An alternative colorimetric RT-LAMP assay for the rapid detection of SARS-CoV-2: development and validation in Thailand.

INTRODUCTION: COVID-19, an emerging infectious disease caused by SARS-CoV-2, continues to be a global public health threat. The development of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) can extend the availability of simple, reliable molecular tests for the rapid detection of COVID-19. METHODOLOGY: The RT-LAMP assay was developed using a new primer set targeting a portion of SARS-CoV-2 orf8. The method was validated at 63 ºC for 60 minutes with naked-eye visualization of the color change. The clinical performance was compared to a real-time reverse transcription-polymerase chain reaction (rtRT-PCR) using 273 RNA samples extracted from nasopharyngeal swab specimens. RESULTS: The developed RT-LAMP was specific to SARS-CoV-2 with a limit of detection at 15 RNA copies per reaction. The assay demonstrated diagnostic accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of 90.48% (95% CI: 86.36-93.68%), 87.00% (95% CI: 81.53-91.33%), 100% (95% CI: 95.07-100%), 100% (95% CI: not available), and 73.74% (95% CI: 66.22-80.07%), respectively, compared to the rtRT-PCR. The greatest sensitivity of 98.03% (95% CI: 94.34-99.59%) was demonstrated in samples with the cycle threshold (Ct) values < 30 cycles. CONCLUSIONS: The RT-LAMP method in this study showed good performance. The assay can increase the scope of laboratory testing for rapidly detecting SARS-CoV-2 in Thailand. Due to a decrease in COVID-19 cases, its application is beneficial when commercial alternatives are unavailable.

Prevalence, Risk Factors, and Result Features in the Detection of Latent Tuberculosis Infection in Thai Healthcare Workers Using QuantiFERON-TB Gold Plus.

Introduction Latent tuberculosis infection (LTBI) is an enormous reservoir for tuberculosis (TB), and healthcare workers (HCWs) are at high risk for TB infection. QuantiFERON-TB Gold Plus (QFT-Plus) is an alternative to the tuberculin skin test for LTBI detection, but data on its application and LTBI detected by QFT-Plus in high TB burden countries are limited. This study aimed to determine the prevalence of LTBI and its risk factors, and to investigate the QFT-Plus results in Thai HCWs. Methods A cross-sectional analytical study was conducted among HCWs at a secondary care hospital in Health Region 5, Thailand. Eligible HCWs were enrolled and underwent QFT-Plus testing. Interferon-gamma (IFN-γ) values in tubes were analysed. The prevalence and associated risk factors for LTBI were assessed based on laboratory and sociodemographic data. Logistic regression analyses were applied to calculate odds ratios (OR, aOR) reported with 95% confidence intervals (CI). Results Of the 269 participants enrolled, their median age was 42 years and 93.31% (n = 251/269) were female. The majority (n = 178/269, 66.17%) were nurses or nurse assistants and 42.75% (n = 115/269) worked in the inpatient medical wards. Overall, the QFT-Plus results showed 110 (40.89%) positive with good agreement (93.68%; κ 0.87) and high correlation (Spearman's ρ 0.91) of IFN-γ concentrations in the two antigen tubes. A true difference in IFN-γ values for predicting a recent infection was found about 7.81% (n = 21/269). By univariate and multivariate analyses, the participants' age > 40 years (OR = 3.21, 95% CI: 1.84-5.64%; aOR = 2.05, 95% CI: 1.07-3.96%), and employment duration > 10 years (OR = 3.19, 95% CI: 1.66-6.37%; aOR = 2.34, 95% CI: 1.05-5.21%) were significantly associated with the increased risk of LTBI (p-value < 0.05). Conclusions The prevalence of LTBI among these HCWs was high, and the increased risk factors for LTBI according to QFT-Plus positivity were age over 40 years and working time in the hospital for more than 10 years. It is important to screen HCWs in this setting for LTBI, particularly those with long employment durations and older ages. The high prevalence of LTBI suggests that LTBI management, such as regular screening and treatment, should be considered together with strengthening preventive measures, especially in high-risk groups.

Clinical Performance of the Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for the Diagnosis of COVID-19 in a Thai Community Hospital at the Thailand-Myanmar Border.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) continues to be a global health threat and is a public health issue in Thailand and other countries. The extensive cross-border between Thailand and Myanmar is considered to be at a potentially high risk for COVID-19 distribution in this region. In this instance, simple and cost-effective tests for rapid and early detection of COVID-19 would be useful for effective patient management and control of the disease. METHODS: This study was conducted at Mae Sot Hospital on the border of Thailand-Myanmar to evaluate the diagnostic performance of a simple colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay developed recently for the rapid detection of SARS-CoV-2. Nasopharyngeal specimens were routinely collected and processed through automated nucleic acid extraction followed by real-time reverse transcription-polymerase chain reaction (rRT-PCR) using the Molaccu® COVID-19 Detection Kit. The RT-LAMP assay was further performed on remnant RNA samples, and the visual results were compared to those of rRT-PCR as a reference. RESULTS: Of the 727 samples tested, the RT-LAMP assay could detect 322 out of 374 samples positive for SARS-CoV-2 by rRT-PCR with 100% (n = 353/353) negative agreement. The comparative analysis demonstrated the overall accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP at 92.85% (n = 675/727, 95% CI: 90.73-94.61), 86.10% (n = 322/374, 95% CI: 82.17-89.44), 100% (n = 353/353, 95% CI: 98.96-100), 100% (n = 322/322, 95% CI: 98.86-100), and 87.16% (n = 353/405, 95% CI: 84.06-89.73), respectively. CONCLUSION: This RT-LAMP assay showed good diagnostic performance in the hospital setting. It can increase laboratory capacity for rapid SARS-CoV-2 testing and has the potential for use as an alternative or a backup assay at the point of need, especially where alternatives are unavailable for any reason, such as a decline in COVID-19 cases.

QuantiFERON-TB Gold Plus and QuantiFERON-TB Gold In-tube assays for detecting latent tuberculosis infection in Thai healthcare workers.

Detecting latent tuberculosis infection (LTBI) is important, especially in high-risk populations including healthcare workers (HCWs). QuantiFERON-TB Gold Plus (QFT-Plus) is a new version of the interferon-gamma release assays (IGRAs) to replace the QuantiFERON-TB Gold In-tube (QFT-GIT). However, data on the use of QFT-Plus for LTBI detection in high TB-burden countries are limited. This study was conducted in a TB-endemic setting in Thailand. HCWs were enrolled in the study and underwent both tests during the annual health screening. The testing results were compared and the concordance was determined. Of 102 HCWs, 11 (10.78%) were positive according to both tests, and 15 (14.71%) were positive according to QFT-Plus. The overall agreement between assays was 96.08%, with Cohen's kappa coefficient (k) at 0.82. All four discordant results occurred with QFT-GIT negative and QFT-Plus positive. The comparison between QFT-GIT and QFT-Plus based on each antigen tube (TB1 or TB2) exhibited similar concordance with 99.02% and 95.10% agreement, respectively. The intra-comparison between TB1 and TB2 of QFT-Plus also showed good concordance at 96.08%. Among this group of HCWs, the LTBI prevalence of any positive results in both tests was low. Overall, the study showed good agreement between QFT-Plus and QFT-GIT (k = 0.82) with a minimal difference, suggesting similar assay performance to that mainly carried out in TB-low incidence countries. The results support the use of QFT-Plus for detecting LTBI in a format similar to QFT-GIT.

Molecular Characterization of Mutations in Isoniazid- and Rifampicin-Resistant Mycobacterium tuberculosis Isolated in Thailand.

The control of drug-resistant tuberculosis (TB) is a major challenge. The frequency and mutation characteristics indicate the efficiency of molecular tests for the rapid detection of TB drug resistance. This study examined the existence of katG and inhA mutations for isoniazid (INH) resistance and rpoB mutations for rifampicin (RFP) resistance. In total, 178 drug-resistant Mycobacterium tuberculosis (MTB) isolates were analyzed. Mutations in katG encoding and inhA regulatory regions were detected in 136/168 (81.0%) and 29/168 (17.3%), respectively, with the most prominent mutation of Ser315Thr substitution in katG in 126/168 (75.0%), and -15 C to T substitution in the regulatory region of the inhA (26/168; 15.5%). Two distinct katG mutations (Tyr337Cys, 1003InsG) were identified. Of 125 RFP-resistant isolates, 118 (94.4%) carried mutations affecting the 81-bp RFP resistance-determining region, with the most commonly affected codons 450, 445, and 435 identified in 74 (59.2%), 26 (20.8%), and 12 (9.6%) isolates, respectively. Genetic mutations were highly associated with phenotypic INH and RFP resistance, and the majority shared similarities with those reported in previous studies in Thailand and other Asian countries. These data are useful for guiding the use and improvement of molecular tests for TB drug resistance.

QuantiFERON-TB Gold Plus and QuantiFERON-TB Gold In-tube assays for detecting latent tuberculosis infection in Thai healthcare workers

ABSTRACT Detecting latent tuberculosis infection (LTBI) is important, especially in high-risk populations including healthcare workers (HCWs). QuantiFERON-TB Gold Plus (QFT-Plus) is a new version of the interferon-gamma release assays (IGRAs) to replace the QuantiFERON-TB Gold In-tube (QFT-GIT). However, data on the use of QFT-Plus for LTBI detection in high TB-burden countries are limited. This study was conducted in a TB-endemic setting in Thailand. HCWs were enrolled in the study and underwent both tests during the annual health screening. The testing results were compared and the concordance was determined. Of 102 HCWs, 11 (10.78%) were positive according to both tests, and 15 (14.71%) were positive according to QFT-Plus. The overall agreement between assays was 96.08%, with Cohen's kappa coefficient (k) at 0.82. All four discordant results occurred with QFT-GIT negative and QFT-Plus positive. The comparison between QFT-GIT and QFT-Plus based on each antigen tube (TB1 or TB2) exhibited similar concordance with 99.02% and 95.10% agreement, respectively. The intra-comparison between TB1 and TB2 of QFT-Plus also showed good concordance at 96.08%. Among this group of HCWs, the LTBI prevalence of any positive results in both tests was low. Overall, the study showed good agreement between QFT-Plus and QFT-GIT (k = 0.82) with a minimal difference, suggesting similar assay performance to that mainly carried out in TB-low incidence countries. The results support the use of QFT-Plus for detecting LTBI in a format similar to QFT-GIT.

Evaluation of an in-house loop-mediated isothermal amplification for Mycobacterium tuberculosis detection in a remote reference laboratory, Thailand.

Loop-mediated isothermal amplification (LAMP) is a simple and efficient nucleic acid amplification method for the rapid diagnosis of infectious diseases. This study assessed the performance of an in-house LAMP for tuberculosis (TB) diagnosis at a remote reference laboratory in the endemic setting of Thailand. As part of the routine service, 1,882 sputum samples were processed for mycobacterial culture in Lowenstein-Jensen and MGIT media. The DNA was extracted from the remaining decontaminated samples after the culture procedure for real-time polymerase chain reaction (PCR) analysis using Anyplex plus MTB/NTM detection kit. 785 (40.28%) were positive by mycobacterial culture. Of these, 222 DNA remnants were available and subjected to LAMP analysis. Based on culture as reference (Mycobacterium tuberculosis; MTB= 209/ non-tuberculous mycobacteria; NTM= 13), the overall sensitivity of LAMP and Anyplex plus assays for MTB detection were 89.95% (188/209; 95% confidential interval [CI]: 85.05-93.67%) and 96.65% (202/209; 95% CI: 93.22-98.64%), and the accuracy values were 88.74% (197/222; 95% CI: 83.83-92.58) and 96.40% (214/222; 93.02-98.43%), respectively. The sensitivity and accuracy of the in-house LAMP and the Anyplex plus real-time PCR assay were high in comparison to culture results. The high sensitivity and accuracy suggested that this in-house LAMP was promising and might be useful for early TB diagnosis.

Evaluation of an in-house loop-mediated isothermal amplification for Mycobacterium tuberculosis detection in a remote reference laboratory, Thailand

ABSTRACT Loop-mediated isothermal amplification (LAMP) is a simple and efficient nucleic acid amplification method for the rapid diagnosis of infectious diseases. This study assessed the performance of an in-house LAMP for tuberculosis (TB) diagnosis at a remote reference laboratory in the endemic setting of Thailand. As part of the routine service, 1,882 sputum samples were processed for mycobacterial culture in Lowenstein-Jensen and MGIT media. The DNA was extracted from the remaining decontaminated samples after the culture procedure for real-time polymerase chain reaction (PCR) analysis using Anyplex plus MTB/NTM detection kit. 785 (40.28%) were positive by mycobacterial culture. Of these, 222 DNA remnants were available and subjected to LAMP analysis. Based on culture as reference (Mycobacterium tuberculosis; MTB= 209/ non-tuberculous mycobacteria; NTM= 13), the overall sensitivity of LAMP and Anyplex plus assays for MTB detection were 89.95% (188/209; 95% confidential interval [CI]: 85.05-93.67%) and 96.65% (202/209; 95% CI: 93.22-98.64%), and the accuracy values were 88.74% (197/222; 95% CI: 83.83-92.58) and 96.40% (214/222; 93.02-98.43%), respectively. The sensitivity and accuracy of the in-house LAMP and the Anyplex plus real-time PCR assay were high in comparison to culture results. The high sensitivity and accuracy suggested that this in-house LAMP was promising and might be useful for early TB diagnosis.

Putative extensive and pre-extensive drug resistant-tuberculosis associated with unusual genotypes on the Thailand-Myanmar border.

Extensive drug-resistant tuberculosis (XDR-TB) is highly life threatening and its diagnosis is usually difficult and time-consuming. Here we present the first two cases of XDR and pre-XDR-TB diagnosed in 2018 on the Thailand-Myanmar border, more specifically in Tak province. Rapid detection of XDR-TB was performed by loop-mediated isothermal amplification (LAMP), Xpert MTB/RIF, and line probe assays. Mutation analyses targeting rpoB, katG, inhA, gyrA and rrs genes showed an association with drug-resistant phenotypes, except for rifampicin resistance. Spoligotyping revealed uncommon Beijing and T2 genotypes and the analysis of M. tuberculosis interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) showed the presence of more polymorphisms. This report highlights the importance of the early detection of drug-resistant tuberculosis by molecular tests followed by phenotyping assays. Based on the up-to-date definition of XDR- and pre-XDR-TB, the susceptibility testing for bedaquiline and linezolid is required and the two reported cases may correspond to putative XDR-TB.

T-SPOT®.TB test and clinical risk scoring for diagnosis of latent tuberculosis infection among Thai healthcare workers.

BACKGROUND: Screening for latent tuberculosis infection (LTBI) is important to identify healthcare workers (HCWs) benefiting from preventive therapy. Interferon-gamma release assays (IGRAs) are sensitive and specific tests for LTBI diagnosis. However, in settings where IGRAs are not available, clinical risk assessment may be used as an alternative to diagnose LTBI. METHODS: A cross-sectional study was conducted among HCWs of a tertiary-care university hospital in Thailand. All HCWs underwent T-SPOT®.TB test (T-SPOT) and assessment of LTBI clinical risks. Clinical risks associated with T-SPOT positivity were determined by multivariable logistic regression analysis and were given scores accordingly. The performance of the clinical risk scoring was evaluated in comparison to T-SPOT. RESULTS: Among 140 enrolled HCWs, 125 (89%) were females, the median age was 27 years and 23 (16%) had T-SPOT positivity. Independent factors associated with T-SPOT positivity were age ≥30 years (adjusted odds ratio [aOR] 3.95; P = 0.002), working duration ≥60 months (aOR 3.75, P = 0.004) and frequency of TB contact ≥6 times (aOR 8.83, P = 0.005). The study's clinical risk scoring had the area under the curve by receiver operating curve analysis of 0.76 (P < 0.001) using T-SPOT positivity as a reference standard. The score of ≥3 had the best performance in diagnosing LTBI with sensitivity, specificity, positive predictive value and negative predictive value of 70%, 71%, 32% and 92%, respectively. CONCLUSIONS: In this setting where LTBI was prevalent among HCWs but IGRAs are not widely available, the clinical risk scoring may be used as an alternative to diagnose LTBI in HCWs.

Pragmatic accuracy of an in-house loop-mediated isothermal amplification (LAMP) for diagnosis of pulmonary tuberculosis in a Thai community hospital.

BACKGROUND: To improve the quality of diagnosing pulmonary tuberculosis (TB), WHO recommends the use of rapid molecular testing as an alternative to conventional microscopic methods. Loop-mediated isothermal amplification assay (LAMP test) is a practical and cost-effective nucleic amplification technique. We evaluated the pragmatic accuracy of an in-house LAMP assay for the diagnosis of TB in a remote health care setting where an advanced rapid molecular test is not available. METHODS: A prospective diagnostic accuracy study was conducted. Patients with clinical symptoms suggestive of TB were consecutively enrolled from April to August 2016. Sputum samples were collected from each patient and were sent for microscopic examination (both acid-fast stain and fluorescence stain), in-house LAMP test, and TB culture. RESULTS: One hundred and seven patients with TB symptoms were used in the final analysis. This included 50 (46.7%) culture-positive TB patients and 57 (53.3%) culture-negative patients. The overall sensitivity of the in-house LAMP based on culture positivity was 88.8% (95/107) with a 95%CI of 81.2-94.1. The sensitivity was 90.9% (40/44) with a 95%CI of 78.3-97.5 for smear-positive, culture-positive patients, and was 16.7% (1/6) with a 95%CI of 0.4-64.1 for smear-negative, culture-positive patients. The overall sensitivity of the in-house LAMP test compared to smear microscopy methods were not significantly different (p = 0.375). The specificity of the in-house LAMP based on non-TB patients (smear-negative, culture-negative) was 94.7% (54/57) with a 95%CI of 85.4-98.9. CONCLUSIONS: The diagnostic accuracy of the in-house LAMP test in a community hospital was comparable to other previous reports in terms of specificity. The sensitivity of the in-house assay could be improved with better sputum processing and DNA extraction method.

The performance of an in-house loop-mediated isothermal amplification for the rapid detection of Mycobacterium tuberculosis in sputum samples in comparison with Xpert MTB/RIF, microscopy and culture.

Simple, low-cost and effective diagnostic tests for tuberculosis (TB) are needed especially in TB-high burden settings. The present study evaluated the performance of an in-house loop-mediated isothermal amplification (LAMP) for diagnosing TB by comparing it to Xpert MTB/RIF, microscopy and culture. In Thailand, a total of 204 excess sputum samples volume after the processing of cultures were used for Mycobacterium tuberculosis (MTB) detection by Xpert MTB/RIF and LAMP. Based on culture results as the gold standard, the overall sensitivity of LAMP and Xpert MTB/RIF were 82.1% (126/153; 95% confidential interval [CI]: 75.4-88.98%) and 86.9 % (133/153; 95% CI: 80.5-90.8%) respectively, and the specificity of both tests was 100% (51/51; 95% CI: 93.0-100.0%). In comparison with Xpert MTB/RIF, the sensitivity and specificity of LAMP were 94.7% (126/133; 95% CI: 89.5-97.9%), and 100.0% (73/73; 95% CI: 94.9-100.0%), respectively. The average threshold cycle (Ct) of Xpert MTB/RIF detection for positive and negative LAMP results was statistically different, of 18.4 and 27.0, respectively (p < 0.05). In comparison with the acid-fast staining technique, and analyzing LAMP and Xpert MTB/RIF in smear-negative/culture-positive specimens, there was an increase of the detection rate by 47.7% (21/44) and 54.6% (24/44). The diagnostic sensitivity and specificity of LAMP appeared to be comparable to those of Xpert MTB/RIF. We claim that this LAMP has potential to provide a sensitive diagnostic test for the rapid TB diagnosis. It allowed a fast detection of MTB before the cultures and it could be used in resource-limited laboratory settings.

Comparison of Loop-Mediated Isothermal Amplification, Microscopy, Culture, and PCR for Diagnosis of Pulmonary Tuberculosis.

The diagnosis of tuberculosis (TB) in endemic countries is challenging due to high caseloads and limited resources. A simple and cost-effective diagnostic test for the rapid detection of Mycobacterium tuberculosis (M. tuberculosis) in clinical specimens is crucially needed. We evaluated the performance of an in-house assay based on loop-mediated isothermal amplification (LAMP) targeting the M. tuberculosis 16S ribosomal RNA (rRNA) gene for the diagnosis of TB in Thailand. A total of 252 sputum samples from suspected cases of pulmonary TB were analyzed. The sensitivity of LAMP was 99.04% (103/104; 95% confidence interval [CI]: 94.76-9.98%) and 72.73% (16/22; 95% CI: 49.78-89.27%) for smear-positive and smear-negative samples with TB-culture positivity, respectively. LAMP detected 20.69% (24/116) of TB culture negative samples but all those were positive by conventional polymerase chain reaction (PCR). The sensitivity of LAMP was higher than that of sputum microscopy while the performance of LAMP was similar to PCR. None of the samples positive for non-tuberculous mycobacteria by culture and PCR were positive by LAMP. Compared to TB culture, the positive predictive value (PPV), negative predictive value (NPV), and kappa coefficient of LAMP were 83.22%, 88.33%, and 0.75 respectively. Based on the diagnostic performance, we propose that LAMP would be suitable as a potential diagnostic test for rapid TB diagnosis in resource-limited laboratory settings.

Loop-Mediated Isothermal Amplification for Rapid Identification of Mycobacterium tuberculosis in Comparison with Immunochromatographic SD Bioline MPT64 Rapid® in a High Burden Setting.

Loop-mediated isothermal amplification (LAMP) was assessed for rapid identification of Mycobacterium tuberculosis complex (MTC) in comparison with an immunochromatographic test (ICT) using SD Bioline Ag MPT64 Rapid®. One hundred and fifty-one MGIT cultures positive for acid-fast bacilli were tested for MTC. DNA was extracted from a small portion of culture samples by heat lysis and subjected to LAMP analysis. Of these, 144 were positive and 5 were negative by both tests. One culture that was ICT negative but was LAMP positive was confirmed to have a mutation in the mpt64 gene. The agreement was 98.68% (95% confidence interval [CI]: 94.80-99.77), and the kappa value was 0.83% (95% CI: 0.59-1.00). Good correlation results suggested that LAMP assay is a reliable molecular test for rapid identification of MTC and is practical for use in resource-limited, high burden settings.

QuantiFERON-TB Gold In-Tube test in active tuberculosis patients and healthy adults.

Interferon-gamma (IFN-γ) release assays have improved latent tuberculosis (TB) detection and have been considered promising for the diagnosis of TB disease. However, diagnosis efficacy data is limited in high burden countries. The aim of this study was to determine the diagnostic potential of the QuantiFERON-TB Gold In-Tube (QFT-GIT) test for the diagnosis of active TB in an endemic setting for TB. A cross-sectional study was conducted in a group of 102 Thai patients with clinical symptoms and chest x-ray findings suggesting of active pulmonary TB and a group of 112 healthy adults. Testing was carried out using sputum microscopy, mycobacterial culture and QFT-GIT test. Of these patients, QFT-GIT was positive in 73 (71.57%), negative in 27 (26.47%), and undetermined in 2 (1.96%) cases. Among healthy controls, QFT-GIT was positive in 18 (16.07%), negative in 93 (83.04%), and undetermined in 1 (0.89%) person. Based on TB culture results, the sensitivity of QFTGIT for diagnosing active TB was 84.21% (95% confidence interval (CI); 72.13-92.52). The positive and negative predictive values were 65.75% (95% CI; 59.26-71.70) and 66.67% (95% CI; 49.94-80.04), respectively. The median IFN-γ level in culture-confirmed TB patients was 3.91 compared to 0.03 IU/mL of the healthy group. QFT-GIT appears to be a useful indirect test for TB diagnosis in Thailand and its use is recommended in association with clinical and radiological assessments for identifying active or latent TB.

Comparison and development of pyrazinamide susceptibility testing methods for tuberculosis in Thailand.

Pyrazinamide (PZA) plays a critical role in shortening tuberculosis treatment duration and in treating multi-drug resistant tuberculosis (MDR-TB). The standard phenotypic MGIT PZA susceptibility testing method is imperfect because it is slow and has potential for false resistance. In this study, we evaluated 2 different phenotypic-based methods, quantitative real-time PCR (qPCR) phage assay, and MTT assay, as well as genotypic sequencing. The assay was evaluated on 71 clinical Mycobacterium tuberculosis isolates (37 MGIT PZA susceptible and 34 MGIT PZA resistant) and compared to the MGIT result. Of these methods, the qPCR phage assay yielded an accuracy of 89% versus standard MGIT while MTT yielded 83%. The genotypic sequencing method yielded 90% accuracy. We conclude that any of these faster PZA susceptibility methods perform reasonably well against a MGIT PZA susceptibility standard.

Use of mycobacteriophage quantitative PCR on MGIT broths for a rapid tuberculosis antibiogram.

Phenotypic culture-based drug susceptibility testing (DST) for Mycobacterium tuberculosis is a valuable tool to identify four to six active drugs for individualized multidrug-resistant (MDR) tuberculosis (TB) regimens. Current culture-based methods are slow, however; therefore, we evaluated a rapid mycobacteriophage-based quantitative PCR (qPCR) assay for use directly on M. tuberculosis-positive MGIT broths. We compared phage qPCRs, using a simple cutoff of 3 for the ΔCq value (where Cq is quantification cycle, and ΔCq is calculated as the Cq of starting phage minus the Cq of TB isolates in drug-containing medium), on 325 clinical M. tuberculosis MGIT broth cultures versus the respective subcultured isolates tested by agar proportion. The median accuracy for the 13 drugs/concentrations tested was 98%, with most discrepancies being false-resistant results. Evaluation of phage qPCR on greater numbers of resistant strains of 393 isolates grown on Löwenstein-Jensen medium showed similar findings, with a median accuracy, sensitivity, and specificity of 97%, 90%, and 99%, respectively. This rapid culture-based DST methodology can be performed for any drug on TB-positive MGIT broths, with a specimen-to-antibiogram turnaround time of approximately 23.9 days, compared with waiting 58.6 days for isolate growth on solid medium followed by agar proportion DST.