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Asymptomatic malarial parasitemia represents the largest reservoir of infection and transmission, and the impact of coinfection with HIV-1 on this reservoir remains incompletely described. Accordingly, we sought to determine the prevalence of asymptomatic malarial parasitemia in Kombewa, Western Kenya, a region that is endemic for both malaria and HIV-1. A total of 1,762 dried blood spots were collected from asymptomatic adults in a cross-sectional study. The presence of parasitemia was first determined by a sensitive Plasmodium genus–specific 18S assay, followed by less sensitive species-specific DNA-based quantitative polymerase chain reaction (PCR) assays. The prevalence of asymptomatic malarial parasitemia by 18S genus-specific PCR assay was 64.4% (1,134/1,762). Of the 1,134 malaria positive samples, Plasmodium falciparum was the most prevalent species (57.4%), followed by Plasmodium malariae (3.8%) and Plasmodium ovale (2.6%) as single or mixed infections. As expected, the majority of infections were below the detection limit of microscopy and rapid diagnostic tests. HIV-1 prevalence was 10.6%, and we observed a significant association with malarial parasitemia by χ2 analysis (P = 0.0475). Seventy-one percent of HIV-1 infected volunteers were positive for Plasmodium 18S (132/186), with only 29% negative (54/186). In HIV-1-negative volunteers, the proportion was lower; 64% were found to be positive for 18S (998/1,569) and 36% were negative (571/1,569). Overall, the prevalence of asymptomatic malarial parasitemia in Western Kenya is high, and knowledge of these associations with HIV-1 infection are critically important for malaria elimination and eradication efforts focused on this important reservoir population.

Background Humans living in regions with high falciparum malaria transmission intensity harbour multi-strain infections comprised of several genetically distinct malaria haplotypes. The number of distinct malaria parasite haplotypes identified from an infected human host at a given time is referred to as the complexity of infection (COI). In this study, an amplicon-based deep sequencing method targeting the Plasmodium falciparum apical membrane antigen 1 (pfama1) was utilized to (1) investigate the relationship between P. falciparum prevalence and COI, (2) to explore the population genetic structure of P. falciparum parasites from malaria asymptomatic individuals participating in the 2007 Demographic and Health Survey (DHS) in the Democratic Republic of Congo (DRC), and (3) to explore selection pressures on geospatially divergent parasite populations by comparing AMA1 amino acid frequencies in the DRC and Mali. Results A total of 900 P. falciparum infections across 11 DRC provinces were examined. Deep sequencing of both individuals, for COI analysis, and pools of individuals, to examine population structure, identified 77 unique pfama1 haplotypes. The majority of individual infections (64.5%) contained polyclonal (COI > 1) malaria infections based on the presence of genetically distinct pfama1 haplotypes. A minimal correlation between COI and malaria prevalence as determined by sensitive real-time PCR was identified. Population genetic analyses revealed extensive haplotype diversity, the vast majority of which was shared across the sites. AMA1 amino acid frequencies were similar between parasite populations in the DRC and Mali. Conclusions Amplicon-based deep sequencing is a useful tool for the detection of multi-strain infections that can aid in the understanding of antigen heterogeneity of potential malaria vaccine candidates, population genetics of malaria parasites, and factors that influence complex, polyclonal malaria infections. While AMA1 and other diverse markers under balancing selection may perform well for understanding COI, they may offer little geographic or temporal discrimination between parasite populations. Electronic supplementary material The online version of this article (10.1186/s12936-017-2137-9) contains supplementary material, which is available to authorized users.

Routine fecal examination revealed novel coccidian oocysts in asymptomatic California sea lions (Zalophus californianus) in a rehabilitation facility. Coccidian oocysts were observed in fecal samples collected from 15 of 410 California sea lions admitted to The Marine Mammal Center between April 2007 and October 2009. Phylogenetic analysis using the full ITS-1 region, partial small subunit 18S rDNA sequence, and the Apicomplexa rpoB region identified 2 distinct sequence clades, referred to as Coccidia A and Coccidia B, and placed them in the Sarcocystidae, grouped with the tissue-cyst-forming coccidia. Both sequence clades resolved as individual taxa at ITS-1 and rpoB and were most closely related to Neospora caninum. Coccidia A was identified in 11 and Coccidia B in 4 of 12 sea lion oocyst samples successfully sequenced (3 of those sea lions were co-infected with both parasites). Shedding of Coccidia A oocysts was not associated with age class, sex, or stranding location, but yearlings represented the majority of shedders (8/15). This is the first study to use molecular phylogenetics to identify and describe coccidian parasites shed by a marine mammal.

Enteric protozoal infection was identified in 5 stranded California sea lions (Zalophus californianus). Microscopically, the apical cytoplasm of distal jejunal enterocytes contained multiple stages of coccidian parasites, including schizonts with merozoites and spherical gametocytes, which were morphologically similar to coccidians. By histopathology, organisms appeared to be confined to the intestine and accompanied by only mild enteritis. Using electron microscopy, both sexual (microgametocytes, macrogamonts) and asexual (schizonts, merozoites) coccidian stages were identified in enterocytes within parasitophorous vacuoles, consistent with apicomplexan development in a definitive host. Serology was negative for tissue cyst-forming coccidians, and immunohistochemistry for Toxoplasma gondii was inconclusive and negative for Neospora caninum and Sarcocystis neurona. Analysis of ITS-1 gene sequences amplified from frozen or formalin-fixed paraffin-embedded intestinal sections identified DNA sequences with closest homology to Neospora sp. (80%); these novel sequences were referred to as belonging to coccidian parasites ‘‘A,’’ ‘‘B,’’ and ‘‘C.’’ Subsequent molecular analyses completed on a neonatal harbor seal (Phoca vitulina) with protozoal lymphadenitis, hepatitis, myocarditis, and encephalitis showed that it was infected with a coccidian parasite bearing the ‘‘C’’ sequence type. Our results indicate that sea lions likely serve as definitive hosts for 3 newly described coccidian parasites, at least 1 of which is pathogenic in a marine mammal intermediate host species.

Background Plasmodium ovale is comprised of two genetically distinct subspecies, P. ovale curtisi and P. ovale wallikeri. Although P. ovale subspecies are similar based on morphology and geographical distribution, allelic differences indicate that P. ovale curtisi and P. ovale wallikeri are genetically divergent. Additionally, potential clinical and latency duration differences between P. ovale curtisi and P. ovale wallikeri demonstrate the need for investigation into the contribution of this neglected malaria parasite to the global malaria burden. Methods In order to detect all P. ovale subspecies simultaneously, we developed an inclusive P. ovale-specific real-time PCR assay based on conserved regions between P. ovale curtisi and P. ovale wallikeri in the reticulocyte binding protein 2 (rbp2) gene. Additionally, we characterized the P. ovale subspecies prevalence from 22 asymptomatic malaria infections using multilocus genotyping to discriminate P. ovale curtisi and P. ovale wallikeri. Results Our P. ovale rbp2 qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 rbp2 plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 rbp2 plasmid copies/microliter. Specificity experiments showed the ability of the rbp2 qPCR assay to detect low-levels of P. ovale in the presence of additional malaria parasite species, including P. falciparum, P. vivax, and P. malariae. We identified P. ovale curtisi and P. ovale wallikeri in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the rbp2 gene. Conclusions Our novel P. ovale rbp2 qPCR assay detects P. ovale curtisi and P. ovale wallikeri simultaneously and can be utilized to characterize the prevalence, distribution, and burden of P. ovale in malaria endemic regions. Using multilocus genotyping, we also provided the first description of the prevalence of P. ovale curtisi and P. ovale wallikeri in Western Kenya, a region holoendemic for malaria transmission., Author Summary Humans can be infected with five malaria parasite species: Plasmodium falciparum, P. vivax, P. malariae, P. knowlesi, and P. ovale. Although the vast majority of malaria morbidity and mortality worldwide can be attributed to P. falciparum, non-falciparum malaria parasites can also cause clinical disease. Researchers use nucleic acid based detection methods, such a polymerase chain reaction (PCR), to detect low-density malaria parasitemias that can evade microscopic detection. P. ovale was recently identified to exist as two subspecies, P. ovale curtisi and P. ovale wallikeri, that look identical but differ genetically. In this study, we developed a novel real-time PCR (qPCR) assay to detect all P. ovale parasites, based on a conserved gene between P. ovale curtisi and P. ovale wallikeri. We also used DNA sequencing to differentiate between P. ovale curtisi and P. ovale wallikeri from a small sample of P. ovale asymptomatic infections in Western Kenya. Through the use of our novel rbp2 qPCR assay, we aim to characterize the prevalence of P. ovale in future epidemiological studies in order to better understand this neglected malaria parasite species.

Background Culex tritaeniorhynchus is the primary vector of Japanese encephalitis virus (JEV), a leading cause of encephalitis in Asia. JEV is transmitted in an enzootic cycle involving large wading birds as the reservoirs and swine as amplifying hosts. The development of a JEV vaccine reduced the number of JE cases in regions with comprehensive childhood vaccination programs, such as in Japan and the Republic of Korea. However, the lack of vaccine programs or insufficient coverage of populations in other endemic countries leaves many people susceptible to JEV. The aim of this study was to predict the distribution of Culex tritaeniorhynchus using ecological niche modeling. Methods/Principal Findings An ecological niche model was constructed using the Maxent program to map the areas with suitable environmental conditions for the Cx. tritaeniorhynchus vector. Program input consisted of environmental data (temperature, elevation, rainfall) and known locations of vector presence resulting from an extensive literature search and records from MosquitoMap. The statistically significant Maxent model of the estimated probability of Cx. tritaeniorhynchus presence showed that the mean temperatures of the wettest quarter had the greatest impact on the model. Further, the majority of human Japanese encephalitis (JE) cases were located in regions with higher estimated probability of Cx. tritaeniorhynchus presence. Conclusions/Significance Our ecological niche model of the estimated probability of Cx. tritaeniorhynchus presence provides a framework for better allocation of vector control resources, particularly in locations where JEV vaccinations are unavailable. Furthermore, this model provides estimates of vector probability that could improve vector surveillance programs and JE control efforts., Author Summary Japanese encephalitis virus (JEV) is transmitted predominately by the mosquito, Culex tritaeniorhynchus. The primary reservoirs of the virus are wading birds, with swine serving as amplifying hosts. Despite the development of a JEV vaccine, people remain unvaccinated in endemic countries and are susceptible to JEV infection. The distribution of the JEV vector(s) provides essential information for preventive measures. This study used an ecological niche modeling program to predict the distribution of Cx. tritaeniorhynchus based on collection records and environmental maps (climate, land cover, and elevation). The model showed that the mean temperatures of the wettest quarter had the greatest impact on the model. Of the 25 countries endemic for Japanese encephalitis (JE) endemic countries, seven possessed greater than 50% land area with an estimated high probability of Cx. tritaeniorhynchus presence. Our model provides a useful tool for JEV surveillance programs that focus on vector control strategies.

Background: A 53-year-old man presented with an acute bilateral posterior uveitis with extensive necrotising retinochoroiditis but without chorioretinal scarring. A thorough workup did not reveal any underlying disease. The possibilities of atypical ocular toxoplasmosis as well as herpetic retinal necrosis were considered and specific therapy instituted, with little improvement. The patient died within 2 months as result of an undifferentiated squamous cell carcinoma. Methods: Histopathological examination, immunohistochemistry and multilocus polymerase chain reaction confirmed Toxoplasma gondii infection of the retina Results: Macroscopic examination of enucleated globe showed extensive retinal necrosis and vitreous detachment. Histological examination of retinal tissue identified numerous round-to-elliptical toxoplasmic cysts within the retina, with retinal necrosis and minimal choroidal inflammation. Immunohistochemical analyses confirmed that the cysts were due to T gondii . DNA extracted from formalin-fixed, paraffin-embedded tissue sections was subjected to multilocus polymerase chain reaction (PCR) analysis at the following typing loci: SAG1, SAG2, SAG3, SAG4, B1, NTS2, GRA6 and GRA7. DNA sequencing of positive PCR products at the NTS2 , SAG1 and GRA7 loci confirmed the presence of a non-archetypal strain of T gondii infecting the eye of the patient experiencing a severe, atypical ocular toxoplasmosis Conclusion: A highly divergent, non-archetypal strain of T gondii was responsible for causing a severe, atypical bilateral retinochoroiditis in a patient from Brazil.

Mycoplasma alligatoris and Mycoplasma crocodyli are closely related siblings, one being highly virulent and the other relatively attenuated. We compared their genomes to better understand the mechanisms and origins of M. alligatoris ' remarkable virulence amid a clade of harmless or much less virulent species. Although its chromosome was refractory to closure, M. alligatoris differed most notably by its complement of sialidases and other genes of the N -acetylneuraminate scavenging and catabolism pathway.