Early Detection Of Mycobacterium Tuberculosis Research Articles

Comparative analysis of CBNAAT (GeneXpert) and Ziehl-Nielsen staining test as diagnostic modalities of tuberculosis.

As centuries and millennia passed, tuberculosis remains the major health threat for millions of people. Due to this resurgence of tuberculosis, scientists around the globe have intensified their research efforts to curb down the transmission of this disease and that is the reason which led to the development of new and efficient diagnostic tools, one such being the rapid cartridge-based nucleic acid amplification test which can give the real-time results and that too in a short period of time. These diagnostic tools which are based on the latest technologies help in paving the way to eliminate tuberculosis by early detection method and further prevent the transmission. In this study, we did a comparative analysis of the effectiveness of CBNAAT technique in the early detection of Mycobacterium tuberculosis over the conventional method of Ziehl-Nielsen microscopy as diagnostic modalities. Comparative analysis of CBNAAT (GeneXpert) and Ziehl-Nielsen staining test was done as diagnostic modalities using standard protocols. Out of total 300 samples, 153 were female, 146 were male, while one was transgender. For all these samples, the positivity rates were 48% for the CBNAAT technique and 21.3% for ZN staining method, respectively. For extrapulmonary samples, positivity rates were 49.1% and 11.86% for CBNAAT and ZN microscopy, respectively, while the same for pulmonary samples was 47.7% and 23.6%, respectively. ZN microscopy and CBNAAT are both initial tests for the detection of Mycobacterium tuberculosis, but the introduction of CBNAAT is a boon for peripheral sites. The higher positivity rate of CBNAAT over ZN staining is the icing on the cake with the ability to detect even those cases that could be missed by the latter.

TO ANALYZE THE TB-PCR POSITIVITY RATE USING REAL-TIME PCR FOR EARLY DETECTION OF TUBERCULOSIS

Objective: Tuberculosis is one of the leading causes of death worldwide. It is present in all the countries and among all age groups and also seen in both the genders. In investigating the early stage, identification, and rapid detection of tuberculosis, the PCR method (polymerase chain reaction) is one of the fast, safest, and reproducible new approaches. It covers all advantages such as the use of closed systems, reduced risk of carryover contamination, improved sensitivity and reproducibility, reduced turnaround time wide dynamic range of target detection, and feasibility for quantitation making it easy and reliable for the early detection of Mycobacterium tuberculosis. Methods: All samples used for the study are confirmed by conventional microscopic observation using acid-fast staining using ZN STAIN. The pulmonary sputum samples are obtained from clinical and radiological evidence of tuberculosis and these samples are selected for DNA extraction. Discussion: As per the statistical analysis using SPSS 22 version, it is found that the TB positivity rate (is 29.75%). Out of 119 positive patients (Male: 60.5% and Female: 39.5%). The average and standard deviation for CT values are 23.6 and 2.9, respectively. The 95% confidence interval of CT values for specimens is (22.7, 24.5). Average and standard deviation for CT values are equal in male patients and female patients. Age group-wise average and standard deviation for CT values are nearly equal. Results: The percentage of 3+ AFB positive grades having CT values between 20 and 24 is more than all other combinations of AFB positive grades and CT values. The average and standard deviation for CT values are 23.6 and 2.9, respectively. The 95% confidence interval of CT values for specimens is (22.7, 24.5). The average and standard deviation for CT values are equal in male patients and female patients. Conclusion: A significant improvement in specificity with high accuracy was achieved by a real-time PCR assay. Real-time PCR tests prove both a high degree of sensitivity in the upper respiratory sputum samples and for the early detection of the TB infection. The MTB real-time PCR test suitably identified the majority of the AFB positive and bacterial culture confirms TB.

USE OF GENE XPERT MTB/RIF ASSAY FOR DETECTING MYCOBACTERIUM TUBERCULOSIS AND RIFAMPICIN RESISTANCE

The current study was designed to assess the specificity and sensitivity of MTB/RIF assay for early detection of Mycobacterium tuberculosis and RIF resistance in smear-positive and smear-negative pulmonary and extrapulmonary samples. This retrospective study was conducted in Bakhtawar Amin Trust Hospital Multan from July 2021 to July 2022. Total 240 pulmonary samples (gastric fluid specimens, post-bronchoscopic sputum, bronchoscopic aspirate, bronchoalveolar lavage and sputum) and 170 extra-pulmonary samples (urine specimens, skin biopsy, pericardial fluid, cerebrospinal fluid, ascitic fluid, disc material, lymph node biopsy and pleural fluid) were collected. After culture growth and identification of species, drug susceptibility testing (DST) was done. All samples underwent Xpert MTB/RIF assay. A total of 240 pulmonary and 170 extra-pulmonary samples from 410 patients were collected. The mean age of the patients was 46.5 ± 10 years. Of 410 subjects, 100 had TB, and 310 had no evidence of TB. Of 100 TB patients, the specificity of the MTB/RIF assay was 100 % (100), sensitivity was 69% (69), PPV was 100% (100), and NPV was 91% (91). According to the MTB/RIF assay, 79 samples were RIF-susceptible, and 1 was RIFresistant; DST confirmed the results. MTB/RIF assay is a valuable tool for detecting Mycobacterium tuberculosis and rifampicin resistance. It is not technique sensitive and provides results in less than three hours.

First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Kuwait and evaluation of REBA MTB-MDR assay for rapid detection of MDR-TB

Early detection of Mycobacterium tuberculosis (Mtb) in clinical specimens, its susceptibility to anti-TB drugs and disruption of infection transmission to new hosts are essential components for global tuberculosis (TB) control efforts. This study investigated major Mtb genotypes circulating in Kuwait and evaluated the performance of REBA MTB-MDR (REBA) test in comparison to GenoType MTBDRplus (gMTBDR+) assay for rapid detection of resistance of Mtb to isoniazid and rifampicin (MDR-TB). M. tuberculosis isolates (n = 256) originating predominantly from expatriate patients during a 6-month period were tested by spoligotyping and a dendrogram was created by UPGMA using MIRU-VNTRplus software. Phenotypic drug susceptibility testing (DST) was performed by MGIT 960 system. Genotypic DST for isoniazid and rifampicin was done by REBA and gMTBDR+ assays. Spoligotyping assigned 188 (73.4%) isolates to specific spoligotype international type (SIT) while 68 isolates exhibited orphan patterns. All major M. tuberculosis lineages were detected and EAI, CAS and Beijing families were predominant. Phylogenetic tree showed 131 patterns with 105 isolates exhibiting a unique pattern while 151 isolates clustered in 26 patterns. Fifteen isolates were resistant to one/more drugs. REBA and gMTBDR+ detected isoniazid resistance in 11/12 and 10/12 and rifampicin resistance in 4/5 and 4/5 resistant isolates, respectively. The diversity of SIT patterns are highly suggestive of infection of most expatriate patients with unique Mtb strains, likely acquired in their native countries before their arrival in Kuwait. Both, REBA and gMTBDR+ assays performed similarly for detection of resistance of Mtb to isoniazid and rifampicin for rapid detection of MDR-TB.

Exploring Targets of Cell Wall Protein Synthesis and Overexpression Mediated Drug Resistance for the Discovery of Potential M. tb Inhibitors.

Tuberculosis is an infectious disease engulfing millions of lives worldwide; it is caused by mammalian Tubercle bacilli, Mycobacterium tuberculosis complex, which may consist of strains viz. M. tuberculosis hominis (human strain), M. microti, M. pinnipedii and M. canettii. The other pathogenic strain is M. africanum, which belongs to the M. tuberculosis complex and it is fully virulent for humans. The non-pathogenic strains in the complex may include M. fortuitum and M.smegmatis. Extensive research has been carried out to combat this dangerous disease. World Health Organization proposed Directly Observed Treatment Short-course regimen (DOTS) for the eradication of TB. In addition, the compounds such as TBA-7371, TBI-166, AZD5847 and PBTZ-169 are under clinical trials whereas the recently FDA-approved anti-tubercular drugs are Pretomanid (PA-824), Bedaquiline (TMC207), Linezolid (PNU-100480) and Delamanid (OPC-67683). The early detection of mycobacterium tuberculosis can be permanently cured by DOTS comprising Pyrazinamide (Z), Isoniazid (H), Rifampin (R) and Ethambutol (E). The duration of treatment depends on the viability of the disease. DOTS can target to disrupt the biosynthesis of mycobacterial cell wall proteins expressed by various genes. Overexpression of these genes may produce drug-resistant due to dose misuse or the intake of quality compromised anti-tubercular drug regimen. Therefore, in the present review, there has been a necessity to report the second line anti-tubercular chemotherapeutics to target various proteins which are the building blocks of M. tb cell wall, overexpression of which may produce drug resistance.

Portable electrochemical immunosensor for detection of Mycobacterium tuberculosis secreted protein CFP10-ESAT6 in clinical sputum samples.

An early detection of Mycobacterium tuberculosis is very important to reduce the number of fatal cases and allow for fast recovery. However, the interpretation of the result from smear microscopy requires skilled personnel due to the propensity of the method to produce false-negative results. In this work, a portable, rapid, and simple sandwich-type immunosensor reader has been developed that is able to detect the presence of M. tuberculosis in sputum samples. By using sandwich-type immunosensor, an anti-CFP10-ESAT6 antibody was immobilized onto the graphene/polyaniline (GP/PANI)-modified gold screen-printed electrode. After incubation with the target CFP10-ESAT6 antigen, the iron/gold magnetic nanoparticles (Fe3O4/Au MNPs) conjugated with anti-CFP10-ESAT6 antibody were used to complete the sandwich format. Differential pulse voltammetry (DPV) technique was used to detect the CFP10-ESAT6 antigen at the potential range of 0.0-1.0V. The detection time isless than 2h. Under optimal condition, CFP10-ESAT6 antigen was detected in a linear range from 10 to 500ngmL-1 with a limit of detection at 1.5ngmL-1. The method developed from this process was then integrated into a portable reader. The performance of the sensor was investigated and compared with the standard methods (culture and smear microscopy). It provides a good correlation (100% sensitivity and 91.7% specificity) with both methods of detection for M. tuberculosis in sputum samples henceforth, demonstrating the potential of the device as a more practical screening tool.Graphical abstract.

Graphdiyne-Based One-Step DNA Fluorescent Sensing Platform for the Detection of Mycobacterium tuberculosis and Its Drug-Resistant Genes.

The accurate and early detection of Mycobacterium tuberculosis (Mtb) is of great significance for the clinical diagnosis and treatment of tuberculosis. In this work, we report a facile method for the controllable synthesis of a novel few-layered two-dimensional graphdiyne nanosheet (GDY NS) with a thickness of only ∼0.9 nm via an electrochemical lithium-intercalation strategy, which possesses a prominent fluorescence quenching effect. The few-layered GDY NS with its strong adsorptivity for single-stranded DNA is first proposed as a new fluorescent sensing platform for the real-time detection of DNA with excellent specificity, multiplicity, and superhigh sensitivity (limit of detection as low as 25 pM). This sensing platform can be further applied for the Mtb detection from clinical samples and the identification of drug-resistant mutants with a low background and a high signal-to-noise ratio. Herein, we provide a potential basis for the clinical development of rapid, sensitive, and accurate substitutes for the molecular diagnosis of Mtb and its drug-resistant genes.

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.

Early detection of Mycobacterium tuberculosis complex in BACTEC MGIT cultures using nucleic acid amplification.

We evaluated the application of nucleic acid amplification (NAA) in liquid cultures for the early detection of Mycobacterium tuberculosis. The Cobas TaqMan MTB test, IS6110 real-time PCR, and hsp65 PCR-restriction fragment length polymorphism (RFLP) analysis were used to detect BACTEC MGIT 960 (MGIT) cultures on days 3, 5, 7, and 14. The procedure was initially tested with a reference strain, H37Rv (ATCC 27294). Subsequently, 200 clinical specimens, including 150 Acid Fast bacillus (AFB) smear-positive and 50 AFB smear-negative samples, were examined. The Cobas TaqMan MTB test and IS6110-based PCR analysis were able to detect M. tuberculosis after 1day when the inoculum of H37Rv was >3 x 10(-2) CFU/ml. After a 5-day incubation in the MGIT system, all three NAA assays had a positive detection regardless of the inoculum size. After a 1-day incubation of the clinical specimens in the MGIT system, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the Cobas TaqMan MTB assay were 70.2%, 100%, 100%, and 82.3% respectively. For IS6110-based PCR analysis, these values were 63.1%, 100%, 100%, and 78.9%, and were 88.1%, 100%, 100%, and 92.1% respectively for hsp65 PCR-RFLP analysis. After a 3-day incubation, the specificity and PPV were 100% for all three NAA tests; the Cobas TaqMan MTB assay had the best sensitivity (97.6%) and NPV (98.3%). The sensitivity, specificity, PPV, and NPV for conventional culture analysis were 98.8%, 100%, 100%, and 99.1%. Thus, NAA may be useful for the early detection of M. tuberculosis after 3 days in MGIT.

Simultaneous and early detection of Mycobacterium tuberculosis resistance to antituberculosis drugs using an indirect series piezoelectric system

Emergence of drug-resistance tuberculosis (DR-TB) represents a serious threat to the world. Most countries, especially the developing countries, lack efficacious and inexpensive tools for early detection of Mycobacterium tuberculosis (M. tuberculosis) resistance to antituberculosis drugs and for preventing the carriers from acquiring further resistance. This report describes an indirect series piezoelectric (ISP) system for simultaneous and early detection of M. tuberculosis resistance to antituberculosis drugs. The ISP system can continually monitor the frequency shift change profiles of 32 detection cells resulting from the changes of volatile metabolites group during M. tuberculosis growth. The quantification detection time (QDT) obtained from the frequency shift change profile was found to be characterisitc of M. tuberculosis resistance. A drug-free chamber was used as a control, in which 1% of M. tuberculosis isolate was added. By comparing the QDT obtained from growth of M. tuberculosis isolate in each drug-containing chamber with that in a drug-free chamber, forty strains of clinical M. tuberculosis isolates were categorized by our method. Comparison was also made using the agar proportion method (APM) and the BACTEC™ MGIT™ 960 system. The mean time for interpretive category of M. tuberculosis resistance measured by the ISP system is (111.7±28.45)h, which is more rapid than by the APM [(600.0±50.30)h] and the BACTEC™ MGIT™ 960 system [(140.3±39.52)h]. The present system is accurate, rapid and cost-effective with a wide application in early detection of DR-TB in the developing countries.

Thin layer agar compared to BACTEC MGIT 960 for early detection of Mycobacterium tuberculosis

We compared the sensitivity and time to detection of growth of Mycobacterium tuberculosis in the thin layer agar (TLA) compared to BACTEC MGIT960. The average time for growth of M. tuberculosis in TLA and BACTEC MGIT960 was 10.6 and 9.6 days, respectively. The sensitivity of detection of M. tuberculosis was 97.3% on TLA and 97% on BACTEC MGIT960 for smear positive samples. TLA showed comparable results to BACTEC MGIT960 and could be an alternative method for low-income countries.

Sensitivity of the Cobas Amplicor system for detection of Mycobacterium tuberculosis in respiratory and extrapulmonary specimens

The Cobas Amplicor PCR system (CA-PCR) was compared with culture and staining for acid-fast bacilli (AFB) for the early detection of Mycobacterium tuberculosis in respiratory clinical specimens and otherwise normal sterile body fluids. The sensitivity, specificity and positive and negative predictive values of CA-PCR were determined with AFB-positive and AFB-negative specimens. The sensitivity of CA-PCR ranged from 73.6% to 100% for AFB-positive samples, while sputa collected after bronchoscopy were the most useful specimens, with 70% sensitivity and 98.6% specificity among the AFB-negative samples.

COMPARATIVE STUDY ON SPECIFIC AND EARLY DETECTION OF PULMONARY MYCOBACTERIA COMPLEX USING SMEAR AND CULTURE METHOD AND SEROLOGICAL PATHOZYME EIA KITS

The objective of this study was to compare the sensitivity and specificity of smear and culture methods with rapid serological EIA myco kits manufactured by Omega diagnostics, for the early detection of Mycobacterium tuberculosis (MTB) complex. Sera from various categories of smear and culture results were compared with the result of 38KDa, 16KDa and purified protein for IgA, IgM and IgG antibodies with sensitivity of 4%, 24% and 76%, respectively and with specificity of100% for IgG in Smear and Culture Positive (S+

Molecular detection of Mycobacterium tuberculosis in tissues showing granulomatous inflammation without demonstrable acid-fast bacilli.

Early diagnosis of tuberculosis (TB) is important for early medical intervention and prevention of spread of the bacteria. It is not uncommon to observe granulomatous inflammation but without demonstrable acid-fast bacilli (AFB) on Ziehl-Neelsen (ZN) staining in tissues sent for histologic examination, and the definitive diagnosis of TB cannot be made because no concurrent tissue is sent for TB culture. In this study, the authors explored the feasibility of using polymerase chain reaction (PCR) for early detection of Mycobacterium tuberculosis (Mtb) in formalin-fixed, paraffin-embedded tissues where a definite diagnosis of TB cannot be made. One hundred fifteen patients (131 paraffin blocks of biopsy specimens) with granulomatous inflammation but ZN-negative for AFB were studied. DNA was extracted from paraffin sections and amplified by PCR with the IS6110 primers (specific for the Mtb complex) and the specific 122-base pairs (bp) PCR product was detected by agarose gel electrophoresis. Sixty-eight of the 115 (59%) patients were TB-PCR positive, thus enabling definite diagnosis of TB in significant numbers of these patients in 3 days. The authors conclude that molecular diagnosis by PCR is useful for early detection of TB in histologic material where morphologic features are suggestive but not confirmatory because of negative staining for AFB.

Early detection of Mycobacterium tuberculosis by culture on BBL ® MGIT™ medium followed by PCR

Early detection of Mycobacterium tuberculosis by culture on BBL ® MGIT™ medium followed by PCR

Rapid Extraction of DNA using Ion Exchange Resin for Early Detection ofMycobacterium tuberculosis by the Polymerase Chain Reaction

연구배경: 본 연구는 결핵의 조기검출을 위하여, PCR을 이용하는데 있어서의 중요한 문제점의 하나인 임상검체로부터 결핵균을 분리 방법을 개선하여, 교차오염의 가능성을 최소화하고 PCR에 의한 결핵균의 DNA검출을 보다 쉽고 안전하게 실시하여 대량의 검체를 처리해야하는 일상 임상검사법으로 정착시키고자 하는데 그 목적이 있다. 방법: 다양한 방법으로 DNA를 검출하여 동일한 방법으로 PCR을 수행하여 이차 PCR 산물의 전기영동 결과를 AFB도말 및 배양검사 결과와 함께 비교하여 감수성, 특이성, 양성예측도 및 음성예측도의 항목으로 비교분석하였다. 실험 1은 Proteinase K 처리후 phenol로 추출하는 방법과 Chelex 100 이온교환 수지를 이용한 InstaGene법을 100예에서 비교하였으며, 실험 2에서는 Microwave 처리후 원침 상청액을 직접 시용하는 방법과 Chelex 100 이온교환 수지를 이용한 InstaGene 법을 98예에서 비교하였다. 결과: 세 DNA 분리법으로 실시한 PCR결과에서 모두 특이성과 양성예측도가 매우 낮았다. 실험 1에서 Proteinase K 법에 의한 경우 보다 Insta Gene을 이용한 경우에 약 20% 높은 감수성과 10%~20% 높은 음성예측도를 보이고 있으며, 특이성은 2%~8% 낮고, 양성예측도는 2.5%~4% 높은 것으로 나타났다. 실험 2에서는 Microwave법과 Insta Gene을 이용한 경우에 거의 비슷한 결과를 보였다. 결론: 결핵진단시 PCR을 위한 객담검체에서의 DNA 분리시에 Microwave법과 $InstaGene^{TM}$ DNA분리 kit가 매우 효율적이며, 특히 InstaGene법이 교차오염의 가능성이 거의 없고, 처리 시간이 짧으며, 조작이 매우 간단하며 매우 경제적인 것으로 나타났다. 다만 특이성을 더욱 높일 수 있도록 연구가 추가되어야 할 것이며 일반 인구집단에서 충분한 임상검체를 대상으로 연구가 추가되면 InstaGene법의 유용성이 더욱 확실히 검증 될 것으로 사료된다. 【Background: The extraction methods of DNA from clinical samples are the major obstacle to use the PCR(polymerase Chain Reaction) in routine labortary for early detection of M. tuberculosis. We tried to improve the extraction method of DNA from sputum for establishment of the PCR in routine labortary by reducing the possibility of cross contamination and performing it easily and safely. Methods: We used the $InstaGene^{TM}$ DNA extraction kit(BioRad Co.) using Chelex 100 ion exchange resin for preparation of DNA. We compared InstaGene method in 100 cases of sputum from proteinase K method which is known as the most commonly used method for DNA purification(Experiment 1). And we compared InstaGene method in 98 cases of sputum from Microwave method developed by a company in Korea(Experiment 2). In experiment 1,245bps of IS6110 were amplified and then 188bps were amplified by nested PCR. In experiment 2,536bps in primary PCR and 276bps in nested PCR were amplified and analysed by agarose gel electrophoresis and EtBr staining. Results: When we chose AFB smear, culture, or AFB smear and culture as a standard test, PCR had low specificity and positive predictive value in both experiments. The InstaGene method has higher value in sensitivity and negative predictive value significantly than proteinase K method. The InstaGene method and the Microwave methods were similar in sensitivity, specificity, positive predictive value and negative predictive value. Conclusion: Even though both methods had lower possibility of cross contamination, shorter time requirement, simplicity, and economic advantages than Proteinase K method, the InstaGene method was a little simpler than the Microwave method. Therefore, in terms of usefulness in clinical application, the Instagene method seems to be the most useful method in DNA extraction for detection of M. tuberculosis using PCR. The reliability of this method will be clarified by further studies with enough clinical samples.】