Predictors of Maternal Serum Concentrations for Selected Persistent Organic Pollutants (POPs) in Pregnant Women and Associations with Birth Outcomes: A Cross-Sectional Study from Southern Malawi.

Population exposure to persistent organic pollutants (POPs) may result in detrimental health effects, especially to pregnant women, developing foetuses and young children. We are reporting the findings of a cross-sectional study of 605 mothers in their late pregnancy, recruited between August 2020 and July 2021 in southern Malawi, and their offspring. The aim was to measure the concentrations of selected POPs in their maternal serum and indicate associations with social demographic characteristics and birth outcomes. A high level of education was the main predictor of p,p'-DDE (p = 0.008), p,p'-DDT (p < 0.001), cis-NC (p = 0.014), o,p'-DDT (p = 0.019) and o,p'-DDE (p = 0.019) concentrations in maternal serum. Multiparity was negatively associated with o,p'-DDE (p = 0.021) concentrations. Maternal age was also positively associated (p,p'-DDE (p = 0.013), o,p'-DDT (p = 0.017) and o,p'-DDE (p = 0.045) concentrations. Living in rural areas was inversely associated with high maternal serum concentrations of p,p'-DDT (p < 0.001). Gestational age was positively associated with p,p'-DDE (p = 0.031), p,p'-DDT (p = 0.010) and o,p'-DDT (p = 0.022) concentrations. Lastly, an inverse association was observed between head circumference and t-NC (p = 0.044), Oxychlordane (p = 0.01) and cis-NC (p = 0.048). These results highlight the need to continue monitoring levels of POPs among vulnerable populations in the southern hemisphere.

Characterization of DNA methylation in Malawian Mycobacterium tuberculosis clinical isolates.

BACKGROUND: Although Mycobacterium tuberculosis (Mtb) strains exhibit genomic homology of >99%, there is considerable variation in the phenotype. The underlying mechanisms of phenotypic heterogeneity in Mtb are not well understood but epigenetic variation is thought to contribute. At present the methylome of Mtb has not been completely characterized. METHODS: We completed methylomes of 18 Mycobacterium tuberculosis (Mtb) clinical isolates from Malawi representing the largest number of Mtb genomes to be completed in a single study using Single Molecule Real Time (SMRT) sequencing to date. RESULTS: We replicate and confirm four methylation disrupting mutations in 4 lineages of Mtb. For the first time we report complete loss of methylation courtesy of C758T (S253L) mutation in the MamB gene of Indo-oceanic lineage of Mtb. Additionally, we report a novel missense mutation G454A (G152S) in the MamA gene of the Euro-American lineage which could potentially be attributed to total disruption of methylation in the CCCAG motif but partial loss in a partner motif. Through a genomic and methylome comparative analysis with a global sample of sixteen, we report previously unknown mutations affecting the pks15/1 locus in L6 isolates. We confirm that methylation in Mtb is lineage specific although some unresolved issues still remain.

Genetic diversity of Mycobacterium tuberculosis clinical isolates in Blantyre, Malawi.

Despite the high burden of tuberculosis (TB) worldwide, specific factors influencing disease transmission remain elusive. Long term epidemiological studies and in vitro experimental models provide evidence of variable relative fitness of Mycobacterium tuberculosis (Mtb) strains but few such studies are available. Large sequence polymorphisms (LSP) are a robust molecular marker and are feasible as an epidemiological investigative tool. Few Mtb molecular epidemiological studies have been reported in Malawi owing to lack of laboratories with molecular tools. We characterized the genetic diversity of Mtb clinical isolates amongst TB patients in Blantyre, Malawi. We genotyped 64 Mtb clinical isolates using LSP-PCR, assigned specific lineages and confirmed 18 of the isolates using SMRT sequencing. The 64 isolates clustered into 4 lineages (L1-L4) with L4 predominating. There were 10/64 (16%) isolates belonging to L1, 6/64 (9%) belonging to L2, 2/64 (3%) belonging to L3 and 46/64 (72%) belonging to L4. Comparison with a previous study done in Karonga revealed concordance in L1 and L4 but discodance in L2 and L3. The phylogenetic tree constructed, comprised of 3/4 lineages present in Blantyre with 3/18 belonging to L1, 3/18 belonging to L2 and 12/18 belonging to L4. Four Mtb lineages were present in Blantyre with L4 predominating. Larger studies are needed to understand the molecular epidemiology of TB in Blantyre in light of increased bi-directional migration with South Africa.

Evaluation of the efficacy of two methods for direct extraction of DNA from Mycobacterium tuberculosis sputum.

INTRODUCTION: Whole genome sequencing (WGS) has shown superiority over other bacterial typing methods and can be used to monitor disease transmission. The long culture period hinders use of WGS as a diagnostic tool for TB. The ideal situation would be to efficiently sequence directly from clinical specimens such as sputum. Attempts to sequence directly from Mtb clinical samples have achieved very low coverage (less than 0.7X). We compared DNA extraction methods for direct extraction from Mycobacterium tuberculosis positive sputum and assessed their suitability for Single Molecule Real Time sequencing. METHODOLOGY: We evaluated the extraction efficiency of the PrimeXtract kit and an in-house CTAB method by extracting DNA from Mtb sputum. We evaluated the methods on these parameters: ease of use, efficiency (quantity and purity) and the cost per extraction. RESULTS: The PrimeXtract kit was able to isolate 5.93 µg/mL ± 0.94, (Mean ± SEM) concentration of DNA and a yield of 0.2975 µg ± 0.04723, (Mean ± SEM). Comparatively, the CTAB method isolated 1.88 µg/mL ± 0.38 DNA and a yield of 0.09 µg ± 0.02. Both concentration and yield from the kit were significantly (p = 0.0002) higher than those from CTAB. The PrimeXtract kit had a DNA purity ratio of 1.69 ± 0.09 compared to the CTAB's 1.73 ± 0.14 and this difference was not statistically different. CONCLUSION: PrimeXtract kit has a superior extraction efficiency than the CTAB method on Mtb sputum in terms of DNA yield although no significant difference by DNA purity was seen.

Serial image analysis of Mycobacterium tuberculosis colony growth reveals a persistent subpopulation in sputum during treatment of pulmonary TB.

Faster elimination of drug tolerant 'persister' bacteria may shorten treatment of tuberculosis (TB) but no method exists to quantify persisters in clinical samples. We used automated image analysis to assess whether studying growth characteristics of individual Mycobacterium tuberculosis colonies from sputum on solid media during early TB treatment facilitates 'persister' phenotyping. As Time to Detection (TTD) in liquid culture inversely correlates with total bacterial load we also evaluated the relationship between individual colony growth parameters and TTD. Sputum from TB patients in Malawi was prepared for solid and liquid culture after 0, 2 and 4 weeks of treatment. Serial photography of agar plates was used to measure time to appearance (lag time) and radial growth rate for each colony. Mixed-effects modelling was used to analyse changing growth characteristics from serial samples. 20 patients had colony measurements recorded at ≥1 time-point. Overall lag time increased by 6.5 days between baseline and two weeks (p = 0.0001). Total colony count/ml showed typical biphasic elimination, but long lag time colonies (>20days) had slower, monophasic decline. TTD was associated with minimum lag time (time to appearance of first colony1). Slower elimination of long lag time colonies suggests that these may represent a persister subpopulation of bacilli.