Analysis of nucleic acids extracted from rapid diagnostic tests reveals a significant proportion of false positive test results associated with recent malaria treatment.

BACKGROUND: Surveillance programmes often use malaria rapid diagnostic tests (RDTs) to determine the proportion of the population carrying parasites in their peripheral blood to assess the malaria transmission intensity. Despite an increasing number of reports on false-negative and false-positive RDT results, there is a lack of systematic quality control activities for RDTs deployed in malaria surveillance programmes. METHODS: The diagnostic performance of field-deployed RDTs used for malaria surveys was assessed by retrospective molecular analysis of the blood retained on the tests. RESULTS: Of the 2865 RDTs that were collected in 2018 on Bioko Island and analysed in this study, 4.7% had a false-negative result. These false-negative RDTs were associated with low parasite density infections. In 16.6% of analysed samples, masked pfhrp2 and pfhrp3 gene deletions were identified, in which at least one Plasmodium falciparum strain carried a gene deletion. Among all positive RDTs analysed, 28.4% were tested negative by qPCR and therefore considered to be false-positive. Analysing the questionnaire data collected from the participants, this high proportion of false-positive RDTs could be explained by P. falciparum histidine rich protein 2 (PfHRP2) antigen persistence after recent malaria treatment. CONCLUSION: Malaria surveillance depending solely on RDTs needs well-integrated quality control procedures to assess the extent and impact of reduced sensitivity and specificity of RDTs on malaria control programmes.

Occurrence and Distribution of Nonfalciparum Malaria Parasite Species Among Adolescents and Adults in Malawi.

BACKGROUND: Plasmodium falciparum malaria dominates throughout sub-Saharan Africa, but the prevalence of Plasmodium malariae, Plasmodium ovale spp., and Plasmodium vivax increasingly contribute to infection in countries that control malaria using P. falciparum-specific diagnostic and treatment strategies. METHODS: We performed quantitative polymerase chain reaction (qPCR) on 2987 dried blood spots from the 2015-2016 Malawi Demographic and Health Survey to identify presence and distribution of nonfalciparum infection. Bivariate models were used to determine species-specific associations with demographic and environmental risk factors. RESULTS: Nonfalciparum infections had broad spatial distributions. Weighted prevalence was 0.025 (SE, 0.004) for P. malariae, 0.097 (SE, 0.008) for P. ovale spp., and 0.001 (SE, 0.0005) for P. vivax. Most infections (85.6%) had low-density parasitemias ≤ 10 parasites/µL, and 66.7% of P. malariae, 34.6% of P. ovale spp., and 40.0% of P. vivax infections were coinfected with P. falciparum. Risk factors for P. malariae were like those known for P. falciparum; however, there were few risk factors recognized for P. ovale spp. and P. vivax, perhaps due to the potential for relapsing episodes. CONCLUSIONS: The prevalence of any nonfalciparum infection was 11.7%, with infections distributed across Malawi. Continued monitoring of Plasmodium spp. becomes critical as nonfalciparum infections become important sources of ongoing transmission.

Neglected malaria parasites in hard-to-reach areas of Odisha, India: implications in elimination programme.

BACKGROUND: Information on the foci of Plasmodium species infections is essential for any country heading towards elimination. Odisha, one of the malaria-endemic states of India is targeting elimination of malaria by 2030. To support decision-making regarding targeted intervention, the distribution of Plasmodium species infections was investigated in hard-to-reach areas where a special malaria elimination drive, namely Durgama Anchalare Malaria Nirakaran (DAMaN) began in 2017. METHODS: A cross-sectional survey was conducted in 2228 households during July to November 2019 in six districts, to evaluate the occurrence of Plasmodium species. The species were identified by polymerase chain reaction (PCR) followed by sequencing, in case of Plasmodium ovale. RESULTS: Of the 3557 blood specimens tested, malaria infection was detected in 282 (7.8%) specimens by PCR. Of the total positive samples, 14.1% were P. ovale spp. and 10.3% were Plasmodium malariae infections. The majority of P. ovale spp. (75.8%) infections were mixed with either Plasmodium falciparum and/or Plasmodium vivax and found to be distributed in three geophysical regions (Northern-plateau, Central Tableland and Eastern Ghat) of the State, while P. malariae has been found in Northern-plateau and Eastern Ghat regions. Speciation revealed occurrence of both Plasmodium ovale curtisi (classic type) and Plasmodium ovale wallikeri (variant type). CONCLUSIONS: In the present study a considerable number of P. ovale spp. and P. malariae were detected in a wide geographical areas of Odisha State, which contributes around 40% of the country's total malaria burden. For successful elimination of malaria within the framework of national programme, P. ovale spp. along with P. malariae needs to be incorporated in surveillance system, especially when P. falciparum and P. vivax spp. are in rapid decline.

Mixed Plasmodium malariae Infections Were Underdetected in a Malaria Endemic Area in the Amazon Region, Brazil.

Plasmodium malariae infections are often asymptomatic and long-lasting. Mixed infections are often underdetected in areas where P. malariae, P. vivax, and P. falciparum are coendemic. In this study, we described the occurrence of these species circulating as single or mixed infections in Pará state, Brazil, in the Amazon region, with the purpose of clarifying the impact of misidentification of parasite species based only on morphological description using thick blood smear. By using real-time polymerase chain reaction based on the amplification of the mitochondrial DNA, we detected a prevalence of 46% (58/126) mixed infections with 33.3% P. malariae/P. vivax which were read as P. vivax monoinfections by microscopy detection. Our findings confirmed the high circulation of P. malariae in a malaria endemic area in the Brazilian Amazon region.

High prevalence of asymptomatic Plasmodium infection in Bandafassi, South-East Senegal.

BACKGROUND: Malaria control and elimination strategies are based on levels of transmission that are usually determined by data collected from health facilities. In endemic areas, asymptomatic Plasmodium infection is thought to represent the majority of infections, though they are not diagnosed nor treated. Therefore, there might be an underestimation of the malaria reservoir, resulting in inadequate control strategies. In addition, these untreated asymptomatic Plasmodium infections maintain transmission, making it difficult or impossible to reach malaria elimination goals. Thus, the aim of this study was to determine the prevalence of asymptomatic Plasmodium infections in southeastern Senegal. METHODS: A cross sectional study was conducted among asymptomatic individuals (N = 122) living in the village of Andiel located in Bandafassi, Kédougou, which consisted of about 200 inhabitants during the malaria transmission season in late October 2019. For each individual without malaria-related symptoms and who consented to participate, a rapid diagnostic test (RDT) was performed in the field. Results were confirmed in the laboratory with photo-induced electron transfer (PET-PCR). RESULTS: Malaria prevalence was 70.3% by PET-PCR and 41.8% by RDT. During the same period, the health post of the area reported 49. 1% test positivity rate by RDT. The majority of the infected study population, 92.9%, was infected with a single species and 7.1% had two or three species of Plasmodium. Plasmodium falciparum was predominant and represented 90.2% of the infections, while 6.5% were due to Plasmodium ovale and 3.3% to Plasmodium malariae. 59.4% of children targeted for SMC (zero to ten years old) were infected. CONCLUSION: In southeastern Senegal, where the transmission is the highest, malaria control strategies should address asymptomatic Plasmodium infections at the community level. The results suggest that this area could be eligible for mass drug administration. Moreover, non-falciparum species could be more common and its prevalence should be determined countrywide.

Quantification of the misidentification of Plasmodium knowlesi as Plasmodium malariae by microscopy: an analysis of 1569 P. knowlesi cases.

BACKGROUND: Plasmodium knowlesi is recognized as the fifth Plasmodium species causing malaria in humans. It is morphologically similar to the human malaria parasite Plasmodium malariae, so molecular detection should be used to clearly discriminate between these Plasmodium species. This study aimed to quantify the rate at which P. knowlesi is misidentified as P. malariae by microscopy in endemic and non-endemic areas. METHODS: The protocol of this systematic review was registered in the PROSPERO International Prospective Register of Systematic Reviews (ID = CRD42020204770). Studies reporting the misidentification of P. knowlesi as P. malariae by microscopy and confirmation of this by molecular methods in MEDLINE, Web of Science and Scopus were reviewed. The risk of bias in the included studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS). The pooled prevalence and 95% confidence interval (CI) of the misidentification of P. knowlesi as P. malariae by microscopy were estimated using a random effects model. Subgroup analysis of the study sites was performed to demonstrate any differences in the misidentification rates in different areas. Heterogeneity across the included studies was assessed and quantified using Cochran's Q and I2 statistics, respectively. Publication bias in the included studies was assessed using the funnel plot, Egger's test and contour-enhanced funnel plot. RESULTS: Among 375 reviewed studies, 11 studies with a total of 1569 confirmed P. knowlesi cases in humans were included. Overall, the pooled prevalence of the misidentification of P. knowlesi as P. malariae by microscopy was estimated at 57% (95% CI 37-77%, I2: 99.3%). Subgroup analysis demonstrated the highest rate of misidentification in Sawarak, Malaysia (87%, 95% CI 83-90%, I2: 95%), followed by Sabah, Malaysia (85%, 95% CI 79-92%, I2: 85.1%), Indonesia (16%, 95% CI 6-38%), and then Thailand (4%, 95% CI 2-9%, I2: 95%). CONCLUSION: Although the World Health Organization (WHO) recommends that all P. malariae-positive diagnoses made by microscopy in P. knowlesi endemic areas be reported as P. malariae/P. knowlesi malaria, the possibility of microscopists misidentifying P. knowlesi as P. malariae is a diagnostic challenge. The use of molecular techniques in cases with malariae-like Plasmodium with high parasite density as determined by microscopy could help identify human P. knowlesi cases in non-endemic countries.

Understanding adherence to reactive treatment of asymptomatic malaria infections in The Gambia.

The impact of different types of reactive case detection and/or treatment strategies for malaria elimination depends on high coverage and participants' adherence. However, strategies to optimise adherence are limited, particularly for people with asymptomatic or no infections. As part of a cluster-randomized trial to evaluate the effect of reactive treatment in The Gambia, all residents in the compound of a diagnosed clinical malaria patient received dihydro-artemisinin-piperaquine (DP). Using a mixed method approach, we assessed which factors contribute to adherence among the contacts of malaria cases that showed no symptoms. Adherence was defined as the proportion of compound members that (1) returned all medicine bags empty and (2) self-reported (3-day) treatment completion. Among the 273 individuals from 14 compounds who received DP, 227 (83.1%) were available for and willing to participate in the survey; 85.3% (233/273) returned empty medicine bags and 91.6% (208/227) self-reported treatment completion. Although clinical malaria was not considered a major health problem, reported adherence was high. The drivers of adherence were the strong sense of responsibility towards protecting the individual, compound and the village. Adherence can be optimised through a transdisciplinary implementation research process of engaging communities to bridge the gap between research goals and social realities.

The hidden Plasmodium malariae in blood donors: a risk coming from areas of low transmission of malaria.

Malaria is an infectious vector-borne disease with other important routes of transmission, such as blood transfusion and organ/tissue transplantation, due to asymptomatic reservoirs of Plasmodium presenting with low parasitemia. Reports of transfusion-transmitted malaria have shown that in immunosuppressed recipients, infections can be fatal if they are not diagnosed and timely treated. All Plasmodium species can survive on blood components at temperatures from 2 to 6 °C for some days or even weeks. This report describes two candidates for blood donation harboring Plasmodium, infected in an area considered non-endemic. Blood samples were collected from donors who attended a blood bank in Sao Paulo and tested by microscopy, qPCR for Plasmodium genus-specific amplification, targeting the parasite 18S ribosomal subunit gene and a multiplex qPCR based on mtDNA of the five species. Under microscopy, only structures resembling Plasmodium were observed. The qPCR whose standard curve tested parasites varying from 2 to 0.1 parasites/ µL, showed the presence of Plasmodium DNA in the two blood donors, as did the multiplex qPCR that revealed the presence of P. malariae. The prevalence of positive donors varies according to the level of transmission, ranging from 0.7 to 55% in endemic areas. In non-endemic regions, prevalences are lower, however, transfusion malaria can evolve to severe cases, due to the lack of suspicion of this transmission route. Asymptomatic donors from low transmission regions pose a risk to blood banks, with particular emphasis on those located in areas with malaria elimination goals.

Prevalence, characteristics and risk factors of imported and local malaria cases in North-Western Province, Zambia: a cross-sectional study.

BACKGROUND: Imported malaria is a major challenge for countries that are in malaria elimination stage such as Zambia. Legitimate cross-border activities add to the risk of transmission, necessitating determination of prevalence, characteristics and risk factors of imported and local malaria. METHODS: This cross-sectional study was conducted in 103 consented child and adult patients with clinical malaria symptoms, from selected health facilities in north-western Zambia. Patient demographic data and blood samples for malaria microscopy and full blood count were obtained. Chi-square and penalized logistic regression were performed to describe the characteristics and assess the risk factors of imported and local malaria in North-Western Province. RESULTS: Overall, malaria prevalence was 78.6% with 93.8% Plasmodium falciparum and 6.2% other species. The local cases were 72 (88.9%) while the imported were 9 (11.1%) out of the 81 positive participants. About 98.6% of the local cases were P. falciparum compared to 55.6% (χ2 = 52.4; p < 0.01) P. falciparum among the imported cases. Among the imported cases, 44% were species other than P. falciparum (χ2 = 48; p < 0.01) while among the local cases only 1.4% were. Gametocytes were present in 44% of the imported malaria cases and only in 2.8% of the local cases (χ2 = 48; p < 0.01). About 48.6% of local participants had severe anaemia compared to 33.3% of participants from the two neighbouring countries who had (χ2 = 4.9; p = 0.03). In the final model, only country of residence related positively to presence of species other than P. falciparum (OR = 39.0, CI [5.9, 445.9]; p < 0.01) and presence of gametocytes (OR = 23.1, CI [4.2, 161.6]; p < 0.01). CONCLUSION: Malaria prevalence in North-Western Province is high, with P. falciparum as the predominant species although importation of Plasmodium ovale and Plasmodium malariae is happening as well. Country of residence of patients is a major risk factor for malaria species and gametocyte presence. The need for enhanced malaria control with specific focus on border controls to detect and treat, for specific diagnosis and treatment according to species obtaining, for further research in the role of species and gametocytaemia in imported malaria, cannot be overemphasized.

High prevalence of asymptomatic malaria infections in adults, Ashanti Region, Ghana, 2018.

BACKGROUND: Ghana is among the high-burden countries for malaria infections and recently reported a notable increase in malaria cases. While asymptomatic parasitaemia is increasingly recognized as a hurdle for malaria elimination, studies on asymptomatic malaria are scarce, and usually focus on children and on non-falciparum species. The present study aims to assess the prevalence of asymptomatic Plasmodium falciparum and non-falciparum infections in Ghanaian adults in the Ashanti region during the high transmission season. METHODS: Asymptomatic adult residents from five villages in the Ashanti Region, Ghana, were screened for Plasmodium species by rapid diagnostic test (RDT) and polymerase chain reaction (PCR) during the rainy season. Samples tested positive were subtyped using species-specific real-time PCR. For all Plasmodium ovale infections additional sub-species identification was performed. RESULTS: Molecular prevalence of asymptomatic Plasmodium infection was 284/391 (73%); only 126 (32%) infections were detected by RDT. While 266 (68%) participants were infected with Plasmodium falciparum, 33 (8%) were infected with Plasmodium malariae and 34 (9%) with P. ovale. The sub-species P. ovale curtisi and P. ovale wallikeri were identified to similar proportions. Non-falciparum infections usually presented as mixed infections with P. falciparum. CONCLUSIONS: Most adult residents in the Ghanaian forest zone are asymptomatic Plasmodium carriers. The high Plasmodium prevalence not detected by RDT in adults highlights that malaria eradication efforts must target all members of the population. Beneath Plasmodium falciparum, screening and treatment must also include infections with P. malariae, P. o. curtisi and P. o. wallikeri.

Fracaso renal agudo anúrico persistente en paciente infectado con Plasmodium malariae: la importancia de la biopsia renal / Persistent acute renal failure in a patient infected with Plasmodium malariae: The importance of renal biopsy

No disponible

Comparative evaluation of two commercial real-time PCR kits (QuantiFast™ and abTES™) for the detection of Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia.

BACKGROUND: The monkey parasite Plasmodium knowlesi is an emerging public health issue in Southeast Asia. In Sabah, Malaysia, P. knowlesi is now the dominant cause of human malaria. Molecular detection methods for P. knowlesi are essential for accurate diagnosis and in monitoring progress towards malaria elimination of other Plasmodium species. However, recent commercially available PCR malaria kits have unpublished P. knowlesi gene targets or have not been evaluated against clinical samples. METHODS: Two real-time PCR methods currently used in Sabah for confirmatory malaria diagnosis and surveillance reporting were evaluated: the QuantiFast™ Multiplex PCR kit (Qiagen, Germany) targeting the P. knowlesi 18S SSU rRNA; and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosed P. knowlesi gene target. Diagnostic accuracy was evaluated using 52 P. knowlesi, 25 Plasmodium vivax, 21 Plasmodium falciparum, and 10 Plasmodium malariae clinical isolates, and 26 malaria negative controls, and compared against a validated reference nested PCR assay. The limit of detection (LOD) for each PCR method and Plasmodium species was also evaluated. RESULTS: The sensitivity of the QuantiFast™ and abTES™ assays for detecting P. knowlesi was comparable at 98.1% (95% CI 89.7-100) and 100% (95% CI 93.2-100), respectively. Specificity of the QuantiFast™ and abTES™ for P. knowlesi was high at 98.8% (95% CI 93.4-100) for both assays. The QuantiFast™ assay demonstrated falsely-positive mixed Plasmodium species at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits for detecting P. vivax, P. falciparum, and P. malariae was comparable to P. knowlesi. The abTES™ assay demonstrated a lower LOD for P. knowlesi of ≤ 0.125 parasites/µL compared to QuantiFast™ with a LOD of 20 parasites/µL. Hospital microscopy demonstrated a sensitivity of 78.8% (95% CI 65.3-88.9) and specificity of 80.4% (95% CI 67.6-89.8) compared to reference PCR for detecting P. knowlesi. CONCLUSION: The QuantiFast™ and abTES™ commercial PCR kits performed well for the accurate detection of P. knowlesi infections. Although the QuantiFast™ kit is cheaper, the abTES™ kit demonstrated a lower LOD, supporting its use as a second-line referral-laboratory diagnostic tool in Sabah, Malaysia.

Characteristics of imported Plasmodium ovale spp. and Plasmodium malariae in Hubei Province, China, 2014-2018.

BACKGROUND: There have been an increasing number of imported cases of malaria in Hubei Province in recent years. In particular, the number of cases of Plasmodium ovale spp. and Plasmodium malariae significantly increased, which resulted in increased risks during the malaria elimination phase. The purpose of this study was to acquire a better understanding of the epidemiological characteristics of P. ovale spp. and P. malariae imported to Hubei Province, China, so as to improve case management. METHODS: Data on all malaria cases from January 2014 to December 2018 in Hubei Province were extracted from the China national diseases surveillance information system (CNDSIS). This descriptive study was conducted to analyse the prevalence trends, latency periods, interval from onset of illness to diagnosis, and misdiagnosis of cases of P. ovale spp. and P. malariae malaria. RESULTS: During this period, 634 imported malaria cases were reported, of which 87 P. ovale spp. (61 P. ovale curtisi and 26 P. ovale wallikeri) and 18 P. malariae cases were confirmed. The latency periods of P. ovale spp., P. malariae, Plasmodium vivax, and Plasmodium falciparum differed significantly, whereas those of P. ovale curtisi and P. ovale wallikeri were no significant difference. The proportion of correct diagnosis of P. ovale spp. and P. malariae malaria cases were 48.3% and 44.4%, respectively, in the hospital or lower-level Centers for Disease Control and Prevention (CDC). In the Provincial Reference Laboratory, the sensitivity of microscopy and rapid diagnostic tests was 94.3% and 70.1%, respectively, for detecting P. ovale spp., and 88.9% and 38.9%, respectively, for detecting P. malariae. Overall, 97.7% (85/87) of P. ovale spp. cases and 94.4% (17/18) of P. malariae cases originated from Africa. CONCLUSION: The increase in the number of imported P. ovale spp. and P. malariae cases, long latency periods, and misdiagnosis pose a challenge to this region. Therefore, more attention should be paid to surveillance of imported cases of P. ovale spp. and P. malariae infection to reduce the burden of public health and potential risk of malaria.

Malaria parasitemia among blood donors in Uganda.

BACKGROUND: Malaria remains a leading transfusion associated infectious risk in endemic areas. However, the prevalence of malaria parasitemia has not been well characterized in blood donor populations. This study sought to determine the prevalence of Plasmodium in red blood cell (RBC) and whole blood (WB) units after the rainy season in Uganda. METHODS AND MATERIALS: Between May and July 2018, blood was collected from the sample diversion pouch of 1000 WB donors in Kampala and Jinja, Uganda. The RBC pellet from ethylenediamine tetraacetic acid (EDTA) anticoagulated blood was stored at -80°C until testing. DNA was extracted and nested PCR was used to screen samples at the genus level for Plasmodium, with positive samples further tested for species identification. RESULTS: Malaria parasitemia among asymptomatic, eligible blood donors in two regions of Uganda was 15.4%; 87.7% (135/154) of infections were with P. falciparum, while P. malariae and P. ovale were also detected. There were 4.3% of blood donors who had mixed infection with multiple species. Older donors (>30 years vs. 17-19 years; aPR = 0.31 [95% CI = 0.17-0.58]), females (aPR = 0.60 [95% CI = 0.42-0.87]), repeat donors (aPR = 0.44 [95% CI = 0.27-0.72]) and those donating near the capital city of Kampala versus rural Jinja region (aPR = 0.49 [95% CI = 0.34-0.69]) had a lower prevalence of malaria parasitemia. CONCLUSIONS: A high proportion of asymptomatic blood donors residing in a malaria endemic region demonstrate evidence of parasitemia at time of donation. Further research is needed to quantify the risk and associated burden of transfusion-transmitted malaria (TTM) in order to inform strategies to prevent TTM.

Screening for malaria antigen and anti-malarial IgG antibody in forcibly-displaced Myanmar nationals: Cox's Bazar district, Bangladesh, 2018.

BACKGROUND: Several refugee settlements in Bangladesh have provided housing and medical care for the forcibly-displaced Myanmar nationals (FDMN, also known as Rohingya) population. The identification of malaria infection status in the refugee settlements is useful in treating infected persons and in developing malaria prevention recommendations. Assays for Plasmodium antigens and human IgG against Plasmodium parasites can be used as indicators to determine malaria infection status and exposure. METHODS: Dried blood spot (DBS) samples (N = 1239) from a household survey performed April-May 2018 in three settlements in Cox's Bazar district, Bangladesh were utilized for a sample population of children from ages 1-14 years of age. The samples were tested using a bead-based multiplex antigen assay for presence of the pan-Plasmodium antigen aldolase as well as Plasmodium falciparum histidine rich protein 2 (HRP2). A bead-based multiplex assay was also used to measure human IgG antibody response to P. falciparum, Plasmodium malariae, and Plasmodium vivax merozoite surface protein 1 antigen (MSP1) isoforms, and P. falciparum antigens LSA1, CSP, and GLURP-R0. RESULTS: There were no detectable Plasmodium antigens in any samples, suggesting no active malaria parasite infections in the tested children. IgG seroprevalence was highest to P. vivax (3.1%), but this was not significantly different from the percentages of children antibody responses to P. falciparum (2.1%) and P. malariae (1.8%). The likelihood of an anti-Plasmodium IgG response increased with age for all three malaria species. Evidence of exposure to any malaria species was highest for children residing 8-10 months in the settlements, and was lower for children arriving before and after this period of time. CONCLUSIONS: Absence of Plasmodium antigen in this population provides evidence that children in these three Bangladeshi refugee settlements did not have malaria at time of sampling. Higher rates of anti-malarial IgG carriage from children who were leaving Myanmar during the malaria high-transmission season indicate these migrant populations were likely at increased risk of malaria exposure during their transit.

Updates on malaria incidence and profile in Malaysia from 2013 to 2017.

BACKGROUND: To date, most of the recent publications on malaria in Malaysia were conducted in Sabah, East Malaysia focusing on the emergence of Plasmodium knowlesi. This analysis aims to describe the incidence, mortality and case fatality rate of malaria caused by all Plasmodium species between Peninsular Malaysia and East Malaysia (Sabah and Sarawak) over a 5-year period (2013-2017). METHODS: This is a secondary data review of all diagnosed and reported malaria confirmed cases notified to the Ministry of Health, Malaysia between January 2013 and December 2017. RESULTS: From 2013 to 2017, a total of 16,500 malaria cases were notified in Malaysia. The cases were mainly contributed from Sabah (7150; 43.3%) and Sarawak (5684; 34.4%). Majority of the patients were male (13,552; 82.1%). The most common age group in Peninsular Malaysia was 20 to 29 years (1286; 35.1%), while Sabah and Sarawak reported highest number of malaria cases in age group of 30 to 39 years (2776; 21.6%). The top two races with malaria in Sabah and Sarawak were Bumiputera Sabah (5613; 43.7%) and Bumiputera Sarawak (4512; 35.1%), whereas other ethnic group (1232; 33.6%) and Malays (1025; 28.0%) were the two most common races in Peninsular Malaysia. Plasmodium knowlesi was the commonest species in Sabah and Sarawak (9902; 77.1%), while there were more Plasmodium vivax cases (1548; 42.2%) in Peninsular Malaysia. The overall average incidence rate, mortality rate and case fatality rates for malaria from 2013 to 2017 in Malaysia were 0.106/1000, 0.030/100,000 and 0.27%, respectively. Sarawak reported the highest average incidence rate of 0.420/1000 population followed by Sabah (0.383/1000). Other states in Peninsular Malaysia reported below the national average incidence rate with less than 0.100/1000. CONCLUSIONS: There were different trends and characteristics of notified malaria cases in Peninsular Malaysia and Sabah and Sarawak. They provide useful information to modify current prevention and control measures so that they are customised to the peculiarities of disease patterns in the two regions in order to successfully achieve the pre-elimination of human-only species in the near future.

Polymorphic markers for identification of parasite population in Plasmodium malariae.

BACKGROUND: Molecular genotyping in Plasmodium serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for Plasmodium malariae, an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of P. malariae population genetics. METHODS: Markers for microsatellite genotyping and pmmsp1 gene polymorphisms were developed and validated in symptomatic P. malariae field isolates from Myanmar (N = 37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, heterozygosity and construct dendrograms. Nucleotide diversity (π), number of haplotypes, and genetic diversity (Hd) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed. RESULTS: Six microsatellite markers were developed and tested in 37 P. malariae isolates which showed sufficient heterozygosity (0.530-0.922), and absence of linkage disequilibrium (IAS=0.03, p value > 0.05) (N = 37). In addition, a tandem repeat (VNTR)-based pmmsp1 INDEL polymorphisms marker was developed and assessed in 27 P. malariae isolates showing a nucleotide diversity of 0.0976, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 and pmmsp1 showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites markers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10-4 and was further reduced to 1.66 × 10-4 when pmmsp1 polymorphisms were included. CONCLUSIONS: Six novel microsatellites genotyping markers and a set of pmmsp1 VNTR-based INDEL polymorphisms markers for P. malariae were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for investigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies.

Role of Anopheles gambiae s.s. and Anopheles coluzzii (Diptera: Culicidae) in Human Malaria Transmission in Rural Areas of Bouaké, in Côte d'Ivoire.

Studies done in Bouaké (Côte d'Ivoire) about 20-yr ago reported that Anopheles gambiae s.l. Giles was the major malaria vector. The present study aimed to update these data and to identify the main vectors. Mosquitoes were collected in Allokokro and Petessou villages between June 2014 and December 2015 using the human landing catching method. Potential breeding sites of An. gambiae s.l. were identified in August and October 2014 and mapped using GPS. Anopheles species were morphologically and molecularly [polymerase chain reaction (PCR)] identified. Ovaries of female were dissected to determine the parity and infection with Plasmodium was detected in head and thorax by quantitative PCR. In Allokokro, the biting rate of An. gambiae s.s was significantly greater than Anopheles coluzzii, whereas, in Petessou, biting rates of both species were comparable. Plasmodium falciparum (Haemosporida: Plasmodiidae), Plasmodium malariae (Haemosporida: Plasmodiidae), and Plasmodium ovale (Haemosporida: Plasmodiidae) identified in both villages. The infection rates of An. gambiae s.s. and An. coluzzii were not significantly different. The entomological inoculation rate (EIR) of An. gambiae s.s. for P. falciparum was 9-fold greater than that of An. coluzzii in Allokokro; however, in Petessou, the EIRs of both species were comparable. In both village, An. gambiae s.s was responsible for P. falciparum and P. ovale transmission whereas An. coluzzii transmitted all three Plasmodium species.

Evaluation of Malaria Blood Smears Sent to National Malaria Reference Laboratory

Objective: Although the disease has been eliminated in Turkey malaria continues to be a threat due to increase in the number of people coming from or going to countries where the disease is endemic. In this study, we aimed to evaluate blood smears sent to the National Malaria Reference Laboratory within the malaria surveillance system. Methods: From March 2016 to July 2018 a retrospective study was conducted to compare the results of Malaria Reference Laboratory and Public Health Laboratories. A total of 16.827 blood stains were sent to our laboratory for approval. Results: In Public Health Laboratories, 315 (1.88%) of the smears were positive, 16.510 (98.12%) were negative, and in the National Malaria Reference Laboratory 252 (1.50%) were positive, 16.466 were negative. In the Public Health Laboratories, one of the two samples considered to be malaria suspected was positive in the National Malaria Reference Laboratory and one was negative. In Public Health Laboratories 35.88% of smears were P. falciparum, 27.30% were Plasmodium spp., 20.96% were P. vivax, 14.92% were mixed infection, 0.63% were P. malariae, 0.31% were P. ovale, and in the Reference Laboratory 49.60% were Plasmodium spp., 29.37% were P. falciparum, 16.27% were P. vivax, 4.36% were mixed infection, 0.40% were P. malariae. Conclusion: In order to malaria surveillance system to be maintained in a healthy manner, preparation, staining, coding, packaging, transportation of blood slides is very important. Also if necessary, continuing training of laboratory staff working in malaria diagnosis is crucial.

Sero-identification of the aetiologies of human malaria exposure (Plasmodium spp.) in the Limu Kossa District of Jimma Zone, South western Ethiopia.

BACKGROUND: Malaria remains a very important public health problem in Ethiopia. Currently, only Plasmodium falciparum and Plasmodium vivax are considered in the malaria diagnostic and treatment policies. However, the existence and prevalence of Plasmodium ovale spp. and Plasmodium malariae in Ethiopia have not been extensively investigated. The objective of this study was to use a multiplex IgG antibody detection assay to evaluate evidence for exposure to any of these four human malaria parasites among asymptomatic individuals. METHODS: Dried blood spots (DBS) were collected from 180 healthy study participants during a 2016 onchocerciasis survey in the Jimma Zone, southwest Ethiopia. IgG antibody reactivity was detected using a multiplex bead assay for seven Plasmodium antigens: P. falciparum circumsporozoite protein (CSP), P. falciparum apical membrane antigen-1 (AMA1), P. falciparum liver stage antigen-1 (LSA1), and homologs of the merozoite surface protein-1 (MSP1)-19kD antigens that are specific for P. falciparum, P. vivax, P. ovale spp. and P. malariae. RESULTS: One hundred six participants (59%) were IgG seropositive for at least one of the Plasmodium antigens tested. The most frequent responses were against P. falciparum AMA1 (59, 33%) and P. vivax (55, 28%). However, IgG antibodies against P. ovale spp. and P. malariae were detected in 19 (11%) and 13 (7%) of the participants, respectively, providing serological evidence that P. malariae and P. ovale spp., which are rarely reported, may also be endemic in Jimma. CONCLUSION: The findings highlight the informative value of multiplex serology and the need to confirm whether P. malariae and P. ovale spp. are aetiologies of malaria in Ethiopia, which is critical for proper diagnosis and treatment.