Avian malaria in a feral-pet pigeon: a case report.

BACKGROUND: Avian malaria is caused by diverse parasite species of the genus Plasmodium, and it affects various bird species. The occurrence of this disease in some wild bird species is sparsely documented due to the scarce availability of samples. Hence the pathogenicity in some hosts is not completely known. In addition, feral birds may act as reservoirs bridging the transmission cycle from wild migratory birds to domestic and zoo-kept bird species. CASE PRESENTATION: An owner of pigeons adopted a feral pigeon (Columba livia forma domestica) and housed it together with his other pet-pigeons. The bird died unexpectedly a few weeks after a surgical procedure and necropsy revealed a severely anaemic carcass, with pale organs and hydropericardium. Histopathologic analysis revealed inflammatory infiltrates in the lung and liver, and monocytes and Kupffer cells contained haemozoin pigment indicative of phagocytosis of Plasmodium-infected erythrocytes. A high erythrocytic infection rate of 18% was evident in tissues and blood vessels in various organs. Furthermore, the thyroid had masses classified as thyroid carcinomas. Immunohistochemistry with anti- Plasmodium falciparum HSP70 antibody revealed positive signals in erythrocytes and intravascular leucocytes. Further microscopy analysis using a Hemacolor-stained impression smear revealed a high parasitaemia with an asynchronous infection showing all erythrocytic stages. Molecular diagnosis by PCR identified Plasmodium relictum, lineage GRW11 as the aetiological agent. The bird presented died most likely due to an acute infection as evidenced by the high blood parasitaemia, leading to major erythrocyte destruction. Further analyses of feral pigeons (n = 22) did not reveal any additional cases of Plasmodium infections. CONCLUSION: This study reports the first mortality associated with P. relictum lineage GRW11. The study supports previous studies, suggesting that Plasmodium infections are not frequent in pigeons. Host conditions like immunosuppression due to the tumour may have influenced the infection outcome in this fatal case. Use of anti-P. falciparum HSP70 antibody for detection of P. relictum antigens for immune assays in blood and tissue samples will be a useful tool for future studies.

Feeding habits and malaria parasite infection of Anopheles mosquitoes in selected agroecological areas of Northwestern Ethiopia.

BACKGROUND: Surveillance of the host-anopheline mosquitoes' interaction is important for assessing malaria transmission risk and guiding vector control. We assume that changes in malaria vector species' feeding habits, as well as the surrounding environment, have a substantial impact on varied malaria transmission. In this study, we determined the vertebrate host feeding patterns of anopheline mosquitoes to characterize entomologic risk factors for malaria in Jabi Tehnan, Northwestern Ethiopia. METHODS: Blood-fed anophelines surveyed during malaria surveillance in Jabi Tehnan district of northwestern Ethiopia were utilized in this study. They were collected using Centers for Disease Control and Prevention (CDC) light traps deployed in selected households per village, placed indoors and outdoors, spanning three agroecological settings (dry mountain, plateau, and semiarid highlands) between June 2020 and May 2021. The engorged mosquitoes were analyzed for host blood meal sources and Plasmodium infection via polymerase chain reaction (PCR) and/or sequencing. Infection rates and bovine and human blood indices were calculated and compared for abundant species; between indoors and outdoors and between agroecology using a chi-squared test for equality of proportion in R package at a significant level of p ≤ 0.05. RESULTS: A total of 246 mosquitoes were successfully typed (indoor, 121; outdoor, 125), with greater relative abundance indoors in mountain and plateau highlands, and outdoors in semiarid areas. Despite ecological differences in blood-fed capture rates, cattle served as the most utilized blood meal source by 11 anopheline species with an overall bovine blood index (BBI) of 74.4%. This trend was dictated by Anopheles gambiae s.l. (198/246; BBI = 73.7%), which exhibited the most plastic feeding habits that included humans (human blood index = 15.7%) and other livestock and rodents. A total of five anopheline species (An. gambiae s.l., An. funestus s.l., An. coustani s.l., An. pretoriensis, and An. pharoensis) fed on humans, of which the first three were found infected with Plasmodium parasites. Most of the infected specimens were An. arabiensis (5.6%, 11/198) and had recently fed mainly on cattle (72.7%, 8/11); one each of infected An. funestus s.l. and An. coustani s.l. had fed on humans and cattle, respectively. CONCLUSIONS: The results demonstrate communal feeding on cattle by anophelines including primary and secondary malaria vectors. This study also indicates the importance of cattle-targeted interventions for sustainable control of malaria vectors in the study areas.

Real-time PCR for malaria diagnosis and identification of Plasmodium species in febrile patients in Cubal, Angola.

BACKGROUND: Malaria is the parasitic disease with the highest morbimortality worldwide. The World Health Organization (WHO) estimates that there were approximately 249 million cases in 2022, of which 3.4% were in Angola. Diagnosis is based on parasite identification by microscopy examination, antigen detection, and/or molecular tests, such as polymerase chain reaction (PCR). This study aimed to evaluate the usefulness of real-time PCR as a diagnostic method for malaria in an endemic area (Cubal, Angola). METHODS: A cross-sectional study was carried out at the Hospital Nossa Senhora da Paz in Cubal, Angola, including 200 patients who consulted for febrile syndrome between May and July 2022. From each patient, a capillary blood sample was obtained by finger prick for malaria field diagnosis [microscopy and rapid diagnostic test (RDT)] and venous blood sample for real-time PCR performed at the Hospital Universitario Vall d'Hebron in Barcelona, Spain. Any participant with a positive result from at least one of these three methods was diagnosed with malaria. RESULTS: Of the 200 participants included, 54% were female and the median age was 7 years. Malaria was diagnosed by at least one of the three techniques (microscopy, RDT, and/or real-time PCR) in 58% of the participants, with RDT having the highest percentage of positivity (49%), followed by real-time PCR (39.5%) and microscopy (33.5%). Of the 61 discordant samples, 4 were only positive by microscopy, 13 by real-time PCR, and 26 by RDT. Plasmodium falciparum was the most frequent species detected (90.63%), followed by P. malariae (17.19%) and P. ovale (9.38%). Coinfections were detected in ten participants (15.63%): six (60%) were caused by P. falciparum and P. malariae, three (30%) by P. falciparum and P. ovale, and one (10%) triple infection with these three species. In addition, it was observed that P. falciparum and P. malariae coinfection significantly increased the parasite density of the latter. CONCLUSIONS: RDT was the technique with the highest positivity rate, followed by real-time PCR and microscopy. The results of the real-time PCR may have been underestimated due to suboptimal storage conditions during the transportation of the DNA eluates. However, real-time PCR techniques have an important role in the surveillance of circulating Plasmodium species, given the epidemiological importance of the increase in non-falciparum species in the country, and can provide an estimate of the intensity of infection.

Transinfection of Wolbachia wAlbB into Culex quinquefasciatus mosquitoes does not alter vector competence for Hawaiian avian malaria (Plasmodium relictum GRW4).

Avian malaria is expanding upslope with warmer temperatures and driving multiple species of Hawaiian birds towards extinction. Methods to reduce malaria transmission are urgently needed to prevent further declines. Releasing Wolbachia-infected incompatible male mosquitoes could suppress mosquito populations and releasing Wolbachia-infected female mosquitoes (or both sexes) could reduce pathogen transmission if the Wolbachia strain reduced vector competence. We cleared Culex quinquefasciatus of their natural Wolbachia pipientis wPip infection and transinfected them with Wolbachia wAlbB isolated from Aedes albopictus. We show that wAlbB infection was transmitted transovarially, and demonstrate cytoplasmic incompatibility with wild-type mosquitoes infected with wPip from Oahu and Maui, Hawaii. We measured vector competence for avian malaria, Plasmodium relictum, lineage GRW4, of seven mosquito lines (two with wAlbB; three with natural wPip infection, and two cleared of Wolbachia infection) by allowing them to feed on canaries infected with recently collected field isolates of Hawaiian P. relictum. We tested 73 groups (Ntotal = 1176) of mosquitoes for P. relictum infection in abdomens and thoraxes 6-14 days after feeding on a range of parasitemias from 0.028% to 2.49%, as well as a smaller subset of salivary glands. We found no measurable effect of Wolbachia on any endpoint, but strong effects of parasitemia, days post feeding, and mosquito strain on both abdomen and thorax infection prevalence. These results suggest that releasing male wAlbB-infected C. quinquefasciatus mosquitoes could suppress wPip-infected mosquito populations, but would have little positive or negative impact on mosquito vector competence for P. relictum if wAlbB became established in local mosquito populations. More broadly, the lack of Wolbachia effects on vector competence we observed highlights the variable impacts of both native and transinfected Wolbachia infections in mosquitoes.

Loop-Mediated Isothermal Amplification for Diagnosis of Zoonotic Malaria.

Zoonotic malaria, caused by Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium coatneyi, and Plasmodium inui, is a significant global health concern. The gold standard microscopy, while widely used for malaria diagnosis, faces limitations in differentiating between malaria species. Polymerase chain reaction (PCR), despite its accuracy, is characterized by high costs and time-consuming procedures. This study aims to develop and validate a rapid and accurate diagnostic test for detecting four simian Plasmodium species by using loop-mediated isothermal amplification (LAMP). Loop-mediated isothermal amplification is a cost-effective and faster molecular testing alternative for malaria diagnosis. The project involved designing specific primers, testing sensitivity and specificity against various parasites (including human Plasmodium species, protozoa, and helminths), and evaluating the LAMP assay using 60 macaque samples infected with simian Plasmodium. The LAMP assay exhibited a sensitivity profile enabling the detection of P. knowlesi, P. coatneyi, and P. cynomolgi across a concentration gradient from 5 × 108 down to 5 × 105 parasites/µL. Notably, P. inui was detectable at 5 × 108 parasites/µL. Furthermore, the specificity of the primer tailored for the four simian Plasmodium species was proven, as it produced a positive amplification exclusively for the respective target species and generated negative results for nontarget species. The results indicated that the LAMP assay is capable of detecting simian Plasmodium within a short span of 60 minutes, without any false positives from other samples. This new test has the potential to revolutionize malaria diagnosis, surveillance, and control, thereby mitigating the impact of zoonotic malaria in regions of endemicity.

A broadly cross-reactive i-body to AMA1 potently inhibits blood and liver stages of Plasmodium parasites.

Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium.

Malaria screening: what is the contribution of molecular biology?

INTRODUCTION: According to the World Health Organization, Microscopy is the gold standard for diagnosing malaria. However, the performance of this examination depends on the experience of the microscopist and the level of parasitemia. Thus, molecular biology detection of malaria could be an alternative technique. AIM: evaluate the contribution of molecular biology in detecting imported malaria. METHODS: This was a descriptive, prospective study, including all students, from the Monastir region, and foreigners, from countries endemic to malaria. The study period was from September 2020 to April 2021. Each subject was screened for malaria by three methods: direct microscopic detection of Plasmodium, detection of plasmodial antigens, and detection of plasmodial DNA by nested PCR. RESULTS: Among the 127 subjects screened, only one had a positive microscopic examination for Plasmodium falciparum. Among the 126 subjects with a negative microscopic examination, twelve students had a positive nested PCR result, i.e. 9.5%. Molecular sequencing allowed the identification of ten isolates of Plasmodium falciparum, one Plasmodium malariae and one Plasmodium ovale. Our study showed that the results of nested PCR agreed with those of microscopy in 90.6% of cases. CONCLUSION: Nested PCR seems more sensitive for the detection of low parasitemias. Hence the importance of including molecular biology as a malaria screening tool to ensure better detection of imported cases.

Orthologs of Plasmodium ICM1 are dispensable for Ca2+ mobilization in Toxoplasma gondii.

Apicomplexan parasites mobilize ionic calcium (Ca2+) from intracellular stores to promote microneme secretion and facilitate motile processes including gliding motility, invasion, and egress. Recently, a multipass transmembrane protein, ICM1, was found to be important for calcium mobilization in Plasmodium falciparum and P. berghei. Comparative genomics and phylogenetics have revealed putative ICM orthologs in Toxoplasma gondii and other apicomplexans. T. gondii possesses two ICM-like proteins, which we have named TgICM1-L (TGGT1_305470) and TgICM2-L (TGGT1_309910). TgICM1-L and TgICM2-L localized to undefined puncta within the parasite cytosol. TgICM1-L and TgICM2-L are individually dispensable in tachyzoites, suggesting a potential compensatory relationship between the two proteins may exist. Surprisingly, mutants lacking both TgICM1-L and TgICM2-L are fully viable, exhibiting no obvious defects in growth, microneme secretion, invasion, or egress. Furthermore, loss of TgICM1-L, TgICM2-L, or both does not impair the parasite's ability to mobilize Ca2+. These findings suggest that additional proteins may participate in Ca2+ mobilization or import in Apicomplexa, reducing the dependence on ICM-like proteins in T. gondii. Collectively, these results highlight similar yet distinct mechanisms of Ca2+ mobilization between T. gondii and Plasmodium.IMPORTANCECa2+ signaling plays a crucial role in governing apicomplexan motility; yet, the mechanisms underlying Ca2+ mobilization from intracellular stores in these parasites remain unclear. In Plasmodium, the necessity of ICM1 for Ca2+ mobilization raises the question of whether this mechanism is conserved in other apicomplexans. Investigation into the orthologs of Plasmodium ICM1 in T. gondii revealed a differing requirement for ICM proteins between the two parasites. This study suggests that T. gondii employs ICM-independent mechanisms to regulate Ca2+ homeostasis and mobilization. Proteins involved in Ca2+ signaling in apicomplexans represent promising targets for therapeutic development.

A Pan Plasmodium lateral flow recombinase polymerase amplification assay for monitoring malaria parasites in vectors and human populations.

Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA-LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10-100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.