ABSTRACT
BACKGROUND: Mixed M. tuberculosis (MTB) infection occurs when one is infected with more than one clonally distinct MTB strain. This form of infection can assist MTB strains to acquire additional mutations, facilitate the spread of drug-resistant strains, and boost the rate of treatment failure. Hence, the presence of mixed MTB infection could affect the performance of some rapid molecular diagnostic tests such as Line Probe Assay (LPA) and GeneXpert MTB/RIF (Xpert) assays. METHODS: This was a cross-sectional study that used sputum specimens collected from participants screened for STREAM 2 clinical trial between October 2017 and October 2019. Samples from 62 MTB smear-positive patients and rifampicin-resistant patients from peripheral health facilities were processed for Xpert and LPA as screening tests for eligibility in the trial. From November 2020, processed stored sputum samples were retrieved and genotyped to determine the presence of mixed-MTB strain infection using a standard 24-locus Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem-Repeat (MIRU-VNTR). Samples with at least 20/24 MIRU-VNTR loci amplified were considered for analysis. Agar proportional Drug Susceptibility Test (DST) was performed on culture isolates of samples that had discordant results between LPA and Xpert. The impact of the presence of mixed-MTB strain on Xpert and LPA test interpretation was analyzed. RESULTS: A total of 53/62 (85%) samples had analyzable results from MIRU-VNTR. The overall prevalence of mixed-MTB infection was 5/53 (9.4%). The prevalence was highest among male's 3/31 (9.7%) and among middle-aged adults, 4/30 (33.3%). Lineage 4 of MTB contributed 3/5 (60.0%) of the mixed-MTB infection prevalence. Having mixed MTB strain infection increased the odds of false susceptible Xpert test results (OR 7.556, 95% CI 0.88-64.44) but not for LPA. Being HIV-positive (P = 0.04) independently predicted the presence of mixed MTB infection. CONCLUSIONS: The presence of mixed-MTB strain infection may affect the performance of the GeneXpert test but not for LPA. For patients with high pre-test probability of rifampicin resistance, an alternative rapid method such as LPA should be considered.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Tuberculosis , Adult , Middle Aged , Humans , Male , Rifampin/pharmacology , Rifampin/therapeutic use , Uganda/epidemiology , Cross-Sectional Studies , Pathology, Molecular , Mycobacterium tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/diagnosis , Tuberculosis, Multidrug-Resistant/drug therapyABSTRACT
BACKGROUND: The World Health Organization endorsed Truenat MTB rapid molecular assay in 2020 and recommended additional in-country evaluation studies before uptake. We evaluated the accuracy and operational feasibility of Truenat MTB assay (Truenat) in comparison with GeneXpert Ultra and culture. METHODS: In a cross-sectional study of 250 presumptive TB patients, participants were requested to provide a sputum sample on the day of their visit to the clinic. The sputum sample was homogenized and a portion was tested using GeneXpert Ultra as per the routine standard procedure and the other portion was tested using Truenat assay at the clinic laboratory. The second sample portion was processed for Concentrated Fluorescent smear Microscopy (CFM), LJ, and MGIT cultures. Truenat sensitivity and specificity were compared to GeneXpert Ultra and culture. Test performance characteristics and operational feasibility assessment data through interview of the study laboratory staff were also collected and summarized as proportions and percentages. RESULTS: Of the 250 participants recruited in the study, the sensitivity and specificity of Truenat was n/N (%, 95%CI); 66/82 (80.5, 70.2-88.4) and 156/159 (98.1, 94.5-99.6) when compared with Ultra, 50/64 (89.3, 66.0-87.4) and 166/180 (92.2, 87.2-95.6) when compared with LJ, 58/71 (81.7,70.7-89.8) and 131/138 (94.9, 89.8-97.9) when compared to MGIT culture and 59/73 (80.8, 69.9-89.1) and 159/169 (94.1,89.3-97.1) when compared to LJ and/or MGIT culture. The sensitivity of Truenat was lower, 14/23 (60.9, 40.6-82.8) among smear-negative compared to 45/50 (90.0, 78.1-96.6) among smear-positive participants but not different by HIV status. There were no special training needs especially among laboratory personnel with previous GeneXpert /molecular test experience, 19/242 (7.8%) error/invalid, and 12 (17,4%) uninterpretable/indeterminate results mainly for rifampicin resistance determination. However, there were 3 (3.5%) of GeneXpert Ultra indeterminate results. CONCLUSION: Among presumptive TB patients in Uganda, the Truenat assay has high sensitivity and specificity. The Truenat assay has acceptable operational feasibility attributes when compared with the GeneXpert Assay.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Humans , Rifampin , Mycobacterium tuberculosis/genetics , Uganda , Cross-Sectional Studies , Sputum , Tuberculosis, Pulmonary/diagnosis , Sensitivity and SpecificityABSTRACT
BACKGROUND: Tuberculosis (TB) is a major cause of mortality worldwide. Children and people living with HIV (PLHIV) have an increased risk of mortality, particularly in the absence of rapid diagnosis. The main challenges of diagnosing TB in these populations are due to the unspecific and paucibacillary disease presentation and the difficulty of obtaining respiratory samples. Thus, novel diagnostic strategies, based on non-respiratory specimens could improve clinical decision making and TB outcomes in high burden TB settings. We propose a multi-country, prospective diagnostic evaluation study with a nested longitudinal cohort evaluation to assess the performance of a new stool-based qPCR, developed by researchers at Baylor College of Medicine (Houston, Texas, USA) for TB bacteriological confirmation with promising results in pilot studies. METHODS: The study will take place in high TB/HIV burden countries (Mozambique, Eswatini and Uganda) where we will enroll, over a period of 30 months, 650 PLHIV (> 15) and 1295 children under 8 years of age (irrespective of HIV status) presenting pressumptive TB. At baseline, all participants will provide clinical history, complete a physical assessment, and undergo thoracic chest X-ray imaging. To obtain bacteriological confirmation, participants will provide respiratory samples (1 for adults, 2 in children) and 1 stool sample for Xpert Ultra MTB/RIF (Cepheid, Sunnyvale, CA, USA). Mycobacterium tuberculosis (M.tb) liquid culture will only be performed in respiratory samples and lateral flow lipoarabinomannan (LF-LAM) in urine following WHO recommendations. Participants will complete 2 months follow-up if they are not diagnosed with TB, and 6 months if they are. For analytical purposes, the participants in the pediatric cohort will be classified into "confirmed tuberculosis", "unconfirmed tuberculosis" and "unlikely tuberculosis". Participants of the adult cohort will be classified as "bacteriologically confirmed TB", "clinically diagnosed TB" or "not TB". We will assess accuracy of the novel qPCR test compared to bacteriological confirmation and Tb diagnosis irrespective of laboratory results. Longitudinal qPCR results will be analyzed to assess its use as treatment response monitoring. DISCUSSION: The proposed stool-based qPCR is an innovation because both the strategy of using a non-sputum based sample and a technique specially designed to detect M.tb DNA in stool. PROTOCOL REGISTRATION DETAILS: ClinicalTrials.gov Identifier: NCT05047315.
Subject(s)
HIV Infections , Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Tuberculosis , Adult , Child , Humans , Eswatini , HIV Infections/complications , HIV Infections/diagnosis , Mozambique , Multicenter Studies as Topic , Prospective Studies , Sensitivity and Specificity , Tuberculosis/diagnosis , Tuberculosis, Pulmonary/diagnosis , UgandaABSTRACT
BACKGROUND: Second-line drug resistance (SLD) among tuberculosis (TB) patients is a serious emerging challenge towards global control of the disease. We characterized SLD-resistance conferring-mutations among TB patients with rifampicin and/or isoniazid (RIF and/or INH) drug-resistance tested at the Uganda National TB Reference Laboratory (NTRL) between June 2017 and December 2019. METHODS: This was a descriptive cross-sectional secondary data analysis of 20,508 M. tuberculosis isolates of new and previously treated patients' resistant to RIF and/or INH. DNA strips with valid results to characterise the SLD resistance using the commercial Line Probe Assay Genotype MTBDRsl Version 2.0 Assay (Hain Life Science, Nehren, Germany) were reviewed. Data were analysed with STATAv15 using cross-tabulation for frequency and proportions of known resistance-conferring mutations to injectable agents (IA) and fluoroquinolones (FQ). RESULTS: Among the eligible participants, 12,993/20,508 (63.4%) were male and median (IQR) age 32 (24-43). A total of 576/20,508 (2.8%) of the M. tuberculosis isolates from participants had resistance to RIF and/or INH. These included; 102/576 (17.7%) single drug-resistant and 474/576 (82.3%) multidrug-resistant (MDR) strains. Only 102 patients had test results for FQ of whom 70/102 (68.6%) and 01/102 (0.98%) had resistance-conferring mutations in the gyrA locus and gyrB locus respectively. Among patients with FQ resistance, gyrAD94G 42.6% (30.0-55.9) and gyrA A90V 41.1% (28.6-54.3) mutations were most observed. Only one mutation, E540D was detected in the gyrB locus. A total of 26 patients had resistance-conferring mutations to IA in whom, 20/26 77.0% (56.4-91.0) had A1401G mutation in the rrs gene locus. CONCLUSIONS: Our study reveals a high proportion of mutations known to confer high-level fluoroquinolone drug-resistance among patients with rifampicin and/or isoniazid drug resistance. Utilizing routinely generated laboratory data from existing molecular diagnostic methods may aid real-time surveillance of emerging tuberculosis drug-resistance in resource-limited settings.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Adult , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Cross-Sectional Studies , Drug Resistance, Multiple, Bacterial/genetics , Female , Fluoroquinolones/therapeutic use , Humans , Isoniazid/pharmacology , Isoniazid/therapeutic use , Male , Microbial Sensitivity Tests , Mutation , Rifampin/pharmacology , Rifampin/therapeutic use , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/epidemiology , Uganda/epidemiology , Young AdultABSTRACT
BACKGROUND: Tuberculosis (TB) and its risk factors are independently associated with cardiovascular disease (CVD). We determined the prevalence and associations of CVD risk factors among people with drug-resistant tuberculosis (DRTB) in Uganda. METHODS: In this cross-sectional study, we enrolled people with microbiologically confirmed DRTB at four treatment sites in Uganda between July to December 2021. The studied CVD risk factors were any history of cigarette smoking, diabetes mellitus (DM) hypertension, high body mass index (BMI), central obesity and dyslipidaemia. We used modified Poisson regression models with robust standard errors to determine factors independently associated with each of dyslipidaemia, hypertension, and central obesity. RESULTS: Among 212 participants, 118 (55.7%) had HIV. Overall, 196 (92.5%, 95% confidence interval (CI) 88.0-95.3) had ≥ 1 CVD risk factor. The prevalence; 95% CI of individual CVD risk factors was: dyslipidaemia (62.5%; 55.4-69.1), hypertension (40.6%; 33.8-47.9), central obesity (39.3%; 32.9-46.1), smoking (36.3%; 30.1-43.1), high BMI (8.0%; 5.0-12.8) and DM (6.5%; 3.7-11.1). Dyslipidaemia was associated with an increase in glycated haemoglobin (adjusted prevalence ratio (aPR) 1.14, 95%CI 1.06-1.22). Hypertension was associated with rural residence (aPR 1.89, 95% CI 1.14-3.14) and previous history of smoking (aPR 0.46, 95% CI 0.21-0.98). Central obesity was associated with increasing age (aPR 1.02, 95%CI 1.00-1.03), and elevated diastolic blood pressure (aPR 1.03 95%CI 1.00-1.06). CONCLUSION: There is a high prevalence of CVD risk factors among people with DRTB in Uganda, of which dyslipidaemia is the commonest. We recommend integrated services for identification and management of CVD risk factors in DRTB.
Subject(s)
Cardiovascular Diseases , Diabetes Mellitus , Dyslipidemias , Hypertension , Tuberculosis, Multidrug-Resistant , Humans , Cardiovascular Diseases/diagnosis , Cardiovascular Diseases/epidemiology , Cardiovascular Diseases/etiology , Risk Factors , Cross-Sectional Studies , Obesity, Abdominal/diagnosis , Obesity, Abdominal/epidemiology , Obesity, Abdominal/complications , Uganda/epidemiology , Tuberculosis, Multidrug-Resistant/diagnosis , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/epidemiology , Hypertension/diagnosis , Hypertension/epidemiology , Hypertension/complications , Dyslipidemias/diagnosis , Dyslipidemias/epidemiology , Dyslipidemias/complications , Heart Disease Risk Factors , Diabetes Mellitus/diagnosis , Diabetes Mellitus/epidemiology , Prevalence , Obesity/complicationsABSTRACT
BACKGROUND: Chest X-ray (CXR) interpretation remains a central component of the current World Health Organization recommendations as an adjuvant test in diagnosis of smear-negative tuberculosis (TB). With its low specificity, high maintenance and operational costs, utility of CXR in diagnosis of smear-negative TB in high HIV/TB burden settings in the Xpert MTB/RIF era remains unpredictable. We evaluated accuracy and additive value of CXR to Xpert MTB/RIF in the diagnosis of TB among HIV-positive smear-negative presumptive TB patients. METHODS: HIV co-infected presumptive TB patients were recruited from the Infectious Diseases Institute outpatient clinic and in-patient medical wards of Mulago Hospital, Uganda. CXR films were reviewed by two independent radiologists using a standardized evaluation form. CXR interpretation with regard to TB was either positive (consistent with TB) or negative (normal or unlikely TB). Sensitivity, specificity and predictive values of CXR and CXR combined with Xpert MTB/RIF for diagnosis of smear-negative TB in HIV-positive patients were calculated using sputum and/or blood mycobacterial culture as reference standard. RESULTS: Three hundred sixty-six HIV co-infected smear-negative participants (female, 63.4%; hospitalized, 68.3%) had technically interpretable CXR. Median (IQR) age was 32 (28-39) years and CD4 count 112 (23-308) cells/mm3. Overall, 22% (81/366) were positive for Mycobacterium tuberculosis (Mtb) on culture; 187/366 (51.1%) had CXR interpreted as consistent with TB, of which 55 (29.4%) had culture-confirmed TB. Sensitivity and specificity of CXR interpretation in diagnosis of culture-positive TB were 67.9% (95%CI 56.6-77.8) and 53.7% (95%CI 47.7-59.6) respectively, while Xpert MTB/RIF sensitivity and specificity were 65.4% (95%CI 54.0-75.7) and 95.8% (95%CI 92.8-97.8) respectively. Addition of CXR to Xpert MTB/RIF had overall sensitivity and specificity of 87.7% (95%CI 78.5-93.9) and 51.6% (95%CI 45.6-57.5) respectively; 86.2% (95%CI 75.3-93.5) and 48.1% (95%CI 40.7-55.6) among inpatients and 93.8% (95%CI 69.8-99.8) and 58.0% (95%CI 47.7-67.8) among outpatients respectively. CONCLUSION: In this high prevalence TB/HIV setting, CXR interpretation added sensitivity to Xpert MTB/RIF test at the expense of specificity in the diagnosis of culture-positive TB in HIV-positive individuals presenting with TB symptoms and negative smear. CXR interpretation may not add diagnostic value in settings where Xpert MTB/RIF is available as a TB diagnostic tool.
Subject(s)
AIDS-Related Opportunistic Infections/complications , Coinfection/diagnosis , HIV/isolation & purification , Mass Chest X-Ray/methods , Mycobacterium tuberculosis/genetics , Nucleic Acid Amplification Techniques/methods , Tuberculosis, Pulmonary/complications , Tuberculosis, Pulmonary/diagnosis , AIDS-Related Opportunistic Infections/epidemiology , AIDS-Related Opportunistic Infections/virology , Adult , CD4 Lymphocyte Count , Coinfection/epidemiology , Coinfection/virology , Data Accuracy , Female , Health Resources , Humans , Male , Sensitivity and Specificity , Sputum/microbiology , Tuberculosis, Pulmonary/epidemiology , Tuberculosis, Pulmonary/microbiology , Uganda/epidemiologyABSTRACT
BACKGROUND: In resource-limited settings, sputum smear conversion is used to document treatment response. Many People living with HIV (PLHIV) are smear-negative at baseline. The Xpert MTB/RIF test can indirectly measure bacterial load through cycle threshold (ct) values. This study aimed to determine if baseline Xpert MTB/RIF could predict time to culture negativity in PLHIV with newly diagnosed TB. METHODS: A subset of 138 PLHIV from the 'SOUTH' study on outcomes related to TB and antiretroviral drug concentrations were included. Bacterial load was estimated by Mycobacterium Growth Indicator Tubes (MGIT) culture time-to-positivity (TTP) and Lowenstein Jensen (LJ) colony counts. Changes in TTP and colony counts were analyzed with Poisson Generalised Estimating Equations (GEE) and multilevel ordered logistic regression models, respectively, while time to culture negativity analysed with Cox proportional hazard models. ROC curves were used to explore the accuracy of the ct value in predicting culture negativity. RESULTS: A total of 81 patients (58.7%) were males, median age 34 (IQR 29 ̶ 40) years, median CD4 cell count of 180 (IQR 68 ̶ 345) cells/µL and 77.5% were ART naive. The median baseline ct value was 25.1 (IQR 21.0 ̶ 30.1). A unit Increase in the ct value was associated with a 5% (IRR = 1.05 95% CI 1.04 ̶ 1.06) and 3% (IRR = 1.03 95% CI 1.03 ̶ 1.04) increase in TTP at week 2 and 4 respectively. With LJ culture, a patient's colony grade was reduced by 0.86 times (0R = 0.86 95% CI 0.74 ̶ 0.97) at week 2 and 0.84 times (OR = 0.84 95% CI 0.79 ̶ 0.95 P = 0.002) at week 4 for every unit increase in the baseline ct value. There was a 3% higher likelihood of earlier conversion to negativity for every unit increase in the ct value. A ct cut point ≥28 best predicted culture negativity at week 4 with a sensitivity of 91. 7% & specificity 53.7% while a cut point ≥23 best predicted culture negativity at week 8. CONCLUSION: Baseline Xpert MTB/RIF ct values predict sputum conversion in PLHIV on anti-TB treatment. Surrogate biomarkers for sputum conversion in PLHIV are still a research priority.
Subject(s)
Bacterial Load/methods , HIV Infections/epidemiology , Mycobacterium tuberculosis/isolation & purification , Sputum/microbiology , Tuberculosis, Pulmonary/diagnosis , Adult , Anti-Retroviral Agents/blood , Antitubercular Agents/therapeutic use , CD4 Lymphocyte Count , Colony Count, Microbial , Female , HIV Infections/blood , Humans , Male , Nucleic Acid Amplification Techniques , Odds Ratio , Retrospective Studies , Sensitivity and Specificity , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/epidemiology , Uganda/epidemiologyABSTRACT
Childhood tuberculosis (TB) presents significant diagnostic challenges associated with paucibacillary disease and requires a more sensitive test. We evaluated the diagnostic accuracy of Xpert MTB/RIF Ultra (Ultra) compared to other microbiological tests using respiratory samples from Ugandan children in the SHINE trial. SHINE is a randomized trial evaluating shorter treatment in 1,204 children with minimal TB disease in Africa and India. Among 352 samples and one cervical lymph node fine needle aspirate, one sample was randomly selected per patient and tested with the Xpert MTB/RIF assay (Xpert) and with Lowenstein-Jensen medium (LJ) and liquid mycobacterial growth indicator tube (MGIT) cultures. We selected only uncontaminated stored sample pellets for Ultra testing. We estimated the sensitivity of Xpert and Ultra against culture and a composite microbiological reference standard (any positive result). Of 398 children, 353 (89%) had culture, Xpert, and Ultra results. The median age was 2.8 years (interquartile range [IQR], 1.3 to 5.3); 8.5% (30/353) were HIV infected, and 54.4% (192/353) were male. Of the 353, 31 (9%) were positive by LJ and/or MGIT culture, 36 (10%) by Ultra, and 16 (5%) by Xpert. Sensitivities (95% confidence intervals [CI]) were 58% (39 to 65% [18/31]) for Ultra and 45% (27 to 64% [14/31]) for Xpert against any culture-positive result, with false positives of <1% and 5.5% for Xpert and Ultra. Against a composite microbiological reference, sensitivities were 72% (58 to 84% [36/50]) for Ultra and 32% (20 to 47% [16/50]) for Xpert. However, there were 17 samples that were positive only with Ultra (majority trace). Among children screened for minimal TB in Uganda, Ultra has higher sensitivity than Xpert. This represents an important advance for a condition which has posed a diagnostic challenge for decades.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Child , Child, Preschool , Female , Humans , India , Male , Mycobacterium tuberculosis/genetics , Sensitivity and Specificity , Sputum , Tuberculosis, Pulmonary/diagnosis , UgandaABSTRACT
BACKGROUND: Smear microscopy lacks sensitivity especially in HIV co-infection, resulting in undiagnosed tuberculosis (TB) and high mortality. The loop-mediated isothermal amplification (TB-LAMP) assay can be staged with minimal infrastructure, is rapid, low cost and detection can be with the naked eye. We assessed feasibility and performance of Eiken TB-LAMP test at point-of-need in TB diagnosis in a high prevalence TB/HIV rural setting in Uganda. METHODS: From October 2013-February 2014, TB-LAMP testing was performed on sputum specimens from outpatient presumptive TB adults at a district hospital and two low-level health centers in Kiboga District where smear microscopy is the available routine diagnostic option. TB-LAMP was performed by a technician after a week of training in the district hospital. The technician had no prior experience in the technology. Samples from the low-level health centers were transported to the district hospital for TB-LAMP. RESULTS: Of the 233 presumptive TB (126 at hospital); 113 (48.5%) were HIV-infected; 129 (55%) male; median age 40 (IQR 30-53). Compared to MTB culture, overall sensitivity and specificity of TB-LAMP were 55.4% (95 CI 44.1-66.3) and 98.0% (95 CI 94.3-99.6) respectively. Among HIV-infected participants, TB-LAMP sensitivity and specificity were 52.3% (95 CI 36.7-67.5%) and 97.1% (95 CI 89.9-99.6) respectively; and 24.4% (95% CI 12.9-39.5) and 98.6% (95% CI 95.1-99.8) respectively among smear-negatives. TB-LAMP sensitivity and specificity were 62.2% (95% CI 44.8-77.5) and 97.8% (95% CI 92.1-99.7) in the hospital setting where central testing occurred compared to 50.0% (95% CI 34.9-65.1) and 98.4% (95% CI 91.2-100) respectively in low-level health centers where specimens were transported centrally. CONCLUSIONS: In this high prevalence TB/HIV rural setting, TB-LAMP performs better than conventional smear microscopy in diagnosis of MTB among presumptive TB patients although the sensitivity is lower than that reported by the World Health Organization. TB-LAMP can easily be performed following a short training period and in absence of sophisticated infrastructure and expertise.
Subject(s)
HIV Infections/diagnosis , Mycobacterium tuberculosis/genetics , Tuberculosis, Pulmonary/diagnosis , Adult , Bacteriological Techniques , Female , HIV Infections/complications , Humans , Male , Middle Aged , Mycobacterium tuberculosis/isolation & purification , Nucleic Acid Amplification Techniques , Prevalence , Rural Population , Sensitivity and Specificity , Sputum/microbiology , Tuberculosis, Pulmonary/complications , Tuberculosis, Pulmonary/epidemiology , Tuberculosis, Pulmonary/microbiology , Uganda/epidemiologyABSTRACT
BACKGROUND: In the context of advanced immunosuppression, M. tuberculosis is known to cause detectable mycobacteremia. However, little is known about the intra-patient mycobacterial microevolution and the direction of seeding between the sputum and blood compartments. METHODS: From a diagnostic study of HIV-infected TB patients, 51 pairs of concurrent blood and sputum M. tuberculosis isolates from the same patient were available. In a previous analysis, we identified a subset with genotypic concordance, based on spoligotyping and 24 locus MIRU-VNTR. These paired isolates with identical genotypes were analyzed by whole genome sequencing and phylogenetic analysis. RESULTS: Of the 25 concordant pairs (49 % of the 51 paired isolates), 15 (60 %) remained viable for extraction of high quality DNA for whole genome sequencing. Two patient pairs were excluded due to poor quality sequence reads. The median CD4 cell count was 32 (IQR; 16-101)/mm(3) and ten (77 %) patients were on ART. No drug resistance mutations were identified in any of the sequences analyzed. Three (23.1 %) of 13 patients had SNPs separating paired isolates from blood and sputum compartments, indicating evidence of microevolution. Using a phylogenetic approach to identify the ancestral compartment, in two (15 %) patients the blood isolate was ancestral to the sputum isolate, in one (8 %) it was the opposite, and ten (77 %) of the pairs were identical. CONCLUSIONS: Among HIV-infected patients with poor cellular immunity, infection with multiple strains of M. tuberculosis was found in half of the patients. In those patients with identical strains, whole genome sequencing indicated that M. tuberculosis intra-patient microevolution does occur in a few patients, yet did not reveal a consistent direction of spread between sputum and blood. This suggests that these compartments are highly connected and potentially seed each other repeatedly.
Subject(s)
Blood/microbiology , Evolution, Molecular , HIV Infections/microbiology , Mycobacterium tuberculosis/genetics , Sputum/microbiology , Tuberculosis/microbiology , AIDS-Related Opportunistic Infections/microbiology , Adult , Bacterial Typing Techniques/methods , Blood/virology , Female , Genome, Bacterial , Genotype , HIV Infections/complications , HIV-1 , Humans , Interspersed Repetitive Sequences , Male , Middle Aged , Minisatellite Repeats , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/isolation & purification , Phylogeny , Polymorphism, Single Nucleotide , Sequence Analysis, DNA/methods , Tuberculosis/complications , Young AdultABSTRACT
BACKGROUND: Tuberculosis control program of Rwanda is currently phasing in light emitting diode-fluorescent microscopy (LED-FM) as an alternative to Ziehl-Neelsen (ZN) smear microscopy. This, alongside the newly introduced Xpert (Cepheid, Sunnyvale, CA, USA) is expected to improve diagnosis of tuberculosis and detection of rifampicin resistance in patients at health facilities. We assessed the accuracy of smear microscopy and the incremental sensitivity of Xpert at tuberculosis laboratories in Rwanda. METHODS: This was a cross-sectional study involving four laboratories performing ZN and four laboratories performing LED-FM microscopy. The laboratories include four intermediate (ILs) and four peripheral (PLs) laboratories. After smear microscopy, the left-over of samples, of a single early-morning sputum from 648 participants, were tested using Xpert and mycobacterial culture as a reference standard. Sensitivity of each test was compared and the incremental sensitivity of Xpert after a negative smear was assessed. RESULTS: A total of 96 presumptive pulmonary tuberculosis participants were culture positive for M. tuberculosis. The overall sensitivity in PL of ZN was 55.1 % (40.2-69.3 %), LED-FM was 37 % (19.4-57.6 %) and Xpert was 77.6 % (66.6-86.4 %) whereas in ILs the same value for ZN was 58.3 % (27.7-84.8 %), LED-FM was 62.5 % (24.5-91.5 %) and Xpert was 90 (68.3-98.8 %). The sensitivity for all tests was significantly higher among HIV-negative individuals (all test p <0.05). The overall incremental sensitivity of Xpert over smear microscopy was 32.3 %; p < 0.0001. The incremental sensitivity of Xpert was statistically significant for both smear methods at PL (32.9 %; p = 0.001) but not at the ILs (30 %; p = 0.125) for both smear methods. CONCLUSIONS: Our study findings of the early implementation of the LED-FM did not reveal significant increment in sensitivity compared to the method being phased out (ZN). This study showed a significant incremental sensitivity for Xpert from both smear methods at peripheral centers where majority of TB patients are diagnosed. Overall our findings support the recommendation for Xpert as an initial diagnostic test in adults and children presumed to have TB.
Subject(s)
Microscopy, Fluorescence/methods , Tuberculosis, Pulmonary/diagnosis , Adolescent , Adult , Cross-Sectional Studies , Diagnostic Tests, Routine , Drug Resistance, Bacterial , Female , Health Facilities , Humans , Laboratories , Male , Middle Aged , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/pathogenicity , Rifampin/therapeutic use , Rwanda , Sensitivity and Specificity , Sputum/microbiology , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/microbiology , Young AdultABSTRACT
The Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection of Mycobacterium tuberculosis in sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 3:1. We evaluated whether a decreased sample reagent/pellet ratio of 2:1 increased Xpert sensitivity compared to the recommended 3:1. The limit of detection was determined by inoculating serial dilutions of M. tuberculosis into sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained from M. tuberculosis culture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 3:1 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 2:1. The proportion of specimens in which M. tuberculosis was detected was greater at 2:1 than at 3:1 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 2:1 was greater than at 3:1 overall (80% versus 72%; P=0.03) and for smear-negative specimens (67% versus 58%; P=0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increased M. tuberculosis detection, especially for paucibacillary specimens. Our study supports use of a 2:1 sample reagent/pellet dilution for Xpert testing of sputum pellets.
Subject(s)
Drug Resistance, Bacterial , Genotyping Techniques/methods , Molecular Diagnostic Techniques/methods , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/isolation & purification , Sputum/microbiology , Tuberculosis, Pulmonary/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Indicators and Reagents , Male , Middle Aged , Sensitivity and Specificity , Tuberculosis, Pulmonary/microbiology , Young AdultABSTRACT
BACKGROUND: Sputum smear microscopy for tuberculosis (TB) diagnosis lacks sensitivity in HIV-infected symptomatic patients and increases the likelihood that mycobacterial infections particularly disseminated TB will be missed; delays in diagnosis can be fatal. Given the duration for MTB growth in blood culture, clinical predictors of MTB bacteremia may improve early diagnosis of mycobacteremia. We describe the predictors and mortality outcome of mycobacteremia among HIV-infected sputum smear-negative presumptive TB patients in a high prevalence HIV/TB setting. METHODS: Between January and November 2011, all consenting HIV-infected adults suspected to have TB (presumptive TB) were consecutively enrolled. Diagnostic assessment included sputum smear microscopy, urine Determine TB lipoarabinomannan (LAM) antigen test, mycobacterial sputum and blood cultures, chest X-ray, and CD4 cell counts in addition to clinical and socio-demographic data. Patients were followed for 12 months post-enrolment. RESULTS: Of 394 sputum smear-negative participants [female, 63.7%; median age (IQR) 32 (28-39) years], 41/394 (10.4%) had positive mycobacterial blood cultures (mycobacteremia); all isolates were M. tuberculosis (MTB). The median CD4 cell count was significantly lower among patients with mycobacteremia when compared with those without (CD4 31 versus 122 cells/µL, p < 0.001). In a multivariate analysis, male gender [OR 3.4, 95%CI (1.4-7.6), p = 0.005], CD4 count <100 cells/µL [OR 3.1, 95% CI (1.1-8.6), p = 0.030] and a positive lateral flow urine TB LAM antigen test [OR 15.3, 95%CI (5.7-41.1), p < 0.001] were significantly associated with mycobacteremia. At 12 months of follow-up, a trend towards increased mortality was observed in patients that were MTB blood culture positive (35.3%) compared with those that were MTB blood culture negative (23.3%) (p = 0.065). CONCLUSIONS: Mycobacteremia occurred in 10% of smear-negative patients and was associated with higher mortality compared with smear-negative patients without mycobacteremia. Advanced HIV disease (CD4 < 100 cells/mm(3)), male gender and positive lateral flow urine TB LAM test predicted mycobacteremia in HIV-infected smear-negative presumptive TB patients in this high prevalence TB/HIV setting.
Subject(s)
HIV Infections/complications , HIV Infections/epidemiology , Tuberculosis/diagnosis , Tuberculosis/etiology , Adult , CD4 Lymphocyte Count , Cytodiagnosis , Female , HIV Infections/diagnosis , HIV Infections/microbiology , Humans , Male , Mycobacterium tuberculosis/isolation & purification , Prevalence , Prognosis , Risk Factors , Sensitivity and Specificity , Sputum/microbiology , Tuberculosis/epidemiology , Tuberculosis/microbiology , Uganda/epidemiologyABSTRACT
BACKGROUND: Prevalence of multidrug resistant tuberculosis (MDR-TB), defined as in vitro resistance to both rifampicin and isoniazid with or without resistance to other TB drugs, in sub-Saharan Africa (SSA) is reportedly low compared to other regions. These estimates are based on data reported to the World Health Organization (WHO) on drug resistance surveys, which may suffer from a reporting bias. We set out to evaluate the variation in prevalence of drug resistant tuberculosis (DR-TB) and its determinants across SSA countries among new and previously treated TB patients. METHODS: The aim was to perform a systematic review and meta-analysis of DR-TB prevalence and associated risk factors in SSA. PubMed, EMBASE, Cochrane and bibliographies of DR-TB studies were searched. Surveys at national or sub-national level, with reported DR-TB prevalence (or sufficient data to calculate a prevalence) to isoniazid (INH), rifampicin (RMP), ethambutol (EMB), and streptomycin (SM) conducted in SSA excluding the Republic of South Africa, published between 2003 and 2013 with no language restriction were considered. Two authors searched and reviewed the studies for eligibility and extracted the data in pre-defined forms. Forest plots of all prevalence estimates by resistance outcome were performed. Summary estimates were calculated using random effects models, when appropriate. Associations between any DR-TB and MDR-TB with potential risk factors were examined through subgroup analyses stratified by new and previously treated patients. RESULTS: A total of 726 studies were identified, of which 27 articles fulfilled the inclusion criteria. Studies reported drug susceptibility testing (DST) results for a total of 13,465 new and 1,776 previously treated TB patients. Pooled estimate of any DR-TB prevalence among the new cases was 12.6% (95% CI 10.6-15.0) while for MDR-TB this was 1.5% (95% CI 1.0-2.3). Among previously treated patients, these were 27.2% (95% CI 21.4-33.8) and 10.3% (95% CI 5.8-17.4%), respectively. DR-TB (any and MDR-TB) did not vary significantly with respect to study characteristics. CONCLUSIONS: The reported prevalence of DR-TB in SSA is low compared to WHO estimates. MDR-TB in this region does not seem to be driven by the high HIV prevalence rates.
Subject(s)
Tuberculosis, Multidrug-Resistant/epidemiology , Africa South of the Sahara/epidemiology , Antitubercular Agents/therapeutic use , Bacteriological Techniques , Ethambutol/therapeutic use , Humans , Isoniazid/therapeutic use , Prevalence , Rifampin/therapeutic use , Risk Factors , Streptomycin/therapeutic use , Tuberculosis, Multidrug-Resistant/drug therapy , World Health OrganizationABSTRACT
BACKGROUND: Slow decline in the incidence of tuberculosis (TB) has been observed in most high TB burden countries. Knowledge of the prevalence of different TB risk factors can help expand TB control strategies. However with the exception of Human Immunodeficiency Virus (HIV) the prevalence of the other TB risk factors are poorly studied in Uganda. We aimed to determine the prevalence of different TB risk factors and TB disease presentation among TB patients in Kampala Uganda. METHODS: We assessed 365 adult TB patients and used descriptive statistics to summarize their socio-demographic, clinical, radiological, sputum mycobacteriology and TB risk factors (HIV, diabetes, TB contact, alcohol use, tobacco smoking, poverty and overcrowding) data. RESULTS: A total of 158 (43.3%) patients were male and the median age was 29 (IQR 28-30). Majority of the patients (89.2%) had pulmonary TB, 86.9% were new and 13.2% were retreatment. Wasting (i.e. body mass index of <18.5 kg/m(2)) was found in 38.5% of the patients and 63% presented with cough. Constitutional symptoms (fever, anorexia, night sweats and weight loss) were reported by 32.1%. Most patients (78.6%) presented with non-cavity lung parenchyma disease (infiltrates, nodules, masses) but 35.2% had cavity disease. Pleural disease was detected in 19.3% of patients. Positive smear microscopy and culture (irrespective of month of treatment) was found in 52.7% and 36.5% of patients respectively. Any drug resistance was detected in 21.1% of patients while multidrug resistance (MDR) TB defined as resistance to rifampicin and isoniazid was detected in 6.3% of patients. All MDR patients were new patients. The prevalence of TB risk factors were as follows: HIV 41.4%, diabetes 5.4%, close contact 11.5%, family history 17.5%, smoking 26.37%, poverty 39.5%, overcrowding 57.3% and alcohol use 50.7%. Overcrowding increased smear positive rate, prevalence ratio 1.22, p = 0.09 but all the other studied risk factors did not affect clinical, radiological and mycobacteriological study patient characteristics. CONCLUSIONS: Among TB patients in Kampala, Uganda, there is high prevalence of the known TB risk factors. Targeting reducing their prevalence may lead to better TB control in the country. Tuberculosis, risk factors, Uganda.
Subject(s)
Primary Prevention/organization & administration , Tuberculosis/drug therapy , Tuberculosis/epidemiology , Adult , Antitubercular Agents/therapeutic use , Female , Humans , Male , Middle Aged , Prevalence , Retreatment , Risk Factors , Sputum/microbiology , Tuberculosis/prevention & control , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/epidemiology , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/epidemiology , Uganda/epidemiology , Young AdultABSTRACT
BACKGROUND: Despite the recent innovations in tuberculosis (TB) and multi-drug resistant TB (MDR-TB) diagnosis, culture remains vital for difficult-to-diagnose patients, baseline and end-point determination for novel vaccines and drug trials. Herein, we share our experience of establishing a BSL-3 culture facility in Uganda as well as 3-years performance indicators and post-TB vaccine trials (pioneer) and funding experience of sustaining such a facility. METHODS: Between September 2008 and April 2009, the laboratory was set-up with financial support from external partners. After an initial procedure validation phase in parallel with the National TB Reference Laboratory (NTRL) and legal approvals, the laboratory registered for external quality assessment (EQA) from the NTRL, WHO, National Health Laboratories Services (NHLS), and the College of American Pathologists (CAP). The laboratory also instituted a functional quality management system (QMS). Pioneer funding ended in 2012 and the laboratory remained in self-sustainability mode. RESULTS: The laboratory achieved internationally acceptable standards in both structural and biosafety requirements. Of the 14 patient samples analyzed in the procedural validation phase, agreement for all tests with NTRL was 90% (P <0.01). It started full operations in October 2009 performing smear microscopy, culture, identification, and drug susceptibility testing (DST). The annual culture workload was 7,636, 10,242, and 2,712 inoculations for the years 2010, 2011, and 2012, respectively. Other performance indicators of TB culture laboratories were also monitored. Scores from EQA panels included smear microscopy >80% in all years from NTRL, CAP, and NHLS, and culture was 100% for CAP panels and above regional average scores for all years with NHLS. Quarterly DST scores from WHO-EQA ranged from 78% to 100% in 2010, 80% to 100% in 2011, and 90 to 100% in 2012. CONCLUSIONS: From our experience, it is feasible to set-up a BSL-3 TB culture laboratory with acceptable quality performance standards in resource-limited countries. With the demonstrated quality of work, the laboratory attracted more research groups and post-pioneer funding, which helped to ensure sustainability. The high skilled experts in this research laboratory also continue to provide an excellent resource for the needed national discussion of the laboratory and quality management systems.
Subject(s)
Clinical Laboratory Techniques/standards , Laboratories/economics , Laboratories/standards , Tuberculosis/diagnosis , Antitubercular Agents/pharmacology , Clinical Laboratory Techniques/economics , Developing Countries/economics , Feasibility Studies , Humans , Laboratories/organization & administration , Microbial Sensitivity Tests/standards , Quality Assurance, Health Care , Tuberculosis/drug therapy , Tuberculosis/epidemiology , Tuberculosis, Multidrug-Resistant/diagnosis , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/epidemiology , Uganda/epidemiologyABSTRACT
For Mycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB for M. tuberculosis drug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archived M. tuberculosis isolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results for M. tuberculosis susceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.
Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Ethambutol/pharmacology , Fluoroquinolones/pharmacology , Isoniazid/pharmacology , Microbial Sensitivity Tests , Moxifloxacin , Rifampin/pharmacologySubject(s)
Microbial Sensitivity Tests , Molecular Diagnostic Techniques , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Tuberculosis/diagnosis , Tuberculosis/microbiology , Antibiotics, Antitubercular/pharmacology , Coinfection , Humans , Mycobacterium tuberculosis/isolation & purification , Rifampin/pharmacology , Sensitivity and Specificity , Tuberculosis/cerebrospinal fluidABSTRACT
Smear microscopy has suboptimal sensitivity, and there is a need to improve its performance since it is commonly used to diagnose tuberculosis (TB). We prospectively evaluated the diagnostic accuracy of the small membrane filtration (SMF) method, an approach that uses a vacuum manifold and is designed to concentrate bacilli onto a filter that can be examined microscopically. We enrolled hospitalized adults suspected to have pulmonary TB in Kampala, Uganda. We obtained a clinical history and three spontaneously expectorated sputum specimens for smear microscopy (direct, concentrated, and SMF), MGIT (mycobacterial growth indicator tube) 960 and Lowenstein-Jensen (LJ) cultures, and Xpert MTB/RIF testing. We performed per-specimen (primary) and per-patient analyses. From October 2012 to June 2013, we enrolled 212 patients (579 sputum specimens). The participants were mostly female (63.2%), and 81.6% were HIV infected; their median CD4 cell count was 47 cells/µl. Overall, 19.0%, 20.4%, 27.1%, 25.2%, and 25.9% of specimens tested positive by direct smear, concentrated smear, MGIT culture, LJ culture, and Xpert test, respectively. In the per-specimen analysis, the sensitivity of the SMF method (48.5%; 95% confidence interval [CI], 37.4 to 59.6) was lower than those of direct smear (60.9%; 51.4 to 70.5 [P = 0.0001]) and concentrated smear (63.3%; 53.6 to 73.1 [P < 0.0001]). Subgroup analyses showed that SMF performed poorly in specimens having a low volume or low bacterial load. The SMF method performed poorly compared to standard smear techniques and was sensitive to sample preparation techniques. The optimal laboratory SMF protocol may require striking a fine balance between sample dilution and filtration failure rate.
Subject(s)
Bacteriological Techniques/methods , Filtration/methods , Microscopy/methods , Mycobacterium tuberculosis/isolation & purification , Specimen Handling/methods , Sputum/microbiology , Tuberculosis, Pulmonary/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Cross-Sectional Studies , Female , Humans , Male , Membranes , Middle Aged , Prospective Studies , Sensitivity and Specificity , Uganda , Young AdultABSTRACT
BACKGROUND: Introduction of Xpert® MTB/RIF assay has revolutionalised the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (rif), a surrogate marker for multi-drug resistant TB (MDR-TB) as well as one of the principal first-line anti-tuberculosis drugs. In general, rpoB mutations can be found in 96.1% of rif-resistant Mycobacterium tuberculosis (MTB) strains worldwide and these mutations usually are located in a region at the 507-533rd amino acid residuals (81 bp) in the MTB rpoB gene, which is referred to as Rifampicin-resistance-determining region (RRDR). In this study, we determined the frequency of MDR-TB in Kampala using Xpert® MTB/RIF in comparison with the agar proportion method using Middlebrook 7H11and further determined the frequency of probes for different rpoB gene mutations using Xpert® MTB/RIF assay in the 81 bp RRDR. METHODS: A total of 1501 specimens received at Mycobacteriology laboratory, Makerere University for Xpert testing between May 2011 and May 2014 were analysed by Xpert® MTB/RIF assay. Specimens that were positive for both MTB and rifampicin resistance were further subjected to a complete first line anti-mycobacterial drug susceptibility testing using Middlebrook 7H11 agar proportion method (APM). RESULTS: Xpert® MTB/RIF assay detected 313 MTB positive specimens and out of which 12 specimens had both MTB and rifampicin- resistance conferred by four different rpoB gene mutations in the 81 bp-RRDR of MTB, further one (1/12), specimen was found to be rifampicin mono-resistant on APM while the 11 were found to be MDR-TB. Probes associated with the observed rif- resistance were as follows: E (7/12), B (3/12), A (1/12), D (1/12) and no rif-resistance was associated with probe C. No specimen yielded rif-resistance associated with more than one probe failure (mutation combinations). Probe D was associated with rifampicin mono-resistant. CONCLUSIONS: MDR-TB was at 3.5% in the studied population. Mutations associated with Probe E (58%) also known as codons 531and 533 are the commonest rpoB gene mutation identified by Xpert® MTB/RIF assay in this setting and mutations identified by probe E of the assay, turned out to be MDR-TB strains by agar proportion method antimicrobial susceptibility testing. No mutation was detected in the codon 522.