Investigating zoonotic infection barriers to ape Plasmodium parasites using faecal DNA analysis

African apes are endemically infected with numerous Plasmodium spp. including close relatives of human Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Although these ape parasites are not believed to pose a zoonotic threat, their ability to colonise humans has not been fully explored. In particular, it remains unknown whether ape parasites are able to initiate exo-erythrocytic replication in human hepatocytes following the bite of an infective mosquito. Since animal studies have shown that liver stage infection can result in the excretion of parasite nucleic acids into the bile, we screened faecal samples from 504 rural Cameroonians for Plasmodium DNA. Using pan-Laverania as well as P. malariae- and P. vivax-specific primer sets, we amplified human P. falciparum (n?=?14), P. malariae (n?=?1), and P. ovale wallikeri (n?=?1) mitochondrial sequences from faecal DNA of 15 individuals. However, despite using an intensified PCR screening approach we failed to detect ape Laverania, ape P. vivax or ape P. malariae parasites in these same subjects. One faecal sample from a hunter-gatherer contained a sequence closely related to the porcupine parasite Plasmodium atheruri. Since this same faecal sample also contained porcupine mitochondrial DNA, but a matching blood sample was Plasmodium-negative, it is likely that this hunter-gatherer consumed Plasmodium-infected bushmeat. Faecal Plasmodium detection was not secondary to intestinal bleeding and/or infection with gastrointestinal parasites, but indicative of blood parasitaemia. Quantitative PCR identified 26-fold more parasite DNA in the blood of faecal Plasmodium-positive than faecal Plasmodium-negative individuals (P?=?0.01). However, among blood-positive individuals only 10% - 20% had detectable Plasmodium sequences in their stool. Thus, faecal screening of rural Cameroonians failed to uncover abortive ape Plasmodium infections, but detected infection with human parasites, albeit with reduced sensitivity compared with blood analysis.     

In vivo study of human Plasmodium knowlesi inMacaca fascicularis

Plasmodium knowlesi is a malaria parasite of Old World monkeys and is infectious to humans. In this study Macaca fascicularis was used as a model to understand the host response to P. knowlesi using parasitological and haematological parameters. Three M. fascicularis of either sex were experimentally infected with P. knowlesi erythrocytic parasites from humans. The pre-patent period for P. knowlesi infection in M. fascicularis ranged from seven to 14 days. The parasitemia observed was 13,686-24,202 parasites per μL of blood for asexual stage and 88-264 parasites per μL of blood for sexual stage. Periodicity analysis adopted from microfilaria periodicity technique of asexual stage showed that the parasitemia peak at 17:39 h while the sexual stage peaked at 02:36 h. Mathematical analysis of the data indicates that P. knowlesi gametocytes tend to display periodicity with a peak (24:00-06:00) that coincides with the peak biting activity (19:00-06:00) of the local vector, Anopheles latens. The morphology of P. knowlesi resembled P. falciparum in early trophozoite and P. malariae in late trophozoite. However, it may be distinguishable by observing the appliqué appearance of the cytoplasm and the chromatin lying inside the ring. Haematological analysis on macaques with knowlesi malaria showed clinical manifestations of hypoglycaemia, anaemia and hyperbilirubinemia. Gross examination of spleen and liver showed malaria pigments deposition in both organs.     

High prevalence of Plasmodium malariae and Plasmodium ovale in co-infections with Plasmodium falciparum in asymptomatic malaria parasite carriers in southwestern Nigeria

Asymptomatic malaria parasite carriers do not seek anti-malarial treatment and may constitute a silent infectious reservoir. In order to assess the level of asymptomatic and symptomatic carriage amongst adolescents in a highly endemic area, and to identify the risk factors associated with such carriage, we conducted a cross-sectional survey of 1032 adolescents (ages 10–19 years) from eight schools located in Ibadan, southwestern Nigeria in 2016. Blood films and blood spot filter paper samples were prepared for microscopy and DNA analysis. The prevalence of asymptomatic malaria was determined using microscopy, rapid diagnostic tests and PCR for 658 randomly selected samples. Of these, we found that 80% of asymptomatic schoolchildren were positive for malaria parasites by PCR, compared with 47% and 9%, determined by rapid diagnostic tests and microscopy, respectively. Malaria parasite species typing was performed using PCR targeting the mitochondrial CoxIII gene, and revealed high rates of carriage of Plasmodium malariae (53%) and Plasmodium ovale (24%). Most asymptomatic infections were co-infections of two or more species (62%), with Plasmodium falciparum + P. malariae the most common (35%), followed by P. falciparum + P. malariae + P. ovale (21%) and P. falciparum + P. ovale (6%). Single infections of P. falciparum, P. malariae and P. ovale accounted for 24%, 10% and 4% of all asymptomatic infections, respectively. To compare the species composition of asymptomatic and symptomatic infections, further sample collection was carried out in 2017 at one of the previously sampled schools, and at a nearby hospital. Whilst the species composition of the asymptomatic infections was similar to that observed in 2016, the symptomatic infections were markedly different, with single infections of P. falciparum observed in 91% of patients, P. falciparum + P. malariae in 5% and P. falciparum + P. ovale in 4%.     

Plasmodium knowlesi Genome Sequences from Clinical Isolates Reveal Extensive Genomic Dimorphism

Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology.     

A new Real Time PCR with species-specific primers from Plasmodium malariae/P. brasilianum mitochondrial cytochrome b gene

Plasmodium malariae mainly causes asymptomatic submicroscopic parasitemia in the endemic Amazon and non-endemic Atlantic Forest, where the number of cases and transmission of malaria through blood transfusion has increased. This study developed a P. malariae/P. brasilianum Real Time PCR (rtPCR) targeting the cytochrome b oxidase (cytb), a highly repetitive gene (20-150 copies/parasite) that should detect more cases than the 18S rRNA (4-8 copies/parasite) gene-based amplification systems. Cytb from human and non-human Plasmodium species (including P. brasilianum) aligned to the only 20 African P. malariae cytb sequences identified polymorphic regions within which we designed P. malariae species-specific primers. Non-human Plasmodium species, related parasites, anemia-causing microorganisms, normal human DNA and 47 blood bank donors samples that were truly negative to malaria accessed rtPCR specificity. Truly positive samples (n = 101) with species identification by semi-nested, nested or TaqMan PCR, and four samples from the Atlantic Forest that were suspected of malaria but three of them had negative genus TaqMan and 18S rRNA nested PCR. The cloned amplification product used in standard curves determined qPCR detection limit (0.5-1 parasite equivalent/μL). The 10 positive P. malariae samples among truly positives yielded positive rtPCR results and more importantly, rtPCR detected the four samples suspected of malaria from the Atlantic Forest. The rtPCR specificity was 100%, reproducibility 11.1% and repeatability 6.7%. In conclusion, the proposed rtPCR is fast, apparently more sensitive than all 18S rRNA amplification systems for detecting extremely low parasitemia. The rtPCR is also specific to P. malariae/P. brasilianum species. This new molecular tool could be applied to the detection of P. malariae/brasilianum infections with submicroscopic parasitemias in the context of epidemiological studies and blood bank safety programs.     

Plasmodium knowlesi infecting humans in Southeast Asia: What’s next?

Plasmodium knowlesi, a simian malaria parasite, has been in the limelight since a large focus of human P. knowlesi infection was reported from Sarawak (Malaysian Borneo) in 2004. Although this infection is transmitted across Southeast Asia, the largest number of cases has been reported from Malaysia. The increasing number of knowlesi malaria cases has been attributed to the use of molecular tools for detection, but environmental changes including deforestation likely play a major role by increasing human exposure to vector mosquitoes, which coexist with the macaque host. In addition, with the reduction in human malaria transmission in Southeast Asia, it is possible that human populations are at a greater risk of P. knowlesi infection due to diminishing cross-species immunity. Furthermore, the possibility of increasing exposure of humans to other simian Plasmodium parasites such as Plasmodium cynomolgi and Plasmodium inui should not be ignored. We here review the current status of these parasites in humans, macaques, and mosquitoes to support necessary reorientation of malaria control and elimination in the affected areas.     

Plasmodium falciparum and P. knowlesi: low temperature preservation using dimethylsulfoxide

Erythrocytic stages of Plasmodium knowlesi and P. falciparum have been successfully recovered from the frozen state (i.e., ?196 C) using dimethylsulfoxide (DMSO: 7.5% final concentration, v/v) as the cryoprotective agent. Quantitated preparations of P. knowlesi, stored for 202 days, yielded patent and synchronous infections as early as 72 hr postinoculation. Similar results were obtained with blood samples with P. falciparum.     

Evolutionary analysis of circumsporozoite surface protein and merozoite surface protein-1 (CSP and MSP-1) sequences of malaria parasites

Malaria, one of the world''s most common diseases, is caused by the intracellular protozoan parasite known as Plasmodium. In this study, we have determined the evolutionary relationship of two single-copy proteins, circumsporozoite protein (CSP) and merozoite surface protein-1 (MSP-1), among Plasmodium species using various bioinformatics tools and softwares. These two proteins are major blood stage antigens of Plasmodium species. This study demonstrates that the circumsporozoite protein of Plasmodium falciparum shows similarity with Plasmodium cynomolgi and Plasmodium knowlesi. The merozoite surface protein-1 of Plasmodium coatneyi forms a monophyletic group with Plasmodium knowlesi, demonstrating their close relationship and these two species also reveal similarity between the human malaria Plasmodium vivax. This Plasmodium phylogenetic arrangement is evidently crucial to identify shared derived characters as well as particular adaptation of plasmodium species from inside and between monophyletic groups.     

Asymptomatic Plasmodium malariae infections in children from suburban areas of Yaoundé, Cameroon

The gold standard for malaria diagnosis is the microscopic examination of Giemsa stained thick blood smears though microscopy mostly may not detect the presence of Plasmodium species infections in asymptomatic samples. In the reported study, we used two diagnostic methods viz. the conventional microscopic examination and polymerase chain reaction (PCR) assay to analyse the asymptomatic malaria samples. PCR assay amplifying 18S small-subunit ribosomal RNA (SSU rRNA) gene of Plasmodium in 122 samples confirmed 68% of isolates as asymptomatic P. falciparum infections; with 87.9% mono-infections. We observed that the P. malariae positive samples were not diagnosed in microscopic examination of the blood smears but the PCR based diagnostic method revealed the presence of 12% P. malariae infections in asymptomatic samples from Yaoundé region of Cameroon where no official cases of P. malariae have been reported for over a decade. The sequence analysis further confirmed the presence of 12% P. malariae in malaria positive samples with three base pair deletions and five substitutions in the SSU rRNA gene.     

Spatial cluster analysis of Plasmodium vivax and P. malariae exposure using serological data among Haitian school children sampled between 2014 and 2016

BackgroundEstimation of malaria prevalence in very low transmission settings is difficult by even the most advanced diagnostic tests. Antibodies against malaria antigens provide an indicator of active or past exposure to these parasites. The prominent malaria species within Haiti is Plasmodium falciparum, but P. vivax and P. malariae infections are also known to be endemic.Methodology/Principal findingsFrom 2014–2016, 28,681 Haitian children were enrolled in school-based serosurveys and were asked to provide a blood sample for detection of antibodies against multiple infectious diseases. IgG against the P. falciparum, P. vivax, and P. malariae merozoite surface protein 19kD subunit (MSP1₁₉) antigens was detected by a multiplex bead assay (MBA). A subset of samples was also tested for Plasmodium DNA by PCR assays, and for Plasmodium antigens by a multiplex antigen detection assay. Geospatial clustering of high seroprevalence areas for P. vivax and P. malariae antigens was assessed by both Ripley’s K-function and Kulldorff’s spatial scan statistic. Of 21,719 children enrolled in 680 schools in Haiti who provided samples to assay for IgG against PmMSP1₁₉, 278 (1.27%) were seropositive. Of 24,559 children enrolled in 788 schools providing samples for PvMSP1₁₉ serology, 113 (0.46%) were seropositive. Two significant clusters of seropositivity were identified throughout the country for P. malariae exposure, and two identified for P. vivax. No samples were found to be positive for Plasmodium DNA or antigens.Conclusions/SignificanceFrom school-based surveys conducted from 2014 to 2016, very few Haitian children had evidence of exposure to P. vivax or P. malariae, with no children testing positive for active infection. Spatial scan statistics identified non-overlapping areas of the country with higher seroprevalence for these two malarias. Serological data provides useful information of exposure to very low endemic malaria species in a population that is unlikely to present to clinics with symptomatic infections.     

A Case of Plasmodium ovale Malaria Imported from West Africa

Malaria is a parasitic infection caused by Plasmodium species. Most of the imported malaria in Korea are due to Plasmodium vivax and Plasmodium falciparum, and Plasmodium ovale infections are very rare. Here, we report a case of a 24-year-old American woman who acquired P. ovale while staying in Ghana, West Africa for 5 months in 2010. The patient was diagnosed with P. ovale malaria based on a Wright-Giemsa stained peripheral blood smear, Plasmodium genus-specific real-time PCR, Plasmodium species-specific nested PCR, and sequencing targeting 18S rRNA gene. The strain identified had a very long incubation period of 19-24 months. Blood donors who have malaria with a very long incubation period could be a potential danger for propagating malaria. Therefore, we should identify imported P. ovale infections not only by morphological findings but also by molecular methods for preventing propagation and appropriate treatment.     

Multiplex real-time PCR detection of P. falciparum, P. vivax and P. malariae in human blood samples

Two duplex real-time PCR assays were developed to diagnose three human parasites: Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. TaqMan duplex real-time PCR was evaluated in 263 blood samples of suspected malaria patients by comparing results against those obtained with microscopy and nested PCR. Compared with nested PCR, duplex real-time PCR assays showed 100% sensitivity and specificity. Duplex real-time PCR detected all mixtures of P. falciparum and P. vivax DNA, except at threshold detection limits for both parasites in which P. vivax was not amplified. Threshold detection limits of real-time PCR were 3.1, 0.3 and 0.8 parasites per microlitre of blood for P. falciparum, P. vivax and P. malariae, respectively. Duplex real-time PCR allows the detection of malarial cases, including mixed species infection, it simplifies analysis and reduces cost. Thus, this protocol may prove invaluable for use in the diagnosis of human infection, trial treatments and epidemiologic studies in which high-throughput analyses are often required.     

Anti-malarial market and policy surveys in sub-Saharan Africa

Background

Malaria microscopy and rapid diagnostic tests are insensitive for very low-density parasitaemia. This insensitivity may lead to missed asymptomatic sub-microscopic parasitaemia, a potential reservoir for infection. Similarly, mixed infections and interactions between Plasmodium species may be missed. The objectives were first to develop a rapid and sensitive PCR-based diagnostic method to detect low parasitaemia and mixed infections, and then to investigate the epidemiological importance of sub-microscopic and mixed infections in Rattanakiri Province, Cambodia.

Methods

A new malaria diagnostic method, using restriction fragment length polymorphism analysis of the cytochrome b genes of the four human Plasmodium species and denaturing high performance liquid chromatography, has been developed. The results of this RFLP-dHPLC method have been compared to 1) traditional nested PCR amplification of the 18S rRNA gene, 2) sequencing of the amplified fragments of the cytochrome b gene and 3) microscopy. Blood spots on filter paper and Giemsa-stained blood thick smears collected in 2001 from 1,356 inhabitants of eight villages of Rattanakiri Province have been analysed by the RFLP-dHPLC method and microscopy to assess the prevalence of sub-microscopic and mixed infections.

Results

The sensitivity and specificity of the new RFLP-dHPLC was similar to that of the other molecular methods. The RFLP-dHPLC method was more sensitive and specific than microscopy, particularly for detecting low-level parasitaemia and mixed infections. In Rattanakiri Province, the prevalences of Plasmodium falciparum and Plasmodium vivax were approximately two-fold and three-fold higher, respectively, by RFLP-dHPLC (59% and 15%, respectively) than by microscopy (28% and 5%, respectively). In addition, Plasmodium ovale and Plasmodium malariae were never detected by microscopy, while they were detected by RFLP-dHPLC, in 11.2% and 1.3% of the blood samples, respectively. Moreover, the proportion of mixed infections detected by RFLP-dHPLC was higher (23%) than with microscopy (8%).

Conclusions

The rapid and sensitive molecular diagnosis method developed here could be considered for mass screening and ACT treatment of inhabitants of low-endemicity areas of Southeast Asia.     

Imported Malaria in United Arab Emirates: Evaluation of a New DNA Extraction Technique Using Nested PCR

Local malaria transmission in the United Arab Emirates (UAE) came to an end in 1997. Nevertheless, UAE has been subjected to substantial importation of malaria cases from abroad, concerning both UAE nationals and immigrants from malarious countries with a total number of 2,119 cases in 2007. To evaluate a new DNA extraction technique using nested PCR, blood samples were collected from 132 individuals who presented to Infectious Diseases Department in Rashid Hospital, Dubai, and Central Department of Malaria Control with fever and persistent headache. Giemsa-stained blood films and ELISA test for malaria antibodies were carried out for detection of Plasmodium infection. Plasmodium infections were identified with the genus-specific primer set and species differentiation using nested PCR. A rapid procedure for diagnosis of malaria infections directly from dried blood spots using for the first time DNA extract from FTA Elute cards was evaluated in contrast to extraction techniques using FTA classic cards and rapid boiling technique. Our new simple technique for DNA extraction using FTA Elute cards was very sensitive giving a sensitivity of 100% compared to 94% using FTA classic cards and 62% in the rapid boiling technique. No complex preparation of blood samples was required prior to the amplification. The production cost of DNA isolation in our PCR assay was much less in comparable to that of other DNA extraction protocols. The nested PCR detected plasmodial infection and could differentiate P. falciparum from P. vivax, and also detected the mixed infection.     

Congenital Plasmodium vivax malaria mimicking neonatal sepsis: a case report

A recently published comment on a report of Plasmodium knowlesi infections in Vietnam states that this may not accurately represent the situation in the study area because the PCR primers used may cross-hybridize with Plasmodium vivax. Nevertheless, P. knowlesi infections have been confirmed by sequencing. In addition, a neighbour-joining tree based on the 18S S-Type SSUrRNA gene shows that the Vietnamese samples clearly cluster with the P. knowlesi isolates identified in Malaysia and are distinct from the corresponding P. vivax sequences. All samples came from asymptomatic individuals who did not consult for fever during the months preceding or following the survey, indicating that asymptomatic P. knowlesi infections occur in this population, although this does not exclude the occurrence of symptomatic cases. Large-scale studies to determine the extent and the epidemiology of P. knowlesi malaria in Vietnam are further needed.     

Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites

The invasion of red blood cells (RBCs) is an essential event in the life cycle of all malaria-causing Plasmodium parasites; however, there are major gaps in our knowledge of this process. Here, we use video microscopy to address the kinetics of RBC invasion in the human malaria parasite Plasmodium falciparum. Under in vitro conditions merozoites generally recognise new target RBCs within 1 min of their release from their host RBC. Parasite entry ensues and is complete on average 27.6 s after primary contact. This period can be divided into two distinct phases. The first is an ∼11 s ‘pre-invasion’ phase that involves an often dramatic RBC deformation and recovery process. The second is the classical ‘invasion’ phase where the merozoite becomes internalised within the RBC in a ∼17 s period. After invasion, a third ‘echinocytosis’ phase commences when about 36 s after every successful invasion a dramatic dehydration-type morphology was adopted by the infected RBC. During this phase, the echinocytotic effect reached a peak over the next 23.4 s, after which the infected RBC recovered over a 5-11 min period. By then the merozoite had assumed an amoeboid-like state and was apparently free in the cytoplasm. A comparison of our data with that of an earlier study of the distantly related primate parasite Plasmodium knowlesi indicated remarkable similarities, suggesting that the kinetics of invasion are conserved across the Plasmodium genus. This study provides a morphological and kinetic framework onto which the invasion-associated physiological and molecular events can be overlaid.     

HIV Treatments Reduce Malaria Liver Stage Burden in a Non-Human Primate Model of Malaria Infection at Clinically Relevant Concentrations In Vivo

We have previously shown that the HIV protease inhibitor lopinavir-ritonavir (LPV-RTV) and the antibiotic trimethoprim sulfamethoxazole (TMP-SMX) inhibit Plasmodium liver stages in rodent malarias and in vitro in P. falciparum. Since clinically relevant levels are better achieved in the non-human-primate model, and since Plasmodium knowlesi is an accepted animal model for the study of liver stages of malaria as a surrogate for P. falciparum infection, we investigated the antimalarial activity of these drugs on Plasmodium knowlesi liver stages in rhesus macaques. We demonstrate that TMP-SMX and TMP-SMX+LPV-RTV (in combination), but not LPV-RTV alone, inhibit liver stage parasite development. Because drugs that inhibit the clinically silent liver stages target parasites when they are present in lower numbers, these results may have implications for eradication efforts.