Publications by authors named "Mark P Nicol"

144 Publications

The association between bacteria colonizing the upper respiratory tract and lower respiratory tract infection in young children: a systematic review and meta-analysis.

Clin Microbiol Infect 2021 Jun 7. Epub 2021 Jun 7.

Division of Medical Microbiology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; Division of Infection and Immunity, School of Biomedical Sciences, University of Western Australia, Perth, Australia.

Background: Bacteria colonizing the upper respiratory tract (URT) of young children play a key role in the pathogenesis of lower respiratory tract infection (LRTI).

Objectives: To systematically review the literature on the association between bacteria colonizing the URT and LRTI among young children.

Data Sources: MEDLINE, Academic Search Premier, Africa-Wide Information and CINAHL, Scopus and Web of Science.

Study Eligibility Criteria: Studies published between 1923 and 2020, investigating URT bacteria from LRTI cases and controls.

Participants: Children under 5 years with and without acute LRTI.

Methods: Three reviewers independently screened titles, abstracts and full texts. Meta-analysis was done using Mantel-Haenszel fixed- or random-effects models.

Results: Most eligible studies (41/50) tested nasopharyngeal specimens when investigating URT bacteria. Most studies were of cross-sectional design (44/50). Twenty-four studies were performed in children in lower- or lower-middle-income countries (LMICs). There was higher prevalence of Haemophilus influenzae (pooled OR 1.60; 95% CI 1.23-2.07) and Klebsiella spp. (pooled OR 2.04; 95% CI 1.17-3.55) from URT specimens of cases versus controls. We observed a positive association between the detection of Streptococcus pneumoniae from URT specimens and LRTI after excluding studies where there was more antibiotic treatment prior to sampling in cases vs. controls (pooled OR 1.41; 95% CI 1.04-1.90). High density colonization with S. pneumoniae (>6.9 log copies/mL) was associated with an increased risk for LRTI. The associations between both Streptococcus and Haemophilus URT detection and LRTI were supported, at genus level, by 16S rRNA sequencing. Evidence for the role of Moraxella catarrhalis and Staphylococcus aureus was inconclusive.

Conclusions: Detection of H. influenzae or Klebsiella spp. in the URT was associated with LRTI, while evidence for association with S. pneumoniae was less conclusive. Longitudinal studies assessing URT microbial communities, together with environmental and host factors are needed to better understand pathogenesis of childhood LRTI.
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http://dx.doi.org/10.1016/j.cmi.2021.05.034DOI Listing
June 2021

Molecular epidemiology of Staphylococcus aureus in African children from rural and urban communities with atopic dermatitis.

BMC Infect Dis 2021 Apr 13;21(1):348. Epub 2021 Apr 13.

Department of Molecular and Cell Biology, Faculty of Science, University of Cape Town, Cape Town, South Africa.

Background: Staphylococcus aureus has been associated with the exacerbation and severity of atopic dermatitis (AD). Studies have not investigated the colonisation dynamics of S. aureus lineages in African toddlers with AD. We determined the prevalence and population structure of S. aureus in toddlers with and without AD from rural and urban South African settings.

Methods: We conducted a study of AD-affected and non-atopic AmaXhosa toddlers from rural Umtata and urban Cape Town, South Africa. S. aureus was screened from skin and nasal specimens using established microbiological methods and clonal lineages were determined by spa typing. Logistic regression analyses were employed to assess risk factors associated with S. aureus colonisation.

Results: S. aureus colonisation was higher in cases compared to controls independent of geographic location (54% vs. 13%, p < 0.001 and 70% vs. 35%, p = 0.005 in Umtata [rural] and Cape Town [urban], respectively). Severe AD was associated with higher colonisation compared with moderate AD (86% vs. 52%, p = 0.015) among urban cases. Having AD was associated with colonisation in both rural (odds ratio [OR] 7.54, 95% CI 2.92-19.47) and urban (OR 4.2, 95% CI 1.57-11.2) toddlers. In rural toddlers, living in an electrified house that uses gas (OR 4.08, 95% CI 1.59-10.44) or utilises kerosene and paraffin (OR 2.88, 95% CI 1.22-6.77) for heating and cooking were associated with increased S. aureus colonisation. However, exposure to farm animals (OR 0.3, 95% CI 0.11-0.83) as well as living in a house that uses wood and coal (OR 0.14, 95% CI 0.04-0.49) or outdoor fire (OR 0.31, 95% CI 0.13-0.73) were protective. Spa types t174 and t1476, and t272 and t1476 were dominant among urban and rural cases, respectively, but no main spa type was observed among controls, independent of geographic location. In urban cases, spa type t002 and t442 isolates were only identified in severe AD, t174 was more frequent in moderate AD, and t1476 in severe AD.

Conclusion: The strain genotype of S. aureus differed by AD phenotypes and rural-urban settings. Continued surveillance of colonising S. aureus lineages is key in understanding alterations in skin microbial composition associated with AD pathogenesis and exacerbation.
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http://dx.doi.org/10.1186/s12879-021-06044-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045247PMC
April 2021

Pertussis among patients with clinically compatible illness in the Amhara Regional State, Ethiopia.

Int J Infect Dis 2021 May 29;106:421-428. Epub 2021 Mar 29.

Department of Medical Microbiology, University of Gondar, Gondar, Amhara Regional State, Ethiopia.

Background: Pertussis is an acute respiratory tract disease caused by Bordetella pertussis. In 2014, 24.1 million pertussis cases, resulting in 160,700 deaths, were estimated to have occurred worldwide. This study aimed to determine the epidemiology of pertussis among patients with clinically compatible illness who visited selected hospitals in the Amhara Regional State of Ethiopia.

Methods: A cross-sectional study design was used to review pertussis patients with clinically compatible illness. Nasopharyngeal swabs were collected from 515 patients from July 2018 through February 2019. DNA was extracted from all nasopharyngeal swabs and samples were analyzed using real-time (RT-) PCR. Crude and adjusted odds ratios with corresponding 95% confidence intervals were estimated using bivariable and multivariable logistic regression analysis, respectively.

Results: The overall prevalence of Bordetella species among the study participants was 156 of 515 (30.3%) [95% CI = 26.4-34.6] as determined by Bordetella RT-PCR, including: 65 (41.7%) B. pertussis, 89 (57.1%) indeterminate B. pertussis, one (0.6%) Bordetella holmesii and one (0.6%) Bordetella parapertussis.

Conclusions: This study found that pertussis is potentially endemic and a common health problem among patients visiting health institutions in the Amhara Regional State of Ethiopia. More data regarding pertussis in Ethiopia could inform development of effective prevention strategies.
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http://dx.doi.org/10.1016/j.ijid.2021.03.073DOI Listing
May 2021

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

Pediatr Pulmonol 2021 Jul 25;56(7):2212-2222. Epub 2021 Mar 25.

Department of Paediatrics and Child Health, Red Cross Childrens Hospital and SA-MRC Unit on Child & Adolescent Health, University of Cape Town, Cape Town, Western Cape, South Africa.

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.
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http://dx.doi.org/10.1002/ppul.25369DOI Listing
July 2021

Prevalence and antimicrobial resistance profiles of respiratory microbial flora in African children with HIV-associated chronic lung disease.

BMC Infect Dis 2021 Feb 25;21(1):216. Epub 2021 Feb 25.

Department of Molecular and Cell Biology & Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa.

Background: HIV-associated chronic lung disease (CLD) is common among children living with HIV (CLWH) in sub-Saharan Africa, including those on antiretroviral therapy (ART). However, the pathogenesis of CLD and its possible association with microbial determinants remain poorly understood. We investigated the prevalence, and antibiotic susceptibility of Streptococcus pneumoniae (SP), Staphylococcus aureus (SA), Haemophilus influenzae (HI), and Moraxella catarrhalis (MC) among CLWH (established on ART) who had CLD (CLD+), or not (CLD-) in Zimbabwe and Malawi.

Methods: Nasopharyngeal swabs (NP) and sputa were collected from CLD+ CLWH (defined as forced-expiratory volume per second z-score < - 1 without reversibility post-bronchodilation with salbutamol), at enrolment as part of a randomised, placebo-controlled trial of azithromycin (BREATHE trial - NCT02426112 ), and from age- and sex-matched CLD- CLWH. Samples were cultured, and antibiotic susceptibility testing was conducted using disk diffusion. Risk factors for bacterial carriage were identified using questionnaires and analysed using multivariate logistic regression.

Results: A total of 410 participants (336 CLD+, 74 CLD-) were enrolled (median age, 15 years [IQR = 13-18]). SP and MC carriage in NP were higher in CLD+ than in CLD- children: 46% (154/336) vs. 26% (19/74), p = 0.008; and 14% (49/336) vs. 3% (2/74), p = 0.012, respectively. SP isolates from the NP of CLD+ children were more likely to be non-susceptible to penicillin than those from CLD- children (36% [53/144] vs 11% [2/18], p = 0.036). Methicillin-resistant SA was uncommon [4% (7/195)]. In multivariate analysis, key factors associated with NP bacterial carriage included having CLD (SP: adjusted odds ratio (aOR) 2 [95% CI 1.1-3.9]), younger age (SP: aOR 3.2 [1.8-5.8]), viral load suppression (SP: aOR 0.6 [0.4-1.0], SA: 0.5 [0.3-0.9]), stunting (SP: aOR 1.6 [1.1-2.6]) and male sex (SA: aOR 1.7 [1.0-2.9]). Sputum bacterial carriage was similar in both groups (50%) and was associated with Zimbabwean site (SP: aOR 3.1 [1.4-7.3], SA: 2.1 [1.1-4.2]), being on ART for a longer period (SP: aOR 0.3 [0.1-0.8]), and hot compared to rainy season (SP: aOR 2.3 [1.2-4.4]).

Conclusions: CLD+ CLWH were more likely to be colonised by MC and SP, including penicillin-non-susceptible SP strains, than CLD- CLWH. The role of these bacteria in CLD pathogenesis, including the risk of acute exacerbations, should be further studied.
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http://dx.doi.org/10.1186/s12879-021-05904-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908671PMC
February 2021

'We had to manage what we had on hand, in whatever way we could': adaptive responses in policy for decentralized drug-resistant tuberculosis care in South Africa.

Health Policy Plan 2021 Apr;36(3):249-259

Department of Infectious Diseases, Livingstone Hospital, Lindsay Rd, Industrial, Port Elizabeth, 6020, South Africa.

In 2011, the South African National TB Programme launched a policy of decentralized management of drug-resistant tuberculosis (DR-TB) in order to expand the capacity of facilities to treat patients with DR-TB, minimize delays to access care and improve patient outcomes. This policy directive was implemented to varying degrees within a rapidly evolving diagnostic and treatment landscape for DR-TB, placing new demands on already-stressed health systems. The variable readiness of district-level systems to implement the policy prompted questions not only about differences in health systems resources but also front-line actors' capacity to implement change in resource-constrained facilities. Using a grounded theory approach, we analysed data from in-depth interviews and small group discussions conducted between 2016 and 2018 with managers (n = 9), co-ordinators (n = 15), doctors (n = 7) and nurses (n = 18) providing DR-TB care. Data were collected over two phases in district-level decentralized sites of three South African provinces. While health systems readiness assessments conventionally map the availability of 'hardware', i.e. resources and skills to deliver an intervention, a notable absence of systems 'hardware' meant that systems 'software', i.e. health care workers (HCWs) agency, behaviours and interactions provided the basis of locally relevant strategies for decentralized DR-TB care. 'Software readiness' was manifest in four areas of DR-TB care: re-organization of service delivery, redressal of resource shortages, creation of treatment adherence support systems and extension of care parameters for vulnerable patients. These strategies demonstrate adaptive capacity and everyday resilience among HCW to withstand the demands of policy change and innovation in stressed systems. Our work suggests that a useful extension of health systems 'readiness' assessments would include definition and evaluation of HCW 'software' and adaptive capacities in the face of systems hardware gaps.
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http://dx.doi.org/10.1093/heapol/czaa147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059133PMC
April 2021

A Longitudinal Study of the Epidemiology of Seasonal Coronaviruses in an African Birth Cohort.

J Pediatric Infect Dis Soc 2021 May;10(5):607-614

Department of Paediatrics and Child Health and SA-MRC Unit on Child & Adolescent Health, Red Cross War Memorial Children's Hospital and University of Cape Town, Cape Town, South Africa.

Background: Since non-epidemic, seasonal human coronaviruses (sHCoV) commonly infect children, an improved understanding of the epidemiology of these infections may offer insights into the context of severe acute respiratory syndrome (SARS)-CoV-2. We investigated the epidemiology of sHCoV infection during the first year of life, including risk factors and association with lower respiratory tract infection (LRTI).

Methods: We conducted a nested case-control study of infants enrolled in a birth cohort near Cape Town, South Africa, from 2012 to 2015. LRTI surveillance was implemented, and nasopharyngeal swabs were collected fortnightly over infancy. Quantitative PCR detected respiratory pathogens, including coronaviruses-229E, -NL63, -OC43, and -HKU1. Swabs were tested from infants at the time of LRTI and from the 90 days prior as well as from age-matched control infants from the cohort over the equivalent period.

Results: In total, 885 infants were included, among whom 464 LRTI events occurred. Of the 4751 samples tested for sHCoV, 9% tested positive, with HCoV-NL63 the most common. Seasonal HCoV detection was associated with LRTI; this association was strongest for coronavirus-OC43, which was also found in all sHCoV-associated hospitalizations. Birth in winter was associated with sHCoV-LRTI, but there were no clear seasonal differences in detection. Co-detection of Streptococcus pneumoniae was weakly associated with sHCoV-LRTI (odds ratio: 1.8; 95% confidence interval: 0.9-3.6); detection of other respiratory viruses or bacteria was not associated with sHCoV status.

Conclusions: Seasonal HCoV infections were common and associated with LRTI, particularly sHCoV-OC43, which is most closely related to the SARS group of coronaviruses. Interactions of coronaviruses with bacteria in the pathogenesis of LRTI require further study.
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http://dx.doi.org/10.1093/jpids/piaa168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928775PMC
May 2021

Breath can discriminate tuberculosis from other lower respiratory illness in children.

Sci Rep 2021 Feb 1;11(1):2704. Epub 2021 Feb 1.

Thayer School of Engineering, Dartmouth College, Hanover, NH, USA.

Pediatric tuberculosis (TB) remains a global health crisis. Despite progress, pediatric patients remain difficult to diagnose, with approximately half of all childhood TB patients lacking bacterial confirmation. In this pilot study (n = 31), we identify a 4-compound breathprint and subsequent machine learning model that accurately classifies children with confirmed TB (n = 10) from children with another lower respiratory tract infection (LRTI) (n = 10) with a sensitivity of 80% and specificity of 100% observed across cross validation folds. Importantly, we demonstrate that the breathprint identified an additional nine of eleven patients who had unconfirmed clinical TB and whose symptoms improved while treated for TB. While more work is necessary to validate the utility of using patient breath to diagnose pediatric TB, it shows promise as a triage instrument or paired as part of an aggregate diagnostic scheme.
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http://dx.doi.org/10.1038/s41598-021-80970-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851130PMC
February 2021

A novel bedside rule-in test for tuberculous meningitis in HIV-infected adults.

Clin Infect Dis 2021 Jan 6. Epub 2021 Jan 6.

Division of Infection and Immunity, School of Biomedical Sciences, University of Western  Australia.

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http://dx.doi.org/10.1093/cid/ciaa1915DOI Listing
January 2021

Effect of Once-Weekly Azithromycin vs Placebo in Children With HIV-Associated Chronic Lung Disease: The BREATHE Randomized Clinical Trial.

JAMA Netw Open 2020 12 1;3(12):e2028484. Epub 2020 Dec 1.

Faculty of Health Sciences, UiT, The Arctic University of Norway, Tromsø, Norway.

Importance: HIV-associated chronic lung disease (HCLD) in children is associated with small airways disease, is common despite antiretroviral therapy (ART), and is associated with substantial morbidity. Azithromycin has antibiotic and immunomodulatory activity and may be effective in treating HCLD through reducing respiratory tract infections and inflammation.

Objective: To determine whether prophylactic azithromycin is effective in preventing worsening of lung function and in reducing acute respiratory exacerbations (AREs) in children with HCLD taking ART.

Design, Setting, And Participants: This double-blind, placebo-controlled, randomized clinical trial (BREATHE) was conducted between 2016 and 2019, including 12 months of follow-up, at outpatient HIV clinics in 2 public sector hospitals in Malawi and Zimbabwe. Participants were randomized 1:1 to intervention or placebo, and participants and study personnel were blinded to treatment allocation. Participants included children aged 6 to 19 years with perinatally acquired HIV and HCLD (defined as forced expiratory volume in 1 second [FEV1] z score < -1) who were taking ART for 6 months or longer. Data analysis was performed from September 2019 to April 2020.

Intervention: Once-weekly oral azithromycin with weight-based dosing, for 48 weeks.

Main Outcomes And Measures: All outcomes were prespecified. The primary outcome was the mean difference in FEV1 z score using intention-to-treat analysis for participants seen at end line. Secondary outcomes included AREs, all-cause hospitalizations, mortality, and weight-for-age z score.

Results: A total of 347 individuals (median [interquartile range] age, 15.3 [12.7-17.7] years; 177 boys [51.0%]) were randomized, 174 to the azithromycin group and 173 to the placebo group; 162 participants in the azithromycin group and 146 placebo group participants had a primary outcome available and were analyzed. The mean difference in FEV1 z score was 0.06 (95% CI, -0.10 to 0.21; P = .48) higher in the azithromycin group than in the placebo group, a nonsignificant difference. The rate of AREs was 12.1 events per 100 person-years in the azithromycin group and 24.7 events per 100 person-years in the placebo groups (hazard ratio, 0.50; 95% CI, 0.27 to 0.93; P = .03). The hospitalization rate was 1.3 events per 100 person-years in the azithromycin group and 7.1 events per 100 person-years in the placebo groups, but the difference was not significant (hazard ratio, 0.24; 95% CI, 0.06 to 1.07; P = .06). Three deaths occurred, all in the placebo group. The mean weight-for-age z score was 0.03 (95% CI, -0.08 to 0.14; P = .56) higher in the azithromycin group than in the placebo group, although the difference was not significant. There were no drug-related severe adverse events.

Conclusions And Relevance: In this randomized clinical trial specifically addressing childhood HCLD, once-weekly azithromycin did not improve lung function or growth but was associated with reduced AREs; the number of hospitalizations was also lower in the azithromycin group but the difference was not significant. Future research should identify patient groups who would benefit most from this intervention and optimum treatment length, to maximize benefits while reducing the risk of antimicrobial resistance.

Trial Registration: ClinicalTrials.gov Identifier: NCT02426112.
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http://dx.doi.org/10.1001/jamanetworkopen.2020.28484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7747021PMC
December 2020

Cost-effectiveness of a novel lipoarabinomannan test for tuberculosis in patients with HIV.

Clin Infect Dis 2020 Nov 17. Epub 2020 Nov 17.

Medical Practice Evaluation Center, Massachusetts General Hospital, Boston, MA, USA.

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 HIV irrespective of symptoms.

Methods: We used a microsimulation model to project clinical and economic outcomes of three 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 modelled cohort matched that of a two-country clinical trial. We applied diagnostic yields from a retrospective study (yields for Xpert/Xpert+AlereLAM/Xpert+FujiLAM among those with CD4<200/µL: 33%/62%/70%; among those with CD4≥200/µL: 33%/35%/47%). Costs of Xpert/AlereLAM/FujiLAM were USD15/3/6 (South Africa) and USD25/3/6 (Malawi). Xpert+FujiLAM was considered cost-effective if its incremental cost-effectiveness ratio (USD/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 five-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.
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http://dx.doi.org/10.1093/cid/ciaa1698DOI Listing
November 2020

Characterization of Pneumococcal Colonization Dynamics and Antimicrobial Resistance Using Shotgun Metagenomic Sequencing in Intensively Sampled South African Infants.

Front Public Health 2020 22;8:543898. Epub 2020 Sep 22.

Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.

There remains a significant proportion of deaths due to pneumococcal pneumonia in infants from low- and middle-income countries despite the marginal global declines recorded in the past decade. Monitoring changes in pneumococcal carriage is key to understanding vaccination-induced shifts in the ecology of carriage, patterns of antimicrobial resistance, and impact on health. We longitudinally investigated pneumococcal carriage dynamics in PCV-13 vaccinated infants by collecting nasopharyngeal (NP) samples at 2-weekly intervals from birth through the first year of life from 137 infants. As a proof of concept, 196 NP samples were retrieved from a subset of 23 infants to explore strain-level pneumococcal colonization patterns and associated antimicrobial-resistance determinants. These were selected on the basis of changes in serotype and antibiogram over time. NP samples underwent short-term enrichment for streptococci prior to total nucleic acid extraction and whole metagenome shotgun sequencing (WMGS). Reads were assembled and aligned to pneumococcal reference genomes for the extraction of pneumococcal and non-pneumococcal bacterial reads. Pneumococcal contigs were aligned to the Antibiotic Resistance Gene-ANNOTation database of acquired AMR genes. pneumococcal capsular and multilocus sequence typing were performed. Of the 196 samples sequenced, 174 had corresponding positive cultures for pneumococci, of which, 152 were assigned an serotype. Metagenomic sequencing detected a single pneumococcal serotype in 85% (129/152), and co-colonization in 15% (23/152) of the samples. Twenty-two different pneumococcal serotypes were identified, with 15B/15C and 16F being the most common non-PCV13 serotypes, while 23F and 19A were the most common PCV13 serotypes. Twenty-six different sequence types (STs), including four novel STs were identified . Mutations in the A and P genes, associated with cotrimoxazole resistance, were detected in 89% (87/98) of cotrimoxazole-non-susceptible pneumococci, as well as in the 1a and 2x genes, in penicillin non-susceptible ST7052 isolates. Metagenomic sequencing of NP samples is a valuable culture-independent technique for a detailed evaluation of the pneumococcal component and resistome of the NP microbiome. This method allowed for the detection of novel STs, as well as co-colonization, with a predominance of non-PCV13 serotypes in this cohort. Forty-eight resistance genes, as well as mutations associated with resistance were detected, but the correlation with phenotypic non-susceptibility was lower than expected.
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http://dx.doi.org/10.3389/fpubh.2020.543898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536305PMC
May 2021

Risk factors for Bordetella pertussis disease in hospitalized children.

PLoS One 2020 15;15(10):e0240717. Epub 2020 Oct 15.

SA-MRC Unit on Child & Adolescent Lung Health, University of Cape Town, Cape Town, South Africa.

Introduction: Despite a resurgence of disease, risk factors for pertussis in children in low and middle-income countries are poorly understood. This study aimed to investigate risk factors for pertussis disease in African children hospitalized with severe LRTI.

Methods: A prospective study of children hospitalized with severe LRTI in Cape Town, South Africa was conducted over a one-year period. Nasopharyngeal and induced sputum samples from child and nasopharyngeal sample from caregiver were tested for Bordetella pertussis using PCR (IS481+/hIS1001). History and clinical details were documented.

Results: 460 children with a median age of 8 (IQR 4-18) months were enrolled. B. pertussis infection was confirmed in 32 (7.0%). The adjusted risk of confirmed pertussis was significantly increased if infants were younger than two months [aRR 2.37 (95% CI 1.03-5.42]), HIV exposed but uninfected (aRR 3.53 [95% CI 1.04-12.01]) or HIV infected (aRR 4.35 [95% CI 1.24-15.29]). Mild (aRR 2.27 [95% CI 1.01-5.09]) or moderate (aRR 2.70 [95% CI 1.13-6.45]) under-nutrition in the children were also associated with higher risk. The highest adjusted risk occurred in children whose caregivers had B. pertussis detected from nasopharyngeal swabs (aRR 13.82 [95% CI 7.76-24.62]). Completion of the primary vaccine schedule (three or more doses) was protective (aRR 0.28 [95% CI 0.10-0.75]).

Conclusions: HIV exposure or infection, undernutrition as well as detection of maternal nasal B. pertussis were associated with increased risk of pertussis in African children, especially in young infants. Completed primary vaccination was protective. There is an urgent need to improve primary pertussis vaccine coverage in low and middle-income countries. Pertussis vaccination of pregnant women, especially those with HIV infection should be prioritized.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240717PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561157PMC
December 2020

Laboratory development of a simple stool sample processing method diagnosis of pediatric tuberculosis using Xpert Ultra.

Tuberculosis (Edinb) 2020 12 3;125:102002. Epub 2020 Oct 3.

IRD UMI 233 TransVIHMI- UM-INSERM U1175, Montpellier, France. Electronic address:

Stool samples are alternatives to respiratory samples for bacteriological confirmation of childhood tuberculosis but require intensive laboratory processing before molecular testing to remove PCR inhibitors and debris. We aimed to develop a centrifuge-free processing method for use in resource-limited settings based on a sucrose-flotation method that showed good sensitivity for childhood tuberculosis diagnosis. In an in vitro study using Xpert MTB/RIF Ultra on stool samples spiked with defined bacterial concentrations of Mycobacterium tuberculosis (MTB), we compared different simplification parameters to the reference sucrose-flotation method. Best methods were selected based on the rate of invalid/error results and on sensitivity, compared to the reference method on stools spiked at 10 colony forming units (CFU)/g MTB. For final selection, we tested the best parameter combinations at 10 CFU/g. Out of 13 different parameter combinations, three were tested at 10 CFU/g. The best combination used 0.5 g stool, manual shaking, no filtration, 30-min sedimentation, and a 1:3.6 dilution ratio. This method gave 10% invalid/error results and a sensitivity of 70% vs 63% at 10 CFU/g and 53% vs 58% at 10 CFU/g compared to the reference method. This pre-clinical study was able to develop a centrifuge-free processing method to facilitate stool Xpert Ultra testing.
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http://dx.doi.org/10.1016/j.tube.2020.102002DOI Listing
December 2020

Co-detection of Bordetella pertussis and other respiratory organisms in children hospitalised with lower respiratory tract infection.

Sci Rep 2020 10 2;10(1):16412. Epub 2020 Oct 2.

SA-MRC Unit On Child and Adolescent Lung Health, University of Cape Town, Cape Town, South Africa.

Multiple potential pathogens are frequently co-detected among children with lower respiratory tract infection (LRTI). Evidence indicates that Bordetella pertussis has an important role in the aetiology of LRTI. We aimed to study the association between B. pertussis and other respiratory pathogens in children hospitalised with severe LRTI, and to assess clinical relevance of co-detection. Nasopharyngeal (NP) swabs and induced sputa (IS) were tested with a B. pertussis specific PCR; additionally, IS was tested for other pathogens using a multiplex PCR. We included 454 children, median age 8 months (IQR 4-18), 31 (7%) of whom tested positive for B. pertussis. Children with B. pertussis had more bacterial pathogens detected (3 versus 2; P < 0.001). While B. pertussis showed no association with most pathogens, it was independently associated with Chlamydia pneumoniae, Mycoplasma pneumoniae and parainfluenza viruses with adjusted risk ratios of 4.01 (1.03-15.64), 4.17 (1.42-12.27) and 2.13 (1.03-4.55), respectively. There was a consistent increased risk of severe disease with B. pertussis. Patterns indicated even higher risks when B. pertussis was co-detected with any of the three organisms although not statistically significant. Improving vaccine coverage against B. pertussis would impact not only the incidence of pertussis but also that of severe LRTI generally.
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http://dx.doi.org/10.1038/s41598-020-73462-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532201PMC
October 2020

Shiga toxin-producing O26:H11 associated with a cluster of haemolytic uraemic syndrome cases in South Africa, 2017.

Access Microbiol 2019 12;1(9):e000061. Epub 2019 Sep 12.

South African Field Epidemiology Training Programme, NICD, NHLS, Johannesburg, South Africa.

Introduction: Shiga toxin-producing (STEC) are foodborne pathogens that may cause diarrhoeal outbreaks and occasionally are associated with haemolytic-uraemic syndrome (HUS). We report on STEC O26:H11 associated with a cluster of four HUS cases in South Africa in 2017.

Methodology: All case-patients were female and aged 5 years and under. Standard microbiological tests were performed for culture and identification of STEC from specimens (human stool and food samples). Further analysis of genomic DNA extracted from bacterial cultures and specimens included PCR for specific virulence genes, whole-genome sequencing and shotgun metagenomic sequencing.

Results: For 2/4 cases, stool specimens revealed STEC O26:H11 containing , and virulence genes. All food samples were found to be negative for STEC. No epidemiological links could be established between the HUS cases. Dried meat products were the leading food item suspected to be the vehicle of transmission for these cases, as 3/4 case-patients reported they had eaten this. However, testing of dried meat products could not confirm this.

Conclusion: Since STEC infection does not always lead to severe symptoms, it is possible that many more cases were associated with this cluster and largely went unrecognized.
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http://dx.doi.org/10.1099/acmi.0.000061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472548PMC
September 2019

Early-life respiratory syncytial virus lower respiratory tract infection in a South African birth cohort: epidemiology and effect on lung health.

Lancet Glob Health 2020 10;8(10):e1316-e1325

Division of Medical Microbiology, University of Cape Town, Cape Town, South Africa; Division of Infection and Immunity, Department of Biomedical Sciences, University of Western Australia, Perth, WA, Australia.

Background: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection (LRTI) in children. Early-life RSV LRTI might affect long-term health but there are few data from low-income and middle-income countries. We investigated the epidemiology and effect of early-life RSV LRTI on lung health in a South African birth cohort.

Methods: We conducted the Drakenstein Child Health Study (DCHS), an ongoing birth cohort longitudinal study in the Western Cape province, South Africa. We enrolled pregnant women aged 18 years or older during their second trimester of pregnancy at two public health clinics. We followed up study children from birth to 2 years. The primary outcome of the study was LRTI and RSV LRTI. LRTI and wheezing episodes were identified through active surveillance; respiratory samples were tested for RSV and other pathogens. Wheezing was longitudinally identified by caregiver report and ascertainment at health facilities. Lung function was measured from 6 weeks to 2 years. We analysed the associations between RSV LRTI and subsequent LRTI, wheezing, and lung function using generalised estimating equations and mixed-effects linear regression.

Findings: We enrolled 1137 mothers between March 5, 2012, and March 31, 2015. Among their 1143 infants, accruing 2093 child-years of follow-up, there were 851 cases of LRTI (incidence 0·41 episodes per child-year, 95% CI 0·38-0·43). Admission to hospital owing to LRTI occurred in 169 (20%) cases (incidence 0·08 episodes per child-year, 0·07-0·09), with a case-fatality ratio of 0·5%. RSV was detected in 164 (21%) of 785 LRTI events with a specimen available for qPCR, an incidence of 0·08 episodes per child-year (0·07-0·09); highest at age 0-6 months (0·15 episodes per child-year, 0·12-0·19). Children with a first RSV LRTI were three times as likely to develop recurrent LRTI compared with those with non-RSV LRTI (0·32 [0·22-0·48] vs 0·10 [0·07- 0·16] episodes per child-year; p<0·0001), particularly following hospitalised RSV LRTI. RSV LRTI and hospitalisation for all-cause LRTI were independently associated with recurrent wheezing (adjusted incident rate ratio 1·41, 95% CI 1·25-1·59, for RSV LRTI and 1·48, 1·30-1·68, for hospitalisation). LRTI or recurrent LRTI was associated with impaired lung function, but a similar outcome was observed following RSV LRTI or non-RSV LRTI. All-cause LRTI was associated with an average 3% higher respiratory rate (95% CI 0·01-0·06; p=0·013) and lower compliance (-0·1, -0·18 to 0·02) at 2 years compared with no LRTI. Recurrent LRTI was associated with further increased respiratory rate (0·01, 0·001-0·02), resistance (0·77 hPa s L, 0·07-1·47), and lower compliance (-0·6 mL hPa, -0·09 to -0·02) with each additional event.

Interpretation: RSV LRTI was common in young infants and associated with recurrent LRTI, particularly after hospitalised RSV. Hospitalisation for all-cause LRTI, especially for RSV-LRTI, was associated with recurrent wheezing. Impairments in lung function followed LRTI or recurrent episodes, but were not specific to RSV. New preventive strategies for RSV might have an effect on long-term lung health.

Funding: Bill & Melinda Gates Foundation; South African Medical Research Council; National Research Foundation South Africa; National Institutes of Health, Human Heredity and Health in Africa; Wellcome Trust.
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http://dx.doi.org/10.1016/S2214-109X(20)30251-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511798PMC
October 2020

Variation in the observed effect of Xpert MTB/RIF testing for tuberculosis on mortality: A systematic review and analysis of trial design considerations.

Wellcome Open Res 2019 17;4:173. Epub 2020 Aug 17.

Division of Pulmonary and Critical Care Medicine, University of California San Francisco Medical Center, San Francisco, California, 94110, USA.

Most studies evaluating the effect of Xpert MTB/RIF testing for tuberculosis (TB) concluded that it did not reduce overall mortality compared to usual care. We conducted a systematic review to assess whether key study design and execution features contributed to earlier identification of patients with TB and decreased pre-treatment loss to follow-up, thereby reducing the potential impact of Xpert MTB/RIF testing. We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, and Scopus for literature published from 1 January 2009 to February 2019. We included all primary intervention studies that had evaluated the effect of Xpert MTB/RIF on mortality compared to usual care in participants with presumptive pulmonary TB. We critically reviewed features of included studies across: Study setting and context, Study population, Participant recruitment and enrolment, Study procedures, and Study follow-up. We included seven randomised and one non-randomised study.  All included studies demonstrated relative reductions in overall mortality in the Xpert MTB/RIF arm ranging from 6% to 40%. However, mortality reduction was reported to be statistically significant in two studies. Study features that could explain the lack of observed effect on mortality included: the higher quality of care at study sites; inclusion of patients with a higher pre-test probability of TB leading to higher than expected empirical rates; performance of additional diagnostic testing not done in usual care leading to increased TB diagnosis or empiric treatment initiation; the recruitment of participants likely to return for follow-up; and involvement of study staff in ensuring adherence with care and follow-up. Most studies of Xpert MTB/RIF were designed and conducted in a manner that resulted in more patients being diagnosed and treated for TB, minimising the potential difference in mortality Xpert MTB/RIF testing could have achieved compared to usual care.
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http://dx.doi.org/10.12688/wellcomeopenres.15412.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438967PMC
August 2020

Accuracy of a Novel Urine Test, Fujifilm SILVAMP Tuberculosis Lipoarabinomannan, for the Diagnosis of Pulmonary Tuberculosis in Children.

Clin Infect Dis 2021 05;72(9):e280-e288

Foundation for Innovative New Diagnostics, Geneva, Switzerland.

Background: An accurate point-of-care test for tuberculosis (TB) in children remains an elusive goal. Recent evaluation of a novel point-of-care urinary lipoarabinomannan test, Fujifilm SILVAMP Tuberculosis Lipoarabinomannan (FujiLAM), in adults living with human immunodeficiency virus (HIV) showed significantly superior sensitivity than the current Alere Determine Tuberculosis Lipoarabinomannan test (AlereLAM). We therefore compared the accuracy of FujiLAM and AlereLAM in children with suspected TB.

Methods: Children hospitalized with suspected TB in Cape Town, South Africa, were enrolled (consecutive admissions plus enrichment for a group of children living with HIV and with TB), their urine was collected and biobanked, and their sputum was tested with mycobacterial culture and Xpert MTB/RIF or Xpert MTB/RIF Ultra. Biobanked urine was subsequently batch tested with FujiLAM and AlereLAM. Children were categorized as having microbiologically confirmed TB, unconfirmed TB (clinically diagnosed), or unlikely TB.

Results: A total of 204 children were enrolled and had valid results from both index tests, as well as sputum microbiological testing. Compared to a microbiological reference standard, the sensitivity of FujiLAM and AlereLAM was similar (42% and 50%, respectively), but lower than that of Xpert MTB/RIF of sputum (74%). The sensitivity of FujiLAM was higher in children living with HIV (60%) and malnourished children (62%). The specificity of FujiLAM was substantially higher than that of AlereLAM (92% vs 66%, respectively). The specificity of both tests was higher in children 2 years or older (FujiLAM, 96%; AlereLAM, 72%).

Conclusions: The high specificity of FujiLAM suggests utility as a "rule-in" test for children with a high pretest probability of TB, including hospitalized children living with HIV or with malnutrition.
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http://dx.doi.org/10.1093/cid/ciaa1052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096212PMC
May 2021

Diagnostic accuracy of 3 urine lipoarabinomannan tuberculosis assays in HIV-negative outpatients.

J Clin Invest 2020 11;130(11):5756-5764

FIND, Geneva, Switzerland.

BACKGROUNDInadequate tuberculosis (TB) diagnostics are a major hurdle in the reduction of disease burden, and accurate point-of-care tests (POCTs) are urgently needed. We assessed the diagnostic accuracy of Fujifilm SILVAMP TB lipoarabinomannan (FujiLAM) POCT for TB diagnosis in HIV-negative outpatients and compared it with Alere Determine TB LAM Ag (AlereLAM) POCT and a laboratory-based ultrasensitive electrochemiluminescence LAM research assay (EclLAM).METHODSIn this multicenter diagnostic test accuracy study, we recruited HIV-negative adults with symptoms suggestive of pulmonary TB presenting to outpatient health care centers in Peru and South Africa. Urine samples were tested using FujiLAM, AlereLAM, and EclLAM, and the diagnostic accuracy was assessed against a microbiological reference standard (MRS) and a composite reference standard.RESULTSThree hundred seventy-two HIV-negative participants were included and the prevalence of microbiologically confirmed TB was 30%. Compared with the MRS, the sensitivities of AlereLAM, FujiLAM, and EclLAM were 10.8% (95% confidence interval [CI] 6.3%-18.0%), 53.2% (95% CI 43.9%-62.1%), and 66.7% (95% CI 57.5%-74.7%), respectively. The specificities of AlereLAM, FujiLAM, and EclLAM were 92.3% (95% CI 88.5%-95.0%), 98.9% (95% CI 96.7%-99.6%), and 98.1% (95% CI 95.6%-99.2%), respectively. Positive likelihood ratios of AlereLAM, FujiLAM, and EclLAM were 1.4, 46.2, and 34.8, respectively, and positive predictive values were 37.5%, 95.2%, and 93.7%, respectively.CONCLUSIONCompared with AlereLAM, FujiLAM detected 5 times more patients with TB in HIV-negative participants, had a high positive predictive value, and has the potential to improve rapid diagnosis of TB at the point-of-care. EclLAM demonstrated that additional sensitivity gains are possible, which highlights LAM's potential as a biomarker. Additional research is required to assess FujiLAM's performance in prospective cohorts, its cost-effectiveness, and its impact in real-world clinical settings.FUNDINGGlobal Health Innovative Technology Fund, the UK Department for International Development, the Dutch Ministry of Foreign Affairs, the Bill and Melinda Gates Foundation, the Australian Department of Foreign Affairs and Trade, the German Federal Ministry of Education and Research through Kreditanstalt für Wiederaufbau, and the NIH and National Institute of Allergy and Infectious Diseases.
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http://dx.doi.org/10.1172/JCI140461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598043PMC
November 2020

Diagnostic limitations of clinical case definitions of pertussis in infants and children with severe lower respiratory tract infection.

PLoS One 2020 17;15(7):e0235703. Epub 2020 Jul 17.

SA-MRC unit on Child & Adolescent Lung Health, University of Cape Town, Cape Town, South Africa.

Introduction: Diagnosis of pertussis is challenging especially in infants. Most low and middle-income countries (LMIC) lack resources for laboratory confirmation, relying largely on clinical diagnosis alone for both case management and surveillance. This necessitates robust clinical case definitions.

Objectives: This study assesses the accuracy of clinical case definitions with and without lymphocytosis in diagnosing pertussis in children with severe lower respiratory tract infection (LRTI) in a LMIC setting.

Methods: Children hospitalized with severe LRTI in a South African hospital were prospectively enrolled and evaluated for pertussis using PCR on respiratory samples. Clinical signs and differential white cell counts were recorded. Sensitivity and specificity of pertussis clinical diagnosis using WHO and Global Pertussis Initiative (GPI) criteria; and with addition of lymphocytosis were assessed with PCR as the reference standard.

Results: 458 children <10 years were enrolled. Bordetella pertussis infection was confirmed in 32 (7.0%). For WHO criteria, sensitivity was 78.1% (95% CI 60.7-89.2%) and specificity 15.5% (95% CI 12.4-19.3%); for GPI sensitivity was 34.4% (95% CI 20.1-52.1) and specificity 64.8% (95% CI 60.1-69.2%). Area under the curve (AUC) on receiver operating character (ROC) analysis was 0.58 (95% CI 0.46-0.70 for WHO criteria, and 0.72 (95% CI 0.56-0.88) for GPI with highest likelihood ratios of 5.33 and 4.42 respectively. Diagnostic accuracy was highest between five and seven days of symptoms for both criteria. Lymphocytosis had sensitivity of 31.3% (95% CI 17.5-49.3%) and specificity of 70.7% (95% CI 66.1-74.8%) and showed a marginal impact on improving clinical criteria.

Conclusion: Clinical criteria lack accuracy for diagnosis and surveillance of pertussis. Non-outbreak settings should consider shorter durations in clinical criteria. New recommendations still fall short of what is required for a viable clinical screening test which means the need to improve access to laboratory diagnostic support remains crucial.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235703PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367487PMC
September 2020

Advances in the diagnosis of pulmonary tuberculosis in children.

Paediatr Respir Rev 2020 Nov 25;36:52-56. Epub 2020 May 25.

Department of Paediatrics and Child Health, and SA-MRC Unit on Child & Adolescent Health, University of Cape Town and Red Cross War Memorial Children's Hospital, Cape Town, South Africa.

Major challenges still exist in the accurate diagnosis of tuberculosis in children. Algorithms based on clinical and radiological features remain in widespread use despite poor performance. Newer molecular diagnostics allow for rapid identification of TB and detection of drug-resistance in a subset of children, but lack sensitivity. Molecular testing of multiple specimens, including non-traditional specimen types, such as nasopharyngeal aspirates and stool and urine, may improve sensitivity, but the optimal combination of specimens requires further research. Novel tests under development or evaluation include a urine lipoarabinomannan test with improved sensitivity and a range of biomarkers measured from stimulated or unstimulated peripheral blood.
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http://dx.doi.org/10.1016/j.prrv.2020.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686111PMC
November 2020

The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles.

Methods Protoc 2020 May 15;3(2). Epub 2020 May 15.

Division of Medical Microbiology, Department of Pathology, Faculty of Health Sciences, Observatory 7925, University of Cape Town, Cape Town 7700, South Africa.

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of and . DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.
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http://dx.doi.org/10.3390/mps3020039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359716PMC
May 2020

Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens.

BMC Microbiol 2020 05 12;20(1):113. Epub 2020 May 12.

Division of Medical Microbiology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.

Background: Careful consideration of experimental artefacts is required in order to successfully apply high-throughput 16S ribosomal ribonucleic acid (rRNA) gene sequencing technology. Here we introduce experimental design, quality control and "denoising" approaches for sequencing low biomass specimens.

Results: We found that bacterial biomass is a key driver of 16S rRNA gene sequencing profiles generated from bacterial mock communities and that the use of different deoxyribonucleic acid (DNA) extraction methods [DSP Virus/Pathogen Mini Kit® (Kit-QS) and ZymoBIOMICS DNA Miniprep Kit (Kit-ZB)] and storage buffers [PrimeStore® Molecular Transport medium (Primestore) and Skim-milk, Tryptone, Glucose and Glycerol (STGG)] further influence these profiles. Kit-QS better represented hard-to-lyse bacteria from bacterial mock communities compared to Kit-ZB. Primestore storage buffer yielded lower levels of background operational taxonomic units (OTUs) from low biomass bacterial mock community controls compared to STGG. In addition to bacterial mock community controls, we used technical repeats (nasopharyngeal and induced sputum processed in duplicate, triplicate or quadruplicate) to further evaluate the effect of specimen biomass and participant age at specimen collection on resultant sequencing profiles. We observed a positive correlation (r = 0.16) between specimen biomass and participant age at specimen collection: low biomass technical repeats (represented by < 500 16S rRNA gene copies/μl) were primarily collected at < 14 days of age. We found that low biomass technical repeats also produced higher alpha diversities (r = - 0.28); 16S rRNA gene profiles similar to no template controls (Primestore); and reduced sequencing reproducibility. Finally, we show that the use of statistical tools for in silico contaminant identification, as implemented through the decontam package in R, provides better representations of indigenous bacteria following decontamination.

Conclusions: We provide insight into experimental design, quality control steps and "denoising" approaches for 16S rRNA gene high-throughput sequencing of low biomass specimens. We highlight the need for careful assessment of DNA extraction methods and storage buffers; sequence quality and reproducibility; and in silico identification of contaminant profiles in order to avoid spurious results.
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http://dx.doi.org/10.1186/s12866-020-01795-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218582PMC
May 2020

Diagnostic accuracy of a novel tuberculosis point-of-care urine lipoarabinomannan assay for people living with HIV: A meta-analysis of individual in- and outpatient data.

PLoS Med 2020 05 1;17(5):e1003113. Epub 2020 May 1.

FIND, Geneva, Switzerland.

Background: Tuberculosis (TB) is the most common cause of death in people living with HIV (PLHIV), yet TB often goes undiagnosed since many patients are not able to produce a sputum specimen, and traditional diagnostics are costly or unavailable. A novel, rapid lateral flow assay, Fujifilm SILVAMP TB LAM (SILVAMP-LAM), detects the presence of TB lipoarabinomannan (LAM) in urine, and is substantially more sensitive for diagnosing TB in PLHIV than an earlier LAM assay (Alere Determine TB LAM lateral flow assay [LF-LAM]). Here, we present an individual participant data meta-analysis of the diagnostic accuracy of SILVAMP-LAM in adult PLHIV, including both published and unpublished data.

Methods And Findings: Adult PLHIV (≥18 years) were assessed in 5 prospective cohort studies in South Africa (3 cohorts), Vietnam, and Ghana, carried out during 2012 to 2017. Of the 1,595 PLHIV who met eligibility criteria, the majority (61%) were inpatients, median age was 37 years (IQR 30-43), 43% had a CD4 count ≤ 100 cells/μl, and 35% were receiving antiretroviral therapy. Most participants (94%) had a positive WHO symptom screen for TB on enrollment, and 45% were diagnosed with microbiologically confirmed TB, using mycobacterial culture or Xpert MTB/RIF testing of sputum, urine, or blood. Previously published data from inpatients were combined with unpublished data from outpatients. Biobanked urine samples were tested, using blinded double reading, with SILVAMP-LAM and LF-LAM. Applying a microbiological reference standard for assessment of sensitivity, the overall sensitivity for TB detection was 70.7% (95% CI 59.0%-80.8%) for SILVAMP-LAM compared to 34.9% (95% CI 19.5%-50.9%) for LF-LAM. Using a composite reference standard (which included patients with both microbiologically confirmed as well as clinically diagnosed TB), SILVAMP-LAM sensitivity was 65.8% (95% CI 55.9%-74.6%), and that of LF-LAM 31.4% (95% CI 19.1%-43.7%). In patients with CD4 count ≤ 100 cells/μl, SILVAMP-LAM sensitivity was 87.1% (95% CI 79.3%-93.6%), compared to 56.0% (95% CI 43.9%-64.9%) for LF-LAM. In patients with CD4 count 101-200 cells/μl, SILVAMP-LAM sensitivity was 62.7% (95% CI 52.4%-71.9%), compared to 25.3% (95% CI 15.8%-34.9%) for LF-LAM. In those with CD4 count > 200 cells/μl, SILVAMP-LAM sensitivity was 43.9% (95% CI 34.3%-53.9%), compared to 10.9% (95% CI 5.2%-18.4%) for LF-LAM. Using a microbiological reference standard, the specificity of SILVAMP-LAM was 90.9% (95% CI 87.2%-93.7%), and that of LF-LAM 95.3% (95% CI 92.2%-97.7%). Limitations of this study include the use of biobanked, rather than fresh urine samples, and testing by skilled laboratory technicians in research laboratories, rather than at the point of care.

Conclusions: In this study, we found that SILVAMP-LAM identified a substantially higher proportion of TB patients in PLHIV than LF-LAM. The sensitivity of SILVAMP-LAM was highest in patients with CD4 count ≤ 100 cells/μl. Further work is needed to demonstrate accuracy when implemented as a point-of-care test.
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http://dx.doi.org/10.1371/journal.pmed.1003113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194366PMC
May 2020

Longitudinal changes in the nasopharyngeal resistome of South African infants using shotgun metagenomic sequencing.

PLoS One 2020 22;15(4):e0231887. Epub 2020 Apr 22.

Division of Medical Microbiology, Faculty of Health Science, University of Cape Town, Cape Town, South Africa.

Introduction: Nasopharyngeal (NP) colonization with antimicrobial-resistant bacteria is a global public health concern. Antimicrobial-resistance (AMR) genes carried by the resident NP microbiota may serve as a reservoir for transfer of resistance elements to opportunistic pathogens. Little is known about the NP antibiotic resistome. This study longitudinally investigated the composition of the NP antibiotic resistome in Streptococcus-enriched samples in a South African birth cohort.

Methods: As a proof of concept study, 196 longitudinal NP samples were retrieved from a subset of 23 infants enrolled as part of broader birth cohort study. These were selected on the basis of changes in serotype and antibiogram over time. NP samples underwent short-term enrichment for streptococci prior to total nucleic acid extraction and whole metagenome shotgun sequencing (WMGS). Reads were assembled and aligned to pneumococcal reference genomes for the extraction of streptococcal and non-streptococcal bacterial reads. Contigs were aligned to the Antibiotic Resistance Gene-ANNOTation database of acquired AMR genes.

Results: AMR genes were detected in 64% (125/196) of the samples. A total of 329 AMR genes were detected, including 36 non-redundant genes, ranging from 1 to 14 genes per sample. The predominant AMR genes detected encoded resistance mechanisms to beta-lactam (52%, 172/329), macrolide-lincosamide-streptogramin (17%, 56/329), and tetracycline antibiotics (12%, 38/329). MsrD, ermB, and mefA genes were only detected from streptococcal reads. The predominant genes detected from non- streptococcal reads included blaOXA-60, blaOXA-22, and blaBRO-1. Different patterns of carriage of AMR genes were observed, with only one infant having a stable carriage of mefA, msrD and tetM over a long period.

Conclusion: This study demonstrates that WMGS can provide a broad snapshot of the NP resistome and has the potential to provide a comprehensive assessment of resistance elements present in this niche.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231887PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176138PMC
July 2020

First clinical assessment of a prototype assay to detect the enzymatic activity of β-lactamase as a marker for pulmonary tuberculosis.

Diagn Microbiol Infect Dis 2020 Jun 19;97(2):115026. Epub 2020 Feb 19.

FIND, Chemin des Mines 9, 1202, Geneva, Switzerland; Division of Tropical Medicine, Center of Infectious Diseases, University Hospital of Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany. Electronic address:

The objective was to evaluate the sensitivity and specificity of a novel prototype test, TB REaD™, a reporter enzyme fluorescence-based assay, for pulmonary tuberculosis and to determine the optimal threshold for test positivity. This blinded, prospective study enrolled 250 patients, of which 23.2% were Mycobacterium tuberculosis complex (MTB) culture-positive. At the manufacturer-set threshold, sensitivity of the assay was 93.1% (95% confidence interval [CI] 83.3-98.1) and specificity was 8.9% (95% CI 5.2-13.8). The highest accuracy was seen at a higher threshold: sensitivity 58.6% (95% CI 44.9-71.4), specificity 59.4% (95% CI 52.1%-66.4%), with sensitivity by smear status being 40.0% (95% CI 21.1-61.3) for smear-negative and 72.7% (95% CI 54.5-86.7) for smear-positive. This study demonstrated limited accuracy of the TB REaD™ prototype for detection of pulmonary TB. Further improvements are necessary, potentially exploring probes that are more specific to MTB.
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http://dx.doi.org/10.1016/j.diagmicrobio.2020.115026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262578PMC
June 2020

Improving the design of studies evaluating the impact of diagnostic tests for tuberculosis on health outcomes: a qualitative study of perspectives of diverse stakeholders.

Wellcome Open Res 2019 21;4:183. Epub 2019 Nov 21.

School of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Western Australia, Perth, WA 6009, Australia.

Studies evaluating the impact of Xpert MTB/RIF testing for tuberculosis (TB) have demonstrated varied effects on health outcomes with many studies showing inconclusive results. We explored perceptions among diverse stakeholders about studies evaluating the impact of TB diagnostic tests, and identified suggestions for improving these studies. We used purposive sampling with consideration for differing expertise and geographical balance and conducted in depth semi-structured interviews. We interviewed English-speaking participants, including TB patients, and others involved in research, care or decision-making about TB diagnostics. We used the thematic approach to code and analyse the interview transcripts. We interviewed 31 participants. Our study showed that stakeholders had different expectations with regard to test impact and how it is measured. TB test impact studies were perceived to be important for supporting implementation of tests but there were concerns about the unrealistic expectations placed on tests to improve outcomes in health systems with many influencing factors. To improve TB test impact studies, respondents suggested conducting health system assessments prior to the study; developing clear guidance on the study methodology and interpretation; improving study design by describing questions and interventions that consider the influences of the health-care ecosystem on the diagnostic test; selecting the target population at the health-care level most likely to benefit from the test; setting realistic targets for effect sizes in the sample size calculations; and interpreting study results carefully and avoiding categorisation and interpretation of results based on statistical significance alone. Researchers should involve multiple stakeholders in the design of studies. Advocating for more funding to support robust studies is essential. TB test impact studies were perceived to be important to support implementation of tests but there were concerns about their complexity. Process evaluations of their health system context and guidance for their design and interpretation are recommended.
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http://dx.doi.org/10.12688/wellcomeopenres.15551.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041361PMC
November 2019

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

BMC Infect Dis 2020 03 2;20(1):187. Epub 2020 Mar 2.

Division of Haematology, Department of Medicine, University of Cape Town, Anzio Rd, Observatory, Cape Town, 7925, South Africa.

After publication of the original article [1], we were notified that there is a mistake in the article note.
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http://dx.doi.org/10.1186/s12879-020-4917-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050142PMC
March 2020

The Determinants of the Human Milk Metabolome and Its Role in Infant Health.

Metabolites 2020 Feb 20;10(2). Epub 2020 Feb 20.

Division of Medical Microbiology, Department of Pathology, University of Cape Town, Observatory, Cape Town 7925, South Africa.

Human milk is needed for optimal growth as it satisfies both the nutritional and biological needs of an infant. The established relationship between breastfeeding and an infant's health is attributable to the nutritional and non-nutritional, functional components of human milk including metabolites such as the lipids, amino acids, biogenic amines and carbohydrates. These components have diverse roles, including protecting the infant against infections and guiding the development of the infant's immature immune system. In this review, we provide an in-depth and updated insight into the immune modulatory and anti-infective role of human milk metabolites and their effects on infant health and development. We also review the literature on potential determinants of the human milk metabolome, including maternal infectious diseases such as human immunodeficiency virus and mastitis.
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http://dx.doi.org/10.3390/metabo10020077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074355PMC
February 2020
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