Molecular Detection Of Mycobacterium Tuberculosis Research Articles

Concordance between microscopic and molecular detection of Mycobacterium tuberculosis in hospitalized patients with suspected pulmonary tuberculosis in Peltier General Hospital, Djibouti

Background: Tuberculosis (TB) remains a significant public health issue, and Djibouti is still one of the nations with a high TB burden, with a forecasted incidence rate of 224 cases per 100,000 people in 2020. Objectives: To compare the Ziehl-Neelsen (ZN) stain direct smear microscopy with the GeneXpert (Xpert MTB/RIF assay) as the gold standard for diagnosing patients with suspected pulmonary tuberculosis (PTB). Material and method: A health facility-based cross-sectional study was conducted on hospitalized patients at Peltier General Hospital from March to July 2022. A total of 153 samples (including bronchoalveolar lavage (BAL) and sputum) collected from presumptive TB patients were tested. Results: Out of the 153 samples analyzed by GeneXpert and ZN smear microscopy, 29 (19.0%) were detected by GeneXpert and 23 (15.0%) were positive by ZN smear microscopy. ZN smear microscopy had sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) that were 79.31%, 100%, 100%, and 95.38%, respectively. Conclusion: This study concluded that ZN smear had a slightly poor sensitivity for determining the presence of PTB; consequently, we continue to advise GeneXpert testing whenever it is possible to prevent prolonged morbidity in those affected and to prevent the direct as well as indirect expenses of missing the diagnosis of smear-negative PTB.

Towards Accurate Point-of-Care Tests for Tuberculosis in Children

In childhood tuberculosis (TB), with an estimated 69% of missed cases in children under 5 years of age, the case detection gap is larger than in other age groups, mainly due to its paucibacillary nature and children’s difficulties in delivering sputum specimens. Accurate and accessible point-of-care tests (POCTs) are needed to detect TB disease in children and, in turn, reduce TB-related morbidity and mortality in this vulnerable population. In recent years, several POCTs for TB have been developed. These include new tools to improve the detection of TB in respiratory and gastric samples, such as molecular detection of Mycobacterium tuberculosis using loop-mediated isothermal amplification (LAMP) and portable polymerase chain reaction (PCR)-based GeneXpert. In addition, the urine-based detection of lipoarabinomannan (LAM), as well as imaging modalities through point-of-care ultrasonography (POCUS), are currently the POCTs in use. Further to this, artificial intelligence-based interpretation of ultrasound imaging and radiography is now integrated into computer-aided detection products. In the future, portable radiography may become more widely available, and robotics-supported ultrasound imaging is currently being trialed. Finally, novel blood-based tests evaluating the immune response using “omic-“techniques are underway. This approach, including transcriptomics, metabolomic, proteomics, lipidomics and genomics, is still distant from being translated into POCT formats, but the digital development may rapidly enhance innovation in this field. Despite these significant advances, TB-POCT development and implementation remains challenged by the lack of standard ways to access non-sputum-based samples, the need to differentiate TB infection from disease and to gain acceptance for novel testing strategies specific to the conditions and settings of use.

Standardization of a stool concentration method for Mycobacterium tuberculosis detection in the pediatric population.

The inability of young children to expectorate sputum and paucibacillary status of Mycobacterium tuberculosis (MTB) increases its diagnostic complexity. In this study, we aimed to standardize a stool concentration method for the detection of MTB and its drug resistance by line probe assay (LPA). The stool from 10 healthy children spiked with H37Rv in five different dilutions (1:1, 1:10, 1:100, 1:1000, and 1:10,000), and stool from 10 confirmed TB and 54 clinically diagnosed TB children were subjected to an in-house stool concentration protocol. All the processed filtrates were subjected to smear microscopy, solid culture, Xpert ultra testing, and LPA. Of 10 control samples, growth was seen in four samples (neat 1:1). In smear microscopy, bacilli could be seen in eight samples (1:1 and 1:10). Xpert ultra testing could detect MTB in eight samples in all dilutions with different loads. LPA could detect MTB in all samples and dilutions. In microbiologically confirmed children, seven out of 10 stool samples tested were positive. Out of 54 children with clinically diagnosed TB, 4 (7.4%) could be confirmed by microbiological diagnosis. The protocol standardized in this study proves to be better working in the molecular detection of MTB.

Molecular Detection of Mycobacterium tuberculosis in Oral Mucosa from Patients with Presumptive Tuberculosis.

Tuberculosis (TB) diagnosis is increasingly based on the detection of Mycobacterium tuberculosis complex (MTBC) DNA in sputum using molecular diagnostic tests as the first test for diagnosis. However, sputum can be difficult to obtain in children, patients without productive cough, and the elderly and approaches testing non-sputum samples are needed. We evaluated whether TB can be detected from the oral mucosa of patients with TB. Adults with presumptive TB were examined using culture, Xpert MTB/RIF, smear microscopy and X-Rays. Oral mucosa swabs collected on PrimeStore-MTM, stored at room temperature if tested within 30 days or at −20 °C if examined at a later time. RT-PCR was performed to detect M. tuberculosis DNA. Eighty patients had bacteriologically-confirmed TB, 34 had bacteriologically-negative TB (negative tests but abnormal X-rays) and 152 were considered not to have TB (not TB). Oral swabs RT-PCR were positive in 29/80 (36.3%) bacteriologically-confirmed, 9/34 (26.5%) bacteriologically-negative and 29/152 (19.1%) not TB. The yield varied among samples stored for less and more than 30 days (p = 0.013) from 61% (11/18) and 29% (18/62) among bacteriologically confirmed, and 30.8% (4/13) and 23.8% (5/21) among bacteriologically-negative participants. Among not TB patients, the specificity was 80.9% (123/152), being 78.3% (18/23) among samples stored less than 30 days and 81.4% (105/129) among samples stored for more than 30 days (p = 0.46). The detection of M. tuberculosis in oral mucosa samples is feasible, but storage conditions may affect the yield.

Molecular detection of Mycobacterium tuberculosis from buccal swabs among adult in Peru

Tuberculosis (TB) diagnosis relies on a sputum sample, which cannot be easily obtained from all symptomatic patients. Mycobacterium tuberculosis DNA can be detected from oral swabs, a noninvasive, safe alternative sample type; however, reported sensitivities have been variable and likely depend on sample collection, processing procedures and host characteristics. We analyzed three buccal swab samples from 123 adults with culture-confirmed TB in Lima, Peru. We compared the sensitivity and specificity of two sample collection devices (OmniSwab and EasiCollect FTA cards) and examined factors associated with detection. DNA was extracted with a commercially available kit and detected via real-time PCR IS6110 amplification. Overall sensitivity for buccal samples was 51% (95% Confidence Interval [CI] 42–60%). Specificity from a single sample among healthy controls was 96.7% (95% CI 83–99.9%). Positive sputum smear and cavitary disease, correlates of disease burden, were associated with detection via buccal swab. Although we observed higher sensitivities with the Omniswab samples, this appeared to be due primarily to differences in patient characteristics (e.g., cavitary disease). Overall, our findings support the potential for a buccal sample-based TB assay. Future work should focus on assay optimization and streamlining the assay workflow.

Molecular detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary samples in a hospital-based study.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a deadly infectious disease. India contributes to one-third of the global TB burden. However, no studies have been carried out in the Telangana (Hyderabad) population using real-time polymerase chain reaction (RT-PCR). Therefore, the current study evaluated the role of RT-PCR as a rapid and non-invasive test to diagnose TB by testing for pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB). This hospital-based study examined 1670 samples (900 EPTB; 770 PTB) comprising tissue (n = 537), peritoneal fluid (n = 420), sputum (n = 166), bronchial fluid (n = 126), cerebrospinal fluid (n = 145), ascetic fluid (n = 76), sputum pus (n = 78), urine (n = 79), and bronchoalveolar fluid (n = 43) samples. DNA from samples was separated using specific isolation kits and subjected to RT-PCR. In this study, we enrolled 1670 subjects and categorized 54.4% as females and 45.6% as males. The collected samples were categorized as 48.5% of fluid samples, followed by tissue (32.2%), sputum (9.9%), urine (4.7%), and pus-swab (4.6%). RT-PCR analysis revealed that 4.7% patients were positive for Mtb. Our results revealed that 61% of the affected patients were male and 39% were female. Among the specimen types, tissue samples gave the highest proportion of positive results (36.3%). The results showed that RT-PCR should be implemented and given top priority in TB diagnosis to save time and facilitate a definitive diagnosis. Tissue samples are highly recommended to screen the Mtb through the technique RTPCR. Future studies should extend the technique to the global population and exome sequencing analysis should be performed to identify TB risk markers.

Comparison evaluation between gene Xpert Mtb/Rif and multiplex PCR for rapid diagnosis of mycobacterium tuberculosis.

To compare the efficacy of Gene Xpert mycobacterium tuberculosis-rifampicin and multiplex polymerase chain reacton for the detection of mycobacterium tuberculosis and Rifampicin resistance. The cross-sectional validation study was conducted at the Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from March to October 2018, and comprised mycobacterium tuberculosis-positive rifampicin-resistant and rifampicin-susceptible samples, with the latter acting as negative controls. Gene Xpert mycobacterium tuberculosis-rifampicin assasy and multiplex polymerase chain reacton were applied simultaneously and compared with gold standard mycobacterium growth indicator tube 960. Data was analysed using SPSS 24. Of the 192 samples, 84(44%) were culture-positive rifampicin-resistant and 108(56%) were culture-positive rifampicin-susceptible. Overall, 84(44%) were found positive. Gene Xpert mycobacterium tuberculosis-rifampicin assay detected all 84(100%) rifampicin-resistant samples, while multiplex polymerase chain reacton detected 44(52.3%) such samples. Sensitivity, specificity, positive predictive value and negative predictive value of Gene Xpert were 100% each respectively, while the corresponding values for multiplex polymerase chain reacton were 52%, 100%, 100% and 72% respectively. Molecular detection of mycobacterium tuberculosis and resistance by Gene Xpert and multiplex polymerase chain reacton simultaneously was found to be a rapid and cost-effective method.

Molecular detection of Mycobacterium tuberculosis by IS 6110 in patients with pulmonary tuberculosis in Babylon Province-Iraq

Molecular detection of Mycobacterium tuberculosis by IS 6110 in patients with pulmonary tuberculosis in Babylon Province-Iraq

Molecular detection of Mycobacterium tuberculosis in poor-quality cough specimens.

Clinical specimens unfit for laboratory processing represent missed opportunities for diagnosing tuberculosis. Poor-quality cough specimens (n=61) from presumptive tuberculosis cases were cultured and GeneXpert MTB/RIF (Xpert) successfully performed on samples transferred by flocked swab into PrimeStore molecular transport medium (PS-MTM). Mycobacterium tuberculosis was grown in culture from 13 (21.3 %) and Xpert reported 15 (24.2 %) positive, of which 10 concordant. RT-PCR of PS-MTM samples showed enhanced sensitivity; three positives were missed by Xpert, five by culture and three more detected for a total of 21 positives (34.4 %).

Diagnostic accuracy of molecular detection of Mycobacterium tuberculosis in pediatric stool samples: A systematic review and meta-analysis

BackgroundStool is a promising specimen option to diagnose pediatric tuberculosis (TB), but studies have reported a wide range of test sensitivities. We conducted a meta-analysis to assess the accuracy of Xpert MTB/RIF or ‘in-house’ molecular tests on stool samples against culture or Xpert MTB/RIF on respiratory samples or clinically-diagnosed unconfirmed TB and aimed to identify factors that contribute to the heterogeneity of reported sensitivity. MethodsWe searched EMBASE and Pubmed databases and conference abstract books for studies reporting molecular stool testing against a clinical or microbiological reference standard among children. ResultsWe identified 16 studies that included 2,481 children in stool test analyses. Pooled specificity was 98% [95%CI: 96–99], pooled sensitivity was 57% [95%CI: 40–72] against culture and 3% [95%CI: 2–6] among children with clinically-diagnosed, unconfirmed TB. There was much heterogeneity. Sensitivity was higher among children with a smear-positive sputum test. Rifampin resistance in stool was reported in two studies and detected in 5/14 children (36%). ConclusionOur results suggest molecular stool tests have potential as diagnostic rule-in tests, but it is challenging to optimize sensitivity due to between-study variation in methodology and test procedures. Therefore, we recommend future research with rigorous study design and standardized results reporting.

Detection of Mycobacterium tuberculosis in pediatric stool samples using TruTip technology

BackgroundRapid and accurate diagnosis of childhood tuberculosis (TB) is challenging because children are often unable to produce the sputum sample required for conventional tests. Stool is an alternative sample type that is easy to collect from children, and studies investigating the use of stool for molecular detection of Mycobacterium tuberculosis (Mtb) have led to promising results. Our objective was to evaluate stool as an alternative specimen to sputum for Mtb detection in children. We did so using the TruTip workstation (Akonni Biosystems), a novel automated lysis and extraction platform.MethodsWe tested stool samples from 259 children aged 0–14 years old, in Lima, Peru who presented with TB symptoms. Following extraction with TruTip, we detected the presence of Mtb DNA by IS6110 real-time PCR. We calculated assay sensitivity in two groups: (1) children with culture confirmed TB (N = 22); and (2) children with clinically-diagnosed unconfirmed TB (N = 84). We calculated specificity among children in whom TB was ruled out (N = 153). Among children who were diagnosed with TB, we examined factors associated with a positive stool test.ResultsAssay sensitivity was 59% (95% confidence interval [CI]: 39–80%) and 1.2% (95% CI: 0.0–6.5%) in children with culture-confirmed and clinically-diagnosed unconfirmed TB, respectively, and specificity was 97% (95% CI: 93–99%). The assay detected Mtb in stool of 7/7 children with smear-positive TB (100% sensitivity; 95% CI: 59–100%), and in 6/15 of children with smear-negative, culture-confirmed TB (40% sensitivity; 95% CI: 16–68%). Older age, smear positivity, culture positivity, ability to produce sputum and cavitary disease were associated with a positive stool result.ConclusionTesting of stool samples with the TruTip workstation and IS6110 amplification yielded sensitivity and specificity estimates comparable to other tests such as Xpert. Future work should include detection of resistance using the TruTip closed amplification system and assay optimization to improve sensitivity in children with low bacillary loads.

Molecular detection of M. tuberculosis and M. bovis and hematological and biochemical analyses in agricultural sprayers exposed to pesticides: A cross-sectional study in Punjab, Pakistan during 2014-2016.

We determined the correlation between a pesticide exposure, physical health and susceptibility toward tuberculosis along with hematological indices and liver enzymes' alterations in sprayers exposed to pesticides. Molecular detection of Mycobacterium tuberculosis and Mycobacterium bovis was detected by targeting histone-like protein (hupB) gene. The WBC (white blood cells) and RBC (red blood cells) levels of male sprayers and non-sprayers were significantly different (P<0.05). In female spray workers, the WBC and neutrophils levels were significantly different as compared with non-sprayers. Overall, in both male and female pesticide-exposed sprayers, mean values of alanine amino transferase and aspartate amino transferase were higher as compared with unexposed workers. M. Tuberculosis were detected in 15% male sprayers and 36% female sprayers while, M. bovis was detected in 5% male sprayers and 10% female sprayers. A χ2-test indicated that there existed a significant different (P<0.05) between positive and negative M. tuberculosis and M. bovis in both male/female spray workers out of total. The susceptibility of pesticide-exposed sprayers to tuberculosis and alterations in hematology and liver enzymes is crucial for health. Toxic effects of pesticides may lead to a weak immune system and increased tuberculosis susceptibility.

Molecular Detection of Mycobacterium tuberculosis (MTB) and Rifampicin Resistant Strain among Subjects Accessing Health Care at Federal Medical Centre, Yenegoa, Bayelsa State; Nigeria

Background: An increasing global incidence of Mycobacterium Tuberculosis (MTB), and emergence of drug resistant strains of MTB, have critically promoted the urgent need to explore better effective methods that can rapidly detect MTB, and identify drug-resistant cases as recommended by World Health Organization report of 2008. Globally, multi-drug-resistant strains of MTB are increasingly becoming a huge public health issue, which has adversely affected the health care delivery of patients, especially in developing communities with limited functioning health care facility. Nonetheless, the aim and focus of the study is to evaluate the trend of rifampicin resistance strains of MTB among patients accessing care at the University of Port Harcourt Teaching Hospital. Methods: This observational cross-sectional study which utilized stratified proportionate random technique, was designed to evaluate the molecular detection, as well as the prevalence of MTB and multidrug resistant TB specifically RIF among the studied population. Results: The study population were 204 subjects recruited randomly, with a mean age of 37.23 ± 16.68 years and were categorized based on sex, 93 (45.6%) males and 111 (54.4%) females respectively. The MTB detected were 21 (10.3%) while 183 (89.7%) were negative. Also, drug resistant test was performed specifically for RIF to ascertain RIF resistant strain and the non-RIF resistant strains; only 6 (2.9%) were resistant to RIF while 197 (96.6%) were not resistant to RIF. Correlation analysis of MTB detected between sex, age and resistant to RIF showed nonsignificant association (p>0.05). The prevalence rates include: 2.9% and 7.4% for RIF Resistance and Non-RIF resistance respectively. Chi square result reported about 90% non-significance and 10% significance which occurred in the counts between RIF resistant and MTB detected (p<0.05). Conclusion: The use of dependable molecular detection of MTB method has been able to provide strong epidemiological impetus for an enhanced MTB diagnosis and management to be encouraged and should be sustained, especially in resources limited regions like Nigeria, with massive challenges of prompt and accurate diagnosis of the infection. However, it is strongly believed that continuous leveraging on the potency of the molecular diagnostic approach will help to uncover the rifampicin resistance strains, which obviously would have been a huge challenge with the routine microscopic smear method.

Molecular detection of Mycobacterium tuberculosis in cattle and buffaloes: a cause for public health concern.

Tuberculosis is a re-emerging disease causing a growing public health burden. The current study was conducted to investigate the occurrence of Mycobacterium tuberculosis among cattle and buffaloes with tuberculous lesions. Typical tuberculous lesions were collected from 34 cattle and 34 buffaloes (Bubalus bubalis) through postmortem examination of slaughtered animals in abattoirs. DNAs were extracted from samples, and M. tuberculosis was identified by PCR. Positive samples were examined for resistance against rifampicin and isoniazid using GenoType MTBDRplus. Moreover, sera from 90 slaughterhouse workers, butchers, or meat inspectors were examined for the presence of M. tuberculosis antibodies using ELISA. Five cattle (14.7%) and three buffaloes (8.8%) tested positive. M. tuberculosis from one cattle was resistant to rifampicin and another was resistant to isoniazid. In addition, the seroprevalence of M. tuberculosis IgG among examined humans was 5.6%. The occurrence of M. tuberculosis in cattle and buffaloes is a public health concern.

Molecular diagnosis of extrapulmonary tuberculosis and sensitivity to rifampicin with an automated real-time method.

Tuberculosis continues to be a global public health problem, the extrapulmonary form being estimated to occur in 10-20% of immunocompetent individuals, increasing in patients who are carriers of the human immunodeficiency virus (HIV); its diagnosis is difficult with conventional methods due to the paucibacillary nature of samples. The Xpert® MTB/RIF test represents an important development in the molecular detection of Mycobacterium tuberculosis and has been used with a variety of non-respiratory clinical samples. To determine the effectiveness of Xpert® MTB/RIF in the detection of M. tuberculosis and sensitivity to rifampicin in patients with suspected extrapulmonary tuberculosis attending Hospital Universitario de San Vicente Fundación in Medellín in 2013-2014. This was a descriptive, cross-sectional ambispective study of 372 consecutive samples from 301 patients with suspected extrapulmonary tuberculosis, who were subjected to bacilloscopy, followed by culture in Ogawa Kudoh and the Xpert® MTB/RIF molecular test. The most frequent base diagnosis (60%) for the 182 patients was infection with HIV. Using the culture as reference, the sensitivity and general specificity of the molecular test was 94% (95% CI: 83-100) and 97% (95% CI: 95-99), respectively; for bacilloscopy it was 38.71(95% CI: 19-57) and 100% (95% CI: 99-100), respectively. Sensitivities higher than 75% were found in analyses stratified by samples. Thirty-seven of the isolates were sensitive and one resistant to rifampicin. Xpert® MTB/RIF performed well in samples from different tissues and liquids, representing a significant advance in support of extrapulmonary tuberculosis diagnosis in terms of time and percentage positivity.

Molecular detection of Mycobacterium tuberculosis from bovine milk samples

Mycobacterium tuberculosis and Mycobacterium bovis are the major causes of tuberculosis. These may infect many animal species, and are likely to be the main source of infection in humans. A total of 181 bovine raw milk samples and 123 pre-scapular lymph node biopsy samples were collected and subjected to acid fast staining, fluorescent staining, isolation and identification. Genus specific PCR to identify the Mycobacterium tuberculosis complex (MTBC) organism, and multiplex PCR (mPCR) were done to differentiate M. tuberculosis and M. bovis. Among the milk samples tested, only one sample was culture-positive for M. tuberculosis. Four samples were positive by MTBC-PCR and mPCR; all these four were proved to be M. tuberculosis. It is quite likely that animals can be infected with human-originated M. tuberculosis, which in turn may act as a source of infection in humans, becoming a reverse zoonosis. Hence, control strategies for human tuberculosis caused by M. tuberculosis should necessarily include the control strategies in animals too.

Setting the Gray Zone of Near-Cutoff Values in a Real-Time PCR Test for the Detection of Mycobacterium tuberculosis in an Intermediate Burden Country.

Recently, molecular detection of Mycobacterium tuberculosis in a respiratory specimens is accepted as one of the standard procedures for diagnosis of tuberculosis in Korea. When detecting tuberculosis using a real-time polymerase chain reaction (PCR), results showed the repeated near-cutoff values in a specimen make it difficult for the laboratory to give definitive reports as positive or negative. We retrospectively evaluated clinical state of ninety-eight patients who were not currently taking antituberculosis medications and had near-cutoff values of respiratory specimens using a real-time PCR test. Sixty-eight percent of the patients had clinical tuberculosis. In subgroup analysis, patients less than the age of 50, 94.3% had tuberculosis while only 55.6% of the patients with the age of equal and over 50 had tuberculosis (p < 0.001). Setting a gray zone for real-time PCR result and give additional tailored information onto the interpretative report would be suggested for the clinical practice in an intermediate burden of tuberculosis country.

Direct molecular detection of Mycobacterium tuberculosis suspected to be the specific infection in a case of recurrent tonsillitis

We report a case of a 21-year-old man with recurrent tonsillitis caused by Mycobacterium tuberculosis. For a period of 5months, the patient had tonsillitis seven times and was treated with several oral or parenteral antibiotics. On one of these occasions, tonsillitis was complicated with a peritonsillar abscess that was treated by incision. According to relevant bibliographic data, this is the first case of Mycobacterium tuberculosis confirmed by direct molecular microbiology methods from the tonsillar tissue of a young immunocompetent male reported in Europe. In a case of recurrent tonsillitis, Mycobacterium tuberculosis infection should be considered as a possible cause.

Molecular detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues and biopsies of gastrointestinal specimens using real-time polymerase chain reaction system

We aimed to perform the molecular detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues and biopsies of gastrointestinal specimens using real-time polymerase chain reaction system. The study included three groups: (A) control (n=24), with no previous signs of M. tuberculosis complex (MTBC), (B) patients (n=28) with known TB origin and (C) patients (n=50) with clinical and histopathological signs of TB but who were culture- and acid-fast bacilli (AFB)-negative. The samples were obtained from the Medical and Surgical Gastroenterology Departments of Bhopal Memorial Hospital and Research Centre. We extracted DNA using DNeasy Blood & Tissue kit (QIAGEN, Germany) and performed realtime assay using Roche LightCycler 2.0 with fluorescence resonance energy transfer (FRET) hybridization probes obtained from Roche Molecular Diagnostics (USA) for the specific amplification of the 159 bp region of the mycobacterium genome. All the samples (n=24) of Group A were found to be negative, while in Group B, 27 out of the 28 cases studied were found to be positive by LightCycler real-time polymerase chain reaction (LC PCR). In Group C, 18 out of the 50 cases studied were found to be positive, showing a positivity of 36%. The overall positive and negative predictive values of the test for clinical TB (Group C) were 100% and 96.9%, respectively. Results of our investigation demonstrated that the real-time detection technology using FRET probes has much higher sensitivity for the detection of MTBC DNA in tissue biopsy samples and formalin-fixed paraffin-embedded surgically resected tissues of the gastrointestinal tract.

Evaluation of reverse transcription loop-mediated isothermal amplification in conjunction with ELISA–hybridization assay for molecular detection of Mycobacterium tuberculosis

Traditional culture, followed by a panel of biochemical tests for the diagnosis of tuberculosis (TB), is time-consuming, and rapid identification of Mycobacterium tuberculosis is crucial for the early administration of appropriate therapy. In this study, the reverse transcription loop-mediated isothermal amplification combined with enzyme-linked immunosorbent hybridization (RT-LAMP–ELISA-hybridization) assay has been designed for the rapid detection of 16S rRNA in clinical isolates of M. tuberculosis. This assay reproducibly detected a single copy, as opposed to 2000 copies of MTB 16S rRNA detected by conventional gel electrophoresis. Among the 150 specimens of sputum analysed, RT-LAMP–ELISA-hybridization assay had a sensitivity of 94.1% in the culture method, compared to the Amplified M. tuberculosis Direct Test (AMTD), 91.1% and the 88.2% sensitivity of acid-fast staining. Furthermore, RT-LAMP–ELISA-hybridization assay is more cost-effective when compared to the real-time TaqMan RT-PCR and AMTD assays. In conclusion, our results suggest that the RT-LAMP–ELISA-hybridization assay is a highly sensitive, low cost diagnostic tool useful for the rapid and accurate direct diagnosis of sputum specimens, and is suitable for routine clinical use.