Molecular Epidemiology of Plasmodium species in Conflicted Federally Administered Tribal Area (FATA) Pakistan

Military conflicts have been significant obstacles in detecting and treating infectious disease diseases due to the diminished public health infrastructure, resulting in malaria endemicity. A variety of violent and destructive incidents were experienced by FATA (Federally Administered Tribal Areas). It was a struggle to pursue an epidemiological analysis due to continuing conflict and Talibanization. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai agencies from May 2017 to May 2018. For Giemsa staining, full blood EDTA blood samples have been collected from symptomatic participants. Malaria-positive microscopy isolates were spotted on filter papers for future Plasmodial molecular detection by nested polymerase chain reaction (nPCR) of small subunit ribosomal ribonucleic acid (ssrRNA) genes specific primers. Since reconfirming the nPCR, a malariometric study of 762 patients found 679 positive malaria cases. Plasmodium vivax was 523 (77%), Plasmodium falciparum 121 (18%), 35 (5%) were with mixed-species infection (P. vivax plus P. falciparum), and 83 were declared negative by PCR. Among the five agencies of FATA, Khyber agency has the highest malaria incidence (19%) with followed by P. vivax (19%) and P. falciparum (4.1%). In contrast, Kurram has about (14%), including (10.8%) P. vivax and (2.7%) P. falciparum cases, the lowest malaria epidemiology. Surprisingly, no significant differences in the distribution of mixed-species infection among all five agencies. P. falciparum and P. vivax were two prevalent FATA malaria species in Pakistan’s war-torn area. To overcome this rising incidence of malaria, this study recommends that initiating malaria awareness campaigns in school should be supported by public health agencies and malaria-related education locally, targeting children and parents alike.

Malaria Dashboard: a digital platform for epidemiological data from India (Preprint)

BACKGROUND A digital dashboard on malaria epidemiological data will be an invaluable resource for the research community and the planning of malaria control. OBJECTIVE To develop a digital Malaria Dashboard (MDB) for malaria epidemiological data METHODS We have developed a digital Malaria Dashboard (MDB) using the R software. A total of thirteen different R packages were used in this process, within which shiny and ggplot2 were used more intensively. The MDB is a web application that can work online as well as offline. Presently it is available in offline mode only. The MS Excel file may be used as an input data source and any personal computer may be used for this application. RESULTS The MDB is a highly versatile interface that allows prompt and interactive analysis of malaria epidemiological data. The primary interface of MDB is like a web page that has 14 tabs (or pages), some more tabs may be added or deleted as per requirement and each tab corresponds to a particular analysis. A user may move from one tab to another via tab icons. Each tab thus allows flexibility in correlating various parameters like SPR, API, AFI, ABER, RT, malaria cases, death due to malaria, BSC, and BSE. The data can be analyzed in required granularity (national, state, district), and its enhanced visualization allows for facile usage. Using the MDB, one can quickly assess national or more granular scenarios in a time series manner and then compare the malaria epidemiology in various states and their constituent districts. CONCLUSIONS This MDB is a highly effective digital tool for studying the malaria situation and strategizing for malaria elimination and researcher may use it as a prototype for developing some other dashboards in their own fields.

A Molecular Investigation of Malaria Infections From High-Transmission Areas of Southern Togo Reveals Different Species of Plasmodium Parasites

Malaria particularly burdens people in poor and neglected settings across the tropics of Africa. Meanwhile, a large proportion of the Togo population have poor understanding of malaria epidemiology and parasites. This study carried out a molecular survey of malaria cases in southern Togo during 2017–2019. We estimated Plasmodium species infection rates and microscopic examination compliance with nested PCR results. Sensitivity and specificity analyses were performed in conjunction with predictive values. Also, phylogenetic characterization of species of malaria parasites was assessed. Plasmodium genus-specific nested PCR identified 565 positive cases including 536/611 (87.8%) confirmed cases from the microscopy-positive group and 29/199 (14.6%) diagnosed malaria cases from the microscopy-negative group. Our findings revealed a disease prevalence (69.8%) higher than that reported (25.5–55.1%) for the country. The diagnostic test had 94.9% sensitivity and 69.4% specificity, i.e., it missed 120 of the people who had malaria and about one-third of the people tested positive for the disease, which they did not have, respectively. In conjunction, the test showed 87.7% positive predictive value and 85.4% negative predictive value, which, from a clinical perspective, indicates the chance that a person with a positive diagnostic test truly has the disease and the probability that a person with a negative test does not have the disease, respectively. Further species-specific nested PCR followed by analysis of gene sequences confirmed species of malaria parasites and indicated infection rates for Plasmodium falciparum (Pf), 95.5% (540/565); P. ovale (Po), 0.5% (3/565); and P. malariae (Pm), 0.4% (2/565). In addition, 20 cases were coinfection cases of Pf-Po (15/565) and Pf-Pm (5/565). This study publicly reports, for the first time, a molecular survey of malaria cases in Togo and reveals the presence of other malaria parasites (Po and Pm) other than Pf. These findings might provide answers to some basic questions on the malaria scenario and, knowledge gained could help with intervention deployment for effective malaria control in Togo.

Malaria Epidemiology Along the Indian Districts Bordering Bhutan and Implications for Malaria Elimination in the Region

It is important for malaria-endemic countries to address malaria control across international borders, and in particular to prioritize appropriate rapid diagnosis, treatment, and surveillance. Bhutan and India aim to achieve malaria elimination by 2023 and 2030, respectively. Malaria elimination along the Indo–Bhutan border is of common concern. We delineated malaria epidemiology along the border to provide a blueprint for focusing malaria control efforts in key foci within this region. Epidemiological data from 2015 to 2019 were analyzed, as the most drastic reductions in malaria burden across most parts of India were witnessed in this time frame. Several areas of concern include low surveillance in most border districts, favorable climatic conditions for perennial malaria transmission, and movement of potential parasite carriers because of the porous borders. India and Bhutan need to control the importation/exportation of malaria cases. We highlight the key foci of concern for which implementing tailor-made malaria control strategies may benefit both countries.

Identifying Plasmodium falciparum transmission patterns through parasite prevalence and entomological inoculation rate

Background:Monitoring malaria transmission is a critical component of efforts to achieve targets for elimination and eradication. Two commonly monitored metrics of transmission intensity are parasite prevalence (PR) and the entomological inoculation rate (EIR). Comparing the spatial and temporal variations in the PR and EIR of a given geographical region and modelling the relationship between the two metrics may provide a fuller picture of the malaria epidemiology of the region to inform control activities.Methods:Using geostatistical methods, we compare the spatial and temporal patterns of Plasmodium falciparum EIR and PR using data collected over 38 months in a rural area of Malawi. We then quantify the relationship between EIR and PR by using empirical and mechanistic statistical models.Results:Hotspots identified through the EIR and PR partly overlapped during high transmission seasons but not during low transmission seasons. The estimated relationship showed a 1-month delayed effect of EIR on PR such that at lower levels of EIR, increases in EIR are associated with rapid rise in PR, whereas at higher levels of EIR, changes in EIR do not translate into notable changes in PR.Conclusions:Our study emphasises the need for integrated malaria control strategies that combine vector and human host managements monitored by both entomological and parasitaemia indices.Funding:This work was supported by Stichting Dioraphte grant number 13050800.

Malaria at Forest Areas in South Kalimantan, Indonesia: Risk Factors and Strategies for Elimination

BACKGROUND: South Kalimantan is one of province in Indonesia which has endemic area, mainly in the villages at forest area. Understanding the risk factors which can increase the risk of malaria in individuals at forest area will enable more effective use for controlling the disease. The identification of risk factors will provide information about local malaria epidemiology and usefull for making appropriate and effective malaria eradication program policies in this area. AIM: To know the risk factors of malaria prevalence in endemic forest areas in South Kalimantan, Indonesia. METHODS: This cross-sectional study was conducted on 107 adult people who lived in Batu Bulan Village and Batu Paha Village, South Kalimantan. Blood samples for malaria microscopy and rapid diagnostic test is taken from cubital vein. Household factors and demographic data were obtained. Chi-square and logistic regression were performed to analyze the factors associated with malaria prevalence in South Kalimantan. This research didn’t do vector survey, only on the prevalence of malaria and risk factor in human and environment. RESULTS: The prevalence of malaria based RDT examination was 35.5% with 23.68% Plasmodium falciparum, 21.05% Plasmodium vivax, and 55.27% mixed infection. The prevalence malaria based on microscopic examination was 17.75% with 47.36% P. falciparum, 26.32% P. vivax, and 26.32% mix infection. Demographic factors influencing the prevalence of malaria were aged below 25-years-old (p = 0.01, 95% CI, OR = 2.289), villages in Batu Paha (p = 0.048, 95% CI, OR = 3.55), and occupation as a forest worker (p = 0.022, 95% CI, OR = 6.38). House factors that influence the prevalence of malaria were the condition of the walls that are open or not tight (p = 0.048 95% CI, OR = 5.205), the roof is made of plastic (p = 0.015 95% CI, OR = 2.831), and the presence of animal cage around the house (p = 0.015 95% CI, OR = 6.292). CONCLUSIONS: Malaria incidence remains occurs with high prevalence in the pupolation in remote forest areas.

GENETIC ARCHITECTURE AND DYNAMICS OF PFKELCH13S PROPELLER DOMAIN IN SENEGALESE PLASMODIUM FALCIPARUMCLINICAL ISOLATES

Plasmodium resistance to Artemisinin Combination-based Therapies (ACT) in Southeast Asia is a major public health concern that is sporadically appearing in Africa. Senegal has shifted from malaria control to elimination plans. Given notable progresses obtained through robust strategic plans,it is still crucial to assess genetic variability of the Plasmodium falciparumartemisinin resistance gene marker Kelch13 (PfKelch13)in circulating field isolates.We herereportan analysis of PfKelch13-propeller polymorphism in clinical isolates collected nine years after ACT introduction in five Senegalese regions with different malaria transmission settings. Sequencing of PfKelch13-propeller domainfrom 280 clinicalisolates reveals that 16% (45/280) of the parasite population harboredvariants. Dynamics of PfKelch13 variants reveals emerging, persistent but also disappearing mutations over time. In addition to the malaria epidemiology, our survey also shows the dynamics of PfKelch13 variants in different malaria transmission settings in Senegal. Despite the absence of PfKelch13associatedartemisinin resistance mutations, a shift from 86% to 68% of PfKelch13WTwas observed when comparing parasites collected prior vs. post ACT intensive usage in Dakar a low malaria transmission area. All together,our data confirms the need to closely monitor PfKelch13 polymorphism to anticipate and or preventemergence ofP. falciparum resistancein Senegal.

A comparison of PCR and ELISA methods to detect different stages of Plasmodium vivax in Anopheles arabiensis

Background In characterizing malaria epidemiology, measuring mosquito infectiousness informs the entomological inoculation rate, an important metric of malaria transmission. PCR-based methods have been touted as more sensitive than the current “gold-standard” circumsporozoite (CSP) ELISA. Wider application of PCR-based methods has been limited by lack of specificity for the infectious sporozoite stage. We compared a PCR method for detecting the parasite’s mitochondrial (mt) cytochrome oxidase I (COX-I) gene with ELISA for detecting circumsporozoite protein for identification of different life stages of the parasite during development within a mosquito. Methods A PCR-based method targeting the Plasmodium mt COX-I gene was compared with the CSP ELISA method to assess infectivity in Anopheles arabiensis colony mosquitoes fed on blood from patients infected with Plasmodium vivax. Mosquitoes were tested at six post-infection time points (days 0.5, 1, 6, 9, 12, 15). The head and thorax and the abdomen for each specimen were tested separately with each method. Agreement between methods at each infection stage was measured using Cohen’s kappa measure of test association. Results Infection status of mosquitoes was assessed in approximately 90 head/thorax and 90 abdomen segments at each time point; in total, 538 head/thorax and 534 abdomen segments were tested. In mosquitoes bisected after 0.5, 1, and 6 days post-infection (dpi), the mt COX-I PCR detected Plasmodium DNA in both the abdomen (88, 78, and 67%, respectively) and head/thorax segments (69, 60, and 44%, respectively), whilst CSP ELISA detected sporozoites in only one abdomen on day 6 post-infection. PCR was also more sensitive than ELISA for detection of Plasmodium in mosquitoes bisected after 9, 12, and 15 dpi in both the head and thorax and abdomen. There was fair agreement between methods for time points 9–15 dpi (κ = 0.312, 95% CI: 0.230–0.394). Conclusions The mt COX-I PCR is a highly sensitive, robust method for detecting Plasmodium DNA in mosquitoes, but its limited Plasmodium life-stage specificity cannot be overcome by bisection of the head and thorax from the abdomen prior to PCR. Thus, the mt COX-I PCR is a poor candidate for identifying infectious mosquitoes. Graphical Abstract

Individual-level variations in malaria susceptibility and acquisition of clinical protection

After decades of research, our understanding of when and why individuals infected with Plasmodium falciparum develop clinical malaria is still limited. Correlates of immune protection are often sought through prospective cohort studies, where measured host factors are correlated against the incidence of clinical disease over a set period of time. However, robustly inferring individual-level protection from these population-level findings has proved difficult due to small effect sizes and high levels of variance underlying such data. In order to better understand the nature of these inter-individual variations, we analysed the long-term malaria epidemiology of children ≤12 years old growing up under seasonal exposure to the parasite in the sub-location of Junju, Kenya. Despite the cohort’s limited geographic expanse (ca. 3km x 10km), our data reveal a high degree of spatial and temporal variability in malaria prevalence and incidence rates, causing individuals to experience varying levels of exposure to the parasite at different times during their life. Analysing individual-level infection histories further reveal an unexpectedly high variability in the rate at which children experience clinical malaria episodes. Besides exposure to the parasite, measured as disease prevalence in the surrounding area, we find that the birth time of year has an independent effect on the individual’s risk of experiencing a clinical episode. Furthermore, our analyses reveal that those children with a history of an above average number of episodes are more likely to experience further episodes during the upcoming transmission season. These findings are indicative of phenotypic differences in the rates by which children acquire clinical protection to malaria and offer important insights into the natural variability underlying malaria epidemiology.