Rationalizing artemisinin-based combination therapies use for treatment of uncomplicated malaria: A situation analysis in health facilities and private pharmacies of Douala 5e-Cameroon.

Artemisinin-based combination therapies (ACTs) represent one of the mainstays of malaria control. Despite evidence of the risk of ACTs resistant infections in resource-limited countries, studies on the rational use of ACTs to inform interventions and prevent their emergence and/or spread are limited. The aim of this study was designed to analyze practices toward ACTs use for treating the treatment of uncomplicated malaria (UM) in an urban community. Between November 2015 and April 2016, a cross-sectional and prospective study was conducted in the 6 health facilities and all pharmacies in the Douala 5e subdivision, Cameroon. Anonymous interviews including both open- and closed-ended questions were conducted with selected participants among drug prescribers, patients attending the health facilities, and customers visiting the pharmacies. Data analysis was performed using StataSE11 software (version 11 SE). A total of 41 prescribers were included in the study. All were aware of national treatment guidelines, but 37.7% reported not waiting for test results before prescribing an antimalarial drug, and the main reason being stock-outs at health facilities. Likewise, artemether+lumefantrine/AL (81%) and dihydroartemisinin+piperaquine (63.5%) were the most commonly used first- and second-line drugs respectively. Biological tests were requested in 99.2% (128/129) of patients in health facilities, 60.0% (74) were performed and 6.2% were rationally managed. Overall 266 (35%) of 760 customers purchased antimalarial drugs, of these, 261 (98.1%) agreed to participate and of these, 69.4% purchased antimalarial drugs without a prescription. ACTs accounted for 90.0% of antimalarials purchased from pharmacies, of which AL was the most commonly prescribed antimalarial drug (67.1%), and only 19.5% of patients were appropriately dispensed. The current data suggest a gap between the knowledge and practices of prescribers as well as patients and customers misconceptions regarding the use of ACTs in Douala 5e subdivision. Despite government efforts to increase public awareness regarding the use of ACTs as first-line treatment for UM, our findings point out a critical need for the development, implementation and scaling-up of control strategies and continuing health education for better use of ACTs (prescription and dispensing) in Cameroon.

Deletions of Histidine-Rich Protein 2/3 Genes in Natural Plasmodium falciparum Populations from Cameroon and India: Role of Asymptomatic and Submicroscopic Infections.

The bulk of malaria rapid diagnostic tests (RDTs) target histidine-rich protein 2 of Plasmodium falciparum, the deadliest malaria species. The WHO considers pfhrp2/3 deletions as one of the main threats to successful malaria control and/or elimination; as such, parasites that lack part or all of the pfhrp2 gene are missed by pfHRP2-targeting RDTs. Such deletions have been reported in several African and Asian countries, but little is known in Cameroon and India. Blood samples were collected from individuals living in four areas of Cameroon (Douala, Maroua, Mayo-Oulo, Pette) and India (Mewat, Raipur, Ranchi, Rourkela). Deletions in pfhrp2/3 genes were confirmed if samples 1) had ≥100 parasites/µL by quantitative polymerase chain reaction (PCR), 2) PCR negative for pfhrp2/3, and 3) PCR positive for at least two single-copy genes. The overall proportion of pfhrp2 and pfhrp3 deletions in Cameroon was 13.5% and 3.1%. In India, the overall proportion was 8% for pfhrp2 and 4% for pfhrp3. The overall proportions of samples with both gene deletions (pfhrp2-/3-) were 3.1% in Cameroon and 1.3% in India. In Cameroon, pfhrp2-/3+ and pfhrp2-/3- deletions were common in Maroua (P = 0.02), in asymptomatic parasitemia (P = 0.006) and submicroscopic parasitemia (P <0.0001). In both countries, pfhrp2/3 deletions, including pfhrp2-/3- deletions, were mainly seen in monoclonal infections. This study outlines that double deletions that result in false negative RDTs are uncommon in our settings, and highlights the importance of active molecular surveillance for pfhrp2/3 deletions in Cameroon and India.

Evolution and spread of Plasmodium falciparum mutations associated with resistance to sulfadoxine-pyrimethamine in central Africa: a cross-sectional study.

BACKGROUND: Efficacy of sulfadoxine-pyrimethamine, the malaria chemoprophylaxis used in pregnant women, and in children when combined with amodiaquine, is threatened by the accumulation of mutations in the Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes. Data on the prevalence of resistant alleles in central Africa and the new pfdhps I431V mutation, particularly associated with other mutations to form the pfdhps vagKgs allele, are scarce. We explored the frequency and geographical distribution of pfdhps and pfdhfr mutations in central Africa in 2014-18, and assessed the evolutionary origin of the vagKgs allele. METHODS: Samples were collected at 18 health-care centres in seven countries (Angola, Cameroon, Central African Republic, Democratic Republic of the Congo, Gabon, Nigeria, and Republic of the Congo) from patients who showed possible symptoms of malaria between March 1, 2014, and Oct 31, 2018. Samples that were positive for P falciparum were transported to a laboratory in Toulouse, France, and genotyped. The frequency of pfdhfr and pfdhps mutations was studied in 1749 samples. Microsatellites in pfdhps flanking regions and whole-genome analysis compared with parasite genomes from the data-sharing network MalariaGEN were performed on samples carrying the vagKgs allele. FINDINGS: Mapping of the prevalence of single nucleotide polymorphisms and corresponding alleles of pfdhfr and pfdhps showed a substantial spread of alleles associated with sulfadoxine-pyrimethamine resistance in central Africa during the 2014-18 period, especially an increase going west to east in pfdhps alleles carrying the K540E and A581G mutations. A high prevalence of the pfdhps I431V mutation was observed in Cameroon (exceeding 50% in the northern region) and Nigeria. Genomic analysis showed a recent African emergence and a clonal expansion of the most frequent pfdhps vagKgs allele. INTERPRETATION: Reduced sulfadoxine-pyrimethamine efficacy due to increased resistance is a worrying situation, especially because the malaria transmission level is high in central Africa. Although the resistance phenotype remains to be confirmed, the emergence and spread of the vagKgs allele in west and central Africa could challenge the use of sulfadoxine-pyrimethamine. FUNDING: Toulouse Institute for Infectious and Inflammatory Diseases.

High Prevalence of Polyclonal Plasmodium falciparum Infections and Association with Poor IgG Antibody Responses in a Hyper-Endemic Area in Cameroon.

Malaria remains a major public health problem worldwide, with eradication efforts thwarted by drug and insecticide resistance and the lack of a broadly effective malaria vaccine. In continuously exposed communities, polyclonal infections are thought to reduce the risk of severe disease and promote the establishment of asymptomatic infections. We sought to investigate the relationship between the complexity of P. falciparum infection and underlying host adaptive immune responses in an area with a high prevalence of asymptomatic parasitaemia in Cameroon. A cross-sectional study of 353 individuals aged 2 to 86 years (median age = 16 years) was conducted in five villages in the Centre Region of Cameroon. Plasmodium falciparum infection was detected by multiplex nested PCR in 316 samples, of which 278 were successfully genotyped. Of these, 60.1% (167/278) were polyclonal infections, the majority (80.2%) of which were from asymptomatic carriers. Host-parasite factors associated with polyclonal infection in the study population included peripheral blood parasite density, participant age and village of residence. The number of parasite clones per infected sample increased significantly with parasite density (r = 0.3912, p < 0.0001) but decreased with participant age (r = -0.4860, p < 0.0001). Parasitaemia and the number of clones per sample correlated negatively with total plasma levels of IgG antibodies to three highly reactive P. falciparum antigens (MSP-1p19, MSP-3 and EBA175) and two soluble antigen extracts (merozoite and mixed stage antigens). Surprisingly, we observed no association between the frequency of polyclonal infection and susceptibility to clinical disease as assessed by the recent occurrence of malarial symptoms or duration since the previous fever episode. Overall, the data indicate that in areas with the high perennial transmission of P. falciparum, parasite polyclonality is dependent on underlying host antibody responses, with the majority of polyclonal infections occurring in persons with low levels of protective anti-plasmodial antibodies.

High risk of virologic failure among HIV-infected children and adolescents routinely followed-up in Littoral region of Cameroon.

Virological response to antiretroviral therapy (ART) remains a challenge for HIV-infected children and adolescents due to non-optimization of pediatric ART for resource-limited settings. In this study, we aimed to investigate factors associated with virologic failure (VF) in HIV-infected-children and adolescents on ART in Cameroon. A prospective patient-based cohort study was conducted among HIV-infected children (0-9 years) and adolescents (10-19 years) followed-up between November 2018 and October 2019 in 38 healthcare centers located in the Littoral region of Cameroon. The 1st viral load (VL) was assessed after 6 months of ART initiation and the 2nd VL between 3 and 6 six months later in patients with VL ≥1000 copies/ml in accordance with the national algorithm using Abbott Real-Time HIV-1 Viral Load Assay. Multivariate analyses were performed to identify the determinants of higher risk of VF. Of 1,029 HIV-infected children and adolescents (393 children and 636 adolescents), 801 (77.8%) cumulatively presented with VL <1000 copies/mL within 12 months on ART. Adolescents were more likely to have VF than children (24.5% vs 18.3%, OR: 1.39; 95%CI: 1.00-1.93; p = 0.047). Patients followed-up in decentralized care units were significantly more likely to have VF compared to those attending the accredited treatment centers (26.1% vs 16.6%, OR: 1.88, 95%CI: 1.37-2.58; p<0.001). Our findings show a high rate of VL suppression (VLS, 77.8%) among HIV-infected children and adolescents, albeit lower than the established target of 90%. Being adolescent and patients followed in the decentralized care units are high risk factors for VF, thereby necessitating routine therapeutic education of patients and guardians in resource limited countries to improve VLS.

Effectiveness of Intermittent Preventive Treatment with Sulfadoxine-Pyrimethamine in Pregnancy: Low Coverage and High Prevalence of Plasmodium falciparum dhfr-dhps Quintuple Mutants as Major Challenges in Douala, an Urban Setting in Cameroon.

Intermittent preventive treatment in pregnancy with sulfadoxine and pyrimethamine (IPTp-SP) is a key component in the malaria control strategy implemented in Africa. The aim of this study was to determine IPTp-SP adherence and coverage, and the impact on maternal infection and birth outcomes in the context of widespread SP resistance in the city of Douala, Cameroon. Clinical and demographic information were documented among 888 pregnant women attending 3 health facilities, from the antenatal care visit to delivery. Positive samples were genotyped for P. falciparum gene (dhfr, dhps, and k13) mutations. The overall IPTp-SP coverage (≥three doses) was 17.5%, and 5.1% received no dose. P. falciparum prevalence was 16%, with a predominance of submicroscopic infections (89.3%). Malaria infection was significantly associated with locality and history of malaria, and it was reduced among women using indoor residual spraying. Optimal doses of IPTp-SP were significantly associated with reduced infection among newborns and women (secundiparous and multiparous), but there was no impact of IPTp-SP on the newborn bodyweight. Pfdhfr-Pfdhps quintuple mutants were over-represented (IRNI-FGKAA, IRNI-AGKAA), and sextuple mutants (IRNI-AGKAS, IRNI-FGEAA, IRNI-AGKGS) were also reported. The Pfk13 gene mutations associated with artemisinin resistance were not detected. This study highlights the role of ANC in achieving optimal SP coverage in pregnant women, the mitigated impact of IPTp-SP on malaria outcomes, and the high prevalence of multiple SP-resistant P. falciparum parasites in the city of Douala that could compromise the efficacy of IPTp-SP.

Chemical composition and repellent activity of essential oils of Tithonia diversifolia (Asteraceae) leaves against the bites of Anopheles coluzzii.

Tithonia diversifolia is widely used in African traditional medicine for the treatment of a large number of ailments and disorders, including malaria. In the present study, we evaluated the repellent activity of essential oils (EO) of this plant against Anopheles coluzzii, a major vector of malaria in Africa. Fresh leaves of T. diversifolia were used to extract EO, which were used to perform repellency assays in the laboratory and in the field using commercially available N,N-Diethyl-meta-toluamide (DEET) and Cymbopogon (C.) citratus EO as positive controls and vaseline as negative control. The repellency rates and durations of protection of the human volunteers involved were used as measures of repellent activity. Chemical composition of the T. diversifolia EO was established further by gas chromatography coupled with mass spectrometry. The moisture content and oil yield were 81% and 0.02% respectively. A total of 29 compounds in the T. diversifolia EO was identified, with D-limonene (20.1%), α-Copaene (10.3%) and o-Cymene (10.0%) as the most represented. In field studies, the mean time of protection against mosquito bites was significantly lower in T. diversifolia EO-treated volunteers compared to treatments with C. citratus EO (71 min versus 125 min, p = 0.04), but significantly higher when compared with the non-treated volunteers (71 min vs 0.5 min, p = 0.03). The same pattern was found in laboratory repellency assays against A. coluzzii. In contrast, repulsion rates were statistically similar between T. diversifolia EO and positive controls. In conclusion, the study suggests promising repellent potential of leaves of T. diversifolia EO against A. coluzzii.

Phytofabrication and characterization of Alchornea cordifolia silver nanoparticles and evaluation of antiplasmodial, hemocompatibility and larvicidal potential.

Purpose: The recent emergence of Plasmodium falciparum (Pf) parasites resistant to current artemisinin-based combination therapies in Africa justifies the need to develop new strategies for successful malaria control. We synthesized, characterized and evaluated medical applications of optimized silver nanoparticles using Alchornea cordifolia (AC-AgNPs), a plant largely used in African and Asian traditional medicine. Methods: Fresh leaves of A. cordifolia were used to prepare aqueous crude extract, which was mixed with silver nitrate for AC-AgNPs synthesis and optimization. The optimized AC-AgNPs were characterized using several techniques including ultraviolet-visible spectrophotometry (UV-Vis), scanning/transmission electron microscopy (SEM/TEM), powder X-ray diffraction (PXRD), selected area electron diffraction (SAED), energy dispersive X-ray spectroscopy (EDX), Fourier transformed infrared spectroscopy (FTIR), dynamic light scattering (DLS) and Zeta potential. Thereafter, AC-AgNPs were evaluated for their hemocompatibility and antiplasmodial activity against Pf malaria strains 3D7 and RKL9. Finally, lethal activity of AC-AgNPs was assessed against mosquito larvae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti which are vectors of neglected diseases such as dengue, filariasis and chikungunya. Results: The AC-AgNPs were mostly spheroidal, polycrystalline (84.13%), stable and polydispersed with size of 11.77 ± 5.57 nm. FTIR revealed the presence of several peaks corresponding to functional chemical groups characteristics of alkanoids, terpenoids, flavonoids, phenols, steroids, anthraquonones and saponins. The AC-AgNPs had a high antiplasmodial activity, with IC50 of 8.05 µg/mL and 10.31 µg/mL against 3D7 and RKL9 Plasmodium falciparum strains. Likewise, high larvicidal activity of AC-AgNPs was found after 24 h- and 48 h-exposure: LC50 = 18.41 µg/mL and 8.97 µg/mL (Culex quinquefasciatus), LC50 = 16.71 µg/mL and 7.52 µg/mL (Aedes aegypti) and LC50 = 10.67 µg/mL and 5.85 µg/mL (Anopheles stephensi). The AC-AgNPs were highly hemocompatible (HC50 > 500 µg/mL). Conclusion: In worrying context of resistance of parasite and mosquitoes, green nanotechnologies using plants could be a cutting-edge alternative for drug/insecticide discovery and development.

Etiology and clinical characteristics of pediatric acute fever among hospitalized children in an endemic malaria transmission area of Cameroon in Central Africa.

Acute fever in the majority of children in resource-limited countries is attributable to malaria and often treated without laboratory evidence. The aim of the study was to characterize acute pediatric infectious fevers (APIF) in the pediatric department of the Douala Laquintinie Hospital. A cross-sectional study was conducted among children aged 2 months to 15 years who were admitted with an acute fever (anal temperature ≥ 37.5°C less than 5 days in infants and 7 days in adolescents). 200 children were included and followed up during their hospitalization. The mean age was 3.7 (IQ25-75: 1-4.6) years. More than 3 out of 5 patients (62.5%) came from another health facility and anemia accounted for 29% of the reasons for consultation associated with fever. The main symptoms were vomiting (28%), cough (26%), convulsions (21%) and diarrhea (20%). Skin-mucosal pallor (43.0%) and hepatosplenomegaly (26.0%) were the most common physical signs encountered. Among febrile children, 116/200 (58%) were infected with at least 1 pathogen, and 1/200 (0.5%) had a fever of unknown etiology. Malaria (53% vs 80.5% presumptive) associated with anemia (95.3% of cases) was the most common pathology associated with APIF, followed by pneumonia (19.5%), meningitis (11.5%) and urinary tract infections (10% vs 54.5% presumptive). Malaria was over-diagnosed on admission and over-treated as well as urinary tract infection. A better understanding of common pathogens carriage, a better capacity for improved diagnosis and a better applied clinical algorithm for febrile illnesses in children are needed.

Non-falciparum species and submicroscopic infections in three epidemiological malaria facets in Cameroon.

BACKGROUND: There are growing reports on the prevalence of non-falciparum species and submicroscopic infections in sub-Saharan African countries but little information is available from Cameroon. METHODS: A hospital-based cross-sectional study was carried out in four towns (Douala, Maroua, Mayo-Oulo, and Pette) from three malaria epidemiological strata (Forest, Sahelian, and Soudanian) of Cameroon. Malaria parasites were detected by Giemsa light microscopy and polymerase chain reaction (PCR) assay. Non-falciparum isolates were characterized and their 18S gene sequences were BLASTed for confirmatory diagnosis. RESULTS: PCR assay detected malaria parasites in 82.4% (98/119) patients, among them 12.2% (12/98) were asymptomatic cases. Three Plasmodium species viz. P. falciparum, P. ovale curtisi and P. vivax, and two co-infection types (P. falciparum + P. vivax and P. falciparum + P. ovale curtisi) were found. The remaining infections were mono-infections with either P. falciparum or P. ovale curtisi. All non-falciparum infections were symptomatic and microscopic. The overall proportion of submicroscopic infections was 11.8% (14/119). Most asymptomatic and submicroscopic infection cases were self-medicated with antimalarial drugs and/or medicinal plants. On analysis, P. ovale curtisi sequences were found to be phylogenetically closer to sequences from India while P. vivax isolates appeared closer to those from Nigeria, India, and Cameroon. No G6PD-d case was found among non-falciparum infections. CONCLUSIONS: This study confirms our previous work on circulation of P. vivax and P. ovale curtisi and the absence of P. knowlesi in Cameroon. More studies are needed to address non-falciparum malaria along with submicroscopic infections for effective malaria management and control in Cameroon.

Rate of virological failure and HIV-1 drug resistance among HIV-infected adolescents in routine follow-up on health facilities in Cameroon.

The objective of this study was to determine the rates of virological failure (VF) and HIV drug resistance (HIVDR) amongst adolescents on antiretroviral Therapy (ART). A retrospectively designed study was conducted in 10 healthcare centers for adolescents living with HIV (ALHIV) in the two main cities of Cameroon (Yaoundé and Douala), from November 2018 to May 2019. Sociodemographic, clinical, therapeutic and laboratory parameters were collected from medical records. All enrolled ALHIV had viral load (VL) measurements following the national guidelines. All patients with a VL ≥ 1000 copies/ml were called to perform genotyping tests. The chi-square test was used to determine the factors associated with VF. Out of the 1316 medical records of ALHIV, we included 1083 ALHIV having a VL result. Among them, 276 (25.5%) were experiencing VF, and VF was significantly higher in ALHIV with suboptimal adherence (p<0.001), older adolescents (p<0.05), those who lived outside the city where they were receiving ART (p<0.006), severely immunocompromised (p<0.01) and started ART at infancy (p<0.02). Among the 45/276 (16.3%) participants with an available genotyping resistance testing (GRT) result, the overall rate of HIVDR was 93.3% (42/45). The most common mutations were K103N (n = 21/42, 52.3%) resulting in high-level resistance to Efavirenz and Nevirapine, followed by M184V (n = 20/42, 47.6%) and thymidine analog mutations (n = 15/42, 35.7%) associated with high-level resistance to Lamivudine and Zidovudine respectively. The high rate of VF and HIVDR among ALHIV regularly followed in health facilities in Cameroon highlights the need to develop interventions adapted to an adolescent-centered approach to preserve future ART options.

Clinical characteristics of Plasmodium falciparum infection among symptomatic patients presenting to a major urban military hospital in Cameroon.

BACKGROUND: Urban malaria has received insufficient attention in the literature. The prevalence and clinical characteristics of Plasmodium falciparum infection amongst patients presenting with suspected malaria were investigated at a major urban hospital in Douala, Cameroon with a particular focus on anaemia. METHODS: A cross-sectional, 18-week demographic and clinical survey was conducted of patients presenting to the Emergency Department of Douala Military Hospital with suspected malaria, largely defined by the presence or recent history of fever. Venous samples were tested for P. falciparum using rapid diagnostic tests and PCR, and anaemia was defined by haemoglobin level according to WHO definitions. Likelihood ratios (LR), odds ratios (OR), and population attributable risk percent (PARP) were calculated. RESULTS: Participants were ages 8 months to 86 years, 51% were women (257/503), and all districts of Douala were represented. Overall, 38.0% (n = 189/497) were anaemic, including 5.2% (n = 26/497) with severe anaemia. Anaemia prevalence was significantly higher (OR: 2.20, 95% CI 1.41-3.45) among children < 15 years (53.1%, n = 52/98) compared to adults (34%, n = 133/392). Plasmodium falciparum was detected in 37.2% by nested PCR. Among all participants, several factors were associated with clinically significant LR for P. falciparum infection, including age 10-14 years (positive LR: 3.73), living in the island district of Douala VI (positive LR: 3.41), travel to any of three northern regions (positive LR: 5.11), and high fever > 40 °C at presentation (positive LR: 4.83). Among all participants, 8.7% of anaemia was associated with P. falciparum infection, while the PARP was 33.2% among those < 15 years of age and 81.0% among 10-14-year-olds. CONCLUSIONS: The prevalence of P. falciparum infection in the urban hospital was high. Mirroring trends in many rural African settings, older children had the highest positivity rate for P. falciparum infection. Anaemia was also common in all age groups, and for those 10-14 years of age, 80% of the risk for anaemia was associated with P. falciparum infection. Malaria rates in major urban population centres can be high, and more research into the multifactorial causes of anaemia across the age spectrum are needed.

Epidemiological and entomological studies of malaria transmission in Tibati, Adamawa region of Cameroon 6 years following the introduction of long-lasting insecticide nets.

BACKGROUND: Malaria remains a serious public health problem in Cameroon. Implementation of control interventions requires prior knowledge of the local epidemiological situation. Here we report the results of epidemiological and entomological surveys carried out in Tibati, Adamawa Region, Cameroon, an area where malaria transmission is seasonal, 6 years after the introduction of long-lasting insecticidal bed nets. METHODS: Cross-sectional studies were carried out in July 2015 and 2017 in Tibati. Thick blood smears and dried blood spots were collected from asymptomatic and symptomatic individuals in the community and at health centers, respectively, and used for the molecular diagnosis of Plasmodium species. Adult mosquitoes were collected by indoor residual spraying and identified morphologically and molecularly. The infection status of Plasmodium spp. was determined by quantitative PCR, and positivity of PCR-positive samples was confirmed by Sanger sequencing. RESULTS: Overall malaria prevalence in our study population was 55.0% (752/1367) and Plasmodium falciparum was the most prevalent parasite species (94.3%), followed by P. malariae (17.7%) and P. ovale (0.8%); 92 (12.7%) infections were mixed infections. Infection parameters varied according to clinical status (symptomatic/asymptomatic) and age of the sampled population and the collection sites. Infection prevalence was higher in asymptomatic carriers (60.8%), but asexual and sexual parasite densities were lower. Prevalence and intensity of infection decreased with age in both the symptomatic and asymptomatic groups. Heterogeneity in infections was observed at the neighborhood level, revealing hotspots of transmission. Among the 592 Anopheles mosquitoes collected, 212 (35.8%) were An. gambiae, 172 (29.1%) were An. coluzzii and 208 (35.1%) were An. funestus (s.s.). A total of 26 (4.39%) mosquito specimens were infected by Plasmodium sp. and the three Anopheles mosquitoes transmitted Plasmodium at equal efficiency. Surprisingly, we found an An. coluzzii specimen infected by Plasmodium vivax, which confirms circulation of this species in Cameroon. The positivity of all 26 PCR-positive Plasmodium-infected mosquitoes was successively confirmed by sequencing analysis. CONCLUSION: Our study presents the baseline malaria parasite burden in Tibati, Adamawa Region, Cameroon. Our results highlight the high malaria endemicity in the area, and hotspots of disease transmission are identified. Parasitological indices suggest low bednet usage and that implementation of control interventions in the area is needed to reduce malaria burden. We also report for the first time a mosquito vector with naturally acquired P. vivax infection in Cameroon.

Preliminary validation of the use of IgG antibody response to Anopheles gSG6-p1 salivary peptide to assess human exposure to malaria vector bites in two endemic areas of Cameroon in Central Africa.

The specific immune response to the Anopheles salivary peptide could be a pertinent and complementary tool to assess the risk of malaria transmission and the effectiveness of vector control strategies. This study aimed to obtain first reliable data on the current state of the Anopheles gSG6-P1 biomarker for assess the level of exposure to Anopheles bites in high malaria endemic areas in Cameroon. Blood smears were collected from people living in the neighborhoods of Youpwe (suburban area, continental) and Manoka (rural area, Island), both areas in the coastal region of Cameroon. Malaria infection was determined using thick blood smear microscopy, whereas the level of specific IgG response to gSG-P1 peptide was assessed by enzyme-linked immunosorbent assay from the dried blood spots. Of 266 (153 from Youpwe, 113 from Manoka) malaria endemic residents (mean age: 22.8±19.8 years, age range: 6 months-94 years, male/female sex ratio: 1/1.2, with Manoka mean age: 23.71±20.53, male/female sex ratio:1/1.13 and Youpwe mean age: 22.12±19.22, male/female sex ratio 1/0.67) randomly included in the study, Plasmodium infection prevalence was significantly higher in Manoka than in Youpwe (64.6% vs 12,4%, p = 0.0001). The anti-gSG6-P1 IgG response showed a high inter-individual heterogeneity and was significantly higher among individuals from Manoka than those from Youpwe (p = 0.023). Malaria infected individuals presented a higher anti-gSG6-P1 IgG antibody response than non-infected (p = 0.0004). No significant difference in the level of specific IgG response to gSG-P1 was observed according to long lasting insecticidal nets use. Taken together, the data revealed that human IgG antibody response to Anopheles gSG-P1 salivary peptide could be also used to assess human exposure to malaria vectors in Central African region. This finding strengthens the relevance of this candidate biomarker to be used for measuring human exposure to malaria vectors worldwide.

EphA2 contributes to disruption of the blood-brain barrier in cerebral malaria.

Disruption of blood-brain barrier (BBB) function is a key feature of cerebral malaria. Increased barrier permeability occurs due to disassembly of tight and adherens junctions between endothelial cells, yet the mechanisms governing junction disassembly and vascular permeability during cerebral malaria remain poorly characterized. We found that EphA2 is a principal receptor tyrosine kinase mediating BBB breakdown during Plasmodium infection. Upregulated on brain microvascular endothelial cells in response to inflammatory cytokines, EphA2 is required for the loss of junction proteins on mouse and human brain microvascular endothelial cells. Furthermore, EphA2 is necessary for CD8+ T cell brain infiltration and subsequent BBB breakdown in a mouse model of cerebral malaria. Blocking EphA2 protects against BBB breakdown highlighting EphA2 as a potential therapeutic target for cerebral malaria.

K-13 propeller gene polymorphisms isolated between 2014 and 2017 from Cameroonian Plasmodium falciparum malaria patients.

The emergence of artemisinin-resistant parasites since the late 2000s at the border of Cambodia and Thailand poses serious threats to malaria control globally, particularly in Africa which bears the highest malaria transmission burden. This study aimed to obtain reliable data on the current state of the kelch13 molecular marker for artemisinin resistance in Plasmodium falciparum in Cameroon. DNA was extracted from the dried blood spots collected from epidemiologically distinct endemic areas in the Center, Littoral and North regions of Cameroon. Nested PCR products from the Kelch13-propeller gene were sequenced and analyzed on an ABI 3730XL automatic sequencer. Of 219 dried blood spots, 175 were sequenced successfully. We identified six K13 mutations in 2.9% (5/175) of samples, including 2 non-synonymous, the V589I allele had been reported in Africa already and one new allele E612K had not been reported yet. These two non-synonymous mutations were uniquely found in parasites from the Littoral region. One sample showed two synonymous mutations within the kelch13 gene. We also observed two infected samples with mixed K13 mutant and K13 wild-type infection. Taken together, our data suggested the circulation of the non-synonymous K13 mutations in Cameroon. Albeit no mutations known to be associated with parasite clearance delays in the study population, there is need for continuous surveillance for earlier detection of resistance as long as ACTs are used and scaled up in the community.

Demographical, hematological and serological risk factors for Plasmodium falciparum gametocyte carriage in a high stable transmission zone in Cameroon.

Presence of mature gametocyte forms of malaria parasites in peripheral blood is a key requirement for malaria transmission. Yet, studies conducted in most malaria transmission zones report the absence of gametocyte in the majority of patients. We therefore sought to determine the risk factors of both all-stage and mature gametocyte carriage in an area with high stable transmission of Plasmodium falciparum in Cameroon. Gametocyte positivity was determined using three complementary methods: thick blood smear microscopy, RT-PCR and RT-LAMP, whereas exposure to the infection was assessed by enzyme-linked immunosorbent assay. Of 361 malaria endemic residents randomly included in the study (mean age: 28±23 years, age range: 2-100 years, male/female sex ratio: 1.1), 87.8% were diagnosed with P. falciparum infection, of whom 45.7% presented with fever (axillary body temperature ≥37.5°C). Mature gametocyte positivity was 1.9% by thick blood smear microscopy and 8.9% by RT-PCR targeting the mature gametocyte transcript, Pfs25. The gametocyte positivity rate was 24.1% and 36.3% by RT-PCR or RT-LAMP, respectively, when targeting the sexual stage marker, Pfs16. Multivariate analyses revealed anemia as a common independent risk factor for both mature and all-stage gametocyte carriage, whereas fever and low anti-gametocyte antibody levels were independently associated with all-stage gametocyte carriage only. Taken together, the data suggest important differences in risk factors of gametocyte carriage depending on stage analyzed, with anemia, fever and low antiplasmodial plasma antibody levels representing the major contributing risk factors.

Knowledge, practices and attitudes on antibiotics use in Cameroon: Self-medication and prescription survey among children, adolescents and adults in private pharmacies.

Benefits of antibiotics are threatened by the self-medication, people's lack of knowledge and inappropriate use of antibiotics, especially in developing countries. This study was designed to determine knowledge; attitudes and practices toward antibiotics use in an urban community, and evaluate the factors that are associated with antibiotic use. Between January and March 2015, a cross sectional and prospective study was conducted in all pharmacies within the Douala IV health district, Cameroon. Anonymous interviews including both open and closed ended questions were conducted in participants selected by convenience sampling Descriptive and logistic regression analysis were performed using StataSE11 software (version 11 SE) and R software (version 3.1.1) in data analysis. Overall 402 (33.7%) of 1,192 customers purchased antibiotics and of these, 47% bought antibiotics without a prescription. 60.7% of purchased antibiotics was for adult 'patients and around 60% of parents carried out self-medication on their children. The vast majority reported that all microbes can be treated with antibiotics (88.3%). The belief that antibiotics are appropriate for bacterial infections was more common among those with a higher level education (OR = 4.03, 95%CI:1.89-8.57, p<0.0001) and among public/private servants (OR = 2.47, 95%CI:1.21-5.08, p = 0.013). Physicians provide less explanations about antibiotics are and their potential side effects than the pharmacy auxiliaries (OR = 0.205, 95%CI = 0.09-0.46, p<0.0001), but more than pharmacists (OR = 3.692, 95%CI:1.44-9.25, p = 0.005). Indications on antibiotics use were 7 times more given to customers with a prescription compared to those without a prescription (OR = 7.37, 95% CI = 2.13-25.43, p = 0.002). Adult male (OR = 2.32, 95%CI:1.24-4.34, p = 0.009) and higher education (OR = 2.05, 95%CI:1.08-3.89, p = 0.027) were significantly associated with self-medication. Misuse, little "practical knowledge" and high self-medication confirm the unsatisfactory prescription and dispensing practices of the antibiotics in our country. These results highlight the important of the development and implementation appropriate guidelines for the responsible use of antibiotics for health care providers and health education targeting community members themselves.

Anopheles coluzzii larval habitat and insecticide resistance in the island area of Manoka, Cameroon.

BACKGROUND: The effectiveness of Long-Lasting Insecticidal Nets and Indoor Residual Spraying in malaria vector control is threatened by vector resistance to insecticides. Knowledge of mosquito habitats and patterns of insecticide resistance would facilitate the development of appropriate vector control strategies. Therefore, we investigated An. coluzzii larval habitats and resistance to insecticides in the Manoka rural island area compared with the Youpwe suburban inland area, in Douala VI and II districts respectively. METHODS: Anopheline larvae and pupae were collected from open water bodies in December 2013 and April 2014 and reared until adult emergence. Two to four day old emerging females were morphologically identified as belonging to the An. gambiae complex and used for WHO susceptibility tests with 4 % DDT, 0.75 % permethrin, and 0.05 % deltamethrin, with or without piperonyl butoxide (PBO) synergist. Control and surviving specimens were identified down to the species using a PCR-RFLP method. Survivors were genotyped for kdr L1014 mutations using Hot Oligonucleotide Ligation Assay. RESULTS: In both study sites, ponds, residual puddles, boats, and drains were identified as the major An. gambiae s.l. larval habitats. A total of 1397 females, including 784 specimens from Manoka and 613 from Youpwe, were used for resistance testing. The two mosquito populations displayed resistance to DDT, permethrin and deltamethrin, with variable mortality rates from 1 % to 90 %. The knock-down times were also significantly increased (at least 2.8 fold). Pre-exposure of mosquitoes to PBO did not impact on their mortality to DDT, conversely the mortality rates to permethrin and deltamethrin were significantly increased (7.56 ≤ X(2) ≤ 48.63, df = 1, p < 0.01), suggesting involvement of P450 oxidases in pyrethroid resistance. A subsample of 400 An. gambiae s.l. specimens including 280 control and 120 survivors from bioassays were all found to be An. coluzzii species. Only the kdr 1014 F mutation was found in survivors, with 88.5 % (N = 76) and 75 % (N = 44) frequencies in Youpwe and Manoka respectively. CONCLUSION: This is the first report of An. coluzzii resistance to insecticides in an insular area in Cameroon. Since permanent larval habitats have been identified, larval source management strategies may be trialed in this area as complementary vector control interventions.

HIV, HBV, HCV and T. pallidum infections among blood donors and Transfusion-related complications among recipients at the Laquintinie hospital in Douala, Cameroon.

BACKGROUND: Transfusion-transmissible infections (TTIs) pose a major health risk in Cameroon given the high prevalence of such pathogens and increased demands for blood donations in the local communities. This study aims at establishing the prevalence of commonly encountered TTIs among blood donors and transfusion-related complications among recipients in an urban center of Cameroon. METHODS: A total of 477 blood donors and 83 blood recipients were recruited by consecutive sampling at the Laquintinie Hospital in Douala (LHD), Cameroon. Serum samples from blood donors were tested by quantitative enzyme-linked immunosorbent assays (ELISA) and/or using various Rapid diagnostic test (RDT) for presence of Hepatits B (HBV) viral antigens, and antibodies to human immunodeficiency (HIV-1/2), Hepatits B (HCV) and Treponema pallidum. Recipient's medical records were also analyzed for possible transfusion-associated complications. RESULTS: The male/female sex ratio of the blood donors was 4/1 with a mean age of 30.2 (Sd = 8.3) years. Of all blood donors, 64/467 (13.7%) were infected by at least one of the four TTIs. Infected volunteer donors represented 8.3% while infected family donors comprised 14.3% of the donor population. The prevalence of HCV, HIV, HBV and T. pallidum were 1.3%, 1.8%, 3.5%, and 8.1%, respectively. More than half of the blood recipients were female (78.3%) and the mean age was 20.6 (SD = 16.1) years. The causes of severe anemia indicative of transfusion in recipients varied with wards (postpartum hemorrhage, caesarean section, uterine or cervical lacerations, abortions, urinary tract infections, severe malaria, vaso-occlusive attacks, wounds and gastrointestinal bleeding). The most frequent complications were chills and hematuria, which represented 46.1% of all observed complications. Other complications such as nausea, vomiting, jaundice, sudden diarrhea, anxiety, tachycardia, or hyperthermia were also found in recipients. Three cases of deaths occurred during the study, including a girl of less than one year. CONCLUSION: This study confirms the presence of blood-borne infectious diseases in blood donors at the LHD, identifying T. pallidum as the greatest threat to blood safety in the region, and hematuria as the most common immunological complications in blood recipients.