB/RIF assay. Out of 317 cases, the rate of TB-positivesamples evaluated by different methods was 33.4% (106/317; 95% CI 28.2-38.6) for smear microscopy,37.9% (120/317; 95% CI 32.5-43.2) for the Xpert MTB/RIF, and 40.7% (129/317; 95% CI 35.3-46.1) forthe PaxView TB/NTM MPCR-ULFA Kit. Compared to the Xpert MTB/RIF as a standard reference, thePaxView TB/NTM MPCR-ULFA Kit was found to possess a 92.5% sensitivity (111/120; 95% CI 87.8-97.2), a 90.8% specificity (179/197; 95% CI 86.8-94.8), 86.0% PPV (111/129; 95% CI 80.0-92.0), anda NPV 95.2% (179/188; 95% CI 92.2-98.3). The PaxView TB/NTM MPCR-ULFA Kit could be a usefulmolecular diagnostic tool to identify MTB in clinical samples in resource-limited countries, as this procedureis more cost-effective and sensitive than the Xpert MTB/RIF, and more convenient than conventional PCRgel electrophoresis approaches.

The clinical diagnostic value of Xpert MTB/RIF for the detection of Mycobacterium tuberculosis in gastric aspirates.

Background: At present, the infection and prevalence rates of tuberculosis (TB) are still high in worldwide. The Xpert MTB/RIF technology has improved the diagnosis speed of Mycobacterium tuberculosis (MTB) and facilitated the rapid treatment of TB patients.Methods: We searched experimental data derived from Xpert MTB/RIF for detecting MTB in gastric aspirates in PubMed, Embase, Web Of Science, and the Cochrane Library databases between January 2012 to April 2019. A summary receiver operating characteristic curve (SROC curve) was used to analyze the pooled sensitivity, pooled specificity, PLR, NLR, and DOR for determining the accuracy of the test.Results: Our database search resulted in 10 relevant articles. The pooled sensitivity of Xpert MTB/RIF for detecting TB in GA was 86% (95% CI, 83–89%), and I2 = 93.4%. The pooled specificity was 92% (95% CI, 90–93%) and I2 = 97.8%. In addition, the positive LR was 12.12 (95% CI, 5.60–26.21), negative LR was 0.20 (95% CI, 0.11–0.36), and the diagnostic odds ratio (DOR) was 147.04 (95% CI, 37.20–581.19). Using the SROC curve, the AUC was 0.9730 and Q* was 0.9248 (SE = 0.0261). The publication bias was P=0.517 (P>0.05).Conclusions: The Xpert MTB/RIF for detecting MTB in gastric aspirates was highly accurate. In addition, we observed that the publication bias in the present study was low. Hence, the Xpert MTB/RIF technology is highly accurate and has the advantage of rapid testing for MTB in clinical samples.

Utility of smear microscopy and GeneXpert for the detection of Mycobacterium tuberculosis in clinical samples

Rapid identification of Mycobacterium tuberculosis (MTB), its resistance to rifampicin and differentiation of MTB from nontuberculous mycobacteria (NTM) is necessary in the management of mycobacterial diseases. Culture, the "gold standard" for the detection of MTB, is time consuming. In spite of its rapidity and low cost, smear microscopy has poor sensitivity for the detection of acid-fast bacilli (AFB). A real-time PCR based rapid diagnostic method like GeneXpert MTB/RIF assay can simultaneously detect M. tuberculosis and rifampicin (RIF) resistance. Hence, we aim to compare the performance of GeneXpert MTB/RIF assay with smear microscopy and culture. In this descriptive cross-sectional study, we compared the performance of GeneXpert in pulmonary (N=127) and extrapulmonary (N=48) clinical specimens with other diagnostic methods like culture, Auramine O (AO), and Ziehl Neelsen (ZN) staining. Rifampicin resistance was detected only by GeneXpert. Demographic data and clinical history of the subjects were collected from the patient's hospital records. AO and ZN staining when compared with mycobacterial growth indicator (MGIT) culture showed the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of 68.6, 95.7, 80, 92.4, 90.3% and 65.7, 95.7, 79.3, 91.8, 89.7%, respectively. The sensitivity, specificity, PPV, NPV and accuracy of GeneXpert was 88.6, 93.6, 77.5, 97.0 and 92.6%, respectively. GeneXpert is the best available rapid diagnostic method as it can detect MTB and rifampicin resistance gene simultaneously. Accuracy and negative predictive value of GeneXpert was found to be better than AFB staining. Thus, a negative GeneXpert test can rule out TB. Further, a negative GeneXpert and a positive smear microscopy results indicate the presence of NTM. However, GeneXpert is expensive and needs sophisticated instrument when compared to smear microscopy.

Comparison of LJ Medium and BACTEC MGIT 960 Culture System for the Diagnosis of Tuberculosis

Introduction: Sputum negative pulmonary Tuberculosis (TB) is a major public health problem. So, the emergence of new techniques for a more precise and rapid microbiological identification of Mycobacterium tuberculosis in clinical samples is of great importance to improve the management of TB. Aim: To determine and compare the sensitivity and turnaround time for Mycobacterium tuberculosis detection by the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system, Lowenstein Jensen (LJ) medium and Ziehl-Neelsen (ZN) staining. Materials and Methods: An Institution based, observational, cross-sectional study was conducted at Rajendra Institute of Medical Sciences (RIMS), Ranchi, Jharkhand, India, from July 2013-March 2016. Sputum, pericardial fluid, pleural fluid, peritoneal fluid, pus and endometrial tissue samples were collected from 80 patients of suspected TB cases. All were Acid-Fast stained by ZN staining method and cultured on solid culture LJ medium and on liquid medium (MGIT). Data was analysed using Statistical Package for the Social Sciences (SPSS) software, Version 20.0 (SPSS Inc, Chicago, IL, USA). Fisher’s-Exact test was used to show association of categorical variables. Non-parametric Mann-Whitney U test was used to show median difference of non-normally distributed continuous variables of two groups. Results: Out of the 80 samples, 41 cases were positive by either of the all methods. The positive specimen for ZN staining, LJ media and MGIT were 21, 29 and 41 cases respectively. The mean Time To Detection (TTD) was shorter for MGIT system than LJ media. Both LJ medium and MGIT 960 detected all cases of sputum smear positive cases and in addition significantly higher number than ZN stain in sputum smear negative cases. MGIT 960 detected significantly higher number of cases of sputum negative cases than LJ Media. The mean TTD was also significantly shorter in case of smear positive cases than the smear negative cases by both the solid and liquid culture mediums. Conclusion: The use of liquid media (MGIT) is more accurate and rapid method for the diagnosis of TB. The combination of more than one method is also highly recommended for rapid detection and early treatment of TB.

Role of Polymerase Chain Reaction in diagnosis of Tuberculosis as compared to routine tests

Introduction: There is a need for rapid and sensitive detection of Mycobacterium tuberculosis in clinical samples. A study was conducted in which the target for the amplification being a segment of IS6110 in the M. tuberculosis chromosome was evaluated using real time PCR and its results were compared with routine tests, using pulmonary and extra-pulmonary specimen.
 Methods: In this descriptive cross-sectional retrospective study, specificity and sensitivity of PCR were analyzed. A total of 293 clinical samples were processed at a tertiary care hospital of Peshawar, during the time period of 2016-2018, from patients suspected of having pulmonary and extra-pulmonary tuberculosis and Follow up patients with DOTS treatment and MDR treatment that are referred by tertiary hospital were also included in this study after taking their informed consent. Patients not willing to participate in the study were excluded. For identification specimens were stained by Ziehl Neelsen staining (ZN), cultured on Lowenstein–Jensen (LJ) medium and then confirmed by PCR for the detection of Mycobacterium tuberculosis (MTB).
 Results: Of the 293 samples, 165(56.3%) were from males and 128(43.7%) females. Mean age was 44 years (2-85 years). Specimen types included: CSF (30.4%), pleural fluid (4.1%), sputum (15%), urine (2.4%), synovial fluid (2.4%), other fluids (33.1%) and biopsies (12.6%). Only 3.1% of specimens were ZN-smear positive for (MTB). LJ culture identified 7.2% whereas PCR method detected (MTB) in 15% of the total specimens. Using PCR as gold standard, ZN microscopy correctly identified 20.5% of total (MTB) positive specimens and LJ culture detected 47.7%.Specimen types showed significant association with PCR test: 42.9% of synovial fluid samples and 41.7% of pleural fluid samples; 28.6% of urine samples were positive for (MTB) by PCR method. This indicates that PCR analysis of these specimens’ exhibit greater positivity rates for (MTB) as opposed to CSF and other fluids and biopsies
 Conclusions: TB PCR is a rapid and reliable test in the diagnosis and management of tuberculosis.

Direct detection of Mycobacterium tuberculosis in clinical samples by a dry methyl green loop-mediated isothermal amplification (LAMP) method

The purpose of this study was to develop a simple visual methyl green (MeG) based dry loop-mediated isothermal amplification (LAMP) method for early detection of Mycobacterium tuberculosis (MTB) from clinical samples. We identified MeG as an indicator of a positive LAMP reaction, where a positive reaction gave a blue-green color while a negative reaction was colorless. The MeG MTB-LAMP system was further simplified by drying all reagents for ease of use, and was then validated for its ability to diagnose TB directly using Nepalese clinical samples. We evaluated the dry MeG MTB-LAMP with 69 new TB suspected samples from patients that did not have a confirmed history of TB treatment and found the sensitivity in culture positive samples as 92.8% (13/14) and specificity in culture negative samples as 96.3% (53/55). Our LAMP system has the potential to be a point of care test for early diagnosis of active TB in developing countries.

Direct detection of rpoB and katG gene mutations of Mycobacterium tuberculosis in clinical samples

ObjectivesTo study the rpoB and katG gene mutation rate and its markers. MethodsCross-sectional study methods were used to study Tuberculosis. A total of 45 sputum samples were collected from Annapurna Neurological Institute and Allied sciences. Then, acid fast bacilli staining were performed. Positive and negative samples were carried for conventional polymerase chain reaction identification and electrophoresis. ResultsOut of 45 samples, 3 were acid fast bacilli positive and the rest were negative. Male participants were more as compare to female participants and the mutation in rpoB and katG gene was found similar i.e. 6.66% among the total samples. ConclusionsWe can conclude that genetic mutation in Mycobacterium tuberculosis can be identified directly from the clinical samples. However, we have carried this study in less sample size and to validate research on large number of sample is recommended.

RNomic identification and evaluation of npcTB_6715, a non-protein-coding RNA gene as a potential biomarker for the detection ofMycobacterium tuberculosis

Technological advances in RNA biology greatly improved transcriptome profiling during the last two decades. Besides the discovery of many small RNAs (sRNA) that are involved in the physiological and pathophysiological regulation of various cellular circuits, it becomes evident that the corresponding RNA genes might also serve as potential biomarkers to monitor the progression of disease and treatment. sRNA gene candidate npcTB_6715 was previously identified via experimental RNomic (unpublished data), and we report its application as potential biomarker for the detection of Mycobacterium tuberculosis (MTB) in patient samples. For proof of principle, we developed a multiplex PCR assay and report its validation with 500 clinical cultures, positive for Mycobacteria. The analysis revealed 98.9% sensitivity, 96.1% specificity, positive and negative predictive values of 98.6% and 96.8%, respectively. These results underscore the diagnostic value of the sRNA gene as diagnostic marker for the specific detection of MTB in clinical samples. Its successful application and the general ease of PCR‐based detection compared to standard bacterial culture techniques might be the first step towards ‘point‐of‐care’ diagnostics of Mycobacteria. To the best of our knowledge, this is the first time for the design of diagnostic applications based on sRNA genes, in Mycobacteria.

Rapid laboratory diagnosis of pulmonary tuberculosis.

Tuberculosis (TB) ranks as the second leading cause of death from an infectious disease worldwide. Early diagnosis of Mycobacterium tuberculosis in clinical samples becomes important in the control of TB both for the treatment of patients and for curbing of disease transmission to the others in the community. The study objective was to perform Ziehl-Neelsen (ZN) staining, fluorochrome staining, line probe assay (LPA), and loop-mediated isothermal amplification (LAMP) assay for rapid detection of pulmonary TB (PTB) and to compare the results of LPA and LAMP in terms of sensitivity, specificity, and turnaround time. A total of 891 sputum samples from clinically diagnosed/suspected cases of TB were subjected to ZN and fluorochrome staining. Smear positive samples were subjected to LPA, and smear negative were cultured on Lowenstein-Jensen media. A total of 177 samples were subjected to liquid culture and LAMP. Conventional culture was considered as "gold standard" for calculation of parameters. Light-emitting diode fluorescence microscopy had the same sensitivity as ZN with similar high specificity. LPA was performed on 548 sputum samples which includes 520 smear positive and 28 smear negative culture positive samples and multidrug-resistant TB was detected in 32.64%. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of TB-LAMP on direct sputum samples was found to be 98.96%, 95%, 96%, and 98.70%, respectively, when compared with ZN smear microscopy. By considering culture as "gold standard," LAMP showed a sensitivity, specificity, PPV, and NPV of 98.94%, 96.34%, 96.90%, and 98.75%, respectively. The sensitivity and PPV of TB-LAMP were 98.97% and 96%, respectively, when compared with LPA. A successful rapid laboratory diagnosis of PTB is possible when one combines the available methodology of microscopy, culture as well as molecular techniques. The LAMP assay was found to be simple, self-contained, and efficacious for early diagnosis of suspected cases of PTB with advantages of having a high throughput, no requirements of sophisticated equipment, and complex biosafety facilities.

Genetic Determinants of Drug Resistance in Mycobacterium tuberculosis and Their Diagnostic Value.

The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs. To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance. We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci. These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs.

Electrochemical Determination of 16s Ribosomal RNA of Mycobacterium Tuberculosis Using Magnetite on Silica with DNA-Functionalized Gold Nanoparticles

ABSTRACTA new electrochemical biosensor for the determination of mycobacterium tuberculosis in clinical samples is reported. A sandwich hybridization layer system with magnetite on a silica nanocomposite was employed as the biosensor. The use of DNA-functionalized gold nanoparticles enhanced the signal of target 16 s ribosomal RNA of mycobacterium tuberculosis in clinical samples. When compared with the commonly used real-time fluorescent polymerase chain reaction, the analysis time was significantly reduced. A linear relationship was obtained for 16 s ribosomal RNA concentrations from 0 to 130 µM. This electrochemical biosensor provides rapid and simple determination of mycobacterium tuberculosis in clinical samples with high sensitivity and low cost.

Comparison of the Diagnostic Performance of Two Real-Time PCR Systems for the Detection of Mycobacterium tuberculosis in Clinical Samples

Comparison of the Diagnostic Performance of Two Real-Time PCR Systems for the Detection of Mycobacterium tuberculosis in Clinical Samples

AID TB resistance line probe assay for rapid detection of resistant Mycobacterium tuberculosis in clinical samples

To determine the sensitivity and specificity of AID TB Resistance line probe assay (AID Diagnostika, Germany) to detect Mycobacterium tuberculosis and its resistance to first- and second-line drugs in clinical samples using BACTEC 460TB as the reference standard. The test consists on three strips to detect resistance to isoniazid/rifampicin, fluoroquinolones/ethambutol, and kanamycin/amikacin/capreomycin/streptomycin, respectively. This test was performed on 65 retrospectively selected clinical samples corresponding to 32 patients. A valid result was obtained for 92.3% (60/65), 90.8% (59/65) and 78.5% (51/65) of the samples tested, considering the three strips, respectively. Global concordance rates between AID and BACTEC for detecting resistance to isoniazid, rifampicin, fluoroquinolones, ethambutol, kanamycin/capreomycin and streptomycin were 98.3% (59/60), 100% (60/60), 91.5% (54/59), 72.9% (43/59), 100% (51/51) and 98.0% (50/51), respectively. Regarding the discordant results obtained between AID and BACTEC, the alternative molecular methods performed (GenoType MTBDRplus, GenoType MTBDRsl [Hain Lifescience, Germany] and/or pyrosequencing) confirmed the genotypic result in 90.9% (20/22) of the cases. AID line probe assay is a useful tool for the rapid detection of drug resistance in clinical samples enabling an initial therapeutic approach. Nevertheless, for a correct management of drug resistant tuberculosis patients, molecular results should be confirmed by a phenotypic method.

Rapid detection of Mycobacterium tuberculosis in clinical samples by multiplex polymerase chain reaction (mPCR)

Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1%) and specificity (89.6%) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify M. tuberculosis from clinical samples and enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.

A historical primer on the US farm bill: Supply management and conservation policy

AIM--To investigate the use of the polymerase chain reaction (PCR) in the routine laboratory for the detection of Mycobacterium tuberculosis in clinical samples. METHODS--Samples were divided and processed separately for the detection of M tuberculosis by microscopy, culture and PCR. After DNA extraction, PCR was performed with primers specific for the insertion element IS6110 and the product was analysed by agarose gel electrophoresis, Southern blotting or dot blotting and hybridisation with a digoxigenin labelled internal probe. Each sample was tested for inhibitors of Taq polymerase with the aid of an internal control. Multiple negative and positive controls were used to monitor each step of the procedure. RESULTS--The data from two laboratories, using the same operating procedures, were combined. Of 1957 specimens, 79 (4%) were culture and PCR positive, while 1839 (93.9%) were negative in both tests. Thirty specimens (1.5%) were PCR positive only and nine (0.5%) were culture positive but PCR negative. CONCLUSION--Using culture and clinical history as the gold standard, sensitivity and specificity for PCR were 92.1% and 99.8%, respectively. With elaborate precautions, PCR is a suitable and reliable method for the detection of M tuberculosis in clinical samples in a routine microbiology laboratory.

Development and evaluation of a multiplex polymerase chain reaction for the detection ofMycobacterium tuberculosisfrom pulmonary specimens

The diagnosis of pulmonary tuberculosis is still a major challenge. Using a polymerase chain reaction (PCR), one can detect Mycobacterium tuberculosis in clinical samples within a few hours. However, single gene targets may result in false negativity due to the absence of target DNA in some M. tuberculosis isolates. The objective of this study was to develop and evaluate a multiplex PCR (M-PCR) using IS6110 and devR primers for the detection of M. tuberculosis in sputum samples. Sputum samples were collected from: (1) 200 confirmed cases of tuberculosis; (2) 100 suspected cases of tuberculosis diagnosed on the basis of clinical and radiological findings; (3) 200 non-tubercular patients suffering from respiratory diseases other than tuberculosis, in whom tuberculosis had been excluded. All 500 sputum samples were subjected to PCR using IS6110 primers, and M-PCR using IS6110 and devR primers; results were compared with conventional techniques. It was found that M-PCR was 97.5% successful in detecting the presence of tuberculosis in the confirmed tuberculosis group as compared to 84.5% by IS6110-based PCR. In the suspected tuberculosis group, M-PCR could detect 45% of cases as compared to 40% by IS6110-based PCR. Overall, the specificities of both the PCR and M-PCR were found to be 96.5%. This study demonstrated that the M-PCR assay is more sensitive than the IS6110-based PCR for the detection of M. tuberculosis in sputum specimens and could be applied in situations of highly suspected tuberculosis when all others tests including IS6110 PCR are negative.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.