OpenUCT :: Browsing by Subject "Mycobacterium tuberculosis"

Browsing by Subject "Mycobacterium tuberculosis"

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Open Access
Altered ratio of IFN-γ/IL-10 in patients with drug resistant Mycobacterium tuberculosis and HIV-tuberculosis immune reconstitution inflammatory syndrome
(Public Library of Science, 2012) Skolimowska, Keira H; Rangaka, Molebogeng X; Meintjes, Graeme; Pepper, Dominique J; Seldon, Ronnett; Matthews, Kerryn; Wilkinson, Robert J; Wilkinson, Katalin A
We have described a clinical relationship between HIV-Tuberculosis Immune Reconstitution Inflammatory Syndrome (TB-IRIS) and anti-tubercular drug resistance. Here we studied the immune response of TB-IRIS patients from whom a drug-resistant (n = 11) or drug-susceptible (n = 25) Mycobacterium tuberculosis (MTB) strain was isolated after presenting with TB-IRIS. ELISpot analysis and multiplex cytokine analysis of the supernatant collected from peripheral blood mononuclear cells stimulated overnight with the heat-killed H37Rv MTB laboratory strain was used. Although there was no statistical difference in IFN-gamma ELISpot responses between the two groups, the results point towards higher bacterial load in the drug-resistant patients, possibly due to failed therapy. The ratio between secreted IFN-gamma/IL-10 and IL-2/IL-10 was significantly lower in TB-IRIS patients in whom the cause of TB was a drug-resistant strain compared to those with a fully sensitive strain (p = 0.02). Since host immune responses are dependent on the bacterial load, we hypothesise that the impaired cytokine balance is likely to be caused by the poorly controlled bacterial growth in these patients.
Open Access
Association of human TLR1 and TLR6 deficiency with altered immune responses to BCG vaccination in South African infants
(Public Library of Science, 2011) Randhawa, April Kaur; Shey, Muki S; Keyser, Alana; Peixoto, Blas; Wells, Richard D; de Kock, Marwou; Lerumo, Lesedi; Hughes, Jane; Hussey, Gregory; Hawkridge, Anthony; Kaplan, Gilla; Hanekom, Willem A; Hawn, Thomas R
The development of effective immunoprophylaxis against tuberculosis (TB) remains a global priority, but is hampered by a partially protective Bacillus Calmette-Guérin (BCG) vaccine and an incomplete understanding of the mechanisms of immunity to Mycobacterium tuberculosis. Although host genetic factors may be a primary reason for BCG's variable and inadequate efficacy, this possibility has not been intensively examined. We hypothesized that Toll-like receptor (TLR) variation is associated with altered in vivo immune responses to BCG. We examined whether functionally defined TLR pathway polymorphisms were associated with T cell cytokine responses in whole blood stimulated ex vivo with BCG 10 weeks after newborn BCG vaccination of South African infants. In the primary analysis, polymorphism TLR6_C745T (P249S) was associated with increased BCG-induced IFN-γ in both discovery (n = 240) and validation (n = 240) cohorts. In secondary analyses of the combined cohort, TLR1_T1805G (I602S) and TLR6_G1083C (synonymous) were associated with increased IFN-γ, TLR6_G1083C and TLR6_C745T were associated with increased IL-2, and TLR1_A1188T was associated with increased IFN-γ and IL-2. For each of these polymorphisms, the hypo-responsive allele, as defined by innate immunity signaling assays, was associated with increased production of TH1-type T cell cytokines (IFN-γ or IL-2). After stimulation with TLR1/6 lipopeptide ligands, PBMCs from TLR1/6-deficient individuals (stratified by TLR1_T1805G and TLR6_C745T hyporesponsive genotypes) secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1/6 genotypes. In contrast, no IL-12p70 was secreted by PBMCs or monocytes. These data support a mechanism where TLR1/6 polymorphisms modulate TH1 T-cell polarization through genetic regulation of monocyte IL-10 secretion in the absence of IL-12. These studies provide evidence that functionally defined innate immune gene variants are associated with the development of adaptive immune responses after in vivo vaccination against a bacterial pathogen in humans. These findings could potentially guide novel adjuvant vaccine strategies as well as have implications for IFN-γ-based diagnostic testing for TB.
Open Access
Bacterial loads measured by the Xpert MTB/RIF assay as markers of culture conversion and bacteriological cure in pulmonary TB
(Public Library of Science, 2016) Shenai, Shubhada; Ronacher, Katharina; Malherbe, Stefanus; Stanley, Kim; Kriel, Magdalena; Winter, Jill; Peppard, Thomas; Barry, Charles E; Wang, Jing; Dodd, Lori E; Via, Laura E; Walzl, Gerhard; Alland, David
Introduction Biomarkers are needed to monitor tuberculosis (TB) treatment and predict treatment outcomes. We evaluated the Xpert MTB/RIF (Xpert) assay as a biomarker for TB treatment during and at the end of the 24 weeks therapy. METHODS: Sputum from 108 HIV-negative, culture-positive pulmonary TB patients was analyzed using Xpert at time points before and during anti-TB therapy. Results were compared against culture. Direct Xpert cycle-threshold (Ct), a change in the Ct (delta Ct), or a novel "percent closing of baseline Ct deficit" (percent closing) were evaluated as classifiers of same-day and end-of-treatment culture and therapeutic outcomes. RESULTS: Xpert was positive in 29/95 (30.5%) of subjects at week 24; and positive one year after treatment in 8/64 (12.5%) successfully-treated patients who remained free of tuberculosis. We identified a relationship between initial bacterial load measured by baseline Xpert Ct and time to culture conversion (hazard ratio 1.06, p = 0.0023), and to the likelihood of being among the 8 treatment failures at week 24 (AUC = 72.8%). Xpert Ct was even more strongly associated with culture conversion on the day the test was performed with AUCs 96.7%, 99.2%, 86.0% and 90.2%, at Day 7, Week 4, 8 and 24, respectively. Compared to baseline Ct measures alone, a combined measure of baseline Ct plus either Delta Ct or percent closing improved the classification of treatment failure status to a 75% sensitivity and 88.9% specificity. CONCLUSIONS: Genome loads measured by Xpert provide a potentially-useful biomarker for classifying same day culture status and predicting response to therapy.
Open Access
Barriers to initiation of antiretrovirals during antituberculosis therapy in Africa
(Public Library of Science, 2011) Pepper, Dominique J; Marais, Suzaan; Wilkinson, Robert J; Bhaijee, Feriyl; De Azevedo, Virginia; Meintjes, Graeme
BACKGROUND: In the developing world, the principal cause of death among HIV-infected patients is tuberculosis (TB). The initiation of antiretroviral therapy (ART) during TB therapy significantly improves survival, however it is not known which barriers prevent eligible TB patients from initiating life-saving ART. Method Setting. A South African township clinic with integrated tuberculosis and HIV services. Design. Logistic regression analyses of a prospective cohort of HIV-1 infected adults (≥18 years) who commenced TB therapy, were eligible for ART, and were followed for 6 months. FINDINGS: Of 100 HIV-1 infected adults eligible for ART during TB therapy, 90 TB patients presented to an ART clinic for assessment, 66 TB patients initiated ART, and 15 TB patients died. 34% of eligible TB patients (95%CI: 25-43%) did not initiate ART. Male gender and younger age (<36 years) were associated with failure to initiate ART (adjusted odds ratios of 3.7 [95%CI: 1.25-10.95] and 3.3 [95%CI: 1.12-9.69], respectively). Death during TB therapy was associated with a CD4+ count <100 cells/µL. CONCLUSION: In a clinic with integrated services for tuberculosis and HIV, one-third of eligible TB patients - particularly young men - did not initiate ART. Strategies are needed to promote ART initiation during TB therapy, especially among young men.
Open Access
Baseline Predictors of Sputum Culture Conversion in Pulmonary Tuberculosis: Importance of Cavities, Smoking, Time to Detection and W-Beijing Genotype
(Public Library of Science, 2012) Visser, Marianne E; Stead, Michael C; Walzl, Gerhard; Warren, Rob; Schomaker, Michael; Grewal, Harleen M S; Swart, Elizabeth C; Maartens, Gary
BACKGROUND: Time to detection (TTD) on automated liquid mycobacterial cultures is an emerging biomarker of tuberculosis outcomes. The M. tuberculosis W-Beijing genotype is spreading globally, indicating a selective advantage. There is a paucity of data on the association between baseline TTD and W-Beijing genotype and tuberculosis outcomes. Aim To assess baseline predictors of failure of sputum culture conversion, within the first 2 months of antitubercular therapy, in participants with pulmonary tuberculosis. Design Between May 2005 and August 2008 we conducted a prospective cohort study of time to sputum culture conversion in ambulatory participants with first episodes of smear and culture positive pulmonary tuberculosis attending two primary care clinics in Cape Town, South Africa. Rifampicin resistance (diagnosed on phenotypic susceptibility testing) was an exclusion criterion. Sputum was collected weekly for 8 weeks for mycobacterial culture on liquid media (BACTEC MGIT 960). Due to missing data, multiple imputation was performed. Time to sputum culture conversion was analysed using a Cox-proportional hazards model. Bayesian model averaging determined the posterior effect probability for each variable. RESULTS: 113 participants were enrolled (30.1% female, 10.5% HIV-infected, 44.2% W-Beijing genotype, and 89% cavities). On Kaplan Meier analysis 50.4% of participants underwent sputum culture conversion by 8 weeks. The following baseline factors were associated with slower sputum culture conversion: TTD (adjusted hazard ratio (aHR) = 1.11, 95% CI 1.02; 1.2), lung cavities (aHR = 0.13, 95% CI 0.02; 0.95), ever smoking (aHR = 0.32, 95% CI 0.1; 1.02) and the W-Beijing genotype (aHR = 0.51, 95% CI 0.25; 1.07). On Bayesian model averaging, posterior probability effects were strong for TTD, lung cavitation and smoking and moderate for W-Beijing genotype. CONCLUSION: We found that baseline TTD, smoking, cavities and W-Beijing genotype were associated with delayed 2 month sputum culture. Larger studies are needed to confirm the relationship between the W-Beijing genotype and sputum culture conversion.
Open Access
Blood neutrophil counts in HIV-infected patients with pulmonary tuberculosis: association with sputum mycobacterial load
(Public Library of Science, 2013) Kerkhoff, Andrew D; Wood, Robin; Lowe, David M; Vogt, Monica; Lawn, Stephen D
BACKGROUND: Increasing evidence suggests that neutrophils play a role in the host response to Mycobacterium tuberculosis . We determined whether neutrophil counts in peripheral blood are associated with tuberculosis (TB) and with mycobacterial load in sputum in HIV-infected patients. METHODOLOGY/PRINCIPAL FINDINGS: Adults enrolling in an antiretroviral treatment (ART) clinic in a Cape Town township were screened for TB regardless of symptoms. Paired sputum samples were examined using liquid culture, fluorescence microscopy, and the Xpert MTB/RIF assay. Absolute neutrophil counts (ANC) were measured in blood samples. Of 602 HIV-infected patients screened, 523 produced one or more sputum samples and had complete results available for analysis. Among these 523 patients, the median CD4 count was 169×10 9 /L (IQR, 96-232) and median ANC was 2.6×10 9 /L (IQR, 1.9-3.6). Culture-positive pulmonary tuberculosis was diagnosed in 89 patients. Patients with TB had a median ANC of 3.4×10 9 /L (IQR, 2.4-5.1) compared to 2.5×10 9 /L (IQR, 1.8-3.4) among those who were culture negative (p<0.0001). In multivariable analyses, having pulmonary TB was associated with an adjusted risk ratio (aRR) of 2.6 (95%CI, 1.5-4.5) for having an ANC level that exceeded the median value (ANC ≥2.6×10 9 /L; p = 0.0006) and an aRR of 6.8 (95%CI, 2.3-20.4) for having neutrophilia defined by a neutrophil count exceeding the upper limit of the normal range (ANC >7.5×10 9 /L; p = 0.0005). Patients were then classified into four mutually exclusive groups with increasing sputum mycobacterial load as defined by the results of culture, Xpert MTB/RIF and sputum smear microscopy. Multivariable analyses demonstrated that increasing sputum mycobacterial load was positively associated with blood ANC ≥2.6×10 9 /L and with neutrophilia. Conclusions/Significance Increased blood neutrophil counts were independently associated with pulmonary TB and sputum mycobacterial burden in this HIV-infected patient group. This observation supports the growing body of literature regarding the potential role for neutrophils in the host response to TB.
Open Access
Burden of new and recurrent tuberculosis in a major South African city stratified by age and HIV-status
(Public Library of Science, 2011) Wood, Robin; Lawn, Stephen D; Caldwell, Judy; Kaplan, Richard; Middelkoop, Keren; Bekker, Linda-Gail
Aim: To describe the burden of tuberculosis (TB) in Cape Town by calculating TB incidence rates stratified by age and HIV-status, assessing the contribution of retreatment disease and estimating the cumulative lifetime TB risk in HIV-negative individuals. METHODS: Details of TB cases were abstracted from the 2009 electronic TB register. Population denominators were estimated from census data and actuarial estimates of HIV prevalence, allowing calculation of age-specific and HIV-stratified TB notification rates. RESULTS: The 2009 mid-year population was 3,443,010 (3,241,508 HIV-negative and 201,502 HIV-positive individuals). There were 29,478 newly notified TB cases of which 56% were laboratory confirmed. HIV status was recorded for 87% of cases and of those with known HIV-status 49% were HIV-negative and 51% were positive. Discrete peaks in the incidence of non-HIV-associated TB occurred at three ages: 511/100,000 at 0-4 years of age, 553/100,000 at 20-24 years and 628/100,000 at 45-49 years with 1.5%, 19% and 45% being due to retreatment TB, respectively. Only 15.5% of recurrent cases had a history of TB treatment failure or default. The cumulative lifetime risks in the HIV-negative population of all new TB episodes and new smear-positive TB episodes were 24% and 12%, respectively; the lifetime risk of retreatment disease was 9%. The HIV-positive notification rate was 6,567/100,000 (HIV-associated TB rate ratio = 17). Although retreatment cases comprised 30% of the HIV-associated TB burden, 88% of these patients had no history of prior treatment failure or default. CONCLUSIONS: The annual burden of TB in this city is huge. TB in the HIV-negative population contributed almost half of the overall disease burden and cumulative lifetime risks were similar to those reported in the pre-chemotherapy era. Retreatment TB contributed significantly to both HIV-associated and non-HIV-associated TB but infrequently followed prior inadequate treatment. This likely reflects ongoing TB transmission to both HIV-negative and positive individuals.
Open Access
Can 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, Keertan
BACKGROUND: 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.
Open Access
Cardio-thoracic ratio is stable, reproducible and has potential as a screening tool for HIV-1 related cardiac disorders in resource poor settings
(Public Library of Science, 2016) Esmail, Hanif; Oni, Tolu; Thienemann, Friedrich; Omar-Davies, Nashreen; Wilkinson, Robert J; Ntsekhe, Mpiko
BACKGROUND: Cardiovascular disorders are common in HIV-1 infected persons in Africa and presentation is often insidious. Development of screening algorithms for cardiovascular disorders appropriate to a resource-constrained setting could facilitate timely referral. Cardiothoracic ratio (CTR) on chest radiograph (CXR) has been suggested as a potential screening tool but little is known about its reproducibility and stability. Our primary aim was to evaluate the stability and the inter-observer variability of CTR in HIV-1 infected outpatients. We further evaluated the prevalence of cardiomegaly (CTR≥0.5) and its relationship with other risk factors in this population. METHODOLOGY: HIV-1 infected participants were identified during screening for a tuberculosis vaccine trial in Khayelitsha, South Africa between August 2011 and April 2012. Participants had a digital posterior-anterior CXR performed as well as history, examination and baseline observations. CXRs were viewed using OsiriX software and CTR calculated using digital callipers. RESULTS: 450 HIV-1-infected adults were evaluated, median age 34 years (IQR 30-40) with a CD4 count 566/mm 3 (IQR 443-724), 70% on antiretroviral therapy (ART). The prevalence of cardiomegaly was 12.7% (95% C.I. 9.6%-15.8%). CTR was calculated by a 2 nd reader for 113 participants, measurements were highly correlated r = 0.95 (95% C.I. 0.93-0.97) and agreement of cardiomegaly substantial κ = 0.78 (95% C.I 0.61-0.95). CXR were repeated in 51 participants at 4-12 weeks, CTR measurements between the 2 time points were highly correlated r = 0.77 (95% C.I 0.68-0.88) and agreement of cardiomegaly excellent κ = 0.92 (95% C.I. 0.77-1). Participants with cardiomegaly had a higher median BMI (31.3; IQR 27.4-37.4) versus 26.9; IQR 23.2-32.4); p<0.0001) and median systolic blood pressure (130; IQR 121-141 versus 125; IQR 117-135; p = 0.01). CONCLUSION: CTR is a robust measurement, stable over time with substantial inter-observer agreement. A prospective study evaluating utility of CXR to identify cardiovascular disorder in this population is warranted.
Open Access
Catching a glimpse: the visualization of Mycobacterium tuberculosis from TB patient bioaerosols
(2023) Dinkele, Ryan; Warner, Digby; Gessner, Sophia
Transmission between hosts is crucial for the success and survival of the obligate human pathogen and aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb). Despite this, little is known about how and when Mtb is aerosolized nor the key metabolic and morphological determinants driving successful transmission. To address these knowledge gaps, my doctoral research sought to develop a microscopic method for the detection of aerosolized Mtb following liquidcapture within the respiratory aerosol sampling chamber (RASC). This was achieved through the combination of the mycobacterial cell wall probe, 4-N,Ndimethylamino-1,8-naphthalimide-trehalose (DMN-tre), with the arraying of bioaerosol samples on bespoke nanowell devices amenable to fluorescence microscopy. With this method, a median of 14 live Mtb bacilli (range 0-36) were detected in 90% of confirmed TB patients following 60 minutes of bioaerosol sampling. Three distinct DMN-tre staining patterns were identified among aerosolized Mtb, strongly suggestive of metabolic heterogeneity. Moreover, a low proportion of patients produced Mtb in small clumps. These observations highlight the advantages of using microscopy over conventional culture- or molecular-based techniques for probing the metabolic and morphological characteristics of aerosolized Mtb. Applying this method in a second study, we sought to understand how and when Mtb is aerosolized. To this end, we aimed to compare the aerosolization of Mtb and total particulate matter from patients with TB during three respiratory manoeuvres: tidal breathing (TiBr), forced vital capacity (FVC), and cough. Although total particle counts were 4.8-fold greater in cough samples than either TiBr or FVC, all three manoeuvres returned similar rates of positivity for Mtb. No correlation was observed between total particle production and Mtb count. Instead, for total Mtb counts, the variability between individuals was greater than the variability between sampling manoeuvres. Finally, when modelled using 24-hour breath and cough frequencies, our data indicate that TiBr might contribute more than 90% of the daily aerosolized Mtb among symptomatic TB patients. Assuming the number of viable Mtb organisms detected provides a proxy measure of patient infectiousness, this method suggests that TiBr is a significant contributor to TB transmission. In developing a novel platform for the detection of aerosolized Mtb, this work has suggested the need to re-examine old assumptions about Mtb transmission.
Open Access
Clinical deterioration during antitubercular treatment at a district hospital in South Africa: the importance of drug resistance and AIDS defining illnesses
(Public Library of Science, 2009) Pepper, Dominique J; Rebe, Kevin; Morroni, Chelsea; Wilkinson, Robert J; Meintjes, Graeme
BACKGROUND: Clinical deterioration on drug therapy for tuberculosis is a common cause of hospital admission in Africa. Potential causes for clinical deterioration in settings of high HIV-1 prevalence include drug resistant Mycobacterium tuberculosis (M.tb) , co-morbid illnesses, poor adherence to therapy, tuberculosis associated-immune reconstitution inflammatory syndrome (TB-IRIS) and subtherapeutic antitubercular drug levels. It is important to derive a rapid diagnostic work-up to determine the cause of clinical deterioration as well as specific management to prevent further clinical deterioration and death. We undertook this study among tuberculosis (TB) patients referred to an adult district level hospital situated in a high HIV-1 prevalence setting to determine the frequency, reasons and outcome for such clinical deterioration. Method A prospective observational study conducted during the first quarter of 2007. We defined clinical deterioration as clinical worsening or failure to stabilise after 14 or more days of antitubercular treatment, resulting in hospital referral. We collected data on tuberculosis diagnosis and treatment, HIV-1 status and antiretroviral treatment, and investigated reasons for clinical deterioration as well as outcome. RESULTS: During this period, 352 TB patients met inclusion criteria; 296 were admitted to hospital accounting for 17% of total medical admissions (n = 1755). Eighty three percent of TB patients (291/352) were known to be HIV-1 co-infected with a median CD4 count of 89cells/mm 3 (IQR 38-157). Mortality among TB patients admitted to hospital was 16% (n = 48). The median duration of hospital admission was 9.5 days (IQR 4-18), longer than routine in this setting (4 days). Among patients in whom HIV-1 status was known (n = 324), 72% of TB patients (n = 232) had an additional illness to tuberculosis; new AIDS defining illnesses (n = 80) were the most frequent additional illnesses (n = 208) in HIV-1 co-infected patients (n = 291). Rifampin-resistant M.tb (n = 41), TB-IRIS (n = 51) and drug resistant bacterial infections (n = 12) were found in 12%, 14% and 3.4% of the 352 cases, respectively. Interpretation In our setting, new AIDS defining illnesses, drug resistant M.tb and other drug resistant bacteria are important reasons for clinical deterioration in HIV-1 co-infected patients receiving antitubercular treatment. HIV-1 co-infected patients may be at increased risk of acquiring nosocomial drug resistant pathogens because profound immune suppression results in co-morbid illnesses that require prolonged inpatient admissions. Routine infection control is essential and needs to be strengthened in our setting.
Open Access
Clinical 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, Rodney
BACKGROUND: 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.
Open Access
Clinical 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, Rodney
BACKGROUND: 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.
Open Access
Comparative genomic and phylogenetic approaches to characterize the role of genetic recombination in mycobacterial evolution
(Public Library of Science, 2012) Smith, Silvia E; Showers-Corneli, Patrice; Dardenne, Caitlin N; Harpending, Henry H; Martin, Darren P; Beiko, Robert G
The genus Mycobacterium encompasses over one hundred named species of environmental and pathogenic organisms, including the causative agents of devastating human diseases such as tuberculosis and leprosy. The success of these human pathogens is due in part to their ability to rapidly adapt to their changing environment and host. Recombination is the fastest way for bacterial genomes to acquire genetic material, but conflicting results about the extent of recombination in the genus Mycobacterium have been reported. We examined a data set comprising 18 distinct strains from 13 named species for evidence of recombination. Genomic regions common to all strains (accounting for 10% to 22% of the full genomes of all examined species) were aligned and concatenated in the chromosomal order of one mycobacterial reference species. The concatenated sequence was screened for evidence of recombination using a variety of statistical methods, with each proposed event evaluated by comparing maximum-likelihood phylogenies of the recombinant section with the non-recombinant portion of the dataset. Incongruent phylogenies were identified by comparing the site-wise log-likelihoods of each tree using multiple tests. We also used a phylogenomic approach to identify genes that may have been acquired through horizontal transfer from non-mycobacterial sources. The most frequent associated lineages (and potential gene transfer partners) in the Mycobacterium lineage-restricted gene trees are other members of suborder Corynebacterinae, but more-distant partners were identified as well. In two examined cases of potentially frequent and habitat-directed transfer ( M. abscessus to Segniliparus and M. smegmatis to Streptomyces ), observed sequence distances were small and consistent with a hypothesis of transfer, while in a third case ( M. vanbaalenii to Streptomyces ) distances were larger. The analyses described here indicate that whereas evidence of recombination in core regions within the genus is relatively sparse, the acquisition of genes from non-mycobacterial lineages is a significant feature of mycobacterial evolution.
Open Access
Comparative performance characteristics of the urine lipoarabinomannan strip test and sputum smear microscopy in hospitalized HIV-infected patients with suspected tuberculosis in Harare, Zimbabwe
(2015) Zijenah, Lynn Sodai; Kadzirange, Gerard; Bandason, Tsitsi; Chipiti, Maria Mary; Gwambiwa, Bevel; Makoga, Forget; Chungu, Pauline; Kaguru, Philip; Sabur, Natasha F
BackgroundIn Zimbabwe, sputum smear microscopy (SSM) is the routinely used TB diagnostic tool in hospitalised HIV-infected patients. However, SSM has poor sensitivity in HIV-infected patients. We compared performance of urine lipoarabinomannan strip test (LAM) and SSM among hospitalized HIV-infected patients with suspected TB.MethodsHospitalized HIV-infected patients with suspected TB were randomized to LAM plus SSM or SSM alone groups as part of a larger multi-country parent study. Here we present a comparison of LAM versus SSM performance from the Zimbabwe study site. LAM analyses (grade 2 cut-off) were conducted using (i) a microbiological reference standard (MRS; culture positivity for M.tb and designated definite TB) and (ii) a composite reference standard (CRS; definite TB plus probable TB i.e. patients with clinical TB excluded from the culture negative group). CRS constituted the primary analysis.Results82/457 (18%) of the patients randomized to the LAM group were M.tuberculosis culture positive. Using CRS, sensitivity (%, 95 % CI) of LAM was significantly higher than SSM [49.2 (42.1-56.4) versus 29.4(23.2-36.3); p < 0.001]. Specificity and PPV were 98.1%, and 95.8%, respectively. By contrast, using MRS, LAM sensitivity was similar to SSM and specificity was significantly lower, however, the combined sensitivity of LAM and SSM was significantly higher than that of SSM alone, p = 0.009. Using CRS, LAM sensitivity (%, CI) was CD4 count dependent [60.6(50.7-69.8) at ≤50 cells/μL; 40.0(22.7-59.4) at 51-100 cells/μL, and 32.8(21.0-46.3) at >100 cells/μL. The combined sensitivity of LAM and SSM was higher than SSM alone being highest at CD4 counts <50 cells/μL [67.6(57.9-76.3); p = <0.001]. Specificity of LAM or SSM alone, or of combined LAM and SSM was >97% in all the 3 CD4 strata.ConclusionAmong hospitalized HIV-infected patients with suspected TB, the sensitivity of LAM is significantly higher than that of SSM, especially at low CD4 counts. LAM and SSM are complimentary tests for diagnosis of TB in HIV-infected patients. We recommend a combination of LAM and SSM for TB diagnosis in HIV-infected patients with low CD4 counts in HIV/TB co-endemic countries, where alternative methods are unavailable.
Open Access
Comparison of mantoux and tine tuberculin skin tests in BCG-vaccinated children investigated for tuberculosis
(Public Library of Science, 2009) Pan, Wenli; Matizirofa, Lyness; Workman, Lesley; Hawkridge, Tony; Hanekom, Willem; Mahomed, Hassan; Hussey, Gregory; Hatherill, Mark
Background: Tuberculin skin tests (TSTs) are long-established screening methods for tuberculosis (TB). We aimed to compare agreement between the intradermal Mantoux and multipuncture percutaneous Tine methods and to quantify risk factors for a positive test result. Methodology/Principal Findings: 1512 South African children younger than 5 years of age who were investigated for tuberculosis (TB) during a Bacille Calmette Guerin (BCG) trial were included in this analysis. Children underwent both Mantoux and Tine tests. A positive test was defined as Mantoux ≥15 mm or Tine ≥ Grade 3 for the binary comparison. Agreement was evaluated using kappa (binary) and weighted kappa (hierarchical). Multivariate regression models identified independent risk factors for TST positivity. The Mantoux test was positive in 430 children (28.4%) and the Tine test in 496 children (32.8%, p<0.0001), with observed binary agreement 87.3% (kappa 0.70) and hierarchical agreement 85.0% (weighted kappa 0.66). Among 173 children culture-positive for Mycobacterium tuberculosis, Mantoux was positive in 49.1% and Tine in 54.9%, p<0.0001 (kappa 0.70). Evidence of digit preference was noted for Mantoux readings at 5 mm threshold intervals. After adjustment for confounders, a positive culture, suggestive chest radiograph, and proximity of TB contact were risk factors for a positive test using both TST methods. There were no independent associations between ethnicity, gender, age, or over-crowding, and TST result. Conclusions/Significance: The Tine test demonstrated a higher positive test rate than the Mantoux, with substantial agreement between TST methods among young BCG-vaccinated children. TB disease and exposure factors, but not demographic variables, were independent risk factors for a positive result using either test method. These findings suggest that the Tine might be a useful screening tool for childhood TB in resource-limited countries.
Open Access
Comparison 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, Keertan
Introduction: 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.
Open Access
Comprehensive definition of Ser/Thr/Tyr phosphorylation in mycobacteria: towards understanding reprogramming of normal macrophage function by pathogenic mycobacteria
(2018) Nakedi, Kehilwe Confidence; Blackburn, Jonathan M; Da Cruz Soares, Nelson
Mycobacterium tuberculosis, the causative agent for the disease Tuberculosis, is a serious public health problem that is responsible for 1.6 million deaths each year. The WHO’s recent report on Tuberculosis estimates that a third of the world’s population is latently infected with the bacteria, and, of those, 10% will progress to active disease. M. tuberculosis is a successful pathogen mainly due to its ability to adapt and survive in changing environments. It can survive a dormant state with limited metabolic activity during latent infection, while also being able to escape the macrophage and disseminate into active disease. Efforts to eradicate the disease must be based on understanding the biology of this organism, and the mechanisms it uses to infect, colonize, and evade the immune system. Understanding the behaviour of pathogenic mycobacteria in the macrophage is also important to the discovery of new drug targets. In this thesis, we employed state of the art mass spectrometry techniques, which allowed us to unpack the biology of this bacterium in different growth environments and expand our understanding of the mechanisms it employs to adapt and survive. We investigated protein regulation by the process of phosphorylation, through sensory kinases, which add a phosphate group to a protein of interest, thereby regulating its function. First, we interrogated the phosphoproteomic landscape between M. bovis BCG and M. smegmatis to explain how differential protein regulation results in the differences between slow and fast growth of mycobacteria. Second, we focused on Protein Kinase G (PknG), which plays an important role in bacterial survival by blocking phagosome/lysosome fusion. We identified the in vivo physiological substrates of this kinase in actively growing M.bovis BCG culture. Our results revealed that this kinase is a regulator of protein synthesis. We then examined the mechanisms of survival in murine RAW 246.7 macrophages mediated by PknG, using M. bovis BCG reference strain and PknG knock-out mutant. Our results indicated strong evidence that pathogenic mycobacteria disrupt the macrophagic cytoskeleton, through phosphorylation of proteins that are involved in cytoskeleton rearrangement. These results explain the strategies that pathogenic mycobacteria employ mediated by PknG to block phagosome-lysosome fusion and evade the host immune system and survive for prolonged periods in the macrophages. The findings of this thesis contribute to our understanding of the physiology of pathogenic mycobacteria and their interaction with the host.
Open Access
Conserved immune recognition hierarchy of mycobacterial PE/PPE proteins during infection in natural hosts
(Public Library of Science, 2012) Vordermeier, H Martin; Hewinson, R Glyn; Wilkinson, Robert J; Wilkinson, Katalin A; Gideon, Hannah P; Young, Douglas B; Sampson, Samantha L
The Mycobacterium tuberculosis genome contains two large gene families encoding proteins of unknown function, characterized by conserved N-terminal proline and glutamate (PE and PPE) motifs. The presence of a large number of PE/PPE proteins with repetitive domains and evidence of strain variation has given rise to the suggestion that these proteins may play a role in immune evasion via antigenic variation, while emerging data suggests that some family members may play important roles in mycobacterial pathogenesis. In this study, we examined cellular immune responses to a panel of 36 PE/PPE proteins during human and bovine infection. We observed a distinct hierarchy of immune recognition, reflected both in the repertoire of PE/PPE peptide recognition in individual cows and humans and in the magnitude of IFN-γ responses elicited by stimulation of sensitized host cells. The pattern of immunodominance was strikingly similar between cattle that had been experimentally infected with Mycobacterium bovis and humans naturally infected with clinical isolates of M. tuberculosis. The same pattern was maintained as disease progressed throughout a four-month course of infection in cattle, and between humans with latent as well as active tuberculosis. Detailed analysis of PE/PPE responses at the peptide level suggests that antigenic cross-reactivity amongst related family members is a major determinant in the observed differences in immune hierarchy. Taken together, these results demonstrate that a subset of PE/PPE proteins are major targets of the cellular immune response to tuberculosis, and are recognized at multiple stages of infection and in different disease states. Thus this work identifies a number of novel antigens that could find application in vaccine development, and provides new insights into PE/PPE biology.
Open Access
Correlation 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, Keertan
BACKGROUND: 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.