Cryptosporidium spp. in captive snakes from 26 provinces in China: Prevalence, molecular characterization, and symptoms.

Snakes are sometimes regarded as pets and are used in traditional Chinese medicine. Cryptosporidium spp. are frequently identified in snakes, representing an important pathogen and causing gastrointestinal diseases. Current data indicate that risk factors for infection and patterns of clinical symptom presentation may differ among Cryptosporidium spp. To better understand the infection status by Cryptosporidium spp., fecal samples were collected from 603 asymptomatic and 147 symptomatic snakes in 26 provinces of China. These samples came from Elaphe guttata, Elaphe obsoleta, Pituophis melanoleucus, Thamnophis sirtalis, Lampropeltis getulus, and Heterodon nasicus. The partial small subunit (SSU) rRNA gene was amplified using nested polymerase chain reaction (PCR) to investigate the infection rate of Cryptosporidium spp., and to assess evolutionary relationships and genetic characterization. A prevalence of 20% was recorded in asymptomatic snakes, with age identified as a significant risk factor. In contrast, 70% of symptomatic snakes were positive for Cryptosporidium spp., with Cryptosporidium serpentis and Cryptosporidium varanii (syn. C. saurophilum). Further analysis revealed a potential association between C. serpentis and regurgitation, and C. varanii and diarrhea, while neither species was linked to flatulence. To our knowledge, this is the first study to report Cryptosporidium spp. and associated clinical signs in symptomatic snakes in China. This study aims to enhance the understanding of Cryptosporidium infections, risk factors, and clinical manifestations in snakes, providing data crucial for the control and prevention of cryptosporidiosis.

Title: Cryptosporidium spp. chez les serpents captifs de 26 provinces de Chine : prévalence, caractérisation moléculaire et symptômes. Abstract: Les serpents sont parfois considérés comme animaux de compagnie et sont utilisés en médecine traditionnelle chinoise. Des Cryptosporidium spp. sont fréquemment identifiés chez les serpents, ont un rôle d'agent pathogène important et provoquent des maladies gastro-intestinales. Les données actuelles indiquent que les facteurs de risque d'infection et les schémas de présentation des symptômes cliniques peuvent varier en fonction des espèces de Cryptosporidium. Pour mieux comprendre l'état d'infection par Cryptosporidium spp., des échantillons fécaux ont été collectés auprès de 603 serpents asymptomatiques et 147 serpents symptomatiques dans 26 provinces de Chine. Ces échantillons provenaient d'Elaphe guttata, Elaphe obsoleta, Pituophis melanoleucus, Thamnophis sirtalis, Lampropeltis getulus et Heterodon nasicus. Le gène de l'ARNr de la petite sous-unité partielle (SSU) a été amplifié à l'aide d'une réaction en chaîne par polymérase (PCR) imbriquée pour étudier le taux d'infection par Cryptosporidium spp. et évaluer les relations évolutives et la caractérisation génétique. Une prévalence de 20 % a été trouvée chez les serpents asymptomatiques, l'âge étant identifié comme un facteur de risque important. En revanche, 70 % des serpents symptomatiques étaient positifs à Cryptosporidium spp. avec Cryptosporidium serpentis et Cryptosporidium varanii (syn. C. saurophilum). Une analyse plus approfondie a révélé une association potentielle entre C. serpentis et la régurgitation, et C. varanii et la diarrhée, alors qu'aucune des deux espèces n'était liée aux flatulences. À notre connaissance, il s'agit ici de la première étude à signaler la présence de Cryptosporidium spp. et les signes cliniques associés chez des serpents symptomatiques en Chine. Cette étude vise à améliorer la compréhension des infections à Cryptosporidium, des facteurs de risque et des manifestations cliniques chez les serpents, en fournissant des données cruciales pour le contrôle et la prévention de la cryptosporidiose.

Tick-borne Apicomplexa in wildlife and ticks of French Guiana.

Tick-borne Apicomplexa encompass a group of parasites responsible for significant medical and veterinary diseases, including babesiosis, theileriosis, and hepatozoonosis. In this study, we investigated the presence and diversity of tick-borne Apicomplexa in wildlife and ticks inhabiting the Amazon rainforests of French Guiana. To this end, we conducted molecular screening and typing using 18S rRNA sequences on a collection of 1161 specimens belonging to 71 species, including 44 species of wild mammals, five species of passerines, and 22 species of ticks. We characterized eight genovariants of Babesia, Theileria, Hemolivia, and Hepatozoon parasites, some matching known species, while others suggested potential novel species. These parasites were detected in wild mammals, including opossums, sloths, armadillos, porcupines, margays, greater grisons, and ticks, but not in passerines. Finally, similarities with surveys conducted in Brazil highlight the specific sylvatic transmission cycles of South American tick-borne Apicomplexa.

Title: Apicomplexes transmis par les tiques chez la faune sauvage et les tiques de Guyane française. Abstract: Les Apicomplexes transmis par les tiques englobent un groupe de parasites responsables de maladies médicales et vétérinaires importantes, notamment la babésiose, la theilériose et l'hépatozoonose. Dans cette étude, nous avons étudié la présence et la diversité des Apicomplexes transmis par les tiques dans la faune sauvage et les tiques habitant les forêts tropicales amazoniennes de Guyane française. À cette fin, nous avons effectué un criblage moléculaire et un typage à l'aide de séquences d'ARNr 18S sur une collection de 1 161 spécimens appartenant à 71 espèces, dont 44 espèces de mammifères sauvages, cinq espèces de passereaux et 22 espèces de tiques. Nous avons caractérisé huit génovariants des parasites Babesia, Theileria, Hemolivia et Hepatozoon, certains correspondant à des espèces connues tandis que d'autres suggéraient de nouvelles espèces potentielles. Ces parasites ont été détectés chez des mammifères sauvages, dont des opossums, des paresseux, des tatous, des porcs-épics, des margays, des grisons et des tiques, mais pas chez des passereaux. Enfin, des similitudes avec des enquêtes menées au Brésil mettent en évidence les cycles de transmission sylvatiques spécifiques des Apicomplexa transmis par les tiques d'Amérique du Sud.

No evidence of pathogenicity of Dientamoeba fragilis following detection in stools: A case-control study.

Dientamoeba fragilis is a ubiquitous intestinal parasite with detection in the stools that has become increasingly frequent following the advent of PCR as a routine screening tool. However, the pathogenicity of this parasite is still much debated. In order to assess the potentially pathogenic nature of this protozoan, a retrospective case-control study was carried out between January and December 2020 on patients from Toulouse University Hospital, with the aim of evaluating the potential clinical effects and changes in laboratory parameters linked to the presence and load of D. fragilis in stools. After matching age, sex and mode of care (consultation or hospitalisation), no significant difference was observed in the frequency of clinical signs between the 36 patients who tested positive for Dientamoeba fragilis PCR in their stools and the 72 control patients who were PCR negative for this protozoan. The presence of D. fragilis in the faeces was not associated with changes in laboratory parameters. Furthermore, a high digestive load of D. fragilis had no identifiable impact on clinical and laboratory parameters. Only the concomitant presence of Blastocystis sp. in stools was significantly more frequent in the D. fragilis group (uni- and multivariate analysis). Finally, this study showed no significant difference in clinical or laboratory signs between patients carrying Dientamoeba fragilis and the control group, regardless of the intestinal parasite load, suggesting that D. fragilis could be considered a commensal of the digestive tract.

Title: Aucune preuve de la pathogénicité de Dientamoeba fragilis détecté dans les selles : une étude cas-témoins. Abstract: Dientamoeba fragilis est un parasite digestif ubiquitaire dont la détection dans les selles est devenue de plus en plus fréquente avec l'avènement de la PCR comme outil de détection de routine. Cependant, la pathogénicité de ce parasite est encore très discutée. Afin d'évaluer le caractère potentiellement pathogène de ce protozoaire, une étude rétrospective cas-témoins a été réalisée entre janvier et décembre 2020 sur des patients du CHU de Toulouse, dans le but d'évaluer les effets cliniques et biologiques potentiels associés à la présence et à la charge de D. fragilis dans les selles. Après appariement sur l'âge, le sexe et le mode de prise en charge (consultation ou hospitalisation), aucune différence significative n'a été observée dans la fréquence des signes cliniques entre les 36 patients testés positifs pour la PCR de Dientamoeba fragilis dans les selles et les 72 patients témoins avec une PCR négative pour ce protozoaire. La présence de D. fragilis dans les selles n'était pas associée à des modifications des paramètres biologiques. De plus, une charge digestive élevée de D. fragilis n'avait pas d'impact identifiable sur les paramètres cliniques et biologiques. Seule la présence concomitante de Blastocystis sp. dans les selles était significativement plus fréquente dans le groupe D. fragilis (analyse uni- et multivariée). En conclusion, cette étude n'a pas montré de différence significative concernant les signes cliniques ou biologiques entre les patients porteurs de Dientamoeba fragilis et le groupe témoin, quelle que soit la charge parasitaire digestive, indiquant que D. fragilis pourrait être considéré comme un commensal du tube digestif.

Cross-species transmission of Cryptosporidium in wild rodents from the southern region of Zhejiang Province of China and its possible impact on public health.

Wild rodents serve as reservoirs for Cryptosporidium and are overpopulated globally. However, genetic data regarding Cryptosporidium in these animals from China are limited. Here, we have determined the prevalence and genetic characteristics of Cryptosporidium among 370 wild rodents captured from three distinct locations in the southern region of Zhejiang Province, China. Fresh feces were collected from the rectum of each rodent, and DNA was extracted from them. The rodent species was identified by PCR amplifying the vertebrate cytochrome b gene. Cryptosporidium was detected by PCR amplification and amplicon sequencing the small subunit of ribosomal RNA gene. Positive samples of C. viatorum and C. parvum were further subtyped by analyzing the 60-kDa glycoprotein gene. A positive Cryptosporidium result was found in 7% (26/370) of samples, involving five rodent species: Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155), and R. tanezumi (86). Their respective Cryptosporidium positive rates were 8.3%, 5.3%, 11.1%, 7.1%, and 7.0%. Sequence analysis confirmed the presence of three Cryptosporidium species: C. parvum (4), C. viatorum (1), and C. muris (1), and two genotypes: Cryptosporidium rat genotype IV (16) and C. mortiferum-like (4). Additionally, two subtypes of C. parvum (IIdA15G1 and IIpA19) and one subtype of C. viatorum (XVdA3) were detected. These results demonstrate that various wild rodent species in Zhejiang were concurrently infected with rodent-adapted and zoonotic species/genotypes of Cryptosporidium, indicating that these rodents can play a role in maintaining and dispersing this parasite into the environment and other hosts, including humans.

Title: Transmission interspécifique de Cryptosporidium chez les rongeurs sauvages de la région sud de la province chinoise du Zhejiang et son impact possible sur la santé publique. Abstract: Les rongeurs sauvages servent de réservoirs à Cryptosporidium et ont des grandes populations à l'échelle mondiale. Cependant, les données génétiques concernant Cryptosporidium chez ces animaux en Chine sont limitées. Ici, nous avons déterminé la prévalence et les caractéristiques génétiques de Cryptosporidium parmi 370 rongeurs sauvages capturés dans trois endroits distincts de la région sud de la province du Zhejiang, en Chine. Des excréments frais ont été collectés dans le rectum de chaque rongeur et l'ADN en a été extrait. L'espèce de rongeur a été identifiée par amplification par PCR du gène du cytochrome b des vertébrés. Cryptosporidium a été détecté par amplification PCR et séquençage d'amplicons de la petite sous-unité du gène de l'ARN ribosomal. Les échantillons positifs de C. viatorum et C. parvum ont ensuite été sous-typés en analysant le gène de la glycoprotéine de 60 kDa. Un résultat positif pour Cryptosporidium a été trouvé dans 7 % (26/370) des échantillons, impliquant cinq espèces de rongeurs : Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155) et R. tanezumi (86). Leurs taux respectifs de positivité pour Cryptosporidium étaient de 8,3 %, 5,3 %, 11,1 %, 7,1 % et 7,0 %. L'analyse des séquences a confirmé la présence de trois espèces de Cryptosporidium : C. parvum (4), C. viatorum (1) et C. muris (1), et de deux génotypes : Cryptosporidium génotype IV de rat (16) et C. mortiferum-like (4). De plus, deux sous-types de C. parvum (IIdA15G1 et IIpA19) et un sous-type de C. viatorum (XVdA3) ont été détectés. Ces résultats démontrent que diverses espèces de rongeurs sauvages du Zhejiang sont simultanément infectées par des espèces/génotypes de Cryptosporidium zoonotiques et adaptés aux rongeurs, ce qui indique que ces rongeurs peuvent jouer un rôle dans le maintien et la dispersion de ce parasite dans l'environnement et d'autres hôtes, y compris les humains.

Phylogenetic inferences based on distinct molecular markers reveals a novel Babesia (Babesia pantanalensis nov. sp.) and a Hepatozoon americanum-related genotype in crab-eating foxes (Cerdocyon thous).

Piroplasmids and Hepatozoon spp. Are apicomplexan protozoa that may cause disease in several canid species. The present study aimed to expand the knowledge on the diversity of piroplasmids and Hepatozoon in crab-eating foxes (Cerdocyon thous; n = 12) sampled in the Pantanal of Mato Grosso do Sul State, central-western Brazil. PCR assays based on the 18S rRNA were used as screening. Three (25%) and 11 (91.7%) were positive for piroplasmids and Hepatozoon spp., respectively. Co-infection was found in three C. thous. Phylogenetic analyses based on the near-complete 18S rRNA, cox-1 and hsp70 genes evidenced the occurrence of a novel of Babesia spp. (namely Babesia pantanalensis nov. sp.) closely related to Rangelia vitalii and Babesia sp. 'Coco'. This finding was supported by the genetic divergence analysis which showed (i) high divergence, ranging from 4.17 to 5.62% for 18 S rRNA, 6.16% for hps70 and 4.91-9.25% for cox-1 and (ii) the genotype network (which displayed sequences separated from the previously described Piroplasmida species by median vectors and several mutational events). Also, phylogenetic analysis based on the 18S rRNA gene of Hepatozoon spp. positioned the sequences obtained herein in a clade phylogenetically related to Hepatozoon sp. 'Curupira 2', Hepatozoon sp. detected in domestic and wild canids from Uruguay and Hepatozoon americanum. The present study described Babesia pantanalensis nov sp. and Hepatozoon closely related to H. americanum in crab-eating foxes from Brazil. Moreover, the coinfection by piroplasmids and Hepatozoon sp. for the first time in crab-eating foxes strongly suggesting that this wild canid species potentially acts as a bio-accumulate of hemoprotozoan in wild environment.

Metabarcoding of protozoa and helminth in black-necked cranes: a high prevalence of parasites and free-living amoebae.

Parasites and free-living amoebae (FLA) are common pathogens that pose threats to wildlife and humans. The black-necked crane (Grus nigricollis) is a near-threatened species and there is a shortage of research on its parasite diversity. Our study aimed to use noninvasive methods to detect intestinal parasites and pathogenic FLA in G. nigricollis using high-throughput sequencing (HTS) based on the 18S rDNA V9 region. A total of 38 fresh fecal samples were collected in Dashanbao, China, during the overwintering period (early-, middle I-, middle II-, and late-winter). Based on the 18S data, eight genera of parasites were identified, including three protozoan parasites: Eimeria sp. (92.1%) was the dominant parasite, followed by Tetratrichomonas sp. (36.8%) and Theileria sp. (2.6%). Five genera of helminths were found: Echinostoma sp. (100%), Posthodiplostomum sp. (50.0%), Euryhelmis sp. (26.3%), Eucoleus sp. (50.0%), and Halomonhystera sp. (2.6%). Additionally, eight genera of FLA were detected, including the known pathogens Acanthamoeba spp. (n = 13) and Allovahlkampfia spp. (n = 3). Specific PCRs were used to further identify the species of some parasites and FLA. Furthermore, the 18S data indicated significant changes in the relative abundance and genus diversity of the protozoan parasites and FLA among the four periods. These results underscore the importance of long-term monitoring of pathogens in black-necked cranes to protect this near-endangered species.

Title: Métabarcoding des protozoaires et des helminthes chez les grues à cou noir : forte prévalence de parasites et d'amibes libres. Abstract: Les parasites et les amibes libres sont des agents pathogènes courants qui constituent une menace pour la faune et les humains. La grue à cou noir (Grus nigricollis) est une espèce quasi menacée et les recherches sur sa diversité parasitaire sont insuffisantes. Notre étude visait à utiliser des méthodes non invasives pour détecter les parasites intestinaux et les amibes libres pathogènes chez G. nigricollis en utilisant le séquençage à haut débit basé sur la région V9 de l'ADNr 18S. Au total, 38 échantillons de matières fécales fraîches ont été collectés à Dashanbao, en Chine, au cours de la période d'hivernage (début, milieu I, milieu II et fin de l'hiver). Sur la base des données 18S, huit genres de parasites ont été identifiés, dont trois parasites protozoaires : Eimeria sp. (92,1 %) était le parasite dominant, suivi de Tetratrichomonas sp. (36,8 %) et Theileria sp. (2,6 %). Cinq genres d'helminthes ont été trouvés : Echinostoma sp. (100 %), Posthodiplostomum sp. (50,0 %), Euryhelmis sp. (26,3 %), Eucoleus sp. (50,0 %) et Halomonhystera sp. (2,6 %). De plus, huit genres d'amibes libres ont été détectés, y compris les agents pathogènes connus Acanthamoeba spp. (n = 13) et Allovahlkampfia spp. (n = 3). Des PCR spécifiques ont été utilisées pour identifier davantage les espèces de certains parasites et amibes libres. En outre, les données 18S ont indiqué des changements significatifs dans l'abondance relative et la diversité des genres des parasites protozoaires et des amibes au cours des quatre périodes. Ces résultats soulignent l'importance de la surveillance à long terme des agents pathogènes chez les grues à cou noir pour protéger cette espèce quasi menacée.

Molecular detection of Toxoplasma gondii and Neospora caninum in seabirds collected along the coast of Santa Catarina, Brazil.

Toxoplasma gondii and Neospora caninum are two closely related protozoans that infect a wide range of animals, including birds. However, the occurrence of N. caninum and T. gondii in seabirds is unknown. Therefore, this study aimed to determine the presence of T. gondii and N. caninum DNA in tissue samples of seabirds. Tissue samples of the pectoral muscles, heart, and brain were collected from 47 birds along the coastline of Santa Catarina State, SC, Brazil. The DNA was extracted from the tissues and screened using nested-PCR (nPCR) targeting internal transcribed spacer 1 (ITS1). T. gondii DNA was detected in tissues from seven seabirds (7/47, 14.8%), kelp gull (Larus dominicanus) (5/21), and Manx shearwater (Puffinus puffinus) (2/8). N. caninum DNA was detected in tissues of nine seabirds (9/47, 19.1%), the kelp gull (L. dominicanus) (4/21), Manx shearwater (P. puffinus) (2/8), neotropic cormorant (Phalacrocorax brasilianus) (1/4), brown booby (Sula leucogaster) (1/5), and white-chinned petrel (Procellaria aequinoctialis) (1/1); however, no co-infection was observed. In conclusion, this study showed the circulation of N. caninum and T. gondii in seabirds along the coastline of Santa Catarina State. Further studies are required to clarify the role of these birds in the epidemiology of neosporosis and toxoplasmosis.

Blood Meal Analysis and Molecular Detection of Leishmania DNA in Wild-Caught Sand Flies in Leishmaniasis Endemic Areas of Turkey and Northern Cyprus.

INTRODUCTION: Phlebotomine sand flies (Diptera: Psychodidae) are known as the vector of diseases such as leishmaniasis, bartonellosis and viral diseases. The aim of this study is to detect the host feeding pattern of sand flies in the endemic areas for leishmaniasis in Turkey (Antalya, Kayseri) and Northern Cyprus (TRNC) as well as the presence of Leishmania DNA in the specimens. METHODS: One-hundred seventy-six blood-fed sand fly specimens were examined for blood meal analysis. A SYBR Green-PCR assay was performed with specific forward primers for each host and a universal reverse primer. Primers of human and goat were used together in multiplex PCR while goat and cow were studied separately. ITS-1 qPCR assay was also performed on both blood-fed and non-blood-fed females to detect Leishmania parasites. RESULTS: Blood sources could be detected in 69 out of 176 blood-fed sand fly specimens. The results of blood meal analysis showed that specimens were fed mostly on cows (22.2%) followed by humans (5.7%), goats (2.8%) and dogs (0.6%). Multiple feeding patterns were also detected as human + cow (3.4%), cow + goat (2.8%) and human + goat (1.7%). Five of the blood-fed specimens were Leishmania spp. positive: P. major s.l. (n = 1), P. tobbi (n = 2) were L. tropica positive from Antalya, P. simici was positive for L. infantum from Kayseri and P. papatasi (n = 1) was positive for L. major from Cyprus. Leishmania infection rates were determined as 3.79%, 1.69% and 2.63% among the blood-fed sand fly specimens in Antalya, Kayseri and TRNC, respectively. CONCLUSION: The SYBR-Green-based multiplex PCR assay is a cost-effective and promising tool for blood meal identification of wild-caught sand flies as well as other blood-sucking arthropods. Feeding patterns of important vector species detected in the present study show the high risk in these endemic areas. As a next step, to identify the blood source in a shorter time and to make the test more sensitive, development of this assay to probe-based and multiplex PCR will be also planned.

Development and evaluation of a molecular based protocol for detection and quantification of Cryptosporidium spp. in wastewater.

Infections caused by protozoan parasites are a major public health concern globally. These infections are commonly diagnosed during water-borne outbreaks, necessitating accurate and highly sensitive detection procedures to assure public health protection. Current molecular techniques are challenged by several factors, such as low parasite concentration, inefficient DNA extraction methods, and inhibitors in environmental samples. This study focused on the development and validation of a molecular protocol for DNA extraction, efficient protozoan (oo)cyst recovery and quantification of protozoan parasites from wastewater using droplet digital polymerase chain reaction (ddPCR). Five DNA extraction methods, including commercial kits, custom phenol-chloroform, and in-house modified methods, were evaluated. The efficiency of each method was assessed via spectrophotometric analysis and ddPCR amplification using specific primers. Lastly, the developed protocol was evaluated for the detection and quantification of Cryptosporidium parvum in wastewater from different regions in South Africa. The conventional phenol-chloroform extraction method yielded the highest DNA concentration of 223 (±0.71) ng/µl and detected the highest number of Cryptosporidium parvum (1807 (±0.30) copies/ddPCR reaction) compared to other methods evaluated in this study. Additionally, the phenol-chloroform method demonstrated high sensitivity in extracting DNA from as few as one cyst/L of Cryptosporidium parvum, corresponding to 5.93 copies/ddPCR reaction. It was also observed that analysis of both the filtered supernatant and pellets after centrifugation improves the recovery efficiency of oocysts from wastewater by 10.5%, resulting in a total recovery of 64.1%. This optimized protocol was successfully applied to measure protozoan concentration in wastewater from different regions in South Africa. The improved DNA extraction and quantification method proposed in this study would be effective in monitoring protozoan concentration in the environment, which will help in instituting mitigation measures to reduce water-borne infections.

Occurrence and subtyping of Blastocystis in coypus (Myocastor coypus) in China.

BACKGROUND: Blastocystis is an anaerobic unicellular protist frequently detected in the gastrointestinal tracts of humans and animals worldwide. However, the prevalence and subtype distribution of Blastocystis in the coypu (Myocastor coypus) population have not been reported so far. The aim of this study was to determine the prevalence, genetic characteristics, and zoonotic potential of Blastocystis isolates detected in coypus in China. RESULTS: A total of 308 fecal samples were collected from coypus in seven regions across China and subsequently examined. Blastocystis was detected in 44 (14.3%) specimens by nested PCR amplification of the small subunit ribosomal rRNA (SSU rRNA) gene. Further DNA sequencing and phylogenetic analyses resulted in the identification of two zoonotic known subtypes, ST4 and ST5, and an unknown subtype. ST4 was the most predominant subtype observed in the samples. ST5 infections were only observed in three coypus. Factors that were associated with prevalence of Blastocystis included age, geographical region and subtype. Interestingly, this is the first report about a potentially novel subtype infecting coypus. CONCLUSIONS: This is the first comprehensive report of Blastocystis in M. coypus across a wide geographic range of China. A moderate degree of genetic divergence was observed. The presence of zoonotic subtypes in farmed M. coypus suggests that these animals have the potential to transmit blastocystosis to both humans and domestic animals. These findings provide a better understanding of the genetic diversity of Blastocystis in rodents and contribute towards the establishment of efficient blastocystosis control strategies in the investigated areas.

Comparative Evaluation of Re 529-Bp Sequence and B1 Gene in the Detection of Toxoplasma gondii Through PCR in Water Samples of Denizli, Turkey.

PURPOSE: While Toxoplasma gondii (T. gondii) infection is asymptomatic in immunocompetent individuals, it is a life-threatening protozoan in immunocompromised individuals. Its water-borne transmission to humans poses a serious public health concern. Polymerase Chain Reaction (PCR) has a considerable potential for the sensitive and specific detection of T. gondii oocysts in waters. METHODS: Comparative evaluation of RE 529-bp sequence and B1 gene to detect T. gondii tachyzoites and oocysts via PCR in agricultural irrigation water taken from downtown Denizli, Turkey and water samples collected from neighborhood fountains was performed for the first time in Turkish context. RESULTS: Based on real-time PCR targeting the B1 genetic markers and RE 529-bp sequence, T. gondii DNA was identified in 6 (16.7%) out of 48 samples collected from agricultural irrigation water. Besides, our PCR analysis did not establish any presence of T. gondii in drinking water samples. CONCLUSION: T. gondii showed lower sensitivity in B1-based PCR than in PCR targeting RE 529-bp sequence.

Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis).

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4-400 parasites/g, DNA-RR: 19-80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10-1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

Contribution to the knowledge on black flies (Diptera: Simuliidae) as vectors of Leucocytozoon (Haemosporida) parasites in Lithuania.

Black flies (Diptera: Simuliidae) are among the most bothersome blood-sucking dipterans causing severe irritation and distress to poultry, wild birds, animals, and humans globally. These insects are vectors of viruses, bacteria, parasitic protozoans, and nematodes of humans and animals. Parasitic protozoa belonging to Haemosporida (Apicomplexa) are distributed worldwide and black flies are the principal vectors of avian haemosporidian parasites of the genus Leucocytozoon, a common parasite of birds. Based on the detection of parasite DNA in insects, 13 black fly species were reported to be potential vectors of Leucocytozoon in Europe. Information about which species of Simulium can play a role in the transmission of Leucocytozoon parasites is insufficient and needs to be developed. The aim of our study was to determine which black fly species are involved in the transmission of Leucocytozoon parasites in the Eastern Europe. The black fly females were collected in Lithuania using entomological net. They were morphologically identified, dissected to prepare salivary glands preparations, and then screened for the presence of Leucocytozoon parasites using microscopy and PCR-based methods. In all, we collected 437 black fly females belonging to eight species. The DNA of Leucocytozoon (genetic lineage lCOCO18) was detected in one of analysed females identified as Simulium maculatum. All salivary gland preparations were negative for the presence of Leucocytozoon sporozoites. Our results included S. maculatum as a potential vector of Leucocytozoon parasites. Increasing the knowledge on vector ecology, behaviour and improving collection methods may be the key to understand the evolution and diversity of these parasites.

Progress in understanding the phylogeny of the Plasmodium vivax lineage.

There has been some controversy about the evolutionary origin of Plasmodium vivax, particularly whether it is of Asian or African origin. Recently, a new malaria species which closely related to ape P. vivax was found in chimpanzees, in addition, the host switches of P. vivax from ape to human was confirmed. These findings support the African origin of P. vivax. Previous phylogenetic analyses have shown the position of P. vivax within the Asian primate malaria parasite clade. This suggested an Asian origin of P. vivax. Recent analyses using massive gene data, however, positioned P. vivax after the branching of the African Old World monkey parasite P. gonderi, and before the branching of the common ancestor of Asian primate malaria parasites. This position is consistent with an African origin of P. vivax. We here review the history of phylogenetic analyses on P. vivax, validate previous analyses, and finally present a definitive analysis using currently available data that indicate a tree in which P. vivax is positioned at the base of the Asian primate malaria parasite clade, and thus that is consistent with an African origin of P. vivax.

Phlebotominae Fauna (Diptera: Psychodidae) and Molecular Detection of Leishmania (Kinetoplastida: Trypanosomatidae) in Urban Caves of Belo Horizonte, Minas Gerais, Brazil.

Sand flies are often collected in urban areas, which has several implications for the risk of transmission of Leishmania Ross, 1903, to humans and other mammals. Given this scenario, we describe the sand fly fauna of caves and their surroundings in Mangabeiras Municipal Park (MMP) and Paredão Serra do Curral Park (PSCP), both located in the urban area of Belo Horizonte, Minas Gerais, Brazil, an endemic focus of visceral and cutaneous leishmaniasis. Collections were conducted monthly from November 2011 to October 2012, using CDC light traps exposed for two consecutive nights in four caves and their surroundings. Nonsystematized collections using Shannon traps and active searches were also performed around the caves. The presence of Leishmania DNA in collected female sand flies was evaluated by ITS1-PCR. A total of 857 sand flies representing fourteen species were collected in MMP, of which Evandromyia edwardsi (Mangabeira, 1941) was the most abundant. Leishmania amazonensis was detected in Brumptomyia nitzulescui (Costa Lima, 1932) and Ev. edwardsi, with the latter also having Leishmania braziliensis, Leishmania infantum, and Leishmania sp. A total of 228 sand flies representing four species were collected in PSCP, of which Sciopemyia microps (Mangabeira, 1942) was the most abundant. No females from PSCP were positive for Leishmania-DNA. Studies aimed at describing sand fly faunas of cave environments and detecting Leishmania are essential to understanding the relationship between these insects and this ecotope and assessing and monitoring areas that may pose risks to the health of visitors and employees.

Safety and Tolerability of Mosquito Bite-Induced Controlled Human Infection with Plasmodium vivax in Malaria-Naive Study Participants-Clinical Profile and Utility of Molecular Diagnostic Methods.

BACKGROUND: Plasmodium vivax controlled human malaria infection (PvCHMI) is an important tool for evaluation of drugs, vaccines, and pathologies associated with this parasite. However, there are few data on safety due to limited numbers of PvCHMIs performed. METHODS: We report clinical and laboratory data, including hematological and biochemical profiles and adverse events (AEs), following mosquito bite-induced PvCHMI in malaria-naive study participants. Malaria diagnosis and treatment initiation was based on microscopic analysis of Giemsa-stained slides. Exploratory molecular assays were used to detect parasites using real-time polymerase chain reaction (PCR). RESULTS: AEs were mild to moderate and no study-related severe AEs were observed in any study participants. The majority of symptoms were transient, resolving within 48 hours. Molecular diagnostic methods detected parasitemia in 100% of study participants before malaria diagnosis using microscopy. Of reported AEs, microscopy detected 67%-100%, quantitative PCR 79%-100%, and quantitative real-time reverse-transcription PCR 96%-100% of study participants prior to appearance of symptoms. Almost all symptoms appeared after initiation of treatment, likely as known consequence of drug treatment. CONCLUSIONS: PvCHMI is safe with the majority of infections being detected prior to appearance of clinical symptoms, which can be further alleviated by using sensitive molecular methods for clinical diagnosis. Clinical Trials Registration. NCT01157897.

Efficacy of artesunate-amodiaquine and artemether-lumefantrine for uncomplicated Plasmodium falciparum malaria in Madagascar, 2018.

BACKGROUND: Since 2005, artemisinin-based combination therapy (ACT) has been recommended to treat uncomplicated falciparum malaria in Madagascar. Artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) are the first- and second-line treatments, respectively. A therapeutic efficacy study was conducted to assess ACT efficacy and molecular markers of anti-malarial resistance. METHODS: Children aged six months to 14 years with uncomplicated falciparum malaria and a parasitaemia of 1000-100,000 parasites/µl determined by microscopy were enrolled from May-September 2018 in a 28-day in vivo trial using the 2009 World Health Organization protocol for monitoring anti-malarial efficacy. Participants from two communes, Ankazomborona (tropical, northwest) and Matanga (equatorial, southeast), were randomly assigned to ASAQ or AL arms at their respective sites. PCR correction was achieved by genotyping seven neutral microsatellites in paired pre- and post-treatment samples. Genotyping assays for molecular markers of resistance in the pfk13, pfcrt and pfmdr1 genes were conducted. RESULTS: Of 344 patients enrolled, 167/172 (97%) receiving ASAQ and 168/172 (98%) receiving AL completed the study. For ASAQ, the day-28 cumulative PCR-uncorrected efficacy was 100% (95% CI 100-100) and 95% (95% CI 91-100) for Ankazomborona and Matanga, respectively; for AL, it was 99% (95% CI 97-100) in Ankazomborona and 83% (95% CI 76-92) in Matanga. The day-28 cumulative PCR-corrected efficacy for ASAQ was 100% (95% CI 100-100) and 98% (95% CI 95-100) for Ankazomborona and Matanga, respectively; for AL, it was 100% (95% CI 99-100) in Ankazomborona and 95% (95% CI 91-100) in Matanga. Of 83 successfully sequenced samples for pfk13, no mutation associated with artemisinin resistance was observed. A majority of successfully sequenced samples for pfmdr1 carried either the NFD or NYD haplotypes corresponding to codons 86, 184 and 1246. Of 82 successfully sequenced samples for pfcrt, all were wild type at codons 72-76. CONCLUSION: PCR-corrected analysis indicated that ASAQ and AL have therapeutic efficacies above the 90% WHO acceptable cut-off. No genetic evidence of resistance to artemisinin was observed, which is consistent with the clinical outcome data. However, the most common pfmdr1 haplotypes were NYD and NFD, previously associated with tolerance to lumefantrine.

Malaria parasite species composition of Plasmodium infections among asymptomatic and symptomatic school-age children in rural and urban areas of Kinshasa, Democratic Republic of Congo.

BACKGROUND: Malaria remains a major public health concern in the Democratic Republic of Congo (DRC), and school-age children are relatively neglected in malaria prevalence surveys and may constitute a significant reservoir of transmission. This study aimed to understand the burden of malaria infections in school-age children in Kinshasa/DRC. METHODS: A total of 634 (427 asymptomatic and 207 symptomatic) blood samples collected from school-age children aged 6 to 14 years were analysed by microscopy, RDT and Nested-PCR. RESULTS: The overall prevalence of Plasmodium spp. by microscopy, RDT and PCR was 33%, 42% and 62% among asymptomatic children and 59%, 64% and 95% in symptomatic children, respectively. The prevalence of Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale spp. by PCR was 58%, 20% and 11% among asymptomatic and 93%, 13% and 16% in symptomatic children, respectively. Among P. ovale spp., P. ovale curtisi, P. ovale wallikeri and mixed P. ovale curtisi + P. ovale wallikeri accounted for 75%, 24% and 1% of infections, respectively. All Plasmodium species infections were significantly more prevalent in the rural area compared to the urban area in asymptomatic infections (p < 0.001). Living in a rural as opposed to an urban area was associated with a five-fold greater risk of asymptomatic malaria parasite carriage (p < 0.001). Amongst asymptomatic malaria parasite carriers, 43% and 16% of children harboured mixed Plasmodium with P. falciparum infections in the rural and the urban areas, respectively, whereas in symptomatic malaria infections, it was 22% and 26%, respectively. Few children carried single infections of P. malariae (2.2%) and P. ovale spp. (1.9%). CONCLUSION: School-age children are at significant risk from both asymptomatic and symptomatic malaria infections. Continuous systematic screening and treatment of school-age children in high-transmission settings is needed.

The sand fly (Diptera: Psychodidae) fauna of the urban area of Lassance, Northeast Minas Gerais, Brazil.

The present study aimed to check the sand flies' fauna on the municipality of Lassance, Minas Gerais, Brazil and detect the presence of Leishmania DNA on the female captured and determine the risk areas of the municipality. Sand flies were collected monthly from May 2018 to April 2019 using automatic light traps for 3 consecutive nights. Eight houses were selected as sample points due its previous reports of tegumentary leishmaniasis and/or canine leishmaniasis. The sand fly's fauna found on the present study it's represented by several medical importance species and the most abundant species found were Lutzomyia longipalpis (77.09%) and Nyssomyia intermedia (10.06%). Leishmania infantum DNA was detected in a pool of Lu. longipalpis resulting on a 2.81% of infection rate. By the frequency of the two most abundant species on this study, we developed a risk area map and it draws attention to sample point 6 due to disparate abundance of sand flies at this site (81.81%). Statistical overview shows Lu. longipalpis as dominant species and, still, Non-Metric Multidimensional Scaling analysis reveal high similarity on fauna's diversity on the study area. Our findings suggest that the diversity of sand flies from the municipality of Lassance may promote the circulation of Leishmania infantum parasites putting in risk the habitants and other mammal's species. Still, our study reinforces the necessity of specific studies focused on breed sites of phlebotomine and its' ecology to expand the knowledge about the behaviour of this group of insects applying directly to leishmaniases' epidemiology.