Dang Thi Minh Ha

Evaluation of GeneXpert MTB/RIF for Diagnosis of Tuberculous Meningitis

ABSTRACT Tuberculous meningitis (TBM) is the most severe form of tuberculosis. Microbiological confirmation is rare, and treatment is often delayed, increasing mortality and morbidity. The GeneXpert MTB/RIF test was evaluated in a large cohort of patients with suspected tuberculous meningitis. Three hundred seventy-nine patients presenting with suspected tuberculous meningitis to the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam, between 17 April 2011 and 31 December 2012 were included in the study. Cerebrospinal fluid samples were tested by Ziehl-Neelsen smear, mycobacterial growth indicator tube (MGIT) culture, and Xpert MTB/RIF. Rifampin (RIF) resistance results by Xpert were confirmed by an MTBDR-Plus line probe assay and all positive cultures were tested by phenotypic MGIT drug susceptibility testing. Overall, 182/379 included patients (48.0%) were diagnosed with tuberculous meningitis. Sensitivities of Xpert, smear, and MGIT culture among patients diagnosed with TBM were 59.3% (108/182 [95% confidence interval {CI}, 51.8 to 66.5%]), 78.6% (143/182 [95% CI, 71.9 to 84.3%]) and 66.5% (121/182 [95% CI, 59.1 to 73.3%]), respectively. There was one false-positive Xpert MTB/RIF test (99.5% specificity). Four cases of RIF resistance (4/109; 3.7%) were identified by Xpert, of which 3 were confirmed to be multidrug-resistant (MDR) TBM and one was culture negative. Xpert MTB/RIF is a rapid and specific test for the diagnosis of tuberculous meningitis. The addition of a vortexing step to sample processing increased sensitivity for confirmed TBM by 20% (P = 0.04). Meticulous examination of a smear from a large volume of cerebrospinal fluid (CSF) remains the most sensitive technique but is not practical in most laboratories. The Xpert MTB/RIF represents a significant advance in the early diagnosis of this devastating condition.

Beijing Genotype of Mycobacterium tuberculosis Is Significantly Associated with High-Level Fluoroquinolone Resistance in Vietnam

ABSTRACT Consecutive fluoroquinolone (FQ)-resistant isolates (n = 109) identified at the Pham Ngoc Thach Hospital for Tuberculosis, Ho Chi Minh City, Vietnam, were sequenced in the quinolone resistance-determining regions of the gyrA and gyrB genes and typed by large sequence polymorphism typing and spoligotyping to identify the Beijing genotype of Mycobacterium tuberculosis. Beijing genotype prevalence was compared with 109 consecutive isolates from newly presenting patients with pulmonary tuberculosis from the hospital outpatient department. Overall, 82.6% (n = 90/109) of isolates had mutations in gyrAB. Nine novel mutations were identified in gyrB (S486F, N538T, T539P, D500A, D500H, D500N, G509A, E540V, and E540D). The influence of these novel gyrB mutations on FQ resistance is not proven. The Beijing genotype was significantly associated with FQ resistance (odds ratio [OR], 2.39 [95% confidence interval {CI}, 1.34 to 4.25]; P = 0.003). Furthermore, Beijing genotype FQ-resistant isolates were significantly more likely than FQ-resistant isolates of other genotypes to have gyrA mutations (OR, 7.75 [95% CI, 2.84 to 21.15]; P = 0.0001) and high-level (>8 μg/ml) FQ resistance (OR, 11.0 [95% CI, 2.6 to 47.0]; P = 0.001). The underlying mechanism of the association of the Beijing genotype with high-level FQ resistance in this setting remains to be determined. The association of the Beijing genotype with relatively high-level FQ resistance conferred by specific gyrA mutations reported here is of grave concern given the epidemic spread of the Beijing genotype and the current hopes for shorter first-line treatment regimens based on FQs.

Evaluation of the MODS culture technique for the diagnosis of tuberculous meningitis.

Background Tuberculous meningitis (TBM) is a devastating condition. The rapid instigation of appropraite chemotherapy is vital to reduce morbidity and mortality. However rapid diagnosis remains elusive; smear microscopy has extremely low sensitivity on cerebrospinal fluid (CSF) in most laboratories and PCR requires expertise with advanced infrastructure and has sensitivity of only around 60% under optimal conditions. Neither technique allows for the microbiological isolation of M. tuberculosis and subsequent drug susceptibility testing. We evaluated the recently developed microscopic observation drug susceptibility (MODS) assay format for speed and accuracy in diagnosing TBM. Methodology/Principal Findings Two hundred and thirty consecutive CSF samples collected from 156 patients clinically suspected of TBM on presentation at a tertiary referal hospital in Vietnam were enrolled into the study over a five month period and tested by Ziehl-Neelsen (ZN) smear, MODS, Mycobacterial growth Indicator tube (MGIT) and Lowenstein-Jensen (LJ) culture. Sixty-one samples were from patients already on TB therapy for >1day and 19 samples were excluded due to untraceable patient records. One hundred and fifty samples from 137 newly presenting patients remained. Forty-two percent (n = 57/137) of patients were deemed to have TBM by clinical diagnostic and microbiological criteria (excluding MODS). Sensitivity by patient against clinical gold standard for ZN smear, MODS MGIT and LJ were 52.6%, 64.9%, 70.2% and 70.2%, respectively. Specificity of all microbiological techniques was 100%. Positive and negative predictive values for MODS were 100% and 78.7%, respectively for HIV infected patients and 100% and 82.1% for HIV negative patients. The median time to positive was 6 days (interquartile range 5–7), significantly faster than MGIT at 15.5 days (interquartile range 12–24), and LJ at 24 days (interquartile range 18–35 days) (P<0.01). Conclusions We have shown MODS to be a sensitive, rapid technique for the diagnosis of TBM with high sensitivity, ease of performance and low cost (0.53 USD/sample).

Microscopic Observation Drug Susceptibility Assay (MODS) for Early Diagnosis of Tuberculosis in Children

MODS is a novel liquid culture based technique that has been shown to be effective and rapid for early diagnosis of tuberculosis (TB). We evaluated the MODS assay for diagnosis of TB in children in Viet Nam. 217 consecutive samples including sputum (n = 132), gastric fluid (n = 50), CSF (n = 32) and pleural fluid (n = 3) collected from 96 children with suspected TB, were tested by smear, MODS and MGIT. When test results were aggregated by patient, the sensitivity and specificity of smear, MGIT and MODS against “clinical diagnosis” (confirmed and probable groups) as the gold standard were 28.2% and 100%, 42.3% and 100%, 39.7% and 94.4%, respectively. The sensitivity of MGIT and MODS was not significantly different in this analysis (P = 0.5), but MGIT was more sensitive than MODS when analysed on the sample level using a marginal model (P = 0.03). The median time to detection of MODS and MGIT were 8 days and 13 days, respectively, and the time to detection was significantly shorter for MODS in samples where both tests were positive (P<0.001). An analysis of time-dependent sensitivity showed that the detection rates were significantly higher for MODS than for MGIT by day 7 or day 14 (P<0.001 and P = 0.04), respectively. MODS is a rapid and sensitive alternative method for the isolation of M.tuberculosis from children.

Evaluation of Xpert MTB/RIF and MODS assay for the diagnosis of pediatric tuberculosis

BackgroundTuberculosis (TB) in children is rarely confirmed due to the lack of effective diagnostic tools; only 10 to 15% of pediatric TB is smear positive due to paucibacillary samples and the difficulty of obtaining high-quality specimens from children. We evaluate here the accuracy of Xpert MTB/RIF in comparison with the Micoroscopic observation drug susceptibility (MODS) assay for diagnosis of TB in children using samples stored during a previously reported evaluation of the MODS assay.MethodsNinety-six eligible children presenting with suspected TB were recruited consecutively at Pham Ngoc Thach Hospital in Ho Chi Minh City Viet Nam between May to December 2008 and tested by Ziehl-Neelsen smear, MODS and Mycobacterial growth Indicator (MGIT, Becton Dickinson) culture. All samples sent by the treating clinician for testing were included in the analysis. An aliquot of processed sample deposit was stored at −20°C and tested in the present study by Xpert MTB/RIF test. 183 samples from 73 children were available for analysis by Xpert. Accuracy measures of MODS and Xpert were summarized.ResultsThe sensitivity (%) in detecting children with a clinical diagnosis of TB for smear, MODS and Xpert were 37.9 [95% CI 25.5; 51.6], 51.7 [38.2; 65.0] and 50.0 [36.6; 63.4], respectively (per patient analysis). Xpert was significantly more sensitive than smear (P=0.046). Testing of additional samples did not increase case detection for MODS while testing of a second sputum sample by Xpert detected only two additional cases. The positive and negative predictive values (%) of Xpert were 100.0 [88.0; 100.0] and 34.1 [20.5; 49.9], respectively, while those of MODS were 96.8 [83.3; 99.9] and 33.3 [19.6; 49.5].ConclusionMODS culture and Xpert MTB/RIF test have similar sensitivities for the detection of pediatric TB. Xpert MTB RIF is able to detect tuberculosis and rifampicin resistance within two hours. MODS allows isolation of cultures for further drug susceptibility testing but requires approximately one week to become positive. Testing of multiple samples by xpert detected only two additional cases and the benefits must be considered against costs in each setting. Further research is required to evaluate the optimal integration of Xpert into pediatric testing algorithms.

Diagnosis of Pulmonary Tuberculosis in HIV-Positive Patients by Microscopic Observation Drug Susceptibility Assay

ABSTRACT The microscopic observation drug susceptibility assay (MODS) is a novel and promising test for the early diagnosis of tuberculosis (TB). We evaluated the MODS assay for the early diagnosis of TB in HIV-positive patients presenting to Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases in southern Vietnam. A total of 738 consecutive sputum samples collected from 307 HIV-positive individuals suspected of TB were tested by smear, MODS, and the mycobacteria growth indicator tube method (MGIT). The diagnostic sensitivity and specificity of MODS compared to the microbiological gold standard (either smear or MGIT) were 87 and 93%, respectively. The sensitivities of smear, MODS, and MGIT were 57, 71, and 75%, respectively, against clinical gold standard (MODS versus smear, P < 0.001; MODS versus MGIT, P = 0.03). The clinical gold standard was defined as patients who had a clinical examination and treatment consistent with TB, with or without microbiological confirmation. For the diagnosis of smear-negative patients, the sensitivities of MODS and MGIT were 38 and 45%, respectively (P = 0.08). The median times to detection using MODS and MGIT were 8 and 11 days, respectively, and they were 11 and 17 days, respectively, for smear-negative samples. The original bacterial/fungal contamination rate of MODS was 1.1%, while it was 2.6% for MGIT. The cross-contamination rate of MODS was 4.7%. In conclusion, MODS is a sensitive, specific, and rapid test that is appropriate for the detection of HIV-associated TB; its cost and ease of use make it particularly useful in resource-limited settings.

Evaluation of microscopic observation drug susceptibility assay for diagnosis of multidrug-resistant Tuberculosis in Viet Nam

BackgroundEarly diagnosis of tuberculosis (TB) and multidrug resistant tuberculosis (MDR TB) is important for the elimination of TB. We evaluated the microscopic observation drug susceptibility (MODS) assay as a direct rapid drug susceptibility testing (DST) method for MDR-TB screening in sputum samplesMethodsAll adult TB suspects, who were newly presenting to Pham Ngoc Thach Hospital from August to November 2008 were enrolled into the study. Processed sputum samples were used for DST by MODS (DST-MODS) (Rifampicin (RIF) 1 μg/ml and Isoniazid (INH) 0.4 μg/ml), MGIT culture (Mycobacterial Growth Indicator Tube) and Lowenstein Jensen (LJ) culture. Cultures positive by either MGIT or LJ were used for proportional DST (DST-LJ) (RIF 40 μg/ml and INH 0.2 μg/ml). DST profiles on MODS and LJ were compared. Discrepant results were resolved by multiplex allele specific PCR (MAS-PCR).ResultsSeven hundred and nine TB suspects/samples were enrolled into the study, of which 300 samples with DST profiles available from both MODS and DST-LJ were analyzed. Cording in MODS was unable to correctly identify 3 Mycobacteria Other Than Tuberculosis (MOTT) isolates, resulting in 3 false positive TB diagnoses. None of these isolates were identified as MDR-TB by MODS. The sensitivity and specificity of MODS were 72.6% (95%CI: 59.8, 83.1) and 97.9% (95%CI: 95.2, 99.3), respectively for detection of INH resistant isolates, 72.7% (95%CI: 30.9, 93.7) and 99.7% (95%CI: 98.1, 99.9), respectively for detecting RIF resistant isolates and 77.8% (95%CI: 39.9, 97.1) and 99.7% (95%CI: 98.1, 99.9), respectively for detecting MDR isolates. The positive and negative predictive values (PPV and NPV) of DST-MODS were 87.5% (95%CI: 47.3, 99.6) and 99.3% (95%CI: 97.5, 99.9) for detection of MDR isolates; and the agreement between MODS and DST-LJ was 99.0% (kappa: 0.8, P < 0.001) for MDR diagnosis. The low sensitivity of MODS for drug resistance detection was probably due to low bacterial load samples and the high INH concentration (0.4 μg/ml). The low PPV of DST-MODS may be due to the low MDR-TB rate in the study population (3.8%). The turnaround time of DST-MODS was 9 days and 53 days for DST-LJ.ConclusionThe DST-MODS technique is rapid with low contamination rates. However, the sensitivity of DST-MODS for detection of INH and RIF resistance in this study was lower than reported from other settings.

Prognostic Models for 9-Month Mortality in Tuberculous Meningitis

Prognostic models developed and validated using data from 1699 adults with tuberculous meningitis (TBM), with or without human immunodeficiency virus infection, performed well and could be used in clinical practice to identify patients with TBM at high risk of death.

Scale-up of diagnostics for multidrug resistant tuberculosis.

656 www.thelancet.com/infection Vol 10 October 2010 How can the number of new HIV infections in MSM living in France and elsewhere be reduced? A combined prevention approach, as proposed by Michel Sidibé (the Executive Director of UNAIDS), is needed. This approach should include targeted structural interventions directed at specifi c populations, behavioural interventions directed at individuals, and new biomedical interventions, including expanded coverage of antiretroviral therapy to all HIV infected individuals who meet eligibility criteria for treatment. Despite a large body of evidence for the secondary preventive value of antiretroviral therapy, the HIV/AIDS community has failed to fully capitalise on the synergy between treatment and prevention. The aim should not be improved treatment or improved prevention, but rather optimisation of both, because treatment is prevention. Recent therapeutic guidelines fully recognise that late initiation of antiretroviral therapy is associated with worse outcomes both at the individual and societal scales. From a public health perspective, expansion of the number of individuals with HIV infection who are eligible for therapy is a unique opportunity to curb the growth of the epidemic. This expansion should decrease HIV/AIDS-related morbidity and mortality in those infected and help to reduce community viral load (and consequently the risk of new infections). The status quo cannot remain.

Frequent transmission of the Mycobacterium tuberculosis Beijing lineage and positive selection for the EsxW Beijing variant in Vietnam

To examine the transmission dynamics of Mycobacterium tuberculosis (Mtb) isolated from tuberculosis patients in Ho Chi Minh City, Vietnam, we sequenced the whole genomes of 1,635 isolates and compared these with 3,144 isolates from elsewhere. The data identify an underlying burden of disease caused by the endemic Mtb lineage 1 associated with the activation of long-term latent infection, and a threefold higher burden associated with the more recently introduced Beijing lineage and lineage 4 Mtb strains. We find that Beijing lineage Mtb is frequently transferred between Vietnam and other countries, and detect higher levels of transmission of Beijing lineage strains within this host population than the endemic lineage 1 Mtb. Screening for parallel evolution of Beijing lineage-associated SNPs in other Mtb lineages as a signal of positive selection, we identify an alteration in the ESX-5 type VII-secreted protein EsxW, which could potentially contribute to the enhanced transmission of Beijing lineage Mtb in Vietnamese and other host populations.Genomic analysis of Mycobacterium tuberculosis (Mtb) isolated from tuberculosis patients identifies the transmission dynamics of Mtb in Vietnam including frequent transmission of Beijing lineage and positive selection for EsxW Beijing variant.