Update on pathology laboratory development and research in advancing regional cancer care in Malawi.

The pathology laboratory at Kamuzu Central Hospital (KCH) in Lilongwe, Malawi was established in 2011. We published our initial experiences in laboratory development and telepathology in 2013 and 2016, respectively. The purpose of this paper is to provide an update on our work by highlighting the positive role laboratory development has played in improving regional cancer care and research. In addition, we provide a summary of the adult pathology data from specimens received between July 1, 2011, and May 31, 2019, with an emphasis on malignant diagnoses. We compare these summaries to estimates of cancer incidence in this region to identify gaps and future needs.

Comparative whole genome analysis reveals re-emergence of human Wa-like and DS-1-like G3 rotaviruses after Rotarix vaccine introduction in Malawi.

G3 rotaviruses rank among the most common rotavirus strains worldwide in humans and animals. However, despite a robust long-term rotavirus surveillance system from 1997 at Queen Elizabeth Central Hospital in Blantyre, Malawi, these strains were only detected from 1997 to 1999 and then disappeared and re-emerged in 2017, 5 years after the introduction of the Rotarix rotavirus vaccine. Here, we analysed representative twenty-seven whole genome sequences (G3P[4], n = 20; G3P[6], n = 1; and G3P[8], n = 6) randomly selected each month between November 2017 and August 2019 to understand how G3 strains re-emerged in Malawi. We found four genotype constellations that were associated with the emergent G3 strains and co-circulated in Malawi post-Rotarix vaccine introduction: G3P[4] and G3P[6] strains with the DS-1-like genetic backbone genes (G3-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2), G3P[8] strains with the Wa-like genetic backbone genes (G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1), and reassortant G3P[4] strains consisting of the DS-1-like genetic backbone genes and a Wa-like NSP2 (N1) gene (G3-P[4]-I2-R2-C2-M2-A2-N1-T2-E2-H2). Time-resolved phylogenetic trees demonstrated that the most recent common ancestor for each ribonucleic acid (RNA) segment of the emergent G3 strains was between 1996 and 2012, possibly through introductions from outside the country due to the limited genetic similarity with G3 strains which circulated before their disappearance in the late 1990s. Further genomic analysis revealed that the reassortant DS-1-like G3P[4] strains acquired a Wa-like NSP2 genome segment (N1 genotype) through intergenogroup reassortment; an artiodactyl-like VP3 through intergenogroup interspecies reassortment; and VP6, NSP1, and NSP4 segments through intragenogroup reassortment likely before importation into Malawi. Additionally, the emergent G3 strains contain amino acid substitutions within the antigenic regions of the VP4 proteins which could potentially impact the binding of rotavirus vaccine-induced antibodies. Altogether, our findings show that multiple strains with either Wa-like or DS-1-like genotype constellations have driven the re-emergence of G3 strains. The findings also highlight the role of human mobility and genome reassortment events in the cross-border dissemination and evolution of rotavirus strains in Malawi necessitating the need for long-term genomic surveillance of rotavirus in high disease-burden settings to inform disease prevention and control.

Acute rotavirus infection is associated with the induction of circulating memory CD4+ T cell subsets.

Strong CD4+ T cell-mediated immune protection following rotavirus infection has been observed in animal models, but its relevance in humans remains unclear. Here, we characterized acute and convalescent CD4+ T cell responses in children who were hospitalized with rotavirus-positive and rotavirus-negative diarrhoea in Blantyre, Malawi. Children presenting with laboratory-confirmed rotavirus infection had higher proportions of effector and central memory T helper 2 cells during acute infection i.e., at disease presentation compared to convalescence, 28 days post-infection defined by a follow-up 28 days after acute infection. However, circulating cytokine-producing (IFN-γ and/or TNF-α) rotavirus-specific VP6-specific CD4+ T cells were rarely detectable in children with rotavirus infection at both acute and convalescent stages. Moreover, following whole blood mitogenic stimulation, the responding CD4+ T cells were predominantly non-cytokine producers of IFN-γ and/or TNF-α. Our findings demonstrate limited induction of anti-viral IFN-γ and/or TNF-α-producing CD4+ T cells in rotavirus-vaccinated Malawian children following the development of laboratory-confirmed rotavirus infection.

Leveraging Beneficial Off-Target Effects of Live-Attenuated Rotavirus Vaccines.

Following the introduction of live-attenuated rotavirus vaccines in many countries, a notable reduction in deaths and hospitalisations associated with diarrhoea in children <5 years of age has been reported. There is growing evidence to suggest that live-attenuated vaccines also provide protection against other infections beyond the vaccine-targeted pathogens. These so called off-target effects of vaccination have been associated with the tuberculosis vaccine Bacille Calmette Guérin (BCG), measles, oral polio and recently salmonella vaccines, and are thought to be mediated by modified innate and possibly adaptive immunity. Indeed, rotavirus vaccines have been reported to provide greater than expected reductions in acute gastroenteritis caused by other enteropathogens, that have mostly been attributed to herd protection and prior underestimation of rotavirus disease. Whether rotavirus vaccines also alter the immune system to reduce non targeted gastrointestinal infections has not been studied directly. Here we review the current understanding of the mechanisms underlying off-target effects of vaccines and propose a mechanism by which the live-attenuated neonatal rotavirus vaccine, RV3-BB, could promote protection beyond the targeted pathogen. Finally, we consider how vaccine developers may leverage these properties to improve health outcomes in children, particularly those in low-income countries where disease burden and mortality is disproportionately high relative to developed countries.

Associations between Gut Microbiota and Intestinal Inflammation, Permeability and Damage in Young Malawian Children.

BACKGROUND: Environmental enteric dysfunction (EED) is common in low- and middle-income countries and associated with childhood undernutrition. The composition of gut microbiota has been implicated in the pathogenesis of EED. Our aim was to assess the associations between gut microbiota and EED biomarkers in rural Malawian children. We hypothesized that there would be an inverse association between microbiota maturity and diversity and fecal concentrations of EED biomarkers. METHODS: We used data from fecal samples collected at 6, 18 and 30 months from 611 children who were followed up during a nutrition intervention trial. The primary time point for analysis was 18 months. Microbiota data were obtained through 16S rRNA sequencing and variables included microbiota maturity and diversity, phylogenetic dissimilarity and relative abundances of individual taxa. EED biomarkers included calprotectin (marker of inflammation), alpha-1 antitrypsin (intestinal permeability) and REG1B (intestinal damage). RESULTS: There was an inverse association between microbiota maturity and diversity and fecal concentrations of all 3 EED biomarkers at 18 months (p≤0.001). The results were similar at 30 months, while at 6 months inverse associations were found only with calprotectin and alpha-1 antitrypsin concentrations. At 18 months, EED biomarkers were not associated with phylogenetic dissimilarity, but at 6 and 30 months several associations were observed. Individual taxa predicting EED biomarker concentrations at 18 months included several Bifidobacterium and Enterobacteriaceae taxa as well as potentially displaced oral taxa. CONCLUSIONS: Our findings support the hypothesis of an inverse association between microbiota maturity and diversity and EED in rural Malawian children.

Chronic childhood undernutrition is an important public health concern that affects about 150 million children, mostly in low- and middle-income countries. Undernutrition is caused by insufficient nutrient intake and frequent infections, but there are also other underlying factors. One of these is a condition called environmental enteric dysfunction (EED), which is characterized by intestinal inflammation and damage without apparent clinical symptoms. EED is thought to be caused by the ingestion of pathogenic bacteria that leads to changes in the intestine such as increased permeability and decreased absorptive capacity. This might make the intestinal wall vulnerable to bacterial invasion and reduce the absorption of nutrients. Besides potentially pathogenic bacteria, there are many commensal bacteria in the gastrointestinal tract that have beneficial functions and that interact with the immune system. The aim of our study was to assess the associations between all these bacteria, that is the intestinal microbiota and biomarkers of EED. We used data from fecal samples collected from young children participating in a nutrition intervention trial in rural Malawi. Our findings support an inverse association between the diversity and maturity of the intestinal microbiota and biomarkers of EED. Additionally, we identified the differences at the level of individual bacterial taxa (groups of bacteria defined by genetic similarity) between participants with different levels of EED biomarkers. Due to the type of study, we cannot determine whether the observed associations represent a causal relationship between the intestinal microbiota and EED. This as well as the exact mechanisms behind these associations should be assessed in further studies.

Rotavirus Genotypes in Hospitalized Children With Acute Gastroenteritis Before and After Rotavirus Vaccine Introduction in Blantyre, Malawi, 1997-2019.

BACKGROUND: Rotavirus vaccine (Rotarix [RV1]) has reduced diarrhea-associated hospitalizations and deaths in Malawi. We examined the trends in circulating rotavirus genotypes in Malawi over a 22-year period to assess the impact of RV1 introduction on strain distribution. METHODS: Data on rotavirus-positive stool specimens among children aged <5 years hospitalized with diarrhea in Blantyre, Malawi before (July 1997-October 2012, n = 1765) and after (November 2012-October 2019, n = 934) RV1 introduction were analyzed. Rotavirus G and P genotypes were assigned using reverse-transcription polymerase chain reaction. RESULTS: A rich rotavirus strain diversity circulated throughout the 22-year period; Shannon (H') and Simpson diversity (D') indices did not differ between the pre- and postvaccine periods (H' P < .149; D' P < .287). Overall, G1 (n = 268/924 [28.7%]), G2 (n = 308/924 [33.0%]), G3 (n = 72/924 [7.7%]), and G12 (n = 109/924 [11.8%]) were the most prevalent genotypes identified following RV1 introduction. The prevalence of G1P[8] and G2P[4] genotypes declined each successive year following RV1 introduction, and were not detected after 2018. Genotype G3 reemerged and became the predominant genotype from 2017 onward. No evidence of genotype selection was observed 7 years post-RV1 introduction. CONCLUSIONS: Rotavirus strain diversity and genotype variation in Malawi are likely driven by natural mechanisms rather than vaccine pressure.

Reduction in Severity of All-Cause Gastroenteritis Requiring Hospitalisation in Children Vaccinated against Rotavirus in Malawi.

Rotavirus is the major cause of severe gastroenteritis in children aged <5 years. Introduction of the G1P[8] Rotarix® rotavirus vaccine in Malawi in 2012 has reduced rotavirus-associated hospitalisations and diarrhoeal mortality. However, the impact of rotavirus vaccine on the severity of gastroenteritis presented in children requiring hospitalisation remains unknown. We conducted a hospital-based surveillance study to assess the impact of Rotarix® vaccination on the severity of gastroenteritis presented by Malawian children. Stool samples were collected from children aged <5 years who required hospitalisation with acute gastroenteritis from December 2011 to October 2019. Gastroenteritis severity was determined using Ruuska and Vesikari scores. Rotavirus was detected using enzyme immunoassay. Rotavirus genotypes were determined using nested RT-PCR. Associations between Rotarix® vaccination and gastroenteritis severity were investigated using adjusted linear regression. In total, 3159 children were enrolled. After adjusting for mid-upper arm circumference (MUAC), age, gender and receipt of other vaccines, all-cause gastroenteritis severity scores were 2.21 units lower (p < 0.001) among Rotarix®-vaccinated (n = 2224) compared to Rotarix®-unvaccinated children (n = 935). The reduction in severity score was observed against every rotavirus genotype, although the magnitude was smaller among those infected with G12P[6] compared to the remaining genotypes (p = 0.011). Each one-year increment in age was associated with a decrease of 0.43 severity score (p < 0.001). Our findings provide additional evidence on the impact of Rotarix® in Malawi, lending further support to Malawi's Rotarix® programme.

Infant gut microbiota characteristics generally do not modify effects of lipid-based nutrient supplementation on growth or inflammation: secondary analysis of a randomized controlled trial in Malawi.

An unhealthy gut microbial community may act as a barrier to improvement in growth and health outcomes in response to nutritional interventions. The objective of this analysis was to determine whether the infant microbiota modified the effects of a randomized controlled trial of lipid-based nutrient supplements (LNS) in Malawi on growth and inflammation at 12 and 18 months, respectively. We characterized baseline microbiota composition of fecal samples at 6 months of age (n = 506, prior to infant supplementation, which extended to 18 months) using 16S rRNA gene sequencing of the V4 region. Features of the gut microbiota previously identified as being involved in fatty acid or micronutrient metabolism or in outcomes relating to growth and inflammation, especially in children, were investigated. Prior to correction for multiple hypothesis testing, the effects of LNS on growth appeared to be modified by Clostridium (p-for-interaction = 0.02), Ruminococcus (p-for-interaction = 0.007), and Firmicutes (p-for-interaction = 0.04) and effects on inflammation appeared to be modified by Faecalibacterium (p-for-interaction = 0.03) and Streptococcus (p-for-interaction = 0.004). However, after correction for multiple hypothesis testing these findings were not statistically significant, suggesting that the gut microbiota did not alter the effect of LNS on infant growth and inflammation in this cohort.