Detection of malaria parasites by nested PCR in south-eastern, Iran: Evidence of highly mixed infections in Chahbahar district

Background

Rapid diagnosis and correct treatment of cases are the main objectives of control programs in malaria-endemic areas.

Methods and results

To evaluate these criteria and in a comparative study, blood specimens were collected from 120 volunteers seeking care at the Malaria Health Center in Chahbahar district. One hundred and seven out of 120 Giemsa-stained slides were positive for malaria parasites by microscopy. Eighty-four (70%) and 20 (16.7%) were identified as having only Plasmodium vivax and Plasmodium falciparum infections, respectively, while only 3 (2.5%) were interpreted as having mixed P. vivax-P. falciparum infections. The target DNA sequence of the 18S small sub-unit ribosomal RNA (ssrRNA) gene was amplified by Polymerase Chain Reaction (PCR) and used for the diagnosis of malaria in south-eastern Iran. One hundred twenty blood samples were submitted and the results were compared to those of routine microscopy. The sensitivity of PCR for detection of P. vivax and P. falciparum malaria was higher than that of microscopy: nested PCR detected 31 more mixed infections than microscopy and parasite positive reactions in 9 out of the 13 microscopically negative samples. The results also confirmed the presence of P. vivax and P. falciparum.

Conclusions

These results suggest that, in places where transmission of both P. vivax and P. falciparum occurs, nested PCR detection of malaria parasites can be a very useful complement to microscopical diagnosis.     

Plasmodium ovale in Bangladesh: genetic diversity and the first known evidence of the sympatric distribution of Plasmodium ovale curtisi and Plasmodium ovale wallikeri in southern Asia

In spite of the high prevalence of malaria in Bangladesh and other southern Asian countries, there remains a substantial shortage of knowledge about the less common human malaria parasites. Recent studies indicate that Plasmodium ovale is made up of two species, namely Plasmodium ovale wallikeri and Plasmodium ovale curtisi. Genus- and species-specific nested PCR analyses of the ssrRNA gene was used to detect P. ovale infections among 2,246 diagnostic samples. Plasmodium ovale infections were further differentiated by nested PCR of the potra gene and multilocus sequence analysis of the cox1, porbp2 and the ssrRNA genes. Both P. ovale curtisi and P. ovale wallikeri occur sympatrically in the Chittagong Hill Tracts, Bangladesh and all patients presented with a mild or asymptomatic symptom complex at the time of diagnosis. The pathogens can be differentiated by nested PCRs targeting the ssrRNA and potra genes, and display dimorphism in multilocus sequence analyses. We believe that we report the first evidence of sympatric P. ovale curtisi and P. ovale wallikeri in southern Asia within a relatively confined study area of less than 5,000 km(2). High rates of mixed infections, the emergence of "new" human malaria parasite species and the evidence of zoonotic capability call for optimised diagnostic strategies for a new era of eradication.     

PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines

Several polymerase chain reaction (PCR)-based methods have recently been developed for diagnosing malarial infections in both birds and reptiles, but a critical evaluation of their sensitivity in experimentally-infected hosts has not been done. This study compares the sensitivity of several PCR-based methods for diagnosing avian malaria (Plasmodium relictum) in captive Hawaiian honeycreepers using microscopy and a recently developed immunoblotting technique. Sequential blood samples were collected over periods of up to 4.4 yr after experimental infection and rechallenge to determine both the duration and detectability of chronic infections. Two new nested PCR approaches for detecting circulating parasites based on P. relictum 18S rRNA genes and the thrombospondin-related anonymous protein (TRAP) gene are described. The blood smear and the PCR tests were less sensitive than serological methods for detecting chronic malarial infections. Individually, none of the diagnostic methods was 100% accurate in detecting subpatent infections, although serological methods were significantly more sensitive (97%) than either nested PCR (61-84%) or microscopy (27%). Circulating parasites in chronically infected birds either disappear completely from circulation or to drop to intensities below detectability by nested PCR. Thus, the use of PCR as a sole means of detection of circulating parasites may significantly underestimate true prevalence.     

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.     

Plasmodium malariae and Plasmodium ovale--the "bashful" malaria parasites

Although Plasmodium malariae was first described as an infectious disease of humans by Golgi in 1886 and Plasmodium ovale identified by Stevens in 1922, there are still large gaps in our knowledge of the importance of these infections as causes of malaria in different parts of the world. They have traditionally been thought of as mild illnesses that are caused by rare and, in case of P. ovale, short-lived parasites. However, recent advances in sensitive PCR diagnosis are causing a re-evaluation of this assumption. Low-level infection seems to be common across malaria-endemic areas, often as complex mixed infections. The potential interactions of P. malariae and P. ovale with Plasmodium falciparum and Plasmodium vivax might explain some basic questions of malaria epidemiology, and understanding these interactions could have an important influence on the deployment of interventions such as malaria vaccines.     

Transmission of Plasmodium vivax in south-western Uganda: report of three cases in pregnant women

Plasmodium vivax is considered to be rare in the predominantly Duffy negative populations of Sub-Saharan Africa, as this red blood cell surface antigen is essential for invasion by the parasite. However, despite only very few reports of molecularly confirmed P. vivax from tropical Africa, serological evidence indicated that 13% of the persons sampled in Congo had been exposed to P. vivax. We identified P. vivax by microscopy in 8 smears from Ugandan pregnant women who had been enrolled in a longitudinal study of malaria in pregnancy. A nested polymerase chain reaction (PCR) protocol was used to detect and identify the Plasmodium parasites present. PCR analysis confirmed the presence of P. vivax for three of the women and analysis of all available samples from these women revealed clinically silent chronic low-grade vivax infections for two of them. The parasites in one woman carried pyrimethamine resistance-associated double non-synonymous mutations in the P. vivax dihydrofolate reductase gene. The three women found infected with P. vivax were Duffy positive as were nine of 68 women randomly selected from the cohort. The data presented from these three case reports is consistent with stable transmission of malaria in a predominantly Duffy negative African population. Given the substantial morbidity associated with vivax infection in non-African endemic areas, it will be important to investigate whether the distribution and prevalence of P. vivax have been underestimated in Sub-Saharan Africa. This is particularly important in the context of the drive to eliminate malaria and its morbidity.     

Plasmodium vivax dhfr mutations among isolates from malarious areas of Iran

The use of sulfadoxine and pyrimethamine (SP) for treatment of vivax malaria is uncommon in most malarious areas, but Plasmodium vivax isolates are exposed to SP because of mixed infections with other Plasmodium species. As P. vivax is the most prevalent species of human malaria parasites in Iran, monitoring of resistance of the parasite against the drug is necessary. In the present study, 50 blood samples of symptomatic patients were collected from 4 separated geographical regions of south-east Iran. Point mutations at residues 57, 58, 61, and 117 were detected by the PCR-RFLP method. Polymorphism at positions 58R, 117N, and 117T of P. vivax dihydrofolate reductase (Pvdhfr) gene has been found in 12%, 34%, and 2% of isolates, respectively. Mutation at residues F57 and T61 was not detected. Five distinct haplotypes of the Pvdhfr gene were demonstrated. The 2 most prevalent haplotypes were F57S58T61S117 (62%) and F57S58T61N117 (24%). Haplotypes with 3 and 4 point mutations were not found. The present study suggested that P. vivax in Iran is under the pressure of SP and the sensitivity level of the parasite to SP is diminishing and this fact must be considered in development of malaria control programs.     

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%.     

Nested cytochrome b polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential

Numerous polymerase chain reaction (PCR)-based methods have been developed and used increasingly to screen vertebrate blood samples for the diagnosis of haemosporidian blood parasites (Sporozoa, Haemosporida), but a rigorous evaluation of the sensitivity of these methods for detecting mixed infections of different haemosporidian species belonging to the same and different genera and subgenera is lacking. This study links the information obtained by nested cytochrome b PCR and traditional microscopy in determining mixed haemosporidian infections in naturally infected birds. Samples from 83 individual passerine birds with single infections of Haemoproteus or Plasmodium spp., as determined by mitochondrial DNA amplification, also were investigated by microscopic examination of stained blood films. Thirty-six samples (43%) were found to harbor mixed Haemoproteus, or Plasmodium spp. infections, or both. Thus, the PCR assays alone underestimate the occurrence of mixed infections of haemosporidian parasites in naturally infected birds. To determine the true species composition of the haemosporidians in each individual host, PCR diagnostics need to be improved. Specific primers for Haemoproteus spp. and Plasmodium spp. should be developed. Ideally, a combination of the approaches of both microscopy and PCR-based methods is recommended for this purpose.     

A cautionary note on the use of nested PCR for parasite screening--an example from avian blood parasites

The use of new powerful nested polymerase chain reaction (PCR) techniques to identify and screen for prevalence of parasites has a huge potential. It allows for the detection and identification of low-intensity infections, but its high sensitivity and technical setup may also induce problems. Here, we report a cautionary note regarding misleading amplification of avian malaria species (Haemoproteus and Plasmodium) during Leucocytozoon spp. detection. We used a previously described nested PCR method for the molecular detection of avian malaria and Leucocytozoon spp. In the first step of the PCR protocol, these parasites are detected simultaneously; in the second PCR, Haemoproteus and Plasmodium spp. are separated from Leucocytozoon spp. However, in certain cases when a bird was infected with avian malaria, we obtained a slightly longer PCR product during the detection of Leucocytozoon spp. Our data imply that these "false" Leucocytozoon fragments are the consequences of strong amplification of certain malaria lineages in the first PCR, which can also be detected after the second PCR amplification that is specific to Leucocytozoon spp. parasites. Because these "false" Leucocytozoon fragments are slightly longer than the normal Leucocytozoon fragments, we suggest the use of well-separating agarose gels, several positive controls, and molecular standards to facilitate their separation. If one obtains a fragment that differs in length from the one expected for Leucocytozoon spp., sequencing is essential. More generally, in order to limit this type of problem with nested PCR protocols, we suggest that the first and the second primer pair be chosen so that they have different annealing temperatures.     

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.     

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.     

Use of PCR for detection of subpatent infections of lizard malaria: implications for epizootiology

The estimated prevalence of a malaria parasite, Plasmodium mexicanum , of western fence lizards, Sceloporus occidentalis , was compared using two techniques: microscopic examination of blood smears, and nested PCR amplification of the 18S small subunit rRNA gene. Two sites in northern California, USA were investigated, one with known long-term high prevalence of the parasite (30% by blood smear scanning), and one with low prevalence (6%). The nested PCR readily detected very low-level infections (< 1 parasite per 10 000 erythrocytes); such infections are often subpatent by normal microscopic examination. False negatives (scored as not infected after scanning the blood smear, but found infected via PCR) were rare at both sites (4% at the high-prevalence site, 6% at the low-prevalence site). However, a greater proportion of infections was detected only by PCR at the low-prevalence site (50% vs. 9%). If 50% of the infections sustain very weak parasitaemia where lizards are rarely infected, this would accord with hypotheses that predict that parasites should reduce infection growth when transmission is uncommon. The study demonstrates that PCR is a powerful tool to detect very low-level malarial infections in vertebrate hosts, including those with nucleated erythrocytes.     

Genetic clonality of Plasmodium falciparum affects the outcome of infection in Anopheles gambiae

Mosquito infections with natural isolates of Plasmodium falciparum are notoriously variable and pose a problem for reliable evaluation of efficiency of transmission-blocking agents for malaria control interventions. Here, we show that monoclonal P. falciparum isolates produce higher parasite loads than mixed ones. Induction of the mosquito immune responses by wounding efficiently decreases Plasmodium numbers in monoclonal infections but fails to do so in infections with two or more parasite genotypes. Our results point to the parasites genetic complexity as a potentially crucial component of mosquito-parasite interactions.     

Mass blood survey for malaria: pooling and realtime PCR combined with expert microscopy in north-west Thailand

ABSTRACT: BACKGROUND: Asymptomatic carriage of Plasmodium falciparum and Plasmodium vivax is common in both low-and high-transmission settings and represents an important reservoir of infection that needs to be targeted if malaria elimination is to succeed. METHODS: Mass blood examinations (475 individuals) were conducted in two villages in Mae Hong Son, an area of endemic but low-transmission malaria in the north-west of Thailand. The microscopist at the local malaria clinic did not detect any infections. Pools of four samples were screened by real-time PCR; individual members of all of the positive pools were then re-examined by expert microscopy and by a second species-specific PCR reaction. RESULTS: Eight subjects were found to be positive by both PCR and expert microscopy and one was found to be positive by PCR alone. The slides contained asexual stage parasites of P. vivax, P. falciparum and Plasmodium malariae, but no gametocytes. The local clinic was notified within two to eight days of the survey. CONCLUSION: A combination of pooling, real-time PCR and expert microscopy provides a feasible approach to identifying and treating asymptomatic malaria infections in a timely manner.     

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.     

Glucose-6-phosphate metabolism in Plasmodium falciparum

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.     

Plasmodium knowlesi: reservoir hosts and tracking the emergence in humans and macaques

Plasmodium knowlesi, a malaria parasite originally thought to be restricted to macaques in Southeast Asia, has recently been recognized as a significant cause of human malaria. Unlike the benign and morphologically similar P. malariae, these parasites can lead to fatal infections. Malaria parasites, including P. knowlesi, have not yet been detected in macaques of the Kapit Division of Malaysian Borneo, where the majority of human knowlesi malaria cases have been reported. In order to extend our understanding of the epidemiology and evolutionary history of P. knowlesi, we examined 108 wild macaques for malaria parasites and sequenced the circumsporozoite protein (csp) gene and mitochondrial (mt) DNA of P. knowlesi isolates derived from macaques and humans. We detected five species of Plasmodium (P. knowlesi, P. inui, P. cynomolgi, P. fieldi and P. coatneyi) in the long-tailed and pig-tailed macaques, and an extremely high prevalence of P. inui and P. knowlesi. Macaques had a higher number of P. knowlesi genotypes per infection than humans, and some diverse alleles of the P. knowlesi csp gene and certain mtDNA haplotypes were shared between both hosts. Analyses of DNA sequence data indicate that there are no mtDNA lineages associated exclusively with either host. Furthermore, our analyses of the mtDNA data reveal that P. knowlesi is derived from an ancestral parasite population that existed prior to human settlement in Southeast Asia, and underwent significant population expansion approximately 30,000-40,000 years ago. Our results indicate that human infections with P. knowlesi are not newly emergent in Southeast Asia and that knowlesi malaria is primarily a zoonosis with wild macaques as the reservoir hosts. However, ongoing ecological changes resulting from deforestation, with an associated increase in the human population, could enable this pathogenic species of Plasmodium to switch to humans as the preferred host.     

Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria

BACKGROUND: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. METHODOLOGY AND SIGNIFICANT FINDINGS: Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. CONCLUSION: This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.