Xpert Assay Research Articles

Evaluation of gene Xpert in detecting suspected pulmonary tuberculosis from stool sample for children

Background: Case detection of pulmonary tuberculosis (PTB) is a challenge, even if different diagnostic methods used. Nowadays, it is improved, after the advancement of the detection method such as gene Xpert assay a prodigious change for adults. But in the pediatric PTB is still a challenge, due to the lack of well-developed physiological capacity. Young children are unable to produce enough amount of sputum to be detected by the gene Xpert. So, a substantial number of children are not diagnosed and fail to get treatment. Methods and materials: A cross-sectional study design was used, to evaluate 422 stool and sputum samples were collected from PTB suspected children (<15 years) by consecutive sampling technique. Both stool and sputum specimens were run in the gene Xpert at AHMC and sputum specimens were cultured using LJ culture medium at Oromia Regional Laboratory, Adama, Ethiopia. Specimen processing and testing were done in accordance with the standard mycobacterial safety precautions, procedures, and manufacturer's instruction. Descriptive statistical analysis was done by SPSS version 20, a chi-square test was used to show their association at 95% CI and P-value <0.05 was considered statistically significant, sensitivity, specificity, PPV and NPV of the tests were also calculated. The study was commenced after obtaining ethical approval and permission letter from respective bodies. All data obtained from the participants kept confidential. Results: A total of 422 children were enrolled. About, 70% were living in urban areas. Male to female proportion was 203/219. The mean age was 10.8 years, the majority (71.8%) of the participant age was less than 5years old. From 422 children investigated, 26 had been detected for PTB and 24 of them are confirmed by the sputum culture, with three invalid and four errors. Two of those PTB detected were rifampicin-resistant. Stool gene Xpert had a sensitivity, specificity, PPV and NPV (96%, 99.5%, 92.3%, 99.7%) respectively. Conclusion: The sensitivity of gene Xpert to detected MTB in the stool sample was higher as 96% (P = 0.000). Therefore, the stool can be used as an alternative sample for the diagnosis of PTB using gene Xpert assay for children unable to give respiratory samples.

Diagnostic accuracy of Xpert MTB/RIF ultra for detection of Mycobacterium tuberculosis in children: a prospective cohort study

The Xpert MTB/RIF Ultra is a recent advancement in molecular diagnostics of tuberculosis (TB) with higher sensitivity compared to its predecessor, the Xpert MTB/RIF assay. Prospective studies evaluating the performance of Xpert MTB/RIF Ultra in children with suspected TB are lacking. In this study, we evaluated the Xpert MTB/RIF Ultra for detection of Mycobacterium tuberculosis in samples from 156 children, of which one was excluded from the analysis. Of the remaining 155 samples, 6·5% (10/155), 21·3% (33/155), 20% (31/155) and 21·9% (34/155) were positive by smear examination, MGIT culture, Xpert MTB/RIF and Xpert MTB/RIF Ultra, respectively. The Xpert MTB/RIF and Xpert MTB/RIF Ultra had a similar overall sensitivity of 81·8% (95% CI: 64·5-93) and 84·8% (95% CI: 68·1-94·9), respectively. In suspected pediatric TB patients, the Xpert MTB/RIF Ultra had higher sensitivity compared to the Xpert MTB/RIF (72·7 vs 63·6). The AUC (area under the curve) of 0·905 for the Xpert MTB/RIF and 0·893 for the Xpert MTB/RIF Ultra indicate similar and good overall performance. Both Xpert assays were found to be equally efficient, however Xpert MTB/RIF Ultra showed better detection rate in suspected TB cases.

Comparison of the Xpert Carba-R and NG-Test CARBA5 for the detection of carbapenemases in an IMP-type carbapenemase endemic region in Japan

IntroductionThe real-time PCR assay Xpert Carba-R and the lateral flow immunoassay NG-Test CARBA5 were developed to detect 5 types of carbapenemase genes (blaIMP, blaKPC, blaVIM, blaOXA-48, and blaNDM). MethodsWe compared the diagnostic performance, turn-around time, and cost of these assays. Carbapenemase genes were defined using the Carba NP test, modified Carbapenem Inactivation Methods (mCIM), multiplex PCR, and whole-genome sequencing. We included clinical Enterobacterales isolates (n = 36) and nonfermenting gram-negative bacilli isolates (n = 17) collected from 16 acute-care hospitals in the Kinki region of Japan. ResultsTwenty-six of these 53 isolates were positive according to both of the Carba NP test and mCIM and, contained the following carbapenemase genes: blaIMP-1 (n = 3), blaIMP-6 (n = 1), blaIMP-19 (n = 12), blaIMP-26 (n = 1), blaIMP-41 (n = 2), blaIMP-66 (n = 2), blaNDM-1 (n = 3), and blaVIM-2 (n = 2). All of the remaining 27 isolates were negative according to the Carba NP test, mCIM, and multiplex PCR. The specificities of both assays were 100%. The sensitivity of the Xpert Carba-R assay was as low as 53.8% and that of the NG-Test CARBA5 was 92.3% because the former failed to detect all isolates with blaIMP-19 (n = 12) and the latter failed to detect isolates with blaIMP-66 (n = 2). Both assays can easily be performed in less than 5 min. ConclusionsThe NG-Test CARBA5 assay was superior with regard to assay time and cost per sample. We propose the use of the NG-Test CARBA5 assay in clinical laboratories where IMP-type carbapenemases are endemic.

Diagnostic implications of bronchial lavage in patients with pleural tuberculosis.

The presence of Mycobacterium tuberculosis in a respiratory specimen is diagnostic in patients with pleural effusion. It is difficult to obtain sputum even after induction in these patients. An alternative method of acquiring respiratory specimens is via bronchial lavage. This study was undertaken to evaluate the diagnostic yield of acid-fast bacilli (AFB) smear, AFB culture, and Xpert assay of bronchial lavage fluid in the workup of pleural tuberculosis patients. All patients who met the inclusion criteria of the study underwent thoracentesis, pleural biopsy, and bronchial lavage. Specimens of pleural fluid, pleural biopsy, and bronchial lavage fluid were sent for acid fast bacilli smear, culture, and Xpert assay. Bronchial lavage AFB smear, culture, and Xpert assay was positive in 9.5%, 17.9%, and 26.2% of patients, respectively. It gave an immediate diagnosis in 22 (26.2%) patients. Bronchial lavage, though not a surrogate to pleural biopsy, offers an additional approach to the early diagnosis of pleural tuberculosis in patients not producing sputum. Besides being diagnostic, this method also has epidemiologic significance in containing the tuberculosis epidemic because detecting Mycobacterium in bronchial lavage confirms that the patient is infectious.

Caregiver experience and perceived acceptability of a novel near point-of-care early infant HIV diagnostic test among caregivers enrolled in the PMTCT program, Myanmar: A qualitative study.

The majority of HIV infection among children occurs through mother-to-child transmission. HIV exposed infants are recommended to have virological testing at birth or 4-6 weeks of age but challenges with centralized laboratory-based testing in Myanmar result in low testing rates and delays in result communication and treatment initiation. Decentralized point-of-care (POC) testing when integrated in prevention of mother-to-child transmission of HIV (PMTCT) services, can be an alternative to increase coverage of early infant diagnosis (EID) and timely engagement in HIV treatment and care. This paper aims to explore experiences of caregivers of HIV-exposed infants enrolled in the PMTCT program in Myanmar and the perceived acceptability of point-of-care EID testing compared to conventional centralised laboratory-based testing. This is a sub-study of the cluster randomised controlled stepped-wedge trial (Trial registration number: ACTRN12616000734460) that assessed the impact of near POC EID testing using Xpert HIV-1 Qual assay in four public hospitals in Myanmar. Caregivers of infants who were enrolled in the intervention phase of the main study, had been tested with both Xpert and standard of care tests and had received the results were eligible for this qualitative study. Semi-structured interviews were conducted with 23 caregivers. Interviews were audio recorded, transcribed verbatim and translated into English. Thematic data analysis was undertaken using NVivo 12 Software (QSR International). The majority of caregivers were satisfied with the quality of care provided by PMTCT services. However, they encountered social and financial access barriers to attend the PMTCT clinic regularly. Mothers had concerns about community stigma from the disclosure of their HIV status and the potential consequences for their infants. While medical care at the PMTCT clinics was free, caregivers sometimes experienced financial difficulties associated with out-of-pocket expenses for childbirth and transportation. Some caregivers had to choose not to attend work (impacting their income) or the adult antiretroviral clinic in order to attend the paediatric PMTCT clinic appointment. The acceptability of the Xpert testing process was high among the caregiver participants and more than half received the Xpert result on the same day as testing. Short turnaround time of the near POC EID testing enabled the caregivers to find out their infants' HIV status quicker, thereby shortening the stressful waiting time for results. Our study identified important access challenges facing caregivers of HIV exposed infants and high acceptability of near POC EID testing. Improving the retention rate in the PMTCT and EID programs necessitates careful attention of program managers and policy makers to these challenges, and POC EID represents a potential solution.

Frequency and Distribution of Rifampicin Resistance in Mycobacterium Tuberculosis Clinical Isolates Using Gene Xpert MTB/RIF in Delta State, South-South Nigeria

The emergence and spread of multi-drug resistant-tuberculosis is a threat, which has complicated the diagnosis, management and control of tuberculosis. In addition to the simultaneous detection of Mycobacterium tuberculosis bacilli and rifampicin resistance, the Gene Xpert assay can also highlight the point of mutation if it occurs around the rifampicin resistance determination region (RRDR) of the rpoB gene, which is responsible for 95% of rifampicin resistance. This study seeks to estimate the prevalence of rifampicin resistance, determine the frequency and distribution of mutations along the rifampicin resistance determination region, and assay for the relationship that exists between these mutations and basic epidemiological variables.

Performance of Xpert/MTB/RIF assay for childhood pulmonary tuberculosis among HIV negative children with real world evidence in China

BackgroundRapid and accurate notification of childhood pulmonary tuberculosis (PTB) is a worldwide challenge. Although the Xpert MTB/RIF assay (Xpert) has been endorsed as the initial test for suspected PTB in many countries, limited studies have reported the performance of Xpert in childhood PTB. The aim of this study is to evaluate the real-world performance of Xpert for the detection of childhood PTB among HIV negative children in China. MethodsWe consecutively extracted the data of all patients ≤14 years with pulmonary disease through the electronic medical record (EMR) systems of Shanghai Public Health Clinical Center from January 2014 to December 2017. The clinical profile, the decision-making tests including AFB smear, solid/liquid culture, pathological examination and Xpert result were matched and assessed. The real diagnostic accuracy and the all-factors case notification rate for childhood PTB with the implementation of Xpert were evaluated. Results519 HIV negative cases ≤14 years with pulmonary disease were extracted from the data base. Of these, 145 had matched results, there were 374 non-matched cases including 346 with incomplete or unavailable data and 28 with NTM, BCG or an unidentified strain. For matched data, the overall sensitivity and specificity of the Xpert assay were 66.7% (32/48, 95%CI 0.52–0.80) and 87.6% (85/97, 95%CI 0.87–0.98) respectively against the gold standard; 34.6% (44/127, 95%CI 26.6–43.7) and 100% against the composite clinical reference standard (CCRS). The all-factors case notification rate by Xpert was 29%. ConclusionXpert/MTB RIF assay has acceptable sensitivity and excellent specificity for rapid diagnosis of children with pulmonary TB as well as for the detection of RIF resistance in China. However, implementation of Xpert for the initial diagnosis of childhood PTB is inadequate to meet the urgent requirement for rapid and accurate detection of childhood PTB.

A descriptive study of smear negative pulmonary tuberculosis in a high HIV burden patient's population in North Central Nigeria.

Tuberculosis (TB) is a serious disease of public health concern, mainly in low- and middle-income countries. Most of these countries have challenges in diagnosis and treatment of TB in people with smear-negative pulmonary tuberculosis (SNPTB), which remains a significant public health challenge because of the global burden of the disease. We evaluated the epidemiology and clinical presentation of SNPTB in a cohort of patients with high HIV burden. The study was a cross-sectional study among patients with SNPTB in four major hospitals that care for TB/HIV patients in north-central Nigeria. All patients 18 years and above who were newly diagnosed as SNPTB, or patients with SNPTB who had not taken TB drugs for up to 2 weeks irrespective of their HIV status were recruited. Demographic data (sex, age), smoking status, and medical history (clinical form of TB, symptoms at admission, diagnostic methods, presence of comorbidities, prior TB treatment) were obtained using a semi-structured questionnaire. Detailed clinical examination was also done on all the study subjects. Baseline results of packed cell volume, HIV test and sputum acid fast bacilli done during TB screening were retrieved from the patients’ case notes and recorded. Also, the base line Chest X-ray films taken during TB screening were reviewed and reported by two radiologists blinded to each other’s reports. The Xpert MTB/RIF tests and sputum culture (using LJ medium) were done in a TB reference laboratory. A total of 150 patients with SNPTB were studied. Majority of the patients were female 93 (62%). The median age of the patients was 36.5 years with greater percentage of the patients within the ages of 25–44 years 92 (61.3%). Twenty-two (14.7%) of the patients had previous TB treatment. History of cigarette smoking was obtained in only 7(4.7%) of the patients while 82 (64.1%) were HIV positive. All the patients had a history of cough for over a period of at least three weeks, while, 27 (18%) reported having hemoptysis. About 87 (58%) had fever and 110 (73.7%) had anemia, while weight loss and night sweat were reported in 98(65.3%) and 82 (54.7%) of the patients respectively. Chest x rays were reported as typical of TB in only 24 (16%) of the patients. Of the 150 sputa sample analyzed, 21/150 (14.0%) and 22/150 (14.7%) where Gene Xpert and sputum culture positive respectively. The sensitivity and specificity of Gene Xpert assay were 81.8% (18/22; 95% CI 61.5 to 92.7%) and 97.4% (112/115; 95% CI 92.6 to 99.1%), respectively. The study found cough, fever and anemia to be the commonest presentation in patient with SNPTB in a high HIV burden patient’s population. There is also relatively high culture positivity among the patients. This underscores the need to expand the facilities for culture and confirmation in TB centers across the country.

Utility of circulating cell-free Mycobacterium tuberculosis DNA for the improved diagnosis of abdominal tuberculosis.

Abdominal tuberculosis (ATB) continues to pose a major diagnostic challenge for clinicians due to its nonspecific clinical presentation, variable anatomical location and lack of sensitive diagnostic tools. In spite of the development of several assays till date; no single test has proved to be adequate for ATB diagnosis. In this study, we for the first time report the detection of circulating cell-free Mycobacterium tuberculosis (M. tuberculosis) DNA (cfMTB-DNA) in ascitic fluid (AF) samples and its utility in ATB diagnosis. Sixty-five AF samples were included in the study and processed for liquid culture, cytological, biochemical and molecular assays. A composite reference standard (CRS) was formulated to categorize the patients into ‘Definite ATB’ (M. tuberculosis culture positive, n = 2), ‘Probable ATB’ (n = 16), ‘Possible ATB’ (n = 13) and ‘Non-TB’ category (n = 34). Two molecular assays were performed, namely, the novel cfMTB-DNA qPCR assay targeting M. tuberculosis devR gene and Xpert MTB/RIF assay (Xpert), and their diagnostic accuracy was assessed using CRS as reference standard. Clinical features such as fever, loss of weight, abdominal distension and positive Mantoux were found to be strongly associated with ATB disease (p<0.05). cfMTB-DNA qPCR had a sensitivity of 66.7% (95% CI:40.9,86.7) with 97.1% specificity (95% CI:84.7,99.9) in ‘Definite ATB’ and ‘Probable ATB’ group collectively. The sensitivity increased to 70.9% (95% CI:51.9,85.8) in the combined ‘Definite’, ‘Probable’ and ‘Possible’ ATB group with similar specificity. The cfMTB-DNA qPCR assay performed significantly better than the Xpert assay which demonstrated a poor sensitivity of ≤16.7% with 100% (95% CI:89.7,100) specificity (p<0.001). We conclude that cfMTB-DNA qPCR assay is an accurate molecular test that can provide direct evidence of M. tuberculosis etiology and has promise to pave the way for improving ATB diagnosis.

Multicenter evaluation of Xpert HIV-1 viral load assay for HIV quantification in China.

New approaches to increase HIV-1 testing and HIV-1 viral load (VL) monitoring are needed for people living with HIV (PLHIV) in China. The Xpert HIV-1 VL assay was prequalified by the World Health Organization in 2017 but has not been evaluated in China. A multicenter evaluation was conducted to assess the accuracy of the Cepheid Xpert HIV-1 VL assay compared to the Abbott RealTime HIV-1 assay in China.Overall agreement was seen in 558 of 562 specimens (99.29%) with a κ value of 0.962. Pearson's coefficient between the two assays was 0.943. Analyzed by the Bland-Altman method, the mean bias was -0.54 log10 copies/mL, and 94.05% results fell within the 95% confidence limit of agreement (-1.248 to 0.168 log10 copies/mL). The coefficient of variation of the Cepheid Xpert HIV-1 VL assay ranged from 0.61% to 1.55%, as determined by testing eight positive plasma specimens with three different lots on different days. Due to its simplicity, random-access, rapid turnaround time, and accuracy, the Xpert HIV-1 VL assay can be used in local hospitals and clinics that bear the burden of identifying and treating HIV patients in China.

Performance Comparison of Xpert HIV-1 Viral Load Assay and Roche Taqman and Abbott M2000 RT in Bamako, Mali.

Routine monitoring of HIV-1 Viral Load (VL) is important in patients on Antiretroviral Therapy (ART) management. Access to HIV VL remains a challenge in resource-limited settings, especially in rural areas. Universal access to VL requires more simplified and less restrictive alternatives to current conventional VL methods. The objective of this study was to evaluate the performance of the new rapid (2-hour turnaround time) Xpert HIV-1VL technique compared to Roche TaqMan and Abbott RT m2000 for HIV-1 RNA quantification in HIV- infected patients. We conducted a cross-sectional study in patients seen for routine VL monitoring between August and November 2018 in a HIV care site in Bamako. The performance of the Xpert HIV-1 VL assay was evaluated against the Roche TaqMan assay and Abbott m2000 RT assay. Performance, utility and reliability/reproducibility were verified using accuracy, sensitivity, specificity, positive and negative predictive values, Diagnostic Odds Ratio (DOR), Kappa coefficient, Pearson correlation coefficient, and Bland-Altman analysis. The Xpert assay compared well with the two current referral assays (Roche TaqMan and Abbott m2000 RT assays). Compared to Roche TaqMan assay the sensitivity was 93.10%, specificity (97.01%) and accuracy (95.20%), the correlation coefficient of Pearson (r) was 0.98 (p <0.01). Bland-Altman analysis showed a mean difference of 0.18 log10 cp/mL; (Standard Deviation) SD=0.33. Compared to the Abbott m2000 RT, the sensitivity, the specificity and the accuracy were respectively 93.44%; 92% and 92.65%. The Xpert HIV-1 VL assay showed a good correlation with a correlation coefficient of Pearson, r=0.99 (p <0.001). The overall mean difference in the HIV-1 VL values obtained by Xpert HIV-1 VL and Abbott m2000 RT assays was 0.08 log10 cp/mL; SD=0.30. Xpert HIV-1 VL showed a good performance compared to Roche TaqMan and Abbott m2000 RT. With the rapid test results (less than 2 h) and ease of testing individual specimens, the Xpert HIV-1 VL assay could be an effective alternative for HIV VL monitoring in resource-limited settings.

Prevalence and drivers of false-positive rifampicin-resistant Xpert MTB/RIF results: a prospective observational study in Rwanda

The Xpert MTB/RIF (Xpert) assay is used globally to rapidly diagnose tuberculosis and resistance to rifampicin. We investigated the frequency and predictors of false-positive findings of rifampicin resistance with Xpert. We did a prospective, observational study of individuals who were enrolled in a Rwandan nationwide diagnostic cohort study (DIAMA trial; NCT03303963). We included patients identified to have rifampicin resistance on initial Xpert testing. We did a repeat Xpert assay and used rpoB Sanger and deep sequencing alongside phenotypic drug susceptibility testing (pDST) to ascertain final rifampicin susceptibility status, with any (hetero)resistant result overriding. We used multivariable logistic regression to assess predictors of false rifampicin resistance on initial Xpert testing, adjusted for HIV status, tuberculosis treatment history, initial Xpert semi-quantitative bacillary load, and initial Xpert probe. Between May 4, 2017, and April 30, 2019, 175 people were identified with rifampicin resistance at initial Xpert testing, of whom 154 (88%) underwent repeat Xpert assay. 54 (35%) patients were confirmed as rifampicin resistant on repeat testing and 100 (65%) were not confirmed with resistance. After further testing and sequencing, 121 (79%) of 154 patients had a final confirmed status for rifampicin susceptibility. 57 (47%) of 121 patients were confirmed to have a false rifampicin resistance result and 64 (53%) had true rifampicin resistance. A high pretest probability of rifampicin resistance did not decrease the odds of false rifampicin resistance (adjusted odds ratio [aOR] 6·0, 95% CI 1·0-35·0, for new tuberculosis patients vs patients who needed retreatment). Ten (16%) of the 64 patients with true rifampicin resistance did not have confirmed rifampicin resistance on repeat Xpert testing, of whom four had heteroresistance. Of 63 patients with a very low bacillary load on Xpert testing, 54 (86%) were falsely diagnosed with rifampicin-resistant tuberculosis. Having a very low bacillary load on Xpert testing was strongly associated with false rifampicin resistance at the initial Xpert assay (aOR 63·6, 95% CI 9·9-410·4). The Xpert testing algorithm should include an assessment of bacillary load and retesting in case rifampicin resistance is detected on a paucibacillary sputum sample. Only when rifampicin resistance has been confirmed on repeat testing should multidrug-resistant tuberculosis treatment be started. When rifampicin resistance has not been confirmed on repeat testing, we propose that patients should be given first-line anti-tuberculosis drugs and monitored closely during treatment, including by baseline culture, pDST, and further Xpert testing. The European & Developing Countries Clinical Trials Partnership 2 programme, and Belgian Directorate General for Development Cooperation.

Evaluation of Loopamp Assay for the Diagnosis of Pulmonary Tuberculosis in Cambodia.

The Loopamp™ MTBC kit (TB-LAMP) is recommended by WHO for Mycobacterium tuberculosis complex detection in low-income countries with a still low drug-resistant tuberculosis (TB) rate. This study is aimed at testing its feasibility in Cambodia on sputa collected from presumptive tuberculosis patients. 499 samples were tested at a smear microscopy center and 200 at a central-level mycobacteriology laboratory. Using mycobacterial cultures as reference, TB-LAMP results were compared with those of LED fluorescent microscopy (LED-FM) and Xpert® MTB/RIF. At the microscopy center, TB-LAMP sensitivity was higher than that of LED-FM (81.5% [95% CI, 74.5-87.6] versus 69.4% [95% CI, 62.2-76.6]), but lower than that of the Xpert assay (95.5% [95% CI 92.3-98.8]). At the central-level laboratory, TB-LAMP sensitivity (92.8% [95% CI, 87.6-97.9]) was comparable to that of Xpert (90.7% [95% CI, 85.0-96.5]) using stored sample. No significant difference in terms of specificity between TB-LAMP and Xpert assays was observed in both study sites. In conclusion, our data demonstrate that TB-LAMP could be implemented at microscopy centers in Cambodia to detect TB patients. In addition, TB-LAMP can be a better choice to replace smear microscopy for rapid TB diagnosis of new presumptive TB patients, in settings with relative low prevalence of drug-resistant TB and difficulties to implement Xpert assay.

Evaluation of the Xpert HBV Viral Load for hepatitis B virus molecular testing.

Accurate molecular methods to detect and quantify hepatitis B virus (HBV) DNA are essential to diagnose chronic infections, guide treatment decisions, assess response to treatment, and determine risk of HBV-related complications. New HBV DNA generations of real-time assay platforms are now available with the availability of results in less than 2-3 h and a continuous loading of specimens with true random access. The aim of this prospective study was to evaluate the performance of the new Xpert HBV Viral Load assay to accurately quantify HBV DNA in plasma and in whole blood collected on dried blood spot (DBS). Plasma and whole blood from 143 patients chronically infected with HBV were tested in parallel using two commercially real-time PCR assay (Cobas AmpliPrep/Cobas Taqman HBV test, version 2.0, and Xpert HBV Viral Load assay). HBV DNA levels in whole blood strongly correlated with those measured in plasma. A positive significant correlation between the HBV DNA levels in plasma measured with the new Xpert HBV Viral Load and CAP/CTM HBV v2.0 assays was found. The newly developed real-time PCR-based assay Xpert HBV Viral Load accurately quantifies HBV DNA in whole blood specimens as well as in plasma samples from patients with chronic HBV infection. Whole blood specimens collected on DBS can be confidently used for replicative HBV infection detection in patients with HBV DNA level above 3 Log using standardized, commercially available methods.

The accuracy of tuberculous meningitis diagnostic tests using Bayesian latent class analysis.

Tuberculous meningitis (TBM) is the most dangerous form of tuberculosis with high mortality and disability rates. However, the delayed diagnostic process is often due to the absence of the gold standard tests leading to a lack of information about the sensitivity and specificity of diagnostic tests. This study aims to estimate the prevalence of TBM and determine the performance of four diagnostic procedures: the mycobacteria growth culture test, Gene Xpert assay, and analysis of protein levels and leukocyte count taken from cerebrospinal fluid. We used a Bayesian latent class analysis to estimate the prevalence of TBM with 95% credible interval (CI), and the specificity and sensitivity of the four diagnostic procedures. The area under the receiver operating characteristic curve (AUC) of the cerebrospinal protein levels and leukocyte count were also compared and estimated using different thresholds. A total of 1,213 patients suspected of having TBM were included. The estimated TBM prevalence was 34.8 % (95% CI: 28.8 - 41.3). The sensitivity of culture test and Gene Xpert assay was 62.7% (95% CI: 52.5 - 74.0), and 57.5% (95% CI: 51.0 - 64.0), and the specificity of Gene-Xpert was 95. 9% (95% CI: 92.0 - 99.8). The AUC for leukocyte count was 76.0%, and for protein level was 73.4%. This study provided better information about the performance of four routine diagnostic tests and the prevalence of TBM which can enhance disease control and improve treatment outcomes.

Apigenin and Ethaverine Hydrochloride Enhance Retinal Vascular Barrier In Vitro and In Vivo.

PurposeThis study aims to develop an impedance-based drug screening platform that will help identify drugs that can enhance the vascular barrier function by stabilizing vascular endothelial cell junctions.MethodsChanges in permeability of cultured human retinal microvascular endothelial cells (HRMECs) monolayer were monitored in real-time with the xCELLigence RTCA system. Using this platform, we performed a primary screen of 2100 known drugs and confirmed hits using two additional secondary permeability assays: the transwell permeability assay and the XPerT assay. The cellular and molecular mechanisms of action and in vivo therapeutic efficacy were also assessed.ResultsEleven compounds blocked interleukin 1 beta (IL-1β) induced hyperpermeability in the primary screen. Two of 11 compounds, apigenin and ethaverine hydrochloride, reproducibly blocked multiple cytokines induced hyperpermeability. In addition to HRMEC monolayers, the two compounds stabilized three other types of primary vascular endothelial cell monolayers. Preliminary mechanistic studies suggest that the two compounds stabilize the endothelium by blocking ADP-ribosylation factor 6 (ARF6) activation, which results in enhanced VE-cadherin membrane localization. The two compounds showed in vivo efficacy in an animal model of retinal permeability.ConclusionsWe developed an impedance-based cellular phenotypic drug screening platform that can identify drugs that enhance vascular barrier function. We found apigenin and ethaverine hydrochloride stabilize endothelial cell junctions and enhance the vascular barrier by blocking ARF6 activation and increasing VE-cadherin membrane localization.Translational RelevanceThe drugs identified from the phenotypic screen would have potential therapeutic efficacy in retinal vascular diseases regardless of the underlying mechanisms that promote vascular leak.

Prevalence of Tuberculosis by Automated GeneXpert Rifampicin Assay and Associated Risk Factors Among Presumptive Pulmonary Tuberculosis Patients at Ataye District Hospital, North East Ethiopia.

BackgroundTuberculosis is a communicable disease that is a major cause of ill health, one of the top 10 causes of death worldwide, and the leading cause of death from a single infectious agent, even ranking above human immuno-deficiency virus (HIV/AIDS).ObjectiveTo assess the prevalence and associated risk factor of Mycobacterium tuberculosis among pulmonary tuberculosis (PTB) suspects attending at Ataye District Hospital from October 1, 2018, to February 30, 2019.MethodologyA facility-based cross-sectional study was conducted among 423 presumptive tuberculosis patients at Ataye District Hospital. Sputum was processed by MTB/RIF Xpert assay. Data were entered into EpiData 3.1 software and exported to SPSS version 20.0 (SPSS, Chicago, IL, USA) for analysis. Univariate and multivariate analyses were used to examine the relationship between the dependent and independent variables. Variables that show significance at P-value of 0.3 during univariate analysis were selected for multivariable analysis. A P-value of less than or equal to 0.05 was used to indicate statistical significance.ResultsOut of the total study participants, about 60% were male, and 39% were aged between 18 and 24 years. Of the total 423 PTB suspected patients, 38 (8.98%) of them were identified as having PTB by GeneXpert and 2/38 (5.3%) were resistant to rifampicin and 3/38 (7.89%) patients were co-infected with HIV. Participant age between 18 and 24 years and between 25 and 34 years, weight loss, chest pain, having contact history with confirmed PTB cases, utilization of congested transportation, and a history of imprisonment were significantly associated with the prevalence of PTB.ConclusionA considerable prevalence of PTB in the area was observed and the magnitude of MDR-TB was low. PTB is still a public health problem in Ethiopia and there is a need for collaborative prevention and control activities in the study area.

Difficulties with the implemented xpert MTB/RIF for determining diagnosis of pulmonary and extrapulmonary tuberculosis in adults and children

Background and aimsHandling of PTB and EPTB patients with adequate standard detection of MTBC and anti-TB drug sensitivity using accurate and rapid methods could provide good TB management and clinical treatment outcomes. The Xpert MTB/RIF assay is an automated, cartridge-based NAAT that can simultaneously detect MTBC and RIF resistance within 2 h. The aim of this study was to evaluate the implementation of Xpert for determining diagnosis of PTB and EPTB in adults and children. MethodsA descriptive study was performed using e-TB Manager data from the MDR-TB Clinic at Dr. Soetomo Academic Hospital. Suspected TB cases were from the areas of East Java Province from January 2016 to December 2018. Xpert assay was conducted using standardized criteria for clinically suspected TB, and MTBC-positive results with RR were examined by the culture method using MGIT 960 BACTEC System. ResultsA total of 1181 (1181/3009, 39.25%) sputum samples from suspected new MDR-PTB cases tested positive for MTBC with 3.02% RR. Among 3893 sputum samples from previously treated probable MDR-PTB cases tested using Xpert, 1936 (49.73%) were MTBC positive with 13.20% RR. Among 59 new suspected MDR-PTB cases tested using MGIT 960 BACTEC System, 55 tested positive for MTBC, although all RR strains were highly sensitive to amikacin (100%), kanamycin (95%), and ofloxacin (89%). A total of 49 children with suspected PTB were tested using Xpert, revealing low positivity (12%) for MTBC, with all RR strains being rifampicin sensitive (RS). Of the 86 suspected EPTB cases tested using Xpert, very few were MTBC-positive (26%), with 91% RS. ConclusionsThis study revealed that in adults and children with PTB and EPTB, the Xpert assay achieved a low positivity detection rate for MTBC in samples from new or previously treated cases, and this could be the result of many factors.

Distribution and Frequency of rpoB Mutations Detected by Xpert MTB/RIF Assay Among Beijing and Non-Beijing Rifampicin Resistant Mycobacterium tuberculosis Isolates in Bangladesh.

BackgroundRifampicin resistance (RR) is a key indicator of multidrug-resistant tuberculosis (MDR-TB) and 95% of the RR is associated with the mutation in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene of Mycobacterium tuberculosis complex (MTBC). The Xpert MTB/RIF (Xpert) assay uses five overlapping molecular beacon probes (A-E) complementary to RRDR region that detect MTBC and mutations associated with RR. The objective of the study was to investigate the distribution and frequency of mutations detected by Xpert assay among Beijing and non-Beijing RR-TB isolates.MethodsA total of 205 randomly selected RR-TB specimens detected by Xpert assay were included in this study. A portion of specimens was further subjected to culture, MTBDRplus test and the positive culture isolates were genotyped by spoligotyping.ResultsWe found that the most frequent mutation occurred at probe E (S531L) binding region in both Beijing and non-Beijing isolates (61.9% and 66.9%, respectively). The Beijing family had higher mutation rates than non-Beijing (19.0% vs 12.4%) at probe B (D516V) while the non-Beijing family had higher mutations at probe D (H526D or H526Y) than the Beijing (13.2% vs 10.7%) family. Mutations at probes Aand C were less common in both Beijing and non-Beijing isolates. There was no significant difference (P=0.36) in the occurrence of mutations at different probes between Beijing and non-Beijing isolates.ConclusionsThe study results revealed that the most frequent mutation occurs in the region of probe E and the least common mutations at probe A and C among both Beijing and non-Beijing RR-TB cases. This first insight into the probe mutation variation and frequencies among the RR-TB cases in Bangladesh forms the baseline information for further investigation.

Diagnosis of Tuberculosis Using Gastric Aspirates in Pediatric Patients in Haiti.

We aimed to determine whether the Xpert MTB/RIF (Xpert) assay is a useful adjunct to culture for the rapid diagnosis of tuberculosis (TB) using gastric lavage aspirates (GLAs) in children aged < 5 years. We reviewed the yield from diagnostic modalities in children suspected of having TB followed at an infectious disease research and treatment center in Port-au-Prince, Haiti, from 2011 to 2016. In 187 children clinically diagnosed with TB, a microbiologic diagnosis could be established in 40 (21%). Cultures, Xpert, and smears were positive in 30 (19%), 28 (17%), and 3 (1.6%) children, respectively. Ten cases that would not have been diagnosed by culture alone were found by the use of the Xpert assay. Collecting 2 GLA samples optimized microbiologic yield. In GLAs, Xpert increased the yield of microbiologically documented cases by 33%. Additionally, the rapidity of diagnosis potentially makes Xpert a valuable adjunct in initiating treatment for TB in children. Smear microscopy has low sensitivity in GLA and did not add to the documented cases. Our findings also highlight the low rate of microbiologic confirmation of clinically diagnosed TB.