Browsing by Author "Nicol, Mark"

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    Open Access
    “A very humiliating illness”: a qualitative study of patient-centered Care for Rifampicin-Resistant Tuberculosis in South Africa
    (2020-01-17) Furin, Jennifer; Loveday, Marian; Hlangu, Sindisiwe; Dickson-Hall, Lindy; le Roux, Sacha; Nicol, Mark; Cox, Helen
    Abstract Background Patient-centered care is pillar 1 of the “End TB” strategy, but little has been documented in the literature about what this means for people living with rifampicin-resistant (RR-TB). Optimizing care for such individuals requires a better understanding of the challenges they face and the support they need. Methods A qualitative study was done among persons living with RR-TB and members of their support network. A purposive sample was selected from a larger study population and open-ended interviews were conducted using a semi-standard interview guide. Interviews were recorded and transcribed and the content analyzed using an iterative thematic analysis based in grounded theory. Results 16 participants were interviewed from three different provinces. Four distinct periods in which support was needed were identified: 1) pre-diagnosis; 2) pre-treatment; 3) treatment; and 4) post-treatment. Challenges common in all four periods included: socioeconomic issues, centralized care, and the need for better counseling at multiple levels. Conclusions Beyond being a “very humiliating illness”, RR-TB robs people of their physical, social, economic, psychological, and emotional well-being far beyond the period when treatment is being administered. Efforts to tackle these issues are as important as new drugs and diagnostics in the fight against TB.
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    Open Access
    Ambulatory and hospitalized childhood pneumonia: a longitudinal study in a peri-urban low-income community with high vaccination coverage in Sub-Saharan Africa
    (2022) le Roux, David; Zar, Heather; Nicol, Mark
    Background Child pneumonia is a substantial cause of childhood mortality and morbidity; it is the largest single cause of under-5 mortality outside the neonatal period. Incidence of child pneumonia, and pneumonia mortality, have decreased substantially due to improved socio-economic attainment, improved HIV programs, coverage of new conjugate vaccines against Streptococcus pneumoniae (PCV) and Haemophilus influenzae type B (Hib), access to early antibiotic therapy, and changing prevalence of pneumonia risk factors. Measurement of community-based pneumonia incidence is difficult; risk factors for pneumonia incidence and factors associated with pneumonia mortality are poorly described in low- and middle-income countries. Careful measurement of pneumonia incidence, and prospective analysis of risk factors is necessary to appreciate the evolving complexities of pneumonia causality and mortality. The aim of this work was to describe the incidence and severity of pneumonia in a birth cohort of children in the first 2 years life; and identify risk factors for pneumonia and for severe outcomes. Methods A prospective birth cohort, the Drakenstein Child Health Study, enrolled mother-infant pairs in two communities outside Cape Town, South Africa. Pregnant women were recruited and followed through pregnancy, labour and delivery, and the first 2 years of the child's life. Comprehensive data collection of risk factors was done through the first 2 years of life. A community pneumonia surveillance system was established; active case finding was used for birth cohort participants over 4 respiratory seasons. Children were examined at scheduled visits and at the time of pneumonia events. Pneumonia or severe pneumonia was diagnosed according to revised World Health Organisation (WHO) guidelines. Chest x-rays were classified according to WHO guidelines. Predictors of ambulatory and hospitalized pneumonia were explored with Poisson regression using generalized estimating equations clustered on mother-infant pairs. Factors associated with death or admission to intensive care unit were analysed with prevalence ratios from modified Poisson regression with robust variance estimation. Findings From March 2012 to March 2015, 1137 pregnant women were enrolled, delivering 1143 live-born infants. Household environmental tobacco smoke exposure was common: 82% of children were exposed in the first 6 months of life. Maternal HIV infection was common: 249 (22%) of 1143 children were HIV-exposed, but only 2 children became HIV-infected. Coverage of primary series of hexavalent vaccine, PCV and Hib was excellent (92%). During the study period (2012 to 2017), there were 795 pneumonia episodes (621 (78%) ambulatory, 274 (22%) hospitalised) in the first 2 years of life. Pneumonia incidence was higher in the first year of life (0.51 episodes per child year (e/cy)) and decreased to 0.25 e/cy in the second year. Active case finding in the birth cohort was more accurate than passive surveillance performed at the community clinics; pneumonia incidence measured by passive surveillance was significantly lower (incidence rate ratio 0.72, 95% CI 0.58 – 0.89) compared to active surveillance. Pneumonia mortality was low: 1.7% of hospitalised cases, and 0.35% of all clinical cases. There was marked variability in pneumonia incidence from year to year during the study. Many risk factors for pneumonia did not have fixed effects, but had different impacts at different ages, and variable effect on ambulatory and hospitalised pneumonia. In multivariable regression, adjusted incidence rate ratios were calculated for 5 risk factors (age< 6 months, male sex, low birth weight (<2500g), maternal smoking, delayed vaccines), which were associated with consistent effects on ambulatory and hospitalised pneumonia. Risk factors for serious outcomes of pneumonia (death or admission to intensive care unit) were identified: age under 2 months, low birth weight and hypoxia. Conclusion In this birth cohort, with low socio-economic status but high vaccination coverage, we demonstrated higher-than expected incidence of pneumonia, but very low mortality, with specific risk factors identified. Active surveillance was important for accurate detection of pneumonia. Children born at low birth weight are at increased risk for pneumonia and for serious outcomes. Pulse oximetry to detect hypoxia, and access to oxygen for children with hypoxic pneumonia, should be included in guidelines. These data will have global applicability for estimation of child pneumonia incidence in regions where direct measurement is impossible. These data can be applied to epidemiology and disease-modelling for child health; they will contribute to long-term morbidity follow-up studies; and they will contribute to understanding the constantly-evolving epidemiology of child pneumonia.
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    Determine 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, Mark
    Detection 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.
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    Dynamics of faecal bacterial populations in early infancy as determined by massively parallel sequencing
    (2015) Claassen, Shantelle; Nicol, Mark; Kaba, Mamadou
    Background: Meconium microbiota have recently gained great interest; however very few studies have included meconium specimens when longitudinally characterizing the infant GIT microbiota. This study therefore aimed to longitudinally characterize meconium microbiota profiles during the first seven months of life and to compare these profiles with those from maternal faecal specimens using quality controlled Illumina MiSeq sequencing data. Methods: We sampled infant meconium and maternal faecal specimens at birth, as well as two subsets of infant faecal specimens at 4-12 and 20-28 weeks of life. We extracted nucleic acid from faecal specimens using the automated QIAsymphony ® SP instrument. Using Illumina MiSeq technology, we sequenced the V4 region of the bacterial 16S rRNA gene. We determined whether sufficient reads were sequenced using accumulation curves; whether any contamination occurred; and whether our sequencing approach was reproducible. The relative abundances of taxonomically classified operational taxonomic units (OTUs), and the Shannon diversity and Bray Curtis dissimilarity indices served to characterize faecal specimens from participants. Log ratio biplots and generalized linear mixed models served to statistically determine differences between faecal bacterial profiles. Results: Faecal specimens were collected from 90 mothers and 107 infants at birth, 72 infants at 4-12 and 36 infants at 20 28 weeks of age. We classified OTUs from two non-template controls which were indicative of potential contamination. Correcting for contamination resulted in a loss of 10 % of OTUs classified. Our reproducibility analysis correlated with increased concentrations of template used during library preparation. Based on diversity measures, meconium specimens harboured the most diverse bacterial profiles. The highest proportions of OTUs classified from meconium belonged to the phylum Proteobacteria (60 %), while the phylum Firmicutes was most abundant at 4-12 weeks (49 %) and 20-28 weeks (64 %) of life. The phylum Actinobacteria was at its highest at 4-12 weeks of age (26 %) and its increased proportions were associated with breastfeeding at 6-10 weeks of life. Firmicutes constituted the majority (79 %) of bacteria from maternal faecal specimens. No mother- infant pairs clustered at any of the time points studied, but infant bacterial profiles became more adult-like with increased age. An increase in infant age significantly affected bacterial proportions of 87 OTUs. Interestingly, we observed that infants exposed to HIV had higher proportions of the genus Leuconostoc and higher diversity indices compared to HIV unexposed infants at 4-12 weeks of age. Conclusion: Our study highlights that reproducibility may be worsened by the use of low template concentrations during library preparation, which may also skew diversity measures. We conclude that meconium is not sterile and that infant faecal bacterial profiles become more adult-like with increased age.
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    Epidemiology of extended spectrum beta-lactamase and carbapenemase-producing bacteria in stool from apparently healthy children, South Africa
    (2015) Manenzhe, Rendani Innocent; Nicol, Mark; Kaba, Mamadou
    Background: The prevalence of extended-spectrum beta-lactamase (ESBL) - and carbapenemase-producing Enterobacteriaceae in healthy humans in the community is largely unknown. We aimed to determine the prevalence and genetic characteristics of ESBL- and carbapenemase-producing Enterobacteriaceae in stools from healthy infants and their mothers, and to determine the risk factors associated with their carriage. Methods: This study was nested within the Drakenstein Child Health Study, a birth cohort in a semi-rural region of Western Cape Province, South Africa. Maternal and infants faecal samples (including the meconium) were collected at birth and at two additional time-points (5-12 and 20-28 weeks) from the infants only. Samples were screened for ESBLs and carbapenemase-producing organisms using ChromID ESBL and ChromID CARBA media, respectively. Identification of suspect ESBL/carbapenemase-producing isolates and antibiotic susceptibility were determined using the Vitek 2 system. ESBL production was confirmed using the combination disc test, and that of carbapenemase using the modified hodge test. Selected ESBL and carbapenemase genes were evaluated by the singleplex conventional polymerase chain reaction and Sanger sequencing. Risk factors were assessed by univariate analysis using the EPI Info version 7 software.
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    High levels of multidrug resistant tuberculosis in new and treatment-failure patients from the Revised National Tuberculosis Control Programme in an urban metropolis (Mumbai) in Western India
    (BioMed Central Ltd, 2009) D'souza, Desiree TB; Mistry, Nerges F; Vira, Tina N; Dholakia, Yatin; Hoffner, Sven; Pasvol, Geoffrey; Nicol, Mark; Wilkinson, Robert J
    BACKGROUND: India, China and Russia account for more than 62% of multidrug resistant tuberculosis (MDRTB) globally. Within India, locations like urban metropolitan Mumbai with its burgeoning population and high incidence of TB are suspected to be a focus for MDRTB. However apart from sporadic surveys at watched sites in the country, there has been no systematic attempt by the Revised National Tuberculosis Control Programme (RNTCP) of India to determine the extent of MDRTB in Mumbai that could feed into national estimates. Drug susceptibility testing (DST) is not routinely performed as a part of programme policy and public health laboratory infrastructure, is limited and poorly equipped to cope with large scale testing. METHODS: From April 2004 to January 2007 we determined the extent of drug resistance in 724 {493 newly diagnosed, previously untreated and 231 first line treatment failures (sputum-smear positive at the fifth month after commencement of therapy)} cases of pulmonary tuberculosis drawn from the RNTCP in four suboptimally performing municipal wards of Mumbai. The observations were obtained using a modified radiorespirometric Buddemeyer assay and validated by the Swedish Institute for Infectious Disease Control, Stockholm, a supranational reference laboratory. Data was analyzed utilizing SPSS 10.0 and Epi Info 2002. RESULTS: This study undertaken for the first time in RNTCP outpatients in Mumbai reveals a high proportion of MDRTB strains in both previously untreated (24%) and treatment-failure cases (41%). Amongst new cases, resistance to 3 or 4 drug combinations (amplified drug resistance) including isoniazid (H) and rifampicin (R), was greater (20%) than resistance to H and R alone (4%) at any point in time during the study. The trend for monoresistance was similar in both groups remaining highest to H and lowest to R. External quality control revealed good agreement for H and R resistance (k = 0.77 and 0.76 respectively). CONCLUSION: Levels of MDRTB are much higher in both previously untreated and first line treatment-failure cases in the selected wards in Mumbai than those projected by national estimates. The finding of amplified drug resistance suggests the presence of a well entrenched MDRTB scenario. This study suggests that a wider set of surveillance sites are needed to obtain a more realistic view of the true MDRTB rates throughout the country. This would assist in the planning of an adequate response to the diagnosis and care of MDRTB.
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    The house dust microbiota in the Drakenstein Child Health Study
    (2015) Duyver, Menna; Nicol, Mark; Ah Tow, Lemese
    Introduction: The indoor home environment comprises many niches that are occupied by bacterial communities. The composition of these bacterial communities may be influenced by numerous factors such as number of occupants, pets, season and location. Understanding the house dust microbial community is vital to understanding its' influence on human respiratory health. Aims: The aims of the studies described in this MSc dissertation were to: 1) evaluate the performance of ten commercial nucleic acid extraction kits on dust samples; 2) optimise dust removal from electrostatic dustfall collectors (EDC); 3) determine the bacterial composition of house dust using 16S rRNA gene sequencing and 4) determine those factors influencing the bacterial composition of house dust by performing bioinformatic and data analysis on the sequenced dust samples. Methods: In order to study the microbial content of house dust, an efficient DNA extraction protocol was required. Ten commercial nucleic acid purification protocols were evaluated on their ability to efficiently extract good quality DNA from very low quantities (20 mg) of wet bulk house dust. For the purpose of this study, EDCs were used to collect settled dust from homes of participants in the Drakenstein Child Health Study (DCHS). Electrostatic Dustfall Collectors were placed twice within the same household, approximately 6 months apart, spanning two seasons. The Z/R Fungal/Bacterial DNA MicroprepTM (ZMC) protocol was used to extract DNA from dust removed from EDCs. The V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform to determine the bacterial taxonomic composition of the house dust samples. A custom python wrapper that meshes a set of tools integrated into a computationally efficient workflow, known as the YAP pipeline was used to classify 16S rRNA sequences into bacterial taxonomies. Based on 97% sequence similarity, the pre-processed sequences were assigned to Operational Taxonomic Units (OTU). R software together with RStudio software was used for all statistical analysis and graphical representations of the data.
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    Impact of a novel molecular TB diagnostic system in patients at high risk of TB mortality in rural South Africa (Uchwepheshe): study protocol for a cluster randomised trial
    (BioMed Central Ltd, 2013) Lessells, Richard; Cooke, Graham; McGrath, Nuala; Nicol, Mark; Newell, Marie-Louise; Godfrey-Faussett, Peter
    BACKGROUND: Tuberculosis control in sub-Saharan Africa has long been hampered by poor diagnostics and weak health systems. New molecular diagnostics, such as the Xpert(R) MTB/RIF assay, have the potential to improve patient outcomes. We present a cluster randomised trial designed to evaluate whether the positioning of this diagnostic system within the health system has an impact on important patient-level outcomes.METHODS/DESIGN:This pragmatic cluster randomised clinical trial compared two positioning strategies for the Xpert MTB/RIF system: centralised laboratory versus primary health care clinic. The cluster (unit of randomisation) is a 2-week time block at the trial clinic. Adult pulmonary tuberculosis suspects with confirmed human immunodeficiency virus infection and/or at high risk of multidrug-resistant tuberculosis are enrolled from the primary health care clinic. The primary outcome measure is the proportion of culture-confirmed pulmonary tuberculosis cases initiated on appropriate treatment within 30 days of initial clinic visit. Univariate logistic regression will be performed as the primary analysis using generalised estimating equations with a binomial distribution function and a logit link. CONCLUSION: Diagnostic research tends to focus only on performance of diagnostic tests rather than on patient-important outcomes. This trial has been designed to improve the quality of evidence around diagnostic strategies and to inform the scale-up of new tuberculosis diagnostics within public health systems in high-burden settings.TRIAL REGISTRATION:Current Controlled Trials ISRCTN18642314; South African National Clinical Trials Registry DOH-27-0711-3568.
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    The innate immune response to Mycobacterium tuberculosis is dependent on strain lineage and on host population
    (2013) Sarkar, Rajesh; Nicol, Mark; Wilkinson, R J
    The genome structure of Mycobacterium tuberculosis is strongly clonal, in the absence of horizontal gene transfer. Thus it is feasible that clonal lineages may exhibit particular phenotypic characteristics, which may, in turn, result in differences in virulence or influence their association with particular host populations. Indeed, the global distribution of M. tuberculosis strains is not uniform and certain strain lineages predominate in particular geographical areas. Further, there is evidence that some strain lineages are emerging, suggesting differences in virulence. Firstly, we investigated the association between strain genotype of M. tuberculosis and in vitro correlates of virulence such as growth phenotype and cytokine induction in the monocyte-derived macrophage (MDM) model.
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    Nasopharyngeal colonization dynamics with Streptococcus pneumoniae and associated antimicrobial resistance in a South African birth cohort
    (2019) Manenzhe, Rendani Innocent; Moodley, Clinton; Nicol, Mark; Dube, Felix
    Introduction: Nasopharyngeal (NP) colonization by Streptococcus pneumoniae (the pneumococcus) precedes the development of respiratory tract infection. Colonization by antimicrobial-resistant pneumococci, especially in infants, is a major public health concern as pneumococcus is a frequent cause of bacterial acute respiratory tract infections among children. This study longitudinally investigated antimicrobial resistance amongst pneumococci colonizing the nasopharynx of South African infants immunized with the 13- valent pneumococcal conjugate vaccine (PCV13). Furthermore, the study explored strainlevel pneumococcal colonization patterns and associated antimicrobial resistance determinants as well as the composition of the NP antibiotic resistome using shotgun metagenomic sequencing. Methods: NP swabs were collected every second week from birth through the first year of life from 137 infants who were immunized with 2+1 doses of PCV13. These were the first 137 infants enrolled in the cohort who had the most complete fortnightly NP sampling (defined as at least 23-26 fortnightly collected NP swabs). Pneumococci were identified and serotyped using conventional techniques, and their antibiotic susceptibility profiles determined by disc diffusion and E-test. A subset of 196 NP samples from 23 infants were selected based on changes in serotype or antimicrobial resistance. These were subjected to broth enrichment, total nucleic acid extraction and subsequent shotgun metagenomic sequencing. Sequence reads were assembled and aligned to reference pneumococcal genomes. In-silico pneumococcal capsular, multilocus sequence typing, and antimicrobial resistance determinants were described. Finally, antibiotic resistance genes were identified from all bacterial contigs, to determine the NP resistome. Results: 1520 pneumococcal (760 non-duplicate) isolates were recovered from 137 infants; including non-typeable (n = 99), PCV13 (n = 133), and non-PCV13 serotypes (n = 528). The prevalence of penicillin, erythromycin, and cotrimoxazole non-susceptibility was 19% (147/760; 95% CI 17-22%) (3% resistant), 18% (136/760; 95% CI 15-21%) (14% resistant) and 45% (344/760; 95% CI 42-49%) (36% resistant), respectively. The predominant penicillin-non-susceptible serotypes included 15B/15C (n = 20), 19A (n = 13), 15A (n = 10), 19F (n = 8), and 21 (n = 8). Multi-drug resistance (MDR) was observed in 9% (68/760; 95% CI 7-11%) of the isolates. PCV13 serotypes were more likely to be non-susceptible, compared to non-PCV13 serotypes, to penicillin (26% vs. 16%, p = 0.007), erythromycin (23% vs. 15%, p = 0.027) and cotrimoxazole (62% vs. 41%, p < 0.001). Non-susceptibility to penicillin, erythromycin, and cotrimoxazole remained relatively constant through the first year of life (X 2 test for trend: p = 0.184, range 0 – 25%; p = 0.171, range 0 – 27%; and p = 0.572, range 0 – 55%, respectively). Overall, penicillin or erythromycin-non-susceptible pneumococci were carried for a shorter duration than susceptible pneumococci (penicillin [mean days, 18 vs. 21, p = 0.013] and erythromycin [mean days, 18 vs. 21, p = 0.035]). Forty-five percentage (61/137) of infants carried the same serotype which acquired or lost resistance over time, and these changes were predominantly for penicillin (76%, 79/104). Of the 196 NP samples sequenced, 174 had corresponding positive cultures for pneumococci and, of these, 152 were assigned an in-silico serotype. Metagenomic sequencing detected a single pneumococcal serotype in 85% (129/152), and co-colonization in 15% (23/152) of NP samples, respectively. In total, 22 different pneumococcal serotypes were identified, with 15B/15C (n = 49) and 16F (n = 21) being the most common non-PCV13 serotypes, while 23F (n = 9) and 19A (n = 8) were the most common PCV13 serotypes. Twenty-six different sequence types (STs), including 4 novel STs were identified. Mutations in the folA and folP genes, associated with cotrimoxazole resistance, were detected in 89% (87/98) of cotrimoxazole-non-susceptible pneumococci and mutations in the pbp1a and pbp2x genes, known to confer beta-lactam resistance, were identified in penicillin nonsusceptible ST705215B/15C isolates. A total of 329 antimicrobial resistance (AMR) genes were detected in 64% (125/196) of the sequenced samples, including 36 non-redundant genes ranging from 1 to 14 genes per sample. The predominant AMR genes detected were those conferring resistance to beta-lactams (52%, 172/329), macrolide-lincosamide-streptogramin (17%, 56/329), and tetracycline (12%, 38/329). The msrD, ermB, and mefA genes were only detected from pneumococcal reads. The predominant resistance genes detected from nonpneumococcal reads included blaOXA-60, blaOXA-22, and blaBRO-1. Conclusion: NP carriage of antibiotic-non-susceptible pneumococci was relatively constant throughout the first year of life. Despite high vaccine coverage levels, PCV13 serotypes were identified and were more commonly non-susceptible to penicillin, erythromycin, and cotrimoxazole. Overall, penicillin or erythromycin-non-susceptible pneumococci were carried for a shorter duration than susceptible pneumococci, however, non-susceptible PCV13 serotypes were carried for a longer duration than non-susceptible non-PCV13 serotypes. Direct shotgun sequencing from enriched NP samples was shown to be a powerful technique for a detailed description of the pneumococcal component of the NP microbiome and resistome, and its use should be explored similarly for other bacteria in this niche.
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    Novel Diagnostic approach for tuberculosis diagnosis
    (2010) Milovic, Ana; Nicol, Mark
    There is a clear need for a rapid, inexpensive point-of-care diagnostic test for tuberculosis (TB). The aim of this study was to analyze the performance of a novel rapid diagnostic test for TB, GeneXpert MTB/RIF, in symptomatic adults and to compare it to other commercially available nucleic acid amplification assays and to standard microbiological smear and culture. The GeneXpert system performs real time, nested PCR from sputum and provides a result within two hours of sampling. The result includes a semi-quantitative assessment of bacillary load in the sample and simultaneously detects rifampicin resistance. This study was part of a cross-sectional, multi-centre clinical trial. The Cape Town component of this study was conducted at three sites, one hospital based and other two community clinics, all with high TB/HIV coinfection rate. Among 43.2% of patients diagnosed with TB during the evaluation study, GeneXpert detected TB in 95.5% of all culture positive cases. In smear positive patients, sensitivity was 99.0% and in smear-negative, culture positive patients, 86.1%. Specificity in patients who were culture negative and clinically diagnosed as non-TB after follow up was 98.4%. Sensitivity and specificity of GeneXpert in detecting rifampicin resistance was 100% comparing to phenotypically detected drug resistance. GeneXpert is a highly promising novel tool for the rapid diagnosis of adult TB. Future studies are needed to establish the performance and impact of GeneXpert when performed at the level of the microscopy centre.
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    Reversion and conversion of Mycobacterium tuberculosis IFN-gamma ELISpot results during anti-tuberculous treatment in HIV-infected children
    (BioMed Central Ltd, 2010) Connell, Tom; Davies, Mary-Ann; Johannisen, Christine; Wood, Kathryn; Pienaar, Sandy; Wilkinson, Katalin; Wilkinson, Robert; Zar, Heather; Beatty, David; Nicol, Mark; Curtis, Nigel; Eley, Brian
    BACKGROUND: Recent interest has focused on the potential use of serial interferon gamma (IFN-gamma) release assay (IGRA) measurements to assess the response to anti-tuberculous (TB) treatment. The kinetics of IFN-gamma responses to Mycobacterium tuberculosis (MTB) antigens in HIV-infected children during treatment have not however been previously investigated. METHODS: IFN-gamma responses to the MTB antigens, ESAT-6, CFP-10 and PPD were measured by an enzyme-linked immunospot assay (IFN-gamma ELISpot) at presentation and at one, two and six months after starting anti-tuberculous treatment in HIV-infected children with definite or probable TB. Responses at different time points were compared using a Mann-Whitney U test with paired data analysed using the Wilcoxon signed rank test. A Fisher's exact or Chi-squared test was used to compare proportions when test results were analysed as dichotomous outcomes. RESULTS: Of 102 children with suspected TB, 22 (21%) had definite TB and 24 (23%) probable TB. At least one follow up IFN-gamma ELISpot assay result was available for 31 (67%) of the 46 children. In children with definite or probable TB in whom the IFN-gamma ELISpot assay result was positive at presentation, anti-tuberculous treatment was accompanied by a significant decrease in both the magnitude of the IFN-gamma response to individual or combined MTB-specific antigens (ESAT-6 median 110 SFCs/106 PBMC (IQR 65-305) at presentation vs. 15 (10-115) at six months, p = 0.04; CFP-10 177 (48-508) vs. 20 (5-165), p = 0.004, ESAT-6 or CFP-10 median 250 SFCs/106 PBMC (IQR 94-508) vs. 25 (10-165), p = 0.004) and in the proportion of children with a positive IFN-gamma ELISpot assay (Fisher's exact test: ESAT-6 15/0 vs 5/11, p = 0.0002, CFP-10 22/0 vs 8/17, p = 0.0001, ESAT-6 or CFP-10 22/0 vs. 9/17, p= 0.002). However almost half of the children had a positive IFN-gamma ELISpot assay after six months of anti-tuberculous treatment. In addition, there was conversion of the IFN-gamma ELISpot assay result during anti-tuberculous therapy in six of 12 children in whom the initial IFN-gamma ELISpot assay was negative. CONCLUSIONS: In HIV-infected children with definite or probable TB, anti-tuberculosis treatment is accompanied by a reduction in the magnitude of the IFN-gamma ELISpot response to MTB-antigens. However, serial IFN-gamma ELISpot measurements appear to have limited clinical utility in assessing a successful response to anti-tuberculous treatment in HIV infected children.
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    The bacteriome and metabolome of human breast milk and their association with infant growth
    (2019) Ojo, Anna Olutoyin; du Toit, Elloise; Nicol, Mark
    Human breast milk is a complex species-specific biological fluid universally known as the optimal postnatal source of nutrition for infants and therefore, recommended by the World Health Organization as the exclusive food for infants in the first six months of life. Despite the importance of human breast milk in infant health, study of its composition, especially the bacteriome (bacterial communities) and metabolome (complete set of metabolites), and their relationship to infant health and growth have not yet been comprehensively characterized. This is particularly true in low- and middle-income countries. We therefore conducted a cross-sectional study, nested within an existing birth cohort, the Drakenstein Child Health Study, to describe the bacteriome and metabolome of human breast milk samples collected between 6-10 weeks postpartum from lactating women living in South Africa. The determinants of these components of human breast milk and their role in infant growth were also investigated. Four commercial DNA extraction kits were compared for DNA extraction from human breast milk samples. The kit showing the best results, including quality and quantity of DNA, as well as best reproducibility, was chosen for further extractions. Using 16S rRNA gene amplicon next generation sequencing, a reproducible bioinformatics sequencing pipeline, and robust multivariate statistical analysis, we confirmed the presence of a diverse bacterial community in human breast milk and identified a core bacteriome, present in 80% of the samples. The bacteriome was shown to cluster into three different profile groups (biotypes) according to the predominant bacterial genus present. Bacterial interactions were suggested by the finding of positive correlations between the relative abundances of bacteria usually found in the oral or skin microbiota. Apart from study site (a proxy for ethnicity in this study), infant birth length and maternal age, no other associations were found between potential sociodemographic and psychosocial determinants and the composition of the human breast milk bacteriome. Using Nuclear Magnetic Resonance spectroscopy, we quantified forty-nine metabolites in all human breast milk samples. A subset of women with low levels of lactose concentrations were identified. Low lactose was associated with an increase in metabolites associated with mixed acid fermentation and microbial dysbiosis (staphylococcal-predominant biotype). Low-lactose (vs normal lactose) human breast milk correlated with a reduced median duration of exclusive breastfeeding and reduced infant growth (reduced weight and length z-scores) during the period of exclusive breastfeeding. These results suggest that bacterial fermentation of lactose results in low-lactose breast milk, which in turn impacts on breastfeeding outcome. Taken together, the results presented in this thesis provide a better understanding of human breast milk composition among lactating mothers living in South Africa, their potential determinants and their role in infant growth. Knowledge about the composition of human breast milk may provide opportunity for diagnostic and therapeutic interventions and help promote (exclusive) breastfeeding for the recommended period to improve infant health.