Tuberculosis In Sputum Samples Research Articles

Devising novel near-infrared aggregation-induced-emission luminogen labeling for point-of-care diagnosis of Mycobacterium tuberculosis.

Detecting and appropriately diagnosing a Mycobacterium tuberculosis infection remains technologically difficult because the pathogen commonly hides in macrophages in a dormant state. Described here is novel near-infrared aggregation-induced-emission luminogen (AIEgen) labeling developed by the current authors' laboratory for point-of-care (POC) diagnosis of an M. tuberculosis infection. The selectivity of AIEgen labeling, the labeling of intracellular M. tuberculosis by AIEgen, and the labeling of M. tuberculosis in sputum samples by AIEgen, along with its accuracy, sensitivity, and specificity, were preliminarily evaluated. Results indicated that this near-infrared AIEgen labeling had satisfactory selectivity and it labeled intracellular M. tuberculosis and M. tuberculosis in sputum samples. It had a satisfactory accuracy (95.7%), sensitivity (95.5%), and specificity (100%) for diagnosis of an M. tuberculosis infection in sputum samples. The current results indicated that near-infrared AIEgen labeling might be a promising novel diagnostic tool for POC diagnosis of M. tuberculosis infection, though further rigorous verification of these findings is required.

Surface-Enhanced Carboxyphenyl Diazonium Functionalized Screen-Printed Carbon Electrode for the Screening of Tuberculosis in Sputum Samples.

Curbing tuberculosis (TB) requires a combination of good strategies, including a proper prevention measure, diagnosis, and treatment. This study proposes an improvised tuberculosis diagnosis based on an amperometry approach for the sensitive detection of MPT64 antigen in clinical samples. An MPT64 aptamer specific to the target antigen was covalently attached to the carboxyphenyl diazonium-functionalized carbon electrode via carbodiimide chemistry. The electrochemical detection assay was adapted from a sandwich assay format to trap the antigen between the immobilized aptamer and horseradish peroxidase (HRP) tagged polyclonal anti-MPT64 antibody. The amperometric current was measured from the catalytic reaction response between HRP, hydrogen peroxide, and hydroquinone, which is used as an electron mediator. From the analysis, the detection limit in the measurement buffer was 1.11 ng mL−1. Additionally, the developed aptasensor exhibited a linear relationship between the current signal and the MPT64 antigen-spiked serum concentration ranging from 10 to 150 ng mL−1 with a 1.38 ng mL−1 detection limit. Finally, an evaluation using the clinical sputum samples from both TB (+) and TB (−) individuals revealed a sensitivity and specificity of 88% and 100%, respectively. Based on the analysis, the developed aptasensor was found to be simple in its fabrication, sensitive, and allowed for the efficient detection and diagnosis of TB in sputum samples.

Diagnostic Accuracy between CBNAAT, TrueNat, and Smear Microscopy for Diagnosis of Pulmonary Tuberculosis in Doda District of Jammu and Kashmir- A Comparative Study

Introduction: The need for fast, precise diagnostic tests to identify active tuberculosis is essential, mainly in endemic nations such as India. An automated real-time Polymerase Chain Reaction (PCR) method for pulmonary tuberculosis (TB) detection known as the Cartridge Based Nucleic Acid Amplification Test (CBNAAT) or GeneXpert assay shows great promise as a complement to the TrueNat and conventional sputum microscopy techniques. Aim: To compare sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) of CBNAAT with TrueNat and smear microscopy in the detection of Mycobacterium tuberculosis. Materials and Methods: A cross-sectional study on 175 patients with suspected pulmonary TB was conducted from June 2021 to November 2021 in a tertiary care hospital at Government Medical College, Doda Jammu and Kashmir. The sensitivity, specificity, PPV, NPV, and diagnostic accuracy for the diagnosis of tuberculosis were calculated for Acid Fast Bacilli (AFB) smear microscopy, TrueNat, and the GeneXpert and compared with each other. Statistical analysis of the data was conducted with Statistical Package for the Social Science (SPSS) version 20.0. Results: Out of the total 175, 168 (96%) patients were TB positive by CBNAAT, 162 (92.6%) by TrueNat, and 148 (84.6%) as per smear microscopy. Sensitivity, specificity, PPV, NPV, and accuracy of Ziehl-Neelsen (ZN) stain in the detection of pulmonary TB in sputum samples were 86.31%, 57.14%, 97.97%, 14.81%, and 85.14%, respectively. Whereas in the case of the TrueNat technique sensitivity, specificity, PPV, NPV, and accuracy were 94.05%, 42.86%, 97.53%, 23.08%, and 92.00%. In the case of CBNAAT sensitivity, specificity, PPV, NPV, and accuracy in the detection of pulmonary TB in sputum samples were 97.02%, 28.57%, 97.02%, 28.57%, and 94.29%, respectively. Conclusion: In respiratory samples, CBNAAT is more sensitive than ZN smear microscopy and TrueNat. Positive CBNAAT, but TrueNat and AFB microscopy negative results should be read cautiously and be well correlated with the clinical and treatment history of the patient.

Aptasensor for the Detection of Mycobacterium tuberculosis in Sputum Utilising CFP10-ESAT6 Protein as a Selective Biomarker.

A portable electrochemical aptamer-antibody based sandwich biosensor has been designed and successfully developed using an aptamer bioreceptor immobilized onto a screen-printed electrode surface for Mycobacterium tuberculosis (M. tuberculosis) detection in clinical sputum samples. In the sensing strategy, a CFP10-ESAT6 binding aptamer was immobilized onto a graphene/polyaniline (GP/PANI)-modified gold working electrode by covalent binding via glutaraldehyde linkage. Upon interaction with the CFP10-ESAT6 antigen target, the aptamer will capture the target where the nano-labelled Fe3O4/Au MNPs conjugated antibody is used to complete the sandwich format and enhance the signal produced from the aptamer–antigen interaction. Using this strategy, the detection of CFP10-ESAT6 antigen was conducted in the concentration range of 5 to 500 ng/mL. From the analysis, the detection limit was found to be 1.5 ng/mL, thereby demonstrating the efficiency of the aptamer as a bioreceptor. The specificity study was carried out using bovine serum albumin (BSA), MPT64, and human serum, and the result demonstrated good specificity that is 7% higher than the antibody–antigen interaction reported in a previous study. The fabricated aptasensor for M. tuberculosis analysis shows good reproducibility with an relative standard deviation (RSD) of 2.5%. Further analysis of M. tuberculosis in sputum samples have shown good correlation with the culture method with 100% specificity and sensitivity, thus making the aptasensor a promising candidate for M. tuberculosis detection considering its high specificity and sensitivity with clinical samples.

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.

Multicentric validation of indigenous molecular test Truenat™ MTB for detection of Mycobacterium tuberculosis in sputum samples from presumptive pulmonary tuberculosis patients in comparison with reference standards.

Background & objectives:Early case detection is essential to interrupt transmission and to prevent further spread of tuberculosis (TB) in high endemic settings. Nucleic acid amplification tests (NAATs) with visual read-outs are ideal as point-of-care tests. Truenat™ MTB is an indigenous chip-based NAAT for detection of Mycobacterium tuberculosis, which involves extraction of DNA and real-time polymerase chain reaction (PCR) using portable, automated, battery-operated instruments. The current multicentric study was aimed to evaluate Truenat for detection of MTB in sputum samples obtained from patients with presumptive pulmonary TB with reference to culture as gold standard and Xpert as a comparator.Methods:The study was conducted at four sites, namely ICMR-National Institute for Research in Tuberculosis, Chennai; All India Institute of Medical Sciences, New Delhi; ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra; and National Institute of TB and Respiratory Diseases, New Delhi. Patients suspected to have TB were screened for eligibility. Two sputum samples were collected from each patient. Tests included smear, Xpert and Truenat directly from the sputum sample and culture by Lowenstein-Jensen (L-J) medium and MGIT960 from decontaminated pellets. Sample used for Truenat assay was coded. Resolution of Truenat false positives was done using an in-house PCR with TRC4 primers.Results:The study enrolled 2419 presumptive TB patients after screening 2465 patients, and 3541 sputum samples were collected from the enrolled patients. Results of 2623 samples were available for analysis. Truenat showed a positivity rate of 48.5 per cent as compared to 37.0 per cent by Xpert. The sensitivities of Truenat and Xpert were was 88.3 and 79.7 per cent, respectively in comparison with culture.Interpretation & conclusions:Truenat MTB identified more positives among culture-confirmed samples than Xpert and had higher sensitivity. In addition, other advantageous operational features of Truenat MTB were identified which would be useful in field settings.

Molecular diagnosis of multidrug-resistant tuberculosis in sputum samples by melting curve analysis

To analyze melting curves for the diagnosis of multidrug-resistant tuberculosis from sputum samples. Sputum samples (n = 250) were collected from patients with clinical suspicion of pulmonary tuberculosis as a result of bacilloscopy and cultured in solid medium Lowenstein Jensen. According to the reference method, 124 samples sensitive to rifampicin and isoniazid, 24 resistant to rifampicin, 33 resistant to isoniazid, and 69 multidrug-resistant were used. It was evaluated by real-time PCR and then by melting curves, the rpoB gene was used as a biomarker of rifampicin resistance, and the katG gene and inhA promoter region were used as biomarkers of isoniazid resistance. The H37Rv strain was considered a drug-sensitive control. The results of the reference method and the results of the melting curve analysis were compared to evaluate the parameters of sensitivity, specificity, positive predictive value and negative predictive value. Rifampicin resistance showed a sensitivity of 90.3%, specificity of 90.4%, positive predictive value of 84.8% and negative predictive value of 94.0%. Isoniazid resistance showed a sensitivity of 90.2%, specificity of 93.9%, positive predictive value of 91.1% and negative predictive value of 93.3%. The detection of multidrug-resistant tuberculosis showed values of 89.9%, 90.6%, 78.5% and 95.9% for sensitivity, specificity, positive predictive value and negative predictive value, respectively. The melting curve analysis showed to be safe and reliable to be used in the rapid diagnosis of multidrug-resistant tuberculosis in sputum samples.

Loop-Mediated Isothermal Amplification Assay for Rapid and Reliable Detection of Mycobacterium tuberculosis in Sputum Samples

Introduction: Tuberculosis (TB) remains a major global health problem. The most common method for diagnosing TB in developing countries is sputum smear microscopy; however, the sensitivity of this test is relatively lower. Detection of Mycobacterium tuberculosis using conventional culture and biochemical-based assays is time-consuming and laborious. Polymerase Chain Reaction (PCR) is also available for diagnosis of Mycobacterium tuberculosis. However, the PCR assay requires an expensive thermal cycler to amplify the DNA fragment in multiple temperature-dependent steps. Therefore, a simple and sensitive method for rapid detection has been anxiously awaited. The loop-mediated isothermal amplification (LAMP) assay is a diagnostic technique which can aid in the fight against TB in resource-poor countries. The LAMP assay can amplify a targeted sequence at a constant temperature. Therefore, a large and costly thermal cycler is not necessary for a LAMP assay.Objectives: The objective of this study was to identify Mycobacterium tuberculosis directly from sputum by LAMP and to compare its efficacy over routinely used methods.Methods: A total of 106 (53 fluorochrome staining positive and 53 fluorochrome staining negative) sputum samples were collected in this study. Mycobacterial DNA was extracted from concentrated sputum samples by freezing and boiling method. LAMP assay using a set of six specific primers targeting the M. tuberculosis 16S rRNA gene with high sensitivity was used to analyze sputum samples. The results were then compared with that of the culture method, which was considered as the gold standard method.Results: Among total of 106 samples studied by microscopy and culture, 53 were positive by both, whole four were positive by culture but negative by microscopy. With reference to culture, the microscopy had sensitivity 92.98%, specificity 100%, and predictive value of positive test 100%, predictive value of negative test 92.5%. Out of 106 samples subjected to culture and LAMP for the diagnosis of TB, 55 samples were positive by both tests and two were positive only in culture, while 48 were negative in both tests and one was negative only in culture. While comparing the LAMP with culture as a gold standard, the sensitivity of LAMP was 96.49%, specificity was 97.95%, predictive value of positive test was 98.21%, predictive value of negative test was 96%.Conclusions: Comparative experiments showed that the LAMP assay is a rapid, sensitive, and specific method to detect M. tuberculosis infection. Indeed, an inexpensive LAMP assay would be potential as a diagnostic test for tuberculosis, especially in resource-limited settings. J-GMC-N | Volume 11 | Issue 01 | January-June 2018, Page: 27-34

COMPARISON OF ZIEHL-NEELSEN, KINYOUN’S AND FLUORESCENT STAINING FOR DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN SPUTUM SAMPLES BEFORE AND AFTER PETROFF’S CONCENTRATION TECHNIQUE

Objectives: The objectives of the study were to find out the presence of Mycobacterium tuberculosis in sputum samples using Ziehl-Neelsen (ZN), Kinyoun’s, and (auramine) fluorescent staining and to compare the three staining techniques with and without Petroff’s concentration and to find out the most preferable staining of M. tuberculosis. Methods: Sputum sample was collected and concentrated by 4% NaOH (Petroff’s concentration). Microscopic examination of the sample was done before concentration and after concentration by ZN staining, Kinyoun’s staining, and fluorescent staining (Auramine). Grading of acid-fast bacilli (AFB) by three staining was done before and after concentration according to RNTCP guidelines.Results: Total of 452 sputum samples were collected and subjected to microscopy examination by ZN, Kinyoun’s, and fluorescent staining methods to compare the presence or absence of AFB with or without concentration. Among the 452 sputum samples, total of 67 (15.6%) sputum samples were positive for the presence of AFB. Majority 40.3% (n=21) of TB positive patients were observed in the age group 51–60 years. The results of Auramine-O staining showed positive diagnoses in 15.9% of the samples; sensitivity was 100% and specificity 95.6%.Conclusion: The use of fluorescent staining significantly increases the diagnostic value of the smear, particularly where there are low-density bacilli which may escape detection on ZN stained smears.

Isoniazid Mono-Resistant Tuberculosis: Impact on Treatment Outcome and Survival of Pulmonary Tuberculosis Patients in Southern Mexico 1995-2010.

BackgroundIsoniazid mono-resistance (IMR) is the most common form of mono-resistance; its world prevalence is estimated to range between 0.0 to 9.5% globally. There is no consensus on how these patients should be treated.ObjectiveTo describe the impact of IMR tuberculosis (TB) on treatment outcome and survival among pulmonary TB patients treated under programmatic conditions in Orizaba, Veracruz, Mexico.Materials and MethodsWe conducted a prospective cohort study of pulmonary TB patients in Southern Mexico. From 1995 to 2010 patients with acid-fast bacilli or culture proven Mycobacterium tuberculosis in sputum samples underwent epidemiological, clinical and microbiological evaluation. We included patients who harbored isoniazid mono-resistant (IMR) strains and patients with strains susceptible to isoniazid, rifampicin, ethambutol and streptomycin. All patients were treated following Mexican TB Program guidelines. We performed annual follow-up to ascertain treatment outcome, recurrence, relapse and mortality.ResultsBetween 1995 and 2010 1,243 patients with pulmonary TB were recruited; 902/1,243 (72.57%) had drug susceptibility testing; 716 (79.38%) harbored pan-susceptible and 88 (9.75%) IMR strains. Having any contact with a person with TB (adjusted odds ratio (aOR)) 1.85, 95% Confidence interval (CI) 1.15–2.96) and homelessness (adjusted odds ratio (aOR) 2.76, 95% CI 1.08–6.99) were associated with IMR. IMR patients had a higher probability of failure (adjusted hazard ratio (HR) 12.35, 95% CI 3.38–45.15) and death due to TB among HIV negative patients (aHR 3.30. 95% CI 1.00–10.84). All the models were adjusted for socio-demographic and clinical variables.ConclusionsThe results from our study provide evidence that the standardized treatment schedule with first line drugs in new and previously treated cases with pulmonary TB and IMR produces a high frequency of treatment failure and death due to tuberculosis. We recommend re-evaluating the optimal schedule for patients harboring IMR. It is necessary to strengthen scientific research for the evaluation of alternative treatment schedules in similar settings.

Prevalence of Tuberculosis and Rifampicin Resistance Among Patients Seeking Medical Care in Nasarawa State, North Central Nigeria

Tuberculosis (TB), caused by Mycobacterium tuberculosis remains a clinical and public health challenge in Nigeria. This study was carried out to determine the prevalence of M. tuberculosis and rifampicin resistance in patients from five selected health facilities in Nasarawa State, Nigeria. The HIV serostatus and the CD4+ count of seropositive individuals were also determined. Using a combination of Zhiel Neelsen staining and WHO approved GeneXpert MTB/Rif device for M. tuberculosis and rifampicin resistance, the presence of M. tuberculosis in sputum samples was detected and confirmed. A total of 346 samples were analysed, out of which M. tuberculosis was detected in 91 (26.3%) samples and rifampicin resistance in 6 (6.6%) of the positive samples. HIV prevalence was found to be 46.8%, HIV and TB co-infection 39.6%, while 83.3% of the rifampicin resistance was from HIV positive cases. Tuberculosis was found to be decreasing with increase in CD4 count. Statistically, there was a significant relationship (P 0.05) between gender, occupation and age with TB was observed. Our results show that tuberculosis still remains endemic in Nasarawa state with a high prevalence of HIV and that the confluence of HIV and tuberculosis increased the rate of rifampicin resistance. Provision of Tuberculosis diagnostics with the capacity to detect TB drug resistance beyond rifampicin is highly recommended.

Evaluation of Giant African Pouched Rats for Detection of Pulmonary Tuberculosis in Patients from a High-Endemic Setting.

BackgroundThis study established evidence about the diagnostic performance of trained giant African pouched rats for detecting Mycobacterium tuberculosis in sputum of well-characterised patients with presumptive tuberculosis (TB) in a high-burden setting.MethodsThe TB detection rats were evaluated using sputum samples of patients with presumptive TB enrolled in two prospective cohort studies in Bagamoyo, Tanzania. The patients were characterised by sputum smear microscopy and culture, including subsequent antigen or molecular confirmation of Mycobacterium tuberculosis, and by clinical data at enrolment and for at least 5-months of follow-up to determine the reference standard. Seven trained giant African pouched rats were used for the detection of TB in the sputum samples after shipment to the APOPO project in Morogoro, Tanzania.ResultsOf 469 eligible patients, 109 (23.2%) were culture-positive for Mycobacterium tuberculosis and 128 (27.3%) were non-TB controls with sustained recovery after 5 months without anti-TB treatment. The HIV prevalence was 46%. The area under the receiver operating characteristic curve of the seven rats for the detection of culture-positive pulmonary tuberculosis was 0.72 (95% CI 0.66–0.78). An optimal threshold could be defined at ≥2 indications by rats in either sample with a corresponding sensitivity of 56.9% (95% CI 47.0–66.3), specificity of 80.5% (95% CI 72.5–86.9), positive and negative predictive value of 71.3% (95% CI 60.6–80.5) and 68.7% (95% CI 60.6–76.0), and an accuracy for TB diagnosis of 69.6%. The diagnostic performance was negatively influenced by low burden of bacilli, and independent of the HIV status.ConclusionGiant African pouched rats have potential for detection of tuberculosis in sputum samples. However, the diagnostic performance characteristics of TB detection rats do not currently meet the requirements for high-priority, rapid sputum-based TB diagnostics as defined by the World Health Organization.

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis

BackgroundModern molecular-based approaches for the detection of Mycobacterium tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore®-Molecular Transport Medium (PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems. MethodsAssessment of the transport medium for application of sputum samples processed for the detection of M. tuberculosis included the inactivation of M. tuberculosis in spiked sputum samples, compatibility of the medium with three commercial nucleic extraction systems and stability of DNA in the medium at ambient temperature over 28days. We further performed a clinical laboratory evaluation on 256 sputum specimens sent for tuberculosis investigation. ResultsComplete inactivation of M. tuberculosis occurred within 30min of exposure at a ratio of 1:3 for sputum to PS-MTM. Sputum specimen in PS-MTM showed very good compatibility with automated bead-based extraction systems, producing high DNA output (estimated lower limits of detection: ~170CFU/ml). Furthermore, PS-MTM samples remained stable over 28days at ambient temperature displaying no significant change over time in Ct-values (<5% on a mean starting value of 22.47). Of the 256 clinical sputum specimens, 10.2% were culture positive and 11.0% were positive by real-time PCR of PS-MTM samples. ConclusionsCollecting and transporting sputum from TB suspects in PS-MTM offer safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.

Association of serum levels of iron, copper, and zinc, and inflammatory markers with bacteriological sputum conversion during tuberculosis treatment.

Iron, copper, and zinc are key micronutrients that play an important role in the immune response to Mycobacterium tuberculosis. The present study aimed to evaluate the association between serum levels of those micronutrients, inflammatory markers, and the smear and culture conversion of M. tuberculosis during 60 days of tuberculosis treatment. Seventy-five male patients with pulmonary tuberculosis (mean age, 40.0 ± 10.7 years) were evaluated at baseline and again at 30 and 60 days of tuberculosis treatment. Serum levels of iron, copper, zinc, albumin, globulin, C-reactive protein, and hemoglobin, and smear and cultures for M. tuberculosis in sputum samples were analyzed. Compared to healthy subjects, at baseline, patients with PTB had lower serum iron levels, higher copper levels and copper/zinc ratio, and similar zinc levels. During the tuberculosis treatment, no significant changes in the serum levels of iron, zinc, and copper/zinc were observed. Lower serum copper levels were associated with bacteriological conversion in tuberculosis treatment (tuberculosis-negative) at 30 days but not at 60 days (tuberculosis-positive). C-reactive protein levels and the C-reactive protein/albumin ratio were lower in tuberculosis-negative patients than in tuberculosis-positive patients at 30 and 60 days after treatment. Albumin and hemoglobin levels and the albumin/globulin ratio in patients with pulmonary tuberculosis increased during the study period, regardless of the bacteriological results. High serum globulin levels did not change among pulmonary tuberculosis patients during the study. Serum copper levels and the C-reactive protein/albumin ratio may be important parameters to evaluate the persistence of non-conversion after 60 days of tuberculosis treatment, and they may serve as predictors for relapse after successful treatment.

Diagnostic accuracy of the PURE-LAMP test for pulmonary tuberculosis at the county-level laboratory in China.

BackgroundEarly and effective detection of Mycobacterium tuberculosis (MTB), particularly in smear-negative tuberculosis (TB), is a priority for global TB control. Loop-mediated isothermal amplification with a procedure for ultra rapid DNA extraction (PURE-LAMP) can detect TB in sputum samples rapidly and with high sensitivity and specificity. However, the PURE-LAMP test has not been effectively evaluated, especially in resource-limited laboratories. In this study, we evaluated the performance of the PURE-LAMP test for TB detection in TB suspects from two county-level TB dispensaries in China.Methodology/Principal FindingsFrom April 2011 to February 2012, patients with suspected TB were continuously enrolled from two county-level TB laboratories in China. Three sputum samples (spot, night, and morning sputum) were collected from each recruited patient. Detection of MTB by PURE-LAMP was compared to a reference standard L-J culture. The results showed that the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection was 70.67%, while the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection in smear positive and culture positive patients and smear negative and culture positive patients was 92.12% and 53.81%, respectively. The specificity of PURE-LAMP based on spot sputum for MTB detection was 98.32%. The sensitivity and specificity of the PURE-LAMP test based on three sputa combination for MTB detection was 88.80% and 96.86%, respectively. The results also showed that the PURE-LAMP test had a significantly lower contamination rate than did solid culture.Conclusions/SignificanceThe study suggested that, in peripheral-level TB laboratories in China, the PURE-LAMP test showed high sensitivity and specificity for TB detection in TB suspects, making it a more effective, rapid, and safe method worthy of broader use in the future.

Assessment of Detection Efficacy of Mycobacterium Tuberculosis in Sputum Samples by Real-Time PCR-Based Method

Tuberculosis (TB) is a major public health problem in Nepal and ranks as one of the most prevalent communicable diseases throughout the country. In Nepal, 45% of the total population is infected with TB, and 40,000 people get TB every year. Twenty thousand new sputum-positive cases are seen annually, with 5000-7000 people dying each year from TB. Thirty sputum samples were collected from Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu, Nepal, and a comparative study of Acid-fast Bacilli (AFB) test and Real-time PCR was conducted separately with the culture test, regarded as the gold standard by WHO. Detection of Mycobacterium tuberculosis through the use of Real-time PCR was found to be higher compared to AFB and culture. The Real-time PCR test showed higher sensitivity (100%) and specificity (94.11%) compared to the AFB test, with sensitivity of 84.61% and specificity of 88.24%. The positive predictive value was found to be 84.61% and 92.86% for AFB and Q-PCR, respectively. The negative predictive value was found to be 88.24% for AFB and 100% for Q-PCR. Our statistics clearly show that TB diagnosis by Q-PCR is highly efficient and reliable over conventional methods of diagnosis, and we recommend its use in the hospitals and clinics of Nepal.

Evaluation of Biochip System in Determining Isoniazid and Rifampicin Resistances of Mycobacterium Tuberculosis in Sputum Samples

ObjectiveTo evaluate a biochip system in determining isoniazid and rifampicin resistances of Mycobacterium tuberculosis in sputum samples in a Chinese population.MethodsWe assembled 907 sputum smeared positive specimens of tuberculosis patients in total. Each sample would be separated into two parts for culture and biochip assay simultaneously. And those cultured positive and having full drug resistance results would be used as reference. The McNemar χ2 test was adopted for evaluating the paired 2×2 table.ResultsCompared with drug sensitivity test, the agreement rates of the two methods in detecting rifampicin and isoniazid resistances were 93.37% and 94.49%, respectively. The sensitivity and specificity of biochip in detecting isoniazid were 74.31% and 96.92%, respectively. Meanwhile, the sensitivity and specificity for rifampicin were 79.76% and 96.53%, respectively. For multi-drug resistance, the sensitivity and specificity were 64.62% and 97.75%, respectively.ConclusionsThe biochip system is a rapid and accurate method for drug resistant tuberculosis diagnosis using sputum samples directly, especially for rifampicin resistance detection.

Association of diabetes and tuberculosis: impact on treatment and post-treatment outcomes

ObjectiveTo determine the clinical consequences of pulmonary tuberculosis (TB) among patients with diabetes mellitus (DM).MethodsWe conducted a prospective study of patients with TB in Southern Mexico. From 1995 to 2010,...

Impact of cigarette smoking on rates and clinical prognosis of pulmonary tuberculosis in Southern Mexico

To examine the relationship between cigarette smoking and incidence and mortality rates of pulmonary tuberculosis (TB) and treatment outcomes. From 1995 to 2010, we analyzed data from 1062 patients with TB and from 2001 to 2004, 2951 contacts in Southern Mexico. Patients with acid-fast bacilli or Mycobacterium tuberculosis in sputum samples underwent epidemiological, clinical and mycobacteriological evaluation and received treatment by the local DOTS program. Consumers of 1-10 (LS) or 11 or more (HS) cigarettes per day incidence (1.75 and 11.79) and mortality (HS, 17.74) smoker-non-smoker rate ratios were significantly higher for smokers. Smoker population was more likely to experience unfavorable treatment outcomes (HS, adjusted OR 2.36) and retreatment (LS and HS, adjusted hazard ratio (HR) 2.14 and 2.37). Contacts that smoked had a higher probability of developing active TB (HR 2.38) during follow up. Results indicate the need of incorporating smoking prevention and cessation, especially among men, into international TB control strategies.

Quality of induced sputum using a human-powered nebuliser in a mobile human immunodeficiency virus testing service in South Africa

To investigate the quality of induced sputum samples using a human-powered (HPN) and an electric-powered nebuliser (EPN). For each participant two sputum samples were induced using the HPN and the EPN. The sequence of the two nebulisers was allocated at random. The proportion of good quality sputum according to different assessment criteria was compared using an exact McNemar test. The difference in time to expectoration was compared using the Wilcoxon matched-pairs signed-rank test. A total of 123 individuals were eligible for the study. Nine individuals refused to participate and five were unable to produce a sputum sample. The proportion of good quality sputum was higher among sputum samples induced by the HPN compared to those obtained using the EPN. The median time to produce a sputum sample was 2.2 min (IQR 1.13-4.1) for the HPN and 2.5 min (IQR 1.4-4.1) for the EPN. The HPN induced good quality sputum within 3 min. The device operates without electricity and is suitable not only for remote clinics with unreliable electricity, but also for mobile services and community-based intensified tuberculosis (TB) case finding. Further research needs to investigate the yield of TB in sputum samples induced by the HPN.