Ziehl-Neelsen Smear Microscopy 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.

Comparison of Light Emitting Diode (LED) Fluorescent Microscopy with Ziehl-Neelsen Microscopy on Sputum Specimens for Diagnosis of Pulmonary Tuberculosis Keeping Culture as ‘Gold Standard’

Objective: To compare LED fluorescence microscopy and Ziehl-Neelsen staining in terms of their diagnostic performance in diagnosing pulmonary tuberculosis, taking sputum specimens from patients suspected of pulmonary tuberculosis.
 Study Design: Prospective longitudinal study.
 Place and Duration of Study: Microbiology Department, Armed Forces Institute of Pathology Rawalpindi, from Jan 2019 to Dec 2019.
 Methodology: Sputum samples from patients with clinical suspicion of pulmonary tuberculosis were stained using Ziehl-Neelsen (ZN) stain, fluorescent stain with Auramine O staining (AO) stain and Mycobacterial culture on Mycobacterial Growth Indicator Tube (MGIT 960), to detect Mycobacterium tuberculosis. WHO guidelines were followed to grade positive smears.
 Results: Among 206 patients with suspicion of tuberculosis, 143 (69%) were male, and 63 (30%) were female patients. The mean age of the patients was 53.67 ± 14.73 years. Out of 206 sputum samples, 64 were negative by all three techniques used. 142 (68%) specimens detected Mycobacterium tuber-culosison MGIT960. Within 142 culture-positive samples, only 40 samples were positive on Ziehl-Neelsen microscopy, whereas 97 samples were detected positive by LED fluorescent microscopy. In culture-negative samples, three were missed on Ziehl-Neelsen staining, which was positive with Fluorescent microscopy. Sensitivity and specificity for Ziehl-Neelsen smear microscopy were 26.7% and 96.8%, respectively. Sensitivity and specificity for Fluorescent smear microscopy were 64.8% and 92.2%, respectively.
 Conclusion: We concluded that the efficacy of LED fluorescence microscopy has proven to have many potential advantages over conventional Ziehl-Neelsen microscopy.

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.

Strengthening the Monitoring and Surveillance System for Bovine Tuberculosis in Post Mortem Material from Slaughtered Cattle in Eswatini: A Review

The main objective of this review study was to examine the validity of test results generated by using the Ziehl-Neelsen smear microscopy as the sole diagnostic tool for the monitoring and surveillance of bovine tuberculosis in cattle slaughtered in Eswatini.
 Methods: A retrospective analysis of available laboratory data of beef carcass condemnations for a study period from 2014 to 2018 was carried out. Literature was reviewed to find out potential sample preparation and concentration methods to improve the sensitivity of current Z-N smear microscopy.
 Results: The limited literature reviewed in this study has briefly highlighted (i) the limitations of using the Z-N direct smear microscopy as a screening and confirmatory laboratory test for bovine tuberculosis suspected carcasses, (ii) that the Z-N smear microscopy method may be inadequate for identifying and differentiating other acid fast bacilli co-existing in a M. bovis infection, (iii) that concentration of lymph node aspirate samples may improve the sensitivity of acid fast bacilli smear microscopy, (iv) that strategic deployment of ancillary laboratory tests, such as the Rapid diagnostic tests (RDT) and the Polymerase chain reaction test (PCR) alongside the Z-N smear microscopy may achieve an adequate level of diagnostic test performance.
 Conclusion: In order to strengthen the surveillance and monitoring of bovine tuberculosis in the country, it is necessary to consider techniques for the concentration of bovine tuberculosis suspect lymph node samples, to use Rapid diagnostic test Kits in parallel to the Z-N direct smear microscopy.

Accuracy of Xpert MTB/RIF assay for the diagnosis of tuberculous pleural effusion.

BackgroundTuberculosis poses a severe threat to human health. At present, compared with the traditional diagnostic methods for tuberculosis pleural effusion, such as Löwenstein–Jensen culture, pleural biopsy, and Ziehl–Neelsen smear microscopy, Xpert MTB/RIF was regarded as an emerging technology for its efficiency. The Xpert MTB/RIF accuracy for tuberculous pleural effusion diagnosis was evaluated in this systematic study.Materials and methodsWe searched the relevant literature published before January 2021 in PubMed, Cochrane, EMBASE, and Web of Science databases. Utilizing Review Manager 5.3 software, the quality of the included literature was evaluated based on the Quality Assessment of Diagnostic Accuracy Studies criteria. Sensitivity, specificity, and the summary receiver operating characteristic curves were plotted and analyzed with Metadisc 1.40 software. We used Stata 12.0 software to evaluate the publication bias of this study.ResultsEighteen articles were identified in total. The sensitivity of Xpert MTB/RIF in the pleural effusion was 0.24, and specificity was 1.00, respectively. The area under the summary receiver operating characteristic curve was 0.9737, which indicated that the overall accuracy of the Xpert MTB/RIF was high. In addition, based on the Deeks funnel plot, no publication bias of the study was found.ConclusionXpert MTB/RIF is a rapid method with high specificity but relatively low sensitivity for detecting Mycobacterium tuberculosis in pleural effusion. Its less sensitivity made it difficult to be used clinically, but the high specificity suggests that it can be used as a specific diagnostic method for tuberculous pleural effusion.

A Retrospective Cross-Sectional Study of the Utility of Cartridge-Based Nucleic Acid Amplification Test in Diagnosis of Pulmonary and Extrapulmonary Tuberculosis in People Living with HIV in Second Highest HIV Prevalent State in India

Abstract Introduction Tuberculosis (TB) in people living with human immunodeficiency virus (PLHIV) is difficult to diagnose due to fewer organisms in sputum and extrapulmonary samples. Sputum culture takes 4 to 8 weeks for growth of the mycobacteria. Delayed treatment for TB in PLHIV leads to increased mortality. This study evaluated cartridge-based nucleic acid amplification test (CBNAAT) as a diagnostic tool for diagnosis of pulmonary TB (PTB) and extrapulmonary TB (EPTB) in PLHIV in the second most HIV prevalent state in India and for comparing its efficacy between Ziehl–Neelsen (ZN) staining sputum smear–positive and sputum smear–negative TB. Methods This cross-sectional study was conducted in RIMS, Imphal, with 167 PLHIV patients, age 15 years or older, having signs and symptoms of TB. Appropriate samples for sputum microscopy and CBNAAT were sent. Conclusion The overall sensitivity of sputum smear for acid-fast bacillus (AFB) was found to be 30.71% and that of CBNAAT was 38.57%. Sensitivity of CBNAAT for sputum smear–positive and sputum smear–negative TB was 100 and 11.3%, respectively. Sensitivity of ZN smear for AFB of EPTB sample was 48.1% and that of CBNAAT was 59.25%. In both PTB and EPTB, CBNAAT showed an increase in diagnosis of microbiologically confirmed PTB cases by 7.8 and 11.1%, respectively, over and above the cases diagnosed by ZN smear microscopy. Rifampicin resistance was detected in five patients. We conclude that CBNAAT is a rapid test with better sensitivity in diagnosis of PTB and EPTB in PLHIV, compared with ZN smear microscopy. It detects rifampicin resistance for multidrug-resistant TB and helps in early treatment intervention.

Diagnostic Accuracy of Ziehl-Neelsen Smear Microscopy in Comparison with GeneXpert in Pulmonary Tuberculosis: A Multi-Center Study in Kandahar Province, Afghanistan

Background: Tuberculosis (TB) is a major global health problem. The early and accurate diagnosis is crucial for disease management and to control disease transmission and the emergence of drug resistance TB.
 Objectives: This study was carried out to determine the diagnostic accuracy of Ziehl Neelsen (ZN) smear microscopy in comparison with GeneXpert MTB/RIF in pulmonary tuberculosis in Kandahar province, Afghanistan.
 Methods: This was a facility-based cross-sectional study. We scrutinized TB registers of three health facilities to include patients who had their sputum tested by both ZN smear microscopy and GeneXpert MTB/RIF. We extracted 734 patients’ data registered during January 2019 - June 2020 in a structured form. Kappa value was analyzed using SPSS version 19 software at 95% Confidence Interval (CI). We calculated sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of ZN smear microscopy against GeneXpert MTB/RIF.
 Results: In comparison with GeneXpert as a reference test, ZN smear microscopy has 67.7% (95% CI:63.44%-70.74%) sensitivity and 100% (95% CI:98.74%-100.00%) specificity. The positive predictive value (PPV) and negative predictive value (NPV) were 100% and 99.89% (95% CI:99.88%-99.90%), respectively. The agreement between ZN smear microscopy and GeneXpert MTB/RIF results was moderate (70.4%) and the Kappa value was 0.45 (95% CI:0.38-0.59).
 Conclusion: This study has found high specificity but moderate sensitivity for the diagnosis of pulmonary TB using sputum ZN smear microscopy test. Hence, GeneXpert MTB/RIF test is more accurate and reliable for the diagnosis of pulmonary TB.

Diagnostic Accuracy of Sputum Microscopy in Comparison With GeneXpert in Pulmonary Tuberculosis.

BackgroundTuberculosis (TB) is a major health problem. In Pakistan, the diagnosis of pulmonary tuberculosis mainly relies on acid-fast bacilli (AFB) smear microscopy and Xpert® MTB/RIF assay (Cepheid Inc., Sunnyvale, CA) - a nucleic acid amplification test - where available. There is a wide variation in the reported sensitivity of Ziehl-Neelsen (ZN) smear microscopy across previous studies. This study aimed to determine the accuracy of sputum ZN smear microscopy in diagnosing pulmonary tuberculosis as compared with the sputum GeneXpert (Xpert MTB/RIF assay) as the reference test.Materials and methodsThis is a retrospective cross-sectional study conducted in the outpatient department of the Pakistan Institute of Medical Sciences. This study included 326 patients, aged 12 years and above, who had their sputum samples tested for ZN smear microscopy and GeneXpert during the period January to June 2019. Patients' demographic details, sputum ZN smear microscopy, and GeneXpert test results were collected for data analysis. A case of pulmonary tuberculosis was defined as a patient with positive sputum GeneXpert test result.ResultsOut of the 326 patients, GeneXpert detected MTB deoxyribonucleic acid (DNA) in 50 patients and ZN smear microscopy detected AFB in 30 patients. There was a marginal male predominance among GeneXpert positive cases. Adolescents were the least affected age group. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of ZN smear microscopy were 60%, 100%, 100%, 93.24%, and 93.87%, respectively. The positive likelihood ratio was infinite whereas the negative likelihood ratio was 0.4. The area under curve (AUC) for ZN smear microscopy was 0.800 and receiver operating characteristic (ROC) curve analysis revealed a diagonal straight line closer to the left upper corner.ConclusionSputum ZN smear microscopy is a highly specific but moderately sensitive test for the diagnosis of pulmonary tuberculosis. This study recommends the sputum GeneXpert MTB/RIF test to avoid a missed diagnosis of smear-negative pulmonary TB.

Association of TIRAP (rs8177374) and MyD88 (rs6853) genetic polymorphisms with susceptibility to pulmonary tuberculosis and treatment response

Toll-like receptors (TLRs) identify some components of Mycobacterium TB, the main causative agent of tuberculosis. The signaling pathways causing activation of the immune response involve the MyD88 and TIRAP genes. We aimed to investigate the role of MyD88 (rs6853) and TIRAP (rs8177374) gene variants in pulmonary TB susceptibility and treatment response in a sample of Egyptian population. A case-control study followed by follow-up was conducted on 220 participants distributed as 110 newly diagnosed PTB patients, and 110 healthy controls. Patients and controls were subjected to genotyping of TIRAP (rs8177374) and MyD88 (rs6853) using real time PCR technique. Our results revealed that, Bacteriological tests Ziehl Neelsen smear microscopy was positive in 46.4% of patients, GeneXpert was positive in 70.9% of patients, bacterial culture of sputum was positive in 100.0% of patients. As regards rs6853, a significant increase of frequency of the mutant GG genotype and G allele observed among patients with pulmonary TB than controls (P = 0.001) with an increased risk of acquiring TB (OR = 4.88, CI95%: (2.31–10.28) and +3.21, CI95% 2.10–4.91 respectively). Additionally C/T polymorphism (rs8177374) at TIRAP gene showed a significant increase of frequency of the heterozygous mutant CT genotype and T allele in the patients than in the controls and showed higher association with TB (OR = 5.46, CI95%: (2.84–10.52) and 3.39, CI95% 2.22–5.19 respectively). MyD88 (rs6853) GG and TIRAP (rs8177374) CT genotypes showed the highest percentage of non-responders to treatment compared with other genetic variants. CT + GG and TT + GG reported high number of no response to treatment than other combinations. TIRAP rs8177374 CT and MyD88 rs6853 GG genotypes are associated with susceptibility to PTB in a sample of Egyptian population. Simultaneous genotyping of both genes could be considered as a genetic risk factor for PTB and predictor of the patient response to treatment.

Declining tuberculosis prevalence in Saharia, a particularly vulnerable tribal community in Central India: evidences for action

BackgroundIn spite of an alarmingly high tuberculosis (TB) burden amongst the Saharia tribe of central India, there is hardly any study to investigate the impact of DOTS implementation on the magnitude of tuberculosis disease and the changes over time. This article present the findings of TB prevalence surveys conducted amongst this indigenous population in two different time periods to know the change in the prevalence of TB.MethodsA cross sectional survey was conducted among Saharia population in Shivpuri district, Madhya Pradesh during February 2013 to May 2013 and resurvey during March 2015 to July 2015. All individuals (≥15 years) were examined for chest symptoms suggestive of TB. Sputum samples were collected from all presumptive TB cases and were confirmed by laboratory examination by Ziehl-Neelsen smear microscopy and solid media culture methods. All detected cases were referred to health facility for anti-tuberculosis treatment as per RNTCP guidelines.ResultsThere was significant reduction (trend Chi square 19.97; OR = 1.521; p = 0.000) in the prevalence of TB at the endline (1995 per 100,000) as compared to baseline (3003 per 100,000). The reduction was significant among males as compared to females (OR 1.55; p = 0.000) and in the age group of 25–34 years (OR 2.0; p = 0.007) and 45–54 years (OR 4.39; p = 0.003). There was significant reduction in the prevalence in both smear (OR 1.29; p = 0.02) and culture positive (OR 1.57; p = 0.000) TB at the endline survey.ConclusionThe study findings highlight a reduction in the prevalence of TB among Saharia tribal population. Further studies are needed to identify the factors associated with reduction in prevalence among this population and also further surveys to monitor the prevalence trend over a period.

Detection of Mycobacterium tuberculosis in AFB smear-negative sputum specimens through MTB culture and GeneXpert® MTB/RIF assay.

Tuberculosis (TB) is an important public health issue around the globe which is a chronic infectious disease and is still one of the major challenges for developing countries. The emergence of drug-resistant TB makes the condition worse and there is an urgent need of fast, highly sensitive diagnostic methods. This study was undertaken to evaluate the performance of GeneXpert® MTB/RIF assay and MTB culture for the detection of Mycobacterium tuberculosis (MTB) in sputum smear-negative pulmonary TB/drug-resistant tuberculosis (DR-TB) suspects. A total of 168 sputum smear-negative TB suspects were recruited for the study. Among the suspected TB cases, 52.98% were male and 47.02% were females with the mean age of 42 ± 17.6 years. All the sputum specimens collected from the study population were subjected to Ziehl–Neelsen (ZN) smear microscopy, GeneXpert MTB/RIF assay, and MTB culture. The results revealed that, out of 168 acid-fast bacilli (AFB)/ZN smear microscopy–negative sputum specimens, 48 (28.57%) and 58 (34.52%) were detected MTB positive by GeneXpert MTB/RIF assay and MTB culture, respectively, while 120 (71.43%) and 110 (65.48%) suspected TB cases were confirmed negative by GeneXpert MTB/RIF assay and MTB culture, respectively. The study concluded that GeneXpert assay was found to be a rapid and accurate tool for MTB detection in smear-negative sputum specimens. GeneXpert has advantage over ZN smear microscopy and MTB culture as it detects MTB and rifampicin resistance simultaneously within 2 h with minimal biohazards.

False Negativity of Ziehl-Neelsen Smear Microscopy: Is the Scale-up the Worth It in Developing Countries?

To evaluate the false negative results of Ziehl-Neelsen (ZN) smear microscopy. Descriptive study. Mycobacteriology Laboratory, Allama Iqbal Medical College (AIMC) and Jinnah Hospital, Lahore (JHL), Pakistan, from February 2014 to August 2016. A total of 3,951 (pulmonary 2,773 and extra-pulmonary 1,178) samples were collected from strong TB suspected patients attending JHL Lahore. Follow-up cases were excluded. Every specimen was processed for ZN smear microscopy, Lowenstein Jensen (LJ) culture. SPSS 21.0 was used; false negative and positive results of ZN smear were calculated keeping LJ culture as gold standard. Out of total 3,951 samples, sputum was most frequently found pulmonary sample 48.4% (n=1915), extra- pulmonary samples, pleural fluid and pus samples were most commonly observed samples 12.0% (n=476) and 8.3% (n=329), respectively. Overall false negativity was 23.1% (pulmonary=19.6%, extra-pulmonary=29.2%) (p<0.001), Maximum false negative results were observed in pericardial, synovial, pleural fluids, and pus samples as 40.0%, 38.0%, 33.0% and 32.0%, respectively. ZN smear microscopy is not a very efficient tool in case of patients with the low mycobacterial load. Therefore, National TB Control programs should consider extending their diagnostic approaches from ZN microscopy to more advanced techniques.

PERFORMANCE OF XPERT MTB/RIF ASSAY FOR DETECTION OF M.TB IN PULMONARY AND EXTRA-PULMONARY SAMPLES IN INDIAN PATIENTS

Introduction: Conventional methods like Ziehl-Neelsen (ZN) staining and liquid culture have been the mainstay for diagnosis of Tuberculosis (TB). The gold standard Liquid Culture method has a longer turnaround time. In the wake of the TB catastrophe, newer rapid and easily accessible methods of detection are the need of the hour. A molecular method like the Xpert MTB/RIF assay has revolutionized the early and rapid diagnosis of TB. Objective of the current study was to assess the performance and utility of Xpert MTB/RIF assay for the diagnosis of M. tuberculosis in pulmonary and extra-pulmonary clinical specimens in a large Indian reference laboratory.Methodology: The reference methods used were MGIT Liquid Culture system and ZN smear microscopy. Our study was performed in Global Reference Laboratory, Metropolis Healthcare, Mumbai, India for a period of 18 months with consecutive one thousand and forty two (518 Pulmonary + 524 extra-pulmonaryspecimens) clinical specimens obtained from the patients with clinical suspicion of tuberculosis. Diagnostic performance (sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the three methods were calculated with standard formulae.Results: In comparison to MGIT Liquid culture, sensitivity of Xpert MTB/RIF assay for pulmonary and extra pulmonary specimens were 87.18% and 68.92%, respectively while in comparison to ZN smear microscopy the sensitivity of Xpert MTB/RIF assay for pulmonary and extra pulmonary specimens were 92.67% and 83.81%, respectively.Conclusion: Our study concludes that in combination with the MGIT culture, Xpert MTB/RIF assay will significantly improve the detection rate of MTB bacteria.SAARC J TUBER LUNG DIS HIV/AIDS, 2017; XIV(1), page: 7-13

Rapid laboratory diagnosis of pulmonary tuberculosis.

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

Role of line probe assay in detection of extra-pulmonary tuberculosis/multidrug-resistant tuberculosis: The experience from Kerala state, India

Background: There are limited data on use of line probe assay (LPA) for detection of TB and Rifampicin resistance among extra-pulmonary tuberculosis (EPTB) patients under specific TB control programme settings. Our aim was to compare the positivity of LPA with Lowenstein–Jensen (LJ) and Bactec MGIT and to test its utility in faster testing and in patients on treatment. Methodology: The data of all 387 EPTB samples processed in two years by the state-level accredited laboratory in 2013–2014 were selected for the study. The laboratory used standard N-acetyl-L-cysteine – sodium hydroxide processing method for all samples except for cerebrospinal fluid and urine samples before conducting Ziehl-Neelsen smear microscopy, LPA and culture on both LJ and MGIT. Those samples with a negative LPA result if culture positive was subjected to a second LPA as per laboratory protocol. Doubtful Rifampicin resistant (RR) results were cleared by phenotypic testing by MGIT. Anti-TB treatment (ATT) duration was correlated with positivity of these tests later. Results and Discussion: LPA done on 321 processed samples out of 387 EPTB samples identified eight RR cases among 136 positive results within two to four days. Performing LPA on culture positive isolates identified an additional seven RR cases, thus reducing turn-around-time by two to eight weeks for the susceptibility results. It also demonstrated far better positivity in smear negative (15.2%) and in smear positive samples (74.3%) compared to culture methods as the test demonstrated clearly, better positivity among patients on ATT for more than two months. Conclusion: LPA in association with culture is an excellent combination with very good results in rapid identification of EPTB along with detection of Rifampicin resistance. LPA gives far superior positivity compared to MGIT and solid cultures among patients under ATT.

What a difference a day makes: same-day vs. 2-day sputum smear microscopy for diagnosing tuberculosis.

Setting: Nine district-level microscopy centres in Assam and Tripura, India. Objective: Same-day sputum microscopy is now recommended for tuberculosis (TB) diagnosis. We compared this method against the conventional 2-day approach in routine programmatic settings. Methods: During October-December 2012, all adult presumptive TB patients were requested to provide three sputum samples (one at the initial visit, the second 1 h after the first sample, and the third the next morning) for examination by Ziehl-Neelsen smear microscopy. Detection of acid-fast bacilli with any sample was diagnostic. The first and second spot sample comprised the same-day approach, and the first spot sample and next-day sample comprised the 2-day approach. Results: Of 2168 presumptive TB patients, 403 (18.6%) were smear-positive according to the same-day method compared to 427 (19.7%) by the 2-day method (McNemar's test, P < 0.001). Of the total 429 TB patients, 26 (6.1%) were missed by the same-day method and 2 (0.5%) by the 2-day method. Conclusion: Same-day specimen collection for microscopy missed more TB than 2-day collection. In India, missing cases by using same-day microscopy would translate into a considerable absolute number, hindering TB control efforts. We question the indiscriminate switch to same-day diagnosis in settings where patients reliably return for testing the next day.

Evaluation of the performance of the microscopic observation drug susceptibility assay for diagnosis of extrapulmonary tuberculosis in China: A preliminary study

While commercial liquid culture techniques have emerged over 20 years ago, Ziehl-Neelsen (ZN) smear microscopy remains the primary method for diagnosis of tuberculosis (TB) in China because of cost considerations. The microscopic observation drug susceptibility (MODS) assay has been evaluated in different parts of the world to determine whether it can give comparable result to commercial liquid techniques. However, most reports detail evaluation of sputum specimens. This study evaluated the performance of MODS assay for detection of Mycobacterium tuberculosis in extrapulmonary specimens in a Chinese population. A total of 173 samples, including pleural fluid (n = 112) and cerebrospinal fluid (CSF, n = 61) samples, were collected from patients suspected to have extrapulmonary TB and tested by ZN smear microscopy, Lowenstein-Jensen (LJ) culture and the MODS assay. Discordant results among MODS assay and the other two methods were resolved by 90-day follow-up evaluation for all suspected patients. The sensitivity of the MODS assay on pleural fluid and CSF samples was 20.5% and 37.5%, respectively, while the specificity of MODS assay on both types of samples approximated 100%. The median time to culture results for the MODS and LJ methods was 14 days, 32 days for pleural fluid, and 9 days and 31 days for CSF samples, respectively. MODS assay is useful to diagnose extrapulmonary TB and may be an effective and affordable method in resource-limited countries.

Prevalence of Pulmonary Tuberculosis - A Baseline Survey In Central India

BackgroundThe present study provides an estimate of the prevalence of bacteriologially positive pulmonary tuberculosis in Jabalpur, a district in central India.Methodology/Principal FindingsA community based cross-sectional survey was undertaken in Jabalpur District of the central Indian state of Madhya Pradesh. A stratified cluster sampling design was adopted to select the sample. All eligible individuals were questioned for pulmonary symptoms suggestive of TB disease. Two sputum samples were collected from all eligible individuals and were examined by Ziehl-Neelsen smear microscopy and solid media culture methods. Of the 99,918 individuals eligible for screening, 95,071 (95.1%) individuals were screened. Of these, 7,916 (8.3%) were found to have symptoms and sputum was collected from 7,533 (95.2%) individuals. Overall prevalence of bacteriologically positive PTB was found to be 255.3 per 100,000 population (95% C.I: 195.3–315.4). Prevalence was significantly higher (p<0.001) amongst males (355.8; 95% C.I: 304.4–413.4) compared with females (109.0; 95% C.I: 81.2–143.3). Prevalence was also significantly higher in rural areas (348.9; 95% C.I: 292.6–412.8) as compared to the urban (153.9; 95% C.I: 123.2–190.1).Conclusions/SignificanceThe TB situation in Jabalpur district, central India, is observed to be comparable to the TB situation at the national level (255.3 versus 249). There is however, a need to maintain and further strengthen TB control measures on a sustained and long term basis in the area to have a significant impact on the disease prevalence in the community.

LED Fluorescence Microscopy for the Diagnosis of Pulmonary Tuberculosis: A Multi-Country Cross-Sectional Evaluation

The diagnosis of tuberculosis (TB) in resource-limited settings relies on Ziehl-Neelsen (ZN) smear microscopy. LED fluorescence microscopy (LED-FM) has many potential advantages over ZN smear microscopy, but requires evaluation in the field. The aim of this study was to assess the sensitivity/specificity of LED-FM for the diagnosis of pulmonary TB and whether its performance varies with the timing of specimen collection. Adults with cough ≥2 wk were enrolled consecutively in Ethiopia, Nepal, Nigeria, and Yemen. Sputum specimens were examined by ZN smear microscopy and LED-FM and compared with culture as the reference standard. Specimens were collected using a spot-morning-spot (SMS) or spot-spot-morning (SSM) scheme to explore whether the collection of the first two smears at the health care facility (i.e., "on the spot") the first day of consultation followed by a morning sample the next day (SSM) would identify similar numbers of smear-positive patients as smears collected via the SMS scheme (i.e., one on-the-spot-smear the first day, followed by a morning specimen collected at home and a second on-the-spot sample the second day). In total, 529 (21.6%) culture-positive and 1,826 (74.6%) culture-negative patients were enrolled, of which 1,156 (49%) submitted SSM specimens and 1,199 (51%) submitted SMS specimens. Single LED-FM smears had higher sensitivity but lower specificity than single ZN smears. Using two LED-FM or two ZN smears per patient was 72.8% (385/529, 95% CI 68.8%-76.5%) and 65.8% (348/529, 95% CI 61.6%-69.8%) sensitive (p<0.001) and 90.9% (1,660/1,826, 95% CI 89.5%-92.2%) and 98% (1,790/1,826, 95% CI 97.3%-98.6%) specific (p<0.001). Using three LED-FM or three ZN smears per patient was 77% (408/529, 95% CI 73.3%-80.6%) and 70.5% (373/529, 95% CI 66.4%-74.4%, p<0.001) sensitive and 88.1% (95% CI 86.5%-89.6%) and 96.5% (95% CI 96.8%-98.2%, p<0.001) specific. The sensitivity/specificity of ZN smear microscopy and LED-FM did not vary between SMS and SSM. LED-FM had higher sensitivity but, in this study, lower specificity than ZN smear microscopy for diagnosis of pulmonary TB. Performance was independent of the scheme used for collecting specimens. The introduction of LED-FM needs to be accompanied by appropriate training, quality management, and monitoring of performance in the field. Current Controlled Trials ISRCTN53339491. Please see later in the article for the Editors' Summary.

Pulmonary tuberculosis: a public health problem amongst the Saharia, a primitive tribe of Madhya Pradesh, Central India

To estimate the prevalence of pulmonary tuberculosis (TB) disease amongst the Saharia, a 'primitive' tribe of Madhya Pradesh, Central India. A community-based cross-sectional TB prevalence survey was undertaken in the Saharia, a 'primitive' tribal community of Madhya Pradesh. A representative random sample of villages predominated by tribal populations was chosen from the selected block of Sheopur District. All eligible individuals were screened for chest symptoms related to TB. Sputum samples were collected from all eligible individuals, transported to the laboratory, and examined by Ziehl-Neelsen smear microscopy and solid media culture methods. Of the 11,468 individuals eligible for screening, 11,116 (96.9%) were screened for symptoms. The overall prevalence of pulmonary TB disease was 1518 per 100,000 population. Prevalence increased with age and the trend was statistically significant (p<0.001). The prevalence of pulmonary TB was also significantly higher in males (2156/100,000) than females (933/100,000) (p<0.001). The findings suggest that TB disease remains a major public health problem in the Saharia 'primitive' tribal community of Madhya Pradesh, Central India.