Non-falciparum malaria infections in Uganda, does it matter? A review of the published literature.

BACKGROUND: Plasmodium falciparum is the dominant malaria species in the sub-Saharan Africa and the main cause of severe disease and death. Notwithstanding, severe malaria and death due to non-falciparum infections have been reported, but at much lower rates than P. falciparum infections. Following increasing use of molecular detection techniques in epidemiological studies, a higher prevalence of non-falciparum species has been reported in the region than previously thought. This article reviews the literature on the prevalence of non-falciparum malaria species in Uganda and the clinical figures of their severe diseases. It aims to elucidate the extent to which mono non-falciparum malaria infections in a highly malaria-endemic country contribute to malaria mortality and outline its policy implications on malaria case management. METHODS: The available English-language published peer-reviewed literature up to March 2024 was sought via PubMed and Google Scholar. The keywords used were severe malaria, AND P. falciparum, P. malariae, P. vivax, P. ovale spp., mixed infections AND Uganda. The review encompassed 53 articles. Articles using molecular diagnosis methods were accounted for analysis. RESULTS: The literature reported a substantial prevalence of non-falciparum infections in Uganda. Plasmodium malariae and Plasmodium ovale spp. were the second and third most prevalent reported malaria species respectively after P. falciparum as dominant species. Non-falciparum malaria infections often occur as mixed infections rather than mono-infections. Besides, molecular diagnostics revealed that 21% of initially reported mono-infections of P. falciparum were, in fact, mixed infections. No article was found on the prevalence of severe malaria or case fatality rate due to mixed or non-falciparum infections. CONCLUSION: A critical knowledge gap exists regarding the impact of mixed and non-falciparum species on severe malaria and death in Uganda. Robust evidence on prevalence, recurrent parasitaemia, and severe clinical manifestations of mixed and non-falciparum malaria infections is crucial for evidence-based and effective policymaking regarding malaria case management.

Evidence of Plasmodium vivax circulation in western and eastern regions of Senegal: implications for malaria control.

BACKGROUND: Malaria elimination in Senegal requires accurate diagnosis of all Plasmodium species. Plasmodium falciparum is the most prevalent species in Senegal, although Plasmodium malariae, Plasmodium ovale, and recently Plasmodium vivax have also been reported. Nonetheless, most malaria control tools, such as Histidine Rich Protein 2 rapid diagnosis test (PfHRP2-RDT,) can only diagnose P. falciparum. Thus, PfHRP2-RDT misses non-falciparum species and P. falciparum infections that fall below the limit of detection. These limitations can be addressed using highly sensitive Next Generation Sequencing (NGS). This study assesses the burden of the four different Plasmodium species in western and eastern regions of Senegal using targeted PCR amplicon sequencing. METHODS: Three thousand samples from symptomatic and asymptomatic individuals in 2021 from three sites in Senegal (Sessene, Diourbel region; Parcelles Assainies, Kaolack region; Gabou, Tambacounda region) were collected. All samples were tested using PfHRP2-RDT and photoinduced electron transfer polymerase chain reaction (PET-PCR), which detects all Plasmodium species. Targeted sequencing of the nuclear 18S rRNA and the mitochondrial cytochrome B genes was performed on PET-PCR positive samples. RESULTS: Malaria prevalence by PfHRP2-RDT showed 9.4% (94/1000) and 0.2% (2/1000) in Diourbel (DBL) and Kaolack (KL), respectively. In Tambacounda (TAM) patients who had malaria symptoms and had a negative PfHRP2-RDT were enrolled. The PET-PCR had a positivity rate of 23.5% (295/1255) overall. The PET-PCR positivity rate was 37.6%, 12.3%, and 22.8% in Diourbel, Kaolack, and Tambacounda, respectively. Successful sequencing of 121/295 positive samples detected P. falciparum (93%), P. vivax (2.6%), P. malariae (4.4%), and P. ovale wallikeri (0.9%). Plasmodium vivax was co-identified with P. falciparum in thirteen samples. Sequencing also detected two PfHRP2-RDT-negative mono-infections of P. vivax in Tambacounda and Kaolack. CONCLUSION: The findings demonstrate the circulation of P. vivax in western and eastern Senegal, highlighting the need for improved malaria control strategies and accurate diagnostic tools to better understand the prevalence of non-falciparum species countrywide.

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.

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.

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.

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.

Clinical Usefulness of LabChip Real-time PCR using Lab-On-a-Chip Technology for Diagnosing Malaria.

As malaria remains a major health problem worldwide, various diagnostic tests have been developed, including microscopy-based and rapid diagnostic tests. LabChip real-time PCR (LRP) is a small and portable device used to diagnose malaria using lab-on-a-chip technology. This study aimed to evaluate the diagnostic performance of LRP for detecting malaria parasites. Two hundred thirteen patients and 150 healthy individuals were enrolled from May 2009 to October 2015. A diagnostic detectability of LRP for malaria parasites was compared to that of conventional RT-PCR. Sensitivity of LRP for Plasmodium vivax, P. falciparum, P. malariae, and P. ovale was 95.5%, 96.0%, 100%, and 100%, respectively. Specificity of LRP for P. vivax, P. falciparum, P. malariae, and P. ovale was 100%, 99.3%, 100%, and 100%, respectively. Cohen's Kappa coefficients between LRP and CFX96 for detecting P. vivax, P. falciparum, P. malariae, and P. ovale were 0.96, 0.98, 1.00, and 1.00, respectively. Significant difference was not observed between the results of LRP and conventional RT-PCR and microscopic examination. A time required to amplify DNAs using LRP and conventional RT-PCR was 27 min and 86 min, respectively. LRP amplified DNAs 2 times more fast than conventional RT-PCR due to the faster heat transfer. Therefore, LRP could be employed as a useful tool for detecting malaria parasites in clinical laboratories.

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.

Polymorphism analysis of propeller domain of k13 gene in Plasmodium ovale curtisi and Plasmodium ovale wallikeri isolates original infection from Myanmar and Africa in Yunnan Province, China.

BACKGROUND: Eighteen imported ovale malaria cases imported from Myanmar and various African countries have been reported in Yunnan Province, China from 2013 to 2018. All of them have been confirmed by morphological examination and 18S small subunit ribosomal RNA gene (18S rRNA) based PCR in YNRL. Nevertheless, the subtypes of Plasmodium ovale could not be identified based on 18S rRNA gene test, thus posing challenges on its accurate diagnosis. To help establish a more sensitive and specific method for the detection of P. ovale genes, this study performs sequence analysis on k13-propeller polymorphisms in P. ovale. METHODS: Dried blood spots (DBS) from ovale malaria cases were collected from January 2013 to December 2018, and the infection sources were confirmed according to epidemiological investigation. DNA was extracted, and the coding region (from 206th aa to 725th aa) in k13 gene propeller domain was amplified using nested PCR. Subsequently, the amplified products were sequenced and compared with reference sequence to obtain CDS. The haplotypes and mutation loci of the CDS were analysed, and the spatial structure of the amino acid peptide chain of k13 gene propeller domain was predicted by SWISS-MODEL. RESULTS: The coding region from 224th aa to 725th aa of k13 gene from P. ovale in 83.3% of collected samples (15/18) were amplified. Three haplotypes were observed in 15 samples, and the values of Ka/Ks, nucleic acid diversity index (π) and expected heterozygosity (He) were 3.784, 0.0095, and 0.4250. Curtisi haplotype, Wallikeri haplotype, and mutant type accounted for 73.3% (11/15), 20.0% (3/15), and 6.7% (1/15). The predominant haplotypes of P. ovale curtisi were determined in all five Myanmar isolates. Of the ten African isolates, six were identified as P. o. curtisi, three were P. o. wallikeri and one was mutant type. Base substitutions between the sequences of P. o. curtisi and P. o. wallikeri were determined at 38 loci, such as c.711. Moreover, the A > T base substitution at c.1428 was a nonsynonymous mutation, resulting in amino acid variation of T476S in the 476th position. Compared with sequence of P. o. wallikeri, the double nonsynonymous mutations of G > A and A > T at the sites of c.1186 and c.1428 leads to the variations of D396N and T476S for the 396th and 476th amino acids positions. For P. o. curtisi and P. o. wallikeri, the peptide chains in the coding region from 224th aa to 725th aa of k13 gene merely formed a monomeric spatial model, whereas the double-variant peptide chains of D396N and T476S formed homodimeric spatial model. CONCLUSION: The propeller domain of k13 gene in the P. ovale isolates imported into Yunnan Province from Myanmar and Africa showed high differentiation. The sequences of Myanmar-imported isolates belong to P. o. curtisi, while the sequences of African isolates showed the sympatric distribution from P. o. curtisi, P. o. wallikeri and mutant isolates. The CDS with a double base substitution formed a dimeric spatial model to encode the peptide chain, which is completely different from the monomeric spatial structure to encode the peptide chain from P. o. curtisi and P. o. wallikeri.

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.

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.

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.

Non-imported malaria in Italy: paradigmatic approaches and public health implications following an unusual cluster of cases in 2017.

BACKGROUND: The European region achieved interruption of malaria transmission during the 1970s. Since then, malaria control programs were replaced by surveillance systems in order to prevent possible re-emergence of this disease. Sporadic cases of non-imported malaria were recorded in several European countries in the past decade and locally transmitted outbreaks of Plasmodium vivax, most probably supported by Anopheles sacharovi, have been repeatedly reported from Greece since 2009. The possibility of locally-transmitted malaria has been extensively studied in Italy where the former malaria vector An. labranchiae survived the control campaign which led to malaria elimination. In this study, we present paradigmatic cases that occurred during a 2017 unusual cluster, which caused strong concern in public opinion and were carefully investigated after the implementation of the updated malaria surveillance system. METHODS: For suspected locally-transmitted malaria cases, alerts to Ministry of Health (MoH) and the National Institute of Health (ISS) were mandated by the Local Health Services (LHS). Epidemiological investigations on the transmission modes and the identification of possible infection's source were carried out by LHS, MoH and ISS. Entomological investigations were implemented locally for all suspected locally-transmitted cases that occurred in periods suitable to anopheline activity. Molecular diagnosis by nested-PCR for the five human Plasmodium species was performed to support microscopic diagnosis. In addition, genotyping of P. falciparum isolate was carried out to investigate putative sources of infection and transmission modalities. RESULTS: In 2017, a cluster of seven non-imported cases was recorded from August through October. Among them, P. ovale curtisi was responsible of one case whereas six cases were caused by P. falciparum. Two cases were proved to be nosocomial while the other five were recorded as cryptic at the end of epidemiological investigations. CONCLUSIONS: The epidemiological evidence shows that the locally acquired events are sporadic, often remain unresolved and classified as cryptic ones despite investigative efforts. The "cluster" of seven non-imported cases that occurred in 2017 in different regions of Italy therefore represents a conscious alert that should lead us to maintain a constant level of surveillance in a former malaria endemic country.

Diagnosis and Molecular Analysis on Imported Plasmodium ovale curtisi and P. ovale wallikeri Malaria Cases from West and South Africa during 2013-2016.

Majority of the imported malaria cases in Korea is attributed to Plasmodium falciparum and P. vivax infections, whereas P. malariae and P. ovale infections are very rare. Falciparum and ovale malaria are mostly imported from Africa, while most of the vivax malaria cases are imported from Southeast Asia. Here, we report 6 Korean imported ovale malaria cases (4 males and 2 females) who had visited in Africa during 2013-2016. These subjects were diagnosed with P. ovale based on microscopic findings, Plasmodium species-specific nested-PCR, and phylogenetic clade using 18S rRNA gene sequences. We identified 2 P. ovale subtypes, 1 P. ovale curtisi (classic type) and 5 P. ovale wallikeri (variant type). All patients were treated with chloroquine and primaquine, and no relapse or recrudescence was reported for 1 year after treatment. With increase of travelers to the countries where existing Plasmodium species, the risk of Plasmodium infection is also increasing. Molecular monitoring for imported malaria parasites should be rigorously and continuously performed to enable diagnosis and certification of Plasmodium spp.

Severity of Plasmodium falciparum and Non-falciparum Malaria in Travelers and Migrants: A Nationwide Observational Study Over 2 Decades in Sweden.

BACKGROUND: The aim was to assess factors affecting disease severity in imported P. falciparum and non-falciparum malaria. METHODS: We reviewed medical records from 2793/3260 (85.7%) of all episodes notified in Sweden between 1995 and 2015 and performed multivariable logistic regression. RESULTS: Severe malaria according to WHO 2015 criteria was found in P. falciparum (9.4%), P. vivax (7.7%), P. ovale (5.3%), P. malariae (3.3%), and mixed P. falciparum episodes (21.1%). Factors associated with severe P. falciparum malaria were age <5 years and >40 years, origin in nonendemic country, pregnancy, HIV, region of diagnosis, and health care delay. Moreover, oral treatment of P. falciparum episodes with parasitemia ≥2% without severe signs at presentation was associated with progress to severe malaria with selected criteria. In non-falciparum, age >60 years, health care delay and endemic origin were identified as risk factors for severe disease. Among patients originating in endemic countries, a higher risk for severe malaria, both P. falciparum and non-falciparum, was observed among newly arrived migrants. CONCLUSIONS: Severe malaria was observed in P. falciparum and non-falciparum episodes. Current WHO criteria for severe malaria may need optimization to better guide the management of malaria of different species in travelers and migrants in nonendemic areas.

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.

Evaluation of automated malaria diagnosis using the Sysmex XN-30 analyser in a clinical setting.

BACKGROUND: Early and accurate diagnosis of malaria is a critical aspect of efforts to control the disease, and several diagnostic tools are available. Microscopic assessment of a peripheral blood smear enables direct visualization of parasites in infected red blood cells and is the clinical diagnostic gold standard. However, it is subjective and requires a high level of skill. Numerous indirect detection methods are in use, but are not ideal since surrogate markers of infection are measured. This study describes the first clinical performance evaluation of the automated Sysmex XN-30 analyser, which utilizes fluorescence flow cytometry to directly detect and quantitate parasite-infected red blood cells. RESULTS: Residual EDTA blood samples from suspected malaria cases referred for routine diagnosis were analysed on the XN-30. Parasitaemia was reported as a percentage, as well as absolute numbers of infected red blood cells, and scattergrams provided a visual image of the parasitized red blood cell clusters. The results reported by the XN-30 correlated with microscopy and the analyser demonstrated 100% sensitivity and specificity. Measurements were reproducible and storage of samples at room temperature did not affect the parameters. Several Plasmodium species were detected, including Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale. The XN-30 also identified the transmissible gametocytes as a separate cluster on the scattergrams. Abnormal red blood cell indices (low haemoglobin and raised reticulocyte counts), haemoglobinopathies and thrombocytopenia did not interfere with the detection of parasites. The XN-30 also generated a concurrent full blood count for each sample. CONCLUSIONS: The novel technology of the Sysmex XN-30 provides a robust, rapid, automated and accurate platform for diagnosing malaria in a clinical setting. The objective enumeration of red blood cells infected with Plasmodium species makes it suitable for global use and allows monitoring of the parasite load once therapy has been initiated, thereby providing an early marker of drug resistance. The automated generation of a full blood count for each sample provides an opportunity for detecting unsuspected cases. Asymptomatic carriers can also be identified, which will be useful in blood transfusion centres, and will enable treatment of these individuals to prevent the spread of the disease.

A comparison of two PCR protocols for the differentiation of Plasmodium ovale species and implications for clinical management in travellers returning to Germany: a 10-year cross-sectional study.

BACKGROUND: To assess the occurrence of Plasmodium ovale wallikeri and Plasmodium ovale curtisi species in travellers returning to Germany, two real-time PCR protocols for the detection and differentiation of the two P. ovale species were compared. Results of parasite differentiation were correlated with patient data. METHODS: Residual nucleic acid extractions from EDTA blood samples of patients with P. ovale spp. malaria, collected between 2010 and 2019 at the National Reference Centre for Tropical Pathogens in Germany, were subjected to further parasite discrimination in a retrospective assessment. All samples had been analysed by microscopy and by P. ovale spp.-specific real-time PCR without discrimination on species level. Two different real-time PCR protocols for species discrimination of P. o. curtisi and P. o. wallikeri were carried out. Results were correlated with patient data on gender, age, travel destination, thrombocyte count, and duration of parasite latency. RESULTS: Samples from 77 P. ovale spp. malaria patients were assessed, with a male:female ratio of about 2:1 and a median age of 30 years. Parasitaemia was low, ranging from few visible parasites up to 1% infected erythrocytes. Discriminative real-time PCRs revealed 41 cases of P. o. curtisi and 36 cases of P. o. wallikeri infections. Concordance of results by the two PCR approaches was 100%. Assessment of travel destinations confirmed co-existence of P. o. curtisi and P. o. wallikeri over a wide range of countries in sub-Saharan Africa. Latency periods for the two P. ovale species were similar, with median values of 56.0 days for P. o. curtisi and 58.0 days for P. o. wallikeri; likewise, there was no statistically significant difference in thrombocyte count with median values of 138.5/µL for patients with P. o. curtisi and 152.0/µL for P. o. wallikeri-infected patients. CONCLUSIONS: Two different real-time PCR protocols were found to be suitable for the discrimination of P. o. curtisi and P. o. wallikeri with only minor differences in sensitivity. Due to the overall low parasitaemia and the lack of differences in severity-related aspects like parasite latency periods or thrombocyte counts, this study supports the use of P. ovale spp. PCR without discrimination on species level to confirm the diagnosis and to inform clinical management of malaria in these patients.

A cluster of the first reported Plasmodium ovale spp. infections in Peru occuring among returning UN peace-keepers, a review of epidemiology, prevention and diagnostic challenges in nonendemic regions.

BACKGROUND: Plasmodium ovale curtisi and Plasmodium ovale wallikeri are regarded as less virulent forms of malaria with a geographic distribution including Southeast Asia, Central and West Africa, and is increasingly reported as an infection in returning travellers. A species of malaria that may have delayed or relapsing presentations similar to Plasmodium vivax, the clinical presentation of P. ovale spp. has been described to have prepatent periods of 2 weeks or slightly longer with reports of relapse following primary infection out to 8-9 months. This presentation may be obscured further in the setting of anti-malarial exposure, with report of delayed primary infection out to 4 years. Presented is a cluster of 4 imported P. ovale spp. cases in returning Peruvian military personnel assigned to United Nations peace-keeping operations in the Central African Republic. CASE PRESENTATION: From January to December 2016, Peruvian peace-keepers were deployed in support of United Nations (UN) operations in the Central African Republic (CAR). While serving abroad, Navy, Army, and Air Force members experienced 223 episodes of Plasmodium falciparum malaria following interruption of prophylaxis with mefloquine. Diagnosis was made using rapid diagnostics tests (RDTs) and/or smear with no coinfections identified. Cases of malaria were treated with locally-procured artemether-lumefantrine. Returning to Peru in January 2017, 200 peace-keepers were screened via thick and thin smear while on weekly mefloquine prophylaxis with only 1 showing nucleic acid within red blood cells consistent with Plasmodium spp. and 11 reporting syndromes of ill-defined somatic complaints. Between a period of 5 days to 11 months post return, 4 cases of P. ovale spp. were diagnosed using smear and polymerase chain reaction (PCR) following febrile complaints. All cases were subsequently treated with chloroquine and primaquine, with cure of clinical disease and documented clearance of parasitaemia. CONCLUSION: These patients represent the first imported cases in Peru of this species of malaria as well as highlight the challenges in implementing population level prophylaxis in a deployed environment, and the steps for timely diagnosis and management in a non-endemic region where risk of introduction for local transmission exists.