Whole-Genome Characterization of SARS-CoV-2 Reveals Simultaneous Circulation of Three Variants and a Putative Recombination (20B/20H) in Pets, Brazzaville, Republic of the Congo.

Following the emergence of SARS-CoV-2, cases of pets infected with variants circulating among humans were reported. In order to evaluate the occurrence of SARS-CoV-2 circulation among pets in the Republic of the Congo, we conducted a ten-month study of dogs and cats living in COVID-19-positive households in Brazzaville and neighboring localities. Real-time PCR and the Luminex platform were used to detect SARS-CoV-2 RNA and antibodies to SARS-CoV-2 RBD and S proteins, respectively. Our results show for the first time the simultaneous circulation of several variants of SARS-CoV-2, including viruses from clades 20A and 20H and a putative recombinant variant between viruses from clades 20B and 20H. We found a high seroprevalence of 38.6%, with 14% of tested pets positive for SARS-CoV-2 RNA. Thirty-four percent of infected pets developed mild clinical signs, including respiratory and digestive signs, and shed the virus for about one day to two weeks. These results highlight the potential risk of SARS-CoV-2 interspecies transmission and the benefits of a "One Health" approach that includes SARS-CoV-2 diagnosis and surveillance of viral diversity in pets. This approach aims to prevent transmission to surrounding wildlife as well as spillback to humans.

Prevalence of SARS-CoV-2 antibodies in the Republic of Congo in mid-2021.

Objectives: To estimate the seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies in the general population in the Republic of Congo. Methods: In this cross-sectional study, conducted from June to July 2021, participants were recruited from the general population in three districts in the Republic of Congo. Eligible participants were tested for anti-SARS-CoV-2 antibodies using a rapid diagnostic assay. Results: Overall, 31.8% [95% confidence interval (CI) 29.5-34.0] of the 1669 participants tested positive for anti-SARS-CoV-2 antibodies. Higher prevalence was observed in the rural region (37.3%, 95% CI 31.0-44.1%) than the urban region (30.9%, 95% CI 28.5-33.3); however, the difference was not significant. The risk of testing positive for anti-SARS-CoV-2 antibodies increased significantly with age, ranging from 22.5% (95% CI 18.1-27.5) in 15-24 year olds to 47.9% (95% CI 39.3-56.5) in 55-64 year olds. Conclusions: The antibody levels observed in this survey correlate with a moderate rate of virus circulation, which correlates with the low number of confirmed cases of coronavirus disease 2019 in the Republic of Congo.

Field evaluation of the diagnostic performance of EasyScan GO: a digital malaria microscopy device based on machine-learning.

BACKGROUND: Microscopic examination of Giemsa-stained blood films remains the reference standard for malaria parasite detection and quantification, but is undermined by difficulties in ensuring high-quality manual reading and inter-reader reliability. Automated parasite detection and quantification may address this issue. METHODS: A multi-centre, observational study was conducted during 2018 and 2019 at 11 sites to assess the performance of the EasyScan Go, a microscopy device employing machine-learning-based image analysis. Sensitivity, specificity, accuracy of species detection and parasite density estimation were assessed with expert microscopy as the reference. Intra- and inter-device reliability of the device was also evaluated by comparing results from repeat reads on the same and two different devices. This study has been reported in accordance with the Standards for Reporting Diagnostic accuracy studies (STARD) checklist. RESULTS: In total, 2250 Giemsa-stained blood films were prepared and read independently by expert microscopists and the EasyScan Go device. The diagnostic sensitivity of EasyScan Go was 91.1% (95% CI 88.9-92.7), and specificity 75.6% (95% CI 73.1-78.0). With good quality slides sensitivity was similar (89.1%, 95%CI 86.2-91.5), but specificity increased to 85.1% (95%CI 82.6-87.4). Sensitivity increased with parasitaemia rising from 57% at < 200 parasite/µL, to ≥ 90% at > 200-200,000 parasite/µL. Species were identified accurately in 93% of Plasmodium falciparum samples (kappa = 0.76, 95% CI 0.69-0.83), and in 92% of Plasmodium vivax samples (kappa = 0.73, 95% CI 0.66-0.80). Parasite density estimates by the EasyScan Go were within ± 25% of the microscopic reference counts in 23% of slides. CONCLUSIONS: The performance of the EasyScan Go in parasite detection and species identification accuracy fulfil WHO-TDR Research Malaria Microscopy competence level 2 criteria. In terms of parasite quantification and false positive rate, it meets the level 4 WHO-TDR Research Malaria Microscopy criteria. All performance parameters were significantly affected by slide quality. Further software improvement is required to improve sensitivity at low parasitaemia and parasite density estimations. Trial registration ClinicalTrials.gov number NCT03512678.

Early Circulation of SARS-CoV-2, Congo, 2020.

To determine when severe acute respiratory syndrome coronavirus 2 arrived in Congo, we retrospectively antibody tested 937 blood samples collected during September 2019-February 2020. Seropositivity significantly increased from 1% in December 2019 to 5.3% in February 2020, before the first officially reported case in March 2020, suggesting unexpected early virus circulation.

Pyronaridine-artesunate real-world safety, tolerability, and effectiveness in malaria patients in 5 African countries: A single-arm, open-label, cohort event monitoring study.

BACKGROUND: In Phase II/III randomized controlled clinical trials for the treatment of acute uncomplicated malaria, pyronaridine-artesunate demonstrated high efficacy and a safety profile consistent with that of comparators, except that asymptomatic, mainly mild-to-moderate transient increases in liver aminotransferases were reported for some patients. Hepatic safety, tolerability, and effectiveness have not been previously assessed under real-world conditions in Africa. METHODS AND FINDINGS: This single-arm, open-label, cohort event monitoring study was conducted at 6 health centers in Cameroon, Democratic Republic of Congo, Gabon, Ivory Coast, and Republic of Congo between June 2017 and April 2019. The trial protocol as closely as possible resembled real-world clinical practice for the treatment of malaria at the centers. Eligible patients were adults or children of either sex, weighing at least 5 kg, with acute uncomplicated malaria who did not have contraindications for pyronaridine-artesunate treatment as per the summary of product characteristics. Patients received fixed-dose pyronaridine-artesunate once daily for 3 days, dosed by body weight, without regard to food intake. A tablet formulation was used in adults and adolescents and a pediatric granule formulation in children and infants under 20 kg body weight. The primary outcome was the hepatic event incidence, defined as the appearance of the clinical signs and symptoms of hepatotoxicity confirmed by a >2× rise in alanine aminotransferase/aspartate aminotransferase (ALT/AST) versus baseline in patients with baseline ALT/AST >2× the upper limit of normal (ULN). As a secondary outcome, this was assessed in patients with ALT/AST >2× ULN prior to treatment versus a matched cohort of patients with normal baseline ALT/AST. The safety population comprised 7,154 patients, of mean age 13.9 years (standard deviation (SD) 14.6), around half of whom were male (3,569 [49.9%]). Patients experienced 8,560 malaria episodes; 158 occurred in patients with baseline ALT/AST elevations >2×ULN. No protocol-defined hepatic events occurred following pyronaridine-artesunate treatment of malaria patients with or without baseline hepatic dysfunction. Thus, no cohort comparison could be undertaken. Also, as postbaseline clinical chemistry was only performed where clinically indicated, postbaseline ALT/AST levels were not systematically assessed for all patients. Adverse events of any cause occurred in 20.8% (1,490/7,154) of patients, most frequently pyrexia (5.1% [366/7,154]) and vomiting (4.2% [303/7,154]). Adjusting for Plasmodium falciparum reinfection, clinical effectiveness at day 28 was 98.6% ([7,369/7,746] 95% confidence interval (CI) 98.3 to 98.9) in the per-protocol population. There was no indication that comorbidities or malnutrition adversely affected outcomes. The key study limitation was that postbaseline clinical biochemistry was only evaluated when clinically indicated. CONCLUSIONS: Pyronaridine-artesunate had good tolerability and effectiveness in a representative African population under conditions similar to everyday clinical practice. These findings support pyronaridine-artesunate as an operationally useful addition to the management of acute uncomplicated malaria. TRIAL REGISTRATION: ClinicalTrials.gov NCT03201770.

Multiple insecticide resistance and Plasmodium infection in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon.

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a village located 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F 1 adult progeny. Bioassays were performed to assess resistance profile to insecticides. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having a similar sporozoite rate. Both species exhibited high levels of resistance to pyrethroids (<40% mortality). An. gambiae s.s. was also resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively . Furthermore, the high pyrethroid/DDT resistances in An. gambiae s.s. corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the high Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas.

Molecular surveillance of the Pfmdr1 N86Y allele among Congolese pregnant women with asymptomatic malaria.

BACKGROUND: Malaria in pregnancy is associated with considerable morbidity and mortality. Regular surveillance of artemisinin-based combination therapy tolerance, or molecular makers of resistance, is vital for effective malaria treatment, control and eradication programmes. Plasmodium falciparum multiple drug resistance-1 gene (Pfmdr1) N86Y mutation is associated with reduced susceptibility to lumefantrine. This study assessed the prevalence of Pfmdr1 N86Y in Brazzaville, Republic of Congo. METHODS: A total 1001 of P. falciparum-infected blood samples obtained from asymptomatic malaria pregnant women having a normal child delivery at the Madibou Integrated Health Centre were analysed. Pfmdr1 N86Y genotyping was conducted using PCR-restriction fragment length polymorphism. RESULTS: The wild type Pfmdr1 N86 allele was predominant (> 68%) in this study, whereas a few isolates carrying the either the mutant allele (Pfmdr1 86Y) alone or both alleles (mixed genotype). The dominance of the wildtype allele (pfmdr1 N86) indicates the plausible decline P. falciparum susceptibility to lumefantrine. CONCLUSION: This study gives an update on the prevalence of Pfmdr1 N86Y alleles in Brazzaville, Republic of Congo. It also raises concern on the imminent emergence of resistance against artemether-lumefantrine in this setting. This study underscores the importance to regular artemether-lumefantrine efficacy monitoring to inform the malaria control programme of the Republic of Congo.

Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo.

BACKGROUND: Acute diarrhea is a leading cause of morbidity and mortality among children under five worldwide. As no published data is available on the occurrence of this infection in the Republic of Congo, this study aimed at (1) determining the prevalence and (2) characterizing genotypes of norovirus strains in Brazzaville. METHODS: From June 2012 to June 2013, stool samples were collected from hospitalized young children with acute gastroenteritis. A total of 545 samples were tested for GI and GII norovirus infections using nested duplex reverse-transcription-polymerase chain reaction and sequencing. RESULTS: The GI and GII norovirus infection were detected in 148 samples. Males (28%) were not significantly more infected than females (25%). Norovirus infection was found exclusively in children aged under 24 months with a higher prevalence (P=0,048) in the age group of 7-12 months, and throughout the year with a peak in August and September. Genetic diversity of norovirus strains revealed that GII was the most prevalent (87%). No risk factor was significantly associated with norovirus infection. CONCLUSION: This study showed that noroviruses are important agents responsible for acute diarrhea in Congolese children and highlights the importance of continued surveillance.

Distribution of the cytochrome P450 CYP2C8*2 allele in Brazzaville, Republic of Congo.

BACKGROUND: Cytochrome P450 (CYP) enzymes are essential in the metabolism of most drugs used today. Single nucleotide polymorphism(s) occurring in CYP genes can adversely affect drug pharmacokinetics, efficacy, and safety. Individuals carrying the CYP2C8*2 c.805A > T (CYP2C8*2; rs11572103) allele have impaired amodiaquine metabolism, increased risk of amodiaquine-related adverse events, and may promote the selection of drug-resistant parasite strains. This study investigated the distribution of the CYP2C8*2 allele in Brazzaville, Republic of Congo, where artesunate + amodiaquine is used as the second-line treatment for uncomplicated Plasmodium falciparum malaria. METHODS: A total of 285 febrile children visiting the Marien Ngouabi paediatric hospital were genotyped for CYP2C8*2 using PCR-restriction fragment length polymorphism (PCR-RFLP). The allele frequencies and genotype distribution were determined. RESULTS: The CYP2C8*2 allele was successfully genotyped in 75% (213/285) of the study participants. The CYP2C8*2A allele had a frequency of 63%, whereas the CYP2C8*2T allele had a frequency of 37%. Genotypes CYP2C8*2AA (rapid metabolizer), CYP2C8*2AT (intermediate metabolizer), and CYP2C8*2TT (poor metabolizer) were observed in 44%, 38%, and 18% of the investigated participants, respectively. CONCLUSIONS: This study gives the first description of CYP2C8*2 allele distribution in the Republic of Congo and highlights the potential risk of amodiaquine-related adverse events. Information from this study will be beneficial during pharmacovigilance investigations.

Cytochrome P450 CYP2B6*6 distribution among Congolese individuals with HIV, Tuberculosis and Malaria infection.

BACKGROUND: The cytochrome P450 CYP2B6*6 (CYP2B6 c.516G>T; rs3745274) is one of the genetic factors that alters the drug metabolism in antimalarial, antiretroviral and TB first-line drugs. In Central African populations, the distribution of the CYP2B6*6 variant is poorly documented. This study investigated the distribution of CYP2B6 c.516G>T variant among Congolese individuals. METHODS: A total of 418 patients with HIV-1 mono-infection, HIV-1 and Tuberculosis coinfection and symptomatic P. falciparum malaria were genotyped for the CYP2B6 c.516G>T SNP using Restriction Fragment Length Polymorphism (RFLP). The allele frequencies and genotype distributions were determined. RESULTS: The CYP2B6 c.516G>T was successfully analysed in 69% (288/418) of the study participants. Among the investigated individuals, the distribution of the major allele CYP2B6*G was 45% and the minor CYP2B6*T allele was 55%. Significant differences in genotype distribution were also observed among the studied individuals. The CYP2B6*GG (rapid metabolizer) genotype was observed in 17% (49/288) followed by CYP2B6*GT (intermediate metabolizer) 55% (159/288) and CYP2B6*TT (poor metabolizers) 28% (80/288). CONCLUSION: This study contributes to increasing understanding on population pharmacogenetics and may help policy makers regulate treatment guidelines in the Congolese population with a high burden of HIV, Malaria and TB.

An update on glucose-6-phosphate dehydrogenase deficiency in children from Brazzaville, Republic of Congo.

BACKGROUND: Malaria transmission-blocking anti-malarial drugs, such as primaquine, offers an effective strategy for reducing the incidence of falciparum malaria. However, this drug induces haemolytic anaemia among glucose-6-phosphate dehydrogenase (G6PD) deficient individuals. The distribution of G6PD deficiency in Brazzaville, Republic of Congo and the association of G6PD deficiency with haemoglobin levels and blood cell counts were investigated. METHODS: A total of 212 febrile children were recruited for this study. Plasmodium falciparum diagnosis was conducted by microscopy and nested PCR. Sanger sequencing was used to assess G6PD deficiency by detecting 202G>A (rs1050828) and 376A>G (rs1050829) single nucleotide polymorphisms. RESULTS: Two hundred and twelve children were successfully genotyped for G6PD variants. Overall, 13% (27/212) of the children were G6PD deficient and 25% (25/100) females were heterozygous (11 BA- and 14 A+A-). The remaining 160 children had a normal G6PD genotype. The mean red blood and mean platelet counts were significantly lower in hemizygous male (G6PD A-) participants than in normal male (G6PD A+ or B) participants (p < 0.05). CONCLUSION: This study gives an update on G6PD deficiency among Congolese children. Understanding the distribution of G6PD deficiency in other geographical regions is recommended before primaquine is adopted in the malaria control programme.

Sub-microscopic Plasmodium falciparum infections in matched peripheral, placental and umbilical cord blood samples from asymptomatic Congolese women at delivery.

In malaria-endemic areas, most pregnant women are susceptible to asymptomatic Plasmodium falciparum infections. We present here the results of a cross-sectional study conducted in Madibou, a southern district of Brazzaville in the Republic of Congo, between March 2014 and April 2015. The main aim was to characterize P. falciparum infections. Blood samples corresponding to peripheral, placental and cord from 370 asymptomatic malaria women at delivery were diagnosed for plasmodium infection by thick blood smears (microscopic infection). Sub-microscopic infection was detected by PCR, using the MSP-2 gene as marker. Microscopic infections were detected in peripheral, placental and cord blood samples with a prevalence of respectively 7.3% (27/370), 2.7% (10/370) and 0%. The negative samples were submitted to sub-microscopic detection, with respective prevalence of 25.4% (87/343), 16.7% (60/360) and 9.4% (35/370) (P < 0.001). We further investigated the genetic diversity of the parasite by characterizing MSP2 allelic families 3D7 (24 distinct alleles) and FC27 (20 distinct alleles). The total number of alleles for these two families were 31, 25 and 19 in peripheral, placental and cord samples respectively. The 3D7 MSP-2 was the predominant allelic family. The multiplicity of infections (MOI) in peripheral (mean 1.4 ± 0.01; range 1-4), placental (mean 1.2 ± 0.01; range 1-3) and cord samples (1.4 ± 0.01; range 1-3) were similar (P = 0.9) and are unaffected by age, gravidity or sulfadoxine-pyrimethamine. These results shown a high prevalence of sub-microscopic infection and a high genetic diversity of Plasmodium falciparum strains in Congo. Age, gravidity and doses of preventive treatment based on sulfadoxine-pyrimethamine do not interfere with the multiplicity of infections.

Plasmodium falciparum merozoite protein-1 genetic diversity and multiplicity of infection in isolates from Congolese children consulting in a pediatric hospital in Brazzaville.

As in many sub-Saharan African countries, the burden of malaria has been reduced in the Republic of Congo as a result of massive deployment of insecticide treated nets and availability of artemisinin-combinations therapies (ACTs). High to moderate genetic diversity of msp-1 gene of Plasmodium falciparum (P. falciparum) has been reported from different parts of the world but limited data are available from Central Africa including the Republic of Congo. For this reason, the aim of study was to investigate the P. falciparum genetic diversity and to determine the multiplicity of infection in P. falciparum isolates from Congolese children in order to dispose of an additional parameter to measure the impact malaria control intervention. A total of 229 blood samples were collected from September 2014 to February 2015 in children aged from one to ten years presenting a paediatric hospital Marien NGOUABI located in Northern part of Brazzaville. Inclusion criterion was fever (axillary temperature ≥ 37.5 °C) or history of fever in the preceding 48 h before inclusion in this study. Then thick and thin blood smears were done to detect malaria parasites, to determine parasite density and to identify plasmodial species. Sub-microscopic infection was detected by PCR using the P. falciparum msp-1 gene as molecular marker. The prevalence of microscopic and sub-microscopic infection in this cohort was 10% and 27.5%, respectively. The K1 allelic family was predominant (45% of isolates) whereas the RO33 and MAD20 represented 35% and 20%, respectively of isolates. In this study 48% (38/79) of isolates harbored more than one parasite clone. Overall the multiplicity of infection (MOI) was 1.7. According to type of infection, the MOI was significantly higher in children with microscopic infection (2.5 vs 1.4 for submicroscopic infection, P = .001). When considering age, hemoglobin genotype (AA or AS) and level and parasite density, no association was observed with the MOI. This study reveals that the P. falciparum genetic diversity in isolates from Congolese children is high but with low multiplicity of infection.

Beyond genome-wide scan: Association of a cis-regulatory NCR3 variant with mild malaria in a population living in the Republic of Congo.

Linkage studies have revealed a linkage of mild malaria to chromosome 6p21 that contains the NCR3 gene encoding a natural killer cell receptor, whereas NCR3-412G>C (rs2736191) located in its promoter region was found to be associated with malaria in Burkina Faso. Here we confirmed the association of rs2736191 with mild malaria in a Congolese cohort and investigated its potential cis-regulatory effect. Luciferase assay results indicated that rs2736191-G allele had a significantly increased promoter activity compared to rs2736191-C allele. Furthermore, EMSAs demonstrated an altered binding of two nuclear protein complexes to the rs2736191-C allele in comparison to rs2736191-G allele. Finally, after in silico identification of transcription factor candidates, pull-down western blot experiments confirmed that both STAT4 and RUNX3 bind the region encompassing rs2736191 with a higher affinity for the G allele. To our knowledge, this is the first report that explored the functional role of rs2736191. These results support the hypothesis that genetic variation within natural killer cell receptors alters malaria resistance in humans.

Molecular surveillance of Plasmodium falciparum drug resistance in the Republic of Congo: four and nine years after the introduction of artemisinin-based combination therapy.

BACKGROUND: Resistance to anti-malarial drugs hinders efforts on malaria elimination and eradication. Following the global spread of chloroquine-resistant parasites, the Republic of Congo adopted artemisinin-based combination therapy (ACT) in 2006 as a first-line treatment for uncomplicated malaria. To assess the impacts after implementation of ACT, a molecular surveillance for anti-malarial drug resistance was conducted in Congo 4 and 9 years after the introduction of ACT. METHODS: Blood samples of 431 febrile children aged 1-10 years were utilized from two previous studies conducted in 2010 (N = 311) and 2015 (N = 120). All samples were screened for malaria parasites using nested PCR. Direct sequencing was used to determine the frequency distribution of genetic variants in the anti-malarial drug-resistant Plasmodium falciparum genes (Pfcrt, Pfmdr1, Pfatp6, Pfk13) in malaria-positive isolates. RESULTS: One-hundred and nineteen (N = 70 from 2010 and N = 49 from 2015) samples were positive for P. falciparum. A relative decrease in the proportion of chloroquine-resistant haplotype (CVIET) from 100% in 2005, 1 year before the introduction and implementation of ACT in 2006, to 98% in 2010 to 71% in 2015 was observed. Regarding the multidrug transporter gene, a considerable reduction in the frequency of the mutations N86Y (from 73 to 27%) and D1246Y (from 22 to 0%) was observed. However, the prevalence of the Y184F mutation remained stable (49% in 2010 compared to 54% in 2015). Isolates carrying the Pfatp6 H243Y was 25% in 2010 and this frequency was reduced to null in 2015. None of the parasites harboured the Pfk13 mutations associated with prolonged artemisinin clearance in Southeast Asia. Nevertheless, 13 new Pfk13 variants are reported among the investigated isolates. CONCLUSION: The implementation of ACT has led to the decline in prevalence of chloroquine-resistant parasites in the Republic of Congo. However, the constant prevalence of the PfMDR1 Y184F mutation, associated with lumefantrine susceptibility, indicate a selective drug pressure still exists. Taken together, this study could serve as the basis for epidemiological studies monitoring the distribution of molecular markers of artemisinin resistance in the Republic of Congo.

Malaria epidemiological research in the Republic of Congo.

BACKGROUND: Reliable and comprehensive information on the burden of malaria is critical for guiding national and international efforts in malaria control. The purpose of this review is to provide an overview of published data and available information on malaria resulting from field studies/investigations conducted in the Republic of Congo (RoC) from 1992 to 2015, as baseline for assisting public health authorities and researchers to define future research priorities as well as interventions. METHODS: This review considers data from peer-reviewed articles and information from the National Malaria Control Programme reports, based on field investigations or samples collected from 1992 to 2015. Peer-reviewed papers were searched throughout online bibliographic databases PubMed, HINARI and Google Scholar using the following terms: "malaria", "Congo", "Brazzaville", "prevalence", "antimalarial", "efficacy", "falciparum", "genetic", "diversity". Original articles and reviews were included and selection of relevant papers was made. RESULTS: Twenty-eight published articles were included in this review and two additional records from the National Malaria Control Programme were also considered. The majority of studies were conducted in Brazzaville and Pointe-Noire. CONCLUSION: The present systematic review reveals that number of studies have been conducted in the RoC with regard to malaria. However, their results cannot formally be generalized at the country level. This suggests a need for implementing regular multisite investigations and surveys that may be representative of the country, calling for the support and lead of the Ministry of Health.

Plasmodium falciparum infection in febrile Congolese children: prevalence of clinical malaria 10 years after introduction of artemisinin-combination therapies.

OBJECTIVES: To investigate the proportion of malaria infection in febrile children consulting a paediatric hospital in Brazzaville, to determine the prevalence of submicroscopic malaria infection, to characterise Plasmodium falciparum infection and compare the prevalence of uncomplicated P. falciparum malaria according to haemoglobin profiles. METHODS: Blood samples were collected from children aged <10 years with an axillary temperature ≥37.5 °C consulting the paediatric ward of Marien Ngouabi Hospital in Brazzaville. Parasite density was determined and all samples were screened for P. falciparum by nested polymerase chain reaction (PCR) using the P. falciparum msp-2 marker to detect submicroscopic infections and characterise P. falciparum infection. Sickle cell trait was screened by PCR. RESULTS: A total of 229 children with fever were recruited, of whom 10% were diagnosed with uncomplicated malaria and 21% with submicroscopic infection. The mean parasite density in children with uncomplicated malaria was 42 824 parasites/µl of blood. The multiplicity of infection (MOI) was 1.59 in children with uncomplicated malaria and 1.69 in children with submicroscopic infection. The mean haemoglobin level was 10.1 ± 1.7 for children with uncomplicated malaria and 12.0 ± 8.6 for children with submicroscopic infection. About 13% of the children harboured the sickle cell trait (HbAS); the rest had normal haemoglobin (HbAA). No difference in prevalence of uncomplicated malaria and submicroscopic infection, parasite density, haemoglobin level, MOI and P. falciparum genetic diversity was observed according to haemoglobin type. CONCLUSION: The low prevalence of uncomplicated malaria in febrile Congolese children indicates the necessity to investigate carefully other causes of fever.

Molecular epidemiology and surveillance of circulating rotavirus and adenovirus in Congolese children with gastroenteritis.

Infectious Diarrhea caused by rotavirus and adenovirus, is a leading cause of death in children in sub-Sahara Africa but there is limited published data on the diverse rotavirus genotypes and adenovirus serotypes circulating in the Republic of Congo. In this study, we investigated the prevalence of severe diarrhea caused by rotavirus A (RVA) and Adenovirus serotype 40 and 41 in Congolese children hospitalized with severe gastroenteritis. Stool samples were collected from 655 Congolese children less than 60 months of age hospitalized with acute gastroenteritis between June 2012 and June 2013. Rotavirus and adenovirus antigens were tested using commercially available ELISA kits and the RVA G- and P- genotypes were identified by seminested multiplex RT-PCR. Three hundred and four (46.4%) children were tested positive for RVA. Adenovirus infection was found in 5.5% of the 564 tested children. Rotavirus infection was frequently observed in children between 6-12 months (55.9%). The dry season months recorded increased RVA infection while no seasonality of adenovirus infection was demonstrated. The most common RVA genotypes were G1 (57.5%), G2 (6.4%), G1G2 mixture (15.5%), P[8] (58%), P[6] (13.2%), and P[8]P[6] mixture (26%). Additionally, the genotype G12P[6] was significantly associated with increased vomiting. This first study on Congolese children demonstrates a high prevalence and clinical significance of existing rotavirus genotypes. Adenovirus prevalence is similar to that of other Central African countries. This baseline epidemiology and molecular characterization study will contribute significantly to the RVA surveillance after vaccine implementation in the country.

Artesunate-amodiaquine versus artemether-lumefantrine for the treatment of acute uncomplicated malaria in Congolese children under 10 years old living in a suburban area: a randomized study.

BACKGROUND: The Republic of Congo adopted a new anti-malarial treatment policy in 2006, with artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) as the first- and second-line anti-malarial drugs, respectively. Only three clinical studies had been conducted before the policy change. A randomized study on these two artemisinin-based combinations was conducted, and the effect that sickle cell trait may have on treatment outcomes was evaluated in children under 10 years old followed during 12 months in Brazzaville in 2010-2011. METHODS: A cohort of 330 children under 10 years of age living in a suburban area in the south of Brazzaville were passively followed for registration of malaria episodes. Uncomplicated Plasmodium falciparum episodes were randomly treated with co-formulated ASAQ (Coarsucam(®)) or AL (Coartem(®)). Patients were followed according to the 2009 World Health Organization protocol for the evaluation of anti-malarial drug efficacy. Plasmodium falciparum recrudescent isolates were compared to pre-treatment isolates by polymerase chain reaction (PCR) to distinguish between re-infection and recrudescence. PCR-uncorrected and PCR-corrected responses to treatment were determined using per protocol analysis. Haemoglobin type (AA, AS, SS) was determined by PCR. RESULTS: Of 282 clinical malaria episodes registered during 1-year follow-up period, 262 children with uncomplicated malaria were treated with ASAQ (129 patients) or AL (133 patients). The PCR-corrected efficacy, expressed as the percentage of adequate clinical and parasitological response, was 97.0 % for ASAQ and 96.4 % for AL. Among ASAQ-treated patients, 112 (86.8 %) carried AA genotype and 17 (13.2 %) were AS carriers. The PCR-corrected efficacy was 96.4 % for AA-carriers and 100 % for AS-carriers treated with ASAQ [relative risk (RR) = 0.96; 95 % confidence interval, 0.93-1.00, p = 0.5]. Among 133 AL-treated children, 109 (82 %) carried AA, and 24 (18 %) AS genotypes. The PCR-corrected efficacy was 96.7 % among AA-carriers and 95.2 % among AS-carriers [RR = 1.01 (0.92-1.12), p = 0.6]. Nausea, jaundice, headache, dizziness, vomiting, pruritus, abdominal pain, and diarrhoea were registered as adverse events in both groups. ASAQ was associated with significantly more frequent adverse events (P < 0.05). CONCLUSION: This first randomized study in Brazzaville confirmed the excellent efficacy of these co-formulated anti-malarial drugs in children. Sickle cell genotype did not influence the treatment efficacy of artemisinin-based combination therapy.

High prevalence of sulphadoxine-pyrimethamine resistance-associated mutations in Plasmodium falciparum field isolates from pregnant women in Brazzaville, Republic of Congo.

Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) has not been evaluated in the Republic of Congo since its implementation in 2006 and there is no published data on molecular markers of SP resistance among Plasmodium falciparum isolates from pregnant women. This first study in this country aimed to describe the prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) point mutations and haplotypes in P. falciparum isolates collected from pregnant women with asymptomatic infection. From March 2012 to December 2013, pregnant women attending Madibou health centre (in Southern Brazzaville) for antenatal visits were enrolled in this study after obtaining their written informed consent. Blood samples were collected and P. falciparum infections were characterized using PCR. A total of 363 pregnant women were enrolled. P. falciparum infection was detected in 67 (18.4%) samples as their PCR amplification of dhfr and dhps genes yielded bands and all the PCR products were successfully digested. Out of these 67 isolates, 59 (88%), 57 (85%) and 53 (79.1%) carried 51I, 59R and 108N dhfr mutant alleles, respectively. The prevalence of dhps 436A, 437G and 540E mutations were 67.1% (45/67), 98.5% (66/67) and 55.2% (37/67), respectively. More than one-half of the isolates carried quintuple mutations, with highly resistant haplotype dhfr51I/59R/108N + dhps437G/540E detected in 33% (22/67) whereas 25% (17/67) were found to carry sextuple mutations. We observed significantly higher frequencies of triple dhps mutations 436A/437G/540E and quintuple mutations dhfr51I/59R/108N+dhps437G/540E in isolates from women who received IPTp-SP than those who did not. Overall, this study shows high prevalence rates of SP-associated resistance mutations in P. falciparum isolates collected from pregnant women. The presence of the dhps mutant allele 540E and the high prevalence of isolates carrying quintuple dhfr/dhps mutations are here reported for the first time in the Republic of Congo. The increasing prevalence of multiple mutant alleles observed in this study is alarming and may present a challenge for the future interventions including IPTp-SP in the country.