Ex vivo RSA and pfkelch13 targeted-amplicon deep sequencing reveal parasites susceptibility to artemisinin in Senegal, 2017.

BACKGROUND: Malaria control is highly dependent on the effectiveness of artemisinin-based combination therapy (ACT), the current frontline malaria curative treatment. Unfortunately, the emergence and spread of parasites resistant to artemisinin (ART) derivatives in Southeast Asia and South America, and more recently in Rwanda and Uganda (East Africa), compromise their long-term use in sub-Saharan Africa, where most malaria deaths occur. METHODS: Here, ex vivo susceptibility to dihydroartemisinin (DHA) was evaluated from 38 Plasmodium falciparum isolates collected in 2017 in Thiès (Senegal) expressed in the Ring-stage Survival Assay (RSA). Both major and minor variants were explored in the three conserved-encoding domains of the pfkelch13 gene, the main determinant of ART resistance using a targeted-amplicon deep sequencing (TADS) approach. RESULTS: All samples tested in the ex vivo RSA were found to be susceptible to DHA (parasite survival rate < 1%). The non-synonymous mutations K189T and K248R in pfkelch13 were observed each in one isolate, as major (99%) or minor (5%) variants, respectively. CONCLUSION: The results suggest that ART is still fully effective in the Thiès region of Senegal in 2017. Investigations combining ex vivo RSA and TADS are a useful approach for monitoring ART resistance in Africa.

Investigating the etiologies of non-malarial febrile illness in Senegal using metagenomic sequencing.

The worldwide decline in malaria incidence is revealing the extensive burden of non-malarial febrile illness (NMFI), which remains poorly understood and difficult to diagnose. To characterize NMFI in Senegal, we collected venous blood and clinical metadata in a cross-sectional study of febrile patients and healthy controls in a low malaria burden area. Using 16S and untargeted sequencing, we detected viral, bacterial, or eukaryotic pathogens in 23% (38/163) of NMFI cases. Bacteria were the most common, with relapsing fever Borrelia and spotted fever Rickettsia found in 15.5% and 3.8% of cases, respectively. Four viral pathogens were found in a total of 7 febrile cases (3.5%). Sequencing also detected undiagnosed Plasmodium, including one putative P. ovale infection. We developed a logistic regression model that can distinguish Borrelia from NMFIs with similar presentation based on symptoms and vital signs (F1 score: 0.823). These results highlight the challenge and importance of improved diagnostics, especially for Borrelia, to support diagnosis and surveillance.

Two mosquito salivary antigens demonstrate promise as biomarkers of recent exposure to P. falciparum infected mosquito bites.

Background: Measuring malaria transmission intensity using the traditional entomological inoculation rate is difficult. Antibody responses to mosquito salivary proteins such as SG6 have previously been used as biomarkers of exposure to Anopheles mosquito bites. Here, we investigate four mosquito salivary proteins as potential biomarkers of human exposure to mosquitoes infected with P. falciparum: mosGILT, SAMSP1, AgSAP, and AgTRIO. Methods: We tested population-level human immune responses in longitudinal and cross-sectional plasma samples from individuals with known P. falciparum infection from low and moderate transmission areas in Senegal using a multiplexed magnetic bead-based assay. Results: AgSAP and AgTRIO were the best indicators of recent exposure to infected mosquitoes. Antibody responses to AgSAP, in a moderate endemic area, and to AgTRIO in both low and moderate endemic areas, were significantly higher than responses in a healthy non-endemic control cohort (p-values = 0.0245, 0.0064, and <0.0001 respectively). No antibody responses significantly differed between the low and moderate transmission area, or between equivalent groups during and outside the malaria transmission seasons. For AgSAP and AgTRIO, reactivity peaked 2-4 weeks after clinical P. falciparum infection and declined 3 months after infection. Discussion: Reactivity to both AgSAP and AgTRIO peaked after infection and did not differ seasonally nor between areas of low and moderate transmission, suggesting reactivity is likely reflective of exposure to infectious mosquitos or recent biting rather than general mosquito exposure. Kinetics suggest reactivity is relatively short-lived. AgSAP and AgTRIO are promising candidates to incorporate into multiplexed assays for serosurveillance of population-level changes in P. falciparum-infected mosquito exposure.

Quality assessment of malaria microscopic diagnosis at the Aristide Le Dantec University Hospital of Dakar, Senegal, in 2020.

BACKGROUND: Following WHO guidelines, microscopy is the gold standard for malaria diagnosis in endemic countries. The Parasitology-Mycology laboratory (LPM) is the National Reference Laboratory and is currently undergoing ISO 15189 accreditation. In this context, we assessed the performance of the laboratory by confirming the reliability and the accuracy of results obtained in accordance with the requirements of the ISO 15189 standards. This study aimed to verify the method of microscopic diagnosis of malaria at the LPM, in the Aristide Le Dantec hospital (HALD) in Dakar, Senegal. METHODS: This is a validation/verification study conducted from June to August 2020. Twenty (20) microscopic slides of thick/thin blood smear with known parasite densities (PD) selected from the Cheick Anta Diop University malaria slide bank in Dakar were used for this assessment. Six (6) were used to assess microscopists' ability to determine PD and fourteen (14) slides were used for detection (positive vs negative) and identification of parasites. Four (4) LPM-HALD microscopists read and recorded their results on prepared sheets. Data analysis was done with Microsoft Excel 2010 software. RESULTS: A minimum threshold of 50% concordance was used for comparison. Of the twenty (20) slides read, 100% concordance was obtained on eight (8) detection (positive vs negative) slides. Four (4) out of the six (6) parasite density evaluation slides obtained a concordance of less than 50%. Thirteen (13) out of the fourteen (14) identification slides obtained a concordance greater than 50%. Only one (1) identification slide obtained zero agreement from the microscopists. For species identification a concordance greater than 80% was noted and the microscopists obtained scores between 0.20 and 0.4 on a scale of 0 to 1 for parasite density reading. The microscopists obtained 100% precision, sensitivity, specificity and both negative and positive predictive values. CONCLUSION: This work demonstrated that the microscopic method of malaria diagnosis used in the LPM/HALD is in accordance with the requirements of WHO and ISO 15189. Further training of microscopists may be needed to maintain competency.

Ex vivo RSA and Pfkelch13 targeted-amplicon deep sequencing reveal parasites susceptibility to artemisinin in Senegal, 2017.

INTRODUCTION: Malaria control is highly dependent on the effectiveness of artemisinin-based combination therapies (ACTs), the current frontline malaria curative treatments. Unfortunately, the emergence and spread of parasites resistant to artemisinin (ART) derivatives in Southeast Asia and South America, and more recently in Rwanda and Uganda (East Africa), compromise their long-term use in Sub-Saharan Africa where most malaria deaths occur. METHODS: Here, we evaluated ex vivo susceptibility to dihydroartemisinin (DHA) from 38 P. falciparum isolates collected in 2017 in Thiès (Senegal) expressed with the Ring-stage Survival Assay (RSA). We explored major and minor variants in the full Pfkelch13 gene, the main determinant of ART resistance using a targeted-amplicon deep sequencing (TADS) approach. RESULTS: All samples tested in the ex vivo RSA were found to be susceptible to DHA. Both non-synonymous mutations K189T and K248R were observed each in one isolate, as major (99%) or minor (5%) variants, respectively. CONCLUSION: Altogether, investigations combining ex vivo RSA and TADS are a useful approach for monitoring ART resistance in Africa.

Improving diagnosis of non-malarial fevers in Senegal: Borrelia and the contribution of tick-borne bacteria.

The worldwide decline in malaria incidence is revealing the extensive burden of non-malarial febrile illness (NMFI), which remains poorly understood and difficult to diagnose. To characterize NMFI in Senegal, we collected venous blood and clinical metadata from febrile patients and healthy controls in a low malaria burden area. Using 16S and unbiased sequencing, we detected viral, bacterial, or eukaryotic pathogens in 29% of NMFI cases. Bacteria were the most common, with relapsing fever Borrelia and spotted fever Rickettsia found in 15% and 3.7% of cases, respectively. Four viral pathogens were found in a total of 7 febrile cases (3.5%). Sequencing also detected undiagnosed Plasmodium, including one putative P. ovale infection. We developed a logistic regression model to distinguish Borrelia from NMFIs with similar presentation based on symptoms and vital signs. These results highlight the challenge and importance of improved diagnostics, especially for Borrelia, to support diagnosis and surveillance.

Plasmodium falciparum genomic surveillance reveals spatial and temporal trends, association of genetic and physical distance, and household clustering.

Molecular epidemiology using genomic data can help identify relationships between malaria parasite population structure, malaria transmission intensity, and ultimately help generate actionable data to assess the effectiveness of malaria control strategies. Genomic data, coupled with geographic information systems data, can further identify clusters or hotspots of malaria transmission, parasite genetic and spatial connectivity, and parasite movement by human or mosquito mobility over time and space. In this study, we performed longitudinal genomic surveillance in a cohort of 70 participants over four years from different neighborhoods and households in Thiès, Senegal-a region of exceptionally low malaria transmission (entomological inoculation rate less than 1). Genetic identity (identity by state, IBS) was established using a 24-single nucleotide polymorphism molecular barcode, identity by descent was calculated from whole genome sequence data, and a hierarchical Bayesian regression model was used to establish genetic and spatial relationships. Our results show clustering of genetically similar parasites within households and a decline in genetic similarity of parasites with increasing distance. One household showed extremely high diversity and warrants further investigation as to the source of these diverse genetic types. This study illustrates the utility of genomic data with traditional epidemiological approaches for surveillance and detection of trends and patterns in malaria transmission not only by neighborhood but also by household. This approach can be implemented regionally and countrywide to strengthen and support malaria control and elimination efforts.

Plasmodium infection is associated with cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein.

Sero-surveillance can monitor and project disease burden and risk. However, SARS-CoV-2 antibody test results can produce false positive results, limiting their efficacy as a sero-surveillance tool. False positive SARS-CoV-2 antibody results are associated with malaria exposure, and understanding this association is essential to interpret sero-surveillance results from malaria-endemic countries. Here, pre-pandemic samples from eight malaria endemic and non-endemic countries and four continents were tested by ELISA to measure SARS-CoV-2 Spike S1 subunit reactivity. Individuals with acute malaria infection generated substantial SARS-CoV-2 reactivity. Cross-reactivity was not associated with reactivity to other human coronaviruses or other SARS-CoV-2 proteins, as measured by peptide and protein arrays. ELISAs with deglycosylated and desialated Spike S1 subunits revealed that cross-reactive antibodies target sialic acid on N-linked glycans of the Spike protein. The functional activity of cross-reactive antibodies measured by neutralization assays showed that cross-reactive antibodies did not neutralize SARS-CoV-2 in vitro. Since routine use of glycosylated or sialated assays could result in false positive SARS-CoV-2 antibody results in malaria endemic regions, which could overestimate exposure and population-level immunity, we explored methods to increase specificity by reducing cross-reactivity. Overestimating population-level exposure to SARS-CoV-2 could lead to underestimates of risk of continued COVID-19 transmission in sub-Saharan Africa.

Genomic investigation of a dengue virus outbreak in Thiès, Senegal, in 2018.

Dengue virus is a major and rapidly growing public health concern in tropic and subtropic regions across the globe. In late 2018, Senegal experienced its largest dengue virus outbreak to date, covering several regions. However, little is known about the genetic diversity of dengue virus (DENV) in Senegal. Here we report complete viral genomes from 17 previously undetected DENV cases from the city of Thiès. In total we identified 19 cases of DENV in a cohort of 198 individuals with fever collected in October and November 2018. We detected 3 co-circulating serotypes; DENV 3 was the most frequent accounting for 11/17 sequences (65%), 4 (23%) were DENV2 and 2 (12%) were DENV1. Sequences were most similar to recent sequences from West Africa, suggesting ongoing local circulation of viral populations; however, detailed inference is limited by the scarcity of available genomic data. We did not find clear associations with reported clinical signs or symptoms, highlighting the importance of testing for diagnosing febrile diseases. Overall, these findings expand the known range of DENV in Senegal, and underscore the need for better genomic characterization of DENV in West Africa.

Plasmodium infection induces cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein.

Individuals with acute malaria infection generated high levels of antibodies that cross-react with the SARS-CoV-2 Spike protein. Cross-reactive antibodies specifically recognized the sialic acid moiety on N-linked glycans of the Spike protein and do not neutralize in vitro SARS-CoV-2. Sero-surveillance is critical for monitoring and projecting disease burden and risk during the pandemic; however, routine use of Spike protein-based assays may overestimate SARS-CoV-2 exposure and population-level immunity in malaria-endemic countries.

Lack of inhibition of the anti-malarial action of sulfadoxine-pyrimethamine by folic acid supplementation when used for intermittent preventive treatment in Gambian primigravidae.

Folic acid is frequently given to pregnant women at the same time as intermittent preventive treatment (IPTp) with sulfadoxine/pyrimethamine (SP), but it is not known if it interferes with the anti-malarial activity of SP. To investigate this concern, 1,035 Gambian primigravidae were randomized to receive either folic acid (500-1,500 microg/day) together with oral iron (522) or oral iron alone (513) for 14 days at the same time as they received IPTp with SP. On presentation, 261 women (25%) had Plasmodium falciparum asexual parasitemia. Prevalences of parasitemia on day 14 after treatment were similar in both groups: 5.7% (26 of 458) in the iron plus folic acid group and 4.9% (22 of 446) in the iron alone group (risk difference = 0.74%, 95% confidence interval [CI] = -2.2% to 3.7%). Parasitologic cure was observed in 116 (91%) of 128 of women who were parasitemic on presentation and who received iron and folic acid and in 122 (92%) of 133 women who received iron alone (difference = 1.1%, 95% CI = -5.6% to 8.0%). Women who received folic acid and iron had a slightly higher mean hemoglobin concentration at day 14 than women who had received iron alone (difference = 0.14 g/dL, 95% CI = 0.01-0.27 g/dL). The results of this study suggest that in an area of low SP resistance, administration of folic acid to pregnant women in a dose of 500-1,500 mug/day will not interfere with the protective effect of SP when used for IPTp.

Does the neuropeptide somatostatin have therapeutic potential against schistosomiasis?

The neuropeptide hormone somatostatin is used to treat bleeding oesophageal varices and to reduce portal pressure, and can prevent progression to severe fibrosis in chronic liver disease. We believe that somatostatin can also have therapeutic potential against schistosomiasis, based on recent observations that severe morbidity symptoms are associated with low levels of host somatostatin in patients infected with Schistosoma mansoni. The administration of exogenous doses of this neuropeptide could therefore alleviate the pathology caused by schistosomiasis.

Determination of ED50 values for praziquantel in praziquantel-resistant and -susceptible Schistosoma mansoni isolates.

The dose of praziquantel required to kill 50% of adult worms in vivo (i.e. the ED50) was estimated for nine different isolates of Schistosoma mansoni in infected mice. Four of the isolates were selected because they had not knowingly been in contact with the drug (i.e. they were putatively praziquantel-susceptible). Five putatively praziquantel-resistant isolates were chosen because they had been selectively bred for drug-resistance in the laboratory and/or had previously been shown to be relatively resistant to praziquantel in the field. The work was performed in three laboratories in different countries using pre-agreed and comparable experimental protocols. All four praziquantel-susceptible isolates had ED50s estimated to be <100 mg/kg (mean=70+/-7 SD; median=68), while all five putatively praziquantel-resistant isolates had estimated ED50s >100 mg/kg (mean=209+/-48 SD; median=192). Thus, the five praziquantel-resistant isolates, including two that had been subjected to drug pressure during more than 20 passages in mice, had drug ED50s that were approximately three times as great as those of the praziquantel-susceptible isolates. Two of the five isolates in the putatively resistant group had previously been passaged 15 or more times in mice without administration of drug-pressure, but had ED50s consistent with the other three isolates in the group, indicating that the trait of praziquantel-resistance did not necessarily impair biological fitness during laboratory passage. The protocols used here to estimate the praziquantel ED50s of S. mansoni isolates should be useful for establishing and monitoring the drug susceptibility/resistance profiles of parasite isolates freshly obtained from endemic areas, particularly those in which increased usage of the drug is likely to occur.

Circulating levels of the neuropeptide hormone somatostatin may determine hepatic fibrosis in Schistosoma mansoni infections.

The neuropeptide hormone somatostatin reduces fibrosis and Schistosoma-caused clinical morbidity in the rodent model. In our study we aimed to delineate an association between fibrosis and the inability to generate critical levels of endogenous somatostatin in S. mansoni infected subjects. In June 2001, 85 subjects from the district dispensary at Richard Toll in the Medical Region of Saint-Louis, Senegal, were selected. Fifty-seven subjects were infected with S. mansoni of whom 32 were suffering from severe morbidity (SM). Twenty-eight subjects showed an inactive disease status with no evident infection at the actual time of study. All subjects were classified according to age, sex, occupation, height, weight, and parasite eggs per gram. All 85 participated in a water contact and morbidity questionnaire, underwent a clinical examination and donated 5ml of peripheral blood for detecting plasma levels of somatostatin. Ultrasonography detected fibrosis grade in all the subjects. To address whether inherent somatostatin levels determined clinically evident disease severity (epg, hepatomegaly, splenomegaly, hematemesis, ascites), the mean somatostatin values of the inactive disease status group and severe morbidity group were compared. Low somatostatin levels were depicted in subjects with severe morbidity symptoms associated with schistosomiasis as compared to exposed but inactive disease status subjects residing in the same region. Logistic regression analysis indicated that with decreasing somatostatin values the probability of severe morbidity increased with age being a confounding factor. To address whether inherent somatostatin levels determined fibrosis and if this association was significant, plasma somatostatin levels of non-fibrotics (ultrasonographic grading A), and fibrotics (ultrasonographic grading B-E) were compared. In all age groups as well as in adults alone, mean somatostatin levels were higher in the non-fibrotic group as compared to the fibrotics group, the difference being significant. The group B comprised of borderline fibrotic cases, therefore a separate analysis was done between groups A (non-fibrotics) and groups C, D (confirmed fibrotics). Mean somatostatin values were higher in the non-fibrotic group as compared to the fibrotics group, the difference being borderline significant. In schistosomiasis patients, circulating levels of somatostatin by binding to hepatic stellate cells (HSC) may modulate fibrosis. This phenomenon is regulated by age whereas gender and prior treatment have no effect on this association. Host specific somatostatin levels may create a 'preset environment' status that can determine progression to severe fibrosis.

Lower levels of the circulating neuropeptide somatostatin in Schistosoma mansoni infected patients may have pathological significance.

In recent years, cases of severe morbidity (fibrosis, haematemesis, hepatosplenomegaly, ascites) caused to Schistosoma mansoni infections are on the rise in Northern Senegal. The neuropeptide somatostatin is reported to decrease portal pressure, control variceal bleeding and fibrosis, and reduce Schistosoma-caused clinical morbidity in the rodent model. The aim of this study was to delineate the role of somatostatin in S. mansoni-caused pathogenesis, by studying host levels of somatostatin in the peripheral blood of uninfected and S. mansoni-infected individuals. Subjects from the district dispensary at Richard Toll, in the Medical Region of Saint-Louis, Senegal, infected with S. mansoni and suffering from severe morbidity were selected. A separate group consisted of individuals resident in the same region but uninfected at the time of the study. Significantly lower somatostatin levels were detected in severe morbidity patients, compared with the exposed but uninfected subjects. In patients with schistosomiasis physiological levels of somatostatin may determine disposition of particular individuals towards severe morbidity, as opposed to others. Host pathology can thus be alleviated by the therapeutic ability of somatostatin to treat bleeding oesophageal varices, reduce portal pressure and prevent progression to severe fibrosis.

Low awareness of intestinal schistosomiasis in northern Senegal after 7 years of health education as part of intense control and research activities.

We evaluated the awareness of and knowledge about intestinal schistosomiasis in a highly infected rural community of northern Senegal where a variety of health information and education activities had taken place for 7 years as a component of different research and control programmes. As the infection had been introduced only recently, an initial 'zero' knowledge can be assumed. Most of the health education activities had been performed with adapted messages through local health and community workers. By a questionnaire, 566 individuals were asked simple questions on symptoms, mode of transmission, the sources of information and health-seeking behaviour. About 86% of the respondents stated that they knew what schistosomiasis was, and 92% that in case of illness they would seek treatment at the health centre. However, only half of the people accurately quoted symptoms associated with intestinal schistosomiasis: diarrhoea, abdominal pain and bloody stools. The majority of respondents realized that the disease was somehow linked with water and (lack of) hygiene, but only 44% of respondents reported water contact as the source of infection. Ultimately, only 30% of the respondents gave adequate answers about both symptoms and mode of transmission. We conclude that even intense and long-lasting education efforts for a specific and straightforward problem as schistosomiasis are not enough to have profound impact on the knowledge of rural traditional communities.

Evaluation of staff performance and material resources for integrated schistosomiasis control in northern Senegal.

BACKGROUND: A project to improve integrated control of schistosomiasis in the primary health care system of northern Senegal was implemented from February 1995 until September 1999, shortly after a Schistosoma mansoni outbreak. The activities included additional training of doctors and nurses in symptom-based treatment and making praziquantel (PZQ) available for an affordable price. OBJECTIVE: To investigate staff performance and the availability and costs of diagnostic materials and PZQ at the end of this intervention project. METHODS: We performed structured interviews with staff from 55 health care facilities in five districts. RESULTS: Respondents from 23 health care facilities reported both S. haematobium and S. mansoni in the coverage area, 32 reported only S. haematobium and three only S. mansoni. The average cost to patients for consultation, diagnosis, treatment and transportation to a referral health care facility was approximately 1.60 Euro. Fifty-seven per cent of the health care facilities with reported S. haematobium in the coverage area treated patients presenting with haematuria on symptoms; 56% of the health care facilities with reported S. mansoni in the coverage area treated patients presenting with blood in stool on symptoms. Thirteen per cent performed a diagnostic test for patients presenting with haematuria and 12% for patients presenting with blood in stool. The remainder, approximately one-third of the health care facilities, referred their patients to another facility for a diagnostic test. Implementation of symptom-based treatment in all health care facilities will reduce the total costs by 0.43 Euro (29%) for patients infected with S. haematobium and 0.78 Euro (46%) for patients infected with S. mansoni. Of the 53 health care facilities with schistosomiasis in their area, 37 had PZQ in stock of which 33 (88%) sold PZQ for the recommended retail price of 0.15 Euro per tablet (or 0.60 Euro per course of four tablets) or lower. CONCLUSION: Four years after the start of the intervention project, patients presenting with schistosomiasis related symptoms can generally expect proper diagnosis and treatment at all levels of the health care system in Northern Senegal, either at the initial visited health care facility or after referral. However, a further reduction of the total costs of treatment is still possible by a better implementation of symptom-based treatment and further reduction of the costs of PZQ.