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.     

Plasmodium vivax: parasitemia determination by real-time quantitative PCR in Aotus monkeys

Plasmodium vivax and Plasmodium falciparum are the two prevalent human malaria species. A Colombian P. vivax wild strain has been adapted in Aotus nancymaae monkeys for use in further biological and immunological studies. We present data validating a real-time PCR assay quantifying P. vivax parasitemia, using the small subunit ribosomal RNA genes as an amplification target. P. vivax species-specific primers were designed on the 18S ribosomal gene V8 region, for amplifying both asexual and sporozoite ssrRNA genes. The assay detects amplification products bound to fluorescent SYBR-Green I dye using Perkin-Elmer GeneAmp-5700-SDS. Linear range standard curves from 6 DNA concentration logs (+0.99 correlation coefficients) were obtained. Standard curves were constructed using a plasmid containing target gene for real-time PCR amplification. This P. vivax specific assay is very sensitive, having a three parasite detection limit, and is reproducible and accurate. It involves a "closed-tube" PCR, avoids time-consuming post-PCR manipulation, and decreases potential PCR contamination.     

Host-cell invasion by malaria parasites: insights from Plasmodium and Toxoplasma

Recent years have seen tremendous progress in our understanding of malaria parasite molecular biology. To a large extent, this progress follows significant developments in genetic, molecular and chemical tools available to study the malaria parasites and related Apicomplexa, in particular Toxoplasma gondii. One area of major advancement has been in understanding parasite host-cell invasion, a process that utilizes several essential molecular mechanisms that are conserved across the different lifecycle stages. Here, we summarize some of the most recent experimental data that shed light on the events underlying preparation and execution of malaria parasite invasion and how these insights might relate to the development of new antimalarial drugs.     

Plasmodium falciparum: rapid quantification of parasitemia in fixed malaria cultures by flow cytometry

A rapid and sensitive method is described for the determination of parasitemia in Plasmodium falciparum cultures using the fluorescence activated cell sorter and DNA-binding fluorochrome, 33258 Hoechst. Conditions were selected to permit its application to the screening of assays with numerous samples. Parasites suspended in culture medium were mixed with an equal volume of aqueous fixative (10% w/v formaldehyde, 4% w/v D-glucose in Tris-saline pH 7.3), stained in a 20 microM final dye concentration, and analyzed with the cell sorter after dilution in Tris-saline. Centrifugation and washing steps were avoided throughout. Close correspondence was obtained between the estimated and actual parasitemia, and fluorescence intensities of infected erythrocytes permitted distinction between ring and schizont stages of the parasites. The ability to store, transport, or assay material rendered not infectious by fixation, and the relative simplicity of this technique are major improvements to methods described previously using living parasites. Reanalysis of fixed material permits reference standards to be used with each assay.     

Prevalence of malaria parasites (Plasmodium floridense and Plasmodium azurophilum) infecting a Puerto Rican lizard (Anolis gundlachi): a nine-year study

The prevalence of malaria parasites was studied in the lizard Anolis gundlachi over a 9-yr period at a site in the wet evergreen forest of eastern Puerto Rico. Three forms of the parasite infected the lizards; these were Plasmodium floridense, Plasmodium azurophilum in erythrocytes, and P. azurophilum in white blood cells. Overall prevalence of infection for 8 samples during the study period was significantly higher for males than females (32% of 3,296 males and 22% of 1,439 females). During the study, the site experienced substantial climatic and physical disturbance including rising temperature, droughts, and hurricanes that severely damaged the forest. Parasite prevalence in the first sample, 8 mo after the massive hurricane Hugo, was slightly, though significantly, lower than for subsequent samples. However, overall prevalence was stable during the 9-yr period. The results show malaria prevalence is more constant at the site than found for 2 studies in temperate forests, and that the Puerto Rico system may be an example of the stable, endemic malaria described by standard models for human malaria epidemiology.     

Relative clonal proportions over time in mixed-genotype infections of the lizard malaria parasite Plasmodium mexicanum

Vertebrate hosts of malaria parasites (Plasmodium) often harbour two or more genetically distinct clones of a single species, and interaction among these co-existing clones can play an important role in Plasmodium biology. However, how relative clonal proportions vary over time in a host is still poorly known. Experimental mixed-clone infections of the lizard malaria parasite, Plasmodium mexicanum, were followed in its natural host, the western fence lizard using microsatellite markers to determine the relative proportions of two to five co-existing clones over time (2-3 months). Results for two markers, and two PCR primer pairs for one of those, matched very closely, supporting the efficacy of the method. Of the 54 infections, 67% displayed stable relative clonal proportions, with the others showing a shift in proportions, usually with one clone outpacing the others. Infections with rapidly increasing or slowly increasing parasitemia were stable, showing that all clones within these infections reproduced at the same rapid or slow rate. Replicate infections containing the same clones did not always reveal the same growth rate, final parasitemia or dominant clone; thus there was no clone effect for these life history measures. The rate of increase in parasitemia was not associated with stable versus unstable relative proportions, but infections with four to five clones were more likely to be unstable than those with two to three clones. This rare look into events in genetically complex Plasmodium infections suggests that parasite clones may be interacting in complex and unexpected ways.     

Search for malaria parasites by PCR and Southern blot in patients with imported malaria in Italy

The present study evaluates the sensitivity, specificity and usefulness of a PCR method with Southern blot hybridization to detect malaria parasites in blood samples from subjects with a suspect clinical diagnosis of malaria imported to Italy. Plasmodia were detected by PCR using a genus-specific primer-set corresponding to the sequences common to P. falciparum, P. vivax, P. malariae and P. ovale, as described by Arai (Arai et al., Nucleosides Nucleotides, 1994, 13, 1363-1364) and Kimura (Kimura et al., Journal of Clinical Microbiology, 1995, 33, 2342-2346). In addition, four distinct tandemly repetitive species-specific probes, described by Kawai (Kawai et al., Analytical Biochimestry, 1993, 209, 63-69), were synthesized to specifically detect the four malaria parasites species by Southern blot hybridization. Fifteen blood samples from 12 patients (7 with malaria) were tested and the genus-specific PCR method showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy, in detecting malaria parasites in the tested blood samples. Fourteen samples (nine were positive and five negative by PCR) were confirmed by Southern blot, whereas only one P. vivax positive sample was not hybridized with the species-specific probes. We conclude that this PCR method with Southern blot hybridization may be useful in detecting malaria parasites in patients with malaria imported to Italy.     

Uptake of L-tryptophan by erythrocytes infected with malaria parasites (Plasmodium falciparum)

The initial rates of uptake of L-tryptophan into normal human red blood cells and into cells infected by the malarial parasite Plasmodium falciparum in vitro, were investigated. We find that transport in non-infected cells, which is mediated by the specific saturable T system and the apparently non-saturable L system (Rosenberg, Young and Ellory (1980) Biochim. Biophys. Acta 598, 375-384) is considerably enhanced by blood preservation and culture conditions. This increase is mostly due to an increase in the maximal velocity of the saturable component and of the rate constant of the linear component. Uptake is further enhanced in non-infected cells by factors released from infected cells into the culture medium and, even more so, in infected cells at the advanced stage of intraerythrocytic parasite development. At these stages the susceptibility of the transport system to the non-specific inhibitor phloretin and to the competitive inhibitor phenylalanine, is virtually lost. The effect of the parasite on L-tryptophan uptake by the host cell membrane is exerted only on the maximal velocity of the T system, which is carrying most of the substrate under physiological conditions. The possible implications of these findings to the life of the intraerythrocytic parasite are briefly discussed.     

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 interspersed repeats: the major multigene superfamily of malaria parasites

Functionally related homologues of known genes can be difficult to identify in divergent species. In this paper, we show how multi-character analysis can be used to elucidate the relationships among divergent members of gene superfamilies. We used probabilistic modelling in conjunction with protein structural predictions and gene-structure analyses on a whole-genome scale to find gene homologies that are missed by conventional similarity-search strategies and identified a variant gene superfamily in six species of malaria (Plasmodium interspersed repeats, pir). The superfamily includes rif in P.falciparum, vir in P.vivax, a novel family kir in P.knowlesi and the cir/bir/yir family in three rodent malarias. Our data indicate that this is the major multi-gene family in malaria parasites. Protein localization of products from pir members to the infected erythrocyte membrane in the rodent malaria parasite P.chabaudi, demonstrates phenotypic similarity to the products of pir in other malaria species. The results give critical insight into the evolutionary adaptation of malaria parasites to their host and provide important data for comparative immunology between malaria parasites obtained from laboratory models and their human counterparts.     

Coinfection with nonlethal murine malaria parasites suppresses pathogenesis caused by Plasmodium berghei NK65

Mixed infection with different Plasmodium species is often observed in endemic areas, and the infection with benign malaria parasites such as Plasmodium vivax or P. malariae has been considered to reduce the risk of developing severe pathogenesis caused by P. falciparum. However, it is still unknown how disease severity is reduced in hosts during coinfection. In the present study, we investigated the influence of coinfection with nonlethal parasites, P. berghei XAT (Pb XAT) or P. yoelii 17X (Py 17X), on the outcome of P. berghei NK65 (Pb NK65) lethal infection, which caused high levels of parasitemia and severe pathogenesis in mice. We found that the simultaneous infection with nonlethal Pb XAT or Py 17X suppressed high levels of parasitemia, liver injury, and body weight loss caused by Pb NK65 infection, induced high levels of reticulocytemia, and subsequently prolonged survival of mice. In coinfected mice, the immune response, including the expansion of B220(int)CD11c(+) cells and CD4(+) T cells and expression of IL-10 mRNA, was comparable to that in nonlethal infection. Moreover, the suppression of liver injury and body weight loss by coinfection was reduced in IL-10(-/-) mice, suggesting that IL-10 plays a role for a reduction of severity by coinfection with nonlethal malaria parasites.     

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.     

Coevolutionary genetics of Plasmodium malaria parasites and their human hosts

Malaria has been invoked, perhaps more than any other infectious disease, as a force for the selection of human genetic polymorphisms. Evidence for genome-shaping interactions can be found in the geographic and ethnic distributions of the hemoglobins, blood group antigens, thalassemias, red cell membrane molecules, human lymphocyte antigen (HLA) classes, and cytokines. Human immune responses and genetic variations can correspondingly influence the structure and polymorphisms of Plasmodium populations, notably in genes that affect the success and virulence of infection. In Africa, where the burden from Plasmodium falciparum predominates, disease severity and manifestations vary in prevalence among human populations. The evolutionary history and spread of Plasmodium species inform our assessment of malaria as a selective force. Longstanding host-pathogen relationships, as well as recent changes in this dynamic, illustrate the selective pressures human and Plasmodium species place on one another. Investigations of malaria protection determinants and virulence factors that contribute to the complexity of the disease should advance our understanding of malaria pathogenesis.     

Species concepts and malaria parasites: detecting a cryptic species of Plasmodium.

Species of malaria parasite (phylum Apicomplexa: genus Plasmodium) have traditionally been described using the similarity species concept (based primarily on differences in morphological or life-history characteristics). The biological species concept (reproductive isolation) and phylogenetic species concept (based on monophyly) have not been used before in defining species of Plasmodium. Plasmodium azurophilum, described from Anolis lizards in the eastern Caribbean, is actually a two-species cryptic complex. The parasites were studied from eight islands, from Puerto Rico in the north to Grenada in the south. Morphology of the two species is very similar (differences are indistinguishable to the eye), but one infects only erythrocytes and the other only white blood cells. Molecular data for the cytochrome b gene reveal that the two forms are reproductively isolated; distinct haplotypes are present on each island and are never shared between the erythrocyte-infecting and leucocyte-infecting species. Each forms a monophyletic lineage indicating that they diverged before becoming established in the anoles of the eastern Caribbean. This comparison of the similarity, biological and phylogenetic species concepts for malaria parasites reveals the limited value of using only similarity measures in defining protozoan species.     

Avian malaria on Madagascar: prevalence,biodiversity and specialization of haemosporidian parasites

Previous studies about geographic patterns of species diversity of avian malaria parasites and others in the Order Haemosporida did not include the avian biodiversity hotspot Madagascar. Since there are few data available on avian malaria parasites on Madagascar, we conducted the first known large-scale molecular-based study to investigate their biodiversity. Samples (1067) from 55 bird species were examined by a PCR method amplifying nearly the whole haemosporidian cytochrome b gene (1063?bp). The parasite lineages found were further characterized phylogenetically and the degree of specialization was determined with a newly introduced host diversity index (Hd). Our results demonstrate that Madagascar indeed represents a biodiversity hotspot for avian malaria parasites as we detected 71 genetically distinct parasite lineages of the genera Plasmodium and Haemoproteus. Furthermore, by using a phylogenetic approach and including the sequence divergence we suspect that the detected haemosporidian lineages represent at least 29 groups i.e. proposed species. The here presented Hd values for each parasite regarding host species, genus and family strongly support previous works demonstrating the elastic host ranges of some avian parsites of the Order Haemosporida. Representatives of the avian parasite genera Plasmodium and Leucocytozoon tend to more often be generalists than those of the genus Haemoproteus. However, as demonstrated in various examples, there is a large overlap and single parasite lineages frequently deviate from this rule.     

Vertebrate host specificity of two avian malaria parasites of the subgenus Novyella: Plasmodium nucleophilum and Plasmodium vaughani

The susceptibility of wild-caught European passeriform birds to naturally isolated malaria parasites, Plasmodium (Novyella) nucleophilum and Plasmodium (Novyella) vaughani, was studied by means of intramuscular subinoculation of infected citrated blood. Plasmodium nucleophilum of the great tit, Parus major, was transmitted to 3 great tits, but 3 blackcaps (Sylvia atricapilla) were not susceptible. Plasmodium vaughani of the robin, Erithacus rubecula, was transmitted to 1 robin and 1 blackcap, but 1 dunnock, Prunella modularis, was not susceptible. The prepatent period was between 8 and 10 days in all experimental infections. Maximum experimental parasitemia (3.4% of red cells) was detected in great tits infected with P. nucleophilum 23 days postexposure. A light (<0.01%) transient parasitemia of P. vaughani developed in the robin and blackcap. This study is in accord with former experimental observations on host specificity of P. nucleophilum and P. vaughani, which are characterized by a wide, but selective, range of avian hosts. Two new host-parasite associations were recorded.     

A new single-step PCR assay for the detection of the zoonotic malaria parasite Plasmodium knowlesi

BACKGROUND: Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. METHODOLOGY AND SIGNIFICANT FINDINGS: We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. CONCLUSIONS: The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi.     

Phylogeography of Caribbean lizard malaria: tracing the history of vector‐borne parasites

The Anolis lizards of the eastern Caribbean islands are parasitized by several species of malaria parasites (Plasmodium). Here I focus on two species of Plasmodium, using molecular data (mitochondrial cytochrome b sequences) to recover the phylogeography of the parasites throughout the Lesser Antilles and Puerto Rico. The two parasites were originally described as a single species, P. azurophilum, which infects both red and white blood cells. Here the two species are termed P. azurophilum Red and P. azurophilum White based on their host cell type. Six haplotypes were found in 100 infections sequenced of P. azurophilum Red and six in 45 infections of P. azurophilum White. Nested clade analysis revealed a significant association of geographical location and clades as well as a pattern of past fragmentation of parasite populations. This is consistent with the hypothesis that vector‐borne parasites such as malaria may be subject to frequent local extinctions and recolonizations. Comparison of the phylogeography of the lizard and parasites shows only weak concordance; that is, the parasites colonized the lizards in the islands, but dispersal events between islands via vectors or failed lizard colonizations were present. The two parasites had different histories, P. azurophilum Red colonized the islands from both the north and south, and P. azurophilum White originated in the central Lesser Antilles, probably from P. azurophilum Red, then moved to both north and south. This is the first study to examine the biogeography of a pair of sibling species of vector‐borne parasites within an island archipelago system.