The Effects of HIV Infection on the Immune Response to Malaria Among Pregnant Women in Kumba, Southwest Cameroon: Protocol for a Cross-sectional Study.

BACKGROUND: Malaria and HIV, 2 of the world's deadliest diseases, share a lot of territory in sub-Saharan Africa. OBJECTIVE: This study seeks to investigate the effect of HIV on the immune response to malaria infection among pregnant women in Kumba in the southwest region (SWR) of Cameroon. The study aims to determine the prevalence of malaria infection, assess the occurrence of Plasmodium falciparum genetic diversity, and evaluate the antibody (immunoglobulin [Ig]G and IgM: apical membrane antigen-1 [AMA1], merozoite surface protein [MSP]1, MSP2, MSP3, and erythrocyte-binding antigen [EBA]175) and cytokine (interleukin [IL]-10, tumor necrosis factor alpha [TNF-α], and interferon gamma [IFNγ]) response to malaria infection among pregnant women with and without HIV in Kumba. METHODS: The study will be a hospital-based cross-sectional design that will run from March 2022 to February 2023. It will recruit pregnant women with and without HIV who are in their third trimester of pregnancy. The study will be carried out in 5 health institutions in Kumba: General Hospital Kumba, Presbyterian Hospital Kumba, District Hospital Kumba-town, Kossala Integrated Health Center Kumba, and Catholic Hospital Kumba. About 3 mL of the mother's venous blood, placental blood, and baby cord blood will be collected from each pregnant women at the point of delivery. Microscopy, rapid diagnostic tests (RDTs), and nested polymerase chain reaction (PCR) will be performed to identify the malaria parasite in all the samples, and nested PCR targeting the different genetic diversity markers for P. falciparum will also be performed. Furthermore, sequencing will be performed to study the nucleotide sequence of different alleles, and the genetic diversity of the alleles responsible for malaria infection among pregnant women will be assessed. A multiplex assay will be conducted to analyze the peripheral blood plasma and cord blood plasma for the cytokine and total antibody response to malaria infection among pregnant women with and without HIV. The questionnaire for data collection will be pretested at the Kumba District Hospital, and ethical clearance will be obtained from the University of Buea and the Regional Delegation of Public Health for the SWR. Data will be analyzed using SPSS Statistics and STATA. All P values <.05 will be considered statistically significant. BioEdit 7.0.0 software will be used to align the nucleotide sequences of different genes after sequencing. Phylogenetic tree searching will be conducted using the maximum-likelihood (ML) method in MEGA V6.0. RESULTS: The project started in March 2022 and will end in February 2023. Presently, three-fourth of the project funding has been disbursed to date. A total of 218 participants have been enrolled: 193 (88.5%) women without HIV and 25 (11.5%) women with HIV. Between February 2023 and March 2024, the following results will be ready for publication: maternal-neonatal malaria prevalence among pregnant women and babies in Kumba, the effect of HIV on (1) P. falciparum genetic diversity among pregnant women in Kumba, (2) the maternal and neonatal immune response to MSP1, MSP2, and EBA175 IgG antibody response to P. falciparum-caused malaria infection among pregnant women, and (3) the maternal and neonatal pro-inflammatory and anti-inflammatory cytokine response to malaria infection. CONCLUSIONS: HIV infection increases the prevalence of malaria infection among pregnant women and also influences the genetic diversity of P. falciparum, with MSP1 alleles being the most prevalent. HIV infection also reduces the antibody response to malaria infection, as well as altering the level of pro-inflammatory and anti-inflammatory responses to malaria infection. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/38213.

Allelic frequencies of mutants of the Plasmodium falciparum, quinoline and folate metabolizing genes in the west region of Cameroon.

The emergence and spread of Plasmodium falciparum (P.f) drug resistance is still a major concern in Sub-Saharan Africa and warrants that its evolution be monitored continuously. The present study aimed at determining the distribution of key P.f drug resistance-mediating alleles in circulating malaria parasites in the West region of Cameroon. A cross sectional hospital-based study was conducted in Dschang and Ngounso in the West region of Cameroon. The Pfcrt, Pfmdr1, and the Pfdhps genes were amplified through nested PCR in 208 malaria-infected samples of the 301 febrile outpatients enrolled. The presence or absence of mutations in the K76T, N86Y, A437G and A581G codons of these P.f. genes respectively were determined through restriction digestion analysis. The proportion of different alleles were estimated as percentages and compared between two study sites using the Chi square test. A p value <0.05 was considered significant. A high prevalence (75.6%) of the 437G allele was observed. It was significantly different between Dschang and Ngounso (62% vs. 89.2%, X2 = 19.6, P = 0.00005). Equally observed was a 19.2% (95%CI: 13.3-25.6) of the dhps-581G mutant allele. Furthermore, we observed the Pfcrt-76T, Pfmdr1-N86 mutations in 73.0% (67.5-79.7) and 87.2% (83.2-91.9), and 3.0% (0.0-9.6) and 12.8% was observed for the Pfcrt-K76T and Pfmdr1-N86Y respectively. When biallelic haplotypes were constructed from alleles of the three genes, same pattern was seen. Overall, 73% and 87% of circulating P. falciparum isolates carried wild type alleles at Pfmdr1-N86Y and Pfcrt-K76T. On the other hand, we found more parasites with mutant alleles at dhps (437G and 581G) loci which may reflect possible drug-related selection of this mutant in the parasite population. Continuous monitoring of these mutations is recommended to pre-empt a loss in sulphadoxine-pyrimethamine efficacy in malaria chemoprevention programs.

Assessing the Tsetse Fly Microbiome Composition and the Potential Association of Some Bacteria Taxa with Trypanosome Establishment.

The tsetse flies, biological vectors of African trypanosomes, harbour a variety of bacteria involved in their vector competence that may help in developing novel vector control tools. This study provides an inventory of tsetse bacterial communities in Cameroon and explores their possible associations with trypanosome establishment in Glossina palpalis palpalis. High throughput sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene, with subsequent metagenomic, multivariate, and association analyses, were used to investigate the levels and patterns of microbial diversity in four tsetse species. Overall, 31 bacterial genera and four phyla were identified. The primary symbiont Wigglesworthia dominated almost all the samples, with an overall relative abundance of 47.29%, and seemed to be replaced by Serratia or Burkholderia in some G. tachinoides flies. Globally, significant differences were observed in the microbiome diversity and composition among tsetse species and between teneral and non-teneral flies, or between flies displaying or not displaying mature trypanosome infections. In addition, differential abundance testing showed some OTUs, or some bacteria taxa, associated with trypanosome maturation in tsetse flies. These bacteria could be further investigated for an understanding of their mechanism of action and alternatively, transformed and used to block trypanosome development in tsetse flies.

Diagnostic Accuracy of CareStart™ Malaria HRP2 and SD Bioline Pf/PAN for Malaria in Febrile Outpatients in Varying Malaria Transmission Settings in Cameroon.

BACKGROUND: There was an increase in the number of malaria cases in Cameroon in 2018 that could reflect changes in provider practice, despite effective interventions. In this study, we assessed the diagnostic performance of two malaria rapid diagnostic tests (mRDTs) for diagnostic confirmation of suspected cases of malaria in public and private health facilities in two malaria transmission settings in Cameroon. METHODS: We evaluated the diagnostic performance of CareStart pf and SD Bioline Pf/PAN mRDT and compared these parameters by RDT type and transmission setting. Nested PCR and blood film microscopy were used as references. The chi square test was used for independent sample comparisons, while the McNemar's test was used to test for the dependence of categorical data in paired sample testing. A p < 0.05 was considered significant in all comparisons. The R (v.4.0.2) software was used for analyses. RESULTS: A total of 1126 participants consented for the study in the four sites. The diagnostic accuracy of the CareStart Pf mRDT was 0.93.6% (0.911-0.961) in Yaoundé, 0.930% (0.90-0.960) in Ngounso, 0.84% (0.794-0.891) in St Vincent Catholic Hospital Dschang and 0.407 (0.345-0.468) in Dschang district hospital. For SD Bioline Pf/PAN the accuracy was 0.759 (0.738-0.846) for St Vincent Catholic Hospital Dschang and 0.426 (0.372-0.496) for the Dschang district hospital. The accuracy was slightly lower in each case but not statistically different when PCR was considered as the reference. The likelihood ratios of the positive and negative tests were high in the high transmission settings of Yaoundé (10.99 (6.24-19.35)) and Ngounso (14.40 (7.89-26.28)) compared to the low transmission settings of Dschang (0.71 (0.37-1.37)) and St Vincent Catholic hospital (7.37 (4.32-12.59)). There was a high degree of agreement between the tests in Yaoundé (Cohen's Kappa: 0.85 ± 0.05 (0.7-0.95)) and Ngounso (Cohen's Kappa: 0.86 ± 0.05 (0.74, 0.97)) and moderate agreement in St Vincent hospital Dschang (k: 0.58 ± 0.06 (0.44-0.71)) and poor agreement in the District Hospital Dschang (Cohen's Kappa: -0.11 ± 0.05 (-0.21-0.01)). The diagnostic indicators of the SD Bioline Pf/PAN were slightly better than for CareStart Pf mRDT in St Vincent Catholic hospital Dschang, irrespective of the reference test. CONCLUSIONS: Publicly procured malaria rapid diagnostic tests in Cameroon have maintained high accuracy (91-94%) in the clinical diagnosis of malaria in high malaria transmission regions of Cameroon, although they failed to reach WHO standards. We observed an exception in the low transmission region of Dschang, West region, where the accuracy tended to be lower and variable between facilities located in this town. These results underscore the importance of the routine monitoring of the quality and performance of malaria RDTs in diverse settings in malaria endemic areas.

Community-based geographical distribution of Mycobacterium ulcerans VNTR-genotypes from the environment and humans in the Nyong valley, Cameroon.

BACKGROUND: Genotyping is a powerful tool for investigating outbreaks of infectious diseases and it can provide useful information such as identifying the source and route of transmission, and circulating strains involved in the outbreak. Genotyping techniques based on variable number of tandem repeats (VNTR) are instrumental in detecting heterogeneity in Mycobacterium ulcerans (MU) and also for discriminating MU from other mycobacteria species. Here, we describe and map the distribution of MU genotypes in Buruli ulcer (BU) endemic communities of the Nyong valley in Cameroon. We also tested the hypothesis of whether the suspected animal reservoirs of BU that share the human microhabitat are shedding contaminated fecal matters and saliva into their surrounding environments. METHODS: Environmental samples from suspected MU-risk factors and lesion swabs from human patients were sampled in BU-endemic communities and tested for the presence of MU by qPCR targeting three independent sequences (IS2404, IS2606, KR-B). Positive samples to MU were further genotyped by VNTR with confirmation by sequencing of four loci (MIRU1, Locus 6, ST1, Locus 19). RESULTS: MU was detected in environmental samples including water bodies (23%), biofilms (14%), detritus (10%), and in human patients (73%). MU genotypes D, W, and C were found both in environmental and human samples. The micro geo-distribution of MU genotypes from communities showed that genotype D is found both in environmental and human samples, while genotypes W and C are specific to environmental samples and human lesions, respectively. No obvious focal grouping of MU genotypes was observed at the community scale. An additional survey in the human microhabitat suggests that domestic and wild animals do not shed MU in their saliva and feces in sampled communities. CONCLUSIONS: VNTR typing uncovered different MU genotypes circulating in the endemic communities of the Akonolinga district. A MU environmental genotype was found in patients, yet the mechanism of contamination remains to be investigated; and recovering MU in culture from the environment remains key priority to enable a better understanding of the mode of transmission of BU. We also conclude that excretions from suspected animals are unlikely to be major sources of MU in the Nyong Valley in Cameroon.

Domestic animals infected with Mycobacterium ulcerans-Implications for transmission to humans.

BACKGROUND: The environmental pathogen, Mycobacterium ulcerans (MU) can infect both humans and animals and cause Buruli ulcer (BU) disease. However, its mode(s) of transmission from the colonized environment to human/animal hosts remain unclear. In Australia, MU can infect both wildlife and domestic mammals. Till date, BU-like lesions have only been reported in wildlife in Africa. This warrants a thorough assessment of possible MU in domestic animals in Africa. Here, we screened roaming domesticated animals that share the human microhabitat in two different BU endemic sites, Sedje-Denou in Benin and Akonolinga in Cameroon, for MU lesions. METHODOLOGY/PRINCIPAL FINDINGS: We screened roaming mammals and birds across 3 endemic villages of Sedje-Denou in Southern Benin and 6 endemic villages of Akonolinga in Cameroon. After approval from relevant authorities, specimens (wound swabs and tissue fragments) were collected from animals with open or active lesion and systematically screened to detect the presence of MU though the diagnostic DNA targets IS2404, IS2606 and KR-B. Out of 397 animals surveyed in Akonolinga, 44 (11.08%) carried skin lesions and all were negative for MU DNA. For Sedje-Denou, only 25 (6.93%) out of 361 animals surveyed carried external skin lesions of which 2 (8%) were positive for MU DNA targets. These MU infected lesions were found in two different villages on a goat (abdominal part) and on a dog (nape area of the neck). Source-tracking of MU isolates within infected animal lesions was performed using VNTR genotyping and further confirmed with sequencing. One MU VNTR genotype (Z) was successfully typed from the goat lesion. The evolutionary history inferred from sequenced data revealed a clustering of animal MU isolates within isolates from human lesions. CONCLUSION/SIGNIFICANCE: This study describes the first report of two MU infected lesions in domestic animals in Africa. Their DNA sequence analyses show close relationship to isolates from human cases. It suggests that MU infection should be suspected in domestic hosts and these could play a role in transmission. The findings further support the hypothesis that MU is a ubiquitous environmental pathogen found in endemic areas, and probably involved in a multiple transmission pathway.

Impact of placental Plasmodium falciparum malaria infection on the Cameroonian maternal and neonate's plasma levels of some cytokines known to regulate T cells differentiation and function.

BACKGROUND: The impact of placental malaria (PM) infection on the expression profile of some cytokines known to regulate T cell differentiation and function and their influence on birth weight remain unclear. Moreover, there are no reports showing the relationship between PM and IL-27 or IL-28A. This study therefore sought to investigate whether placental P. falciparum infection alters the expression profile of the cytokines IL-28A, IL-27, IL-17E and IL-6 in mothers and their new born. METHODS: In a cross-sectional study conducted between 2013 and 2015 in Yaoundé, Cameroon, peripheral, placental and cord blood samples were collected from 108 women at delivery. Parasitaemia was determined microscopically and haemoglobin levels determined using a Coulter counter. Plasma levels of cytokines (IL-28A, IL-27, IL-17E and IL-6) were measured by Luminex magnetic screening assay. RESULTS: Malaria parasite density in placenta impression smear associated negatively with maternal haemoglobin level (P < 0.0001) and baby birth weight (P = 0.016). While IL-17E, IL-27 and IL-28A levels were significantly higher in placental and cord plasma than in peripheral (P < 0.0001, < 0.001 and P = 0.026, respectively), an opposite relationship was observed with IL-6 (P = 0.0018). Multivariate analysis confirmed results of univariate analysis where the presence of malaria parasites or pigments in placenta tissue impression smears correlated with decrease levels of maternal IL-17E, IL-27 and IL-28A and neonate levels of IL-28A and IL-17E (0.0001 ≤ P ≤ 0.02). Placental and peripheral parasitaemias also correlated positively with peripheral plasma levels of IL-6 (rs = 0.18, P = 0.05 and rs = 0.17, P = 0.07, respectively). In addition, high maternal haemoglobin level associated with increasing levels of IL-17E, IL-27 and IL-28A in peripheral plasma (0.002 ≤ P ≤ 0.018) and high placental and cord plasma levels of these cytokines associated with increasing birth weight (0.0001 ≤ P ≤ 0.0027). CONCLUSIONS: Placental malaria downregulates maternal plasma levels of IL-17E, IL-27 and IL-28A and neonates' plasma levels of IL-17E and IL-28A cytokines, which could help for parasite clearance and increase child birth weight. The study is expected to provide leads that should help identify potential biomarkers for improved birth weight and therapeutic interventions.

Impact of placental Plasmodium falciparum malaria on the profile of some oxidative stress biomarkers in women living in Yaoundé, Cameroon.

BACKGROUND: Impact of the pathophysiology of Plasmodium falciparum placental malaria (PM) on the profile of some oxidative stress biomarkers and their relationship with poor pregnancy outcomes in women remain unknown. METHODS: Between 2013 and 2014, peripheral blood and placenta tissue from 120 Cameroonian women at delivery were assessed for maternal haemoglobin and, parasitaemia respectively. Parasite accumulation in the placenta was investigated histologically. The levels of oxidative stress biomarkers Malondialdehyde (MDA), Nitric Oxide (NO), Superoxide dismutase (SOD), Catalase (CAT) and Gluthatione (GSH) in the supernatant of teased placenta tissues were determined by Colorimetric enzymatic assays. RESULTS: Parasitaemia was inversely related to haemoglobin levels and birth weight (P <0.001 and 0.012, respectively). The level of lipid peroxide product (MDA) was significantly higher in the malaria infected (P = 0.0047) and anaemic (P = 0.024) women compared to their non-infected and non-anaemic counterparts, respectively. A similar trend was observed with SOD levels, though not significant. The levels of MDA also correlated positively with parasitaemia (P = 0.0024) but negatively with haemoglobin levels (P = 0.002). There was no association between parasitaemia, haemoglobin level and the other oxidative stress biomarkers. From histological studies, levels of MDA associated positively and significantly with placenta malaria infection and the presence of malaria pigments. The levels of SOD, NO and CAT increased with decreasing leukocyte accumulation in the intervillous space. Baby birth weight increased significantly with SOD and CAT levels, but decreased with levels of GSH. CONCLUSIONS: Placental P. falciparum infection may cause oxidative stress of the placenta tissue with MDA as a potential biomarker of PM, which alongside GSH could lead to poor pregnancy outcomes (anaemia and low birth weight). This finding contributes to the understanding of the pathophysiology of P. falciparum placental malaria in women.

Co-infections of malaria and geohelminthiasis in two rural communities of Nkassomo and Vian in the Mfou health district, Cameroon.

BACKGROUND: Human co-infection with malaria and helmimths is ubiquitous throughout Africa. Nevertheless, its public health significance on malaria severity remains poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: To contribute to a better understanding of epidemiology and control of this co-infection in Cameroon, a cross-sectional study was carried out to assess the prevalence of concomitant intestinal geohelminthiasis and malaria, and to evaluate its association with malaria and anaemia in Nkassomo and Vian. Finger prick blood specimens from a total of 263 participants aged 1-95 years were collected for malaria microscopy, assessment of haemoglobin levels, and molecular identification of Plasmodium species by PCR. Fresh stool specimens were also collected for the identification and quantification of geohelminths by the Kato-Katz method. The prevalence of malaria, geohelminths, and co-infections were 77.2%, 28.6%, and 22.1%, respectively. Plasmodium falciparum was the only malaria parasite species identified with mean parasite density of 111 (40; 18,800) parasites/µl of blood. The geohelminths found were Ascaris lumbricoides (21.6%) and Trichuris trichiura (10.8%), with mean parasite densities of 243 (24; 3,552) and 36 (24; 96) eggs/gram of faeces, respectively. Co-infections of A. lumbricoides and P. falciparum were the most frequent and correlated positively. While no significant difference was observed on the prevalences of single and co-infections between the two localities, there was a significant difference in the density of A. lumbricoides infection between the two localities. The overall prevalence of anaemia was 42%, with individuals co-infected with T. trichiura and P. falciparum (60%) being the most at risk. While the prevalence of malaria and anaemia were inversely related to age, children aged 5-14 years were more susceptible to geohelminthiasis and their co-infections with malaria. CONCLUSION/SIGNIFICANCE: Co-existence of geohelminths and malaria parasites in Nkassomo and Vian enhances the occurrence of co-infections, and consequently, increases the risk for anaemia.