Measuring protective efficacy and quantifying the impact of drug resistance: A novel malaria chemoprevention trial design and methodology.

BACKGROUND: Recently revised WHO guidelines on malaria chemoprevention have opened the door to more tailored implementation. Countries face choices on whether to replace old drugs, target additional age groups, and adapt delivery schedules according to local drug resistance levels and malaria transmission patterns. Regular routine assessment of protective efficacy of chemoprevention is key. Here, we apply a novel modelling approach to aid the design and analysis of chemoprevention trials and generate measures of protection that can be applied across a range of transmission settings. METHODS AND FINDINGS: We developed a model of genotype-specific drug protection, which accounts for underlying risk of infection and circulating genotypes. Using a Bayesian framework, we fitted the model to multiple simulated scenarios to explore variations in study design, setting, and participant characteristics. We find that a placebo or control group with no drug protection is valuable but not always feasible. An alternative approach is a single-arm trial with an extended follow-up (>42 days), which allows measurement of the underlying infection risk after drug protection wanes, as long as transmission is relatively constant. We show that the currently recommended 28-day follow-up in a single-arm trial results in low precision of estimated 30-day chemoprevention efficacy and low power in determining genotype differences of 12 days in the duration of protection (power = 1.4%). Extending follow-up to 42 days increased precision and power (71.5%) in settings with constant transmission over this time period. However, in settings of unstable transmission, protective efficacy in a single-arm trial was overestimated by 24.3% if recruitment occurred during increasing transmission and underestimated by 15.8% when recruitment occurred during declining transmission. Protective efficacy was estimated with greater precision in high transmission settings, and power to detect differences by resistance genotype was lower in scenarios where the resistant genotype was either rare or too common. CONCLUSIONS: These findings have important implications for the current guidelines on chemoprevention efficacy studies and will be valuable for informing where these studies should be optimally placed. The results underscore the need for a comparator group in seasonal settings and provide evidence that the extension of follow-up in single-arm trials improves the accuracy of measures of protective efficacy in settings with more stable transmission. Extension of follow-up may pose logistical challenges to trial feasibility and associated costs. However, these studies may not need to be repeated multiple times, as the estimates of drug protection against different genotypes can be applied to different settings by adjusting for transmission intensity and frequency of resistance.

Effectiveness and safety of artesunate-amodiaquine versus artemether-lumefantrine for home-based treatment of uncomplicated Plasmodium falciparum malaria among children 6-120 months in Yaoundé, Cameroon: a randomized trial.

BACKGROUND: Many studies have reported high efficacy and safety of artesunate-amodiaquine (AS-AQ) and artemether-lumefantrine (AL) when administered under direct observation in Cameroon. There is paucity of data to support their continuous use in home-based treatment of uncomplicated Plasmodium falciparum malaria in Cameroon. Hence, this study aimed to assess the effectiveness and safety of AS-AQ versus AL for home-based treatment of uncomplicated P. falciparum malaria among children 6-120 months in Yaoundé, Cameroon. METHODS: A two-arm, open-label, randomized, controlled trial comparing the equivalence of AS-AQ (experimental group) and AL (control group) was carried out from May 2019 to April 2020 at two secondary hospitals in Yaoundé. Participants were randomized to receive either AS-AQ or AL. After the first dose, antimalarial drugs were given at home, rather than under direct observation by a study staff. The conventional on-treatment and post-treatment laboratory and clinical evaluations were not done until day 3 of the full antimalarial treatment course. The evaluation of effectiveness was mainly based on per protocol polymerase chain reaction adjusted adequate clinical and parasitological response (PP PCR adjusted ACPR) on day 28 post-treatment. Safety was based on assessment of adverse events (AEs) and severe adverse events (SAEs) from day 1 to day 28. RESULTS: A total of 242 children were randomized to receive AS-AQ (n = 114) and AL (n = 128). The PP PCR adjusted day 28 cure rates were [AS-AQ = 96.9% (95% CI, 91.2-99.4) versus AL = 95.5% (95% CI, 89.9-98.5), P = 0.797]. Expected mild to moderate adverse events were reported in both arms [AS-AQ = 83 (84.7%) versus AL = 99 (86.1%), P = 0.774]. The most common adverse events included: transient changes of hematologic indices and fever. CONCLUSIONS: This study demonstrated that AS-AQ and AL are effective and safe for home management of malaria in Yaoundé. The evidence from this study supports the parallel use of the two drugs in routine practice. However, the findings from this study do not describe the likely duration of antimalarial effectiveness in holoendemic areas where multiple courses of treatment might be required. TRIAL REGISTRATION: This study is a randomized controlled trial and it was retrospectively registered on 23/09/2020 at ClinicalTrials.gov with registration number NCT04565184.

Drug resistance markers within an evolving efficacy of anti-malarial drugs in Cameroon: a systematic review and meta-analysis (1998-2020).

BACKGROUND: Malaria remains highly endemic in Cameroon. The rapid emergence and spread of drug resistance was responsible for the change from monotherapies to artemisinin-based combinations. This systematic review and meta-analysis aimed to determine the prevalence and distribution of Plasmodium falciparum drug resistance markers within an evolving efficacy of anti-malarial drugs in Cameroon from January 1998 to August 2020. METHODS: The PRISMA-P and PRISMA statements were adopted in the inclusion of studies on single nucleotide polymorphisms (SNPs) of P. falciparum anti-malarial drug resistance genes (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfatp6, Pfcytb and Pfk13). The heterogeneity of the included studies was evaluated using the Cochran's Q and I2 statistics. The random effects model was used as standard in the determination of heterogeneity between studies. RESULTS: Out of the 902 records screened, 48 studies were included in this aggregated meta-analysis of molecular data. A total of 18,706 SNPs of the anti-malarial drug resistance genes were genotyped from 47,382 samples which yielded a pooled prevalence of 35.4% (95% CI 29.1-42.3%). Between 1998 and 2020, there was significant decline (P < 0.0001 for all) in key mutants including Pfcrt 76 T (79.9%-43.0%), Pfmdr1 86Y (82.7%-30.5%), Pfdhfr 51I (72.2%-66.9%), Pfdhfr 59R (76.5%-67.8%), Pfdhfr 108 N (80.8%-67.6%). The only exception was Pfdhps 437G which increased over time (30.4%-46.9%, P < 0.0001) and Pfdhps 540E that remained largely unchanged (0.0%-0.4%, P = 0.201). Exploring mutant haplotypes, the study observed a significant increase in the prevalence of Pfcrt CVIET mixed quintuple haplotype from 57.1% in 1998 to 57.9% in 2020 (P < 0.0001). In addition, within the same study period, there was no significant change in the triple Pfdhfr IRN mutant haplotype (66.2% to 67.3%, P = 0.427). The Pfk13 amino acid polymorphisms associated with artemisinin resistance were not detected. CONCLUSIONS: This review reported an overall decline in the prevalence of P. falciparum gene mutations conferring resistance to 4-aminoquinolines and amino alcohols for a period over two decades. Resistance to artemisinins measured by the presence of SNPs in the Pfk13 gene does not seem to be a problem in Cameroon. Systematic review registration PROSPERO CRD42020162620.

The effect of improved housing on indoor mosquito density and exposure to malaria in the rural community of Minkoameyos, Centre Region of Cameroon.

BACKGROUND: This study evaluated the effectiveness of improved housing on indoor residual mosquito density and exposure to infected Anophelines in Minkoameyos, a rural community in southern forested Cameroon. METHODS: Following the identification of housing factors affecting malaria prevalence in 2013, 218 houses were improved by screening the doors and windows, installing plywood ceilings on open eaves and closing holes on walls and doors. Monthly entomological surveys were conducted in a sample of 21 improved and 21 non-improved houses from November 2014 to October 2015. Mosquitoes sampled from night collections on human volunteers were identified morphologically and their parity status determined. Mosquito infectivity was verified through Plasmodium falciparum CSP ELISA and the average entomological inoculation rates determined. A Reduction Factor (RF), defined as the ratio of the values for mosquitoes collected outdoor to those collected indoor was calculated in improved houses (RFI) and non-improved houses (RFN). An Intervention Effect (IE = RFI/RFN) measured the true effect of the intervention. Chi square test was used to determine variable significance. The threshold for statistical significance was set at P < 0.05. RESULTS: A total of 1113 mosquitoes were collected comprising Anopheles sp (58.6%), Culex sp (36.4%), Aedes sp (2.5%), Mansonia sp (2.4%) and Coquillettidia sp (0.2%). Amongst the Anophelines were Anopheles gambiae sensu lato (s.l.) (95.2%), Anopheles funestus (2.9%), Anopheles ziemanni (0.2%), Anopheles brohieri (1.2%) and Anopheles paludis (0.5%). Anopheles gambiae sensu stricto (s.s.) was the only An. gambiae sibling species found. The intervention reduced the indoor Anopheles density by 1.8-fold (RFI = 3.99; RFN = 2.21; P = 0.001). The indoor density of parous Anopheles was reduced by 1.7-fold (RFI = 3.99; RFN = 2.21; P = 0.04) and that of infected Anopheles by 1.8-fold (RFI = 3.26; RFN = 1.78; P = 0.04). Indoor peak biting rates were observed between 02 a.m. to 04 a.m. in non-improved houses and from 02 a.m. to 06 a.m. in improved houses. CONCLUSION: Housing improvement contributed to reducing indoor residual anopheline density and malaria transmission. This highlights the need for policy specialists to further evaluate and promote aspects of house design as a complementary control tool that could reduce indoor human-vector contact and malaria transmission in similar epidemiological settings.

Field evaluation of the 22 rapid diagnostic tests for community management of malaria with artemisinin combination therapy in Cameroon.

BACKGROUND: All suspected cases of malaria should receive a diagnostic test prior to treatment with artemisinin-based combinations based on the new WHO malaria treatment guidelines. This study compared the accuracy and some operational characteristics of 22 different immunochromatographic antigen capture point-of- malaria tests (RDTs) in Cameroon to inform test procurement prior to deployment of artemisinin-based combinations for malaria treatment. METHODS: One hundred human blood samples (50 positive and 50 negative) collected from consenting febrile patients in two health centres at Yaoundé were used for evaluation of the 22 RDTs categorized as "Pf Only" (9) or "Pf + PAN" (13) based on parasite antigen captured [histidine rich protein II (HRP2) or lactate dehydrogenase (pLDH) or aldolase]. RDTs were coded to blind technicians performing the tests. The sensitivity, specificity, and predictive values of the positive and negative tests (PPV and NPV) as well as the likelihood ratios were assessed. The reliability and some operational characteristics were determined as the mean values from two assessors, and the Cohen's kappa statistic was then used to compare agreement. Light microscopy was the referent. RESULTS: Of all RDTs tested, 94.2 % (21/22) had sensitivity values greater than 90% among which 14 (63.6%) were 'Pf + PAN' RDTs. The specificity was generally lower than the sensitivity for all RDTs and poorer for "Pf Only" RDTs. The predictive values and likelihood ratios were better for non-HRP2 analytes for "Pf + PAN" RDTs. The Kappa value for most of the tests obtained was around 67% (95% CI 50-69%), corresponding to a moderate agreement. CONCLUSION: Overall, 94.2% (21/22) of RDTs tested had accuracy within the range recommended by the WHO, while one performed poorly, below acceptable levels. Seven "Pf + PAN" and 3 "Pf Only" RDTs were selected for further assessment based on performance characteristics. Harmonizing RDT test presentation and procedures would prevent mistakes of test performance and interpretation.

Prevalence of malaria, typhoid, toxoplasmosis and rubella among febrile children in Cameroon.

BACKGROUND: The current roll-out of rapid diagnostic tests (RDTs) in many endemic countries has resulted in the reporting of fewer cases of malaria-attributed illnesses. However, lack of knowledge of the prevalence of other febrile illnesses and affordable diagnostic tests means that febrile patients are not managed optimally. This study assessed the prevalence of commonly treatable or preventable febrile illnesses in children between 6 months and 15 years using rapid diagnostic tests at the point-of-care. METHODS: Febrile children were enrolled between February-April 2014 at a health facility after obtaining informed consent from parent. Eligible participants were aged 6 months-15 years with a history of fever in the last 24 h or axillary temperature ≥38 °C at consultation. All participants were tested using RDTs for malaria, typhoid, toxoplasmosis and rubella. Malaria parasites were further identified by microscopy and PCR. Clinical and household characteristics were recorded and association with pathogens determined. RESULTS: Of the 315 children enrolled, the mean age was 5.8 ± 3.8 years. Stomach pain (41.2 %) was the most reported symptom. Prior to attending the health facility, 70.8 % had taken antipyretics, 27.9 % antimalarials, 11.4 % antibiotics and 13.3 % antifungal drugs. Among 315 children with fever, based on RDTs, 56.8 % were infected with malaria, 4.4 % with typhoid, 3.2 % with acute toxoplasmosis, and 1.3 % with rubella (all positive for rubella were in the same family and not vaccinated). All non-malarial infections were co-infections and approximately 30 % of the fever cases went un-diagnosed. Malaria prevalence by microscopy and PCR was 43.4 and 70.2 % respectively. The sensitivity and specificity of RDTs for the diagnosis of malaria were 75.98 and 100 % respectively, with 0.73 measurement agreement between RDTs and microscopy while that of RDT and PCR were 81 and 100 % respectively with a K value of 0.72. The use of Insecticide Treated Bednets was 44 %. There was a significant association between ITN non-usage and malaria (p = 0. 029) as well as drinking water and presence of typhoid (p = 0.047). No association was observed between type of housing and malaria, or toxoplasmosis and raising cats. CONCLUSION: Though malaria still remains the major cause of fever in children, using RDTs for other treatable febrile illnesses like typhoid and toxoplasmosis could facilitate the optimal management of febrile illnesses in children especially when these occur as co-infections with malaria.

Randomized non-inferiority and safety trial of dihydroartemisin-piperaquine and artesunate-amodiaquine versus artemether-lumefantrine in the treatment of uncomplicated Plasmodium falciparum malaria in Cameroonian children.

BACKGROUND: Artemether-lumefantrine and artesunate-amodiaquine are first-line treatment for uncomplicated malaria in Cameroon. No study has yet compared the efficacy of these drugs following the WHO recommended 42-day follow-up period. The goal of this study was to compare the clinical efficacy, tolerability and safety of artesunate-amodiaquine (ASAQ), artemether-lumefantrine (AL) and dihydroartemisinin piperaquine (DHAP) among children aged less than ten years in two malaria-endemic ecological regions of Cameroon. METHODS: A three-arm, randomized, controlled, non-inferiority trial was conducted among children of either gender aged six months (>5 kg) to ten years (n = 720) with acute uncomplicated Plasmodium falciparum infection. Parents/guardians of children provided consent prior to randomization to receive ASAQ, DHAP or AL in the ratio of 2:2:1, respectively. Treatment outcome was assessed based on standard WHO 2003 classification after 42 days of follow-up. The primary outcome was PCR-corrected day-42 cure rates. The non-inferiority, one-sided, lower limit asymptotic 97.5% confidence interval (CI) on the difference in PCR-corrected cure rates of ASAQ and DHAP when compared to AL was accepted if the lower limit of the CI was greater than -10%. Secondary outcomes were parasite and fever clearances and day 7 haemoglobin changes. RESULTS: PCR-corrected PP cure rates of 96.7, 98.1 and 96.3, respectively, for AL, ASAQ and DHAP was observed. The lower bound of the one-sided 97.5% CI calculated around the difference between day-42 cure rate point estimates in AL and ASAQ groups, AL and DHAP groups were, -6% and -4% respectively. There were no statistical significant differences in parasite or fever clearance times between treatments, although fever clearance pattern was different between ASAQ and DHAP. No statistical significant differences were observed in the occurrence of adverse events among treatment groups. CONCLUSION: ASAQ and DHAP are considered safe and tolerable and are not inferior to AL in the treatment of uncomplicated P. falciparum malaria in Cameroonian children. TRIAL REGISTRATION: NCT01845701.

Trends in malaria admissions at the Mbakong Health Centre of the North West Region of Cameroon: a retrospective study.

BACKGROUND: Malaria is the leading cause of death worldwide. It is urgent to assess the impact of interventions and scaled-up control efforts. Despite reported reduction in malaria prevalence in Africa, the trends in Cameroon are not yet fully understood. The aim of this study was to investigate the trends in malaria admissions among febrile patients seeking treatment over a seven-year period (2006-2012) in an endemic area in Cameroon, hypothesizing a declining trend. This period followed changes in malaria treatment policy. The objectives were to identify possible trends in malaria admissions and to evaluate the impact of changes to treatment guidelines on the prevalence. METHODS: Data was collected through consultation and perusal of laboratory and prescription registers of the Mbakong Health Centre. Data analysis was conducted using SPSS and SAS Statistics. RESULTS: Analysis revealed that 4,230 febrile patients were received from 2006-2012. Of these febrile cases, 29.30% were confirmed positive. Between 2006 and 2012 confirmed malaria positive cases of those tested fluctuated, dropping from 53.21% in 2006 to 17.20% in 2008; then rising to 35.00% in 2011 and, finally, dropping to 18.2% of those tested in 2012. The prevalence in females and males across all age groups were similar: a slightly higher risk of males to have malaria (OR = 1.08, 95% CI 0.94-1.25) were not practically significant. Of those tested, the 5 to < 15 years and the 1 to < 5 years age groups were the hardest hit by malaria in the area. A practically visible and significant association was observed between the age and gender with regards to the number of malaria positive results (Pearson ×2 = 153.675, p < 0.00001, Cramer's V = 0.352). Malaria prevalence exhibited a fluctuating yet declining trend, as observed over the 28 quarters between January, 2006 and December, 2012. CONCLUSIONS: The changes to the treatment guidelines appear to result in a declining trend as was observed between 2006 and 2008. However, malaria admissions fluctuated between 2008 and 2012. There is, therefore, a need to step up control efforts of especially the vulnerable groups, such as the very young.

Designing and implementing interventions to change clinicians' practice in the management of uncomplicated malaria: lessons from Cameroon.

BACKGROUND: Effective case management of uncomplicated malaria is a fundamental pillar of malaria control. Little is known about the various steps in designing interventions to accompany the roll out of rapid diagnostic tests (RDTs) and artemisinin-based combination therapy (ACT). This study documents the process of designing and implementing interventions to change clinicians' practice in the management of uncomplicated malaria. METHODS: A literature review combined with formative quantitative and qualitative research were carried out to determine patterns of malaria diagnosis and treatment and to understand how malaria and its treatment are enacted by clinicians. These findings were used, alongside a comprehensive review of previous interventions, to identify possible strategies for changing the behaviour of clinicians when diagnosing and treating uncomplicated malaria. These strategies were discussed with ministry of health representatives and other stakeholders. Two intervention packages - a basic and an enhanced training were outlined, together with logic model to show how each was hypothesized to increase testing for malaria, improve adherence to test results and increase appropriate use of ACT. The basic training targeted clinicians' knowledge of malaria diagnosis, rapid diagnostic testing and malaria treatment. The enhanced training included additional modules on adapting to change, professionalism and communicating effectively. Modules were delivered using small-group work, card games, drama and role play. Interventions were piloted, adapted and trainers were trained before final implementation. RESULTS: Ninety-six clinicians from 37 health facilities in Bamenda and Yaounde sites attended either 1-day basic or 3-day enhanced training. The trained clinicians then trained 632 of their peers at their health facilities. Evaluation of the training revealed that 68% of participants receiving the basic and 92% of those receiving the enhanced training strongly agreed that it is not appropriate to prescribe anti-malarials to a patient if they have a negative RDT result. CONCLUSION: Formative research was an important first step, and it was valuable to engage stakeholders early in the process. A logic model and literature reviews were useful to identify key elements and mechanisms for behaviour change intervention. An iterative process with feedback loops allowed appropriate development and implementation of the intervention. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01350752.

Maternal Socio-Demographic Factors and Mother-to-Child Transmission of HIV in the North Region of Cameroon.

Background and Objective: Socio-demographic factors are important risk factors for HIV infection. Maternal socio-demographic factors associated with HIV transmission from mother to child are not well elucidated to our knowledge. This study aimed to assess the maternal socio-demographic factors associated with HIV vertical transmission. Methods: A matched case-control study was conducted among children under 15 years of age born to HIV-infected mothers; using a structured questionnaire. The study was conducted in four health facilities in the North Region of Cameroon from July 2015 to October 2016. HIV- infected children were the cases, and HIV-uninfected children were the controls. One case was matched to nearly 4 controls according to age and sex. A total of 113 HIV-infected mothers of children under 15 years of age were purposively enrolled in the study. A questionnaire was administered to mothers and socio-demographic characteristics were collected. Blood samples were collected from the mother and her child for the determination or confirmation of HIV status. Univariate and multiple logistic regressions were used to assess associations between socio-demographic variables and HIV transmission from mother to child. Results: A total of 113 HIV-infected mothers and 113 children under 15 years of age were enrolled in this study. The majority of the mothers were between the age ranges of 25 years to 34 years. Of the 113 HIV-infected mothers, 69 (61%) were Muslims, 33 (32.1%) were not educated, 88 (77.8%) were unemployed, 80 (70.9%) were married, out of which 49 (61.6%) were engaged in a monogamous union. Of the 113 children (49.6%) were female, 25 (22.1%) were HIV-infected and 88 (77.9%) were HIV-exposed uninfected. At the univariate level, mothers who achieved a primary level of education were less likely to transmit HIV to infants compared to uneducated mothers [OR=0.28; CI (0.08-0.95); p=0.04]; and widows had a higher likelihood of HIV transmission to infants compared to married mothers [OR=4.65; CI (1.26-17.20); p=0.02]. Using multiple logistic regression, the maternal primary education level [aOR=0.32; CI (0.08-0.90); p=0.03] and widowerhood [aOR=7.05; CI (1.49-33.24); p=0.01] remained highly associated with the likelihood of HIV transmission to infants. Conclusion and Global Health Implications: Uneducated mothers and widows had a higher likelihood of mother-to-child transmission of HIV. Our findings should prompt reinforcement of prevention strategies targeting uneducated women and widows.

Evolution of Plasmodium falciparum antimalarial drug resistance markers post-adoption of artemisinin-based combination therapies in Yaounde, Cameroon.

OBJECTIVES: This study aimed to investigate the evolution of Plasmodium falciparum antimalarial drug resistance markers by comparing the pre- and post-adoption of artemisinin-based combination therapies (ACTs) in Yaounde, Cameroon. METHODS: The molecular characterization of known antimalarial drug resistance markers (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and Pfk13) in P. falciparum-positive samples collected in 2014 and 2019-2020 was achieved using nested polymerase chain reaction, followed by targeted amplicon deep sequencing on the Illumina MiSeq platform. Data derived were compared with those published during the pre-ACT adoption period from 2004 to 2006. RESULTS: A high prevalence of Pfmdr1 184F, Pfdhfr 51I/59R/108N, and Pfdhps 437G mutant alleles was observed during the post-ACT adoption period. The Pfcrt 76T and Pfmdr1 86Y mutant alleles significantly declined between 2004 and 2020 (P <0.0001). Conversely, the resistance markers to antifolates, Pfdhfr 51I/59R/108N and Pfdhps 437G, significantly increased during the same study period (P <0.0001). We identified nine mutations in the propeller domains of Pfk13; although they were all present in single parasite isolates, none of them are known to confer artemisinin resistance. CONCLUSION: This study documented a near-complete reversion to sensitive parasites for markers conferring resistance to the 4-aminoquinolines and arylamino alcohols in Yaounde. In contrast, the Pfdhfr mutations associated with pyrimethamine resistance are moving toward saturation.

Malaria prevalence and treatment of febrile patients at health facilities and medicine retailers in Cameroon.

OBJECTIVE: To investigate the quality of malaria case management in Cameroon 5 years after the adoption of artemisinin-based combination therapy (ACT). Treatment patterns were examined in different types of facility, and the factors associated with being prescribed or receiving an ACT were investigated. METHODS: A cross-sectional cluster survey was conducted among individuals of all ages who left public and private health facilities and medicine retailers in Cameroon and who reported seeking treatment for a fever. Prevalence of malaria was determined by rapid diagnostic tests (RDTs) in consenting patients attending the facilities and medicine retailers. RESULTS: Among the patients, 73% were prescribed or received an antimalarial, and 51% were prescribed or received an ACT. Treatment provided to patients significantly differed by type of facility: 65% of patients at public facilities, 55% of patients at private facilities and 45% of patients at medicine retailers were prescribed or received an ACT (P = 0.023). The odds of a febrile patient being prescribed or receiving an ACT were significantly higher for patients who asked for an ACT (OR = 24.1, P < 0.001), were examined by the health worker (OR = 1.88, P = 0.021), had not previously sought an antimalarial for the illness (OR = 2.29, P = 0.001) and sought treatment at a public (OR = 3.55) or private facility (OR = 1.99, P = 0.003). Malaria was confirmed in 29% of patients and 70% of patients with a negative result were prescribed or received an antimalarial. CONCLUSIONS: Malaria case management could be improved. Symptomatic diagnosis is inefficient because two-thirds of febrile patients do not have malaria. Government plans to extend malaria testing should promote rational use of ACT; though, the introduction of rapid diagnostic testing needs to be accompanied by updated clinical guidelines that provide clear guidance for the treatment of patients with negative test results.

Compounds from Sorindeia juglandifolia (Anacardiaceae) exhibit potent anti-plasmodial activities in vitro and in vivo.

BACKGROUND: Discovering new lead compounds against malaria parasites is a crucial step to ensuring a sustainable global pipeline for effective anti-malarial drugs. As far as we know, no previous phytochemical or pharmacological investigations have been carried out on Sorindeia juglandifolia. This paper describes the results of an anti-malarial activity-driven investigation of the fruits of this Cameroonian plant. METHODS: Air-dried fruits were extracted by maceration using methanol. The extract was fractionated by flash chromatography followed by column chromatography over silica gel, eluting with gradients of hexane-ethyl acetate mixtures. Resulting fractions and compounds were tested in vitro against the Plasmodium falciparum chloroquine-resistant strain W2, against field isolates of P. falciparum, and against the P. falciparum recombinant cysteine protease falcipain-2. Promising fractions were assessed for acute toxicity after oral administration in mice. One of the promising isolated compounds was assessed in vivo against the rodent malaria parasite Plasmodium berghei. RESULTS: The main end-products of the activity-guided fractionation were 2,3,6-trihydroxy benzoic acid (1) and 2,3,6-trihydroxy methyl benzoate (2). Overall, nine fractions tested against P. falciparum W2 and falcipain-2 were active, with IC50 values of 2.3-11.6 µg/ml for W2, and 1.1-21.9 µg/ml for falcipain-2. Purified compounds (1) and (2) also showed inhibitory effects against P. falciparum W2 (IC50s 16.5 µM and 13.0 µM) and falcipain-2 (IC50s 35.4 and 6.1 µM). In studies of P. falciparum isolates from Cameroon, the plant fractions demonstrated IC50 values of 0.14-19.4 µg/ml and compounds (1) and (2) values of 6.3 and 36.1 µM. In vivo assessment of compound (1) showed activity against P. berghei strain B, with mean parasitaemia suppressive dose and curative dose of 44.9 mg/kg and 42.2 mg/kg, respectively. Active fractions were found to be safe in mice after oral administration of 7 g/kg body weight. CONCLUSIONS: Fractions of Sorindeia juglandifolia and two compounds isolated from these fractions were active against cultured malaria parasites, the P. falciparum protease falcipain-2, and in a rodent malaria model. These results suggest that further investigation of the anti-malarial activities of natural products from S. juglandifolia will be appropriate.

Host candidate gene polymorphisms and clearance of drug-resistant Plasmodium falciparum parasites.

BACKGROUND: Resistance to anti-malarial drugs is a widespread problem for control programmes for this devastating disease. Molecular tests are available for many anti-malarial drugs and are useful tools for the surveillance of drug resistance. However, the correlation of treatment outcome and molecular tests with particular parasite markers is not perfect, due in part to individuals who are able to clear genotypically drug-resistant parasites. This study aimed to identify molecular markers in the human genome that correlate with the clearance of malaria parasites after drug treatment, despite the drug resistance profile of the protozoan as predicted by molecular approaches. METHODS: 3721 samples from five African countries, which were known to contain genotypically drug resistant parasites, were analysed. These parasites were collected from patients who subsequently failed to clear their infection following drug treatment, as expected, but also from patients who successfully cleared their infections with drug-resistant parasites. 67 human polymorphisms (SNPs) on 17 chromosomes were analysed using Sequenom's mass spectrometry iPLEX gold platform, to identify regions of the human genome, which contribute to enhanced clearance of drug resistant parasites. RESULTS: An analysis of all data from the five countries revealed significant associations between the phenotype of ability to clear drug-resistant Plasmodium falciparum infection and human immune response loci common to all populations. Overall, three SNPs showed a significant association with clearance of drug-resistant parasites with odds ratios of 0.76 for SNP rs2706384 (95% CI 0.71-0.92, P = 0.005), 0.66 for SNP rs1805015 (95% CI 0.45-0.97, P = 0.03), and 0.67 for SNP rs1128127 (95% CI 0.45-0.99, P = 0.05), after adjustment for possible confounding factors. The first two SNPs (rs2706384 and rs1805015) are within loci involved in pro-inflammatory (interferon-gamma) and anti-inflammatory (IL-4) cytokine responses. The third locus encodes a protein involved in the degradation of misfolded proteins within the endoplasmic reticulum, and its role, if any, in the clearance phenotype is unclear. CONCLUSIONS: The study showed significant association of three loci in the human genome with the ability of parasite to clear drug-resistant P. falciparum in samples taken from five countries distributed across sub-Saharan Africa. Both SNP rs2706384 and SNP1805015 have previously been reported to be associated with risk of malaria infection in African populations. The loci are involved in the Th1/Th2 balance, and the association of SNPs within these genes suggests a key role for antibody in the clearance of drug-resistant parasites. It is possible that patients able to clear drug-resistant infections have an enhanced ability to control parasite growth.

Incidence of adverse drug events among patients on second line anti-tuberculosis regimen in the littoral region of cameroon.

Background: An adverse drug event (ADE) is an injury resulting from medical intervention associated with a drug. This study assesses the incidence of ADEs among participants on second-line drugs for tuberculosis (TB) in Cameroon. Methods: This was a longitudinal observational study including 65 participants and carried out from January 2017 to December 2017. Markers of ADEs were obtained from creatinine, transaminase audiogram, and clinical data. Multivariate analysis was used to determine the association between predictors and ADEs. Results: Forty-eight (73.8%) of the 65 participants developed 72 ADEs. Fifty-four (75%), 11 (15.3%), and 7 (9.7%) of the 72 ADEs were classified as Grades 1, 2, and 3, respectively. Gastrointestinal disorders were most common (35 [46.6%]) followed by auditory injuries (16 [22.2%]), hepatotoxicity (11 [15.3%]), neurological disorders (6 [8.3%]), and kidney disorders (4 [5.6%]). The follow-up duration of this study was 11,250-person day (PDY). The incidence rate for ADEs was 4.3/1000 PDY and that for gastrointestinal disorders, auditory injuries, hepatotoxicity, neurological disorders, and kidney disorders was 3.1, 1.4, 1.0, 0.5, and 0.2 (/1000PDY), respectively. Kanamycin (65 [90.3%]), isoniazid (4 [5.6%]), and ethambutol (3 [4.2%]) were incriminated with ADEs. Most (29 [60.4%]) of the ADEs occurred during the first 2 months of drug initiation. There was an association between poor treatment outcome and ADEs (P = 0.04, odds ratio = 1.20, 95% confidence of interval = 0.21-6.80]. Conclusions: The incidence of ADEs is associated with several factors and most of them occurred during the intensive phase of treatment. Kanamycin was the most associated drug linked to ADEs requiring its replacement with a less toxic one.

Discrete Survival Model Analysis of Plasmodium falciparum Response to Artemisinin-Based Combination Therapies among Children in Regions of Varying Malaria Transmission in Cameroon.

The need to monitor changes in parasite clearance following treatment with artemisinin-based combination therapies (ACTs) is important in the containment of drug resistance. This study aimed to model Plasmodium falciparum response to ACTs among children in two different transmission settings (Mutengene and Garoua) in Cameroon. Using the step function, a discrete-time survival model was fitted with all the covariates included that might play a role in parasite clearance. The probability of clearing parasites within 24 h following treatment was 21.6% and 70.3% for younger children aged 6 to 59 months and 29.3% and 59.8% for older children aged 60 to 120 months in Mutengene and Garoua, respectively. After two days of treatment, the conditional probability of clearing parasites given that they were not cleared on day 1 was 76.7% and 96.6% for children aged 6-59 months and 83.1% and 93.5% for children aged 60-120 months in Mutengene and Garoua, respectively. The model demonstrated that the ecological setting, age group and pretreatment serum levels of creatinine and alanine aminotransferase were the main factors that significantly influenced parasite clearance in vivo after administration of ACTs (p < 0.05). The findings highlight the need for further investigations on host differential response to ACTs in current practice.

Optimization of the Formulation of Rice Biscuits Supplemented with D. Edulis (L.) Powder Using Response Surface Methodology.

Trends in the food industry are nowadays directed towards the reduction of the level of trans fatty acids in food intended for human consumption. The present study was designed and aimed at valorizing Dacryodes edulis (L.) powder as a substitute for margarine in the production of functional rice biscuits. The effect of substituting margarine and refined wheat flour with D. edulis powder locally called safou and rice flour, respectively, at different proportions was assessed for the sensory and physicochemical properties of the formulated biscuits. For this, statistical models were developed, validated, and optimized using the response surface methodology with the Doehlert design as a tool. The results showed that an increase in the substitution rate of margarine with of D. edulis powder enhanced the aroma while the substitution of refined wheat flour with rice flour led to an improvement of the overall quality of the biscuits. The optimal composition of dough for the production of biscuits with satisfying sensory properties was 20.24% of wheat flour, 24.51% of rice flour, 19.09% of margarine, and 2.47% of D. edulis powder. The optimized biscuit which scored the highest overall acceptability contained proteins (10.33 g/100 g DM), fat (27.66 g/100 g DM), crude fibers (2.5 g/100 g DM), ash (3.55 g/100 g DM), and carbohydrates (54.01 g/100 g DM). It has an energy density of 506.3 ± 0.1 kcal/g and could therefore be suitable for the management of malnutrition. Mineral analysis revealed that the biscuit contained sodium (0.200 mg/100 g), potassium (0.192 mg/100 g), phosphorus (0.123 mg/100 g), iron (33.60 ppm), and zinc (26.81 ppm) at levels satisfying the recommended daily intakes. The results of this study demonstrated the suitability of safou as substitute of margarine in the rice biscuit formulation and suggests the potential of the formulated biscuits in the management of malnutrition and noncommunicable diseases such as hypertension, obesity, and cardiovascular diseases.

Efficacy of amodiaquine, sulphadoxine-pyrimethamine and their combination for the treatment of uncomplicated Plasmodium falciparum malaria in children in Cameroon at the time of policy change to artemisinin-based combination therapy.

BACKGROUND: The efficacy of amodiaquine (AQ), sulphadoxine-pyrimethamine (SP) and the combination of SP+AQ in the treatment of Cameroonian children with clinical malaria was investigated. The prevalence of molecular markers for resistance to these drugs was studied to set the baseline for surveillance of their evolution with time. METHODS: Seven hundred and sixty children aged 6-59 months with uncomplicated falciparum malaria were studied in three ecologically different regions of Cameroon - Mutengene (littoral equatorial forest), Yaoundé (forest-savannah mosaic) and Garoua (guinea-savannah). Study children were randomized to receive either AQ, SP or the combination AQ+SP. Clinical outcome was classified according to WHO criteria, as either early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) or adequate clinical and parasitological response (ACPR). The occurrence of mutations in pfcrt, pfmdr1, dhfr and dhps genes was studied by either RFLP or dot blot techniques and the prevalence of these mutations related to parasitological and therapeutic failures. RESULTS: After correction for the occurrence of re-infection by PCR, ACPRs on day 28 for AQ, SP and AQ+SP were 71.2%, 70.1% and 80.9%, in Garoua, 79.2%, 62.5%, and 81.9% in Mutengene, and 80.3%, 67.5% and 76.2% in Yaoundé respectively. High levels of Pfcrt 76T (87.11%) and Pfmdr1 86Y mutations (73.83%) were associated with quinoline resistance in the south compared to the north, 31.67% (76T) and 22.08% (86Y). There was a significant variation (p < 0.001) of the prevalence of the SGK haplotype between Garoua in the north (8.33%), Yaoundé (36.29%) in the savannah-forest mosaic and Mutengene (66.41%) in the South of Cameroon and a weak relation between SGK haplotype and SP failure. The 540E mutation on the dhps gene was extremely rare (0.3%) and occurred only in Mutengene while the pfmdr1 1034K and 1040D mutations were not detected in any of the three sites. CONCLUSION: In this study the prevalence of molecular markers for quinoline and anti-folate resistances showed high levels and differed between the south and north of Cameroon. AQ, SP and AQ+SP treatments were well tolerated but with low levels of efficacy that suggested alternative treatments were needed in Cameroon since 2005.

Physical, nutritional, and sensory quality of rice-based biscuits fortified with safou (Dacryodes edulis) fruit powder.

The reduction of postharvest losses in rice and safou is imperative to increase productivity in their respective value chains. In this study, fine broken rice grains were used to produce rice flour and subsequently rice-based biscuits. The biscuits were further fortified with safou powder, and the physical, nutritional, and sensory quality and stability during storage of the different types of biscuits were analyzed using standard methods. Fine or nonsandy biscuits had peak particle size of 500 µm, while medium (slightly sandy) and large (sandy) biscuits had peak particle sizes of 1,000 µm and 1,400 µm, respectively. The hardness varied from 5.7 ± 2.3 N for biscuits with large particles to 16.1 ± 4.4 N for biscuits with fine particles. Fortification of biscuits with sour safou increased the protein and amino acid content of the biscuits. Tryptophan was absent in both safou and the biscuits produced. There was an increase in phosphorus, potassium, calcium, magnesium, copper, iron, manganese, and aluminum following fortification with safou. Nonsandy biscuits dissolved faster in the mouth (melt) during consumption than the other biscuits although most of the biscuits were perceived to be low in melting and buttery. Nonsandy biscuits were rated as "very good," while slightly sandy and sandy were rated as "good." Safou rice-based biscuits were perceived as "very good," while simple rice biscuits were perceived as "good." Fortification of rice biscuits with safou increased the protein, essential amino acid, and mineral contents of the biscuits with very appreciable taste. These biscuits can be used to help fight protein, iron, and zinc malnutrition and in mitigating postharvest losses of underutilized broken rice and safou especially sour safou.

Comparison of the Accuracy of Four Malaria Diagnostic Methods in a High Transmission Setting in Coastal Cameroon.

BACKGROUND: Despite recommendation from the World Health Organization that all malaria suspected patients undergo a parasitological confirmation using rapid diagnostic test or light microscopy prior to treatment, health facilities in remote malaria endemic settings sometimes resort to presumptive diagnosis of malaria for clinical management for various reasons. Following observation of this practice, we undertook a cross-sectional study aimed at comparing presumptive diagnosis based on axillary temperature, SD Bioline™ rapid test, and light microscopy as strategies for malaria diagnosis in the coastal region of Mutengene in the South West of Cameroon with the overall goal of supporting improved malaria diagnosis at local levels. METHODOLOGY: Venous blood from 320 participants was used to detect the presence of malaria parasite using SD Bioline™ mRDT and Giemsa stained microscopy or spotted on filter paper for PCR amplification of the 18s rRNA gene of Plasmodium sp following standard procedures. The axillary temperature of each participant was also measured. The sensitivity, specificity, and predictive values and their confidence intervals were determined for each of the methods with PCR as the reference. The area under the curve was used to estimate accuracy of diagnostic method and compared between test method using the X2 test with P<0.05 considered significant. RESULTS: The overall diagnostic sensitivities of presumptive diagnosis using axillary temperature, light microscopy, and SD Bioline™ were observed to be 74.30% (95%CI: 67.90-80.01), 94.86% (95%CI: 90.99-97.41), and 95.33% (95%CI: 91.57-97.74), respectively, and their respective diagnostic specificities were 53.77% (95%CI: 43.82-63.51), 94.34% (95%CI: 88.09-97.87), and 94.34%(95%CI: 88.09-97.89). SD Bioline™ had a diagnostic sensitivity of 91.80% [95%CI: 81.90-97.28] at a parasitaemia of less than 500 parasites/µl of blood but a sensitivity of 100% for parasite counts above 500 parasites/µl of blood. The predictive values of the positive test were highly comparable between light microscopy (90.09%, [95%CI: 83.61-94.18]) and SD Bioline™ mRDT (90.91%, [95%CI: 84.50-94.83]), P=0.98 with kappa values of 0.898 but lower for presumptive diagnosis (50.89%, [95%CI: 43.72-58.03]), P<0.0001, and kappa value of 0.277. Perfect agreement was observed between SD Bioline™ mRDT and light microscopy (Cohen kappa= 0.924). CONCLUSIONS: The study showed that SD Bioline™ was as good as light microscopy in the diagnosis of malaria in remote areas of perennial transmission in South West Cameroon. This study equally revealed the limitations of presumptive diagnosis of malaria (as opposed to the use of RDTs or microscopy). Efforts should be made in such areas to promote parasitological confirmation of malaria using quality assured rapid tests or light microscopy for case management of malaria. The presence of nonnegligible levels of Plasmodium ovale in this study area indicate that treatment guidelines may require revision if same trend is proven in several other areas of same ecology.