A new morphologically distinct avian malaria parasite that fails detection by established polymerase chain reaction-based protocols for amplification of the cytochrome B gene

Plasmodium polymorphum n. sp. (Haemosporida, Plasmodiidae) was found in the skylark, Alauda arvensis (Passeriformes: Alaudidae), during autumnal migration in southern Italy. This organism is illustrated and described based on the morphology of its blood stages. The most distinctive feature of this malaria parasite is the clear preference of its blood stages (trophozoites, meronts, and gametocytes) for immature red blood cells, including erythroblasts. Based on preference of erythrocytic meronts for immature red blood cells, P. polymorphum is most similar to species of the subgenus Huffia . This parasite can be readily distinguished from all other bird malaria parasites, including Plasmodium ( Huffia ) spp., due to preferential development and maturation of its gametocytes in immature red blood cells, a unique character for avian Plasmodium spp. In addition, the margins of nuclei in blood stages of P. polymorphum are markedly smooth and distinct; this is also a distinct diagnostic feature of this parasite. Plasmodium polymorphum has been recorded only in the skylark; it is probably a rare parasite, whose host range and geographical distribution remain unclear. Microscopic examination detected a light infection of Plasmodium relictum (lineage GRW11, parasitemia of <0.01%) in the same sample with P. polymorphum ; the latter parasite clearly predominated (3.5% parasitemia). However, experienced researchers were unable to detect sequences of mitochondrial cytochrome b gene (cyt b ) of P. polymorphum from the microscopically positive sample by using published and newly designed primers for DNA amplification of avian Plasmodium spp. The light parasitemia of P. relictum was easily detectable using several polymerase chain reaction (PCR)-based assays, but P. polymorphum was undetectable in all applied assays. Quantitative PCR also showed the presence of light parasitemia (0.06%) of the lineage GRW11 in this sample. This supports the conclusion that the morphologically distinct parasite observed along with P. relictum and predominant in the sample is genetically dissimilar from the lineage GRW11 based on cyt b sequence. In samples with co-infections, general PCR protocols tend to favor the amplification of the parasite with the higher parasitemia or the amplification with the best matching sequence to the primers. Because the parasitemia of P. polymorphum was >50-fold higher than that of P. relictum and several different primers were tested, we suggest that the failure to amplify P. polymorphum is a more complex problem than why co-infections are commonly overlooked in PCR-based studies. We suggest possible explanations of these results and call for additional research on evolution of mitochondrial genome of hemosporidian parasites.     

No evidence for avian malaria infection during the nestling phase in a passerine bird

One of many uncertainties concerning the epidemiology of avian malaria in wild bird populations is the age at first infection. While nestlings, being naked and presumably immunologically na?ve would seem a likely stage of first infection, most age-stratified prevalence studies have not examined the nestling cohort, whereas those that have use relatively insensitive blood smear examination to diagnose infection. In the study presented here, we used sensitive nested polymerase chain reaction methods to screen blood samples from 195, 14-day-old blue tit (Cyanistes caeruleus) nestlings for avian malaria parasites (species of Plasmodium and Haemoproteus). Adults in this population are commonly infected with Plasmodium spp. (prevalence c. 30%). No avian malaria infections were found in nestlings, but a single positive identification of the related hematozoan parasite, Leucocytozoon sp., was made. Our results suggest either that the nestlings were infected but the disease had not yet reached patency, or that young birds in the nest are not bitten by the insect vectors of the disease.     

Coamplification of Leucocytozoon by PCR diagnostic tests for avian malaria: a cautionary note

A number of PCR assays have now been described for detecting species of the avian malaria parasites Plasmodium and Haemoproteus from blood samples. The published protocols amplify both genera simultaneously, owing to the high degree of sequence similarity between them in target genes. However, the potential for coamplification in these assays of a third, closely related hematozoan parasite, Leucocytozoon spp. has been largely overlooked. In this paper, we highlight the importance of this issue, showing that coamplification of Leucocytozoon spp. occurs in several of the protocols designed to amplify avian malaria parasites. This leads not only to scoring of false positives but, in cases of mixed Leucocytozoon/malaria infections, may also lead to scoring of false negatives. We, therefore, advocate the use of a post-PCR diagnostic step, such as RFLP analysis or sequencing, to assess the contribution of Leucocytozoon spp. to overall prevalence.     

Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia

We have optimized a faster and cheaper real-time PCR and developed a conventional genus specific PCR based on 18S rRNA gene to detect malaria parasites in low-grade parasitemias. Additionally, we compared these PCRs to the OptiMAL-IT test. Since there is no consensus on choice of standard quantitative curve in real-time assays, we decided to investigate the performance of parasite DNA from three different sources: "genome", amplicon and plasmid. The amplicon curve showed the best efficiency in quantifying parasites. Both PCR assays detected 100% of the clinical samples tested; the sensitivity threshold was 0.5 parasite/mul and no PCR positive reaction occurred when malaria parasites were not present. Conversely, if OptiMAL-IT were employed for malaria diagnosis, 30% of false-negative results could be expected. We conclude that PCR assays have potential for detecting malaria parasites in asymptomatic infections, in evaluation of malaria vaccine molecule candidates, for screening blood donors, especially in endemic areas, or even in monitoring malaria therapy.     

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.     

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.     

Association between Knops blood group polymorphisms and susceptibility to malaria in an endemic area of the Brazilian Amazon

Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the genotype and allele and haplotype frequencies of single-nucleotide polymorphisms (SNPs) of Knops blood group antigens and examine their association with susceptibility to malaria in an endemic area of Brazil. One hundred and twenty-six individuals from the Brazilian Amazon were studied. The CR1-genomic fragment was amplified by PCR and six SNPs and haplotypes were identified after DNA sequence analysis. Allele and haplotype frequencies revealed that the Kn(b) allele and H8 haplotype were possibly associated with susceptibility to Plasmodium falciparum. The odds ratios were reasonably high, suggesting a potentially important association between two Knops blood antigens (Kn(b) and KAM(+)) that confer susceptibility to P. falciparum in individuals from the Brazilian Amazon.     

Evaluation of a new plate hybridization assay for the laboratory diagnosis of imported malaria in Italy

A new molecular diagnostic method "Malaria-IBRIDOGEN" (Amplimedical S.p.A.--Bioline Division, Turin, Italy) based on a plate-hybridization assay for the simultaneous detection and identification of human malaria parasites was evaluated in this study. A target DNA sequence of the plasmodial 18S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) and hybridized in microtiter wells with five biotinylated probes each specific for Plasmodium falciparum, P. vivax, P. malariae, P. ovale and the beta-globine human gene, respectively. Compared to the nested-PCR actually used in our laboratory for the molecular diagnosis of malaria, "Malaria-IBRIDOGEN" revealed an overall sensitivity of 100% (51/51) for the four human Plasmodium species testing 100 whole blood samples from people with malaria-like symptoms and fever. Specificity was 92% (45/49) considering four discordant samples as "false positive" by "Malaria-IBRIDOGEN". The assay showed a threshold of parasite density (detection limit) of 0.07 P. falciparum parasites/microliter, 0.15-1.5 P. vivax parasites/microliter, 0.3 P. malariae parasites/microliter and 0.4 P. ovale parasites/microliter of whole blood, respectively. This assay could be successfully applied to the laboratory diagnosis of malaria as a useful aid to microscopy.     

Nonspecific patterns of vector, host and avian malaria parasite associations in a central African rainforest

Malaria parasites use vertebrate hosts for asexual multiplication and Culicidae mosquitoes for sexual and asexual development, yet the literature on avian malaria remains biased towards examining the asexual stages of the life cycle in birds. To fully understand parasite evolution and mechanism of malaria transmission, knowledge of all three components of the vector-host-parasite system is essential. Little is known about avian parasite-vector associations in African rainforests where numerous species of birds are infected with avian haemosporidians of the genera Plasmodium and Haemoproteus. Here we applied high resolution melt qPCR-based techniques and nested PCR to examine the occurrence and diversity of mitochondrial cytochrome b gene sequences of haemosporidian parasites in wild-caught mosquitoes sampled across 12 sites in Cameroon. In all, 3134 mosquitoes representing 27 species were screened. Mosquitoes belonging to four genera (Aedes, Coquillettidia, Culex and Mansonia) were infected with twenty-two parasite lineages (18 Plasmodium spp. and 4 Haemoproteus spp.). Presence of Plasmodium sporozoites in salivary glands of Coquillettidia aurites further established these mosquitoes as likely vectors. Occurrence of parasite lineages differed significantly among genera, as well as their probability of being infected with malaria across species and sites. Approximately one-third of these lineages were previously detected in other avian host species from the region, indicating that vertebrate host sharing is a common feature and that avian Plasmodium spp. vector breadth does not always accompany vertebrate-host breadth. This study suggests extensive invertebrate host shifts in mosquito-parasite interactions and that avian Plasmodium species are most likely not tightly coevolved with vector species.     

2019年辽宁省3例输入性三日疟基因型别实验室诊治与分析

对辽宁省2019年的3例境外输入性三日疟疟疾病例进行了实验室检测与诊断分析。 收集并进行流行病学调查与资料汇总。根据疟疾实验室现有最新执行诊断标准《疟疾的诊断》（WS259-2015）的要求，对临床诊断的疑似三日疟患者采集抗凝血制作血涂片镜检、进行疟疾快速诊断检测（RDT），上送全血到辽宁省疾病预防控制中心进行病例复核，巢氏PCR检测并进行测序比对。3份病例患者外周血血涂片镜检薄厚血膜，虫体分期主要为环状体期、大滋养体期、配子体期和成熟裂殖体期，其中大滋养体期中疟色素呈深棕色、较大、沿边缘分布，发现寄生的红细胞通常不胀大甚至会缩小，配子体小而圆，根据镜下特点初步判定为三日疟原虫；RDT结果提示为感染除恶性疟以外的其他3种疟疾（三日疟、卵形疟、间日疟）的单一感染，省级参比实验室对于上送全血利用巢氏PCR检测技术进行复核检测；将扩增出的三日疟的目的片段产物序列送至上海维基基因测序公司进行序列分析比对，基因序列同源性达到了100%。 同时使用血涂片镜检、进行疟疾快速诊断检测（RDT）和PCR进行检测，实验结果均鉴定为三日疟，根据病例的流行病学调查和临床表现确定为境外输入性三日疟病例。     

Metabarcoding options to study eukaryotic endoparasites of birds

There is growing interest in the study of avian endoparasite communities, and metabarcoding is a promising approach to complement more conventional or targeted methods. In the case of eukaryotic endoparasites, phylogenetic diversity is extreme, with parasites from 4 kingdoms and 11 phyla documented in birds. We addressed this challenge by comparing different primer sets across 16 samples from 5 bird species. Samples consisted of blood, feces, and controlled mixes with known proportions of bird and nematode DNA. Illumina sequencing revealed that a 28S primer set used in combination with a custom blocking primer allowed detection of various plasmodiid parasites and filarioid nematodes in the blood, coccidia in the feces, as well as two potentially pathogenic fungal groups. When tested on the controlled DNA mixes, these primers also increased the proportion of nematode DNA by over an order of magnitude. An 18S primer set, originally designed to exclude metazoan sequences, was the most effective at reducing the relative number of avian DNA sequences and was the only one to detect Trypanosoma in the blood. Expectedly, however, it did not allow nematode detection and also failed to detect avian malaria parasites. This study shows that a 28S set including a blocking primer allows detection of several major and very diverse bird parasite clades, while reliable amplification of all major parasite groups may require a combination of markers. It helps clarify options for bird parasite metabarcoding, according to priorities in terms of the endoparasite clades and the ecological questions researchers wish to focus on.     

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.     

Seasonal changes in the feeding pattern of Culex pipiens pallens govern the transmission dynamics of multiple lineages of avian malaria parasites in Japanese wild bird community

Heterogeneity in the transmission of mosquito-borne pathogens is determined largely by distribution patterns of mosquito bites among wild animal populations. Although mosquitoes are crucial for transmission of avian malaria parasites, little is known about the ecology of natural vectors. We examined bloodmeal and parasite incidence in Culex pipiens pallens by a polymerase chain reaction (PCR)-based procedure to determine how the feeding pattern of mosquitoes govern transmission dynamics of avian malaria parasites in Japanese wild birds. We collected 881 unfed and 486 blood-fed Cx. pipiens pallens resting on vegetation in a park in Tokyo. The mosquitoes were separated into abdomen and thorax prior to PCR screening. Abdomens of unfed mosquitoes were combined into 95 pools. From these, we amplified Plasmodium DNA in 32 (33.7%) pools. Among blood-fed mosquitoes, 371 individuals were screened for blood-sources and Plasmodium parasites. Plasmodium DNA was amplified from mosquitoes fed on 6 of 13 avian species identified as blood-sources. Ten Plasmodium lineages were identified on the basis of 478 bp of the cytochrome b gene, with 0.2-10% sequence divergence. The three commonest Plasmodium lineages (CXPIP09, SGS1 and PADOM02) were detected in both the abdomens and thoraxes of mosquitoes, strongly suggesting transmission of these lineages. Jungle crow (Corvus macrorhynchos) served as a natural host for the three commonest Plasmodium lineages and made up 63.8% of blood-sources. As a significant increase in feeding of vector mosquitoes on jungle crows coincided with their breeding season, jungle crows were considered to be the primary reservoir of Plasmodium transmission in this study.     

Screening and purification for novel cytochrome b5 from uncultured environmental micro-organisms

AIMS: We describe a sequence-based PCR method suitable for the isolation of a novel soluble heme-binding domain of cytochrome b(5) (cyt b(5)) gene directly from metagenomic DNA is described. METHODS AND RESULTS: Using the degenerate primer set, a cyt b(5) gene was isolated directly from metagenomic DNA. Based on the sequence-based PCR method, the similar conserved motif of cyt b(5) from Rhodopseudomonas palustris strain makes the novel target gene. The gene encoding cyt b(5) was cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized. CONCLUSIONS: Sequence-based strategy is an effective method for application of the novel gene from metagenomic DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigation of novel genes from metagenome, most of the micro-organism species are largely untapped, could represent an interesting and useful reservoir for biological processes.     

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.     

First report of avian malaria in a Manx shearwater (Puffinus puffinus)

This study reports the case of a Manx shearwater (Puffinus puffinus) that died from avian malaria while under care at a rehabilitation center in Espírito Santo, Brazil. The bird was rescued on October 2018, and remained under care until it died suddenly on January 2019. A blood smear produced 8 days before death was negative for parasites, whereas a blood smear produced post-mortem revealed a high parasitemia by a parasite resembling Plasmodium cathemerium. The sequence of a 412 bp segment of the cyt-b gene was identical to that of lineage PADOM09, and phylogenetic analysis corroborated that this parasite was closely-related to known lineages of P. cathemerium. The acuteness and severity of the infection documented in this case suggest that seabirds of the order Procellariiformes might be highly susceptible to Plasmodium infections, raising the concern that avian malaria may present a significant threat to their conservation.     

A Colorimetric DNA Diagnostic Method for Falciparum Malaria and Vivax Malaria: A Field Trial in the Solomon Islands

Abstract

We have developed a colorimetric assay, “microtiter plate-hybridization”, for the detection of malaria parasites Plasmodium falciparum and P. vivax in human blood, in which the target DNA sequences (18s small subunit ribosomal RNA gene) amplified by polymerase chain reaction (PCR) are hybridized with the species-specific probes immobilized on a microtiter well. This assay system was tested in Guadalcanal, Solomon Islands, where malaria is highly endemic. We obtained blood samples by finger puncture from 130 asymptomatic donors. Among the 130 samples, 30 (23 %) were P. falciparum positive, 28 (22 %) were P. vivax positive, and 8 (6 %) were mixed infections. The results of our DNA diagnostic method showed good correlation with those of acridine orange microscopy.