Effect of mixed Mycobacterium tuberculosis infection on rapid molecular diagnostics among patients starting MDR-TB treatment in Uganda.

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.

Cardiovascular risk factors among people with drug-resistant tuberculosis in Uganda.

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.

Chest X-ray interpretation does not complement Xpert MTB/RIF in diagnosis of smear-negative pulmonary tuberculosis among TB-HIV co-infected adults in a resource-limited setting.

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.

Prevalence and predictors of ocular complications among children undergoing nephrotic syndrome treatment in a resource-limited setting.

BACKGROUND: Nephrotic syndrome is the most common glomerulopathy among children aged 2-18 years and high dose corticosteroids are the backbone of its management. Potentially blinding ocular complications often result from nephrotic syndrome and/or its treatment. We conducted a study to determine the prevalence and predictors of ocular complications among children undergoing nephrotic syndrome treatment at Mulago National Referral Hospital. METHODS: This was a cross-sectional study conducted for three [3] months at the pediatric renal unit of Mulago National Referral Hospital (MNRH). Data from a consecutive sample of 100 children was collected using a semi-structured questionnaire, entered into Epi-data 4.4.2 and exported to STATA 14 for analysis at univariate, bivariate and multivariate levels. A robust Poisson regression model was used to identify predictors of ocular complications. RESULTS: Out of 100 patients examined, 80(80%) had ocular complications. The median age was 10 (IQR: 7-12) and 52 (52%) were girls. The most frequent complications were hypertrichosis and refractive errors in 71% (95%CI 61.1-79.6) and 56% (95%CI 45.7-65.9) of the patients respectively. Age above 10 years was the predictor for ocular complications with a RR = 1.37 (95%CI:1.14-1.64) p = 0.001. CONCLUSIONS: We found a high prevalence of ocular complications among children with nephrotic syndrome in this tertiary hospital. The predictor of ocular complications was age greater than 10 years. We recommend that all children with nephrotic syndrome undergo a baseline ocular examination prior to commencement of treatment and be reviewed periodically by an ophthalmologist.

Multicenter Study of the Accuracy of the BD MAX Multidrug-resistant Tuberculosis Assay for Detection of Mycobacterium tuberculosis Complex and Mutations Associated With Resistance to Rifampin and Isoniazid.

BACKGROUND: Tuberculosis (TB) control is hindered by absence of rapid tests to identify Mycobacterium tuberculosis (MTB) and detect isoniazid (INH) and rifampin (RIF) resistance. We evaluated the accuracy of the BD MAX multidrug-resistant (MDR)-TB assay (BD MAX) in South Africa, Uganda, India, and Peru. METHODS: Outpatient adults with signs/symptoms of pulmonary TB were prospectively enrolled. Sputum smear microscopy and BD MAX were performed on a single raw sputum, which was then processed for culture and phenotypic drug susceptibility testing (DST), BD MAX, and Xpert MTB/RIF (Xpert). RESULTS: 1053 participants with presumptive TB were enrolled (47% female; 32% with human immunodeficiency virus). In patients with confirmed TB, BD MAX sensitivity was 93% (262/282 [95% CI, 89-95%]); specificity was 97% (593/610 [96-98%]) among participants with negative cultures on raw sputa. BD MAX sensitivity was 100% (175/175 [98-100%]) for smear-positive samples (fluorescence microscopy), and 81% (87/107 [73-88%]) in smear-negative samples. Among participants with both BD MAX and Xpert, sensitivity was 91% (249/274 [87-94%]) for BD MAX and 90% (246/274 [86-93%]) for Xpert on processed sputa. Sensitivity and specificity for RIF resistance compared with phenotypic DST were 90% (9/10 [60-98%]) and 95% (211/222 [91-97%]), respectively. Sensitivity and specificity for detection of INH resistance were 82% (22/27 [63-92%]) and 100% (205/205 [98-100%]), respectively. CONCLUSIONS: The BD MAX MDR-TB assay had high sensitivity and specificity for detection of MTB and RIF and INH drug resistance and may be an important tool for rapid detection of TB and MDR-TB globally.

Accuracy of Xpert Ultra in Diagnosis of Pulmonary Tuberculosis among Children in Uganda: a Substudy from the SHINE Trial.

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.

Performance of loop-mediated isothermal amplification assay in the diagnosis of pulmonary tuberculosis in a high prevalence TB/HIV rural setting in Uganda.

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.

"Tuberculosis in advanced HIV infection is associated with increased expression of IFNγ and its downstream targets".

BACKGROUND: Tuberculosis (TB) is the major cause of death in Human Immunodeficiency Virus (HIV)-infected individuals. However, diagnosis of TB in HIV remains challenging particularly when HIV infection is advanced. Several gene signatures and serum protein biomarkers have been identified that distinguish active TB from latent infection. Our study was designed to assess if gene expression signatures and cytokine levels would distinguish active TB in advanced HIV. METHODS: We conducted a case-control study of whole blood RNA-Seq and plasma cytokine/chemokine analysis in HIV-infected with CD4+ T cell count of ≤ 100 cells/µl, with and without active TB. Next, the overlap of the differentially expressed genes (DEG) with the published signatures was performed and then receiver operator characteristic (ROC) analysis was done on small gene discriminators to determine their performance in distinguishing TB in advanced HIV. ELISA was performed on plasma to evaluate cytokine and chemokine levels. RESULTS: Hierarchical clustering of the transcriptional profiles showed that, in general, HIV-infected individuals with TB (TB-HIV) clustered separately from those without TB. IPA indicated that the TB-HIV signature was characterized by an increase in inflammatory signaling pathways. Analysis of overlaps between DEG in our data set with published TB signatures revealed that significant overlap was seen with one TB signature and one TB-IRIS signature. ROC analysis revealed that transcript levels of FcGR1A (AUC = 0.85) and BATF2 (AUC = 0.82), previously reported as consistent single gene classifiers of active TB irrespective of HIV status, performed successfully even in advanced HIV. Plasma protein levels of IFNγ, a stimulator of FcGR1A and BATF2, and CXCL10, also up-regulated by IFNγ, accurately classified active TB (AUC = 0.98 and 0.91, respectively) in advanced HIV. Neither of these genes nor proteins distinguished between TB and TB-IRIS. CONCLUSIONS: Gene expression of FcGR1A and BATF2, and plasma protein levels of IFNγ and CXCL10 have the potential to independently detect TB in advanced HIV. However, since other lung diseases were not included in this study, these final candidates need to be validated as specific to TB in the advanced HIV population with TB.

Possible misdiagnosis of HIV associated lymphoma as tuberculosis among patients attending Uganda Cancer Institute.

BACKGROUND: Early diagnosis of HIV associated lymphoma is challenging because the definitive diagnostic procedure of biopsy, requires skills and equipment that are not readily available. As a consequence, diagnosis may be delayed increasing the risk of mortality. We set out to determine the frequency and risk factors associated with the misdiagnosis of HIV associated lymphoma as tuberculosis (TB) among patients attending the Uganda Cancer Institute (UCI). METHODS: A retrospective cohort study design was used among HIV patients with associated lymphoma patients attending the UCI, Kampala, Uganda between February and March 2015. Eligible patient charts were reviewed for information on TB treatment, socio-demographics, laboratory parameters (Hemoglobin, CD4cells count and lactate dehydrogenase) and clinical presentation using a semi structured data extraction form. RESULTS: A total of 183 charts were reviewed; 106/183 were males (57.9%), the median age was 35 (IQR, 28-45). Fifty six (30.6%) patients had a possible misdiagnosis as TB and their median time on TB treatment was 3.5 (1-5.3) months. In multivariate analysis the presence of chest pain had an odd ratio (OR) of 4.4 (95% CI 1.89-10.58, p < 0.001) and stage III and IV lymphoma disease had an OR of 3.22 (95% CI 1.08-9.63, p < 0.037) for possible misdiagnosis of lymphoma as TB. CONCLUSION: A high proportion of patients with HIV associated lymphoma attending UCI are misdiagnosed and treated as TB. Chest pain and stage III and IV of lymphoma were associated with an increased risk of a possible misdiagnosis of lymphoma as TB.

Analytical and Clinical Evaluation of the Epistem Genedrive Assay for Detection of Mycobacterium tuberculosis.

The Epistem Genedrive assay rapidly detects the Mycobacterium tuberculosis omplex from sputum and is currently available for clinical use. However, the analytical and clinical performance of this test has not been fully evaluated. The analytical limit of detection (LOD) of the Genedrive PCR amplification was tested with genomic DNA; the performance of the complete (sample processing plus amplification) system was tested by spiking M. tuberculosismc(2)6030 cells into distilled water andM. tuberculosis-negative sputum. Specificity was tested using common respiratory pathogens and nontuberculosis mycobacteria. A clinical evaluation enrolled adults with suspected pulmonary tuberculosis, obtained three sputum samples from each participant, and compared the accuracy of the Gene drive to that of the Xpert MTB/RIF assay using M. tuberculosiscultures as the reference standard. The Genedrive assay had an LOD of 1 pg/µl (100 genomic DNA copies/reaction). The LODs of the system were 2.5 × 10(4)CFU/ml and 2.5 × 10(5)CFU/ml for cells spiked into water and sputum, respectively. False-positiverpoBprobe signals were observed in 3/32 (9.4%) of the negative controls and also in few samples containing Mycobacterium abscessus,Mycobacterium gordonae, o rMycobacterium thermoresistibile In the clinical study, among 336 analyzed participants, the overall sensitivities for the tuberculosis case detection of Gene drive, Xpert, and smear microscopy were 45.4% (95% confidence interval [CI], 35.2% to 55.8%), 91.8% (95% CI, 84.4% to 96.4%), and 77.3% (95% CI, 67.7% to 85.2%), respectively. The sensitivities of Gene drive and Xpert for the detection of smear-microscopy-negative tuberculosis were 0% (95% CI, 0% to 15.4%) and 68.2% (95% CI, 45.1% to 86.1%), respectively. The Genedrive assay did not meet performance standards recommended by the World Health Organization for a smear microscopy replacement tuberculosis test. Epistem is working on modifications to improve the assay.

Predictors and outcomes of mycobacteremia among HIV-infected smear- negative presumptive tuberculosis patients in Uganda.

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.

Tuberculosis risk factors among tuberculosis patients in Kampala, Uganda: implications for tuberculosis control.

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.

Prospective cross-sectional evaluation of the small membrane filtration method for diagnosis of pulmonary tuberculosis.

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.

Prospective multicentre accuracy evaluation of the FUJIFILM SILVAMP TB LAM test for the diagnosis of tuberculosis in people living with HIV demonstrates lot-to-lot variability.

There is an urgent need for rapid, non-sputum point-of-care diagnostics to detect tuberculosis. This prospective trial in seven high tuberculosis burden countries evaluated the diagnostic accuracy of the point-of-care urine-based lipoarabinomannan assay FUJIFILM SILVAMP TB LAM (FujiLAM) among inpatients and outpatients living with HIV. Diagnostic performance of FujiLAM was assessed against a mycobacterial reference standard (sputum culture, blood culture, and Xpert Ultra from urine and sputum at enrollment, and additional sputum culture ≤7 days from enrollment), an extended mycobacterial reference standard (eMRS), and a composite reference standard including clinical evaluation. Of 1637 participants considered for the analysis, 296 (18%) were tuberculosis positive by eMRS. Median age was 40 years, median CD4 cell count was 369 cells/ul, and 52% were female. Overall FujiLAM sensitivity was 54·4% (95% CI: 48·7-60·0), overall specificity was 85·2% (83·2-87·0) against eMRS. Sensitivity and specificity estimates varied between sites, ranging from 26·5% (95% CI: 17·4%-38·0%) to 73·2% (60·4%-83·0%), and 75·0 (65·0%-82·9%) to 96·5 (92·1%-98·5%), respectively. Post-hoc exploratory analysis identified significant variability in the performance of the six FujiLAM lots used in this study. Lot variability limited interpretation of FujiLAM test performance. Although results with the current version of FujiLAM are too variable for clinical decision-making, the lipoarabinomannan biomarker still holds promise for tuberculosis diagnostics. The trial is registered at clinicaltrials.gov (NCT04089423).

Xpert MTB/RIF Ultra versus mycobacterial growth indicator tube liquid culture for detection of Mycobacterium tuberculosis in symptomatic adults: a diagnostic accuracy study.

BACKGROUND: Xpert MTB/RIF Ultra (Ultra) is an automated molecular test for the detection of Mycobacterium tuberculosis in sputum. We compared the sensitivity of Ultra to that of mycobacterial growth indicator tube (MGIT) liquid culture, considered the most sensitive assay in routine clinical use. METHODS: In this prospective, multicentre, cross-sectional diagnostic accuracy study, we used a non-inferiority design to assess whether the sensitivity of a single Ultra test was non-inferior to that of a single liquid culture for detection of M tuberculosis in sputum. We enrolled adults (age ≥18 years) with pulmonary tuberculosis symptoms in 11 countries and each adult provided three sputum specimens with a minimum volume of 2 mL over 2 days. Ultra was done directly on sputum 1, and Ultra and MGIT liquid culture were done on resuspended pellet from sputum 2. Results of MGIT and solid media cultures done on sputum 3 were considered the reference standard. The pre-defined non-inferiority margin was 5·0%. FINDINGS: Between Feb 18, 2016, and Dec 4, 2019, we enrolled 2906 participants. 2600 (89%) participants were analysed, including 639 (25%) of 2600 who were positive for tuberculosis by the reference standard. Of the 2357 included in the non-inferiority analysis, 877 (37%) were HIV-positive and 984 (42%) were female. Sensitivity of Ultra performed directly on sputum 1 was non-inferior to that of sputum 2 MGIT culture (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; 95% CI -2·8 to 1·1). Sensitivity of Ultra performed on sputum 2 pellet was also non-inferior to that of sputum 2 MGIT (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; -2·7 to 1·0). INTERPRETATION: For the detection of M tuberculosis in sputum from adults with respiratory symptoms, there was no difference in sensitivity of a single Ultra test to that of a single MGIT culture. Highly sensitive, rapid molecular approaches for M tuberculosis detection, combined with advances in genotypic methods for drug resistance detection, have potential to replace culture. FUNDING: US National Institute of Allergy and Infectious Diseases.

Effect of mixed Mycobacterium tuberculosis infection on rapid molecular diagnostics among patients starting MDR-TB treatment in Uganda.

Background: We evaluated the effect of mixed-MTB strain infection on the performance of Line Probe Assay (LPA) and GeneXpert MTB/RIF (Xpert) assays among patients initiating MDR-TB treatment in Uganda. Methods: This was a cross-sectional study using 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 the 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 males 3/33 (9.7%) and among middle-aged adults, 4/30 (13.3%). Lineage 4 of MTB contributed 3/33 (9.1%) 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.

Determinants of QuantiFERON Plus-diagnosed tuberculosis infection in adult Ugandan TB contacts: A cross-sectional study.

BACKGROUND: The tuberculin skin test is commonly used to diagnose latent tuberculosis infection (LTBI) in resource-limited settings, but its specificity is limited by factors including cross-reactivity with BCG vaccine and environmental mycobacteria. Interferon-gamma release assays (IGRA) overcome this problem by detecting M. tuberculosis complex-specific responses, but studies to determine risk factors for IGRA-positivity in high TB burden settings are lacking. METHODS: We conducted a cross-sectional study to determine factors associated with a positive IGRA by employing the QuantiFERON-TB® Gold-plus (QFT Plus) assay in a cohort of asymptomatic adult TB contacts in Kampala, Uganda. Multivariate logistic regression analysis with forward stepwise logit function was employed to identify independent correlates of QFT Plus-positivity. RESULTS: Of the 202 participants enrolled, 129/202 (64%) were female, 173/202 (86%) had a BCG scar, and 67/202 (33%) were HIV-infected. Overall, 105/192 (54%, 95% CI 0.48-0.62) participants had a positive QFT Plus result. Increased risk of QFT-Plus positivity was independently associated with casual employment/unemployment vs. non-casual employment (adjusted odds ratio (aOR) 2.18, 95% CI 1.01-4.72), a family vs. non-family relation to the index patient (aOR 2.87, 95% CI 1.33-6.18), living in the same vs. a different house as the index (aOR 3.05, 95% CI 1.28-7.29), a higher body mass index (BMI) (aOR per additional kg/m2 1.09, 95% CI 1.00-1.18) and tobacco smoking vs. not (aOR 2.94, 95% CI 1.00-8.60). HIV infection was not associated with QFT-Plus positivity (aOR 0.91, 95% CI 0.42-1.96). CONCLUSION: Interferon Gamma Release Assay positivity in this study population was lower than previously estimated. Tobacco smoking and BMI were determinants of IGRA positivity that were previously unappreciated.

Factors associated with ocular injuries among adult road traffic accident patients presenting at Mulago National Referral Hospital, Uganda.

Background: Ocular trauma is the leading cause of unilateral blindness globally. Road traffic accidents are among the top risk factors for ocular trauma. Objectives: This study aimed to evaluate factors associated with ocular injuries among adult road traffic accident patients at Mulago Hospital, Uganda. Methods: A cross-sectional study was conducted among adult road traffic accident patients. History taking and ophthalmological examination were performed on consenting participants. Data was analysed using STATA version 14.0. Results: Overall, 428 road traffic accident cases were enrolled, of which majority (84.3%) were male. Age 30-39years (aOR = 0.58, 0.36 - 0.94, p = 0.027), being male (aOR = 2.64, 1.21 - 5.13, p = 0.004) and being a passenger of motor vehicle/cycle (aOR = 3.85, 1.49 - 9.93, p = 0.005) were the factors associated with ocular injuries among the participants. Conclusions: Age 30-39 years, male gender and being a passenger of motor vehicle/cycle were the factors associated with ocular injuries among the adult road traffic accident patients. Ocular injuries were more common among the road users who did not use safety measures. Use of safety measure by passengers of motor vehicles and cycles is recommended.

Mycobacterium tuberculosis disease associates with higher HIV-1-specific antibody responses.

Mycobacterium tuberculosis (Mtb) is the most common infection among people with HIV (PWH). Mtb disease-associated inflammation could affect HIV-directed immune responses in PWH. We show that HIV antibodies are broader and more potent in PWH in the presence as compared to the absence of Mtb disease. With co-existing Mtb disease, the virus in PWH also encounters unique antibody selection pressure. The Mtb-linked HIV antibody enhancement associates with specific mediators important for B cell and antibody development. This Mtb humoral augmentation does not occur due to cross-reactivity, a generalized increase in all antibodies, or differences in duration or amount of antigen exposure. We speculate that the co-localization of Mtb and HIV in lymphatic tissues leads to the emergence of potent HIV antibodies. PWH's Mtb disease status has implications for the future use of HIV broadly neutralizing antibodies as prophylaxis or treatment and the induction of better humoral immunity.

Diagnostic accuracy of the NOVA Tuberculosis Total Antibody Rapid test for detection of pulmonary tuberculosis and infection with Mycobacterium tuberculosis.

Background: The NOVA Tuberculosis Total Antibody Rapid Test is a commercially available lateral flow serological assay that is intended to be used as an aid in the diagnosis of tuberculosis. We conducted a study to estimate diagnostic accuracy of this assay for diagnosis of active pulmonary tuberculosis disease and for detection of M. tuberculosis infection. Methods: This study used existing frozen plasma specimens that had been obtained previously from consenting HIV-negative adults in Cambodia, South Africa, and Vietnam whose tuberculosis status was rigorously characterized using sputum mycobacterial cultures and blood interferon gamma release assay. The investigational assay was performed in a single laboratory by laboratory staff specifically trained to conduct the assays according to the manufacturer's procedures. In addition, intensity of the test band was subjectively assessed. Results: Plasma specimens from 150 participants were tested. All testing attempts yielded a determinate result of either positive or negative. For diagnosis of active pulmonary tuberculosis disease, test sensitivity and specificity were 40.0 % (20/50, 95 % confidence interval [CI] 27.6 % to 53.8 %) and 85.0 % (95 % CI 76.7 % to 90.7 %), respectively. For detection of M. tuberculosis infection, test sensitivity and specificity were 28.0 % (95 % CI 20.5 % to 37.2 %) and 86.0 % (95 % CI 73.8 % to 93.0 %), respectively. Among the 35 positive tests, no statistically significant band intensity trend was found across participant groups (p = 0.17). Conclusion: Study findings do not support a role for the NOVA Tuberculosis Test in current tuberculosis diagnostic algorithms.