Determinants of lung function development from birth to age 5 years: an interrupted time series analysis of a South African birth cohort.

BACKGROUND: Early life is a key period that determines long-term health. Lung development in childhood predicts lung function attained in adulthood and morbidity and mortality across the life course. We aimed to assess the effect of early-life lower respiratory tract infection (LRTI) and associated risk factors on lung development from birth to school age in a South African birth cohort. METHODS: We prospectively followed children enrolled in a population-based cohort from birth (between March 5, 2012 and March 31, 2015) to age 5 years with annual lung function assessment. Data on multiple early-life exposures, including LRTI, were collected. The effect of early-life risk factors on lung function development from birth to age 5 years was assessed using the Generalised Additive Models for Location, Scale and Shape and Interrupted Time Series approach. FINDINGS: 966 children (475 [49·2%] female, 491 [50·8%] male) had lung function measured with oscillometry, tidal flow volume loops, and multiple breath washout. LRTI occurred in 484 (50·1%) children, with a median of 2·0 LRTI episodes (IQR 1·0-3·0) per child. LRTI was independently associated with altered lung function, as evidenced by lower compliance (0·959 [95% CI 0·941-0·978]), higher resistance (1·028 [1·016-1·041]), and higher respiratory rate (1·018 [1·063-1·029]) over 5 years. Additional impact on lung function parameters occurred with each subsequent LRTI. Respiratory syncytial virus (RSV) LRTI was associated with lower expiratory flow ratio (0·97 [0·95-0·99]) compared with non-RSV LRTI. Maternal factors including allergy, smoking, and HIV infection were also associated with altered lung development, as was preterm birth, low birthweight, female sex, and coming from a less wealthy household. INTERPRETATION: Public health interventions targeting LRTI prevention, with RSV a priority, are vital, particularly in low-income and middle-income settings. FUNDING: UK Medical Research Council Grant, The Wellcome Trust, The Bill & Melinda Gates Foundation, US National Institutes of Health Human Heredity and Health in Africa, South African Medical Research Council, Hungarian Scientific Research Fund, and European Respiratory Society.

Skin-Microbiome Assembly in Preterm Infants during the First Three Weeks of Life and Impact of Topical Coconut Oil Application.

The structure and function of infant skin is not fully developed until 34 weeks of gestation, and this immaturity is associated with risk of late-onset sepsis (LOS). Topical coconut oil improves preterm-infant skin integrity and may reduce LOS. However, data on early-life skin-microbiome succession and potential effects of emollient skin care in preterm infants are scarce. We therefore collected skin-microbiome samples from the ear, axilla, and groin on days 1, 7, 14, and 21 from preterm infants born <30 weeks of gestation as part of a randomized clinical trial of standard skin care vs. topical coconut oil. We found that within-sample microbiome diversity was highest on day 1 after birth, with a subsequent decline and emergence of Staphylococcus genus dominance from day 7. Moreover, microbiome assembly was less diverse in infants receiving coconut oil vs. standard skin care. Our study provides novel data on preterm-infant skin-microbiome composition and highlights the modifying potential of emollient skin care.

Succession and determinants of the early life nasopharyngeal microbiota in a South African birth cohort.

BACKGROUND: Bacteria colonizing the nasopharynx play a key role as gatekeepers of respiratory health. Yet, dynamics of early life nasopharyngeal (NP) bacterial profiles remain understudied in low- and middle-income countries (LMICs), where children have a high prevalence of risk factors for lower respiratory tract infection. We investigated longitudinal changes in NP bacterial profiles, and associated exposures, among healthy infants from low-income households in South Africa. METHODS: We used short fragment (V4 region) 16S rRNA gene amplicon sequencing to characterize NP bacterial profiles from 103 infants in a South African birth cohort, at monthly intervals from birth through the first 12 months of life and six monthly thereafter until 30 months. RESULTS: Corynebacterium and Staphylococcus were dominant colonizers at 1 month of life; however, these were rapidly replaced by Moraxella- or Haemophilus-dominated profiles by 4 months. This succession was almost universal and largely independent of a broad range of exposures. Warm weather (summer), lower gestational age, maternal smoking, no day-care attendance, antibiotic exposure, or low height-for-age z score at 12 months were associated with higher alpha and beta diversity. Summer was also associated with higher relative abundances of Staphylococcus, Streptococcus, Neisseria, or anaerobic gram-negative bacteria, whilst spring and winter were associated with higher relative abundances of Haemophilus or Corynebacterium, respectively. Maternal smoking was associated with higher relative abundances of Porphyromonas. Antibiotic therapy (or isoniazid prophylaxis for tuberculosis) was associated with higher relative abundance of anerobic taxa (Porphyromonas, Fusobacterium, and Prevotella) and with lower relative abundances of health associated-taxa Corynebacterium and Dolosigranulum. HIV-exposure was associated with higher relative abundances of Klebsiella or Veillonella and lower relative abundances of an unclassified genus within the family Lachnospiraceae. CONCLUSIONS: In this intensively sampled cohort, there was rapid and predictable replacement of early profiles dominated by health-associated Corynebacterium and Dolosigranulum with those dominated by Moraxella and Haemophilus, independent of exposures. Season and antibiotic exposure were key determinants of NP bacterial profiles. Understudied but highly prevalent exposures prevalent in LMICs, including maternal smoking and HIV-exposure, were associated with NP bacterial profiles. Video Abstract.

Early childhood wheezing phenotypes and determinants in a South African birth cohort: longitudinal analysis of the Drakenstein Child Health Study.

BACKGROUND: Developmental trajectories of childhood wheezing in low-income and middle-income countries (LMICs) have not been well described. We aimed to derive longitudinal wheeze phenotypes from birth to 5 years in a South African birth cohort and compare those with phenotypes derived from a UK cohort. METHODS: We used data from the Drakenstein Child Health Study (DCHS), a longitudinal birth cohort study in a peri-urban area outside Cape Town, South Africa. Pregnant women (aged ≥18 years) were enrolled during their second trimester at two public health clinics. We followed up children from birth to 5 years to derive six multidimensional indicators of wheezing (including duration, temporal sequencing, persistence, and recurrence) and applied Partition Around Medoids clustering to derive wheeze phenotypes. We compared phenotypes with a UK cohort (the Avon Longitudinal Study of Parents and Children [ALSPAC]). We investigated associations of phenotypes with early-life exposures, including all-cause lower respiratory tract infection (LRTI) and virus-specific LRTI (respiratory syncytial virus, rhinovirus, adenovirus, influenza, and parainfluenza virus) up to age 5 years. We investigated the association of phenotypes with lung function at 6 weeks and 5 years. FINDINGS: Between March 5, 2012, and March 31, 2015, we enrolled 1137 mothers and there were 1143 livebirths. Four wheeze phenotypes were identified among 950 children with complete data: never (480 children [50%]), early transient (215 children [23%]), late onset (104 children [11%]), and recurrent (151 children [16%]). Multivariate adjusted analysis indicated that LRTI and respiratory syncytial virus-LRTI, but not other respiratory viruses, were associated with increased risk of recurrent wheeze (odds ratio [OR] 2·79 [95% CI 2·05-3·81] for all LTRIs; OR 2·59 [1·30-5·15] for respiratory syncytial virus-LRTIs). Maternal smoking (1·88 [1·12-3·02]), higher socioeconomic status (2·46 [1·23-4·91]), intimate partner violence (2·01 [1·23-3·29]), and male sex (2·47 [1·50-4·04]) were also associated with recurrent wheeze. LRTI and respiratory syncytial virus-LRTI were also associated with early transient and late onset clusters. Wheezing illness architecture differed between DCHS and ALSPAC; children included in ALSPAC in the early transient cluster wheezed for a longer period before remission and late-onset wheezing started at an older age, and no persistent phenotype was identified in DCHS. At 5 years, airway resistance was higher in children with early or recurrent wheeze compared with children who had never wheezed. Airway resistance increased from 6 weeks to 5 years among children with recurrent wheeze. INTERPRETATION: Effective strategies to reduce maternal smoking and psychosocial stressors and new preventive interventions for respiratory syncytial virus are urgently needed to optimise child health in LMICs. FUNDING: UK Medical Research Council; The Bill & Melinda Gates Foundation; National Institutes of Health Human Heredity and Health in Africa; South African Medical Research Council; Wellcome Trust.

Human microbiota research in Africa: a systematic review reveals gaps and priorities for future research.

BACKGROUND: The role of the human microbiome in health and disease is an emerging and important area of research; however, there is a concern that African populations are under-represented in human microbiome studies. We, therefore, conducted a systematic survey of African human microbiome studies to provide an overview and identify research gaps. Our secondary objectives were: (i) to determine the number of peer-reviewed publications; (ii) to identify the extent to which the researches focused on diseases identified by the World Health Organization [WHO] State of Health in the African Region Report as being the leading causes of morbidity and mortality in 2018; (iii) to describe the extent and pattern of collaborations between researchers in Africa and the rest of the world; and (iv) to identify leadership and funders of the studies. METHODOLOGY: We systematically searched Medline via PubMed, Scopus, CINAHL, Academic Search Premier, Africa-Wide Information through EBSCOhost, and Web of Science from inception through to 1st April 2020. We included studies that characterized samples from African populations using next-generation sequencing approaches. Two reviewers independently conducted the literature search, title and abstract, and full-text screening, as well as data extraction. RESULTS: We included 168 studies out of 5515 records retrieved. Most studies were published in PLoS One (13%; 22/168), and samples were collected from 33 of the 54 African countries. The country where most studies were conducted was South Africa (27/168), followed by Kenya (23/168) and Uganda (18/168). 26.8% (45/168) focused on diseases of significant public health concern in Africa. Collaboration between scientists from the United States of America and Africa was most common (96/168). The first and/or last authors of 79.8% of studies were not affiliated with institutions in Africa. Major funders were the United States of America National Institutes of Health (45.2%; 76/168), Bill and Melinda Gates Foundation (17.8%; 30/168), and the European Union (11.9%; 20/168). CONCLUSIONS: There are significant gaps in microbiome research in Africa, especially those focusing on diseases of public health importance. There is a need for local leadership, capacity building, intra-continental collaboration, and national government investment in microbiome research within Africa. Video Abstract.

The child ecosystem and childhood pulmonary tuberculosis: A South African perspective.

INTRODUCTION: This study investigates drivers of childhood pulmonary tuberculosis (PTB) using a childhood ecosystem approach in South Africa. An ecosystem approach toward identifying risk factors for PTB may identify targeted interventions. METHODS: Data were collected as part of a prospective cohort study of children presenting at a primary care facility or tertiary hospital with possible TB. Characterization of the childhood ecosystem included proximal, medial, and distal determinants. Proximal determinants included child characteristics that could impact PTB outcomes. Medial determinants included relational factors, such as caregiver health, which might impact interactions with the child. Distal determinants included macro-level determinants of disease, such as socioeconomic status and food insecurity. Children who started on TB treatment were followed for up to 6 months. Multivariate regression models tested independent associations between factors associated with PTB in children. RESULTS: Of 1202 children enrolled, 242 (20%) of children had confirmed PTB, 756 (63%) were started on TB treatment, and 444 (37%) had respiratory conditions other than TB. In univariate analyses, childhood malnutrition and caregiver smoking were associated with treated or confirmed PTB. In multivariate analyses, proximal factors, such as male gender and hospitalization, as well as low socioeconomic status as a distal factor, were associated with PTB. CONCLUSIONS: Interventions may need to target subgroups of children and families with elevated proximal, medial, and distal risk factors for PTB. Screening for risk factors, such as caregiver's health, may guide targeting. The provision of social protection programs to bolster economic security may be an important intervention for attenuating childhood exposure to risk factors.

Cost-effectiveness of a Novel Lipoarabinomannan Test for Tuberculosis in Patients With Human Immunodeficiency Virus.

BACKGROUND: A novel urine lipoarabinomannan assay (FujiLAM) has higher sensitivity and higher cost than the first-generation AlereLAM assay. We evaluated the cost-effectiveness of FujiLAM for tuberculosis testing among hospitalized people with human immunodeficiency virus (HIV), irrespective of symptoms. METHODS: We used a microsimulation model to project clinical and economic outcomes of 3 testing strategies: (1) sputum Xpert MTB/RIF (Xpert), (2) sputum Xpert plus urine AlereLAM (Xpert+AlereLAM), (3) sputum Xpert plus urine FujiLAM (Xpert+FujiLAM). The modeled cohort matched that of a 2-country clinical trial. We applied diagnostic yields from a retrospective study (yields for Xpert/Xpert+AlereLAM/Xpert+FujiLAM among those with CD4 <200 cells/µL: 33%/62%/70%; among those with CD4 ≥200 cells/µL: 33%/35%/47%). Costs of Xpert/AlereLAM/FujiLAM were US$15/3/6 (South Africa) and $25/3/6 (Malawi). Xpert+FujiLAM was considered cost-effective if its incremental cost-effectiveness ratio (US$/year-of-life saved) was <$940 (South Africa) and <$750 (Malawi). We varied key parameters in sensitivity analysis and performed a budget impact analysis of implementing FujiLAM countrywide. RESULTS: Compared with Xpert+AlereLAM, Xpert+FujiLAM increased life expectancy by 0.2 years for those tested in South Africa and Malawi. Xpert+FujiLAM was cost-effective in both countries. Xpert+FujiLAM for all patients remained cost-effective compared with sequential testing and CD4-stratified testing strategies. FujiLAM use added 3.5% (South Africa) and 4.7% (Malawi) to 5-year healthcare costs of tested patients, primarily reflecting ongoing HIV treatment costs among survivors. CONCLUSIONS: FujiLAM with Xpert for tuberculosis testing in hospitalized people with HIV is likely to increase life expectancy and be cost-effective at the currently anticipated price in South Africa and Malawi. Additional studies should evaluate FujiLAM in clinical practice settings.

Diagnostic accuracy of the Xpert MTB/Rif Ultra for tuberculosis adenitis.

BACKGROUND: The WHO recently recommended the new Xpert MTB/RIF Ultra assay (Ultra) instead of the Xpert MTB/RIF assay because Ultra has improved sensitivity. We report the diagnostic accuracy of Ultra for tuberculous adenitis in a tuberculosis and HIV endemic setting. METHODS: We obtained fine-needle aspirates (FNA) and lymph node tissue by core-needle biopsy in adult patients with peripheral lymphadenopathy of >20 mm. Ultra and mycobacterial culture were performed on FNA and tissue specimens, with histological examination of tissue specimens. We assessed the diagnostic accuracy of Ultra against a composite reference standard of 'definite tuberculosis' (microbiological criteria) or 'probable tuberculosis' (histological and clinical criteria). RESULTS: We prospectively evaluated 99 participants of whom 50 were HIV positive: 21 had 'definite tuberculosis', 15 'probable tuberculosis' and 63 did not have tuberculosis (of whom 38% had lymphoma and 19% disseminated malignancy). Using the composite reference standard the Ultra sensitivity on FNA was 70% (95% CI 51-85; 21 of 30), and on tissue was 67% (45-84; 16/24) these were far superior to the detection of acid-fast bacilli on an FNA (26%; 7/27); AFB on tissue (33%; 8/24); or tissue culture (39%; 9/23). The detection of granulomas on histology had high senstivity (83%) but the lowest specficity. When compared with culture the Ultra on FNA had a sensitvity of 78% (40-97; 7/9) and tissue 90% (55-100; 9/10). CONCLUSIONS: Ultra performed on FNA or tissue of a lymph node had good sensitivity and high specificity. Ultra had a higher yield than culture and has the advantage of being a rapid test. Ultra on FNA would be an appropriate initial investigation for lymphadenopathy in tuberculosis endemic areas followed by a core biopsy for histopathology with a repeat Ultra on tissue if granulomas are present.

"SILVAMP TB LAM" Rapid Urine Tuberculosis Test Predicts Mortality in Patients Hospitalized With Human Immunodeficiency Virus in South Africa.

Reducing diagnostic delay is key toward decreasing tuberculosis-associated deaths in people living with human immunodeficiency virus. In tuberculosis patients with retrospective urine testing, the point-of-care Fujifilm SILVAMP TB LAM (FujiLAM) could have rapidly diagnosed tuberculosis in up to 89% who died. In FujiLAM negative patients, the probability of 12-week survival was 86-97%.

Composition of Gut Microbiota of Children and Adolescents With Perinatal Human Immunodeficiency Virus Infection Taking Antiretroviral Therapy in Zimbabwe.

BACKGROUND: Human immunodeficiency virus (HIV) infection causes impairment of the gastrointestinal barrier, with substantial depletion of CD4+ T cells in the gut. Antiretroviral therapy (ART) restores CD4+ counts and may have beneficial effects on gut microbiota in adults. Little is known about effect of long-term ART on gut microbiome in HIV-infected children. We investigated composition of gut microbiota in HIV-infected and -uninfected children and assessed associations between gut microbiota and patient characteristics. METHODS: In a cross-sectional study, rectal swabs were collected from 177 HIV-infected and 103 HIV-uninfected controls. Gut microbial composition was explored using 16S ribosomal ribonucleic acid sequencing. RESULTS: Human immunodeficiency virus-infected children had significantly lower alpha-diversity and higher beta-diversity compared to HIV-uninfected. No association was observed between microbiome diversity and CD4+ T-cell count, HIV viral load, or HIV-associated chronic lung disease. We found enriched levels of Corynebacterium (P < .01), Finegoldia (P < .01), and Anaerococcus (P < .01) in HIV-infected participants and enrichment of Enterobacteriaceae (P = .02) in participants with low CD4+ counts (<400 cells/mm3). Prolonged ART-treatment (≥10 years) was significantly associated with a richer gut microbiota by alpha diversity. CONCLUSIONS: Human immunodeficiency virus-infected children have altered gut microbiota. Prolonged ART may restore the richness of the microbiota closer to that of HIV-uninfected children.

Lower Respiratory Tract Infections in Children in a Well-vaccinated South African Birth Cohort: Spectrum of Disease and Risk Factors.

BACKGROUND: Childhood lower respiratory tract infections (LRTIs) cause substantial morbidity and under-5 child mortality. The epidemiology of LRTI is changing in low- and middle-income countries with expanding access to conjugate vaccines, yet there are few data on the incidence and risk factors for LRTI in these settings. METHODS: A prospective birth cohort enrolled mother-infant pairs in 2 communities near Cape Town, South Africa. Active surveillance for LRTI was performed for the first 2 years of life over 4 respiratory seasons. Comprehensive data collection of risk factors was done through 2 years of life. World Health Organization definitions were used to classify clinical LRTI and chest radiographs. RESULTS: From March 2012 to February 2017, 1143 children were enrolled and followed until 2 years of age. Thirty-two percent of children were exposed to antenatal maternal smoking; 15% were born at low birth weights. Seven hundred ninety-five LRTI events occurred in 429 children by February 2017; incidence of LRTI was 0.51 and 0.25 episodes per child-year in the first and second years of life, respectively. Human immunodeficiency virus (HIV)-exposed, uninfected infants (vs HIV-unexposed infants) were at increased risk of hospitalized LRTI in the first 6 months of life. In regression models, male sex, low birth weight, and maternal smoking were independent risk factors for both ambulatory and hospitalized LRTI; delayed or incomplete vaccination was associated with hospitalized LRTI. CONCLUSIONS: LRTI incidence was high in the first year of life, with substantial morbidity. Strategies to ameliorate harmful exposures are needed to reduce LRTI burden in vulnerable populations.

Indoor air pollution and tobacco smoke exposure: impact on nasopharyngeal bacterial carriage in mothers and infants in an African birth cohort study.

Indoor air pollution (IAP) or environmental tobacco smoke (ETS) exposure may influence nasopharyngeal carriage of bacterial species and development of lower respiratory tract infection (LRTI). The aim of this study was to longitudinally investigate the impact of antenatal or postnatal IAP/ETS exposure on nasopharyngeal bacteria in mothers and infants. A South African cohort study followed mother-infant pairs from birth through the first year. Nasopharyngeal swabs were taken at birth, 6 and 12 months for bacterial culture. Multivariable and multivariate Poisson regression investigated associations between nasopharyngeal bacterial species and IAP/ETS. IAP exposures (particulate matter, carbon monoxide, nitrogen dioxide, volatile organic compounds) were measured at home visits. ETS exposure was measured through maternal and infant urine cotinine. Infants received the 13-valent pneumococcal and Haemophilus influenzae B conjugate vaccines. There were 881 maternal and 2605 infant nasopharyngeal swabs. Antenatal ETS exposure was associated with Streptococcus pneumoniae carriage in mothers (adjusted risk ratio (aRR) 1.73 (95% CI 1.03-2.92)) while postnatal ETS exposure was associated with carriage in infants (aRR 1.14 (95% CI 1.00-1.30)) Postnatal particulate matter exposure was associated with the nasopharyngeal carriage of H. influenzae (aRR 1.68 (95% CI 1.10- 2.57)) or Moraxella catarrhalis (aRR 1.42 (95% CI 1.03-1.97)) in infants. Early-life environmental exposures are associated with an increased prevalence of specific nasopharyngeal bacteria during infancy, which may predispose to LRTI.

HIV-exposure, early life feeding practices and delivery mode impacts on faecal bacterial profiles in a South African birth cohort.

There are limited data on meconium and faecal bacterial profiles from African infants and their mothers. We characterized faecal bacterial communities of infants and mothers participating in a South African birth cohort. Stool and meconium specimens were collected from 90 mothers and 107 infants at birth, and from a subset of 72 and 36 infants at 4-12 and 20-28 weeks of age, respectively. HIV-unexposed infants were primarily exclusively breastfed at 4-12 (49%, 26/53) and 20-28 weeks (62%, 16/26). In contrast, HIV-exposed infants were primarily exclusively formula fed at 4-12 (53%; 10/19) and 20-28 weeks (70%, 7/10). Analysis (of the bacterial 16S rRNA gene sequences of the V4 hypervariable region) of the 90 mother-infant pairs showed that meconium bacterial profiles [dominated by Proteobacteria (89%)] were distinct from those of maternal faeces [dominated by Firmicutes (66%) and Actinobacteria (15%)]. Actinobacteria predominated at 4-12 (65%) and 20-28 (50%) weeks. HIV-exposed infants had significantly higher faecal bacterial diversities at both 4-12 (p = 0.026) and 20-28 weeks (p = 0.002). HIV-exposed infants had lower proportions of Bifidobacterium (p = 0.010) at 4-12 weeks. Maternal faecal bacterial profiles were influenced by HIV status, feeding practices and mode of delivery. Further longitudinal studies are required to better understand how these variables influence infant and maternal faecal bacterial composition.

A clinical scoring system to prioritise investigation for tuberculosis among adults attending HIV clinics in South Africa.

BACKGROUND: The World Health Organization (WHO) recommendation for regular tuberculosis (TB) screening of HIV-positive individuals with Xpert MTB/RIF as the first diagnostic test has major resource implications. OBJECTIVE: To develop a diagnostic prediction model for TB, for symptomatic adults attending for routine HIV care, to prioritise TB investigation. DESIGN: Cohort study exploring a TB testing algorithm. SETTING: HIV clinics, South Africa. PARTICIPANTS: Representative sample of adult HIV clinic attendees; data from participants reporting ≥1 symptom on the WHO screening tool were split 50:50 to derive, then internally validate, a prediction model. OUTCOME: TB, defined as "confirmed" if Xpert MTB/RIF, line probe assay or M. tuberculosis culture were positive; and "clinical" if TB treatment started without microbiological confirmation, within six months of enrolment. RESULTS: Overall, 79/2602 (3.0%) participants on ART fulfilled TB case definitions, compared to 65/906 (7.2%) pre-ART. Among 1133/3508 (32.3%) participants screening positive on the WHO tool, 1048 met inclusion criteria for this analysis: 52/515 (10.1%) in the derivation and 58/533 (10.9%) in the validation dataset had TB. Our final model comprised ART status (on ART > 3 months vs. pre-ART or ART < 3 months); body mass index (continuous); CD4 (continuous); number of WHO symptoms (1 vs. >1 symptom). We converted this to a clinical score, using clinically-relevant CD4 and BMI categories. A cut-off score of ≥3 identified those with TB with sensitivity and specificity of 91.8% and 34.3% respectively. If investigation was prioritised for individuals with score of ≥3, 68% (717/1048) symptomatic individuals would be tested, among whom the prevalence of TB would be 14.1% (101/717); 32% (331/1048) of tests would be avoided, but 3% (9/331) with TB would be missed amongst those not tested. CONCLUSION: Our clinical score may help prioritise TB investigation among symptomatic individuals.

Highly sensitive sequence specific qPCR detection of Mycobacterium tuberculosis complex in respiratory specimens.

Nucleic acid amplification tests for Mycobacterium tuberculosis (MTB) detection from sputum are highly sensitive and specific with smear microscopy positive specimens, but their sensitivity with smear-negative/culture-positive specimens is much lower; therefore, these tests cannot rule out a tuberculosis diagnosis. Co-extraction of PCR inhibitors may be a cause of decreased test sensitivity. Here the design and early validation of a MTB screening assay with sample preparation and qPCR methods designed to specifically address this diagnostic gap is reported. First, human genomic DNA is identified as a significant qPCR inhibitor. To circumvent this problem, a novel, streamlined sample preparation method utilizing detergent and proteolysis to thin the sputum and DNA sequence specific MTB DNA isolation was developed. Additionally, a multiplexed qPCR assay targeting two MTB complex-specific loci: the potentially multi-copy IS6110 and the single-copy senX3-regX3, combined with the cotJC gene from Bacillus atrophaeus spores amplified as a process control was developed. The limit of detection of the test was estimated to be 20 cfu/ml which is significantly lower than the Xpert® MTB/RIF assay. In a preliminary field study of 60 de-identified blinded sputa, a test sensitivity of 96% and specificity of 100% was observed when compared to the Xpert® MTB/RIF assay.

Respiratory microbes present in the nasopharynx of children hospitalised with suspected pulmonary tuberculosis in Cape Town, South Africa.

BACKGROUND: Lower respiratory tract infection in children is increasingly thought to be polymicrobial in origin. Children with symptoms suggestive of pulmonary tuberculosis (PTB) may have tuberculosis, other respiratory tract infections or co-infection with Mycobacterium tuberculosis and other pathogens. We aimed to identify the presence of potential respiratory pathogens in nasopharyngeal (NP) samples from children with suspected PTB. METHOD: NP samples collected from consecutive children presenting with suspected PTB at Red Cross Children's Hospital (Cape Town, South Africa) were tested by multiplex real-time RT-PCR. Mycobacterial liquid culture and Xpert MTB/RIF was performed on 2 induced sputa obtained from each participant. Children were categorised as definite-TB (culture or qPCR [Xpert MTB/RIF] confirmed), unlikely-TB (improvement of symptoms without TB treatment on follow-up) and unconfirmed-TB (all other children). RESULTS: Amongst 214 children with a median age of 36 months (interquartile range, [IQR] 19-66 months), 34 (16 %) had definite-TB, 86 (40 %) had unconfirmed-TB and 94 (44 %) were classified as unlikely-TB. Moraxella catarrhalis (64 %), Streptococcus pneumoniae (42 %), Haemophilus influenzae spp (29 %) and Staphylococcus aureus (22 %) were the most common bacteria detected in NP samples. Other bacteria detected included Mycoplasma pneumoniae (9 %), Bordetella pertussis (7 %) and Chlamydophila pneumoniae (4 %). The most common viruses detected included metapneumovirus (19 %), rhinovirus (15 %), influenza virus C (9 %), adenovirus (7 %), cytomegalovirus (7 %) and coronavirus O43 (5.6 %). Both bacteria and viruses were detected in 73, 55 and 56 % of the definite, unconfirmed and unlikely-TB groups, respectively. There were no significant differences in the distribution of respiratory microbes between children with and without TB. Using quadratic discriminant analysis, human metapneumovirus, C. pneumoniae, coronavirus 043, influenza virus C virus, rhinovirus and cytomegalovirus best discriminated children with definite-TB from the other groups of children. CONCLUSIONS: A broad range of potential respiratory pathogens was detected in children with suspected TB. There was no clear association between TB categorisation and detection of a specific pathogen. Further work is needed to explore potential pathogen interactions and their role in the pathogenesis of PTB.

Diagnostic Test Accuracy in Childhood Pulmonary Tuberculosis: A Bayesian Latent Class Analysis.

Evaluation of tests for the diagnosis of childhood pulmonary tuberculosis (CPTB) is complicated by the absence of an accurate reference test. We present a Bayesian latent class analysis in which we evaluated the accuracy of 5 diagnostic tests for CPTB. We used data from a study of 749 hospitalized South African children suspected to have CPTB from 2009 to 2014. The following tests were used: mycobacterial culture, smear microscopy, Xpert MTB/RIF (Cepheid Inc.), tuberculin skin test (TST), and chest radiography. We estimated the prevalence of CPTB to be 27% (95% credible interval (CrI): 21, 35). The sensitivities of culture, Xpert, and smear microscopy were estimated to be 60% (95% CrI: 46, 76), 49% (95% CrI: 38, 62), and 22% (95% CrI: 16, 30), respectively; specificities of these tests were estimated in accordance with prior information and were close to 100%. Chest radiography was estimated to have a sensitivity of 64% (95% CrI: 55, 73) and a specificity of 78% (95% CrI: 73, 83). Sensitivity of the TST was estimated to be 75% (95% CrI: 61, 84), and it decreased substantially among children who were malnourished and infected with human immunodeficiency virus (56%). The specificity of the TST was 69% (95% CrI: 63%, 76%). Furthermore, it was estimated that 46% (95% CrI: 42, 49) of CPTB-negative cases and 93% (95% CrI: 82; 98) of CPTB-positive cases received antituberculosis treatment, which indicates substantial overtreatment and limited undertreatment.

Aetiology of childhood pneumonia in a well vaccinated South African birth cohort: a nested case-control study of the Drakenstein Child Health Study.

BACKGROUND: Pneumonia is a leading cause of mortality and morbidity in children globally. The cause of pneumonia after introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) has not been well studied in low-income and middle-income countries, and most data are from cross-sectional studies of children admitted to hospital. We aimed to longitudinally investigate the incidence and causes of childhood pneumonia in a South African birth cohort. METHODS: We did a nested case-control study of children in the Drakenstein Child Health Study who developed pneumonia from May 29, 2012, to Dec 1, 2014. Children received immunisations including acellular pertussis vaccine and PCV13. A nested subgroup had nasopharyngeal swabs collected every 2 weeks throughout infancy. We identified pneumonia episodes and collected blood, nasopharyngeal swabs, and induced sputum specimens. We used multiplex real-time PCR to detect pathogens in nasopharyngeal swabs and induced sputum of pneumonia cases and in nasopharyngeal swabs of age-matched and site-matched controls. To show associations between organisms and pneumonia we used conditional logistic regression; results are presented as odds ratios (ORs) with 95% CIs. FINDINGS: 314 pneumonia cases occurred (incidence of 0·27 episodes per child-year, 95% CI 0·24-0·31; median age 5 months [IQR 3-9]) in 967 children during 1145 child-years of follow-up. 60 (21%) cases of pneumonia were severe (incidence 0·05 episodes per child-year [95% CI 0·04-0·07]) with a case fatality ratio of 1% (three deaths). A median of five organisms (IQR 4-6) were detected in cases and controls with nasopharyngeal swabs, and a median of six organisms (4-7) recorded in induced sputum (p=0·48 compared with nasopharyngeal swabs). Bordetella pertussis (OR 11·08, 95% CI 1·33-92·54), respiratory syncytial virus (8·05, 4·21-15·38), or influenza virus (4·13, 2·06-8·26) were most strongly associated with pneumonia; bocavirus, adenovirus, parainfluenza virus, Haemophilus influenzae, and cytomegalovirus were also associated with pneumonia. In cases, testing of induced sputum in addition to nasopharyngeal swabs provided incremental yield for detection of B pertussis and several viruses. INTERPRETATION: Pneumonia remains common in this highly vaccinated population. Respiratory syncytial virus was the most frequently detected pathogen associated with pneumonia; influenza virus and B pertussis were also strongly associated with pneumonia. Testing of induced sputum increases the yield for detection of several organisms. New vaccines and strategies are needed to address the burden of childhood pneumonia. FUNDING: Bill & Melinda Gates Foundation, Medical Research Council South Africa, National Research Foundation South Africa, National Institute of Health, and H3Africa.