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Wilson Lewis Mandala1– 3 1Academy of Medical Sciences, Malawi University of Science and Technology (MUST), Thyolo, Malawi; 2Malawi Liverpool Wellcome Trust Clinical Research Programme, Blantyre, Malawi; 3Basic Sciences Department, College of Medicine, University of Malawi, Blantyre, MalawiCorrespondence: Wilson Lewis Mandala Tel +265 8-8885-8454Email wmandala2002@gmail.comBackground: Malaria in individuals who have never had an infection before is usually characterized by an inflammatory response that is linked to the expression of specific activation markers on cells of the innate immune system.Methods: This study investigated absolute white blood cell (WBC) and neutrophil counts and expression of several adhesion markers on neutrophils from HIV-uninfected children who were suffering from cerebral malaria (n=35), severe malarial anemia (SMA, n=39), and uncomplicated malaria (n=49) and healthy aparasitemic children (n=33) in Blantyre, Malawi.Results: All clinical malaria groups had higher WBC and neutrophil counts compared to healthy controls, with the acute SMA group having significantly (p< 0.0001) higher WBC counts than the controls. These elevated counts normalized during recovery. Surprisingly, in all clinical malaria groups, the surface expression of CD11b, CD11c, and CD18 on neutrophils was lower than in healthy controls, again normalizing during convalescence.Conclusion: In areas where Plasmodium falciparum malaria is hyperendemic, such as where this study was conducted, neutrophils have reduced expression of adhesion molecules and activation markers during acute stages of the infection, regardless of the clinical type of malaria. This reduced expression could be due to an individual’s past exposure to P. falciparum or other parasite-related factors that manifest during active malaria that still need to be investigated.Keywords: white blood cells, neutrophils, malaria, integrins, receptors

Wilson Mandala,1– 3 Alinane Munyenyembe,2 Innocent Sulani,2 Monica Soko,2 Jane Mallewa,4 Jasmin Hiestand4 1Basic Sciences Department, Kamuzu University of Health Sciences, Blantyre, Malawi; 2Malawi Liverpool Wellcome Trust Clinical Research Programme, Blantyre, Malawi; 3Academy of Medical Sciences, Malawi University of Science and Technology, Thyolo, Malawi; 4Medicine Department, Kamuzu University of Health Sciences, Blantyre, MalawiCorrespondence: Wilson Mandala, Tel +265 888858454, Email wmandala2002@gmail.comBackground: Plasmodium falciparum malaria has been linked with significant perturbations of the peripheral cell-mediated immune system during acute phase. Some of these changes include lower than normal platelet counts. Although the exact mechanisms that drive thrombocytopenia in P. falciparum malaria are not fully known, a number of hypotheses have been proposed. We conducted two sets of studies with one aimed at determining platelet counts in Malawian children, and the other in adults during acute P. falciparum malaria and a month post treatment.Materials and Methods: We recruited a total of 113 HIV-uninfected children with acute malaria [n=54 with uncomplicated malaria (UCM), n=30 with severe malarial anemia (SMA), n=29 presenting with cerebral malaria (CM)]. We also recruited 42 HIV-uninfected healthy controls. Out of the 113 participants with malaria, 73 (65%) [n=34 (63%) UCM, n=21 (70%) SMA and n=18 (62%) CM] were successfully followed-up one month after treatment. A 5mL peripheral blood sample was collected for platelet count using HMX Haematological Analyzer analysis both at baseline (acute malaria) and at follow-up a month later. Platelet counts were also determined in blood samples of 106 HIV-uninfected adults, 47 of whom presented with UCM and 29 with severe malaria (SM) and these counts were compared to those of 30 healthy controls. Of the malaria cases, platelet counts for 44 UCM and 21 SM were determined again during follow-up a month after treatment.Results: In both children and adults, platelet counts were significantly lower during acute disease compared to the levels in the healthy controls with the lowest levels observed in CM (children) or SM (adults). These lower than normal levels increased close to normal levels a month post treatment.Conclusion: P. falciparum malaria in Malawian children and adults was characterized by profound thrombocytopenia which recovered during convalescence.Keywords: acute malaria, platelet counts, children and adults, convalescence

Background: Listeria monocytogenes is one of the food borne pathogens that cause an illness known as Listeriosis upon ingestion of contaminated food. Outbreaks of Listeriosis have been reported in Canada, USA, Europe, South Africa, Ghana and other countries but there is limited data on outbreaks due to Listeria monocytogenes and its isolation from contaminated foods in Malawi. This prompted our interest to determine the presence of Listeria monocytogenes in cow milk from Goliati area, Thyolo district in Malawi. Objectives: To determine the presence and concentration of Listeria monocytogenes in cow milk from daily farmers around Goliati area in Thyolo district in Malawi. Methodology: Nine raw milk samples from three bulk centers and two processed milk samples were cultured on both Nutrient Agar and Brain Heart Infusion agar as primary cultures. A pure culture of Listeria monocytogenes was obtained by sub culturing the primary culture on Brain Heart Infusion agar media. Colonies from the pure culture were further identified using Gram staining, Catalase test, Motility test and Beta hemolysis test on blood agar to phenotypically identify Listeria monocytogenes. Results: There was a 100% growth rate of colonies suggestive of Listeria monocytogenes on all the nine samples. These colonies were observed to contain Gram positive, purple, short, rod shaped bacteria which was motile and catalase positive and it caused complete beta hemolysis on blood agar. Conclusion: In this study, isolates of Listeria monocytogenes were phenotypically identified in cow milk that is produce from Goliath Area in Thyolo district. This is a major public health concern for milk farmers and consumers in the area and beyond. The findings from this work calls for improved hygienic practices in the handling of milk and milk products to reduce contamination.

Although substantial progress has been made in increasing access to care during childbirth, reductions in maternal and neonatal mortality have been slower. Poor-quality care may be to blame. In this study, we measure the quality of labour and delivery services in Kenya and Malawi using data from observations of deliveries and explore factors associated with levels of competent and respectful care.We used data from nationally representative health facility assessment surveys. A total of 1100 deliveries in 392 facilities across Kenya and Malawi were observed and quality was assessed using two indices: the quality of the process of intrapartum and immediate postpartum care (QoPIIPC) index and a previously validated index of respectful maternity care. Data from standardised observations of care were analysed using descriptive statistics and multivariable random-intercept regression models to examine factors associated with variation in quality of care. We also quantified the variance in quality explained by each domain of covariates (patient-, provider- and facility-level and subnational divisions).Only 61-66% of basic elements of competent and respectful care were performed. In adjusted models, better-staffed facilities, private hospitals and morning deliveries were associated with higher levels of competent and respectful care. In Malawi, younger, primipara and HIV-positive women received higher-quality care. Quality also differed substantially across regions in Kenya, with a 25 percentage-point gap between Nairobi and the Coast region. Quality was also higher in higher-volume facilities and those with caesarean section capacity. Most of the explained variance in quality was due to regions in Kenya and to facility, and patient-level characteristics in Malawi.Our findings suggest considerable scope for improvement in quality. Increasing staffing and shifting births to higher-volume facilities - along with promotion of respectful care in these facilities - should be considered in sub-Saharan Africa to improve outcomes for mothers and newborns.Bien que des progrès substantiels aient été accomplis dans l'amélioration de l'accès aux soins pendant l'accouchement, les réductions de la mortalité maternelle et néonatale ont été plus lentes. Des soins de mauvaise qualité peuvent être à blâmer. Dans cette étude, nous mesurons la qualité de la main-d'œuvre et des services d'accouchement au Kenya et au Malawi en utilisant les données des observations des accouchements et explorons les facteurs associés aux niveaux de la compétence et du respect dans les soins. MÉTHODES: Nous avons utilisé les données d'enquêtes d'évaluation des établissements de santé représentatives au niveau national. 1100 accouchements dans 392 établissements au Kenya et au Malawi ont été observés et la qualité a été évaluée à l'aide de deux indices: l'indice de qualité du processus de soins intra-partum et postpartum immédiat (QoPIIPC) et un indice précédemment validé de soins maternels respectueux. Les données des observations normalisées des soins ont été analysées à l'aide de statistiques descriptives et de modèles de régression à interceptions aléatoires multivariables pour examiner les facteurs associés à la variation de la qualité des soins. Nous avons également quantifié la variance de la qualité expliquée par chaque domaine de covariables (divisions au niveau des patients, des prestataires et des établissements, et infranationales). RÉSULTATS: Seuls 61% à 66% des éléments de base de soins compétents et respectueux ont été réalisés. Dans les modèles ajustés, des établissements mieux dotés en personnel, des hôpitaux privés et des accouchements le matin étaient associés à des niveaux plus élevés de soins compétents et respectueux. Au Malawi, les femmes plus jeunes, primipares et VIH positives ont reçu des soins de meilleure qualité. La qualité différait également considérablement d'une région à l'autre au Kenya, avec un écart de 25 points de pourcentage entre Nairobi et la région côtière. La qualité était également plus élevée dans les établissements avec un volume plus élevé et ceux ayant une capacité de césarienne. La majeure partie des raisons de la variance dans la qualité était liée aux régions du Kenya et à l'établissement et aux caractéristiques des patients au Malawi.Nos résultats suggèrent une marge considérable pour l'amélioration de la qualité. L'augmentation du personnel et le déplacement des naissances vers des établissements de plus grand volume - ainsi que la promotion de soins respectueux dans ces établissements - devraient être envisagés en Afrique subsaharienne pour améliorer les résultats pour les mères et les nouveau-nés.

Since its emergence in 2019 SARS-CoV-2 has proven to have a higher level of morbidity and mortality compared to the other prevailing coronaviruses. Although initially most African countries were spared from the devastating effect of SARS-CoV-2, at present almost every country has been affected. Although no association has been established between being HIV-1-infected and being more vulnerable to contracting COVID-19, HIV-1-infected individuals have a greater risk of developing severe COVID-19 and of COVID-19 related mortality. The rapid development of the various types of COVID-19 vaccines has gone a long way in mitigating the devastating effects of the virus and has controlled its spread. However, global vaccine deployment has been uneven particularly in Africa. The emergence of SARS-CoV-2 variants, such as Beta and Delta, which seem to show some subtle resistance to the existing vaccines, suggests COVID-19 will still be a high-risk infection for years. In this review we report on the current impact of COVID-19 on HIV-1-infected individuals from an immunological perspective and attempt to make a case for prioritising COVID-19 vaccination for those living with HIV-1 in Sub-Saharan Africa (SSA) countries like Malawi as one way of minimising the impact of COVID-19 in these countries.

Corn flour-based porridge like dough, ugali, is the staple food of low-income population in sub-Saharan Africa. Lack of vitamin A, carotenoids, and dietary fibers brings about serious health issues to this population. In this study, vegetables including bok choy, broccoli, cabbage, carrot, Chinese onion stalk (C_onion), mushroom, are added during the cooking of ugali, as nutritional supplements. The freeze-dried powder of each vegetable was used for its long storage, stable nutrients, and similar particle size. Sub-Saharan African assessors were trained and sensory evaluated the six different vegetable fortified ugali with the plain, unfortified as the control on five attributes. The plain ugali was indistinguishable with the C_onion stalk fortified in color, with the carrot and C_onion stalk fortified in odor, with all vegetables (except broccoli and mushroom) fortified ugali in taste, with carrot and C_onion stalk fortified in granularity, and with cabbage, carrot, C_onion stalk fortified in viscosity. Preference ranking analysis showed that the C_onion stalk fortified ugali is even more favorably preferred than the plain, unfortified ugali, probably due to the umami components in C_onion that serve as the taste enhancer. This study indicates that Chinese onion stalk is a potential vegetable supplement to population in the sub-Saharan Africa.

Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella bacteraemia is poorly understood, in part due to the low sensitivity of stool culture and the lack of validated molecular diagnostic tests for the detection of Salmonella in the stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimised an in-house monoplex real-time polymerase chain reaction (PCR) for the detection of Salmonella ttr and InvA genes in stool by including a selenite broth pre-culture step for Salmonella before DNA extraction and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same ttr and InvA genes and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. Results: Ttr and InvA primers were both able to detect all the different Salmonella serovars tested and had superior limits of detection when DNA was extracted after selenite pre-culture. Ttr sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99%, respectively. Culture showed the highest PPV (99.73%), and monoplex-ttr had the highest NPV (99.67%). Conclusion: Test methods demonstrated high concordance, although stool culture and monoplexed ttr primers had superior specificity and sensitivity, respectively. The use of selenite pre-enrichment step increased Salmonella detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic Salmonella exposure events.

Highlights • Although substantial gains have been made in reducing malaria burden through various initiatives, it is still a major public health challenge. • Unlike other infectious diseases, there had not been any malaria vaccine until RTS,S/AS01 was approved for pilot trial in three countries in 2015. • However, RTS,S/AS01 still has low efficacy in some age groups, poor immunogenicity and requires three boosters to attain a reasonable efficacy. • The search for a more effective malaria vaccine therefore still continues, hence the need for a better understanding of the host immunity. • This review therefore compiles what is known about the basic biology of P. falciparum, the host immune response and progress on malaria vaccines., Plasmodium falciparum malaria still remains a major global public health challenge with over 220 million new cases and well over 400,000 deaths annually. Most of the deaths occur in sub-Saharan Africa which bears 90 % of the malaria cases. Such high P. falciparum malaria-related morbidity and mortality rates pose a huge burden on the health and economic wellbeing of the countries affected. Lately, substantial gains have been made in reducing malaria morbidity and mortality through intense malaria control initiatives such as use of effective antimalarials, intensive distribution and use of insecticide-treated nets (ITNs), and implementation of massive indoor residual spraying (IRS) campaigns. However, these gains are being threatened by widespread resistance of the parasite to antimalarials, and the vector to insecticides. Over the years the use of vaccines has proven to be the most reliable, cost-effective and efficient method for controlling the burden and spread of many infectious diseases, especially in resource poor settings with limited public health infrastructure. Nonetheless, this had not been the case with malaria until the most promising malaria vaccine candidate, RTS,S/AS01, was approved for pilot implementation programme in three African countries in 2015. This was regarded as the most important breakthrough in the fight against malaria. However, RTS,S/AS01 has been found to have some limitations, the main ones being low efficacy in certain age groups, poor immunogenicity and need for almost three boosters to attain a reasonable efficacy. Thus, the search for a more robust and effective malaria vaccine still continues and a better understanding of naturally acquired immune responses to the various stages, including the transmissible stages of the parasite, could be crucial in rational vaccine design. This review therefore compiles what is currently known about the basic biology of P. falciparum and the natural malaria immune response against malaria and progress made towards vaccine development.

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.

AlthoughMycobacterium tuberculosis (Mtb)strains exhibit genomic homology of >99%, there is considerable variation in the phenotype. The underlying mechanisms of phenotypic heterogeneity inMtbare not well understood but epigenetic variation is thought to contribute. At present the methylome ofMtbhas not been completely characterized. We completed methylomes of 18Mycobacterium tuberculosis(Mtb) clinical isolates from Malawi representing the largest number ofMtbgenomes to be completed in a single study using Single Molecule Real Time (SMRT) sequencing to date. We replicate and confirm four methylation disrupting mutations in lineages ofMtb. For the first time we report complete loss of methylation courtesy of C758T (S253L) mutation in theMamBgene of Indo-oceanic lineage ofMtb. We also conducted a genomic and methylome comparison of the Malawian samples against a global sample. We confirm that methylation inMtbis lineage specific although some unresolved issues still remain.

Presently, it is difficult to accurately diagnose sepsis, a common cause of childhood death in sub-Saharan Africa, in malaria-endemic areas, given the clinical and pathophysiological overlap between malarial and non-malarial sepsis. Host biomarkers can distinguish sepsis from uncomplicated fever, but are often abnormal in malaria in the absence of sepsis. To identify biomarkers that predict sepsis in a malaria-endemic setting, we retrospectively analyzed data and sera from a case-control study of febrile Malawian children (aged 6-60 months) with and without malaria who presented to a community health center in Blantyre (January-August 2016). We characterized biomarkers for 29 children with uncomplicated malaria without sepsis, 25 without malaria or sepsis, 17 with malaria and sepsis, and 16 without malaria but with sepsis. Sepsis was defined using systemic inflammatory response criteria; biomarkers (interleukin-6 [IL-6], tumor necrosis factor receptor-1, interleukin-1 β [IL-1β], interleukin-10 [IL-10], von Willebrand factor antigen-2, intercellular adhesion molecule-1, and angiopoietin-2 [Ang-2]) were measured with multiplex magnetic bead assays. IL-6, IL-1β, and IL-10 were elevated, and Ang-2 was decreased in children with malaria compared with non-malarial fever. Children with non-malarial sepsis had greatly increased IL-1β compared with the other subgroups. IL-1β best predicted sepsis, with an area under the receiver operating characteristic (AUROC) of 0.71 (95% CI: 0.57-0.85); a combined biomarker-clinical characteristics model improved prediction (AUROC of 0.77, 95% CI: 0.67-0.85). We identified a distinct biomarker profile for non-malarial sepsis and developed a sepsis prediction model. Additional clinical and biological data are necessary to further explore sepsis pathophysiology in malaria-endemic regions.

The study was funded by the Helse Nord Tuberculosis Initiative (HNTI). 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 mu g/mL ± 0.94, (Mean ± SEM) concentration of DNA and a yield of 0.2975 mu g ± 0.04723, (Mean ± SEM). Comparatively, the CTAB method isolated 1.88 mu g/mL ± 0.38 DNA and a yield of 0.09 mu g ± 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. Publisher PDF