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BACKGROUND: Extrapulmonary TB (EPTB) comprises approximately 15–20% of TB cases worldwide, and its diagnosis is difficult. The sensitivity of Xpert® MTB/RIF (Xpert) in the diagnosis of EPTB is low on account of its paucibacillary nature. Xpert® MTB/RIF Ultra (Ultra) was developed to improve sensitivity.OBJECTIVE: To compare the sensitivity of Ultra test with that of Xpert against MGIT™ (Mycobacteria Growth Indicator Tube) culture and a composite reference standard (CRS).METHODS: We recruited consecutive treatment-naïve patients with suspected EPTB. Demographic information, clinical and relevant laboratory data were collected.RESULTS: From January 2019 to November 2019, 210 patients provided 250 samples. Against MGIT culture, the sensitivity of Ultra was significantly higher than Xpert (72% vs. 51.1%; P = 0.04), the specificity was lower (87.8% vs. 95.1%). Against the CRS also, the sensitivity of Ultra was significantly higher than Xpert (45.4% vs. 25.2%; P = 0.002); however, the specificities were similar (98.2% vs. 99.1%). The trend towards higher sensitivity of Ultra compared to Xpert was seen in most of the individual samples. The sensitivities against MGIT and CRS were as follows: lymph node (68.1% vs. 31.8%; P = 0.01) and (59.5% vs. 23.8%; P = 0.001), pleural biopsy (80.0% on both; P = NS) and (53.8% vs. 46.2%; P = NS) and pleural fluid (66.7% vs. 50%; P = NS) and (22.5% vs. 9.6%; P = NS), respectively.CONCLUSIONS: Xpert Ultra showed a significantly higher sensitivity in diagnosing EPTB than Xpert.

The QuantaMatrix Microfluidic Agarose Channel (QMAC) system was used for rapid drug susceptibility testing (DST). Here, we performed DST using QMAC integrated with the mycobacteria growth indicator tube (MGIT) liquid culture employing a specially designed cross agarose channel for the tuberculosis chip. MGIT-, QMAC-, and Löwenstein-Jensen (LJ)-DSTs were performed using 13 drugs. The protocol for QMAC-DST was optimized using the inoculum obtained after the disaggregation of Mycobacterium tuberculosis clumps in MGIT culture. The completion times of QMAC-DST and MGIT-DST were analyzed, and the results of all three DSTs were compared. Discrepant results were analyzed using line probe assays and DNA sequencing. Nontuberculous mycobacteria were distinguished using the ρ-nitrobenzoic acid inhibition test. The overall agreement rate of QMAT-DST and LJ-DST was 97.0% and that of QMAT-DST and MGIT-DST was 86.3%. An average turnaround time for DST was 5.4 days, which was considerably less than the time required for MGIT-DST. The overall time required to obtain DST results using QMAC-DST integrated with MGIT culture was an average of 18.6 days: 13.2 days for culture and identification and 5.4 days for DST. Hence, QMAC-DST integrated with liquid culture can be used to perform DSTs with short turnaround times and effective detection. KEY POINTS: • QMAC system can simultaneously perform phenotypic DST with 13 anti-TB drugs and PNB. • An optimized DST protocol led to a marked decrease in clumping in MGIT culture. • QMAC system integrated with MGIT liquid culture system reduced the turnaround time.

Highlights (1) Blood culture is the gold standard for the diagnosis of bacterial infections. (2) Bone marrow culture is more sensitive than blood culture even in patients with enteric fever receiving antibiotics. (3) Microscopic agglutination test is considered the gold standard for diagnosing leptospirosis; however, now IgM ELISA and polymerase chain reaction (PCR) are more frequently used for diagnosis. (4) Tuberculosis is diagnosed with the help of nucleic acid amplification tests like Xpert MTB/RIF Ultra which also detects rifampicin resistance. Other tests include microscopy, Lowenstein–Jensen and mycobacteria growth indicator tube culture, line probe assay. (5) Tropical rickettsial infections are diagnosed by serological reactions (Weil–Felix, ELISA for antibodies) and PCR. (6) For Brucellosis culture from blood, bone marrow or tissue specimens remain the mainstay in diagnosis. (7) Dengue, Zika, Crimean–Congo hemorrhagic fever, Ebola, hantavirus, rabies are diagnosed with reverse transcriptase-polymerase chain reaction. Serological tests like IgM ELISA or paired sera samples for IgG are also used for diagnosis. How to cite this article Basu S, Shetty A. Laboratory Diagnosis of Tropical Infections. Indian J Crit Care Med 2021;25(Suppl 2):S122–S126.

Early case detection by sputum smear microscopy is a crucial step in the control of pulmonary tuberculosis in high burden countries. Due to low sensitivity of this rapid and cost effective method, culture of Mycobacterium tuberculosis (MTB) is considered as the gold standard. Modified Petroff's method using 2%-4% sodium hydroxide (NaOH) and N-acetyl- l-cysteine (NALC) to digest and at the same time to decontaminate the specimen is widely used in developing countries prior to culture. This method is considered tedious and cumbersome. A novel ReaSLR (ReaMetrix's Sputum Liquefying Reagent) methodology has been proposed as a simple and low-cost method for sputum processing. This study was undertaken to evaluate the performance of the ReaSLR method of sputum processing prior to culture in comparison to the modified Petroff's method.Early morning sputum samples, collected from suspected TB patients, were divided into two equal halves and processed by two different methods i.e modified Petroff's method and ReaSLR method. After processing with different methods, each sample was inoculated in Lowenstein Jensen (LJ) medium and Mycobacteria growth indicator tube (MGIT). Smears were also prepared from the samples processed with modified Petroff's method and graded according to Centers for Disease Control and Prevention (CDC) grading after microscopic examination. Culture results of both the methods were recorded and analysed using SPSS 20.0 version.On comparing different methods of sputum processing for culture in solid and liquid media, the rate of contamination in both the media was significantly high with ReaSLR method as compared with modified Petroff's method. Also, the mean time-to-detection of MTB growth in LJ medium was significantly less with modified Petroff's method i.e 30.21 days as compared to ReaSLR method (34.23 days; p 0.001). However, the mean time-to-detection of MTB growth in MGIT was similar with both the methods.Due to the high contamination rate in solid and liquid culture media, ReaSLR method cannot be considered as an alternative to modified Petroff's method for sputum processing prior to culture. The detection of growth of MTB in LJ media was also earlier with modified Petroff's method than ReaSLR method.

AIM Key to the successful management of paediatric pulmonary tuberculosis (PTB) lies in the early detection and proper treatment. We evaluated the performances of modern diagnostic tests: loop-mediated isothermal amplification (LAMP-IS6110), Xpert MTB/RIF (Cepheid) and mycobacteria growth indicator tube (BACTEC MGIT 960 culture) against a modified version of international consensus diagnostic definition (i.e. composite reference standard (CRS)). METHODS A cross-sectional analytical study was conducted in a tertiary care hospital in North India from July 2016 to December 2017 involving 100 children

Background The National Tuberculosis Elimination Programme (NTEP) of India is aiming to eliminate TB by 2025. The programme has increased its services and resources to strengthen the accurate and early detection of TB. It is important to estimate the cost of TB diagnosis in India considering the advancement and implementation of new diagnostic tools under the NTEP. The objective of this study was to estimate the unit costs of providing TB diagnostic services at different levels of public health facilities with different algorithms implemented under the NTEP in Chennai, Tamil Nadu, South India. Methods This costing study was conducted from the perspective of the health system. This study used only secondary data and information that were available in the public domain. Data were collected with the approval of health authorities. The patient's diagnostic path from the point of registration until the final diagnosis was considered in the costing exercise. The unit costs of different diagnostic tools used in the NTEP implemented by Chennai Corporation were calculated. Results We estimated the unit cost of the eight laboratory tests (Ziehl–Neelsen [ZN], fluorescence microscopy [FM], x-ray, digital x-ray, gene Xpert MTB/RIF (cartridge-based nucleic acid amplification test [NAAT] that identifies rifampicin resistant Mycobacterium Tuberculosis) Mycobacterium Tuberculosis/Rifampicin [MTB/RIF], mycobacteria growth indicator tube [MGIT], line probe assay [LPA] and Lowenstein Jensen [LJ] culture) for diagnosis of drug-sensitive and drug-resistant TB. The unit costs included fixed and variable costs for smear examination by ZN microscopy (₹ [Indian Rupee] 326 [US\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\$}$\end{document}4.72], FM (₹104 [US\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\$}$\end{document}1.5]), x-ray (₹218 [US\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\$}$\end{document}3.15]), digital X-ray (₹281 [US\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\$}$\end{document}4.07]), gene Xpert MTB/RIF (₹1137 [US\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\$}$\end{document}16.47]), MGIT (₹7038 [US\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\$}$\end{document}102]), LPA (₹6448 [US\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\$}$\end{document}93.44]) and LJ culture (₹4850 [US\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\$}$\end{document}70.28]). Out of 10 diagnostic algorithms used for TB diagnosis, algorithms using only smear microscopy had the lowest cost, followed by smear microscopy with x-ray for drug-sensitive TB (₹104 [US\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\$}$\end{document}1.5] to ₹544 [US\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\$}$\end{document}7.88]). Diagnostic algorithms for drug-resistant TB involving LPA and gene Xpert MTB/RIF were the most expensive. Conclusions Understanding the various costs contributing to TB diagnosis in India provides crucial evidence for policymakers, programme managers and researchers to optimise programme spending and efficiently use resources.

Introduction. COVID-19 is responsible for the current global pandemic. Globally, over 15 million people are currently infected, and just over 600,000 have died due to being infected. It is known that people with chronic illnesses and compromised immune systems can develop more severe clinical presentation. Tuberculosis (TB) is still one of the biggest epidemiological problems worldwide. Both of these diseases can be misdiagnosed and can manifest in a similar way. We will present a case study of a patient who was initially treated as a COVID-19 infection, with TB being diagnosed later on. The recovery began only after being treated for both diseases simultaneously. Case report. The patient is a 27-year-old male, non-smoker, with no history of any significant diseases. He presented with fever, fatigue and hemoptysis. Computed tomography pulmoangiography had shown massive consolidations and excavations, which could be caused by COVID-19. Despite being treated for COVID-19, there was no clinical improvement. On the follow-up chest X-ray, beside signs of COVID-19, there were also changes that could indicate TB. TB was detected in sputum, using PCR and Mycobacteria Growth Indicator Tube, and only after being treated for both diseases did his condition improve. Conclusion. There are a few reported cases of COVID-19 and TB coinfections, and we believe that there are many more patients with this coinfection being unrecognized

Objective/Introduction: Tuberculosis is a overwhelming public health worry in India. The Indian Subcontinent accounts for about 25% of the TB burden worldwide. Across the world, India has the highest burden of both TB and MDR-TB. The aim of this study was to study the resistance pattern of Mycobacterium Tuberculosis to first and second line anti-TB drugs among specimens from a hospital in Tertiary Care Hospital in Western Maharashtra. Materials and Method: This record based study was conducted at Tertiary Care Hospital in Western Maharashtra, from January 2017 .Records of Sputum from 2013 to 2017 were obtained from patients with suspected TB. Drug-sensitivity testing was performed by the Mycobacteria Growth Indicator Tube (MGIT) method. Sensitivity to First line and second line drugs and Quinolones were tested. Results: Among 734 patients, 701(95.5%) were males and 33(4.5%) were females. Analysis of our Study result showed that 21.7% of patients were mono-drug resistant. Out of 734 isolates, 56 (7.62 %) showed multi-drug resistance (resistance to INH and RIF at least) and 33 out of 734 (4.5%) whereas 39 (5.6%) out of the 734 samples showed XDR. Conclusion:This study confirms that drug resistance, including MDR, observed against all first-line TB drugs was a real threat in the management of TB infection. The patterns of resistance in this study will assist clinicians in providing the right treatment regimen to TB patients and bring about an improvement in their clinical condition. Keywords: Pulmonary Tuberculosis, Drug resistance.

Culture remains the gold standard for tuberculosis (TB) diagnosis, and the mycobacteria growth indicator tube (MGIT), endorsed by the World Health Organization (WHO), is widely used. Further identification of a positive culture is done with the help of an immunochromatography assay, which often shows faint bands that are difficult to interpret. We analysed 125 BACTEC MGIT culture positive results, of which 11/16 (68.7%) of the doubtful assays, analysed by MGIT™ TBc Identification test (TBcId), were positive for Mycobacterium tuberculosis complex (MTBC), the remaining being non-tuberculous mycobacteria as determined by an in-house duplex polymerase chain reaction and line probe assay. Guidelines on faint or doubtful bands in immunochromatography assays are important so as not to overlook true-positive cases of TB.

Summary Background Zoonotic tuberculosis is defined as human infection with Mycobacterium bovis. Although globally, India has the largest number of human tuberculosis cases and the largest cattle population, in which bovine tuberculosis is endemic, the burden of zoonotic tuberculosis is unknown. The aim of this study was to obtain estimates of the human prevalence of animal-associated members of the Mycobacterium tuberculosis complex (MTBC) at a large referral hospital in India. Methods We did a molecular epidemiological surveillance study of 940 positive mycobacteria growth indicator tube (MGIT) cultures, collected from patients visiting the outpatient department at Christian Medical College (Vellore, India) with suspected tuberculosis between Oct 1, 2018, and March 31, 2019. A PCR-based approach was applied to subspeciate cultures. Isolates identified as MTBC other than M tuberculosis or as inconclusive on PCR were subject to whole-genome sequencing (WGS), and phylogenetically compared with publicly available MTBC sequences from south Asia. Sequences from WGS were deposited in the National Center for Biotechnology Information Sequence Read Archive, accession number SRP226525 (BioProject database number PRJNA575883). Findings The 940 MGIT cultures were from 548 pulmonary and 392 extrapulmonary samples. A conclusive identification was obtained for all 940 isolates; wild-type M bovis was not identified. The isolates consisted of M tuberculosis (913 [97·1%] isolates), Mycobacterium orygis (seven [0·7%]), M bovis BCG (five [0·5%]), and non-tuberculous mycobacteria (15 [1·6%]). Subspecies were assigned for 25 isolates by WGS, which were analysed against 715 MTBC sequences from south Asia. Among the 715 genomes, no M bovis was identified. Four isolates of cattle origin were dispersed among human sequences within M tuberculosis lineage 1, and the seven M orygis isolates from human MGIT cultures were dispersed among sequences from cattle. Interpretation M bovis prevalence in humans is an inadequate proxy of zoonotic tuberculosis. The recovery of M orygis from humans highlights the need to use a broadened definition, including MTBC subspecies such as M orygis, to investigate zoonotic tuberculosis. The identification of M tuberculosis in cattle also reinforces the need for One Health investigations in countries with endemic bovine tuberculosis. Funding Bill & Melinda Gates Foundation, Canadian Institutes for Health Research.

Background The detection of Mycobacterium tuberculosis (MTB) in the intensive care unit (ICU) presents several challenges, mainly associated to the clinical state of the patient. The presence of HIV infection further aggravates this scenario, requiring a reliable collection method, with better performance in the microbiological/molecular techniques to be used. We evaluated the performance of two methods for sample collection, mini bronchoalveolar lavage (Mini-BAL) and endotracheal aspirate (ETA), for diagnosis of pulmonary tuberculosis (PTB) in critically ill patients. Methods This prospective study involved 26 HIV positive ICU internalized patients, with presumptive PTB who required mechanical ventilation. Two samples were obtained prospectively from 26 HIV ICU patients with presumptive PTB by Mini-BAL and ETA. The samples were processed for smear microscopy, Löwenstein-Jensen medium and the BACTEC Mycobacteria Growth Indicator Tube 960 system®. We define as confirmed PTB patients with positive MTB culture. Furthermore, all samples obtained through the Mini-BAL were analyzed by Xpert® MTB/RIF. Results Our results demonstrated that the respiratory samples obtained by Mini-BAL were able to increase MTB detection in critically ill patients with presumptive PTB. The Mini-BAL allowed 30% increased recovery and guaranteed enough sample volume for processing in all methods. In addition, the larger volume of the samples obtained with this technique enabled the Xpert® MTB/RIF molecular test for diagnosis of TB. Conclusions The Mini-BAL showed be an acceptable alternative to ETA in this population, since these critically ill and often-immunocompromised patients are more likely to develop complications related to invasive procedures.

Background: Appropriate technology tests are needed for Mycobacterium tuberculosis drug-susceptibility testing (DST) in resource-constrained settings. This study was performed to evaluate the MDR/XDR-TB Colour Test (a colour platethin-layer agar test; TB-CX) for M. tuberculosis DST by directly testing sputum at University of Gondar Hospital. Methods: Sputum samples were each divided into two aliquots. One aliquot was mixed with disinfectant and applied directly to the TB-CX quadrant petri-plate containing culture medium with and without isoniazid, rifampicin, or ciprofloxacin. Concurrently, the other aliquot was decontaminated with sodium hydroxide, centrifuged, and cultured on Lӧwenstein–Jensen medium; the stored M. tuberculosis isolates were then sub-cultured in BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 for reference DST. Results: The TB-CX test yielded DST results for 94% (123/131) of positive samples. For paired DST results, the median number of days from sputum processing to DST was 12 for TB-CX versus 35 for LJ-MGIT (p

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis. Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

The gold standard of antituberculosis susceptibility testing is based on culture method which takes weeks. Rapid detection of resistance to isoniazid (INH) and rifampin (RIF) to avoid inappropriate regimens and to prevent transmission of resistant strains are important. A membrane array (BluePoint MTBDR) was developed to identify Mycobacterium tuberculosis complex (MTBC) and the genetic mutations responsible for resistance to RIF and INH. We aimed to evaluate the performance of this array for diagnosing drug‐resistant MTBC. A total of 261 acid‐fast bacilli positive sputum specimens, 1025 positive mycobacteria growth indicator tube (MGIT) cultures and 544 clinical isolates were analyzed. Antituberculosis susceptibility testing was the gold standard and was performed on MTBC isolated from positive MGIT cultures and on 544 clinical isolates. The sensitivity and specificity of the array to detect MTBC were 62.2% and 88.1% for sputum specimens, 100% and 97.9% for MGIT cultures. For detection of drug‐resistant MTBC in positive MGIT tubes, the sensitivities of the array were 100% for RIF and 97.1% for INH, while the specificities were 99.7% and 100%, respectively. Interestingly, we noticed four genotypically RIF‐resistant but phenotypically RIF‐susceptible isolates and eight genotypically INH resistant but phenotypically INH‐susceptible isolates. Comparing with conventional culture methods for species identification and drug susceptibility testing, the BluePoint MTBDR assay demonstrated to be a rapid test with high sensitivity and specificity to identify MTBC and to detect isoniazid and rifampin resistance when it is applied to broth culture specimens and clinical isolates.

Objective To compare the diagnostic performance of the manual BACTEC™ Mycobacteria Growth Indicator Tube (MGIT™) system (M-MGIT) with the automated BACTEC™ MGIT™ 960 system (A-MGIT) and Löwenstein-Jensen (L-J) culture method in detecting mycobacteria in sputum specimens from patients with suspected pulmonary tuberculosis (TB). Methods For this cross-sectional study, sputum samples were taken from patients aged ≥18 years attending a TB clinic in Beijing, China between July 2015 and October 2016. Processed sputum samples were inoculated into the MGIT systems and L-J medium for up to 6 and 8 weeks, respectively. Results The M-MGIT and A-MGIT methods detected significantly more Mycobacterium tuberculosis complex (MTC) isolates than L-J culture from the 565 sputum samples (39%, 40% and 32%, respectively). Using a positive result from any of the three culture systems as reference, the sensitivity of M-MGIT, A-MGIT and L-J methods were 92%, 94%, and 74%, respectively. The time-to-detection of mycobacteria was 12.9±4.2 days for M-MGIT, 11.8±5.2 days for A-MGIT and 24.2±8.7 days for L-J. Conclusions M-MGIT has a similar diagnostic performance to A-MGIT, and is a fast and reliable alternative to conventional culture methods in the diagnosis of pulmonary TB in a developing country.

Background Microscopic observation drug susceptibility (MODS) assay has been suggested as a low cost method for rapid, accurate detection of tuberculosis (TB) and multidrug resistant tuberculosis (MDR-TB). Methods A total of 2424 samples collected from 1063 eligible patients of suspected pulmonary or extrapulmonary TB were subjected to MODS assay. Performance of MODS was compared with culture and drug susceptibility testing (DST) by conventional solid Lowenstein–Jensen (LJ) media or liquid Mycobacteria Growth Indicator Tube (MGIT) culture. Results When compared to reference gold standard of positivity in either solid or liquid reference culture, the MODS assay had sensitivity, specificity, positive predictive value and negative predictive value of 91.3%, 98.2%, 96.0% and 95.9% respectively. MODS took a median time of 10.3 days to culture positivity as compared to 13.8 days using MGIT and 30.5 days using LJ culture. Culture and DST being concurrent in MODS, the median turnaround time for DST was the same as that for culture i.e. 10.3 days. The overall median turn around time for culture positivity and DST using manual MGIT and LJ medium was 23.6 days and 61.2 days respectively. The concordance between MODS culture and the reference susceptibility method was 97.7% for rifampicin, 95.6% for isoniazid, 98.5% for rifampicin and isoniazid. The cost of performing a single MODS assay was INR 200. Conclusion MODS is a rapid and sensitive, yet simple and inexpensive test that may be helpful to enhance diagnostic accuracy, and case detection of TB and MDR-TB in resource constrained settings.

Objectives Culture-based assays are currently the reference standard for drug susceptibility testing for Mycobacterium tuberculosis. They provide good sensitivity and specificity but are time consuming. The objective of this study was to evaluate whether whole genome sequencing (WGS), combined with software tools for data analysis, can replace routine culture-based assays for drug susceptibility testing of M. tuberculosis. Methods M. tuberculosis cultures sent to the Finnish mycobacterial reference laboratory in 2014 (n = 211) were phenotypically tested by Mycobacteria Growth Indicator Tube (MGIT) for first-line drug susceptibilities. WGS was performed for all isolates using the Illumina MiSeq system, and data were analysed using five software tools (PhyResSE, Mykrobe Predictor, TB Profiler, TGS-TB and KvarQ). Diagnostic time and reagent costs were estimated for both methods. Results The sensitivity of the five software tools to predict any resistance among strains was almost identical, ranging from 74% to 80%, and specificity was more than 95% for all software tools except for TGS-TB. The sensitivity and specificity to predict resistance to individual drugs varied considerably among the software tools. Reagent costs for MGIT and WGS were €26 and €143 per isolate respectively. Turnaround time for MGIT was 19 days (range 10–50 days) for first-line drugs, and turnaround time for WGS was estimated to be 5 days (range 3–7 days). Conclusions WGS could be used as a prescreening assay for drug susceptibility testing with confirmation of resistant strains by MGIT. The functionality and ease of use of the software tools need to be improved.

Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treatment of drug-resistant and multi-drug resistant (MDR) tuberculosis. Discordant RIF phenotype/genotype susceptibility results remain a challenge due to the presence of rpoB mutations which do not confer high levels of RIF resistance as have been exhibited in strains with mutations such as Ser450Leu. These strains, termed low-level RIF resistant, exhibit elevated RIF minimum inhibitory concentrations (MICs) compared to fully susceptible strains, however remain phenotypically susceptible by mycobacteria growth indicator tube (MGIT) testing and have been associated with poor patient outcomes. Here we assess RIF resistance prediction by whole-genome sequencing (WGS) among a set of 1779 prospectively tested strains by both prevalence of rpoB gene mutation and phenotype as part of routine clinical testing during a 21/2-year period. During this time, 139 strains were found to have nonsynonymous rpoB mutations, 53 of which were associated with RIF resistance, including both low-level and high-level resistance. Resistance to RIF (1.0 μg/mL in MGIT) was identified in 43 (81.1%) isolates. The remaining 10 (18.9%) strains were susceptible by MGIT, however were confirmed to be low-level RIF resistant by MIC testing. Full rpoB gene sequencing overcame the limitations of critical concentration phenotyping, probe-based genotyping, and partial-gene sequencing methods. Universal clinical WGS with concurrent phenotypic testing provided a more complete understanding of the prevalence and type of rpoB mutations and their association with RIF resistance in New York.

We present a case of a 59-year-old man, who on being evaluated for abdominal pain and headache, was found to have a pancreatic head mass and inflammatory hypophysitis. Xpert MTB/Rif of the pancreatic mass biopsy showed the presence of tuberculosis (TB) with a very low load, and rifampicin resistance was detected with absence of probes A and B. Pyrosequencing (a novel genotypic test for TB) of the Xpert MTB/Rif isolate detected a single, rare, high-confidence mutation (S512T) in the rpoB region (rifampicin resistance determining region in the MTB genome). The TB mycobacteria growth indicator tube (TBMGIT) phenotypic drug susceptibility test (DST), however, showed rifampicin susceptibility. Incidentally, he was unable to tolerate rifampicin and responded well to a non-rifampicin-based regimen. We discuss a possible hypothesis of the Xpert-DST discordance in accordance with a recent literature review on phenotypic DST methods. We also discuss the utility of pyrosequencing in clinical practice for the diagnosis of TB and its resistance patterns.

BACKGROUND The authors report a case of tumor-like colonic tuberculosis revealed by PCR in a 32-year-old patient with a low-level peritoneal effusion on CT scan with negative histological study on colonic biopsy. METHODS The colonic biopsy received at the laboratory after grinding in a porcelain mortar, was the object of a molecular study by GeneXpert MT/RIF (Cepheid, Sunnyvale, CA, USA) using the automated real-time PCR technique and a conventional study based on Ziehl-Nielsen staining and culture on Lowenstein-Jensen® solid medium (LJ) and Mycobacteria Growth Indicator Tube (MGIT®) liquid medium. RESULTS The patient was a 32-year-old male without any personal or family history of tuberculosis and without signs of tuberculosis impregnation. He had a story of ingestion of non-pasteurized dairy products including milk and cheese. For 45 days he had constipation with abdominal pain and feeling of heaviness. Physical examination of the patient revealed abdominal tenderness without adenopathy. The laboratory workup showed a normal blood count, CRP, liver and kidney function tests. The HIV test was negative. Medical imaging revealed a low-level peritoneal effusion that could not be punctured. Colonoscopy showed a thickening of the colon. The colonic biopsy, after crushing and sonication, was searched for the Mycobacterium tuberculosis complex by both molecular biology and conventional methods. Molecular research by GeneXpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) using the automated real-time PCR technique, revealed the presence of the Mycobacterium tuberculosis complex without detection of rifampicin resistance. On the other hand, the direct examination after special Ziehl-Nielsen staining was positive (Figure 2) and the cultures on Lowenstein-Jensen® solid medium (LJ) and Mycobacteria Growth Indicator Tube (MGIT®) liquid medium were also positive after two and three weeks, confirming the molecular diagnosis. The histology study showed moderate non-specific chronic colitis with no histological arguments for tuberculosis or malignancy. The patient was placed on curative tuberculosis treatment according to the national protocol, with a favorable clinical-radiological course. CONCLUSIONS Colonic tuberculosis is a disease that may mimic many other diseases; therefore, a correct approach is necessary for the correct diagnosis and treatment.

Background: Tuberculosis (TB) is one of the major global public health concern particularly affecting population of low-income countries. Early detection of disease coupled with other parameters help in treatment and reducing disease transmission. Methods: The current study was conducted to assess the sensitivity and specificity of the GeneXpert MTB/RIF (Cepheid Sunnyvale, CA, United States) in comparison to conventional techniques used for the diagnosis of TB. Our study is one of the first ones from Pakistan investigating and assessing the performance of GeneXpert. We recruited eight hundred clinically TB suspects initially and included seven hundred and sixteen clinically TB suspects in the final analysis. Results: The results of GeneXpert were compared with Mycobacteria Growth Indicator Tube (MGIT) and Ziehl-Neelsen (ZN) staining. In comparison to MGIT and ZN staining the sensitivity of GeneXpert with 95 % confidence interval (CI) was (99.7 %, CI 0.98-0.99) and (95.1 %, CI 0.92-0.97) respectively. The positive and negative predictive values with 95 % CI were (97.1 %, CI 0.94-0.98) and (99.7 %, CI 0.98-0.99) when results of GeneXpert were compare with MGIT results. Conclusion: The results of this study confirms the performance of GeneXpert. With high sensitivity and rapid detection, GeneXpert is ready to be considered as preferred diagnostic tool for TB.

Objectives We evaluated the performance of the MicroIDSys Elite system, a newly developed matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry system for identification of mycobacteria directly from positive MGIT liquid cultures. Methods Analytical specificity was evaluated with 63 reference strains grown in mycobacteria growth indicator tube media. Prospective performance evaluation was conducted with primary liquid cultures of sputum samples for identification of mycobacteria, and results were compared to multigenerational sequencing as the reference method. Liquid media subcultures were also analyzed. Results The accuracy for the 63 reference strains was 98.4% (62/63). A total of 167 paired mycobacterial primary cultures and subcultures in liquid media, comprised of seven Mycobacterium tuberculosis isolates, 109 slowly growing nontuberculous mycobacterial isolates, and 51 rapidly growing nontuberculous mycobacterial isolates, was identified by the MicroIDSys Elite system. Using primary liquid cultures, the MicroIDSys Elite system correctly identified 143 (85.6%) isolates; 21 (12.6%) resulted in “no identification”; and three (1.8%) isolates were misidentified. Using liquid media subcultures with this system, 159 (95.2%) isolates were correctly identified; seven (4.2%) resulted in “no identification”; and one (0.6%) isolate was misidentified. Conclusion The MicroIDSys Elite system is a useful routine diagnostic tool for identification of mycobacterial species from liquid culture.

This study aimed to evaluate the performance of real-time PCR (qPCR) and MALDI-TOF for accurate and timely detection of nontuberculous mycobacterium (NTM) from clinical isolates. We collected fifty NTM suspected Mycobacteria Growth Indicator Tube (MGIT) cultures and analysed the diagnostic performance of qPCR and VITEK MS using Line Probe Assay (LPA) GenoType CM (Common Mycobacteria) as gold standard. The qPCR assays targeting 16S rRNA, ITS and IS6110 genes were developed for the identification of NTM and Mycobacterium tuberculosis complex (MTBC). LPA GenoType CM, a PCR technique targeting 23S rRNA gene, followed by reverse hybridization and line probe technology identified 90% of Mycobacterium species including M. fortuitum (16%,n = 8), M. intracellulare (10%,n = 5), M. gordonae (10%,n = 5), M. xenopi (4%,n = 2), M. scrofulaceum (4%,n = 2), Mycobacterium additional species (AS) (32%,n = 16) and MTBC (14%,n = 7), qPCR detected 80% of Mycobacterium species (NTM, 66% (n = 33) and MTBC, 14% (n = 7)) and MALDI-TOF, 52% (M. fortuitum (12%,n = 6), M. intracellulare (10%, n = 5), M. simiae (8%,n = 4), M. gordonae (8%,n = 4), and MTBC (14%,n = 7)). Sensitivity of qPCR and MALDI-TOF was 88.9% and 57.8%, respectively with 100% specificity. The combination of qPCR and MALDI-TOF remains an appropriate test for timely diagnosis of Mycobacterium species. This may eventually assist to detect the cases that may have been missed by phenotypic tests and enhance the NTM diagnosis capability to improve effective patient management.

Tuberculosis (TB) is a major public health problem worldwide. Contamination rate and poor recovery of Mycobacterium tuberculosis complex (MTBC) in MGIT960 culture may affect the early diagnosis of TB. Evidence is needed to determine the factors associated with contamination rates and MTBC recovery in MGIT960. Hence, we undertook this study to compare the factors influencing MTBC culture positivity and contamination rates in MGIT960 in patients with Pulmonary tuberculosis (PTB).A total of 849 sputum samples from newly diagnosed smear-positive TB cases enrolled into the Regional Prospective Observational Research for Tuberculosis India cohort between May 2014 to March 2017 were analyzed. Samples were inoculated into MGIT960 and positive cultures were examined for the presence of MTBC by immunochromatographic test for detection of MPT64 antigen.Of the 849 cases, 811 (95.5%) were culture positive for MTBC, 23 (2.7%) were culture negative and 15 (1.8%) were contaminated. Salivary sputum showed significantly less culture yield compared to mucopurulent/blood stained samples (p = 0.021). Sputum from individuals20 or ≥60 years showed lower culture yield of 93.9%, compared to those aged 20-59years (98.2%) (p = 0.002). Based on smear grading, culture isolation of MTBC by MGIT960 was 86.1%, 93.6% and 99.5% for negative, scanty and positive (1+/2+/3+) samples, respectively (p ≤ 0.0001). Sputum from HIV negative patients showed higher culture yield, compared to HIV positive patients (p ≤ 0.0001). Chest X-Ray revealed that patient with cavity showed higher culture isolation of MTBC compared to patients without cavity (p = 0.035). Contamination rates were higher in smear negatives (6.0%), compared to scanty (2.1%) and smear positives (1.1%) (p = 0.007). However, delay in transport of the specimen to the laboratory was the only independent factor significantly associated with increase in culture contamination.Our results highlight that extremes of age, smear negativity, HIV infection, sputum quality and cavitation significantly influence the culture yield of MTBC, whereas transport duration and smear grading affected the contamination rates in MGIT960. Hence, addressing these factors may improve the diagnostic performance of MGIT960.

Background and Aim To evaluate the role of multiplex polymerase chain reaction (PCR) for diagnosis of gastrointestinal tuberculosis (GITB). Methods This was a prospective observational study conducted from July 2015 to November 2016 at a tertiary care teaching institution in north India. Fifty individuals with clinically suspected GITB and older than 18 years of age were recruited. Patients underwent radiological investigations, esophagogastroduodenoscopy, or colonoscopy as clinically indicated. Multiple biopsies for tissue diagnosis and PCR were taken. All specimens were subjected to Ziehl Neelsen staining, histopathology, and multiplex PCR using specific primers for genes IS6110, MPB64, and Protein b. The performance of the assay was assessed using a composite reference standard for diagnosis of tuberculosis. It comprised a combination of clinical characteristics and microbiological methods, including smear, Bactenecin (BACTEC) culture, histopathology, and response to antitubercular therapy. Results A final diagnosis of tuberculosis was made in 32 cases (Duodenal-4, Ileo-colonic-28). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of histopathology for the diagnosis of tuberculosis was 28.12, 100, 100, and 43.9%, respectively. The sensitivity, specificity, PPV and NPV of BACTEC Mycobacteria Growth Indicator Tube (MGIT) culture for the diagnosis of tuberculosis was 9.3, 100, 100, and 38.29%, respectively. The sensitivity, specificity, PPV, and NPV of multiplex PCR for the diagnosis of tuberculosis was 87.5, 100, 100, and 86.2%, respectively. Conclusion Multiplex PCR using specific primers for genes IS6110, MPB64, and Protein b had a higher sensitivity compared to conventional techniques for the diagnosis of GITB.

OBJECTIVES The aim of the study was to clarify how HIV infection affects tuberculosis liquid and solid culture results in a resource-limited setting. METHODS We used baseline data from the Study on Outcomes Related to Tuberculosis and HIV Drug Concentrations in Uganda (SOUTH), which included 268 HIV/tuberculosis (TB)-coinfected individuals. Culture results from Lowenstein-Jensen (LJ) solid culture and mycobacteria growth indicator tube (MGIT) liquid culture systems and culture-based correlates for bacillary density from the sputum of HIV/TB-coinfected individuals at baseline were analysed. RESULTS Of 268 participants, 243 had a CD4 cell count available and were included in this analysis; 72.2% of cultures showed growth on solid culture and 82.2% in liquid culture systems (P

We evaluated the microscopic and macroscopic characteristics of mycobacteria growth indicator tube (MGIT) cultures for the presumptive identification of the Mycobacterium tuberculosis complex (MTBC) and assessed the reliability of this strategy for correctly directing isolates to drug susceptibility testing (DST) or species identification. A total of 1526 isolates of mycobacteria received at the Instituto Adolfo Lutz were prospectively subjected to presumptive identification by the observation of growth characteristics along with cord formation detection via microscopy. The presumptive identification showed a sensitivity, specificity and accuracy of 98.8, 92.5 and 97.9 %, respectively. Macroscopic analysis of MTBC isolates that would have been erroneously classified as non-tuberculous mycobacteria based solely on microscopic morphology enabled us to direct them rapidly to DST, representing a substantial gain to patients. In conclusion, the growth characteristics of mycobacteria in MGIT, when considered along with cord formation, increased the reliability of the presumptive identification, which has a great impact on the laboratory budget and turnaround times.

Summary Background WHO recommends Xpert MTB/RIF as initial diagnostic testing for tuberculous meningitis. However, diagnosis remains difficult, with Xpert sensitivity of about 50–70% and culture sensitivity of about 60%. We evaluated the diagnostic performance of the new Xpert MTB/RIF Ultra (Xpert Ultra) for tuberculous meningitis. Methods We prospectively obtained diagnostic cerebrospinal fluid (CSF) specimens during screening for a trial on the treatment of HIV-associated cryptococcal meningitis in Mbarara, Uganda. HIV-infected adults with suspected meningitis (eg, headache, nuchal rigidity, altered mental status) were screened consecutively at Mbarara Regional Referral Hospital. We centrifuged CSF, resuspended the pellet in 2 mL of CSF, and tested 0·5 mL with mycobacteria growth indicator tube culture, 1 mL with Xpert, and cryopreserved 0·5 mL, later tested with Xpert Ultra. We assessed diagnostic performance against uniform clinical case definition or a composite reference standard of any positive CSF tuberculous test. Findings From Feb 27, 2015, to Nov 7, 2016, we prospectively evaluated 129 HIV-infected adults with suspected meningitis for tuberculosis. 23 participants were classified as probable or definite tuberculous meningitis by uniform case definition, excluding Xpert Ultra results. Xpert Ultra sensitivity was 70% (95% CI 47–87; 16 of 23 cases) for probable or definite tuberculous meningitis compared with 43% (23–66; 10/23) for Xpert and 43% (23–66; 10/23) for culture. With composite standard, we detected tuberculous meningitis in 22 (17%) of 129 participants. Xpert Ultra had 95% sensitivity (95% CI 77–99; 21 of 22 cases) for tuberculous meningitis, which was higher than either Xpert (45% [24–68]; 10/22; p=0·0010) or culture (45% [24–68]; 10/22; p=0·0034). Of 21 participants positive by Xpert Ultra, 13 were positive by culture, Xpert, or both, and eight were only positive by Xpert Ultra. Of those eight, three were categorised as probable tuberculous meningitis, three as possible tuberculous meningitis, and two as not tuberculous meningitis. Testing 6 mL or more of CSF was associated with more frequent detection of tuberculosis than with less than 6 mL (26% vs 7%; p=0·014). Interpretation Xpert Ultra detected significantly more tuberculous meningitis than did either Xpert or culture. WHO now recommends the use of Xpert Ultra as the initial diagnostic test for suspected tuberculous meningitis. Funding National Institute of Neurologic Diseases and Stroke, Fogarty International Center, National Institute of Allergy and Infectious Disease, UK Medical Research Council/DfID/Wellcome Trust Global Health Trials, Doris Duke Charitable Foundation.

Introduction: The aim was to study the accuracy of Xpert® (Cepheid Inc., Sunnyvale, CA, USA) Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay as compared to a composite gold standard (urine culture, imaging, and biopsy) and to asses its utility as the initial test compared to smear microscopy to diagnose urinary tuberculosis. Methods: This prospective study included adult patients suspected to have urinary tuberculosis from March 2014 to December 2017. Three urine samples were collected from each patient and were subjected to Xpert MTB/RIF assay, acid-fast bacillus (AFB) smear microscopy, and liquid media (BACTEC Mycobacteria Growth Indicator Tube [MGIT] 960) culture. Imaging and tissue biopsies were performed as clinically indicated. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated using the bootstrap method for 95% confidence intervals for the Xpert assay. Results: Xpert MTB/RIF assay was found to be superior to the currently best available light-emitting diode fluorescent smear microscopy as the initial test for urinary tuberculosis (sensitivity of 69.09% vs. 32.72%). The Xpert MTB/RIF polymerase chain reaction test was found to have a moderate sensitivity (69.09%) and high specificity (100%) as compared to the composite reference standard. The sensitivity of liquid AFB culture MGIT 960 as compared to the reference standard was 90.32%. Conclusions: Xpert MTB/RIF assay on an early morning first void urine specimen can replace smear microscopy as the initial diagnostic test for urinary tuberculosis.

BACKGROUND: The purpose of this study was to assess the utility of Mycobacteria Growth Indicator Tube (MGIT) 960 culture medium for the diagnosis of genital tuberculosis (GTB) in women presenting with infertility. METHODS: The premenstrual endometrial biopsy samples in 300 women presenting with primary and secondary infertility were subjected to AFB smear method, histopathological examination and culture on LJ and MGIT 960 media. Detection rates were compared for diagnostic modalities and their combinations. RESULTS: In total, 30 cases were positive for genital tuberculosis by either of the four tests employed. The detection rates for AFB smear, MGIT culture, LJ culture and HPE were 50, 46.7, 3.3 and 33.3%, respectively. A combination of smear examination for AFB, MGIT 960 culture and histopathological examination was able to detect all the positive cases. A combination of MGIT and LJ media provided no added advantage over MGIT alone since the only case where LJ culture was positive had been detected by positive MGIT culture. In as many as five positive cases (16.7%), only MGIT culture was positive. CONCLUSION: The addition of MGIT 960 culture medium to routine battery of investigations in infertility patients significantly improves the diagnosis.

Tuberculosis (TB) is an often neglected, epidemic disease that remains to be controlled by contemporary techniques of medicine and biotechnology. In this study, a nanoscale sensing system, referred to as magnetophoretic immunoassay (MPI) was designed to capture culture filtrate protein (CFP)-10 antigens effectively using two different types of nanoparticles (NPs). Two specific monoclonal antibodies against CFP-10 antigen were used, including gold NPs for signaling and magnetic particles for separation. These results were carefully compared with those obtained using the commercial mycobacteria growth indicator tube (MGIT) test via 2 sequential clinical tests (with ca. 260 clinical samples). The sensing linearity of MPI was shown in the range of pico- to micromoles and the detection limit was 0.3pM. MPI using clinical samples shows robust and reliable sensing while monitoring Mycobacterium tuberculosis (MTB) growth with monitoring time 3-10 days) comparable to that with the MGIT test. Furthermore, MPI distinguished false-positive samples from MGIT-positive samples, probably containing non-tuberculous mycobacteria. Thus, MPI shows promise in early TB diagnosis.

OMNIgene·SPUTUM (OM-S) is a sample transport reagent designed to work with all tuberculosis diagnostics while eliminating the need for cold chain. OM-S-treated sputum samples were assayed in several tests after multiday holds. Raw sputa from 100 patients underwent direct smear microscopy, were manually split and assigned to the OM-S group [OM-S added at collection (no other processing required) and tested after 0- to 5-day holds at room temperature] or standard-of-care (SOC) group (NaOH/N-acetyl l-cysteine decontamination, all tested on day of collection). Concentrated smear microscopy, Lowenstein Jensen (LJ) culture, and mycobacteria growth indicator tube (MGIT) culture were performed. For patients with negative direct smear, a second sample was split, with SOC (raw sputum) and OM-S portions (sediment) tested in the Xpert MTB/RIF (Xpert) assay. OM-S group and SOC group results were strongly concordant on all four tests [range, 89% (MGIT)-97% (Xpert)]. OM-S MGIT, LJ, and Xpert tests were in statistical agreement with SOC MGIT as reference. OM-S specimens had lower culture contamination rates (3% vs. 10% LJ; 2% vs. 5% MGIT) but required, on average, 5.6 additional days to become MGIT-positive. The findings suggest that samples held/transported in OM-S are compatible with smear microscopy, LJ or MGIT culture, and Xpert, and perform comparably to fresh sputum samples. Larger feasibility studies are warranted.

We compared different methods for their ability to isolate Mycobacterium bovis from tissue samples from animals with lesions resembling bovine tuberculosis. In the first trial, M. bovis was isolated from 86 of 200 tissue samples that were cultured using 2 liquid media, BACTEC 12B and BBL mycobacteria growth indicator tube (MGIT), and a solid medium, Middlebrook 7H11 supplemented with pyruvate (7H11P). M. bovis was isolated from 2 samples with MGIT but not BACTEC 12B. M. bovis was isolated from 9 samples with BACTEC but not MGIT; these 9 samples came from the North Canterbury/Marlborough region of New Zealand. The proportion of tissues from which M. bovis was isolated with BACTEC 12B or MGIT and the mean time for isolation was different for samples from the North Canterbury/Marlborough region but not the rest of New Zealand. In the second trial, M. bovis was isolated from 401 of 1,033 tissues that were cultured using MGIT, Middlebrook 7H9 broth, or solid 7H11P. The proportion of isolates of M. bovis and the mean time for their isolation with MGIT was different for the North Canterbury/Marlborough and the rest of New Zealand. The reason for this difference was not determined but may be related to the genotypes present in this region. Genotyping using variable number tandem repeats (VNTRs) of 197 isolates of M. bovis revealed that the 44 isolates from North Canterbury/Marlborough were represented by 2 closely related VNTR types that were not found in 153 isolates from the remainder of New Zealand.

Objectives To characterize rifampicin-resistant strains missed by the Mycobacteria Growth Indicator Tube (MGIT) 960 system but not by egg-based media in the UK and Ireland and to ascertain their prevalence. Methods All strains sent for second-line susceptibility testing were prospectively collected. Drug Susceptibility Testing was performed by Resistance Ratio (RR), Proportion Method (PM), MGIT 960 and MIC determination by microdilution. Rifampicin-resistance-conferring mutations were detected with line probe assays and sequencing. At the end of the study period, retrospective archived strains from 2010 to 2014 showing key mutations were analysed phenotypically and genotypically. Results Seventeen of 7234 prospective isolates were included. All of them were susceptible by MGIT. One was borderline by RR (MIC to rifampicin of 4 mg/L) and was resistant by PM. Eight were resistant and eight were highly resistant on RR. These 16 isolates had MICs between 1 and 8 mg/L on microdilution. With PM, 16/17 were susceptible to rifampicin. 17/17 had mutations in the rpoB gene. D516Y was the mutation most frequently found (13/17). Retrospectively, ten additional strains with key genotypes were found in our collection: 6/10 were susceptible in the MGIT and resistant in RR. Of the 27 studied strains, the MGIT only detected resistance in four. Conclusions Rifampicin resistance is missed by the MGIT system. In the UK and Ireland the prevalence of these strains is low. The introduction of routine molecular testing would detect false susceptibility. Further research is needed to ascertain the role of these strains in clinical failure and their prevalence in other settings.

Tuberculosis represents a main concern for public health worldwide. In poor countries, the most prevalent method for bacteriological confirmation re- mains Smear Sputum Microscopy (SSM). This study objective was to assess clinical performances of Loop Mediated Isothermal Amplification for TB detection (Lamp-TB). Sputum of patients presenting symptoms consistent with tuberculosis were collected according to the National Tuberculosis Control Programme guidelines in Centre Antituberculeux de Yopougon. SSM after Ziehl-Neelsen staining and TB-Lamp were blindly performed with spot sputum specimen. Samples, transported at Institut Pasteur de Cote d’Ivoire were decontaminated according to N-acetyl-L-cystein (NALC) method. In Mycobacteria Growth Indicator Tube (MGIT), 500 μl of pellet were inoculated and incubated in MGIT 960 instrument. MPT64 antigen was detected on positive culture. Of 500 patients enrolled, 469 were included. Clinical isolates of M. tuberculosis Complex were detected for 157 (33.5%). Comparatively to culture, Sensitivity and Specificity of SSM were 86% (95% Confidence interval (CI): 81% - 91%) 96% (95%IC: 94% - 98%) respectively. TB-Lamp Sensitivity was 92% (95%CI: 88% - 96%), and Specificity 94% (95%CI: 91% - 97%). Positive Predictive Value of SSM and TB-Lamp was 91.8% and 88.8% respectively. Negative Predictive Value of TB-Lamp assay was 95.7% whereas this of SSM was 93.3%. Positive Likelihood Ratio was 15.3 for TB-Lamp and 21.5 for SSM 21.5 whereas negative Likelihood of TB-Lamp was lower than SSM. Active tuberculosis was detected in162/469 (34.5%) with TB-Lamp and 147 (31.3%) with SSM. TB-Lamp assay performances estimated from sputum samples may improve detection of active TB cases in routine.

Background Cartridge-based nucleic acid amplification test (CB-NAAT) has been recommended for diagnosis of tuberculosis (TB) in children, but its wide use is limited by high cost and the need for well-equipped laboratories. This study was conducted in children with pulmonary TB to compare the diagnostic yield of TB-LAMP (loop-mediated isothermal amplification test) with CB-NAAT and other conventional methods. Methods Patients ≤ 14 years of age diagnosed with probable pulmonary TB were included in the study. Induced sputum/gastric aspirate was obtained and subjected to acid-fast bacilli (AFB) microscopy, mycobacteria growth indicator tube (MGIT) culture, CB-NAAT, and TB-LAMP. The TB-LAMP assay was performed using 2 different primers, IS6110 and mpb64, for detection of Mycobacterium tuberculosis (MTB). TB-LAMP assays were compared to other assays using appropriate statistical tests. Results One hundred fourteen subjects were recruited in the study. AFB microscopy, MGIT culture, CB-NAAT, TB-LAMP IS6110, and TB-LAMP mpb64 showed positivity of 32 (28.1%), 59 (51.7%), 66 (57.9%), 75 (65.8%), and 81 (71%), respectively. TB-LAMP IS6110 showed significantly higher MTB detection in comparison to AFB microscopy and MGIT culture (P = .0001 and P = .03, respectively), and showed no significant difference in MTB detection in comparison with CB-NAAT (P = .219). TB-LAMP mpb64 showed significantly higher MTB detection as compared to AFB microscopy, MGIT culture, and CB-NAAT (P = .0001, P = .003, and P = .037, respectively). TB-LAMP mpb64 and IS6110 showed sensitivity of 94.9% (95% confidence interval [CI], 85.9%–98.9%) and 89.8% (95% CI, 79.7%–96.2%), respectively, in reference to MGIT culture. The degree of agreement between TB-LAMP (mpb64 and IS6110) with CB-NAAT showed κ values of 0.718 and 0.834, respectively. Conclusions TB-LAMP assay can be a useful alternative test in diagnosis of pulmonary TB in children.