CRISPR/Cas12a-based on-site diagnostics of Cryptosporidium parvum IId-subtype-family from human and cattle fecal samples

Background Cryptosporidium parvum is an enteric protozoan parasite with zoonotic importance and can cause cryptosporidiosis in humans as well as domestic and wild animals worldwide. The IId subtype family (SF) is one of the most prevalent subtypes of C. parvum. Some clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been developed to detect nucleic acid with high flexibility, sensitivity and specificity. Methods By integrating recombinase polymerase amplification and the Cas12a/crRNA trans-cleavage system (termed ReCTC), we established end-point diagnostics by observing fluorescence readouts with the naked eye under blue light and on-site diagnostics using a lateral flow strip (LFS) biosensor. Results Our ReCTC-based diagnoses can detect as little as a single copy of a cloned C. parvum 60-kDa glycoprotein (GP60) gene, 10 oocysts per gram (OPG), clinical fecal sample without tedious extraction of genomic DNA and have no cross-reactivity with other SFs of C. parvum or other common enteric parasitic protozoa. Conclusions This study provided a new strategy for direct identification of the IId SF of C. parvum free of highly trained operators and expensive special equipment. Graphic Abstract

Infection rate and genetic diversity of Giardia duodenalis assemblage C in Iranian stray dogs, targeting the glutamate dehydrogenase gene

Background and Aim: Giardia duodenalis is one of the most common enteric protozoan parasites in vertebrates, such as humans, domestic and wild animals, causing giardiasis. To the best of our knowledge, little is known about the genetic diversity of G. duodenalis assemblages. This study aimed to identify genetic diversity of G. duodenalis assemblages in Iranian stray dogs. Materials and Methods: A total of 450 fecal samples were collected from 2015 to 2016 from stray dogs of Northwest Iran. All specimens were observed microscopically following concentration and flotation techniques. Subsequently, DNA samples were extracted, amplified, and sequenced targeting the glutamate dehydrogenase gene. Results: The overall prevalence of G. duodenalis in infected dogs was estimated at 1.6%, based on microscopic and molecular diagnoses. Sequencing and phylogenetic analyses indicated a high level of genetic diversity of assemblage C (haplotype diversity; 0.802). Conclusion: The pairwise sequence distances between the identified isolates of assemblage C showed an intradiversity of 0.3%-1.3% and identity of 98.7%-100%. Current findings indicate that a significant genetic diversity of G. duodenalis assemblage C haplotypes is unequivocally circulates among stray dogs in Northwest Iran.

Prevalence and Subtype Distribution of Blastocystis sp. in Senegalese School Children

Blastocystis sp. is an enteric protozoan that frequently colonizes humans and many animals. Despite impacting on human health, data on the prevalence and subtype (ST) distribution of Blastocystis sp. remain sparse in Africa. Accordingly, we performed the first multicenter and largest epidemiological survey ever conducted on Blastocystis sp. for this continent. A total of 731 stool samples collected from healthy school children living in 10 villages of the northwestern region of Senegal were tested for the presence of Blastocystis sp. by real-time polymerase chain reaction followed by subtyping of positive samples. Considerable variation in prevalence between villages (51.7 to 100%) was evident with the overall prevalence being 80.4%. Mixed infections were identified in 23% of positive individuals. Among 453 school children with a single infection, ST2 was predominant, followed by ST1, ST3, ST7, ST10, and ST14; this is the first report of ST10 and ST14 in humans. Genetic polymorphisms were evident at the intra-ST level with the identification of numerous ST1 to ST3 genotypes. ST1 showed the greatest intra-ST diversity followed by ST2 and ST3. The prevalence and distribution of STs and genotypes varied among target villages, pointing to several potential infection sources, including human-to-human, zoonotic, and waterborne transmission.

Update on the major imported protozoan infections in travelers and migrants

Globalization has contributed to the emergence of specific parasitic diseases in novel geographical areas, and in these regions, these infections in travelers and immigrants may cause a considerable burden of disease. Timely diagnosis and treatment of protozoan infections to decrease mortality and prevent associated complications are essential. In this respect, the increased availability of specific DNA-detection procedures has improved the diagnosis of many imported parasitic infections. Travelers and immigrants with associated comorbidities or immunosuppression may pose a special challenge regarding management. An updated review of the main protozoan infections in mobile populations (malaria, Chagas disease, leishmaniasis, enteric protozoan infections) is provided, focusing on the changing epidemiology of these diseases, recent developments in diagnosis and management and the possibility of local transmission of imported infections.

Differential detection of Entamoeba species in stool samples collected from children in District Swat, Khyber Pakhtunkhwa Pakistan

BackgroundAmoebiasis is an intestinal disease caused by enteric protozoan called Entamoeba histolytica belongs to the Genus Entamoeba. The main reason of infection is the contamination of food and water due to the poor sanitation. Among Entamoeba species, Entamoeba histolytica is highly pathogenic while the other species are non-pathogenic and needs no medical treatment.MethodologyA total of 400 stool samples were collected from different areas of district Swat and were processed for screening of amoebic cells. Microscopically identified samples containing amoebic cells were stored at −20 °C till DNA extraction. Extracted DNA was used in a PCR reaction with specific reference primers to amplify the target DNA.ResultsOut of all 400 stool samples 111 (27.7%) were found positive through microscopy while PCR reaction confirmed 80 out of microscope positive samples. Among 80 PCR positive samples, the infection with Entamoeba dispar was most common (57.5%) followed by E. histolytica (47.5%) and Entamoeba moshkovskii (20%). The positive cases for mono-infection of E. dispar were 33 (41.25%), followed by E.histolytica 25 (31.25%) and E. moshkovskii 7 (8.75%). The co-infection of E. histolytica with E. dispar and E. moshkovskii was 6 (7.5%) and 2 (2.5%), respectively. Similarly the co-infection of Entamoeba dispar with Entamoeba moshkovskii was also 2 (2.5%) while 5 (6.25%) samples were observed with mixed infection of E. histolytica, E. dispar and E. moshkovskii.Significance of the studyThe aim of the study was to detect and differentiate the E. histolytica, Entamoeba dispar and Entamoeba moshkovskii using conventional microscopy and polymerase chain reaction. The results suggested that the use of PCR is necessary to differentiate E. histolytica from E. dispar and E. moshkovskii and therefore, to avoid unnecessary treatment the present study recommend the use of PCR for the routine diagnosis of amoebiasis in the study area. It is also suggested that further studies from this area may also facilitate the understanding of genetic diversity of these pathogens.

Socio-Behavioral Risk Factors Associated with Cryptosporidiosis in HIV/AIDS Patients Visiting the HIV Referral Clinic at Cape Coast Teaching Hospital, Ghana

Objective:To identify the socio-behavioral risk factors associated with cryptosporidiosis among HIV/AIDS patients with chronic diarrhea symptoms visiting the HIV referral clinic at Cape Coast Teaching Hospital, Ghana.Methods:A cross-sectional study was conducted among 50 HIV/AIDS patients with recurrent diarrhea. Questionnaires were administered to collect social and behavioral risk factors associated withCryptosporidiumand other opportunistic protozoan parasitic infections in HIV patients. Stool samples were collected for the diagnosis of enteric protozoan pathogens using modified Ziehl-Neelsen and acid-fast staining methods. CD4+cells counts of study subjects were obtained from patients clinical records. The data obtained were analyzed using Pearson chi-square and multivariate-adjusted statistics tool on SPSS 16 for Windows.Results:Twenty-seven (54%) of the subjects were infected with enteric protozoan pathogens. The prevalences ofCryptosporidium,CyclosporaandMicrosporidiuminfections were 46%, 32% and 16%, respectively.Cryptosporidiuminfection was significantly associated with drinking water (×2=13.528, p<0.001),Cyclosporawas associated with the type of drinking water (×2=14.931, p<0.001) and toilet facilities used by the study subjects (×2=12.463, p<0.01), whilesMicrosporidiuminfection was associated with hand washing behavior (×2=12.463, p<0.01). Enteric protozoans were frequently encountered among subjects with CD4+ T-cell count <200 cells/mm3. However, coinfection ofCyclospora spp&Cryptosporidiumspp was not observed in CD4+cell count <200 and >500 cells/mm3.Multivariate analysis showed that the risk factor forCryptosporidiuminfection among HIV/AIDS patients was the source of drinking water (pipe borne water 76.2% prevalence: sachet water 25%; OR=0.10, 95%CI: 0.03-0.39, p<0.001).Conclusion:We report the risk factor for exposure ofCryptosporidiuminfection among HIV/AIDS patients for the first time in Ghana. The contamination of drinking water by protozoan parasites should be a public health concern. These results provide the stepping block to understand the transmission dynamics ofCryptosporidiumand other opportunistic pathogens in HIV/AIDS infected patients in Ghana.