Epigenetic studies in children at risk of stunting and their parents in India, Indonesia and Senegal: a UKRI GCRF Action Against Stunting Hub protocol paper.

INTRODUCTION: In 2020, an estimated 150 million children under the age of 5 years were stunted. Stunting results from early-life adversity and it is associated with significant physical and cognitive deficit, lifelong socioeconomic disadvantage and reduced life expectancy. There is a need to understand the causes of stunting and its effects in order to develop strategies to avoid it and to mitigate the consequences once stunting has occurred. Epigenetics is an important mechanism through which early-life factors are thought to influence biological function, with long-term consequences. We describe a series of epigenetic studies designed to understand how early-life adversity results in stunting and to inform the development of practical tools such as predictive markers and therapeutic targets. This work is part of the UKRI GCRF Action Against Stunting Hub. METHODS AND ANALYSIS: The project-in India, Indonesia and Senegal-comprises an observational study of mothers, fathers, and offspring (n=500) spanning the first 1000 days of life, and an intervention study in each country. Epigenetic status (DNA methylation) is determined in saliva from babies collected within 1 month of birth and again at 18 months of age, and from mothers and fathers around the time of birth. Epigenome-wide analysis is carried out using the Illumina EPIC array, augmented by high-definition sequencing approaches. Statistical analysis is carried out at the level of candidate genes/regions, higher dimensional epigenetic states and epigenome-wide association. Data analysis focuses on the determinants of stunting, the effectiveness of interventions, population comparisons and the link between epigenetics and other thematic areas, which include anthropometry, microbiome, gut health, parasitology, cognition, nutrition, food hygiene and water sanitation, food systems and the home environment. ETHICS AND DISSEMINATION: This study has been approved by the relevant Ethics Committees in Indonesia, India and Senegal, and the UK. Research data will be published and posted in public repositories.

Prevalence of Malaria Infection in Pregnant Women Attending Antenatal Clinics in Southern Senegal.

Despite marked progress in Senegal, three regions in the southeast part continue to have a high burden of malaria, but there have been no recent studies assessing the prevalence of malaria associated with pregnancy. This study aimed to determine the prevalence of malaria infection in pregnant women attending antenatal clinics in Senegal. During the malaria transmission season of 2019, pregnant women attending 11 health care facilities for a scheduled visit and those presenting unwell with signs of malaria were invited to participate in a malaria screening study. A finger prick blood sample was taken for malaria diagnosis by rapid diagnosis test (RDT) and polymerase chain reaction (PCR). A total of 877 pregnant women were enrolled, 787 for a scheduled antenatal consultation and 90 for an unscheduled consultation with signs of malaria. The prevalence of Plasmodium falciparum among the first group was 48% by PCR and 20% by RDT, and that among the second group was 86% by PCR and 83% by RDT. RDT sensitivity in capturing asymptomatic, PCR-positive infections was 9.2% but ranged from 83% to 94% among febrile women. The prevalence of infection by PCR in women who reported having received at least three doses of sulfadoxine pyrimethamine (SP) was 41.9% compared with 58.9% in women who reported they had not received any SP doses (prevalence ratio adjusted for gravidity and gestational age, 0.54; 95% CI, 0.41-0.73). The burden of P. falciparum infections remains high among pregnant women, the majority of which are not captured by RDT. More effective measures to prevent malaria infection in pregnancy are needed.

Cytobrush and cotton swab as sampling tools for molecular diagnosis of female genital schistosomiasis in the uterine cervix.

Female genital schistosomiasis (FGS) caused by Schistosoma haematobium is a neglected chronic parasitic disease. Diagnosis relies mainly on a colposcopy, which reveals non-specific lesions. This study aimed to assess the performance of two sampling methods for the molecular diagnosis of FGS in the uterine cervix. We conducted a descriptive cross-sectional study in women of reproductive age in Saint Louis, Senegal, who presented for cervical cancer screening. Cotton swab and cytobrush samples were collected from the cervix and examined by real-time PCR. The PCR results obtained using the cotton swabs were compared with those obtained using cytobrush. Of the 189 women recruited, 56 (30%) were found to be positive for S. haematobium infection via real-time PCR. Women aged 40-54 years were predominantly infected (45%) followed by those aged 25-39 years (36%). Numerically more PCR-positive specimens were identified using cytobrush sampling. Of the 89 women who underwent both cytobrush and cotton swab sampling, 27 were PCR-positive in the cytobrush sampling vs 4 in the swab sampling. The mean Ct-value was 31.0 ± 3.8 for cytobrush-based PCR vs 30.0 ± 4.4 for swab-based PCR. The results confirm that real-time PCR can detect Schistosoma haematobium DNA in the uterine cervix. The next step will be to compare PCR with the other diagnostic methods of FGS.

A Case of Visceral Leishmaniasis in a 4-Year-Old Child Living in Nonendemic Area Located in Suburbs of Dakar, Senegal.

Visceral leishmaniasis (VL) is an infectious disease caused by protozoa of the genus Leishmania. Sporadic cases are observed in nonendemic areas and often associated with limited foci; therefore, the disease is easily overlooked. In addition, other diseases have similar clinical symptoms, which make it difficult for clinicians to make an accurate diagnosis and to provide effective treatment. We identified visceral leishmaniasis in a 4-year-old child in Pikine, Senegal. The patient was admitted to the Pikine National Teaching Hospital for haemorrhagic, tumoral, and infectious syndromes. At admission, the patient presented with epistaxis and gingivorrhagia, a severe anaemic syndrome poorly tolerated, a systemic inflammatory response syndrome with fever at 39.5°C, a tumoral syndrome with 11 cm of hepatomegaly and 12 cm of type IV splenomegaly, and noninflammatory macropoly adenopathies. A spinal cord puncture was performed, and direct microscopy examination of the sample after GIEMSA staining revealed amastigote forms of Leishmania. The PCR amplification of extracted DNA from the bone marrow aspiration using specific primers for VL (forward and reverse) confirmed that VL was responsible for the infection. A treatment with meglumine antimoniate (Glucantime) was given and it gave a successful outcome with remission of clinical symptoms and favourable evolution with 3 months hindsight. Conclusion. This visceral leishmaniasis case diagnosis in Senegal has shown that, apart from haematological malignancies, this disease must be considered in combination with a tumor syndrome, haemorrhagic syndrome, and infectious syndrome.

Malaria-associated risk factors among adolescents living in areas with persistent transmission in Senegal: a case-control study.

BACKGROUND: In Senegal, malaria morbidity has sharply decreased over these past years. However, malaria epidemiology remains heterogeneous with persistent transmission in the southeastern part of the country and many cases among older children and adolescents. Little is known about factors associated with clinical malaria among this group. A better understanding of malaria transmission among this newly emerging vulnerable group will guide future interventions targeting this population group. This study aimed to identify factors associated with clinical malaria among adolescents in Senegal. METHODS: A case-control study was conducted from November to December 2020 in four health posts located in the Saraya district. Cases were defined as adolescents (10-19 years) with an uncomplicated malaria episode with fever (temperature > 37.5°) or a history of fever and positive malaria rapid diagnostic test (RDT). Controls were from the same age group, living in the neighbourhood of the case, presenting a negative RDT. A standardized, pre-tested questionnaire was administered to each study participant followed by a home visit to assess the participant's living conditions. Factors associated with clinical malaria were assessed using stepwise logistic regression analysis. RESULTS: In total, 492 individuals were recruited (246 cases and 246 controls). In a multivariate analysis, factors associated with clinical malaria included non-use of long-lasting insecticidal net (LLIN) (aOR = 2.65; 95% CI 1.58-4.45), non-use of other preventive measures (aOR = 2.51; 95% CI 1.53-4.11) and indoor sleeping (aOR = 3.22; 95% CI 1.66-6.23). Protective factors included 15-19 years of age (aOR = 0.38; 95% CI 0.23-0.62), absence of stagnant water around the house (aOR = 0.27; 95% CI 0.16-0.44), having a female as head of household (aOR = 0.47; 95% CI 0.25-0.90), occupation such as apprentice (OR = 0.24; 95% CI 0.11-0.52). CONCLUSIONS: The study revealed that environmental factors and non-use of malaria preventive measures are the main determinants of malaria transmission among adolescents living in areas with persistent malaria transmission in Senegal. Strategies aimed at improving disease awareness and access to healthcare interventions, such as LLINs, are needed to improve malaria control and prevention among these vulnerable groups.

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.

Malaria parasite carriage before and two years after the implementation of seasonal malaria chemoprevention: a case study of the Saraya health district, southern Senegal.

Background : Seasonal malaria chemoprevention (SMC) has been adopted and implemented in the southern regions of Senegal in children aged between three and 120 months since 2013. Scaling up this strategy requires its evaluation to assess the impact. This study was carried out to determine the dynamics of Plasmodium falciparum carriage before and after two years of SMC implementation. Methods : Four household surveys were conducted in villages in the health district of Saraya, which is a SMC implementation area in Senegal. These villages were selected using probability proportional to size sampling. Each selected village was divided into segments containing at least 50 children. In each segment, a household questionnaire was administered to the parents or legal representatives of children aged three to 120 months. Blood smears were collected to determine P. falciparum prevalence by microscopy one month before the first round of SMC, one month after the last round of the first SMC campaign and two years after the start of the implementation. Results : A total of 2008 children were included with a mean average age of 4.81 (+/-2.73) years. Of the study population, 50.33% were more than five years old and 50.3% were male. In 2013, mosquito net ownership was 99.4 % before the SMC campaign and 97.4% after. In 2015, it was 36.6% before and 45.8% after the campaign. In 2013, the prevalence of plasmodium carriage was 11.8% before and 6.1% after the SMC campaign. In 2015, the prevalence was 4.9% before the administration of SMC and this increased up to 15.3% after. Malaria prevalence was high among children over five years old and in boys. Conclusions : The decrease in Plasmodium falciparum parasite prevalence, which subsequently increased after two years of SMC implementation in this study, suggests adding an extra cycle of the SMC or adjusting the administration period.

Anemia, Thrombocytopenia, and Changes in Biochemical Parameters Occurring in Patients with Uncomplicated Plasmodium falciparum Malaria: Data Analysis from Antimalarial Efficacy-Randomized Trials in Dakar and Kaolack Regions, Senegal.

Background: Artemisinin-based Combination Therapies (ACTs) are widely used in the treatment of uncomplicated malaria. Plasmodium falciparum infection is often accompanied by disturbances of hematological and biochemical parameters. The objective of this study was to evaluate the changes in biochemical and hematological parameters during uncomplicated malaria in patients treated with ACTs. Methods: Data from patient with uncomplicated Plasmodium falciparum malaria were pooled from different open-randomized trial evaluating the efficacy of Artesunate-Mefloquine (ASMQ), Artesunate-Amodiaquine (ASAQ), Artemether-Lumefantrine (AL), and Dihydro-artemisinin-Piperaquine (DHAPQ) combinations. Biochemical (transaminases, creatinine, and bilirubin) and hematological (hemoglobin and platelet levels) parameters were performed at baseline (D0) and at day 7 after treatment (D7). Data were analyzed as both continuous and categorical variables with 95% confidence interval. Risks and trends were calculated using multivariate logistic random effect models. Results: A total of 720 patients with completed biological data were included in the analysis (320 in the AL arm, 160 in the ASMQ arm, 120 in the DHAPQ arm, and 88 in the ASAQ arm). The mean age of the patients was 9.43 ± 9.1 years. Male subjects represented 58.47% (sex ratio was 1.4 for males). The mean hemoglobin level at inclusion (D0) was 9.79 g/dl and anemia (Hb < 11 g/dl) was 71.43% (aOR = 1.16 [0.68 - 1.98]p = 0.57). At D7, hemoglobin level was 9.63 g/dl and anemia was significantly more frequent (78.29% [p = 0.002]). The mean platelet count at day 0 was 154075.5 platelets/mm3 of blood and 339328.7 platelets/mm3 at day 7. Thrombocytopenia was about 53.61% and was associated with malaria (aOR = 3.4 [2.18 - 5.3]p < 10-3). 19.58% of patients had abnormal ALT and 40.28% had abnormal AST at D0. 27.22% of patients had normal bilirubin at D0. Renal function was normal in all patients in the study. Normalization of transaminases was noted between D0 and D7. The percentage of subjects with normal bilirubin increased between D0 and D7. Renal function did not vary significantly between D0 and D7. Conclusion: Results from this analysis showed that subjects with high parasitaemia had a greater risk of anemia and thrombocytopenia. Artemisinin combinations were well-tolerated as no major biological disturbances were noted. The effects of ACTs on hematologic and biochemical parameters were not different.

Specificity of SARS-CoV-2 Antibody Detection Assays against S and N Proteins among Pre-COVID-19 Sera from Patients with Protozoan and Helminth Parasitic Infections.

We aimed to assess the specificity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody detection assays among people with tissue-borne parasitic infections. We tested three SARS-CoV-2 antibody-detection assays (cPass SARS-CoV-2 neutralization antibody detection kit [cPass], Abbott SARS-CoV-2 IgG assay [Abbott Architect], and Standard Q COVID-19 IgM/IgG combo rapid diagnostic test [SD RDT IgM/SD RDT IgG]) among 559 pre-COVID-19 seropositive sera for several parasitic infections. The specificity of assays was 95 to 98% overall. However, lower specificity was observed among sera from patients with protozoan infections of the reticuloendothelial system, such as human African trypanosomiasis (Abbott Architect; 88% [95% CI, 75 to 95]) and visceral leishmaniasis (SD RDT IgG; 80% [95% CI, 30 to 99]), and from patients with recent malaria in areas of Senegal where malaria is holoendemic (ranging from 91% for Abbott Architect and SD RDT IgM to 98 to 99% for cPass and SD RDT IgG). For specimens from patients with evidence of past or present helminth infection overall, test specificity estimates were all ≥96%. Sera collected from patients clinically suspected of parasitic infections that tested negative for these infections yielded a specificity of 98 to 100%. The majority (>85%) of false-positive results were positive by only one assay. The specificity of SARS-CoV-2 serological assays among sera from patients with tissue-borne parasitic infections was below the threshold required for decisions about individual patient care. Specificity is markedly increased by the use of confirmatory testing with a second assay. Finally, the SD RDT IgG proved similarly specific to laboratory-based assays and provides an option in low-resource settings when detection of anti-SARS-CoV-2 IgG is indicated.

Effectiveness of seasonal malaria chemoprevention administered in a mass campaign in the Kedougou region of Senegal in 2016: a case-control study.

Background: Seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine plus amodiaquine (SPAQ) is a malaria prevention strategy recommended since 2012 by the World Health Organization (WHO) for children under 5 years. In Senegal, the scaling up of SMC started in 2013 in the south-eastern regions of the country with an extension of the target to 10 years old children. The scaling up of SMC requires regular evaluation of the strategy as recommended by the WHO. This study was conducted to evaluate the effectiveness of SMC. Methods: A case-control study was conducted in some villages of the health districts of Saraya and Kedougou in the Kedougou region from July to December 2016. A case was a sick child, aged 3 months to 10 years, seen in consultation and with a positive malaria rapid diagnostic test (RDT). The control was a child of the same age group with a negative RDT and living in the same compound as the case or in a neighbouring compound. Each case was matched with two controls. Exposure to SMC was assessed by interviewing the mothers/caretakers and by checking the SMC administration card. Results: Overall, 492 children, including 164 cases and 328 controls, were recruited in our study. Their mean ages were 5.32 (+/- 2.15) and 4.44 (+/-2.25) years for cases and controls, respectively. The number of boys was higher in both cases (55.49%; CI 95%=47.54-63.24%) and controls (51,22%; CI 95%=45.83-56.58%). Net ownership was 85.80% among cases and 90.85% among controls (p=0,053). The proportion of controls who received SMC was higher than that of cases (98.17% vs 85.98% and p=1.10 -7). The protective effectiveness of SMC was 89% (OR= 0.12 (CI 95%=0.04-0.28)). Conclusions: SMC is an effective strategy in the control of malaria in children. Case-control studies are a good approach for monitoring the efficacy of drugs administered during SMC.

Identification of Single-Nucleotide Polymorphisms in the Mitochondrial Genome and Kelch 13 Gene of Plasmodium falciparum in Different Geographical Populations.

The emergence of artemisinin-resistant Plasmodium falciparum parasites in Southeast Asia threatens malaria control and elimination. The interconnectedness of parasite populations may be essential to monitor the spread of resistance. Combining a published barcoding system of geographically restricted single-nucleotide polymorphisms (SNPs), mainly mitochondria of P. falciparum with SNPs in the K13 artemisinin resistance marker, could elucidate the parasite population structure and provide insight regarding the spread of drug resistance. We explored the diversity of mitochondrial SNPs (bp position 611-2825) and identified K13 SNPs from malaria patients in the districts of India (Ranchi), Tanzania (Korogwe), and Senegal (Podor, Richard Toll, Kaolack, and Ndoffane). DNA was amplified using a nested PCR and Sanger-sequenced. Overall, 199 K13 sequences (India: N = 92; Tanzania: N = 48; Senegal: N = 59) and 237 mitochondrial sequences (India: N = 93; Tanzania: N = 48; Senegal: N = 96) were generated. SNPs were identified by comparisons with reference genomes. We detected previously reported geographically restricted mitochondrial SNPs (T2175C and G1367A) as markers for parasites originating from the Indian subcontinent and several geographically unrestricted mitochondrial SNPs. Combining haplotypes with published P. falciparum mitochondrial genome data suggested possible regional differences within India. All three countries had G1692A, but Tanzanian and Senegalese SNPs were well-differentiated. Some mitochondrial SNPs are reported here for the first time. Four nonsynonymous K13 SNPs were detected: K189T (India, Tanzania, Senegal); A175T (Tanzania); and A174V and R255K (Senegal). This study supports the use of mitochondrial SNPs to determine the origin of the parasite and suggests that the P. falciparum populations studied were susceptible to artemisinin during sampling because all K13 SNPs observed were outside the propeller domain for artemisinin resistance.

Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal.

BACKGROUND: Malaria surveillance requires powerful tools and strategies to achieve malaria elimination. Rapid diagnostic tests for malaria (RDTs) are easily deployed on a large scale and are helpful sources of parasite DNA. The application of sensitive molecular techniques to these RDTs is a modern tool for improving malaria case detection and drug resistance surveillance. Several studies have made it possible to extract the DNA of Plasmodium falciparum from RDTs. The knowledge of gametocyte carriage in the population is important to better assess the level of parasite transmission in elimination settings. The aim of this study was to detect P. falciparum gametocytes from used RDTs by quantitative PCR for molecular monitoring of malaria transmission. METHODS: DNA was extracted from 303 RDT devices (SD Bioline Malaria Pf) using the Chelex-100 protocol. qPCR was performed in a 20 µL reaction to detect and quantify transcripts of the pfs25 gene. The cycle threshold (Ct) was determined by the emission fluorescence corresponding to the initial amount of amplified DNA. RESULTS: The study found an overall prevalence of 53.47% with an average Ct of 32.12 ± 4.28 cycles. In 2018, the prevalence of gametocytes was higher in the Ranérou district (76.24%) than in the Saint-Louis district (67.33%) where an increase in the number of gametocyte carriers in 2018 was noted, in comparison with 2017. CONCLUSIONS: RDTs are a good source of DNA for molecular monitoring of gametocyte carriage. This method is a simple and effective tool to better understand the level of malaria transmission with a view to elimination.

Entomological impact of indoor residual spraying with pirimiphos-methyl: a pilot study in an area of low malaria transmission in Senegal.

BACKGROUND: Scaling-up of effective anti-malarial control strategies in Central-West region of Senegal has resulted in the sharp decline in malaria prevalence in this area. However, despite these strategies, residual malaria transmission has been observed in some villages (hot spots). The objective of this study was to assess the impact of indoor residual spraying (IRS) with pirimiphos-methyl on malaria transmission in hot spot areas. METHODS: The malaria vector population dynamics were monitored in each of the six selected villages (4 of which used IRS, 2 were unsprayed control areas) using overnight human landing catches (HLC) and pyrethrum spray catches (PSC). The host source of blood meals from freshly fed females collected using PSC was identified using the direct ELISA method. Females caught through HLC were tested by ELISA for the detection of Plasmodium falciparum circumsporozoite protein and Anopheles gambiae complex was identified using PCR. RESULTS: Preliminary data shown that the densities of Anopheles populations were significantly lower in the sprayed areas (179/702) compared to the control. Overall, malaria transmission risk was 14 times lower in the intervention zone (0.94) compared to the control zone (12.7). In the control areas, three Anopheles species belonging to the Gambiae complex (Anopheles arabiensis, Anopheles coluzzii and Anopheles melas) maintained the transmission, while only An. coluzzii was infective in the sprayed areas. CONCLUSION: The preliminary data from this pilot study showed that IRS with the CS formulation of pirimiphos-methyl is likely very effective in reducing malaria transmission risk. However, additional studies including further longitudinal entomological surveys as well as ecological and ethological and genetical characterization of vectors species and their populations are needed to better characterize the entomological impact of indoor residual spraying with pirimiphos-methyl in the residual transmission areas of Senegal.

Country-Wide Surveillance of Molecular Markers of Antimalarial Drug Resistance in Senegal by Use of Positive Malaria Rapid Diagnostic Tests.

In Senegal, antimalarial drugs used in treatment and prevention of malaria are one of the main reasons for the current success in controlling malaria. However, the successful control of malaria is highly dependent on continued effectiveness of these drugs which may be compromised by the spread of drug resistance. Therefore, surveillance of drug resistance in the malaria parasites is essential. The objective of this pilot study was to test the feasibility of routinely sampled malaria rapid diagnostic tests (RDTs) at a national scale to assess the temporal changes in the molecular profiles of antimalarial drug resistance markers of Plasmodium falciparum parasites. Overall, 9,549 positive malaria RDTs were collected from 14 health facilities across the country. A limited random set of RDTs were analyzed regarding Pfcrt gene polymorphisms at codon 72-76. Overall, a high but varied prevalence (> 50%) of the wild-type CVMNK haplotype was observed including a higher CVMNK prevalence in the northern part (75%) compared with the southern part of the country (59%). With caution, the study provides a proof of concept that reuse of discarded P. falciparum positive RDTs can be applied in large-scale surveillance of antimalarial drug resistance.

Comparison of Para-Selles Bailenger/Kop-Color Fumouze, Para-Selles-Iodésine/Kop-Color II Fumouze diagnostic kits with conventional microscopic methods in identifying intestinal parasitic diseases in Senegal.

In the context of controlling intestinal parasites, accurate diagnosis is essential. Our objective was to evaluate the performance of new diagnostic kits compared to conventional microscopic methods in identifying intestinal parasites. Faeces collected in rural area in Senegal were subjected to several detection techniques. Thus, the sensitivity, specificity, positive and negative predictive values of new diagnostic techniques were compared to conventional merthiolate-iodine-formalin, conventional Bailenger and modified Ritchie. Furthermore, the kappa coefficient was calculated to evaluate the correlation between the new kit and those of modified Ritchie. Out of the 117 patients examined, 102 presented with a parasite, or prevalence of 87.1%. The Fumouze techniques proved to be as effective as the conventional methods in detecting flagellates and helminths with sensitivities ranging from 97 to 100%. However, conventional techniques were slightly more sensitive in identifying Endolimax nana and Blastocystis hominis. The correlation was nearly perfect (k = 0.83 and 1), respectively between Bailenger Fumouze, Iodesine Fumouze and modified Ritchie in identifying helminths while it was just acceptable (k = 0.27 and 0.28) in identifying B. hominis. The modified Ritchie technique routinely used in our laboratory remains a good diagnostic tool. However, the use of kit techniques was interesting when reading the pellet after concentration and the Colour KOP staining was a considerable contribution to the diagnosis of the vegetative forms. Therefore, it would be interesting to determine the cost of a stool test using Fumouze kit techniques to provide the most cost effective way.

Performance of Real-Time Polymerase Chain Reaction Assays for the Detection of 20 Gastrointestinal Parasites in Clinical Samples from Senegal.

Gastrointestinal parasite infections represent one of the biggest public health problems in the world. Therefore, appropriate innovative tools are needed for assessing interventions to control these infections. This study aims to compare the performance of real-time polymerase chain reaction (PCR) assays to microscopic examination for detection of intestinal parasites. A direct microscopic examination and stool concentration was performed on 98 stool samples from patients attending Senegalese hospitals. Negative microscopic control samples were also collected in Nice and Marseille (France). Species-specific primers/probes were used to detect 20 common gastrointestinal protozoans and helminths. Positive frequency and the sensitivity of each real-time PCR assay were compared with conventional microscopic examination. Real-time PCR was positive in 72 of 98 samples (73.5%), whereas microscopic examination was positive in 37 (37.7%) samples (P < 0.001). The real-time PCR assays were more sensitive than microscopy, with 57.4% (31/54) versus 18.5% (10/54), respectively, in the detection of parasites in asymptomatic patients (P < 0.05). In terms of polyparasitism, there were more coinfections detected by real-time PCR assays compared with microscopic methods (25.5% versus 3.06%). In comparison to parasite prevalence on individual samples, the results showed a perfect agreement (100%) between the two techniques for seven species, whereas discrepancies were observed for the others (agreement percentage varying from 64.2% to 98.9%). Real-time PCR appeared to be superior to microscopic examination for the detection of parasites in stool samples. This assay will be useful in diagnostic laboratories and in the field for evaluating the efficacy of mass drug administration programs.

Safety and Efficacy of Adding a Single Low Dose of Primaquine to the Treatment of Adult Patients With Plasmodium falciparum Malaria in Senegal, to Reduce Gametocyte Carriage: A Randomized Controlled Trial.

Introduction: More information is needed about the safety of low-dose primaquine in populations where G6PD deficiency is common. Methods: Adults with Plasmodium falciparum malaria were randomized to receive 1 of 3 artemisinin combination therapies (ACTs) with or without primaquine (0.25 mg/kg). Glucose-6-phosphate dehydrogenase (G6PD) status was determined using a rapid test. Patients were followed for 28 days to record hemoglobin concentration, adverse events, and gametocyte carriage. The primary end point was the change in Hb at day 7. Results: In sum, 274 patients were randomized, 139 received an ACT alone, and 135 received an ACT + primaquine. The mean reduction in Hb at day 7 was similar in each group, a difference in the ACT + PQ versus the ACT alone group of -0.04 g/dL (95% confidence interval [CI] -0.23, 0.31), but the effect of primaquine differed according to G6PD status. In G6PD-deficient patients the drop in Hb was 0.63 g/dL (95% CI 0.03, 1.24) greater in those who received primaquine than in those who received an ACT alone. In G6PD-normal patients, the reduction in Hb was 0.22 g/dL (95% CI -0.08, 0.52) less in those who received primaquine (interaction P = .01). One G6PD normal patient who received primaquine developed moderately severe anaemia (Hb < 8 g/dL). Dark urine was more frequent in patients who received primaquine. Primaquine was associated with a 73% (95% CI 24-90) reduction in gametocyte carriage (P = .013). Conclusion: Primaquine substantially reduced gametocyte carriage. However, the fall in Hb concentration at day 7 was greater in G6PD-deficient patients who received primaquine than in those who did not and one patient who received primaquine developed moderately severe anemia. Clinical Trial registration: PACTR201411000937373 (www.pactr.org).

Safety, Immunogenicity and Efficacy of Prime-Boost Vaccination with ChAd63 and MVA Encoding ME-TRAP against Plasmodium falciparum Infection in Adults in Senegal.

Malaria transmission is in decline in some parts of Africa, partly due to the scaling up of control measures. If the goal of elimination is to be achieved, additional control measures including an effective and durable vaccine will be required. Studies utilising the prime-boost approach to deliver viral vectors encoding the pre-erythrocytic antigen ME-TRAP (multiple epitope thrombospondin-related adhesion protein) have shown promising safety, immunogenicity and efficacy in sporozoite challenge studies. More recently, a study in Kenyan adults, similar to that reported here, showed substantial efficacy against P. falciparum infection. One hundred and twenty healthy male volunteers, living in a malaria endemic area of Senegal were randomised to receive either the Chimpanzee adenovirus (ChAd63) ME-TRAP as prime vaccination, followed eight weeks later by modified vaccinia Ankara (MVA) also encoding ME-TRAP as booster, or two doses of anti-rabies vaccine as a comparator. Prior to follow-up, antimalarials were administered to clear parasitaemia and then participants were monitored by PCR for malaria infection for eight weeks. The primary endpoint was time-to-infection with P. falciparum malaria, determined by two consecutive positive PCR results. Secondary endpoints included adverse event reporting, measures of cellular and humoral immunogenicity and a meta-analysis of combined vaccine efficacy with the parallel study in Kenyan adults.We show that this pre-erythrocytic malaria vaccine is safe and induces significant immunogenicity, with a peak T-cell response at seven days after boosting of 932 Spot Forming Cells (SFC)/106 Peripheral Blood Mononuclear Cells(PBMC) compared to 57 SFC/ 106 PBMCs in the control group. However, a vaccine efficacy was not observed: 12 of 57 ME-TRAP vaccinees became PCR positive during the intensive monitoring period as compared to 13 of the 58 controls (P = 0.80). This trial confirms that vaccine efficacy against malaria infection in adults may be rapidly assessed using this efficient and cost-effective clinical trial design. Further efficacy evaluation of this vectored candidate vaccine approach in other malaria transmission settings and age-de-escalation into the main target age groups for a malaria vaccine is in progress.

Effectiveness of Seasonal Malaria Chemoprevention in Children under Ten Years of Age in Senegal: A Stepped-Wedge Cluster-Randomised Trial.

BACKGROUND: Seasonal Malaria Chemoprevention (SMC) with sulfadoxine-pyrimethamine (SP) plus amodiaquine (AQ), given each month during the transmission season, is recommended for children living in areas of the Sahel where malaria transmission is highly seasonal. The recommendation for SMC is currently limited to children under five years of age, but, in many areas of seasonal transmission, the burden in older children may justify extending this age limit. This study was done to determine the effectiveness of SMC in Senegalese children up to ten years of age. METHODS AND FINDINGS: SMC was introduced into three districts over three years in central Senegal using a stepped-wedge cluster-randomised design. A census of the population was undertaken and a surveillance system was established to record all deaths and to record all cases of malaria seen at health facilities. A pharmacovigilance system was put in place to detect adverse drug reactions. Fifty-four health posts were randomised. Nine started implementation of SMC in 2008, 18 in 2009, and a further 18 in 2010, with 9 remaining as controls. In the first year of implementation, SMC was delivered to children aged 3-59 months; the age range was then extended for the latter two years of the study to include children up to 10 years of age. Cluster sample surveys at the end of each transmission season were done to measure coverage of SMC and the prevalence of parasitaemia and anaemia, to monitor molecular markers of drug resistance, and to measure insecticide-treated net (ITN) use. Entomological monitoring and assessment of costs of delivery in each health post and of community attitudes to SMC were also undertaken. About 780,000 treatments were administered over three years. Coverage exceeded 80% each month. Mortality, the primary endpoint, was similar in SMC and control areas (4.6 and 4.5 per 1000 respectively in children under 5 years and 1.3 and 1.2 per 1000 in children 5-9 years of age; the overall mortality rate ratio [SMC: no SMC] was 0.90, 95% CI 0.68-1.2, p = 0.496). A reduction of 60% (95% CI 54%-64%, p < 0.001) in the incidence of malaria cases confirmed by a rapid diagnostic test (RDT) and a reduction of 69% (95% CI 65%-72%, p < 0.001) in the number of treatments for malaria (confirmed and unconfirmed) was observed in children. In areas where SMC was implemented, incidence of confirmed malaria in adults and in children too old to receive SMC was reduced by 26% (95% CI 18%-33%, p < 0.001) and the total number of treatments for malaria (confirmed and unconfirmed) in these older age groups was reduced by 29% (95% CI 21%-35%, p < 0.001). One hundred and twenty-three children were admitted to hospital with a diagnosis of severe malaria, with 64 in control areas and 59 in SMC areas, showing a reduction in the incidence rate of severe disease of 45% (95% CI 5%-68%, p = 0.031). Estimates of the reduction in the prevalence of parasitaemia at the end of the transmission season in SMC areas were 68% (95% CI 35%-85%) p = 0.002 in 2008, 84% (95% CI 58%-94%, p < 0.001) in 2009, and 30% (95% CI -130%-79%, p = 0.56) in 2010. SMC was well tolerated with no serious adverse reactions attributable to SMC drugs. Vomiting was the most commonly reported mild adverse event but was reported in less than 1% of treatments. The average cost of delivery was US$0.50 per child per month, but varied widely depending on the size of the health post. Limitations included the low rate of mortality, which limited our ability to detect an effect on this endpoint. CONCLUSIONS: SMC substantially reduced the incidence of outpatient cases of malaria and of severe malaria in children, but no difference in all-cause mortality was observed. Introduction of SMC was associated with an overall reduction in malaria incidence in untreated age groups. In many areas of Africa with seasonal malaria, there is a substantial burden in older children that could be prevented by SMC. SMC in older children is well tolerated and effective and can contribute to reducing malaria transmission. TRIAL REGISTRATION: ClinicalTrials.gov NCT00712374.