Simple fecal flotation is a superior alternative to guadruple Kato Katz smear examination for the detection of hookworm eggs in human stool

Microscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnostic parameters of the Kato-Katz (KK) and simple sodium nitrate flotation technique (SNF) in terms of detection and quantification of hookworm eggs, with PCR as an additional reference test in stool, collected as part of a baseline cross-sectional study in Cambodia.; Fecal samples collected from 205 people in Dong village, Rovieng district, Preah Vihear province, Cambodia were subjected to KK, SNF and PCR for the detection (and in case of microscopy-based methods, quantification) of hookworm eggs in stool. The prevalence of hookworm detected using a combination of three techniques (gold standard) was 61.0%. PCR displayed a highest sensitivity for hookworm detection (92.0%) followed by SNF (44.0%) and quadruple KK smears (36.0%) compared to the gold standard. The overall eggs per gram feces from SNF tended to be higher than for quadruple KK and the SNF proved superior for detecting low egg burdens.; As a reference, PCR demonstrated the higher sensitivity compared to SNF and the quadruple KK method for detection of hookworm in human stool. For microscopic-based quantification, a single SNF proved superior to the quadruple KK for the detection of hookworm eggs in stool, in particular for low egg burdens. In addition, the SNF is cost-effective and easily accessible in resource poor countries

Immunoproteomics to identify species-specific antigens in Neospora caninum recognised by infected bovine sera

Bovine neosporosis is a disease of concern due to its global distribution and significant economic impact through massive losses in the dairy and meat industries. To date, there is no effective chemotherapeutic drug or vaccine to prevent neosporosis. Control of this disease is therefore dependent on efficient detection tests that may affect treatment management strategies. This study was conducted to identify the specific immunoreactive proteins of Neospora caninum tachyzoites recognised by sera from cattle infected with N. caninum, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis and B. bigemina, and by sera from uninfected cattle using two-DE dimensional gel electrophoresis (2-DE) combined with immunoblot and mass spectrometry (LC-MS/MS). Among 70 protein spots that reacted with all infected sera, 20 specific antigenic spots corresponding to 14 different antigenic proteins were recognised by N. caninum-positive sera. Of these immunoreactive antigens, proteins involved in cell proliferation and invasion process were highly immunogenic, including HSP90-like protein, putative microneme 4 (MIC4), actin, elongation factor 1-alpha and armadillo/beta-catenin-like repeat-containing protein. Interestingly, we discovered an unnamed protein product, rhoptry protein (ROP1), possessing strong immunoreactivity against N. caninum but with no data on function available. Moreover, we identified cross-reactive antigens among these apicomplexan parasites, especially N. caninum, T. gondii and C. parvum. Neospora caninum-specific immunodominant proteins were identified for immunodiagnosis and vaccine development. The cross-reactive antigens could be evaluated as potential common vaccine candidates or drug targets to control the diseases caused by these apicomplexan protozoan parasites

Development and evaluation of a multiplex quantitative real-time Polymerase Chain Reaction for hookworm species in human stool

Hookworm disease caused by; Necator americanus; ,; Ancylostoma duodenale; , and; Ancylostoma ceylanicum; affects half a billion people worldwide. The prevalence and intensity of infection of individual hookworm species are vital for assessing morbidity and generating targeted intervention programs for their control. The present study aims to evaluate a multiplex real-time quantitative PCR (qPCR) assay to determine the prevalence and egg intensity of all three hookworm species and compare this with standard microscopy and published genus-based conventional and real-time multiplex qPCRs. Performance of the diagnostic assays was evaluated using DNA extracted from 192 fecal samples collected as part of a soil-transmitted helminth (STH) survey in northern Cambodia. The prevalence of hookworms as detected by the multiplex hookworm qPCR of 84/192 (43.8%) was significantly higher than that using microscopy of 49/192 (25.5%). The hookworm multiplex qPCR showed very good agreement for the detection of both; N. americanus; (Kappa 0.943) and; Ancylostoma; spp. (Kappa 0.936) with a multiplex STH qPCR. A strong and moderate quantitative correlation between cycle threshold and eggs per gram (EPG) feces was obtained for the hookworm qPCR for seeded DNA egg extracts (; R; 2; ≥ 0.9004) and naturally egg-infected individuals (; R; 2; = 0.6848), respectively. The newly developed hookworm quantitative multiplex qPCR has the potential for application in anthelmintic efficacy trials and for monitoring the success of mass deworming programs targeting individual species of anthroponotic and zoonotic hookworms

Semi-domesticated dogs as a potential reservoir for zoonotic hookworms in Bangkok, Thailand

Background and Aim: Hookworms are parasitic nematodes that live in the small intestine of their mammalian hosts including humans, dogs, and cats. This study was conducted to determine the prevalence and perform genetic characterization of hookworms using molecular techniques and to elucidate the risk factors associated with hookworm infections among semi-domesticated dogs residing in temples in the Bangkok Metropolitan Area, Thailand. Materials and Methods: A total of 500 fecal samples were collected from semi-domesticated dogs from 91 temples in 48 districts of Bangkok. DNA was extracted and screened using internal transcribed spacer polymerase chain reaction-restriction fragment length polymorphism. In addition, samples positive for Ancylostoma ceylanicum were further characterized at the haplotype level based on the analysis of the mitochondrial cytochrome oxidase-1 gene (cox1). Results: The prevalence of hookworm infections in semi-domesticated dogs was 6.2% (31/500). Hookworm infections were detected in temple-community dogs in 12 of 48 districts (25.0%), with Bang Khen and Lak Si districts having the highest proportion of infected dogs (22.6%). Regarding molecular characterization of hookworm species, 21 positive samples (67.74%) were infected with A. ceylanicum and 10 (32.26%) with Ancylostoma caninum. Characterization of cox1 in A. ceylanicum isolates revealed the presence of a mixture of human and dog isolates. Conclusion: Semi-domesticated dogs act as a potential source of hookworm infections for human and animal populations in Bangkok, Thailand

Prevalence and genotyping of Cryptosporidium SPP from dairy cow fecal samples in western Thailand

The aims of this study were to determine the prevalence of Cryptosporidium spp in dairy cows in central Thailand and to investigate the genotype of Cryptosporidium spp in this population. A total of 200 fecal samples from dairy cows were collected and examined by the acid-fast staining technique and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). The prevalence of Cryptosporidium infection in dairy cows was 7% (95% CI 3.5-10.5) by acid-fast staining, and 15.5% (95% CI 10.5-20.5) by PCR-RFLP. This is the first report of genetic identification of the C. parvum bovine genotype in dairy cows in Thailand. PCR-RFLP analysis showed all positive samples were C. parvum (bovine genotype). C. andersoni was not found in this study. The only significant risk factor for Cryptosporidium infection in dairy cows was age. Calves less than 2 months old were more frequently infected by Cryptosporidium than others (OR 13.82, 95% CI 3.67-51.97, p = 0.001). Cattle may be a potential source of human cryptosporidiosis

Molecular Detection of Bartonella quintana among Long-Tailed Macaques (Macaca fascicularis) in Thailand

Bartonella quintana is a zoonotic pathogen with a worldwide distribution. Humans and non-human primates are considered to be natural reservoir hosts for B. quintana. However, information on the molecular epidemiology of this organism is very limited in regard to long-tailed macaques (Macaca fascicularis) in Thailand. Therefore, this study aimed to investigate the occurrence and genetic diversity of Bartonella spp. among long-tailed macaques in Thailand. In total, 856 blood samples were collected from long-tailed macaques in Thailand. All specimens were screened for Bartonella spp. using a polymerase chain reaction (PCR) assay targeting the 16S rRNA, gltA and ftsZ genes. All positive samples were further analyzed based on nucleotide sequencing, phylogenetic analysis and multiple sequence alignment analysis. Only one macaque showed a positive result in the PCR assays based on the 16S rRNA, gltA and ftsZ genes. Nucleotide sequencing and phylogenetic analysis revealed that the obtained sequences were closely related to B. quintana previously detected in non-human primates. Single-nucleotide polymorphisms (SNPs) were detected in the gltA and ftsZ gene sequences. This study revealed that long-tailed macaques in Thailand carried B. quintana. Despite the low infection rate detected, long-tailed macaques may be a reservoir of B. quintana

Low risk for transmission of zoonotic Giardia duodenalis from dogs to humans in rural Cambodia

A number of epidemiological studies have demonstrated Giardia as prevalent in both humans and dogs worldwide and have postulated the occurrence of anthroponotic, zoonotic and animal-specific cycles of transmission, which may be geographically and regionally unique in its epidemiology. The aim of this study was to utilise molecular tools to determine the prevalence and compare genotypes of Giardia duodenalis infecting humans and dogs living in a previously identified Giardia-endemic village in rural Cambodia in order to ascertain zoonotic transmission risk.; The prevalence of G. duodenalis in humans and dogs was 18.3% (40/218) and 10.6% (10/94) by PCR, respectively. Molecular characterisation of the small subunit of ribosomal RNA (SSU rRNA) gene, triose phosphate isomerase (TPI) gene and sub-assemblage characterisation of the glutamate dehydrogenase (gdh) gene placed 27.5% (11/40) of Giardia positive humans into assemblage AII and 72.5% (29/40) into assemblage BIII of G. duodenalis. In dogs, 20.0% (2/10) of Giardia-positive samples were characterised as G. duodenalis assemblage BIII, 40.0% (4/10) as assemblage C and 40.0% (4/10) as mix infection between assemblage C and D.; Overall, just over 2% of dogs harboured potentially zoonotic assemblages of G. duodenalis in the studied communities and hence pose a minimal zoonotic risk for the transmission of Giardia to humans