Culture Drug Susceptibility Testing Research Articles

Diagnostic Performance and Comparative Analysis of Conventional Drug Susceptibility Testing, Line Probe Assay, and GeneXpert in Extrapulmonary Tuberculosis Cases: A Cross-sectional Observational Study

Introduction: Tuberculosis (TB), a known threat to mankind for ages, causes a significant burden on healthcare worldwide. The alarming rise of Extrapulmonary Tuberculosis (EPTB) has led healthcare professionals to opt for molecular diagnostics. Despite the availability of rapid diagnostics, conventional culture Drug Susceptibility Testing (DST) is still considered the gold standard in diagnosing TB. Aim: To assess the diagnostic accuracy of culture DST which is considered the gold standard. Materials and Methods: This cross-sectional observational study included 150 suspected EPTB patients and was conducted over a period of one year (September 2018 to August 2019) at Lokmanya Tilak Municipal Medical College and Sion Hospital (LTMMC), a tertiary care hospital in central Mumbai, Maharashtra, India. The hospital is located adjacent to Dharavi, the largest slum area with a high population density, which contributes to the majority of patients attending clinics. Patients attending the pulmonary medicine Outpatient Department (OPD) with extrapulmonary manifestations and suspected cases of EPTB were enrolled in the study. Samples, excluding sputum, were sent to the Department of Microbiology, LTMMC, Sion Hospital for GeneXpert analysis. The samples were further evaluated by microscopy, GeneXpert, and culture DST to detect the presence of Mycobacterium tuberculosis (M. tuberculosis) and drug susceptibility, respectively. Patients positive for Pulmonary Tuberculosis (PTB) were excluded from the study. Categorical variables were described using percentages. A Chi-square test was applied, and a p-value of <0.05 was considered significant. All statistical analyses were performed using SPSS statistical software (IBM SPSS version 26.0, Armonk, N.Y.). Results: Out of the 150 extrapulmonary samples, 23 (15.33%) samples were culture positive for M. tuberculosis and were subjected to DST using the 1% proportion method and GeneXpert assay. The MDR isolates were tested using the firstline Line Probe Assay (LPA). Eleven (47.8%) showed resistance to first-line antitubercular drugs. Among the 12 new cases, only 2 (16.7%) showed resistance compared to 9 (81.8%) in previously treated cases. A discordance of 8.7% was observed between DST with GeneXpert and LPA. Additionally, a discordance of 8.7% was observed between DST and LPA for rifampicin resistance and 4.3% for isoniazid resistance. Conclusion: The paucibacillary nature of extrapulmonary samples contributes to the challenging diagnosis of EPTB cases, leading to increased drug resistance. Highlighting the importance of the conventional solid culture DST method, this study strongly recommends the use of conventional DST accompanied by LPA for extrapulmonary cases.

Crystal violet decolorization assay: a simplified colorimetric test for the rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates

The increased prevalence of multi-drug resistant M. tuberculosis is quite possibly the direst and most difficult task for the early diagnosis and treatment. A rapid, reliable, and inexpensive diagnostic method is the need of the hour. The current study on crystal violet decolorization assay explores the possibility to develop a rapid and simple detection method to detect multi-drug-resistant tuberculosis isolates by comparing the results with the traditional liquid culture drug susceptibility testing method based on their sensitivity, specificity, positive predictive value, and negative predictive value. 70 isolates were used for the study and were detected as multi-drug resistant, mono drug-resistant, and sensitive by using crystal violet decolourization assay and further compared with the results of DST and using H37Rv as the standard control strain. The sensitivity, specificity, positive predictive value, and negative predictive value of crystal violet decolorization assay (Rifampicin: 100%, 94.60%, 100% and 82.40%; isoniazid: 100%, 94.10%, 100%, 86.40%) are calculated and the percentage were compared with the conventional liquid culture drug susceptibility testing for M. tuberculosis using rifampicin and isoniazid. Crystal violet decolourization assay is rapid, reproducible, and doesn't require any highly experienced personal or sophisticated laboratory instruments for interpretation. This assay is found to be nearly as reliable as conventional liquid culture drug susceptibility testing and may thus be of great help in phenotypic confirmation of multi-drug resistant tuberculosis by providing results more rapidly.

Utility of laboratory diagnostic tests in women suspected of genital tuberculosis attending a tertiary care teaching hospital.

Genital Tuberculosis is a form of extrapulmonary tuberculosis, which if not diagnosed early can lead to complications. The objective of this study was to determine the sensitivity and specificity of Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF) assay in genital tuberculosis (TB) in comparison with culture as a gold standard. The results of the Xpert MTB/RIF assay performed from January 2020 to August 2021 were compared with the results of culture by Mycobacterium Growth Indicator Tube (MGIT) 960. Out of 75 specimens, fluorescent microscopy and liquid culture using MGIT and Xpert assay were positive in 3 (4%), 21 (28%), and 14 (18%), respectively. The sensitivity and specificity of the Xpert MTB/RIF assay were 66.67% and 100%. All smear-positive specimens were positive by culture and Xpert assay. Three specimens were positive by all the tests, i.e., microscopy, culture, and Xpert assay. Fifty-four specimens were negative by microscopy, culture, and Xpert assay. Discordance between the results of culture and Xpert assay was observed in seven specimens which were culture positive and Xpert assay negative. Three (21.42%) out of 21 culture-positive specimens showed monoresistance to rifampicin by Xpert MTB/RIF assay and culture drug susceptibility testing. Xpert MTB/RIF assay showed good sensitivity and specificity compared to liquid culture in genital TB. This test is easy to perform, provides results in 2 h, and can also detect rifampicin resistance, which is a surrogate marker for multidrug-resistant TB. Hence, the Xpert assay can be used under the National TB Elimination Program for early and rapid diagnosis of TB in endometrial specimens to prevent complications like infertility.

Cartridge-based nucleic acid amplification test in drug-resistant Mycobacterium tuberculosis as compared to solid cultures: Is it time to look beyond cartridge-based nucleic acid amplification test?

Background: There is a need to study the performance, validity, and accuracy of cartridge-based nucleic acid amplification test (CB-NAAT) for accessing drug resistance among pulmonary tuberculosis (TB) compared with the solid culture drug susceptibility test (DST).Methods: Patients with symptoms of cough for more than 2 weeks with anyone symptoms such as night sweats, fever, and unintentional weight loss were studied. Cases with previously diagnosed drug-resistant pulmonary TB by sputum CB-NAAT having constitutional symptoms but not on any ATT for a minimum of 2 months were also included in the study. The patient's information, including age, immune surveillance status, clinical features, and chest X-rays, were recorded. Each sputum sample was divided into three aliquots and tested for smear microscopy, liquid culture (LC), and genotypic DST. Results of all three diagnostic modalities were compared with CB-NAAT reports.Results: Of 236 patients with sputum-positive CB-NAAT (n = 236), 49.4% (n = 117) were rifampicin resistant, while 50. 4% (n = 119) were rifampicin sensitive. The genotypic DST assays carried out of all enrolled patients showed that 76.3% (n = 181) patients were resistant to one or more first-line antitubercular drugs (FL ATTs) or second-line (SL) ATTs, while 23.7% (n = 55) patients were sensitive to all ATTs. Among all the study participants, 56.4% (n = 133) of patients had sputum smear-positive by ZN stain, while 88.6% (n = 209) showed growth on LC (BACTEC) media. On concordant analysis of CB-NAAT with DST assays, we found that among 119 CB-NAAT rifampicin-sensitive patients, 66 patients were drug-resistant (DR) TB to any of the FL or SL ATTs. The sensitivity, specificity, positive predictive value, and negative predictive value of CB-NAAT for detecting rifampicin resistance on sputum for pulmonary TB when compared with the gold-standard DST assays were 97.67%, 76.67%, 70.59%, and 98.29%, respectively.Conclusions: This study found that the use of rapid molecular technique (CB-NAAT) in screening DRTB at the community level is suboptimal compared to the gold-standard solid culture method. Although CB-NAAT's sensitivity in detecting DR pulmonary TB is significantly higher, the specificity is lower in that population who have received ATT earlier.

Comparison of xpert MTB/RIF assay, line probe assay, and culture in diagnosis of pulmonary tuberculosis on bronchoscopic specimen.

In patients unable to expectorate good quality sputum or with minimal to none sputum production, bronchoscopic specimens may be collected. The objective of the study is to determine the use of Xpert MTB/RIF assay and line probe assay (LPA) in the diagnosis of pulmonary TB (PTB) using specimens collected by bronchoscopy in a tertiary care center. Bronchoscopy specimens received in the TB laboratory were processed by microscopy, Xpert MTB/RIF assay, LPA, and mycobacteria growth indicator tube (MGIT) culture. Results of MGIT culture are considered gold standard. Of the 173 specimens tested, MTB was detected in 48 (27.74%) samples by any of the above methods. Positivity in bronchoalveolar lavage was 31.4% (44/140) and in bronchial wash was 12.1% (4/33). Detection by microscopy, Xpert assay, and culture was 20 (11.56%), 45 (26.01%), and 38 (21.96%), respectively. Culture detected MTB in three additional specimens compared to Xpert assay. Xpert assay detected MTB in 45 (26%) specimens which include 10 specimens which were negative by culture. LPA detected MTB in 18 (90%) out of 20 smear-positive specimens. RIF resistance was detected in 20 (41.7%) specimens by Xpert and/or MGIT culture drug susceptibility testing (DST). Isoniazid (INH) resistance was detected in 19 specimens by LPA and MGIT culture DST. Bronchoscopy can provide alternative respiratory specimens for diagnosing PTB in patients with difficulty to expectorate sputum. The utility of Xpert MTB/RIF as a rapid, sensitive, and specific test should always be supplemented with culture in difficult-to-obtain and precious respiratory specimens. LPA plays an important role in rapid detection of INH monoresistance.

A Systematic Review on the Diagnostic Accuracy of Line Probe Assay in the Rapid Diagnosis of Drug Resistant Tuberculosis in Indian Scenario

Owing to the drastic increase in the number of patients with drug resistant TB around the world, it is important to increase the testing for it. Line probe assay (LPA) is the rapid diagnostic tool to detect drug resistant TB and it was endorsed by WHO for testing first line drugs such as Isoniazid (INH) and Rifampicin (RIF). This systematic review evaluated the accuracy of this LPA by analysing its sensitivity and specificity against the phenotypic drug susceptibility testing (DST) methods like LJ and liquid culture DST. A total of 4774 samples were included in this review from 19 articles. The average sensitivity and specificity for the detection of RIF resistance from 17 articles was 95.79% and 96.71% and for INH resistance it was 89.85% and 97.33% respectively when compared to phenotypic DST. Out of 19 articles included, 2 articles have mentioned the sensitivity and specificity for multi-drug resistant TB (MDR TB) and the average was 98.50 % and 97 % respectively. The accuracy for RIF resistance detection through first line LPA was good and the sensitivity detection for INH was less across the studies. This could be improved further in future generation assays. Our finding supports the use of LPA especially on smear positive specimens but use on smear negative specimens still be considered as studies have shown some interpretable results.

Discordance of the Repeat GeneXpert MTB/RIF Test for Rifampicin Resistance Detection Among Patients Initiating MDR-TB Treatment in Uganda.

BackgroundThe Global Laboratory Initiative (GLI) guidelines recommend repeat for GeneXpertMTB/RIF (XpertMTB/RIF) in patients with a low pretest probability of rifampicin resistance (RR).MethodsThis was a cross-sectional study using results of sputum specimens collected from participants screened for the STREAM 2 trial. We recruited all patients with XpertMTB/RIF RR-TB detected who were referred for RR/multidrug-resistant (MDR) TB treatment initiation at Mulago National Referral Hospital, Kampala, between September 2017 and October 2019. At baseline, smear microscopy, repeat XpertMTB/RIF, Xpert Ultra, and MTBDRplus assays were done on sputum specimens. Culture-based drug susceptibility testing (DST) was performed on discordant specimens. We analyzed the prevalence and factors associated with discordance between initial and repeat XpertMTB/RIF RR and false XpertMTB/RIF RR. False XpertMTB/RIF RR was defined as no RR detected by any of Xpert Ultra, LPA, or culture DST (reference comparator).ResultsA total of 126/130 patients had repeat XpertMTB/RIF results, of whom 97 (77.0%) had M. tuberculosis detected, 81 (83.5%) had RR detected, and 1 (1.0%) had RR indeterminate. The prevalence of discordant XpertMTB/RIF RR was 15/96 (15.6%), whereas false XpertMTB/RIF RR prevalence was 10/96 (10.4%).Low–bacillary load sputum specimens were more likely to have discordant XpertMTB/RIF RR and false XpertMTB/RIF RR results (adjusted odds ratio [aOR], 0.04; 95% CI, 0.00–0.37; P = .01; aOR, 0.02; 95% CI, 0.01–0.35; P = .01, respectively).ConclusionsOur findings show a high false-positive rifampicin resistance rate in low–TB burden patients, which calls for repeat testing in order to prevent unnecessary prescription of anti-MDR-TB therapy.

Detection of a novel mutation in the rpoB gene in a multidrug resistant Mycobacterium tuberculosis isolate using whole genome next generation sequencing

BackgroundMycobacterium tuberculosis (Mtb) drug resistance is a global concern. Moreover, multiple drug resistant (MDR), extensively drug resistant (XDR), and totally drug resistant (TDR) Mtb cases are on the rise in developing countries like India. Most of these cases are identified only 3–6 months after initiation of treatment owing to incomplete/failed clinical response and incomplete information from phenotypic drug resistance assays and/or targeted Mtb mutation analysis. Here, we report the development of an in-house whole genome sequencing (WGS) assay and bioinformatics pipeline that helped resolve the phenotype-genotype discrepancy in a clinical isolate. Methodology and resultsA sample from a suspected drug resistant Mtb case tested by line probe assay (LPA) showed the absence of both the mutant and wild type alleles for an rpoB gene mutation site. An in-house next generation sequencing (NGS) assay was used for WGS of this isolate. Bioinformatics analysis revealed that the isolate harboured a novel insertional mutation in the 81-bp hotspot region of the rpoB gene and a S315T mutation in the katG gene, which could explain resistance to rifampicin and isoniazid, respectively. These results correlated with the clinical diagnosis, LPA, solid culture drug susceptibility testing, and pyrosequencing carried out on the sample. The WGS data also provided information regarding the isolate’s lineage and indicated an absence of known mutations conferring resistance to other antitubercular drugs. ConclusionWGS is a highly sensitive, specific, and unbiased approach for identification of all possible drug resistance-conferring mutations, which can help clinicians make more informed treatment-related decisions.

Evaluation of rapid diagnostic tests and assessment of risk factors in drug-resistant pulmonary tuberculosis.

Background:Early diagnosis and treatment of drug-resistant tuberculosis (TB) is crucial to halt the spread of drug resistance in the community.Aim:The aim of the study was to compare rapid diagnostic tests (GeneXpert and line probe assay, LPA) with conventional liquid culture for the diagnosis of drug-resistant TB and to assess the risk factors for it.Method:This cross-sectional study recruited 229 multidrug-resistant TB suspects who were sputum smear positive. They were evaluated by the rapid diagnostic tests and sensitivity, specificity, positive predictive value and negative predictive value were calculated for drug resistance detection as compared to liquid culture drug susceptibility testing. The risk factors for the development of drug resistance were also assessed and the P value of < 0.05 was considered significant.Results:In the final comparison, 193 samples were included. The sensitivity and specificity of GeneXpert for detection of drug resistance (rifampicin) was 100% (95% confidence interval, CI: 88.8–100%) and 99.4% (95% CI: 96.6–99.9%), respectively. Whereas sensitivity and specificity of LPA was 94.3% (95% CI: 80.8–99.3%) and 100% (95% CI: 97.7–100%), respectively. Only three discordant samples were observed. Defaulting to antitubercular therapy, contact with resistant TB, and disseminated disease were found to be significant risk factors for the development of drug-resistant TB with high statistical significance (P value < 0.05).Conclusion:Both rapid diagnostic tests have very high sensitivity and specificity for detection of drug resistance in sputum smear positive with the advantage of short turn-around time. Defaulting to antitubercular therapy, contact with resistant TB, and disseminated disease are significant risk factors for drug resistance.

Tackling drug-resistant tuberculosis: we need a critical synergy of product and process innovations.

The End TB Strategy diagnostic pillar calls for access to high-sensitivity diagnostic testing and universal rapid drug susceptibility testing (DST). The recommended diagnostic technologies available in low and middle-income, high-burden countries for multidrug-resistant tuberculosis (MDR-TB) are essentially limited to Xpert® MTB/RIF and MTB/RIF Ultra assays, culture DST and the line-probe assays. The primary reasons for slow scale-up are insufficient political will, and therefore, insufficient funding for qualified human resources, and safe laboratory and health system infrastructure. Innovative approaches to enable the private health sector to provide high-quality diagnosis are also needed. The Essential Diagnostics List provides impetus and a standard benchmark for the rational implementation of MDR-TB diagnostics, but the epidemic will ultimately only be favorably impacted by complete end-to-end solutions to patients that address the complete cascade of care, including patient-centered diagnosis and treatment of TB and MDR-TB, management of comorbidities and social protection. By scaling up access to the currently available diagnostics, we lay the groundwork for future innovations for rapid accurate diagnosis of MDR-TB, which in turn will bring us closer to meeting the targets in the End TB Strategy.

Presence of Fluoroquinolone mono-resistance among drug-sensitive Mycobacterium tuberculosis isolates: An alarming trend and implications

BackgroundThe phenomenon of Drug Resistant Tuberculosis (DRTB) has been evolving aggressively and is posing great threat to tuberculosis control programs worldwide. But what really is fueling drug resistance in high disease burden countries apart from other well known risk factors, with this intent in mind the present study was developed and carried out to. The aim was to capture the presence of Fluoroquinolone (FQ) mono-resistance among drug-sensitive TB cases. MethodsA total of 1280 sputum smear-positive patients were enrolled in the study and were subjected for Drug Susceptibility Testing (DST) using First-Line Drugs (FLD's; Rifampicin, Streptomycin, Isoniazid and Ethambutol) using liquid culture DST and Line Probe Assay. These samples were further subjected to second-line drugs DST (ofloxacin and kanamycin) and DNA sequencing to confirm FQ mono-resistance. ResultsThe occurrence of FQ mono-resistance among FLD sensitive cases was found to be 3.2% (35/1099). Xpert MTB/RIF assay and rpoB gene sequencing were showed 100% concordance with FLD DST. A total of 35 FQ mono-resistant isolates were further sequenced for gyrA, gyrB and rrs genes. The gene sequencing was found to be in agreement with the DST results for 34 (3.1%) isolates with gyrA and gyrB genes. ConclusionPresence of FQ resistance among drug sensitive TB cases is a red flag sign. The findings of the present study suggest that, second-line DST should be routinely performed not only for drug-resistant cases but also for drug-sensitive cases so as to capture prevailing true drug resistance at an initial stage.

Has TB CARE I sputum transport improved access to culture services for retreatment tuberculosis patients in Zimbabwe?

Setting: Retreatment tuberculosis (TB) patients in Zimbabwe are investigated using microscopy, Xpert® MTB/RIF and culture + drug susceptibility testing (CDST). TB CARE I, a sputum transport service using motorcycles, was introduced to transport specimens between peripheral health facilities and laboratories, including National Reference Laboratories (NRLs). Objectives: To compare access to CDST and treatment outcomes among retreatment TB patients in facilities with and those without TB CARE I support. Design: This was a retrospective cohort study. Results: There were 187 patients from TB CARE I-supported facilities and 116 from non-TB CARE I facilities, with no difference in demographic characteristics. Altogether, specimens from 22 (12%) retreatment TB patients had successful CDST from TB CARE I facilities, which was not statistically significantly different from non-supported facilities (n = 14, 12%; P = 0.94). The median number of days from sputum collection to receipt at the NRL was lower in TB CARE I facilities than in non-supported facilities (median 6, interquartile range [IQR] 4-8 vs. median 8, IQR 6-13.5; P = 0.000). Favourable treatment outcomes were documented in 65% of patients under TB CARE I, significantly more than among patients in non-supported facilities (47%, P < 0.01). Conclusion: The process of sputum specimen collection for CDST was not different between TB CARE I and non-TB CARE I-supported health facilities, apart from a slightly shorter time. Ways to improve the current system are discussed.

Association of gyrA and rrs gene mutations detected by MTBDRsl V1 on Mycobacterium tuberculosis strains of diverse genetic background from India

There is limited data on the use of Genotype MTBDRslVersion 1 (MTBDRsl V1) as an initial rapid screening test to rule out XDR-TB and most importantly its performance in various genotypes of Mycobacterium tuberculosis is scarcely studied. A total of 359 MDR-TB isolates were tested for gene mutations representing second line drug resistance, using the MTBDRsl_V.1 and the results were compared with phenotypic method (Bactec MGIT-960 system) for second-line drug (SLD) susceptibility testing. Genetic lineages of all these isolates were also determined using spoligotyping and SITVIT2 WEB database. The MTBDRsl V1 detected mutations in the gyrA, rrs, and emb genes in 108 (30%), 2 (0.5%) and 129 (35.9%) isolates, respectively. Remaining 120 (33.4%) had no second line drug (SLD) resistance. In 17 (4.7%) isolates mutations were detected in both gyrA and rrs genes. Its concordance with MGIT-960 culture drug susceptibility testing (DST) was 97% and 94.1%, 93.5%, 60.5% and 50% for the detection of XDR-TB, pre-XDR, Ethambutol, and Aminoglycosides/Cyclopeptides resistance. The Beijing lineage was predominant (46%) between both the pre-XDR/XDR-TB isolates. We conclude that MTBDRsl is useful for rapid detection of SLD resistance. Also in pre-XDR and XDR-TB isolates the frequency of relevant genetic mutations was significantly higher in the Beijing strains.

Utility of GeneXpert in Diagnosis of Multidrug-resistant Extrapulmonary Tuberculosis

ABSTRACT Background: The Xpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) assay (GeneXpert) is a rapid semi-quantitative nucleic acid amplification test with established role in the diagnosis of pulmonary tuberculosis (PTB) and multidrug-resistant (MDR) PTB. We determined the performance of the GeneXpert assay for the diagnosis of extrapulmonary tuberculosis (EPTB) MDR cases. Aims and objectives: To study the role of GeneXpert in the diagnosis of EPTB MDR. Materials and methods: A retrospective study was conducted over a period of 2 years at a tertiary care hospital after Ethics Committee permission. Data of 44 consecutive patients of diagnosed EPTB MDR were retrieved for GeneXpert and culture drug susceptibility test (DST). Sensitivity of GeneXpert in the diagnosis of EPTB MDR was calculated comparing culture DST results. Results: Various EPTB MDR cases studied were lymph node TB (n = 23) 51%, pleural effusion (n = 14) 32%, central nervous system TB/spinal TB/psoas abscess/gluteal abscess (n = 7) 17%. Sensitivity for GeneXpert was found to be 91.30, 57, 100% respectively. True positive and false negative were 36 and 8 cases respectively. The overall sensitivity of GeneXpert in diagnosing EPTB MDR was 81.80%. The sensitivity among lymph node, pleural effusions, and spinal/psoas abscess/gluteal abscess was 91.30, 57.14, and 100% respectively. Conclusion: GeneXpert sensitivity for the diagnosis of EPTB MDR varied with site of extrapulmonary involvement, with lower sensitivity in pleural fluid as compared with higher sensitivity among lymph node and spine TB. Nevertheless, given the rapid turnaround time and simplicity, it is a useful tool in the diagnosis of EPTB MDR when used in correct clinical context. Subsequent confirmation with culture DST, however, is recommended to diagnose false negatives.

Evaluation of Genotype MTBDRplus Line Probe Assay in Detection of Rifampicin and Isoniazid Resistance in Comparison to Solid Culture Drug Susceptibility Testing in a Tertiary Care Centre of Western Uttar Pradesh

Background: Isoniazid (INH) and rifampicin (Rif) are the key first-line antituberculosis drugs, and resistance to these drugs i.e., multi-drug-resistant tuberculosis (MDR-TB), is likely to result in treatment failure and poor clinical outcomes. India has the highest burden of TB and MDR-TB in the world, disproportionately high even for India’s population. The GenoType® MTBDRplus molecular method allows rapid detection of Rif and INH resistance. Aim: The present study was done to compare the performance of line probe assay test (GenoType® MTBDRplus) with solid culture method for an early diagnosis of MDR-TB. Methods: Totally 1503 sputum samples of MDR-TB suspects were subjected to fluorescent microscopy. Decontamination was done by N-acetyl-L-cysteine and sodium hydroxide method. Fluorescent microscopy-positive samples were subjected to GenoType® MTBDRplus (HAIN Lifescience) assay. Sixty-two random samples were compared with phenotypic drug susceptibility testing (DST) (1% proportion method) using solid culture method by Lowenstein–Jensen media. Results: The sensitivity, specificity, positive predictive value and negative predictive value for detection of resistance to Rif were 94.74%, 95.35%, 90% and 97.62% and to INH were 92.00%, 91.89%, 88.46% and 94.44%, respectively, in comparison with the phenotypic DST. Conclusion: GenoType® MTBDRplus has good sensitivity and specificity in detecting MDR-TB cases with a significantly lesser turnaround time as compared to conventional DST method and simultaneous detection of Rif and INH resistance. This technique saves several weeks of time required for culture and DST.

Comparison of Histological, Microbiological, and Molecular Methods in Diagnosis of Patients with TBLN Having Different Anti-TB Treatment Background

ObjectiveThe influence of anti-tuberculosis (TB) treatment history on tuberculous lymphadenitis (TBLN) diagnosis is unclear. Therefore, this study aims to evaluate the diagnostic methods, including histology, microbiology, and molecular tests, used for TBLN. MethodsIn this study, suspected patients with TBLN and having different anti-TB treatment background were enrolled. All the samples were tested simultaneously by histology, Ziehl-Neelsen (ZN) staining, mycobacterial culture (culture), Xpert MTB/RIF (xpert), real-time PCR, and high-resolution melting curve PCR (HRM). Thereafter, the performance of these methods on samples with different anti-TB treatment background was assessed. ResultsIn our study, 89 patients were prospectively included 82 patients with TBLN and 7 with other diseases. The overall sensitivities of Xpert, real-time PCR, histology, ZN staining, and culture were 86.6%, 69.5%, 58.5%, 43.9%, and 22.0%, respectively. The anti-TB treatment history revealed dramatic influences on the sensitivity of culture (P < 0.0001). In fact, the treatment that lasted over 3 months also influenced the sensitivity of Xpert (P < 0.05). However, the treatment history did not affect the performance of remaining tests (P > 0.05). For rifampicin drug susceptibility test (DST), the anti-TB treatment showed only significant influence on the success rate of culture DST (P = 0.001), but not on those of Xpert and HRM tests (P > 0.05). ConclusionOther tests as well as culture should be considered for patients with TBLN having retreatment history or over 1-month treatment to avoid false negative results.

Systematic review, meta-analysis and economic modelling of molecular diagnostic tests for antibiotic resistance in tuberculosis.

Drug-resistant tuberculosis (TB), especially multidrug-resistant (MDR, resistance to rifampicin and isoniazid) disease, is associated with a worse patient outcome. Drug resistance diagnosed using microbiological culture takes days to weeks, as TB bacteria grow slowly. Rapid molecular tests for drug resistance detection (1 day) are commercially available and may promote faster initiation of appropriate treatment. To (1) conduct a systematic review of evidence regarding diagnostic accuracy of molecular genetic tests for drug resistance, (2) conduct a health-economic evaluation of screening and diagnostic strategies, including comparison of alternative models of service provision and assessment of the value of targeting rapid testing at high-risk subgroups, and (3) construct a transmission-dynamic mathematical model that translates the estimates of diagnostic accuracy into estimates of clinical impact. A standardised search strategy identified relevant studies from EMBASE, PubMed, MEDLINE, Bioscience Information Service (BIOSIS), System for Information on Grey Literature in Europe Social Policy & Practice (SIGLE) and Web of Science, published between 1 January 2000 and 15 August 2013. Additional 'grey' sources were included. Quality was assessed using quality assessment of diagnostic accuracy studies version 2 (QUADAS-2). For each diagnostic strategy and population subgroup, a care pathway was constructed to specify which medical treatments and health services that individuals would receive from presentation to the point where they either did or did not complete TB treatment successfully. A total cost was estimated from a health service perspective for each care pathway, and the health impact was estimated in terms of the mean discounted quality-adjusted life-years (QALYs) lost as a result of disease and treatment. Costs and QALYs were both discounted at 3.5% per year. An integrated transmission-dynamic and economic model was used to evaluate the cost-effectiveness of introducing rapid molecular testing (in addition to culture and drug sensitivity testing). Probabilistic sensitivity analysis was performed to evaluate the impact on cost-effectiveness of diagnostic and treatment time delays, diagnosis and treatment costs, and associated QALYs. A total of 8922 titles and abstracts were identified, with 557 papers being potentially eligible. Of these, 56 studies contained sufficient test information for analysis. All three commercial tests performed well when detecting drug resistance in clinical samples, although with evidence of heterogeneity between studies. Pooled sensitivity for GenoType® MTBDRplus (Hain Lifescience, Nehren, Germany) (isoniazid and rifampicin resistance), INNO-LiPA Rif.TB® (Fujirebio Europe, Ghent, Belgium) (rifampicin resistance) and Xpert® MTB/RIF (Cepheid Inc., Sunnyvale, CA, USA) (rifampicin resistance) was 83.4%, 94.6%, 95.4% and 96.8%, respectively; equivalent pooled specificity was 99.6%, 98.2%, 99.7% and 98.4%, respectively. Results of the transmission model suggest that all of the rapid assays considered here, if added to the current diagnostic pathway, would be cost-saving and achieve a reduction in expected QALY loss compared with current practice. GenoType MTBDRplus appeared to be the most cost-effective of the rapid tests in the South Asian population, although results were similar for GeneXpert. In all other scenarios GeneXpert appeared to be the most cost-effective strategy. Rapid molecular tests for rifampicin and isoniazid resistance were sensitive and specific. They may also be cost-effective when added to culture drug susceptibility testing in the UK. There is global interest in point-of-care testing and further work is needed to review the performance of emerging tests and the wider health-economic impact of decentralised testing in clinics and primary care, as well as non-health-care settings, such as shelters and prisons. This study is registered as PROSPERO CRD42011001537. The National Institute for Health Research Health Technology Assessment programme.

Use of the Xpert® MTB/RIF assay for diagnosing pulmonary tuberculosis comorbidity and multidrug‐resistant TB in obstetrics and gynaecology inpatient wards at the University Teaching Hospital, Lusaka, Zambia

OBJECTIVESIn high-tuberculosis (TB)-endemic countries, comorbidity of pulmonary TB in hospitalised patients with non-communicable diseases is well documented. In this study, we evaluated the use of the Xpert® MTB/RIF assay for the detection of concomitant pulmonary TB in patients admitted to the University Teaching Hospital, Lusaka, Zambia, with a primary obstetric or gynaecological condition.METHODSThe Study population were inpatients admitted with a primary obstetric or gynaecological problem who had a concomitant cough and were able to expectorate a sputum sample. Sputum samples from 94 patients were analysed for the presence of Mycobacterium tuberculosis (M.tb) by standard smear microscopy, MGIT culture, MGIT drug-susceptibility testing (DST) and the Xpert® MTB/RIF assay. The sensitivity and specificity of the Xpert® MTB/RIF assay were evaluated against the culture gold standard.RESULTSTwenty-six of 94 (27.7%) patients had culture-confirmed pulmonary TB. The Xpert® MTB/RIF assay had a sensitivity of 80.8% [95% CI: 60.0–92.7%]) compared against MGIT culture. The Xpert® MTB/RIF assay was more sensitive than sputum smear microscopy (21/26 (80.8%) vs. 13/26 (50.0%), P = 0.02) and detected an additional eight culture-confirmed cases. Culture DST analysis identified two monoresistant M.tb strains: one resistant to rifampicin (rifampicin sensitive by the Xpert® MTB/RIF assay) and one to ethambutol. HIV infection was linked with a 3-fold increase in risk of TB, accounting for 87.5% (21/24) of TB cases. 50% of cases presented as comorbidities with other communicable diseases (CDs) and non-communicable diseases (NCDs).CONCLUSIONSAs an alternative to sputum microscopy, the Xpert® MTB/RIF assay provides a sensitive, specific and rapid method for the diagnosis of pulmonary TB in obstetric or gynaecological inpatients. Pulmonary TB is an important cause of concomitant comorbidity to the obstetric or gynaecological condition necessitating admission. TB and HIV comorbidities with other communicable and non-communicable diseases were also common. More proactive screening for TB comorbidity is required in obstetric and gynaecological wards.

New and improved diagnostics for detection of drug-resistant pulmonary tuberculosis

Tuberculosis (TB) remains a global emergency and continues to kill 1.7 million people globally each year. Drug-resistant TB is now well established throughout the world and most TB patients are not being screened for drug resistance due to lack of laboratory resources and rapid accurate point-of-care tests. Accurate and rapid diagnosis of TB and drug-resistant TB is of paramount importance in establishing appropriate clinical management and infection control measures. During the past decade, there have been significant advances in diagnostic technologies for TB and drug-resistant TB. The purpose of this article is to review the current data, recommendations and evidence base for these tests. Second-line drug susceptibility testing (DST) is complex and expensive. Automated liquid culture systems and molecular line probe assays are recommended by the WHO as the current 'gold standard' for first-line DST. Liquid culture DST for aminoglycosides, polypeptides and fluoroquinolones has been shown to have relatively good reliability and reproducibility for diagnosis of extensively drug-resistant TB; however, DST for other second-line drugs (ethionamide, prothionamide, cycloserine, terizidone, para-aminosalicylic acid, clofazimine, amoxicillin-clavulanate, clarithromycin, linezolid) is not recommended. Automated liquid culture systems are currently recommended by the WHO as the 'gold standard' for second-line DST. In this review, we describe the phenotypic and genotypic methods currently available for the diagnosis of TB and drug-resistant forms of Mycobacterium tuberculosis and discuss future prospects for TB diagnostics. Current technologies for the detection of drug resistant M. tuberculosis vary greatly in terms of turnaround time, cost and complexity. Ultimately, the 'holy grail' diagnostic for TB must fulfil all technical specifications for a good point-of-care test, screen for drug resistance concurrently and be adaptable to the various health system levels and to countries with diverse economic status and TB burden.

Microscopic-observation drug susceptibility and thin layer agar assays for the detection of drug resistant tuberculosis: a systematic review and meta-analysis

Simple, rapid, and affordable tests are needed to detect drug resistance in Mycobacterium tuberculosis. We did a systematic review and meta-analysis to investigate the accuracy of microscopic-observation drug susceptibility (MODS) and thin layer agar (TLA) assays for rapid screening of patients at risk of drug-resistant tuberculosis. In accordance with protocols and methods recommended by the Cochrane Diagnostic Test Accuracy Working Group, we systematically searched PubMed, Embase, and Biosis for reports published between January, 1990, and February, 2009. We included studies investigating detection of drug resistance in M tuberculosis with the MODS or TLA assay, and in which an accepted reference standard was used. Data extracted from the studies were combined by use of bivariate random-effects regression models and hierarchical summary receiver operating characteristic curves to estimate sensitivity and specificity for detection of resistance to specific drugs. We identified 12 studies, of which nine investigated the MODS assay and three investigated the TLA assay. For the MODS assay of rifampicin resistance, pooled estimates were 98·0% (95% CI 94·5-99·3) for sensitivity and 99·4% (95·7-99·9) for specificity. For the MODS assay of isoniazid resistance with a 0·1 μg/mL cutoff, pooled sensitivity was 97·7% (94·4-99·1) and pooled specificity was 95·8% (88·1-98·6), but with a 0·4 μg/mL cutoff, sensitivity decreased to 90·0% (84·5-93·7) and specificity increased to 98·6% (96·9-99·4). All assessments of rifampicin and isoniazid resistance with the TLA assay yielded 100% accuracy. Mean turnaround time was 9·9 days (95% CI 4·1-15·8) for the MODS assay and 11·1 days (10·1-12·0) for the TLA assay. MODS and TLA assays are inexpensive, rapid alternatives to conventional methods for drug susceptibility testing of M tuberculosis. Our data and expert opinion informed WHO's recommendation for use of selected non-commercial drug susceptibility tests, including MODS, as an interim solution until capacity for genotypic or automated liquid culture drug susceptibility testing is developed. Stop TB Department of WHO.