Unresolved haemosporidia of the Australian skink, Egernia stokesii.

The Australian skink Egernia stokesii had been recognised as a host of two species of Plasmodium, Plasmodium mackerrasae and P. circularis; nevertheless, molecular data are available for only a single haemosporidian species of this host. Its sequences are labelled as "Plasmodium sp." or "Plasmodium mackerrasae", but morphological characteristics of this isolate are unavailable. Phylogenetic analyses of these sequences placed them into the clade of the genus Haemocystidium. In this study, blood samples of six E. stokesii were analysed by both, molecular and microscopic methods to clarify the haemosporidia of this lizard. Application of these approaches offered discordant results. Whereas sequence analysis clustered our isolates with lizard species of Haemocystidium, morphology of blood stages is more akin to Plasmodium than Haemocystidium. However, limited sampling, indistinguishable nuclei/merozoites and risk of possible hidden presence of mixed infection prevent reliable species identification of detected parasites or their description as new species of Haemocystidium.

Recombinase polymerase amplification - lateral flow dipstick for rapid and visual detection of Blastocystis spp.

Blastocystis spp. is a ubiquitous protozoon in the intestinal tract of human and many animals. Microscopic examination is the main method of clinical diagnosis for Blastocystis spp., which is prone to false negative. A simple and rapid diagnosis of Blastocystis spp. infection is an important step to prevent and control blastocystosis. Here, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay was developed for rapid visual detection of Blastocystis spp. DNA amplification could be performed within 18 min at 37°C. The minimum DNA detection limit was 1 pg/µL, and there was no cross-reactivity with 12 other non-target pathogens, which was consistent with the sensitivity of conventional PCR (cPCR). Furthermore, 56 fecal samples from the Third Affiliated Hospital of Xinxiang Medical University were tested using RPA and cPCR methods respectively, and the results were completely consistent. The results show that RPA-LFD method has high accuracy and visual results, which provides a new choice for the differential diagnosis and rapid field detection of Blastocystis spp.

Plasmodium falciparum AMA1 and CSP antigen diversity in parasite isolates from southern Ghana.

Introduction: Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity. Methods: The study involved 300 subjects, whose P. falciparum infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA. Results: Of the 300 subjects, 171 (57%) had P. falciparum infection, with 165 of the 171 (96.5%) being positive for either or both of the msp2 allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the Pfama1 gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons. Discussion: These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the Pfama1 gene.

Diversity of Sarcocystis parasites in southeastern Baltic Sea catchment ecosystems.

Currently, research on apicomplexan Sarcocystis parasites is mainly carried out by analyzing animal carcasses. However, environmental studies would not only allow faster detection of possible sources of infection but also avoid the use of animals for investigations. Therefore, in the current study, we aimed to identify tested Sarcocystis species in sediment collected from water bodies located in the southeastern Baltic countries. A total of 99 sediment samples were collected during the summer from different types of water bodies in Estonia, Latvia, Lithuania, and Poland. Species-specific nested PCR targeting cox1 gene was used for the detection of selected Sarcocystis species (S. cruzi, S. bovifelis, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. miescheriana, and S. bertrami) infecting livestock. The results showed a statistically lower (p < 0.05) occurrence of Sarcocystis parasites in Estonia (50%) compared to three countries, where the detection rate of Sarcocystis spp. DNA was remarkably higher, ranging from 88 to 100%. Among Sarcocystis species tested, S. cruzi (83.8%) and S. arieticanis (55.6%) using cattle and sheep as their intermediate hosts were most commonly identified. The detection rates of some of the analyzed Sarcocystis species were significantly different in southeastern Baltic countries. It is discussed that the detection rates of certain Sarcocystis species depend not only on the number of animals per 1 km2 but also on various ecological factors and farming practices that differ in the amount of contact domestic animals have with predators and the potential for animals to become infected through natural water or food sources.

Molecular detection of Hepatozoon species (Apicomplexa: Hepatozoidae) infecting snakes in the Northeastern region of Argentina.

The genus Hepatozoon Miller (1908) contains a wide range of obligate parasitic organisms with complex life cycles involving vertebrates and hematophagous invertebrates. Despite over 300 species being described, only a small percentage has been characterized in snakes using morphological and molecular techniques. The prevalence of these parasites in snakes is significant, highlighting the need for molecular descriptions in such elusive hosts. Thus, the objective of this study was to determine molecularly the presence of Hepatozoon species in snakes from the Northeastern region of Argentina. Thirty-two specimens of eight snake species (Bothrops alternatus, Dryophylax hypoconia, Erythrolamprus jaegeri coralliventris, Erythrolamprus poecilogyrus, Erythrolamprus semiaureus, Philodryas olfersii latirostris, Pseudablabes (ex Philodryas) patagoniensis and Palusophis (ex Mastigodryas) bifossatus were collected and examined. PCR analysis of the 18S rRNA locus detected four samples (12% prevalence) positive for the presence of Hepatozoon DNA. Phylogenetic analysis positioned the 18S rRNA Hepatozoon sequences obtained in three different clades, one with Hepatozoon musa, another with sequences of Hepatozoon cuestensis, while the third was placed as a sister taxon to a clade including Hepatozoon cevapii and Hepatozoon massardi. This study presents the first documentation of Hepatozoon infecting snakes in Argentina, thereby expanding their distribution within southern South America. Additionally, B. alternatus and Pa. bifossatus are reported as new hosts of Hepatozoon.

Meteorological, demographic, and environmental risk factors for Leishmania infantum in wildlife.

Leishmania infantum is an important and neglected vector-borne zoonotic protozoa endemic in the Mediterranean basin. Several wild and domestic mammals can contribute to maintaining its circulation but their importance as effective reservoirs is still under discussion and varies depending on local ecological communities. By combining environmental, climatic, and individual information, this study assessed the presence of L. infantum DNA in a set of wild species from Northwestern Italy and the potential ecological factors related to the risk of infection. From 2020 to 2022, 304 free-ranging wild animals were analyzed for the detection of L. infantum DNA in the spleen and popliteal lymph node (when available). The prevalence obtained in wild boar (Sus scrofa) and roe deer (Capreolus capreolus) was higher than those previously reported (% ± confidence interval 95%; 42.9 ± 18.4% and 27 ± 6.6% in wild boar and roe deer, respectively), and this is the first report of this parasite infecting the coypu Myocastor coypus (60 ± 34.7%). L. infantum DNA was detected in all the seasons including those free of adult sandflies and seasonal differences were minimal, suggesting a long course of infection. The models revealed that animals from rainy areas with higher greenness during the summer, highly populated by humans and predominantly covered by water surfaces had a higher risk of L. infantum. This study contributes to confirming previous findings on the existence of a sylvatic cycle for L. infantum in certain regions of Italy, as well as on the potential epidemiological role of roe deer for this parasite given the elevated prevalence found.

Challenging diagnosis of Plasmodium ovale malaria in a Colombian traveler: the importance of including P. ovale wallikeri in molecular screening.

This study reports a challenging diagnosis of Plasmodium ovale malaria in a Colombian citizen returning from Cameroon. Initial microscopy screenings conducted at two private hospitals yielded conflicting results, with the first showing negative smears and the second diagnosing P. vivax. Subsequent microscopy examinations at two government laboratories identified P. ovale, although the routine species-specific PCR strategy was negative. PCR confirmation was finally obtained when P. ovale wallikeri primers were used. Although P. ovale is not frequently found in Colombia, there is a clear need to include both P. ovale curtisi and P. ovale wallikeri in the molecular diagnostic strategy. Such need stems primarily from their extended latency period, which affects travelers, the increasing number of African migrants, and the importance of accurately mapping the distribution of Plasmodium species in Colombia.

Adipose and skin distribution of African trypanosomes in natural animal infections.

BACKGROUND: Animal African trypanosomiasis, which is caused by different species of African trypanosomes, is a deadly disease in livestock. Although African trypanosomes are often described as blood-borne parasites, there have been recent reappraisals of the ability of these parasites to reside in a wide range of tissues. However, the majority of those studies were conducted on non-natural hosts infected with only one species of trypanosome, and it is unclear whether a similar phenomenon occurs during natural animal infections, where multiple species of these parasites may be present. METHODS: The infective trypanosome species in the blood and other tissues (adipose and skin) of a natural host (cows, goats and sheep) were determined using a polymerase chain reaction-based diagnostic. RESULTS: The animals were found to harbour multiple species of trypanosomes. Different patterns of distribution were observed within the host tissues; for instance, in some animals, the blood was positive for the DNA of one species of trypanosome and the skin and adipose were positive for the DNA of another species. Moreover, the rate of detection of trypanosome DNA was highest for skin adipose and lowest for the blood. CONCLUSIONS: The findings reported here emphasise the complexity of trypanosome infections in a natural setting, and may indicate different tissue tropisms between the different parasite species. The results also highlight the need to include adipose and skin tissues in future diagnostic and treatment strategies.

Morphological and molecular characteristics of a Trypanosoma sp. from triatomines (Triatoma rubrofasciata) in China.

BACKGROUND: Triatomines (kissing bugs) are natural vectors of trypanosomes, which are single-celled parasitic protozoans, such as Trypanosoma cruzi, T. conorhini and T. rangeli. The understanding of the transmission cycle of T. conorhini and Triatoma rubrofasciata in China is not fully known. METHODS: The parasites in the faeces and intestinal contents of the Tr. rubrofasciata were collected, and morphology indices were measured under a microscope to determine the species. DNA was extracted from the samples, and fragments of 18S rRNA, heat shock protein 70 (HSP70) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) were amplified and sequenced. The obtained sequences were then identified using the BLAST search engine, followed by several phylogenetic analyses. Finally, laboratory infections were conducted to test whether Tr. rubrofasciata transmit the parasite to rats (or mice) through bites. Moreover, 135 Tr. rubrofasciata samples were collected from the Guangxi region and were used in assays to investigate the prevalence of trypanosome infection. RESULTS: Trypanosoma sp. were found in the faeces and intestinal contents of Tr. rubrofasciata, which were collected in the Guangxi region of southern China and mostly exhibited characteristics typical of epimastigotes, such as the presence of a nucleus, a free flagellum and a kinetoplast. The body length ranged from 6.3 to 33.9 µm, the flagellum length ranged from 8.7 to 29.8 µm, the nucleus index was 0.6 and the kinetoplast length was -4.6. BLAST analysis revealed that the 18S rRNA, HSP70 and gGAPDH sequences of Trypanosoma sp. exhibited the highest degree of similarity with those of T. conorhini (99.7%, 99.0% and 99.0%, respectively) and formed a well-supported clade close to T. conorhini and T. vespertilionis but were distinct from those of T. rangeli and T. cruzi. Laboratory experiments revealed that both rats and mice developed low parasitaemia after inoculation with Trypanosoma sp. and laboratory-fed Tr. rubrofasciata became infected after feeding on trypanosome-positive rats and mice. However, the infected Tr. rubrofasciata did not transmit Trypanosoma sp. to their offspring. Moreover, our investigation revealed a high prevalence of Trypanosoma sp. infection in Tr. rubrofasciata, with up to 36.3% of specimens tested in the field being infected. CONCLUSIONS: Our study is the first to provide a solid record of T. conorhini from Tr. rubrofasciata in China with morphological and molecular evidence. This Chinese T. conorhini is unlikely to have spread through transovarial transmission in Tr. rubrofasciata, but instead, it is more likely that the parasite is transmitted between Tr. rubrofasciata and mice (or rats). However, there was a high prevalence of T. conorhini in the Tr. rubrofasciata from our collection sites and numerous human cases of Tr. rubrofasciata bites were recorded. Moreover, whether these T. conorhini strains are pathogenic to humans has not been investigated.

First report on prevalence, molecular characterization and phylogenetic study of Toxoplasma gondii infecting sheep of the Malakand Division of Pakistan.

BACKGROUND: Toxoplasma gondii is an apicomplexan protozoan parasite that infects one-third of the population of the world, including humans, animals, birds, and other vertebrates. The present investigation is the first molecular attempt in the Malakand Division of Pakistan to determine the epidemiology and phylogenetic study of Toxoplasma gondii infecting small ruminants. METHODOLOGY: A total of (N = 450) blood samples of sheep were randomly collected during the study period (December 2020 to November 2021), and DNA detection was done using PCR by amplifying ITS-1 genes. SPSS.20 and MEGA-11 software were used for statistical significance and phylogenetic analysis. RESULTS: The overall prevalence of T. gondii infection among sheep was 14.44 % (65/450). A high infection rate was found in more than five-year-olds at 18.33 % (11/60). Sequencing and BLAST analysis of PCR-positive samples confirmed the presence of T. gondii. Randomly, three isolates were sequenced and submitted to GenBank under accession numbers (PP028089-PP028091), respectively. The BLAST analysis of the obtained sequences based on the ITS-1 gene showed 99 % similarities with reported genotypes found in goats of Malakand, Pakistan (PP028089) and dogs of Brazil (MF766454). The study concludes that T. gondii is notably prevalent among the sheep population in the region, emphasizing the significant role of risk factors in disease transmission across animals and potentially to humans. Further research, zoonotic potential analysis, and targeted control measures are warranted to address and manage this parasitic infection effectively.

Enteric parasites Cyclospora cayetanensis and Cryptosporidium hominis in domestic and wildlife animals in Ghana.

BACKGROUND: Enteric parasitic infections remain a major public health problem globally. Cryptosporidium spp., Cyclospora spp. and Giardia spp. are parasites that cause diarrhea in the general populations of both developed and developing countries. Information from molecular genetic studies on the speciation of these parasites and on the role of animals as vectors in disease transmission is lacking in Ghana. This study therefore investigated these diarrhea-causing parasites in humans, domestic rats and wildlife animals in Ghana using molecular tools. METHODS: Fecal samples were collected from asymptomatic school children aged 9-12 years living around the Shai Hills Resource Reserve (tourist site), from wildlife (zebras, kobs, baboons, ostriches, bush rats and bush bucks) at the same site, from warthogs at the Mole National Park (tourist site) and from rats at the Madina Market (a popular vegetable market in Accra, Ghana. The 18S rRNA gene (18S rRNA) and 60-kDa glycoprotein gene (gp60) for Cryptosporidium spp., the glutamate dehydrogenase gene (gdh) for Giardia spp. and the 18S rDNA for Cyclospora spp. were analyzed in all samples by PCR and Sanger sequencing as markers of speciation and genetic diversity. RESULTS: The parasite species identified in the fecal samples collected from humans and animals included the Cryptosporidium species C. hominis, C. muris, C. parvum, C. tyzzeri, C. meleagridis and C. andersoni; the Cyclopora species C. cayetanensis; and the Gardia species, G. lamblia and G. muris. For Cryptosporidium, the presence of the gp60 gene confirmed the finding of C. parvum (41%, 35/85 samples) and C. hominis (29%, 27/85 samples) in animal samples. Cyclospora cayetanensis was found in animal samples for the first time in Ghana. Only one human sample (5%, 1/20) but the majority of animal samples (58%, 51/88) had all three parasite species in the samples tested. CONCLUSIONS: Based on these results of fecal sample testing for parasites, we conclude that animals and human share species of the three genera (Cryptosporidium, Cyclospora, Giardia), with the parasitic species mostly found in animals also found in human samples, and vice-versa. The presence of enteric parasites as mixed infections in asymptomatic humans and animal species indicates that they are reservoirs of infections. This is the first study to report the presence of C. cayetanensis and C. hominis in animals from Ghana. Our findings highlight the need for a detailed description of these parasites using high-throughput genetic tools to further understand these parasites and the neglected tropical diseases they cause in Ghana where such information is scanty.

Unveiling genotypic diversity of Theileria orientalis in lethal outbreaks among bovines in Karnataka, India.

Theileria orientalis, the causal agent of oriental theileriosis, is known to cause mild disease in cattle and buffalo across the world. Recently, different genotypes of T. orientalis have emerged as pathogenic, causing high reported morbidity in cattle. This study focuses on investigating three suspected outbreaks of oriental theileriosis that resulted in fatalities among crossbred and indigenous bulls in Karnataka, India. Examination of blood smears revealed the presence of T. orientalis piroplasms within erythrocytes. The genetic characterization of T. orientalis was conducted by targeting specific markers, including the mpsp gene, p23 gene, and ribosomal DNA markers (18S rRNA gene, ITS-1, and ITS-2). Analysis based on the 18S rRNA gene unveiled the presence of both Type A and Type E genotypes of T. orientalis in the outbreaks. The mpsp gene-based analysis identified genotype 7 of T. orientalis in crossbred cows, whereas genotype 1 (Chitose B) was found to be present in indigenous bulls. Haplotype network analysis based on the mpsp gene revealed the presence of 39 distinct haplotypes within the 12 defined genotypes of T. orientalis with a high haplotype diversity of 0.9545 ± 0.017. Hematological and biochemical analysis revealed a decrease in calcium, hemoglobin levels, red blood cell counts, and phosphorus. This study constitutes the initial documentation of a clinical outbreak of oriental theileriosis in indigenous bulls with genotype 1 (Chitose 1B). Substantial epidemiological investigations are imperative to gain a comprehensive understanding of the geographical distribution of distinct genotypes and the diverse clinical manifestations of the disease across various hosts.

Morphological and molecular characterization of Eimeria haematopusi n. sp. (Apicomplexa: Eimeriidae) in an Australian Pied Oystercatcher (Haematopus longirostris) (Aves: Charadriiformes).

A novel Eimeria Schneider, 1875 species is described from an Australian pied oystercatcher Haematopus longirostris Vieillot, in Western Australia. The pied oystercatcher was admitted to the Kanyana Wildlife Rehabilitation Centre (KWRC), Perth, Western Australia in a poor body condition, abrasion to its right hock and signs of partial delamination to its lower beak. Investigation into potential medical causes resulted in a faecal sample being collected and screened for gastrointestinal parasites. Unsporulated coccidian oocysts were initially observed in the faeces and identified as Eimeria upon sporulation. The sporulated oocysts (n = 20) are ellipsoidal, 20-21 × 12-13 µm in shape and have thick bi-layered walls which are c.2/3 of the total thickness. Micropyle is present, robust and protruding, and occasionally has a rounded polar body attached to the micropyle. Within the oocyst, a residuum, in addition, two to five polar granules are present. There are four ellipsoidal sporocysts 9-11 × 5-6 µm with flattened to half-moon shaped Stieda bodies. Sub-Stieda body and para-Stieda body are absent. The sporocysts contain sporocyst residuums composed of a few spherules scattered among the sporozoites. Within the sporozoites, anterior and posterior refractile bodies are present, but the nucleus is indiscernible. To further characterise the novel Eimeria species from H. longirostris, molecular analysis was conducted at the 18S ribosomal RNA locus, using PCR amplification and cloning. Two cloned sequences from the novel Eimeria were compared with those from other Eimeria spp. with the highest genetic similarity of 97.6% and 97.2% from Clone 1 and 2, respectively with Eimeria reichenowi (AB544308) from a hooded crane (Grus monacha Temminck) in Japan. Both sequences grouped in a clade with the Eimeria spp. isolated from wetland birds, which include Eimeria paludosa (KJ767187) from a dusky moorhen (Gallinula tenebrosa Gould) in Western Australia, Eimeria reichenowi (AB544308) and Eimeria gruis (AB544336) both from hooded cranes. Based on the morphological and molecular data, this Eimeria sp. is a new species of coccidian parasite and is named Eimeria haematopusi n. sp. after its host H. longirostris.

Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites.

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.

Susceptibility of Leishmania to novel pentavalent organometallics: Investigating impact on DNA and membrane integrity in antimony(III)-sensitive and -resistant strains.

The aim the present study was to investigate the impact of novel pentavalent organobismuth and organoantimony complexes on membrane integrity and their interaction with DNA, activity against Sb(III)-sensitive and -resistant Leishmania strains and toxicity in mammalian peritoneal macrophages. Ph3M(L)2 type complexes were synthesized, where M = Sb(V) or Bi(V) and L = deprotonated 3-(dimethylamino)benzoic acid or 2-acetylbenzoic acid. Both organobismuth(V) and organoantimony(V) complexes exhibited efficacy at micromolar concentrations against Leishmania amazonensis and L. infantum but only the later ones demonstrated biocompatibility. Ph3Sb(L1)2 and Ph3Bi(L1)2 demonstrated distinct susceptibility profiles compared to inorganic Sb(III)-resistant strains of MRPA-overexpressing L. amazonensis and AQP1-mutated L. guyanensis. These complexes were able to permeate the cell membrane and interact with the Leishmania DNA, suggesting that this effect may contribute to the parasite growth inhibition via apoptosis. Taken altogether, our data substantiate the notion of a distinct mechanism of uptake pathway and action in Leishmania for these organometallic complexes, distinguishing them from the conventional inorganic antimonial drugs.

Simplified molecular diagnosis of visceral leishmaniasis: Laboratory evaluation of miniature direct-on-blood PCR nucleic acid lateral flow immunoassay.

BACKGROUND: Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. METHODOLOGY/PRINCIPLE FINDINGS: The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. CONCLUSIONS/SIGNIFICANCE: Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.

Hepatozoon spp. in stray cats from the metropolitan area of Rio de Janeiro, Brazil.

In the last few years, the number of studies on feline hepatozoonosis has increased, but our knowledge on the actual species of Hepatozoon and/or different genotypes affecting felines is still incipient. At least three species, namely Hepatozoon felis, H. canis, and H. silvestris, have been isolated from domestic cats in various countries. Additionally, there are indications that other species and genotypes may affect felines in given geographic areas. This study was carried out to investigate the occurrence of Hepatozoon spp. in cats from Niterói, a municipality within the metropolitan area of Rio de Janeiro, Brazil. Individual blood samples were collected from 28 cats enrolled in a spaying/castration program. DNA was extracted from all samples and subjected to sequencing specific for Hepatozoon spp. DNA of H. felis was found in 21/28 cats (75%), and four genetic polymorphisms never described thus far were detected. This is the first report of H. felis in cats living in the State of Rio de Janeiro, and the present data confirm that H. felis is a species complex encompassing different genotypes circulating within cat populations. Further studies are warranted to investigate whether different genotypes have different biology or pathogenicity for felids.

Title: Hepatozoon spp. chez les chats errants de la zone métropolitaine de Rio de Janeiro, Brésil. Abstract: Au cours des dernières années, le nombre d'études sur l'hépatozoonose féline a augmenté, mais nos connaissances sur les espèces d'Hepatozoon et/ou différents génotypes affectant les félins sont encore naissantes. Au moins trois espèces, à savoir Hepatozoon felis, H. canis et H. silvestris, ont été isolées chez des chats domestiques dans divers pays. De plus, il semble que d'autres espèces et génotypes puissent affecter les félins dans des zones géographiques données. Cette étude a été réalisée pour étudier la présence d'Hepatozoon spp. chez des chats de Niterói, une municipalité de la zone métropolitaine de Rio de Janeiro, au Brésil. Des échantillons de sang ont été prélevés individuellement sur 28 chats d'un programme de castration. L'ADN a été extrait de tous les échantillons et soumis à un séquençage spécifique de Hepatozoon spp. L'ADN de H. felis a été trouvé chez 21 chats sur 28 (75%) et quatre polymorphismes génétiques, jamais décrits jusqu'à présent, ont été détectés. Il s'agit du premier signalement de H. felis chez des chats vivant dans l'État de Rio de Janeiro et les données actuelles confirment que H. felis est un complexe d'espèces englobant différents génotypes circulant au sein des populations de chats. Des études supplémentaires sont nécessaires pour déterminer si les différents génotypes ont une biologie ou une pathogénicité différente pour les félidés.

Atypical acquired toxoplasmosis.

A 33-year-old male presented with unilateral painless vision loss with a history of sub-tenon steroid for the same. The fundus showed an elevated focus of retinochoroiditis with vitritis. On investigating for the cause, polymerase chain reaction test on the anterior chamber tap was found to be positive for Toxoplasma. Such confusing and atypical cases usually produce a clinical dilemma and should be managed in a stepwise manner. Ancillary investigations usually provide a clue to the clinician and should be performed without any hesitation.

The Development of a One-Step RT-qPCR for the Detection and Quantification of Viable Forms of Trypanosoma cruzi in Açai Samples from Areas at Risk of Chagas Disease through Oral Transmission.

Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of T. cruzi DNA in açaí, the evaluation of parasites' viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various T. cruzi DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for T. cruzi DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable T. cruzi. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of T. cruzi in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption.