Browsing by Author "Dheda, Keertan"
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- ItemOpen AccessA landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic(2020-02-21) Klopper, Marisa; Heupink, Tim H; Hill-Cawthorne, Grant; Streicher, Elizabeth M; Dippenaar, Anzaan; de Vos, Margaretha; Abdallah, Abdallah M; Limberis, Jason; Merker, Matthias; Burns, Scott; Niemann, Stefan; Dheda, Keertan; Posey, James; Pain, Arnab; Warren, Robin MAbstract Background Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. Methods We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. Results Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. Conclusion The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.
- ItemOpen AccessA landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic(2020-02-04) Klopper, Marisa; Heupink, Tim H; Hill-Cawthorne, Grant; Streicher, Elizabeth M; Dippenaar, Anzaan; de Vos, Margaretha; Abdallah, Abdallah M; Limberis, Jason; Merker, Matthias; Burns, Scott; Niemann, Stefan; Dheda, Keertan; Posey, James; Pain, Arnab; Warren, Robin MAbstract Background Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. Methods We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. Results Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. Conclusion The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.
- ItemOpen AccessApproaches to the diagnosis of smear-negative and sputum-scarce TB in South Africa(2013) Peter, Jonathan G; Dheda, KeertanIn South Africa, an HIV-endemic setting, the burden of smear-negative (SN) and sputum-scarce (SS) TB is a major public health catastrophe. Diagnostic delay or failure is a key bottleneck. We hypothesised that i) assisted sputum smpling using sputum induction (SI), and ii) the use of the newer diagnostic tools (Xpert MTB/RIF and urine LAM strip), could improve diagnosis of SN or SSTB and impact clinical care. We investigated the accuracy and impact of these approaches at different levels in the health-system.
- ItemOpen AccessCan point-of-care urine LAM strip testing for tuberculosis add value to clinical decision making in hospitalised HIV-infected persons(Public Library of Science, 2013) Peter, Jonathan G; Theron, Grant; Dheda, KeertanBACKGROUND: The urine lipoarabinomannan (LAM) strip-test (Determine®-TB) can rapidly rule-in TB in HIV-infected persons with advanced immunosuppression. However, given high rates of empiric treatment amongst hospitalised patients in high-burden settings (∼50%) it is unclear whether LAM can add any value to clinical decision making, or identify a subset of patients with unfavourable outcomes that would otherwise have been missed by empiric treatment. METHODS: 281 HIV-infected hospitalised patients with suspected TB received urine LAM strip testing, and were categorised as definite (culture-positive), probable-, or non-TB. Both the proportion and morbidity of TB cases identified by LAM testing, early empiric treatment (initiated prior to test result availability) and a set of clinical predictors were compared across groups. RESULTS: 187/281 patients had either definite- (n = 116) or probable-TB (n = 71). As a rule-in test for definite and probable-TB, LAM identified a similar proportion of TB cases compared to early empiric treatment (85/187 vs. 93/187, p = 0.4), but a greater proportion than classified by a set of clinical predictors alone (19/187; p<0.001). Thirty-nine of the 187 (21%) LAM-positive patients who had either definite- or probable-TB were missed by early empiric treatment, and of these 25/39 (64%) would also have been missed by smear microscopy. Thus, 25/187 (8%) of definite- or probable-TB patients with otherwise delayed initiation of TB treatment could be detected by the LAM strip test. LAM-positive patients missed by early empiric treatment had a lower median CD4 count (p = 0.008), a higher median illness severity score (p = 0.001) and increased urea levels (p = 0.002) compared to LAM-negative patients given early empiric treatment. CONCLUSIONS: LAM strip testing outperformed TB diagnosis based on clinical criteria but in day-to-day practice identified a similar proportion of patients compared to early empiric treatment. However, compared to empiric treatment, LAM identified a different subset of patients with more advanced immunosuppression and greater disease severity.
- ItemOpen AccessCigarette smoke and human pulmonary immune responses to mycobacteria(2011) Van Zyl-Smit, Richard Nellis; Dheda, Keertan; Bateman, Eric DRecent epidemiological evidence suggests that up to 15% of worldwide tuberculosis (TB) cases may be attributable to tobacco smoking. The aim of the studies reported here was to gain insights into the effects of exposure to cigarette smoke on human cells that form part of the innate immune system of host defence in the lung. The experiments on the pulmonary effects of cigarette smoke confirm that exposure has a significant effect upon innate host defences. Significant reductions in the production of key cytokines implicated in defences against mycobacteria were observed, not attributable to impairment of mycobacterial uptake by cigarette smoke extract exposure. Furthermore, control of intracellular mycobacterial growth was impaired by cigarette smoke extract exposure.
- ItemOpen AccessClinical diagnostic utility of IP-10 and LAM antigen levels for the diagnosis of tuberculous pleural effusions in a high burden setting(Public Library of Science, 2009) Dheda, Keertan; Van-Zyl Smit, Richard N; Sechi, Leonardo A; Badri, Motasim; Meldau, Richard; Symons, Gregory; Khalfey, Hoosein; Carr, Igshaan; Maredza, Alice; Dawson, RodneyBACKGROUND: Current tools for the diagnosis of tuberculosis pleural effusions are sub-optimal. Data about the value of new diagnostic technologies are limited, particularly, in high burden settings. Preliminary case control studies have identified IFN-γ-inducible-10kDa protein (IP-10) as a promising diagnostic marker; however, its diagnostic utility in a day-to-day clinical setting is unclear. Detection of LAM antigen has not previously been evaluated in pleural fluid. METHODS: We investigated the comparative diagnostic utility of established (adenosine deaminase [ADA]), more recent (standardized nucleic-acid-amplification-test [NAAT]) and newer technologies (a standardized LAM mycobacterial antigen-detection assay and IP-10 levels) for the evaluation of pleural effusions in 78 consecutively recruited South African tuberculosis suspects. All consenting participants underwent pleural biopsy unless contra-indicated or refused. The reference standard comprised culture positivity for M. tuberculosis or histology suggestive of tuberculosis. Principal FINDINGS: Of 74 evaluable subjects 48, 7 and 19 had definite, probable and non-TB, respectively. IP-10 levels were significantly higher in TB vs non-TB participants (p<0.0001). The respective outcomes [sensitivity, specificity, PPV, NPV %] for the different diagnostic modalities were: ADA at the 30 IU/L cut-point [96; 69; 90; 85], NAAT [6; 93; 67; 28], IP-10 at the 28,170 pg/ml ROC-derived cut-point [80; 82; 91; 64], and IP-10 at the 4035 pg/ml cut-point [100; 53; 83; 100]. Thus IP-10, using the ROC-derived cut-point, missed ∼20% of TB cases and mis-diagnosed ∼20% of non-TB cases. By contrast, when a lower cut-point was used a negative test excluded TB. The NAAT had a poor sensitivity but high specificity. LAM antigen-detection was not diagnostically useful. CONCLUSION: Although IP-10, like ADA, has sub-optimal specificity, it may be a clinically useful rule-out test for tuberculous pleural effusions. Larger multi-centric studies are now required to confirm our findings.
- ItemOpen AccessClinical utility of a commercial LAM-ELISA assay for TB diagnosis in HIV-infected patients using urine and sputum samples(Public Library of Science, 2010) Dheda, Keertan; Davids, Virginia; Lenders, Laura; Roberts, Teri; Meldau, Richard; Ling, Daphne; Brunet, Laurence; Smit, Richard van Zyl; Peter, Jonathan; Green, Clare; Badri, Motasim; Sechi, Leonardo; Sharma, Surendra; Hoelscher, Michael; Dawson, RodneyBACKGROUND: The accurate diagnosis of TB in HIV-infected patients, particularly with advanced immunosuppression, is difficult. Recent studies indicate that a lipoarabinomannan (LAM) assay (Clearview-TB®-ELISA) may have some utility for the diagnosis of TB in HIV-infected patients; however, the precise subgroup that may benefit from this technology requires clarification. The utility of LAM in sputum samples has, hitherto, not been evaluated. METHODS: LAM was measured in sputum and urine samples obtained from 500 consecutively recruited ambulant patients, with suspected TB, from 2 primary care clinics in South Africa. Culture positivity for M. tuberculosis was used as the reference standard for TB diagnosis. RESULTS: Of 440 evaluable patients 120/387 (31%) were HIV-infected. Urine-LAM positivity was associated with HIV positivity (p = 0.007) and test sensitivity, although low, was significantly higher in HIV-infected compared to uninfected patients (21% versus 6%; p<0.001), and also in HIV-infected participants with a CD4 <200 versus >200 cells/mm 3 (37% versus 0%; p = 0.003). Urine-LAM remained highly specific in all 3 subgroups (95%-100%). 25% of smear-negative but culture-positive HIV-infected patients with a CD4 <200 cells/mm 3 were positive for urine-LAM. Sputum-LAM had good sensitivity (86%) but poor specificity (15%) likely due to test cross-reactivity with several mouth-residing organisms including actinomycetes and nocardia species. CONCLUSIONS: These preliminary data indicate that in a high burden primary care setting the diagnostic usefulness of urine-LAM is limited, as a rule-in test, to a specific patient subgroup i.e. smear-negative HIV-infected TB patients with a CD4 count <200 cells/mm 3 , who would otherwise have required further investigation. However, even in this group sensitivity was modest. Future and adequately powered studies in a primary care setting should now specifically target patients with suspected TB who have advanced HIV infection.
- ItemOpen AccessComparison of a clinical prediction rule and a LAM antigen-detection assay for the rapid diagnosis of TBM in a high HIV prevalence setting(Public Library of Science, 2010) Patel, Vinod B; Singh, Ravesh; Connolly, Cathy; Kasprowicz, Victoria; Zumla, Allimudin; Ndungu, Thumbi; Dheda, KeertanBackground/Objective: The diagnosis of tuberculous meningitis (TBM) in resource poor TB endemic environments is challenging. The accuracy of current tools for the rapid diagnosis of TBM is suboptimal. We sought to develop a clinical-prediction rule for the diagnosis of TBM in a high HIV prevalence setting, and to compare performance outcomes to conventional diagnostic modalities and a novel lipoarabinomannan (LAM) antigen detection test (Clearview-TB®) using cerebrospinal fluid (CSF). METHODS: Patients with suspected TBM were classified as definite-TBM (CSF culture or PCR positive), probable-TBM and non-TBM. RESULTS: Of the 150 patients, 84% were HIV-infected (median [IQR] CD4 count = 132 [54; 241] cells/µl). There were 39, 55 and 54 patients in the definite, probable and non-TBM groups, respectively. The LAM sensitivity and specificity (95%CI) was 31% (17;48) and 94% (85;99), respectively (cut-point ≥0.18). By contrast, smear-microscopy was 100% specific but detected none of the definite-TBM cases. LAM positivity was associated with HIV co-infection and low CD4 T cell count (CD4<200 vs. >200 cells/µl; p = 0.03). The sensitivity and specificity in those with a CD4<100 cells/µl was 50% (27;73) and 95% (74;99), respectively. A clinical-prediction rule ≥6 derived from multivariate analysis had a sensitivity and specificity (95%CI) of 47% (31;64) and 98% (90;100), respectively. When LAM was combined with the clinical-prediction-rule, the sensitivity increased significantly (p<0.001) to 63% (47;68) and specificity remained high at 93% (82;98). CONCLUSIONS: Despite its modest sensitivity the LAM ELISA is an accurate rapid rule-in test for TBM that has incremental value over smear-microscopy. The rule-in value of LAM can be further increased by combination with a clinical-prediction rule, thus enhancing the rapid diagnosis of TBM in HIV-infected persons with advanced immunosuppression.
- ItemOpen AccessComparison of quantitative techniques including Xpert MTB/RIF to evaluate mycobacterial burden(Public Library of Science, 2011) van Zyl-Smit, Richard N; Binder, Anke; Meldau, Richard; Mishra, Hridesh; Semple, Patricia L; Theron, Grant; Peter, Jonathan; Whitelaw, Andrew; Sharma, Suren K; Warren, Robin; Bateman, Eric D; Dheda, KeertanIntroduction: Accurate quantification of mycobacterial load is important for the evaluation of patient infectiousness, disease severity and monitoring treatment response in human and in-vitro laboratory models of disease. We hypothesized that newer techniques would perform as well as solid media culture to quantify mycobacterial burden in laboratory specimens. METHODS: We compared the turn-around-time, detection-threshold, dynamic range, reproducibility, relative discriminative ability, of 4 mycobacterial load determination techniques: automated liquid culture (BACTEC-MGIT-960), [ 3 H]-uracil incorporation assays, luciferase-reporter construct bioluminescence, and quantitative PCR(Xpert -MTB/RIF) using serial dilutions of Mycobacterium bovis and Mycobacterium tuberculosis H37RV. Mycobacterial colony-forming-units(CFU) using 7H10-Middlebrook solid media served as the reference standard. RESULTS: All 4 assays correlated well with the reference standard, however, bioluminescence and uracil assays had a detection threshold ≥1×10 3 organisms. By contrast, BACTEC-MGIT-960 liquid culture, although only providing results in days, was user-friendly, had the lowest detection threshold (<10 organisms), the greatest discriminative ability (1 vs. 10 organisms; p = 0.02), and the best reproducibility (coefficient of variance of 2% vs. 38% compared to uracil incorporation; p = 0.02). Xpert-MTB/RIF correlated well with mycobacterial load, had a rapid turn-around-time (<2 hours), was user friendly, but had a detection limit of ∼100 organisms. CONCLUSIONS: Choosing a technique to quantify mycobacterial burden for laboratory or clinical research depends on availability of resources and the question being addressed. Automated liquid culture has good discriminative ability and low detection threshold but results are only obtained in days. Xpert MTB/RIF provides rapid quantification of mycobacterial burden, but has a poorer discrimination and detection threshold.
- ItemOpen AccessComparison of same day diagnostic tools including Gene Xpert and unstimulated IFN-γ for the evaluation of pleural tuberculosis: a prospective cohort study(BioMed Central, 2014-04-08) Meldau, Richard; Peter, Jonny; Theron, Grant; Calligaro, Greg; Allwood, Brian; Symons, Greg; Khalfey, Hoosain; Ntombenhle, Gina; Govender, Ureshnie; Binder, Anke; van Zyl-Smit, Richard; Dheda, KeertanBackground: The accuracy of currently available same-day diagnostic tools (smear microscopy and conventional nucleic acid amplification tests) for pleural tuberculosis (TB) is sub-optimal. Newer technologies may offer improved detection. Methods: Smear-microscopy, adenosine deaminase (ADA), interferon gamma (IFN-γ), and Xpert MTB/RIF [using an unprocessed (1 ml) and centrifuged (~20 ml) sample] test accuracy was evaluated in pleural fluid from 103 consecutive patients with suspected pleural TB. Culture for M.tuberculosis and/or histopathology (pleural biopsy) served as the reference standard. Patients were followed prospectively to determine their diagnostic categorisation. Results: Of 93 evaluable participants, 40 had definite-TB (reference positive), 5 probable-TB (not definite but treated for TB) and 48 non-TB (culture and histology negative, and not treated for TB). Xpert MTB/RIF sensitivity and specificity (95% CI) was 22.5% (12.4 - 37.6) and 98% (89.2 - 99.7), respectively, and centrifugation did not improve sensitivity (23.7%). The Xpert MTB/RIF internal positive control showed no evidence of inhibition. Biomarker specific sensitivity, specificity, PPV, and NPVs were: ADA (48.85 IU/L; rule-in cut-point) 55.3% (39.8 - 69.9), 95.2% (83.9 - 98.7), 91.4 (73.4 - 95.4), 69.7% (56.7 - 80.1); ADA (30 IU/L; clinically used cut-point) 79% (63.7 - 89), 92.7% (80.6 - 97.5), 91.0 (73.4 - 95.4), 82.7% (69.3 - 90.1); and IFN-γ (107.7 pg/ml; rule-in cut-point) 92.5% (80.2 - 97.5), 95.9% (86.1 - 98.9), 94.9% (83.2 - 98.6), 93.9% (83.5 - 97.9), respectively (IFN-γ sensitivity and NPV better than Xpert [p < 0.05] and rule-in ADA [p < 0.05]). Conclusion: The usefulness of Xpert MTB/RIF to diagnose pleural TB is limited by its poor sensitivity. IFN-γ is an excellent rule-in test and, compared to ADA, has significantly better sensitivity and rule-out value in a TB-endemic setting.
- ItemOpen AccessCorrelation of mycobacterium tuberculosis specific and non-specific quantitative Th1 T-cell responses with bacillary load in a high burden setting(Public Library of Science, 2012) Theron, Grant; Peter, Jonny; Lenders, Laura; van Zyl-Smit, Richard; Meldau, Richard; Govender, Ureshnie; Dheda, KeertanBACKGROUND: Measures of bacillary load in patients with tuberculosis (TB) may be useful for predicting and monitoring response to treatment. The relationship between quantitative T-cell responses and mycobacterial load remains unclear. We hypothesised that, in a HIV-prevalent high burden setting, the magnitude of mycobacterial antigen-specific and non-specific T-cell IFN-γ responses would correlate with (a) bacterial load and (b) culture conversion in patients undergoing treatment. METHODS: We compared baseline (n = 147), 2 (n = 35) and 6 month (n = 13) purified-protein-derivative (PPD) and RD1-specific (TSPOT.TB and QFT-GIT) blood RD1-specific (TSPOT.TB; QFT-GIT) responses with associates of sputum bacillary load in patients with culture-confirmed TB in Cape Town, South Africa. RESULTS: IFN-γ responses were not associated with liquid culture time-to-positivity, smear-grade, Xpert MTB/RIF-generated cycle threshold values or the presence of cavities on the chest radiograph in patients with culture-confirmed TB and irrespective of HIV-status. 2-month IGRA conversion rates (positive-to-negative) were negligible [<11% for TSPOT.TB (3/28) and QFT-GIT (1/29)] and lower compared to culture [60% (21/35); p<0.01]. CONCLUSIONS: In a high burden HIV-prevalent setting T-cell IFN-γ responses to M. tuberculosis- specific and non-specific antigens do not correlate with bacillary load, including Xpert MTB/RIF-generated C T values, and are therefore poorly suited for monitoring treatment and prognostication.
- ItemOpen AccessDetermine TB-LAM lateral flow urine antigen assay for HIV-associated tuberculosis: recommendations on the design and reporting of clinical studies(BioMed Central Ltd, 2013) Lawn, Stephen; Dheda, Keertan; Kerkhoff, Andrew; Peter, Jonathan; Dorman, Susan; Boehme, Catharina; Nicol, MarkDetection of the Mycobacterium tuberculosis cell wall antigen lipoarabinomannan (LAM) in urine permits diagnoses of tuberculosis (TB) to be made in HIV-infected patients with advanced immunodeficiency. This can be achieved at the point-of-care within just 30 minutes using the Determine TB-LAM, which is a commercially available, lateral-flow urine 'strip test' assay. The assay has been shown to have useful diagnostic accuracy in patients enrolling in antiretroviral treatment services or in HIV-infected patients requiring admission to hospital medical wards in sub-Saharan Africa. Such patients have high mortality risk and have most to gain from rapid diagnosis of TB and immediate initiation of treatment. However, few studies using this assay have yet been reported and many questions remain concerning the correct use of the assay, interpretation of results, the role of the assay as an add-on test within existing diagnostic algorithms and the types of further studies needed. In this paper we address a series of questions with the aim of informing the design, conduct and interpretation of future studies. Specifically, we clarify which clinical populations are most likely to derive benefit from use of this assay and how patients enrolled in such studies might best be characterised. We describe the importance of employing a rigorous microbiological diagnostic reference standard in studies of diagnostic accuracy and discuss issues surrounding the specificity of the assay in different geographical areas and potential cross-reactivity with non-tuberculous mycobacteria and other organisms. We highlight the importance of careful procedures for urine collection and storage and the critical issue of how to read and interpret the test strips. Finally, we consider how the assay could be used in combination with other assays and outline the types of studies that are required to build the evidence base concerning its use.
- ItemOpen AccessDevelopment of a simple reliable radiographic scoring system to aid the diagnosis of pulmonary tuberculosis(Public Library of Science, 2013) Pinto, Lancelot M; Dheda, Keertan; Theron, Grant; Allwood, Brian; Calligaro, Gregory; van Zyl-Smit, Richard; Peter, Jonathan; Schwartzman, Kevin; Menzies, Dick; Bateman, Eric; Pai, Madhukar; Dawson, RodneyRationale: Chest radiography is sometimes the only method available for investigating patients with possible pulmonary tuberculosis (PTB) with negative sputum smears. However, interpretation of chest radiographs in this context lacks specificity for PTB, is subjective and is neither standardized nor reproducible. Efforts to improve the interpretation of chest radiography are warranted. Objectives To develop a scoring system to aid the diagnosis of PTB, using features recorded with the Chest Radiograph Reading and Recording System (CRRS). METHODS: Chest radiographs of outpatients with possible PTB, recruited over 3 years at clinics in South Africa were read by two independent readers using the CRRS method. Multivariate analysis was used to identify features significantly associated with culture-positive PTB. These were weighted and used to generate a score. RESULTS: 473 patients were included in the analysis. Large upper lobe opacities, cavities, unilateral pleural effusion and adenopathy were significantly associated with PTB, had high inter-reader reliability, and received 2, 2, 1 and 2 points, respectively in the final score. Using a cut-off of 2, scores below this threshold had a high negative predictive value (91.5%, 95%CI 87.1,94.7), but low positive predictive value (49.4%, 95%CI 42.9,55.9). Among the 382 TB suspects with negative sputum smears, 229 patients had scores <2; the score correctly ruled out active PTB in 214 of these patients (NPV 93.4%; 95%CI 89.4,96.3). The score had a suboptimal negative predictive value in HIV-infected patients (NPV 86.4, 95% CI 75,94). CONCLUSIONS: The proposed scoring system is simple, and reliably ruled out active PTB in smear-negative HIV-uninfected patients, thus potentially reducing the need for further tests in high burden settings. Validation studies are now required.
- ItemOpen AccessDevelopment of an Autologous Human Dendritic Cell Vaccine against Mycobacterium tuberculosis in Patients with Extensively Drug-Resistant Tuberculosis(2022) Londt, Rolanda Sabrina; Dheda, Keertan; Tomasicchio, MicheleIntroduction: Extensively drug-resistant tuberculosis (XDR-TB), and resistance beyond XDR-TB (often untreatable), is an increasing public health concern globally. Drug resistance has outpaced the drug development pipeline. Therefore, alternative immunotherapeutic approaches are urgently needed. In this proof-of-concept study, and based on precedents in breast and prostate cancer, we aimed to develop an autologous therapeutic dendritic cell (DC) vaccine by evaluating two TB antigen-specific multi-peptide pools in combination with different adjuvants (such a cellular vaccine would require ex-vivo manipulation in a clean room and reinfusion back into the patient). Vaccine efficacy was evaluated using an in vitro mycobacterial containment model. Methods: DCs were derived from monocytes isolated from the peripheral blood of patients with XDR-TB (n=30) and participants with presumed latent TB infection (LTBI; n=15). DCs were matured with a differential combination of cytokines and pattern-recognition receptor agonists (maturation cocktail) together with different TB antigen combinations. The complete cocktail contained interferon-, interferon-, CD40L, IL-1 and TLR-3, TLR-7 and TLR-8 agonists, whilst in the limited cocktail the TLR agonists were absent. Two M.tb-specific multi peptide pools suited to GMP-grade vaccine manufacture were evaluated: (i) an immunodominant peptide pool (ESAT6 + CFP10 + Ag85B + TB10.4; referred to as ECAT) and (ii) a PE/PPE peptide pool. PPD and the lysate of a clinical M.tb strain (HN878), though not amenable to GMP-grade vaccine development, served as controls representing the entire antigenic repertoire of M.tb. Thus, there were four major comparator groups (unstimulated DCs, limited cocktail-only stimulated DCs, antigen-only stimulated DCs, and antigen + complete cocktail stimulated DCs). As the two latter groups were interrogated using the two multi-peptide pools and the two broad-spectrum antigen controls, a total of 10 different experimental groups were generated (see overview figure 3.2). DCs were assessed for the expression of key maturation markers using flow cytometry and the secretion of Th1-polarising cytokines by ELISA. The ability of DC-primed peripheral blood mononuclear cells (PBMCs) to restrict the growth of M.tb-infected monocyte derived-macrophages was evaluated using an in vitro validated mycobacterial containment assay. Results: In patients with XDR-TB, DCs matured with any M.tb-antigen + complete cocktail, compared to DCs matured with M.tb-antigen only, showed significantly higher upregulation of CD80, CD83, CD86, and CCR7 (p< 0.001 for all comparisons), and higher secreted levels of IL12p70 (0.67 versus 0.01 ng/mL per 106 cells; p< 0.001). A similar pattern was seen in the containment experiments: mycobacterial stasis within the XDR-TB group was significantly better with antigen + complete cocktail versus antigen alone (p≤0.0002 for PE/PPE and PPD), and the limited cocktail did not show this effect. Furthermore, PE/PPE + complete cocktail matured DCs achieved a higher magnitude of mycobacterial containment compared to ECAT + complete cocktail-matured DCs (50%, IQR:39-75, versus 46%, IQR: 15-62, p= 0.02). Using PPD and the HN878 lysate did not improve the containment effect. Furthermore, the improved containment effect of the PE/PPE + complete cocktail, versus ECAT + complete cocktail, was only seen in the XDR-TB and not in the LTBI group. Conclusion: In patients with XDR-TB, an effector response primed by PE/PPE antigen and complete cocktail-matured DCs was able to better restrict the growth of M.tb in vitro. These data indicate proof-of-concept feasibility to generate a DC-based immunotherapeutic intervention for therapeutically destitute patients with DR-TB. Further mechanistic studies and future phase 1 and 2 human clinical studies are warranted.
- ItemOpen AccessThe development of aptamer-based probes for the detection of TB antigens ESAT-6.CFP-10 potential TB diagnostic tools(2013) Maserumule, Matsopiane Charlotte; Dheda, Keertan; Khati, Makobetsa; Gresh, LionelLack of point-of-care (PoC) diagnostic tools for TB hinders control of the disease, particularly in resource-limited, high HIV and TB prevalence countries. Therefore, there is a need for simple, rapid, accurate, and affordable PoC diagnostics to detect active TB early enough for opportune intervention. To develop TB detection probes that will constitute such diagnostics, our research group recently isolated DNA aptamers that bind to a putative marker for active TB; the ESAT-6.CFP-10 heterodimer. Aptamers are highly specific artificial mimics of antibodies that have shown great prospects in diagnostic applications. The aim of this study was to characterise the anti-ESAT-6.CFP-10 aptamers, and to optimise them into more specific and affordable detection probes for the development of potential PoC TB diagnostic tools.
- ItemOpen AccessDiagnosing tuberculosis in hospitalized HIV-infected individuals who cannot produce sputum: is urine lipoarabinomannan testing the answer?(BioMed Central, 2017-12-28) Sabur, Natasha F; Esmail, Aliasgar; Brar, Mantaj S; Dheda, KeertanBackground: Up to one third of HIV-infected individuals with suspected TB are sputum-scarce. The Alere Determine™ TB LAM Ag lateral flow strip test can be used to diagnose TB in HIV-infected patients with advanced immunosuppression. However, how urine LAM testing should be incorporated into testing algorithms and in the context of specific patient sub-groups remains unclear. Methods: This study represents a post hoc sub-group analysis of data from a randomized multi-center parent study. The study population consisted of hospitalized HIV-infected patients with suspected TB who were unable to produce sputum and who underwent urine LAM testing. The diagnostic utility of urine LAM for TB in this group was compared to the performance of urine LAM in patients who did produce a sputum sample in the parent study. Results: There were a total of 187 and 2341 patients in the sputum-scarce and sputum-producing cohorts, respectively. 80 of the sputum-scarce patients underwent testing with urine LAM. In comparison to those who did produce sputum, sputum-scarce patients had a younger age, a lower Karnofsky performance score, and a lower weight and BMI at admission. A greater proportion of sputum-scarce patients were urine LAM positive, compared to those who were able to produce sputum (31% vs. 21%, p = 0.04). A higher proportion of sputum-scarce patients died within 8 weeks of admission (32% vs. 24%, p = 0.013). We inferred that 19% of HIV-infected sputum-scarce patients suspected of TB were diagnosed with tuberculosis by urine LAM testing, with an estimated positive predictive value of 63% (95% CI 43–82%). Conclusions: Urine LAM testing can effectively identify tuberculosis in HIV-infected patients who are at a higher risk of mortality yet are unable to generate a sputum sample for diagnostic testing. Our findings support the use of urine LAM testing in sputum-scarce hospitalized HIV-infected patients, and its incorporation into diagnostic algorithms for this patient population.
- ItemOpen AccessDiagnostic accuracy of a liquid chromatography-tandem mass spectrometry assay in small hair samples for rifampin-resistant tuberculosis drug concentrations in a routine care setting(2021-01-22) Metcalfe, John; Bacchetti, Peter; Esmail, Ali; Reckers, Andrew; Aguilar, David; Wen, Anita; Huo, Shu; Muyindike, Winnie R; Hahn, Judith A; Dheda, Keertan; Gandhi, Monica; Gerona, RoyBackground Treatment monitoring of drug-resistant tuberculosis (DR-TB) in resource-limited settings is challenging. We developed a multi-analyte assay for eleven anti-TB drugs in small hair samples as an objective metric of drug exposure. Methods Small hair samples were collected from participants at various timepoints during directly observed RR-TB treatment at an inpatient tertiary referral facility in South Africa (DR-TB cohort). We assessed qualitative determination (i.e., detection above limit of detection) of bedaquiline, linezolid, clofazimine, pretomanid, levofloxacin, moxifloxacin, pyrazinamide, isoniazid, ethambutol, ethionamide, and prothionamide in an LC-MS/MS index panel assay against a reference standard of inpatient treatment records. Because treatment regimens prior to hospitalization were not available, we also analyzed specificity (for all drugs except isoniazid) using an external cohort of HIV-positive patients treated for latent TB infection with daily isoniazid (HIV/LTBI cohort) in Uganda. Results Among the 57 DR-TB patients (58% with pre-XDR/XDR-TB; 70% HIV-positive) contributing analyzable hair samples, the sensitivity of the investigational assay was 94% or higher for all drugs except ethionamide (58.5, 95% confidence interval [CI], 40.7–99.9). Assay specificity was low across all tested analytes within the DR-TB cohort; conversely, assay specificity was 100% for all drugs in the HIV/LTBI cohort. Conclusions Hair drug concentrations reflect long-term exposure, and multiple successive regimens commonly employed in DR-TB treatment may result in apparent false-positive qualitative and falsely elevated quantitative hair drug levels when prior treatment histories within the hair growth window are not known.
- ItemOpen AccessDiagnostic accuracy of quantitative PCR (Xpert MTB/RIF) for tuberculous meningitis in a high burden setting: a prospective study(Public Library of Science, 2013) Patel, Vinod B; Theron, Grant; Lenders, Laura; Matinyena, Brian; Connolly, Cathy; Singh, Ravesh; Coovadia, Yacoob; Ndung'u, Thumbi; Dheda, KeertanBackground: Tuberculous meningitis (TBM) is difficult to diagnose promptly. The utility of the Xpert MTB/RIF test for the diagnosis of TBM remains unclear, and the effect of host- and sample-related factors on test performance is unknown. This study sought to evaluate the sensitivity and specificity of Xpert MTB/RIF for the diagnosis of TBM. Methods and Findings: 235 South-African patients with a meningeal-like illness were categorised as having definite (culture or Amplicor PCR positive), probable (anti-TBM treatment initiated but microbiological confirmation lacking), or non-TBM. Xpert MTB/RIF accuracy was evaluated using 1 ml of uncentrifuged and, when available, 3 ml of centrifuged cerebrospinal fluid (CSF). To evaluate the incremental value of MTB/RIF over a clinically based diagnosis, test accuracy was compared to a clinical score (CS) derived using basic clinical and laboratory information. Of 204 evaluable patients (of whom 87% were HIV-infected), 59 had definite TBM, 64 probable TBM, and 81 non-TBM. Overall sensitivity and specificity (95% CI) were 62% (48%–75%) and 95% (87%–99%), respectively. The sensitivity of Xpert MTB/RIF was significantly better than that of smear microscopy (62% versus 12%; p = 0.001) and significantly better than that of the CS (62% versus 30%; p = 0.001; C statistic 85% [79%–92%]). Xpert MTB/RIF sensitivity was higher when centrifuged versus uncentrifuged samples were used (82% [62%–94%] versus 47% [31%–61%]; p = 0.004). The combination of CS and Xpert MTB/RIF (Xpert MTB/RIF performed if CS<8) performed as well as Xpert MTB/RIF alone but with a ∼10% reduction in test usage. This overall pattern of results remained unchanged when the definite and probable TBM groups were combined. Xpert MTB/RIF was not useful in identifying TBM among HIV-uninfected individuals, although the sample was small. There was no evidence of PCR inhibition, and the limit of detection was ∼80 colony forming units per millilitre. Study limitations included a predominantly HIV-infected cohort and the limited number of culture-positive CSF samples. Conclusions: Xpert MTB/RIF may be a good rule-in test for the diagnosis of TBM in HIV-infected individuals from a tuberculosis-endemic setting, particularly when a centrifuged CSF pellet is used. Further studies are required to confirm these findings in different settings.
- ItemOpen AccessDiagnostic accuracy of quantitative PCR (Xpert MTB/RIF) for tuberculous pericarditis compared to adenosine deaminase and unstimulated interferon-γ in a high burden setting: a prospective study(2014-06-18) Pandie, Shaheen; Peter, Jonathan G; Kerbelker, Zita S; Meldau, Richard; Theron, Grant; Govender, Ureshnie; Ntsekhe, Mpiko; Dheda, Keertan; Mayosi, Bongani MBackground: Tuberculous pericarditis (TBP) is associated with high morbidity and mortality, and is an important treatable cause of heart failure in developing countries. Tuberculous aetiology of pericarditis is difficult to diagnose promptly. The utility of the new quantitative PCR test (Xpert MTB/RIF) for the diagnosis of TBP is unknown. This study sought to evaluate the diagnostic accuracy of the Xpert MTB/RIF test compared to pericardial adenosine deaminase (ADA) and unstimulated interferon-gamma (uIFNγ) in suspected TBP. Methods: From October 2009 through September 2012, 151 consecutive patients with suspected TBP were enrolled at a single centre in Cape Town, South Africa. Mycobacterium tuberculosis culture and/or pericardial histology served as the reference standard for definite TBP. Receiver-operating-characteristic curve analysis was used for selection of ADA and uIFNγ cut-points. Results: Of the participants, 49% (74/151) were classified as definite TBP, 33% (50/151) as probable TBP and 18% (27/151) as non TBP. A total of 105 (74%) participants were human immunodeficiency virus (HIV) positive. Xpert-MTB/RIF had a sensitivity and specificity (95% confidence interval (CI)) of 63.8% (52.4% to 75.1%) and 100% (85.6% to 100%), respectively. Concentration of pericardial fluid by centrifugation and using standard sample processing did not improve Xpert MTB/RIF accuracy. ADA (≥35 IU/L) and uIFNγ (≥44 pg/ml) both had a sensitivity of 95.7% (88.1% to 98.5%) and a negative likelihood ratio of 0.05 (0.02 to 0.10). However, the specificity and positive likelihood ratio of uIFNγ was higher than ADA (96.3% (81.7% to 99.3%) and 25.8 (3.6 to 183.4) versus 84% (65.4% to 93.6%) and 6.0 (3.7 to 9.8); P = 0.03) at an estimated background prevalence of TB of 30%. The sensitivity and negative predictive value of both uIFNγ and ADA were higher than Xpert-MT/RIF (P < 0.001). Conclusions: uIFNγ offers superior accuracy for the diagnosis of microbiologically confirmed TBP compared to the ADA assay and the Xpert MTB/RIF test.
- ItemOpen AccessThe diagnostic accuracy of urine-based Xpert MTB/RIF in HIV-infected hospitalized patients who are smear-negative or sputum scarce(Public Library of Science, 2012) Peter, Jonathan G; Theron, Grant; Muchinga, Tapuwa E; Govender, Ureshnie; Dheda, KeertanBACKGROUND: Hospitals in sub-Saharan Africa are inundated with HIV-infected patients and tuberculosis (TB) is the commonest opportunistic infection in this sub-group. Up to one third of TB-HIV co-infected patients fail to produce a sputum sample (sputum scarce) and diagnosis is thus often delayed or missed. We investigated the sensitivity of urine-based methods (Xpert MTB/RIF, LAM strip test and LAM ELISA) in such patients. METHODOLOGY/PRINCIPAL FINDINGS: 281 HIV-infected hospitalised patients with clinically suspected TB provided a spot urine sample. The reference standard was culture positivity for Mycobacterium tuberculosis on ≥1 sputum or extra-pulmonary sample. MTB/RIF was performed using 1 ml of both unprocessed and, when possible, concentrated urine. Each unconcentrated urine sample was also tested using the Clearview LAM ELISA and Alere LAM strip test. 42% (116/242) of patients had culture-proven TB. 18% (20/54) were sputum scarce. In sputum-scarce patients, the sensitivity of urine MTB/RIF and LAM ELISA was 40% (95%CI: 22-61) and 60% (95%CI: 39-78), respectively. Urine MTB/RIF specificity was 98% (95%CI: 95-100). Combined sensitivity of urine LAM ELISA and MTB/RIF was better than MTB/RIF alone [MTB/RIF and LAM: 70% (95%CI: 48-85) vs. MTB/RIF: 40% (95%CI: 22-61), p = 0.03]. Significant predictors of urine MTB/RIF positivity were CD4<50 cells/ml (p = 0.001), elevated protein-to-creatinine ratio (p<0.001) and LAM ELISA positivity (p<0.001). Urine centrifugation and pelleting significantly increased the sensitivity of MTB/RIF over unprocessed urine in paired samples [42% (95%CI: 26-58) vs. 8% (95%CI: 0-16), p<0.001]. Urine MTB/RIF-generated C T values correlated poorly with markers of bacillary burden (smear grade and time-to-positivity). Conclusions/Significance This preliminary study indicates that urine-based MTB/RIF, alone or in combination with LAM antigen detection, may potentially aid the diagnosis of TB in HIV-infected patients with advanced immunosuppression when sputum-based diagnosis is not possible. Concentration of urine prior to MTB/RIF-testing significantly improves sensitivity.
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