Analysis of breast milk fatty acid composition using dried milk samples.

BACKGROUND: The effect of breast milk fatty acid (FA) composition, particularly levels of docosahexaenoic acid (DHA), on infant health outcomes is unclear. Part of the reason for this is difficulties in collecting, storing and shipping milk samples to the laboratory. Here we report the validation of a dried milk spot (DMS) system to measure FA composition to help overcome these obstacles. Milk FA were measured by gas chromatography and reported as percent of total FA; the FA of primary interest in this study were DHA and industrially produced trans FA (iTFA). Experiments were carried out using pooled milk samples from US (n = 5) and Malawian women (n = 50). Experiments compared liquid vs. DMS samples (n = 55), assessed stability of FA composition under different storage conditions (n = 5), and compared the results from two different labs using the same methods (n = 5). RESULTS: Both % DHA and % iTFA levels in liquid and DMS samples were strongly correlated (R(2) = 0.99 and 0.99, respectively, P < 0.0001). The % DHA in DMS samples was stable for up to four weeks at room temperature and up to three years at -80 °C; only slight deviations from the acceptable range of variability (±15 %) occurred in the 4 °C and -20 °C conditions for % DHA. The % iTFA was stable under all conditions. All % DHA and % iTFA were within 15 % of the referent when analyzed in two laboratories. CONCLUSIONS: Valid FA composition values can be obtained from DMS samples using this robust collection and transport system which should facilitate studies of the role of milk FA composition in infant development

Suppression of host gene expression is associated with latent TB infection : a possible diagnostic biomarker

Funding:This work was Supported by the Wellcome Trust Institutional Strategic Support fund of the University of St Andrews, grant code 204821/Z/16/Z. Additional funding was obtained from Helse Nord Tuberculosis Initiative (HNTI), Pathology Department, Kamuzu University of Health Sciences and the Africa Centre for Public Health and Herbal Medicines (ACEPHEM), Kamuzu University of Health Sciences.The World Health Organization End TB strategy aims for a 90% reduction of tuberculosis (TB) incidence by 2035. Systematic testing and treatment of latent TB infection (LTBI) among contacts of active TB patients is recommended as one of the ways to curtail TB incidence. However, there is a shortage of tools to accurately diagnose LTBI. We assessed the appropriateness of whole blood host transcriptomic markers (TM) to diagnose LTBI among household contacts of bacteriologically confirmed index cases compared to HIV negative healthy controls (HC). QuantiFERON-TB Gold Plus Interferon gamma release assay (IGRA) and reverse-transcriptase quantitative PCR were used to determine LTBI and quantify TM expression respectively. Association between TM expression and LTBI was evaluated by logistic regression modelling. A total of 100 participants, 49 TB exposed (TBEx) household contacts and 51 HC, were enrolled. Twenty-five (51%) TBEx individuals tested positive by IGRA, and were denoted as LTBI individuals, and 37 (72.5%) HC were IGRA-negative. Expression of 11 evaluated TM was significantly suppressed among LTBI compared to HC. Out of the 11 TM, ZNF296 and KLF2 expression were strongly associated with LTBI and successfully differentiated LTBI from HC. Paradoxically, 21 (49%) TBEx participants who tested IGRA negative exhibited the same pattern of suppressed TM expression as IGRA positive (LTBI-confirmed individuals). Results suggest that suppression of gene expression underlies LTBI and may be a more sensitive diagnostic biomarker than standard-of-care IGRA.Peer reviewe

A fast extraction-free isothermal LAMP assay for detection of SARS-CoV-2 with potential use in resource-limited settings

BACKGROUND: To retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR. METHODS: Four previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay. RESULTS: In this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies/µL of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site. CONCLUSION: SARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management

Lack of Associations between Environmental Exposures and Environmental Enteric Dysfunction among 18-Month-Old Children in Rural Malawi

Environmental enteric dysfunction (EED) is common and contributes to linear growth faltering (stunting) and mortality among children in low-resource settings. A few studies on the environmental causes of EED have been conducted but the exact exposures that cause or predispose children to EED are context-specific and not clear. This study aimed to assess associations between selected environmental exposures and EED markers among 620 18-month-old children. This was a secondary analysis of data from Malawian children who participated in a randomized controlled trial (iLiNS-DYAD, registered at clinicaltrials.gov as NCT01239693) from birth to 18 months of age. Data on environmental exposures, including drinking water source, sanitation, exposure to animals, housing materials, season, residential area, and food insecurity were collected at enrolment. Biomarkers of EED included concentrations of calprotectin, regenerating 1B protein (REG1B), and alpha-1-antitrypsin from stool samples to assess intestinal inflammation, repair, and permeability, respectively. We performed bivariate and multivariable analyses to assess associations between environmental exposures and EED biomarkers. Adjusting for possible confounders, we did not find associations between the selected environmental exposures and the three biomarkers. These results do not provide support for our hypothesis that the studied adverse environmental exposures are associated with increased concentrations of children’s EED markers in rural Malawi.publishedVersionPeer reviewe

Biannual Administrations of Azithromycin and the Gastrointestinal Microbiome of Malawian Children: A Nested Cohort Study Within a Randomized Controlled Trial.

Community-level mass treatment with azithromycin has been associated with a mortality benefit in children. However, antibiotic exposures result in disruption of the gut microbiota and repeated exposures may reduce recovery of the gut flora. We conducted a nested cohort study within the framework of a randomized controlled trial to examine associations between mass drug administration (MDA) with azithromycin and the gut microbiota of rural Malawian children aged between 1 and 59 months. Fecal samples were collected from the children at baseline and 6 months after two or four biannual rounds of azithromycin treatment. DNA was extracted from fecal samples and V4-16S rRNA sequencing used to characterize the gut microbiota. Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were the dominant phyla while Faecalibacterium and Bifidobacterium were the most prevalent genera. There were no associations between azithromycin treatment and changes in alpha diversity, however, four biannual rounds of treatment were associated with increased abundance of Prevotella. The lack of significant changes in gut microbiota after four biannual treatments supports the use of mass azithromycin treatment to reduce mortality in children living in low- and middle-income settings

Fecal biomarkers of environmental enteric dysfunction and the gut microbiota of rural Malawian children: An observational study

Environmental enteric dysfunction (EED) is a subclinical condition of the gut characterized by changes in morphology and function with underlying chronic inflammatory responses. This study characterized composition and diversity of the gut microbiota in rural Malawian children with and without signs of EED. Fecal samples were collected from children aged 1-59 months. Neopterin, myeloperoxidase and alpha-1 antitrypsin concentrations were quantified by ELISA and combined to form a composite EED score using principal component analysis. DNA was extracted from fecal samples and V4-16S rRNA gene sequencing was used to characterize the gut microbiota. The concentrations of all three biomarkers decreased with increasing age, which is consistent with other studies of children living in similar low-income settings. Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were the dominant phyla while Faecalibacterium and Bifidobacterium were the most prevalent genera. Increased alpha diversity was associated with a reduction in neopterin concentration. Microbiota composition was different between fecal samples with low and high composite EED scores; increased abundance of Succinivibrio was associated with reduced composite EED scores

Validation of a direct-to-PCR COVID-19 detection protocol utilizing mechanical homogenization: a model for reducing resources needed for accurate testing

Efficient and effective viral detection methodologies are a critical piece in the global response to COVID-19, with PCR-based nasopharyngeal and oropharyngeal swab testing serving as the current gold standard. With over 100 million confirmed cases globally, the supply chains supporting these PCR testing efforts are under a tremendous amount of stress, driving the need for innovative and accurate diagnostic solutions. Herein, the utility of a direct-to-PCR method of SARS-CoV-2 detection grounded in mechanical homogenization is examined for reducing resources needed for testing while maintaining a comparable sensitivity to the current gold standard workflow of nasopharyngeal and oropharyngeal swab testing. In a head-to-head comparison of 30 patient samples, this initial clinical validation study of the proposed homogenization-based workflow demonstrated significant agreeability with the current extraction-based method utilized while cutting the total resources needed in half

Child Aflatoxin Exposure is Associated with Poor Child Growth Outcomes : A Prospective Cohort Study in Rural Malawi

Background: Aflatoxin (AF) exposure is associated with child growth faltering in cross-sectional studies, with limited findings from longitudinal studies. Objectives: To evaluate the relationship between maternal AF B1-lysine adduct concentration, child AF B1-lysine adduct concentration, and child growth in the first 30 mo of life. Methods: AF B1-lysine adduct was measured in mother-child dyad plasma samples using isotope dilution mass spectrometry. Using linear regression, we assessed the relationship between AF B1-lysine adduct concentration and child weight, height, and head and mid-upper arm circumferences at 1 wk, 6, 12, 18, 24, and 30 mo of age. Results: In adjusted models, maternal prenatal AF B1-lysine adduct (pg/μL) was positively associated with newborn anthropometric outcomes; largest beta coefficients for associations between standardized values were for newborn weight-for-age z-score [β = 0.13; 95% confidence interval (CI): 0.02, 0.24; P < 0.05 and β = 0.11; 95% CI: 0.00, 0.22; P < 0.05 for second and third trimester AF, respectively]. Child AF B1-lysine adduct (pg/μL) at 6 mo was negatively associated with head circumference-for-age z-score at 6, 18, 24, and 30 mo, with beta coefficients ranging from β = –0.15; 95% CI: –0.28, –0.02 to β = –0.17; 95% CI: –0.31, –0.03; P < 0.05); 18-mo AF was negatively associated with anthropometric outcomes at 18, 24, and 30 mo, most consistently with length-for-age z-score (β = –0.18; 95% CI: –0.32, –0.04, β = –0.21; 95% CI: –0.35, –0.07, β = –0.18; 95% CI: –0.32, –0.03 at 18, 24 and 30 mo, respectively). Conclusions: Child AF exposure was associated with impaired child growth, but maternal AF exposure was not. Exposure during infancy was linked to persistent deficit in head circumference, implying reduced brain size lasting beyond the age of 2 years. Exposure at 18 mo was linked to persistent linear growth deficit. Further research should elucidate mechanisms through which AF affects child growth.Peer reviewe

Characterising protective immune responses to SARS-CoV-2 in urban and rural Malawi between February 2021 and April 2022

SARS-CoV-2 transmission in Malawi remains unclear due to high proportions of mild/asymptomatic infections and limited diagnostics. Existing seroprevalence studies in Malawi have primarily used convenience samples and enzyme-linked immunosorbent assays (ELISAs). We assessed SARSCoV-2 neutralisation in a longitudinal Malawian population-based cohort, assessing protective immunity post-infection and vaccination. Sera were obtained from rural (Karonga, n = 958) and urban (Lilongwe, n = 918) based participants at three-monthly intervals (February 2021-April 2022). Neutralising antibodies against SARS-CoV-2 were measured using human immunodeficiency (HIV)based pseudotype assays in HIV-uninfected participants, and vesicular stomatitis virus-based assays in HIV-infected participants and an HIV-uninfected subset. Nucleocapsid ELISAs identified vaccinated participants also infected. SARS-CoV-2 neutralisation profiles increased in complexity over time from rising vaccination coverage and emerging variants. Neutralising antibody prevalence was higher in Lilongwe than Karonga (68.1% (CI 63.5–72.4) vs. 45.4% (CI 41.6–49.3), Survey 4). Hybrid immune and solely vaccinated participants exhibited higher titres than those solely infected. Children &lt; 15 years had the lowest neutralising antibody titres among infected (not vaccinated) participants. People living with HIV had lower neutralising responses than those HIV-uninfected, particularly post vaccination. We therefore recommend surveillance of children and people living with HIV as low neutralisation responses increase reinfection risk. COVID-19 vaccination should be prioritised for HIV-infected individuals