Xpert® MTB/RIF assay testing on stool for the diagnosis of paediatric pulmonary TB in Tanzania

SETTING: Six health facilities in Dar es Salaam, Tanzania.OBJECTIVE: To evaluate the use of stool specimens in the diagnostic workup of paediatric TB using the Xpert® MTB/RIF assay.DESIGN: Between December 2018 and May 2019, we performed a cross-sectional diagnostic study of children aged between 1 month and 14 years with presumptive TB. A single stool specimen was tested using Xpert. The result was compared with the reference microbiological standard for respiratory or gastric specimens tested using Xpert and/or solid culture. The sensitivity, specificity and predictive values of stool Xpert assay were assessed.RESULTS: A total of 225 children with a median age of 2.17 years (IQR 1.16–5.19) were enrolled; 165/225 (73.3%) were aged <5 years. Of 225 children, 8 (3.6%) were diagnosed with TB as they were culture- or Xpert-positive on sputum/gastric aspirate. The stool Xpert assay showed a sensitivity of 62.5% (95% CI 25–92) and specificity of 100% (95% CI 98–100) against the reference standard.CONCLUSION: Use of the Xpert assay on stool specimens had a moderate sensitivity and high specificity in the diagnosis of pulmonary TB in children. Our data adds to the body of evidence for the use of Xpert assay on stool as a non-respiratory specimen to complement conventional methods used to diagnose the disease.

The Simple One-Step (SOS) stool processing method for use with the Xpert MTB/RIF assay for a child-friendly diagnosis of tuberculosis closer to the point-of-care

Young children cannot easily produce sputum for diagnosis of pulmonary tuberculosis. Alternatively, Mycobacterium tuberculosis complex (MTB) bacilli can be detected in stool by using the Xpert MTB/RIF (Ultra) assay (Xpert). Published stool processing methods contain somewhat complex procedures and additional supplies. The aim of this study was to develop a simple one step (SOS) stool processing method, based on gravity sedimentation only, similar to sputum Xpert testing for the detection of MTB in stool. We first assessed if the SOS stool method could provide valid Xpert results without the need of bead-beating, dilution and filtration steps. We concluded that this was the case and, subsequently, validated the SOS stool method by testing spiked stool samples. By using the SOS stool method, of the 29 spiked samples, 27 gave valid Xpert results and MTB was recovered from all 27. The proof of principle of the SOS stool method was demonstrated in a routine setting in Addis Ababa, Ethiopia. Nine of 123 children with presumptive TB were MTB positive on nasogastric aspirate (NGA), and seven (77.8%) of these also had an MTB positive Xpert result on stool. Additionally, MTB was detected in the stool but not on the NGA of two children. The SOS stool processing method makes use of the standard Xpert assay kit, without the need for additional supplies or equipment. The method can potentially be rolled out to any Xpert site, bringing a bacteriologically confirmed diagnosis of TB in children closer to the point of care.

Performance Evaluation of Xpert MTB/RIF Assay and Prevalence of rpoB Gene Mutation in Mycobacterium Tuberculosis in Clinical Isolates Quetta, Pakistan: A Cross Sectional Retrospective Cohort Study

With the use of Xpert assay, the detection mechanism of tuberculosis has completely transformed. Concurrently detecting mycobacterium and its resistance to rifampin (rif) which is also one of the primary marker for MDR-TB in addition to its surrogate activity against MTB in first line drugs. At large, resistance of mycobacterium strains against rifampicin is caused by rpoB gene mutations about 96.1 % globally while these mutations are generally situated at a site of 507-533rd residuals of amino acid in the rpoB gene of Mycobacterium, this region is also termed as Rifampicin resistance determining region (RRDR). In this study, we determined the mutations of various rpoB genes in different probes of 81 bp of RRD Region. A total of 6353 different specimens obtained at provincial reference laboratory of Fatima Jinnah general and chest hospital, Quetta (FJHQ), between Jan 2016 and Apr 2018 were examined by Xpert MTB/RIF assay. Altogether positive and negative samples for MTB were further examined with the light emitting diode (LED) fluorescence microscope. Pearson Chi square test was used to find out the associations between probe type and gender, treatment history and age group of the patients. Out of 6353 specimens 1297 MTB positive cases were detected by Xpert assay including 184 samples having both mycobacterium and RIF-resistance advised by mutations in 81 bp-RRD Region of rpoB gene which is further divided into five different probes. Further LED fluorescence microscope detected and confirmed 327 positive MTB samples out of 1297 specimens of Xpert assay. The associated probes mutation for rifampicin resistance were as follow: E (98/184), D (23/184), B (15/184), C (5/184), A (3/184), two probe mutation (5/184) in D&E (3/184) and C&D (2/184) and all probe mutation (35/184). The percentage of rpoB gene mutation in the studied population was 14.187 %. The commonest mutation associated with rpoB gene was in Probe E (53.2608%) also termed as 531 and 533 codons, additionally two probe mutation (2.7173%) in D&E (1.6304%) and C&D (1.0869%) and all probe mutation (19.0217%) were also detected that has never been detected before globally.

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.

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.

Performance evaluation of Xpert HBV viral load (VL) assay: Point-of-care molecular test to strengthen and decentralize management of chronic hepatitis B (CHB) infection

IntroductionEstimation of hepatitis B (HBV) viral load (VL) is critical in hepatitis-B cascade-of-care and currently there is no point of care (POC) molecular assay available for that. This study evaluated the performance of a new near point of care molecular assay Xpert HBV-VL assay against FDA approved Real time PCR assays.Materials & methodsIn this retrospective study 119 archived plasma samples from HBV infected patients, and 53 hepatitis B surface antigen (HBsAg) patients were simultaneously tested for HBV DNA quantification on 2 real time PCR conventional assays and Xpert assay. The routine method for reporting to patient was Abbott Real Time PCR.ResultsThe range of HBV DNA load in samples was 1 to 8.76 log10IU/ml with a median load of 4.46 (IQR: 1-8.76) log10IU/ml as detected by routine assay (Abbott Real-Time HBV VL assay). Genotyping could be done in 95 (79.8%) samples and genotype D (83; 87.37%) was found commonest. The Xpert assay demonstrated good correlation with Abbott (R2= 0.944) and Roche (R2= 0.963). On comparison the mean difference (95% Confidence Interval) in average viral load was −0.018 log10 IU/ml and −0.043 log10 IU/ml when Xpert was compared with the Abbott and Roche assay, respectively. The overall sensitivity, specificity, negative predictive value and positive predictive value of the Xpert assay was found 97.5%, 100%, 94.65 & 100% respectively.ConclusionXpert HBV-VL assay which has a potential for near point of care molecular testing has shown excellent performance and found to be a reliable method for HBV DNA quantification.

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

Introduction: 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. Methodology: 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. Results: 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%. Conclusions: 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.

Development of Drug Resistance among Comorbid and Non-Comorbid HIV-Negative Relapsed Cases of Tuberculosis in Lahore, Pakistan

Background: Mycobacterium tuberculosis sometimes become resistant to the drugs that are used to treat it. Drug resistant TB (DR-TB) is spread in the same way as drug susceptible TB. DR-TB is a public health crisis. This study aims to find the pattern of drug resistance and correlations between drug resistance and comorbid/non-comorbid conditions in patients with a relapse of TB. Methodology: A cross-sectional study was conducted among 200 HIV-negative relapsed TB patients from 2016-2017 in Mayo Hospital Lahore. The patients’ sputum samples were tested by Ziehl-Neelsen staining to observe acid-fast bacilli. The demographics and medical history of patients was recorded, who were positive for AFB in their sputum samples. Molecular procedure of Gene-Xpert assay was conducted to detect the presence of MTB and rifampicin resistance in the samples. Whereas, the drug susceptibility test (DST) was conducted on the LJ culture medium containing drugs.Results: Out of 200 relapsed TB cases; 97 were comorbid, 99 were non-comorbid. The most prevalent comorbidities were hypertension (42 cases- 43.3%), diabetes (45 cases-46.4%) and hepatitis B (14 cases-14.4%). Among 97 comorbid patients; 37 worked as laborers, 43 earned less than 20,000 PKR and 23 were found to have a history of imprisonment. Whereas in non-comorbid patients; 20 worked as laborers, 28 earned less than 20,000 PKR and 12 had been in prison before. The Gene-Xpert test detected rifampicin resistance (RR) in 20 comorbid (20.6%) and 33 non-comorbid (33.3%) patients. Whereas, the drug susceptibility test (DST) showed that 22 comorbid (22.7%) and 33 non-comorbid (33.3%) patients were RR. A contrast was seen in the results of Gene-Xpert and DST; Gene-Xpert detected 3 cases of RR-negative whereas the same 3 cases were found to be RR-positive on DST. Only 1 case was RR-positive on Gene-Xpert but RR-negative on DST. 17 comorbid patients (17.5%) were diagnosed with MDR-TB and 5 (5.2%) with XDR-TB. Whereas, in non-comorbid patients, there were 26 cases of MDR-TB (26.3%) and 5 cases of XDR-TB (5.1%). There were 2 patients (2.1%) resistant to all drugs.Conclusion: There was a deviation in the results of molecular Gene-Xpert assay compared to the conventional culture methods. Drug resistance was relatively higher in non-comorbid patients than comorbid patients, however, the difference between the two is not very significant.