Diagnostic Accuracy and Turnaround Time of the Xpert MTB/RIF Assay in Routine Clinical Practice

The Xpert MTB/RIF assay was introduced for timely and accurate detection of tuberculosis (TB). The aim of this study was to determine the diagnostic accuracy and turnaround time (TAT) of Xpert MTB/RIF assay in clinical practice in South Korea. We retrospectively reviewed the medical records of patients in whom Xpert MTB/RIF assay using sputum were requested. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of pulmonary tuberculosis (PTB) and detection of rifampicin resistance were calculated. In addition, TAT of Xpert MTB/RIF assay was compared with those of other tests. Total 681 patients in whom Xpert MTB/RIF assay was requested were included in the analysis. The sensitivity, specificity, PPV and NPV of Xpert MTB/RIF assay for diagnosis of PTB were 79.5% (124/156), 100.0% (505/505), 100.0% (124/124) and 94.0% (505/537), respectively. Those for the detection of rifampicin resistance were 57.1% (8/14), 100.0% (113/113), 100.0% (8/8) and 94.9% (113/119), respectively. The median TAT of Xpert MTB/RIF assay to the report of results and results confirmed by physicians in outpatient settings were 0 (0–1) and 6 (3–7) days, respectively. Median time to treatment after initial evaluation was 7 (4–9) days in patients with Xpert MTB/RIF assay, but was 21 (7–33.5) days in patients without Xpert MTB/RIF assay. Xpert MTB/RIF assay showed acceptable sensitivity and excellent specificity for the diagnosis of PTB and detection of rifampicin resistance in areas with intermediate TB burden. Additionally, the assay decreased time to the initiation of anti-TB drugs through shorter TAT.     

Enhancing TB Case Detection: Experience in Offering Upfront Xpert MTB/RIF Testing to Pediatric Presumptive TB and DR TB Cases for Early Rapid Diagnosis of Drug Sensitive and Drug Resistant TB

Background

Diagnosis of pulmonary tuberculosis (PTB) in children is challenging due to difficulties in obtaining good quality sputum specimens as well as the paucibacillary nature of disease. Globally a large proportion of pediatric tuberculosis (TB) cases are diagnosed based only on clinical findings. Xpert MTB/RIF, a highly sensitive and specific rapid tool, offers a promising solution in addressing these challenges. This study presents the results from pediatric groups taking part in a large demonstration study wherein Xpert MTB/RIF testing replaced smear microscopy for all presumptive PTB cases in public health facilities across India.

Methods

The study covered a population of 8.8 million across 18 programmatic sub-district level tuberculosis units (TU), with one Xpert MTB/RIF platform established at each study TU. Pediatric presumptive PTB cases (both TB and Drug Resistant TB (DR-TB)) accessing any public health facilities in study area were prospectively enrolled and tested on Xpert MTB/RIF following a standardized diagnostic algorithm.

Results

4,600 pediatric presumptive pulmonary TB cases were enrolled. 590 (12.8%, CI 11.8–13.8) pediatric PTB were diagnosed. Overall 10.4% (CI 9.5–11.2) of presumptive PTB cases had positive results by Xpert MTB/RIF, compared with 4.8% (CI 4.2–5.4) who had smear-positive results. Upfront Xpert MTB/RIF testing of presumptive PTB and presumptive DR-TB cases resulted in diagnosis of 79 and 12 rifampicin resistance cases, respectively. Positive predictive value (PPV) for rifampicin resistance detection was high (98%, CI 90.1–99.9), with no statistically significant variation with respect to past history of treatment.

Conclusion

Upfront access to Xpert MTB/RIF testing in pediatric presumptive PTB cases was associated with a two-fold increase in bacteriologically-confirmed PTB, and increased detection of rifampicin-resistant TB cases under routine operational conditions across India. These results suggest that routine Xpert MTB/RIF testing is a promising solution to present-day challenges in the diagnosis of PTB in pediatric patients.     

Use of Xpert MTB/RIF in Decentralized Public Health Settings and Its Effect on Pulmonary TB and DR-TB Case Finding in India

BackgroundXpert MTB/RIF, the first automated molecular test for tuberculosis, is transforming the diagnostic landscape in high-burden settings. This study assessed the impact of up-front Xpert MTB/RIF testing on detection of pulmonary tuberculosis (PTB) and rifampicin-resistant PTB (DR-TB) cases in India.MethodsThis demonstration study was implemented in 18 sub-district level TB programme units (TUs) in India in diverse geographic and demographic settings covering a population of 8.8 million. A baseline phase in 14 TUs captured programmatic baseline data, and an intervention phase in 18 TUs had Xpert MTB/RIF offered to all presumptive TB patients. We estimated changes in detection of TB and DR-TB, the former using binomial regression models to adjust for clustering and covariates.ResultsIn the 14 study TUs, which participated in both phases, 10,675 and 70,556 presumptive TB patients were enrolled in the baseline and intervention phase, respectively, and 1,532 (14.4%) and 14,299 (20.3%) bacteriologically confirmed PTB cases were detected. The implementation of Xpert MTB/RIF was associated with increases in both notification rates of bacteriologically confirmed TB cases (adjusted incidence rate ratio [aIRR] 1.39; CI 1.18-1.64), and proportion of bacteriological confirmed TB cases among presumptive TB cases (adjusted risk ratio (aRR) 1.33; CI 1.6-1.52). Compared with the baseline strategy of selective drug-susceptibility testing only for PTB cases at high risk of drug-resistant TB, Xpert MTB/RIF implementation increased rifampicin resistant TB case detection by over fivefold. Among, 2765 rifampicin resistance cases detected, 1055 were retested with conventional drug susceptibility testing (DST). Positive predictive value (PPV) of rifampicin resistance detected by Xpert MTB/RIF was 94.7% (CI 91.3-98.1), in comparison to conventional DST.ConclusionIntroduction of Xpert MTB/RIF as initial diagnostic test for TB in public health facilities significantly increased case-notification rates of all bacteriologically confirmed TB by 39% and rifampicin-resistant TB case notification by fivefold.     

Catching the Missing Million: Experiences in Enhancing TB & DR-TB Detection by Providing Upfront Xpert MTB/RIF Testing for People Living with HIV in India

BackgroundA critical challenge in providing TB care to People Living with HIV (PLHIV) is establishing an accurate bacteriological diagnosis. Xpert MTB/RIF, a highly sensitive and specific rapid tool, offers a promising solution in addressing these challenges. This study presents results from PLHIV taking part in a large demonstration study across India wherein upfront Xpert MTB/RIF testing was offered to all presumptive PTB cases in public health facilities.MethodThe study covered a population of 8.8 million across 18 sub-district level tuberculosis units (TU), with one Xpert MTB/RIF platform established at each TU. All HIV-infected patients suspected of TB (both TB and Drug Resistant TB (DR-TB)) accessing public health facilities in study area were prospectively enrolled and provided upfront Xpert MTB/RIF testing.Result2,787 HIV-infected presumptive pulmonary TB cases were enrolled and 867 (31.1%, 95% Confidence Interval (CI) 29.4‒32.8) HIV-infected TB cases were diagnosed under the study. Overall 27.6% (CI 25.9–29.3) of HIV-infected presumptive PTB cases were positive by Xpert MTB/RIF, compared with 12.9% (CI 11.6–14.1) who had positive sputum smears. Upfront Xpert MTB/RIF testing of presumptive PTB and DR-TB cases resulted in diagnosis of 73 (9.5%, CI 7.6‒11.8) and 16 (11.2%, CI 6.7‒17.1) rifampicin resistance cases, respectively. Positive predictive value (PPV) for rifampicin resistance detection was high 97.7% (CI 89.3‒99.8), with no significant difference with or without prior history of TB treatment.ConclusionThe study results strongly demonstrate limitations of using smear microscopy for TB diagnosis in PLHIV, leading to low TB and DR-TB detection which can potentially lead to either delayed or sub-optimal TB treatment. Our findings demonstrate the usefulness and feasibility of addressing this diagnostic gap with upfront of Xpert MTB/RIF testing, leading to overall strengthening of care and support package for PLHIV.     

A dot-immunobinding assay for the laboratory diagnosis of tuberculous meningitis and its comparison with enzyme-linked immunosorbent assay.

In an attempt to establish an alternative to standard bacteriological methods in the laboratory diagnosis of tuberculous meningitis (TBM), a simple dot-immunobinding assay (Dot-Iba) was standardized to detect Mycobacterium tuberculosis antigen 5 and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of patients with TBM. Sensitivity and specificity of Dot-Iba was compared with conventional enzyme-linked immunosorbent assay (ELISA) and standard bacteriological techniques. The Dot-Iba showed excellent correlation with indirect ELISA for the detection of antimycobacterial antibody in CSF and showed 60% sensitivity and 100% specificity in culture-negative patients with TBM. However Dot-Iba was less sensitive for the detection of antigen 5 in CSFs and showed false negative results (60%) in culture-positive patients with TBM.     

Point of Care Tuberculosis Sero-Diagnosis Kit for Wild Animals: Combination of Proteins for Improving the Diagnostic Sensitivity and Specificity

Tuberculosis is a significant problem globally for domestic animals as well as captive and free ranging wild life. Rapid point of care (POC) serology kits are well suited for the diagnosis of TB in wild animals. However, wild animals are invariably exposed to environmental non-pathogenic mycobacterium species with the development of cross reacting antibodies. In the present study, POC TB diagnosis kit was developed using a combination of pathogenic Mycobacteria specific recombinant antigens and purified protein derivatives of pathogenic and non-pathogenic Mycobacteria. To benchmark the TB antibody detection kit, particularly in respect to specificity which could not be determined in wildlife due to the lack of samples from confirmed uninfected animals, we first tested well-characterized sera from 100 M. bovis infected and 100 uninfected cattle. Then we investigated the kit’s performance using sera samples from wildlife, namely Sloth Bears (n = 74), Elephants (n = 9), Cervidae (n = 14), Felidae (n = 21), Cape buffalo (n = 2), Wild bear (n = 1) and Wild dog (n = 1).In cattle, a sensitivity of 81% and a specificity of 90% were obtained. The diagnostic sensitivity of the kit was 94% when the kit was tested using known TB positive sloth bear sera samples. 47.4% of the in-contact sloth bears turned seropositive using the rapid POC TB diagnostic kit. Seropositivity in other wild animals was 25% when the sera samples were tested using the kit. A point of care TB sero-diagnostic kit with the combination of proteins was developed and the kit was validated using the sera samples of wild animals.     

A dot-immunobinding assay for the laboratory diagnosis of tuberculous meningitis and its comparison with enzyme-linked immunosorbent assay

V.V. RADHAKRISHNAN AND A. MATHAI. 1991. In an attempt to establish an alternative to standard bacteriological methods in the laboratory diagnosis of tuberculous meningitis (TBM), a simple dot-immunobinding assay (Dot-Iba) was standardized to detect Mycobacterium tuberculosis antigen 5 and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of patients with TBM. Sensitivity and specificity of Dot-Iba was compared with conventional enzyme-linked immunosorbent assay (ELISA) and standard bacteriological techniques. The Dot-Iba showed excellent correlation with indirect ELISA for the detection of antimycobacterial antibody in CSF and showed 60% sensitivity and 100% specificity in culture-negative patients with TBM. However Dot-Iba was less sensitive for the detection of antigen 5 in CSFs and showed false negative results (60%) in culture-positive patients with TBM.     

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.     

结核分枝杆菌分泌蛋白Ag85B、Hsp16.3和ESAT6在血清学检测中的初步应用

目的：探讨结核分枝杆菌分泌蛋白Hsp16.3、Ag85B以及融合蛋白ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6用于TB病人血清学检测的意义。方法：将已构建的含5种目的基因的表达载体（pProEXHTb-Hsp16.3、pProEXHTa-Ag85B、pProEXHTb-ESAT6-CFP10、pProEXHTa-Ag85B-Hsp16.3、pProEXHTa-Ag85B-ESAT6）,分别转入宿主菌E.coli DH5α中,诱导表达后分别获得Hsp16.3、Ag85B、ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6五种蛋白,采用Ni2＋亲和层析柱进行纯化,并用透析方法进行目的蛋白的复性。将经过复性的5种蛋白分别作为抗原,采用间接ELISA方法检测待测的血清样本,经OPD显色,测定各孔OD490值并判定结果。结果：五种蛋白被成功纯化并复性,通过ELISA方法共检测了22例TB病人血清、10例非结核病人血清和6例正常对照血清,Hsp16.3、Ag85B、ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6这5种抗原的灵敏度分别为36.4%、90.9%、77.3%、95.5%、100%,特异性分别为100%、75%、100%、93.8%、93.8%。统计分析显示,ESAT6-CFP10和Ag85B、Ag85B-Hsp16.3、Ag85B-ESAT6这三种蛋白ELISA检测的结果无差异,而与Hsp16.3和痰涂片检测结果有显著差异。结论：Ag85B-Hsp16.3和Ag85B-ESAT6可作为结核分枝杆菌ELISA检测的初选抗原。     

Evaluating the Diagnostic Accuracy of Xpert MTB/RIF Assay in Pulmonary Tuberculosis

Pulmonary tuberculosis still remains a major communicable disease worldwide. In 2013, 9 million people developed TB and 1.5 million people died from the disease. India constitutes 24% of the total TB burden. Early detection of TB cases is the key to successful treatment and reduction of disease transmission. Xpert MTB/RIF, an automated cartridge-based molecular technique detects Mycobacterium tuberculosis and rifampicin resistance within two hours has been endorsed by WHO for rapid diagnosis of TB. Our study is the first study from India with a large sample size to evaluate the performance of Xpert MTB/RIF assay in PTB samples. The test showed an overall sensitivity and specificity of 95.7% (430/449) and 99.3% (984/990) respectively. In smear negative-culture positive cases, the test had a sensitivity of 77.7%. The sensitivity and specificity for detecting rifampicin resistance was 94.5% and 97.7% respectively with respect to culture as reference standard. However, after resolving the discrepant samples with gene sequencing, the sensitivity and specificity rose to 99.0% and 99.3% respectively. Hence, while solid culture still forms the foundation of TB diagnosis, Xpert MTB/RIF proposes to be a strong first line diagnostic tool for pulmonary TB cases.     

上海口岸2014-2015年入境人员肺结核筛查情况

为了解上海口岸入境人员肺结核的筛查情况及后续处理,防止结核病通过口岸跨境传播,本研究于2014年1月—2015年12月对所有在上海口岸办理入境体检的14岁以上人员进行结核病筛查,通过病史、体格检查和胸部X线摄影筛查疑似肺结核患者;对疑似肺结核患者进行痰细菌学检测、T‐SPOT ．TB和 Xpert MTB／RIF检测。结果显示,2014—2015年上海口岸入境人员共检出疑似肺结核患者215例,总检出率为229．76／10万;确诊肺结核患者33例,总检出率为35．27／10万,确诊率为15．3％。对210例疑似肺结核患者进行痰细菌学检测,结果显示结核分枝杆菌培阳率为14．3％,非结核分枝杆菌培阳率为17．1％。有95例和78例疑似肺结核患者分别接受 T‐SPOT ．TB和 Xpert MTB／RIF 检测,以痰细菌学检测为“金标准”,T‐SPOT ．TB的灵敏度为100％,特异度为49．4％;Xpert MTB／RIF的灵敏度为87．5％,特异度为96．8％。33例确诊肺结核患者中,25例（75．8％）离境,15例（45．5％）在离境前接受抗结核治疗,8例（24．2％）失访。本研究显示,上海口岸入境人员中肺结核确诊率仍有待提高。筛查与诊断中,T‐SPOT ．TB具备较高灵敏度, Xpert MTB／RIF具备较高特异度,两种方法均有较高应用价值,两者联用可提高检出率,缩短检出时间。对确诊病例或未确诊的可疑病例应加强后续监管。     

Smear positive pulmonary tuberculosis and associated factors among homeless individuals in Dessie and Debre Birhan towns,Northeast Ethiopia

Background

Tuberculosis (TB) remains one of the globe’s deadliest communicable diseases. The homeless individuals are at high risk to acquire TB and multi-drug resistant TB (MDR-TB), because of their poor living conditions and risky behaviors. Tuberculosis and MDR-TB in the homeless individuals can pose a risk to entire communities. However, the magnitude of the problem is not known in Ethiopia. Therefore, the aim of this study was to determine the prevalence and associated factors of smear positive pulmonary TB (PTB) and MDR-TB among homeless individuals in Dessie and Debre Birhan towns, Northeast Ethiopia.

Methods

A community based cross-sectional study design was conducted from September 2014 to June 2015. Using an active screening with cough of ≥2 weeks, 351 TB suspects homeless individuals were participated in this study. Data were collected by using pre-tested and structured questionnaire. Spot-morning-spot sputum sample was collected and examined for acid-fast bacilli (AFB) using fluorescence microscopy by Auramine O staining technique. All AFB positive sputum was further analyzed by GeneXpert for detection of Mycobacterium tuberculosis complex and rifampicin resistant gene. Univariate and multivariate logistic regressions were applied to identify factors associated with smear positive PTB and P value <0.05 was considered as statistically significant.

Results

The prevalence of smear positive PTB was 2.6 % (95 % CI 1.3–5) among TB suspect homeless individuals. Extrapolation of this study finding implies that there were 505 smear positive PTB per 100,000 homeless individuals. All smear positive PTB sputum specimens were further analyzed by GeneXpert assay, the assay confirmed that all were positive for MTBC but none were resistant to RIF or MDR. Smoking cigarette regularly for greater than 5 years (AOR 10.1, 95 % CI 1.1, 97.7), body mass index lower than 18.5 (AOR 6.9, 95 % CI 1.12, 41.1) and HIV infection (AOR 6.8, 95 % CI 1.1, 40.1) were significantly associated with smear positive PTB.

Conclusion

The prevalence of smear positive PTB among TB suspect homeless individuals was 2.6 %. Among smear positive PTB, prevalence of HIV co-infection was very high 5 (55.5 %). Smoking cigarette regularly for greater than 5 years, BMI lower than 18.5 and HIV infection were factors associated with smear positive PTB. Special emphasis is needed for homeless individuals to exert intensive effort to identify undetected TB cases to limit the circulation of the disease into the community.

     

Differential expression of CD64 in patients with Mycobacterium tuberculosis infection: A potential biomarker for clinical diagnosis and prognosis

To evaluate the clinical utility of neutrophil (n)CD64 index to diagnose pulmonary tuberculosis (PTB) and extrapulmonary TB (ePTB) and to predict the outcome of Mycobacterium tuberculosis infection. We recruited 189 patients with active TB and 140 controls and measured the differential expression of nCD64 index using flow cytometry. The receiver operating characteristics (ROC) curve analysis was performed to estimate the diagnostic performance of the nCD64 index and T‐SPOT.TB assay for the diagnosis of TB. Furthermore, we analysed whether the nCD64 index in patients with TB was correlated with inflammatory indicators. Finally, we assessed the prognosis of patients by following the dynamic changes of the nCD64 index once a week. The nCD64 index was significantly higher in active TB group (PTB and ePTB), than in the anti‐TB and healthy controls (HC) groups. The sensitivity and specificity of nCD64 index for the differential diagnosis of PTB and pneumonia (PN) patients were 68.33% and 77.55%, respectively. The sensitivity and specificity of nCD64 index for the diagnosis of tuberculous meningitis (TBM) were 53.85% and 100%, respectively. Furthermore, there was a weak correlation between the nCD64 index and inflammatory indicators. More importantly, with the improvement in patient condition, the nCD64 index started to decline in the first week of anti‐TB therapy and significantly decreased at 4 weeks after treatment. Our study demonstrated that the CD64 assay is a rapid, non‐invasive and stable method for clinical application, and the nCD64 index can serve as a potential biomarker for the diagnosis and prognosis of TB.     

Potential application of dot-immunobinding assay as a rapid diagnostic test in tuberculous meningitis

A simple dot-immunobinding assay (Dot-Iba) in nitrocellulose paper was developed for the detection of specific IgG antibody to Mycobacterium tuberculosis antigen 5 and mycobacterial antigen in cerebrospinal fluid of patients with tuberculous meningitis (TBM). The assay gave 77.1% sensitivity for the detection of IgG antibody to M. tuberculosis antigen 5 and 48.6% sensitivity for the detection of mycobacterial antigen in patients with TBM.     

结核病实验诊断技术

结核病诊断一直是控制结核病疫情的关键,快速准确、敏感特异、简便低廉的诊断方法是目前迫切需要的.从结核分枝杆菌快速诊断噬菌体法、AMPLICOR(R) MTB试验、Gen-Probe分子生物学诊断方法到T-SPOT.TB和QuantiFeron-Gold Test免疫学检测方法,结核病实验诊断方法在不断改进和完善.近年来...     

Diagnostic Accuracy of the PURE-LAMP Test for Pulmonary Tuberculosis at the County-Level Laboratory in China

Background

Early and effective detection of Mycobacterium tuberculosis (MTB), particularly in smear-negative tuberculosis (TB), is a priority for global TB control. Loop-mediated isothermal amplification with a procedure for ultra rapid DNA extraction (PURE-LAMP) can detect TB in sputum samples rapidly and with high sensitivity and specificity. However, the PURE-LAMP test has not been effectively evaluated, especially in resource-limited laboratories. In this study, we evaluated the performance of the PURE-LAMP test for TB detection in TB suspects from two county-level TB dispensaries in China.

Methodology/Principal Findings

From April 2011 to February 2012, patients with suspected TB were continuously enrolled from two county-level TB laboratories in China. Three sputum samples (spot, night, and morning sputum) were collected from each recruited patient. Detection of MTB by PURE-LAMP was compared to a reference standard L-J culture. The results showed that the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection was 70.67%, while the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection in smear positive and culture positive patients and smear negative and culture positive patients was 92.12% and 53.81%, respectively. The specificity of PURE-LAMP based on spot sputum for MTB detection was 98.32%. The sensitivity and specificity of the PURE-LAMP test based on three sputa combination for MTB detection was 88.80% and 96.86%, respectively. The results also showed that the PURE-LAMP test had a significantly lower contamination rate than did solid culture.

Conclusions/Significance

The study suggested that, in peripheral-level TB laboratories in China, the PURE-LAMP test showed high sensitivity and specificity for TB detection in TB suspects, making it a more effective, rapid, and safe method worthy of broader use in the future.     

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections. Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains. The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB. The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB. In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.Download video file.^{(243M, mp4)}     

emPAI-assisted strategy enhances screening and assessment of Mycobacterium tuberculosis infection serological markers

Discovering new serological markers of Mycobacterium tuberculosis (MTB) infection and establishing a rapid and efficient detection technology is of great significance for the prevention and control of tuberculosis. In this study, we established an exponentially modified protein abundance index (emPAI) value-assisted strategy to investigate and improve the screening efficiency of serological biomarkers of tuberculosis. First, we used LC-MS/MS to analyse MTB culture filtrate proteins (MTB-CFPs), and 632 MTB proteins were identified. Then, the characteristic values of MTB-CFPs – including emPAI value, molecular weight (Mw), isoelectric point (pI), grand average of hydropathy (GRAVY), transmembrane domain (TMD) and functional groups were calculated. Next, we successfully prepared 10 MTB proteins with emPAI value > 1.0 and recombinantly expressed these proteins in Escherichia coli. At the same time, 3 MTB proteins with emPAI between 0.1 and 0.5 were randomly selected as the control groups, and the immunogenicity of the recombinant MTB proteins was detected using ELISA. The sensitivity and receiver operating characteristic (ROC) curves were calculated for each recombinant MTB protein. The results showed that the areas under the curve (AUC) value of Rv2031c, Rv0577, Rv0831c, Rv0934 and Rv3248c were all higher than those of Rv3875 (AUC, 0.6643). Further analysis of the relationship between emPAI value and antibody sensitivity, AUC value and antibody affinity in mice immunized with recombinant MTB protein showed that emPAI values were positively correlated with them, and R-squared value ranged from 0.64 to 0.79. The only exception was ESAT-6 (encoded by the Rv3875 gene), which AUC value was relatively low owing to its strong immunosuppressive properties. This study provides a rationale for the serological marker screening of emPAI-assisted tuberculosis clinical test. The results also provide new technical support for the screening of candidate serological markers of infectious diseases in the future.     

Screening for HIV-associated tuberculosis and rifampicin resistance before antiretroviral therapy using the Xpert MTB/RIF assay: a prospective study

Background

The World Health Organization has endorsed the Xpert MTB/RIF assay for investigation of patients suspected of having tuberculosis (TB). However, its utility for routine TB screening and detection of rifampicin resistance among HIV-infected patients with advanced immunodeficiency enrolling in antiretroviral therapy (ART) services is unknown.

Methods and Findings

Consecutive adult HIV-infected patients with no current TB diagnosis enrolling in an ART clinic in a South African township were recruited regardless of symptoms. They were clinically characterised and invited to provide two sputum samples at a single visit. The accuracy of the Xpert MTB/RIF assay for diagnosing TB and drug resistance was assessed in comparison with other tests, including fluorescence smear microscopy and automated liquid culture (gold standard) and drug susceptibility testing. Of 515 patients enrolled, 468 patients (median CD4 cell count, 171 cells/µl; interquartile range, 102–236) produced at least one sputum sample, yielding complete sets of results from 839 samples. Mycobacterium tuberculosis was cultured from 81 patients (TB prevalence, 17.3%). The overall sensitivity of the Xpert MTB/RIF assay for culture-positive TB was 73.3% (specificity, 99.2%) compared to 28.0% (specificity, 100%) using smear microscopy. All smear-positive, culture-positive disease was detected by Xpert MTB/RIF from a single sample (sensitivity, 100%), whereas the sensitivity for smear-negative, culture-positive TB was 43.4% from one sputum sample and 62.3% from two samples. Xpert correctly identified rifampicin resistance in all four cases of multidrug-resistant TB but incorrectly identified resistance in three other patients whose disease was confirmed to be drug sensitive by gene sequencing (specificity, 94.1%; positive predictive value, 57%).

Conclusions

In this population of individuals at high risk of TB, intensive screening using the Xpert MTB/RIF assay increased case detection by 45% compared with smear microscopy, strongly supporting replacement of microscopy for this indication. However, despite the ability of the assay to rapidly detect rifampicin-resistant disease, the specificity for drug-resistant TB was sub-optimal. Please see later in the article for the Editors'' Summary