Rapid Detection Of Mycobacterium Tuberculosis Research Articles

Study of rapid detection of Mycobacterium Tuberculosis through GeneXpert MTB/RIF assay from acid fast bacilli smear-negative specimens in a Tertiary Care Hospital, Biratnagar Nepal

Introduction: Tuberculosis (TB) is an important public health concern around the world. It is well known that acid-fast bacilli (AFB) smear-negative TB cases are a major source of spreading TB to others when left untreated.
 Objectives: Our study aim to detect Mycobacterium tuberculosis (MTB) in AFB smear-negative samples through GeneXpert MTB/RIF assay and also to evaluate the drug resistance patterns towards Mycobacterium tuberculosis in hospitals.Methodology: This descriptive cross-sectional study was conducted among smear-negative presumptive TB patients at a tertiary care center with effect from September 2022 to September 2023 after approval from the Institutional Review Committee of the college. A convenience sampling method was used.
 Results: Out of 653 smear-negative samples, 71 (10.9%) were positive for TB and 17 (2.6%) were rifampicin resistance cases detected by GeneXpert MTB/RIF assay. The cases of tuberculosis were more in age groups between 46->60 years and least in 17-30 years of age. Pulmonary samples yielded a higher number of MTB 66 (92.95%) as compared to extra-pulmonary samples 5 (7.04%). There is an almost equal proportion of mycobacterium infection in males 37(52.11%) and females 34(47.88%). All the rifampicin resistance cases detected in our study were from pulmonary samples.
 Conclusion: Our study shows that the sensitivity of GeneXpert MTB/RIF assay is higher than AFB smear microscopy. We are missing actual TB cases while using AFB smear microscopy only. Hence the GeneXpert MTB/RIF assay is important for the rapid detection of MTB in smear-negative as well as smear-positive drug resistance MTB cases.

Rapid detection of Mycobacterium tuberculosis in sputum using CRISPR-Cas12b combined with cross-priming amplification in a single reaction.

In this study, we successfully established a new One-Pot method, named TB One-Pot, for detecting Mtb in sputum by combining CRISPR-cas12b-mediated trans-cleavage with cross-priming amplification (CPA). Our study evaluated the diagnostic performance of TB One-Pot in clinical sputum samples for tuberculosis. The findings provide evidence for the potential of TB One-Pot as a diagnostic tool for tuberculosis.

Rapid detection of Mycobacterium tuberculosis using recombinase polymerase amplification: A pilot study.

Tuberculosis remains one of the leading causes of death worldwide, especially in low- and middle-income countries. Tuberculosis treatment and control efforts are hindered by the difficulty in making the diagnosis, as currently available diagnostic tests are too slow, too expensive, or not sufficiently sensitive. Recombinase polymerase amplification (RPA) is a novel technique that allows for the amplification of DNA rapidly, at constant temperature, and with minimal expense. We calculated and compared the limit of detection, sensitivity, and specificity of two RPA-based assays for the diagnosis of pulmonary tuberculosis, using two sets of published primers. We also calculated and compared the assays' limits of detection and compared their performance using two different DNA extraction methods prior to amplification (a commercially available DNA extraction kit vs. the chelex method). The RPA-lateral flow assay had a limit of detection of 5 fg/μL of DNA, a sensitivity of 53.2%, and a specificity of 93.3%, while the real time-RPA assay had a limit of detection of 25 fg/μL of DNA, a sensitivity of 85.1%, and a specificity of 93.3%. There was no difference in assay performance when DNA extraction was carried out using the commercial kit vs. the chelex method. The real-time RPA assay has adequate sensitivity and specificity for the diagnosis of pulmonary tuberculosis and could be a viable diagnostic tool in resource-limited settings, but the lateral flow assay did not perform as well, perhaps due to the fact we used stored sputum specimens from a biorepository. More work is needed to optimize the RPA-lateral flow assay, to get a more accurate estimate of its specificity and sensitivity using prospectively collected specimens, and to develop both assays into point-of-care tests that can be easily deployed in the field.

Diagnostic accuracy of gold nanoparticle combined with molecular method for detection of Mycobacterium tuberculosis: A systematic review and meta-analysis study

The importance of rapid detection of Mycobacterium tuberculosis (MTB) has become greater than ever. Among biosensors as a rapid test, gold nanoparticles (GNPs) can play an important role in accelerating the diagnosis of TB. This systematic review and meta-analysis study were conducted to evaluate the diagnostic accuracy of GNPs for the diagnosis of MTB. All cross sectional and case control studies as original article about rapid detection of MTB using GNPs combined with molecular methods published in PubMed, Web of Science, and Scopus databases were selected, retrieved and appraised up to September 2021. The sensitivity and specificity of GNPs in different studies ranges from 83.7% to 100% for sensitivity and from 86.6% to 100% for specificity. The highest sensitivity and specificity of GNPs combined with molecular methods was related to the LAMP method. Therefore, GNPs are a simpler, low cost and more efficient way in the clinical diagnosis of TB.

FLASH-TB: an Application of Next-Generation CRISPR to Detect Drug Resistant Tuberculosis from Direct Sputum

Offering patients with tuberculosis (TB) an optimal and timely treatment regimen depends on the rapid detection of Mycobacterium tuberculosis (Mtb) drug resistance from clinical samples. Finding Low Abundance Sequences by Hybridization (FLASH) is a technique that harnesses the efficiency, specificity, and flexibility of the Cas9 enzyme to enrich targeted sequences. Here, we used FLASH to amplify 52 candidate genes probably associated with resistance to first- and second-line drugs in the Mtb reference strain (H37Rv), then detect drug resistance mutations in cultured Mtb isolates, and in sputum samples. 92% of H37Rv reads mapped to Mtb targets, with 97.8% of target regions covered at a depth ≥ 10X. Among cultured isolates, FLASH-TB detected the same 17 drug resistance mutations as whole genome sequencing (WGS) did, but with much greater depth. Among the 16 sputum samples, FLASH-TB increased recovery of Mtb DNA compared with WGS (from 1.4% [IQR 0.5-7.5] to 33% [IQR 4.6-66.3]) and average depth reads of targets (from 6.3 [IQR 3.8-10.5] to 1991 [IQR 254.4-3623.7]). FLASH-TB identified Mtb complex in all 16 samples based on IS1081 and IS6110 copies. Drug resistance predictions for 15/16 (93.7%) clinical samples were highly concordant with phenotypic DST for isoniazid, rifampicin, amikacin, and kanamycin [15/15 (100%)], ethambutol [12/15 (80%)] and moxifloxacin [14/15 (93.3%)]. These results highlighted the potential of FLASH-TB for detecting Mtb drug resistance from sputum samples.

Modified gold nanoparticle colorimetric probe-based biosensor for direct and rapid detection of Mycobacterium tuberculosis in sputum specimens

The incidence of Mycobacterium tuberculosis (MTB) is increasing due to lack of appropriate diagnostic and therapeutic methods. Therefore, early and accurate detection of this bacteria plays a significant role in controlling tuberculosis. This study aimed to design, develop, and implement a direct and rapid detection method of MTB using modified gold nanoparticle (AuNP) colorimetric probe-based biosensor in sputum specimens. Spherical AuNPs were synthesized by the citrate reduction method and were functionalized using thiol-modified oligonucleotides (AuNP-biosensor). AuNP-biosensor and IS6110 PCR were compared to the gold standard in terms of analytical and clinical sensitivity and specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic odds ratio (DOR), and accuracy in 52 clinical specimens. Gold standard was defined as a positive result in concentrated sputum smear microscopy (SSM), culture, or Xpert MTB/RIF.The AuNP-biosensor had 100% sensitivity and specificity for detection of total sputum DNA in less than 15min with ready-to-use AuNP-biosensor. PPV, NPV, DOR and accuracy of this method were 100%, 100%, 2325 and 100%, respectively. Considering the promising results of the diagnostic value indices of the AuNP-biosensor, the designed method is an affordable, rapid, reliable, and cost-beneficial way for direct detection of MTB in sputum specimens.

A Comparative Study on Visual Detection of Mycobacterium tuberculosis by Closed Tube Loop-Mediated Isothermal Amplification: Shedding Light on the Use of Eriochrome Black T.

Loop-mediated isothermal amplification is a promising candidate for the rapid detection of Mycobacterium tuberculosis. However, the high potential for carry-over contamination is the main obstacle to its routine use. Here, a closed tube LAMP was intended for the visual detection of Mtb to compare turbidimetric and two more favorable colorimetric methods using calcein and hydroxy naphthol blue (HNB). Additionally, a less studied dye (i.e., eriochrome black T (EBT)) was optimized in detail in the reaction for the first time. Mtb purified DNA and 30 clinical specimens were used to respectively determine the analytical and diagnostic sensitivities of each method. The turbidimetric method resulted in the best analytical sensitivity (100 fg DNA/reaction), diagnostic sensitivity and specificity (100%), and time-to-positivity of the test (15 min). However, this method is highly prone to subjective error in reading the results. Moreover, HNB-, calcein-, and EBT-LAMP could respectively detect 100 fg, 1 pg, and 1 pg DNA/reaction (the analytical sensitivities) in 30, 15, and 30 min, while the diagnostic sensitivity and specificity were respectively 93.3% and 100% for them all. Interestingly, EBT-LAMP showed the lowest potential for subjective error in reading the results. This report helps judiciously choose the most appropriate visual method, taking a step forward toward the field applicability of LAMP for the detection of Mtb, particularly in resource-limited settings.

Utility of line probe assay in detecting drug resistance and the associated mutations in patients with extrapulmonary tuberculosis in Addis Ababa, Ethiopia.

Introduction:Molecular tests allow rapid detection of Mycobacterium tuberculosis and drug resistance in a few days. Identifying the mutations in genes associated with drug resistance may contribute to the development of appropriate interventions to improve tuberculosis control. So far, there is little information in Ethiopia about the diagnostic performance of line probe assay (LPA) and the M. tuberculosis common gene mutations associated with drug resistance in extrapulmonary tuberculosis. Thus, this study aimed to assess the frequency of drug resistance-associated mutations in patients with extrapulmonary tuberculosis (EPTB) and to compare the agreement and determine the utility of the genotypic in the detection of drug resistance in Addis Ababa, Ethiopia.Methods:A cross-sectional study was conducted on stored M. tuberculosis isolates. The genotypic and phenotypic drug susceptibility tests were performed using LPA and BACTEC-MGIT-960, respectively. The common mutations were noted, and the agreement and the utility of the LPA were determined using the BACTEC-MGIT-960 as a gold standard.Results:Of the 151 isolates, the sensitivity and specificity of MTBDRplus in detecting isoniazid resistance were 90.9% and 100%, respectively. While for rifampicin, it was 100% and 99.3% for sensitivity and specificity, respectively. The katG S315Tl was the most common mutation observed in 85.7% of the isoniazid-resistant isolates. In the case of rifampicin, the most common mutation (61.9%) was observed at position rpoB S531L. Mutations in the gyrA promoter region were strongly associated with Levofloxacin and Moxifloxacin resistance.Conclusion:Line probe assay has high test performance in detecting resistance to anti-TB drugs in EPTB isolates. The MTBDRplus test was slightly less sensitive for the detection of isoniazid resistance as compared to the detection of rifampicin. The most prevalent mutations associated with isoniazid and rifampicin resistance were observed at katG S315Tl and rpoB S531L respectively. Besides, all the fluoroquinolone-resistant cases were associated with gyrA gene. Finally, a validation study with DNA sequencing is recommended.

Increased Detection of Mycobacterium tuberculosis Disease Using a Tissue-Based Laboratory-Developed Polymerase Chain Reaction Assay Compared to Standard Diagnostics.

Standard diagnostics for Mycobacterium tuberculosis (MTB) including acid-fast bacilli (AFB) smear and culture, and Xpert™ MTB/RIF real-time Polymerase Chain Reaction (RT-PCR; Xpert) have variable sensitivity and/or long turnaround times. We describe the clinical performance of a laboratory-developed tissue-based MTB PCR compared with AFB culture and Xpert using a composite reference standard (CRS). Over an 8-year period, MTB PCR was performed on pulmonary, pleural, or lymph node specimens for 36 patients. Of these, 11 met criteria for confirmed/probable MTB using CRS. MTB PCR was positive in 100% (11/11), AFB cultures were positive in 73% (8/11), and Xpert in 0% (0/4). MTB PCR was negative in 25 cases of "No MTB" (100% specific). The MTB PCR assay resulted faster than positive AFB culture (mean time 4.3 versus 21.2 days). Tissue-based MTB PCR was associated with increased and rapid detection of MTB, improving clinical sensitivity in strongly suspected MTB cases.

MPT64 assays for the rapid detection of Mycobacterium tuberculosis

BackgroundTuberculosis (TB) is a serious infectious disease caused by Mycobacterium tuberculosis (MTB). An estimated 1.7 billion people worldwide are infected with Mycobacterium tuberculosis (LTBI) during the incubation period without any obvious symptoms. Because of MTB’s high infection and mortality rates, there is an urgent need to develop a fast, portable, and sensitive diagnostic technology for its detection.MethodsWe included research from PubMed, Cochrane Library, Web of Science, and Embase and extracted the data. MetaDisc and STATA were used to build forest plots, Deek’s funnel plot, Fagan plot, and bivariate boxplot for analysis.ResultsForty-six articles were analyzed, the results of which are as follows: sensitivity and specificity were 0.92 (0.91–0.93) and 0.95 (0.94–0.95) respectively. The NLR and PLR were 0.04 (95% CI 0.03–0.07) and 25.32 (95% CI 12.38–51.78) respectively. DOR was 639.60 (243.04–1683.18). The area under the SROC curve (AUC) was 0.99.ConclusionsMPT64 exhibits good diagnostic efficiency for MTB. There is no obvious heterogeneity between the three commercial kits.

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification.

Tuberculosis (TB), as a common infectious disease, still remains a severe challenge to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real-time recombinase polymerase amplification (RPA) technology for identifying M. tuberculosis within 20min at 39°C via custom-designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4-R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify M. tuberculosis with the detection limit of 4·0 copies per μl, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B-based RPA could determine the genomic DNA extracted from M. tuberculosis with high reliability (100%, 22/22). More importantly, when testing clinical sputum samples, the real-time RPA displayed an admirable sensitivity (90%, 95% CI: 80·0-96·0%) and specificity (98%, 95% CI: 89·0-100·0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real-time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.

Rapid detection of Mycobacterium tuberculosis in pus samples of suspected cases of extrapulmonary tuberculosis by GeneXpert MTB/RIF assay and its comparison with conventional methods

Introduction: GeneXpert MTB/RIF or cartridge-based nucleic acid amplification test is a rapid diagnostic tool used for the detection of Mycobacterium tuberculosis and its resistance to rifampicin. It can be offered as a first-line diagnostic modality in suspected cases of extrapulmonary tuberculosis (TB). This study was done to evaluate the diagnostic accuracy of the Xpert MTB/RIF assay for the detection of M. tuberculosis in extrapulmonary specimens and its resistance to rifampicin.Materials and Methods: This study was done in the Department of Microbiology at Indira Gandhi Institute of Medical Sciences, Patna, Bihar, over a period 18 months from January 2018 to June 2019 in 508 patients. The analysis and interpretation of the data were performed using Microsoft excel. The quantitative data obtained were expressed as percentage in tabular form.Results: Out of a total of 508 pus samples in suspected extrapulmonary TB patients, the percentage of males was more than females with male-to-female ratio being 1.6:1. Among the testing of Mycobacterium tuberculosis by various methods, maximum detection was done by Xpert MTB/RIF assay in 37% of cases. The least number of cases were detected by Ziehl–Neelsen staining (8.1%). Rifampicin resistance was detected in 14.9% of cases (28/188) while its resistance was not detected in 85.1% of cases (160/188) among cases of M. tuberculosis detected. Many of the negative samples on fluorescent and Ziehl–Neelsen staining came to be positive with GeneXpert testing assuring the sensitivity and specificity of Xpert MTB/RIF assay.Conclusion: In today's era when we are going for TB elimination, not only rapid TB case detection but also the early determination of multidrug resistance status is of prime value. This will not only assist in the identification of the patient with disease and drug resistance but also initiate prompt early intervention and treatment and moving a step closer in achieving the goal of TB elimination.

Clinical efficacy of metagenomic next-generation sequencing for rapid detection of Mycobacterium tuberculosis in smear-negative extrapulmonary specimens in a high tuberculosis burden area

ObjectiveThe aim of this study was to assess the clinical utility of metagenomic next-generation sequencing (mNGS) on smear-negative extrapulmonary specimens collected in China. MethodsSpecimens were tested by mNGS and other routine tests for tuberculosis (TB). The diagnostic accuracy of mNGS was calculated and compared with the final clinical diagnosis. ResultsThe sensitivity of mNGS was found to be significantly higher than the sensitivities of the other routine TB tests. Receiver operating characteristic curve analysis showed that mNGS achieved the highest area under the curve (AUC) value of 0.79. The mNGS positive rate was highest for tuberculous meningitis. All non-tuberculous extrapulmonary pathogens were directly and simultaneously detected. ConclusionsmNGS appeared to be superior to all previous etiological tests for TB on smear-negative extrapulmonary specimens and could identify all possible pathogens at once within 48 h.

Role of automated GenexpertMTb/Rif system for rapid detection of Mycobacterium tuberculosis and Rifampicin resistance in extrapulmonary tuberculosis: A prospective study

Introduction: Extrapulmonary Tuberculosis is infection of mycobacterium tuberculosis involving organ other than lung. It account for 15-20% of all TB cases. Various combined approach used for diagnosis of Extrapulmonary TB. GeneXpert MTB/RIF System is cartridge based nucleic acid amplification test to detect tuberculosis and rifampicin resistance in two hours. Materials and Methods: This hospital based prospective study was carried out for period of 6 months with objective:-1) To evaluate the efficacy of GeneXpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis (EPTB) by comparing it with composite reference standard which include smear microscopy, clinical findings at time of presentation and three months follow up and radiological investigations.2) To look for Rifampicin Resistance by GeneXpert. Results: Out of total of 168 enrolled patients 135 patients met the criteria of composite reference standard (CRS). Among total 135 CRS+ve EPTB Cases, 23 were paediatric (below 12 years of age) and 112 were Adults. Smear Microscopy was positive in 18 patients while GeneXpert gave positive results in 38 patients (8 paediatric and 30 adult patients). Rifampicin resistance was detected in 4 patients. Pleural effusion is the most commonly obtained sample followed by CSF, lymphnode, ascitic, urine, other fluid. Conclusions: Sensitivity of GeneXpert is more as compared to smear microscopy. GeneXpert showed better sensitivity in CSF, tissue, lymphnode, pleural and urine. So early microbiological diagnosis and Rifampicin drug sensitivity in Extrapulmonary sample lead to early treatment initiation of Tuberculosis and decrease morbidity and mortality due to tuberculosis. RNTCP has also recommended use of GeneXpert in suspected TB in Extrapulmonary sites. Keywords: GeneXpert, Extrapulmonary Tuberculosis, Smear Microscopy, Sensitivity.

Whole genome enrichment approach for rapid detection of Mycobacterium tuberculosis and drug resistance-associated mutations from direct sputum sequencing

Tuberculosis is the leading cause of death among infectious diseases worldwide. Detection of Mycobacterium tuberculosis (Mtb), using routine culture-based methods is time consuming resulting in delayed diagnosis and poor treatment outcomes. Currently available molecular tests provide faster diagnosis but are able to screen only limited hot-spot mutations. Whole genome sequencing from direct sputum offers a potential solution, however, due to the presence of other microbes and host DNA its use in diagnostic testing remains challenging.In this study, we present a targeted Mtb-enrichment assay for lineage-4 coupled with an improved analysis pipeline that uses 1657 bacterial taxa as background for reducing non-Mtb genome from sputum DNA. This method drastically improved the recovery of Mtb DNA from sputum (Mtb alignment increased from 3% to >65%) as compared to non-enrichment-based sequencing. We obtained >99% Mtb genome coverage as compared to 49% in non-enriched sputum sequencing. We were able to identify Mtb positive samples from controls with 100% accuracy using Mpt64 gene coverage. Our method not only achieved 100% sensitivity to resistance variants profiled by line probe assay (LPA), but also outperformed LPA in determining drug resistance based on phenotypic drug susceptibility tests for 6 anti-tuberculosis drugs (accuracy of 97.7% and 92.8% by enriched WGS and LPA, respectively).

Rapid detection of Mycobacterium tuberculosis in children using blood and urine specimens.

INTRODUCTION: Laboratory and clinical features of childhood tuberculosis (TB) are non-specific and establishing an accurate diagnosis remains a challenge. This study evaluated a Single tube nested-PCR (STNPCR) to detect genomic DNA of Mycobacterium tuberculosis complex in blood and urine. METHODS: Biological samples were obtained from children (<15 years old) with clinical suspicion of pulmonary and extrapulmonary TB at public hospitals in Recife-Pernambuco, Brazil. Cultures yielded negative results in a majority of childhood TB cases, which are generally paucibacillary. A set of clinical, epidemiological, radiological, and laboratory criteria with evident clinical improvement after anti-TB treatment were frequently used to define childhood TB cases. RESULTS: Ninety children with clinical suspicion were enrolled in this study (44 with TB and 46 without TB). The pulmonary TB group had 20 confirmed cases and 46 negative controls, while the extrapulmonary TB group had 24 confirmed cases. The STNPCR showed sensitivities to pulmonary and extrapulmonary TB of 47.4% and 52.2% (blood) and 38.8% and 20% (urine), respectively. Considering the low performance of STNPCR on separate samples, we decided to perform a combined analysis (parallel sensitivity analysis) of the results from blood and urine samples. The parallel sensitivity increased to 65% in blood and 62.5% in urine. The specificity in both samples ranged from 93.5-97.8%. CONCLUSIONS: Although STNPCR showed moderate sensitivity, the specificity is high; therefore, the test can be used as an auxiliary tool to diagnose TB in children. It is a rapid test that demonstrated better performance than other diagnostic tests in paucibacillary samples as it does in childhood tuberculosis.

Comparison of Led Fluorescent Microscopy and the Gene Xpert MTB/RIF Assay in Diagnosis of Pulmonary and Extrapulmonary Tuberculosis

Objectives: The objective of this study was to evaluate Gene Xpert MTB/RIF Assay and anid fast staining (AFB) for rapid detection of Mycobacterium tuberculosis in specimen of patients suspected of pulmonary tuberculosis (PTB) and extra pulmonary tuberculosis (EPTB).&#x0D; Methods: A comparative cross-sectional study of 400 samples (PTB-365 and EPTB-35) of patients visiting National Tuberculosis Centre (NTC) was conducted from July 2018 to December 2018. Gene Xpert MTB/ RIF Assay, smear microscopy were performed under standard guideline inside biosafety cabinet class II. The result obtained from both the tests were analyzed using SPSS 20.0 software and Excel 2019.&#x0D; Results: Of the total samples, 18% (72/400) and 39% (156/400) were positive by AFB smear microscopy and Xpert MTB/RIF assay respectively. Prevalence of MTB positive was highest in the age group 35-44 years, 33 cases (17.74%) were detected in total, with a male to female ratio of 2.3:1. Pleural fluid, pus, and CSF fluid also yielded positive results with the Gene Xpert MTB/RIF assay accounting 1.28%, 0.64% and 1.28% of MTB positive case respectively. Rifampicin resistance was observed in 1.28% of the cases.&#x0D; Conclusion: The key findings of this study suggest that Gene Xpert test should be implemented as primary diagnostic test for PTB and EPTB.

Mycobacterium tuberculosis strain H37Rv Electrochemical Sensor Mediated by Aptamer and AuNPs-DNA.

The accurate and rapid detection of Mycobacterium tuberculosis ( M. tuberculosis) is essential for the effective treatment of tuberculosis. In this article, we propose an electrochemical sensor to detect M. tuberculosis reference strain H37Rv. The sensor contains an H37Rv aptamer and oligonucleotides modified with gold nanoparticles (AuNPs-DNA). An H37Rv aptamer screened by our laboratory was used as the recognition probe. The change in frequency shift mediated by AuNPs-DNA in the presence of H37Rv was detected using a multichannel series piezoelectric quartz crystal (MSPQC) system. Three oligonucleotides modified with gold nanoparticles were designed. These oligonucleotides contained 12, 12, and 13 bases that hybridized with the 37-nt H37Rv aptamer. H37Rv aptamer was immobilized on the gold electrode surface by Au-S bonds. A conductive-layer was then formed by sequential hybridization of the aptamer with the three designed AuNPs-DNAs. When H37Rv was present, it specifically bound to the aptamer, resulting in the detachment of AuNPs-DNA from the electrode. The conductive layer was thereby replaced by a nonconductive complex of aptamer and bacteria. These changes were monitored by the MSPQC system. The proposed sensor is rapid, specific and sensitive, the detection time was 2 h. The detection limit was 100 cfu/mL. This sensor would be of great benefit for the early clinical diagnosis of tuberculosis.

Role of GeneXpert in Rapid Molecular Detection of Extrapulmonary Tuberculosis in Tertiary Care Hospital

Introduction: Tuberculosis, the most common infectious disease with prevalence of 9.6 million globally. Most prevalent (23%) in India. Extrapulmonary tuberculosis (EPTB) accounts for 20% of total burden of tuberculosis. Rapid detection of Mycobacterium Tuberculosis (MTB) is essential for effective disease management. CBNAAT (Cartridge Based NucleicAcid Amplification Test) or GeneXpert MTB/RIF assay - novel diagnostic tool to detect MTB and RIF resistance simultaneously. WHO recommends its utility for non-respiratory samples also. Burden of EPTB and drug resistance vary from place to place. Objective: Study was conducted to gather information about burden of disease in our locality and to assesutility of CBNAAT in detecting MTB and rifampicin resistance in suspected EPTB cases. Methods: Retrospective analysis of 281 samples from suspected cases collected in falcon tubes and processed using CBNAAT. Result: Total of 281 extrapulmonary samples received, 67(23.8%) were positive and 214(76.1%) were negative for MTB. Of 67 positives, RIF resistance detected in 1(1.49%) case. Maximum number of MTB detected in the age group 21-30 years (n=23, 34.3%). Among 165 males and 116 females, MTB detected in 44(26.6%) and 23(19.8%) respectively. Out of 281 patients, 24(8.54%) were HIV positive. Of these 24, only 8(33.3%) found positive for MTB. Among 257 non-HIV patients, MTB detected in 59(22.9%). Among different samples received, maximum number were Pleural fluid n=115(40.9%) and Maximum MTB positives found in FNAC (of lymphnodes) samples [n=35(52.2%)]. : CBNAAT is a rapid test to detect MTB and rifampicin resistance simultaneously in EPTB and it reduced the treatment abuse in suspected cases.

Direct Detection of Mycobacterium tuberculosis in Clinical Specimens Using Nucleic Acid Amplification Tests

Tuberculosis continues to be a significant cause of morbidity and mortality worldwide. Rapid detection of Mycobacterium tuberculosis directly in clinical specimens using nucleic acid amplification tests enables infected patients to be placed on appropriate therapy much sooner than when results of conventional culture methods are used. The availability of rapid results also facilitates infection control measures to interrupt transmission of tuberculosis in healthcare settings. The era of commercially available molecular diagnostics for detection of M. tuberculosis began 25 years ago and now encompasses multiple assays that can detect organisms in the M. tuberculosis complex. Several assays also detect chromosomal mutations associated with antimicrobial resistance to further refine therapeutic strategies early in the course of disease. NAATs have revolutionized the diagnosis of TB across the globe and have become the standard of care in many high-burden developing countries.