Phylogenomic reconstruction of Cryptosporidium spp. captured directly from clinical samples reveals extensive genetic diversity.

Cryptosporidium is a leading cause of severe diarrhea and mortality in young children and infants in Africa and southern Asia. More than twenty Cryptosporidium species infect humans, of which C. parvum and C. hominis are the major agents causing moderate to severe diarrhea. Relatively few genetic markers are typically applied to genotype and/or diagnose Cryptosporidium. Most infections produce limited oocysts making it difficult to perform whole genome sequencing (WGS) directly from stool samples. Hence, there is an immediate need to apply WGS strategies to 1) develop high-resolution genetic markers to genotype these parasites more precisely, 2) to investigate endemic regions and detect the prevalence of different genotypes, and the role of mixed infections in generating genetic diversity, and 3) to investigate zoonotic transmission and evolution. To understand Cryptosporidium global population genetic structure, we applied Capture Enrichment Sequencing (CES-Seq) using 74,973 RNA-based 120 nucleotide baits that cover ~92% of the genome of C. parvum. CES-Seq is sensitive and successfully sequenced Cryptosporidium genomic DNA diluted up to 0.005% in human stool DNA. It also resolved mixed strain infections and captured new species of Cryptosporidium directly from clinical/field samples to promote genome-wide phylogenomic analyses and prospective GWAS studies.

Prevalence and risk factors associated with gastrointestinal parasites in ruminant livestock in the Coastal Savannah zone of Ghana.

Gastrointestinal (GIT) parasite infections result in significant economic losses to ruminant livestock production. To determine the prevalence and risk factors associated with GIT parasite infections in livestock from Ghana, a cross-sectional survey was conducted in cattle and small ruminants kept under different management systems in the Coastal Savannah zone from October 2014 to February 2015. Faecal samples were collected from 328 cattle and 502 small ruminants (sheep and goats) and examined by formal ether concentration microscopy. The management systems and environmental conditions of the farm or household were observed, and a questionnaire administered to the livestock owners. Overall, 90.8% (754/830) of livestock were infected with at least one of ten different parasites (Eimeria, Strongylid nematodes, Toxocara, Trichuris, Schistosoma, Dicrocoelium, Paramphistomum, Fasciola, Moniezia and Thysaniezia), with Eimeria the most prevalent (78.4%). Most (64.5%) livestock had coinfections with two to five parasites with parasite intensity mostly light and at least one parasite was found in 98.6% (140/142) of the herds. Binary logistic regression models were generated to assess the risk factors associated with infection. Earthen floor was positively associated with strongylid infection, multiple ruminant species with Paramphistomum infection and flock size (>25 animal) with Thysaniezia, Dicrocoelium and Fasciola infections. Separating young animals from older animals was negatively associated with Strongylid infection, feed supplementation with Thysaniezia infection and small ruminant species with Paramphistomum and Toxocara infections. The findings from this study suggests that good sanitation, proper husbandry practices and improved nutrition can improve livestock health and production in Ghana.

Sero-Epidemiology of Toxocara Canis Infection in Children Attending Four Selected Health Facilities in the Central Region of Ghana.

OBJECTIVE: The study determined the seroprevalence of Toxocara canis infection among children attending four selected health facilities in the Central Region of Ghana. DESIGN: Cross-sectional study. METHOD: Sera from 566 children aged 1-15 years attending four selected health facilities in the Central Region of Ghana between July and September 2012 was used in a Toxocara excretory-secretory antigen-based ELISA to detect serum IgG. A short questionnaire was designed to obtain data on respondents as to age, gender, educational level, locality of residence, habits of washing of fruits, vegetable and hands before eating, keeping of pet (dogs or cats), and history of playing with soil and pets. Clinical information was also collected. Associations between sero-positivity and age group, gender, risk factors, educational level and other variables were determined by Chi square test. RESULTS: The overall sero-prevalence was 53.5% (n=566). Age, educational level and hospital visited were significantly associated with sero-positivity (p< 0.05). Children with history of playing with soil (χ(2)=9.03, p=0.003), pet-keeping (χ(2)=14.77, p=0.001) and not washing hands with soap before eating (χ(2)=5.82, p=0.016) were significantly associated with sero-positivity. CONCLUSION: The sero-prevalence of T. canis infection in children in the study was high. The children should be educated to desist from risk factors such as playing with soil and pets and be encouraged to ensure proper personal hygiene.

Infection risk factors associated with seropositivity for Toxoplasma gondii in a population-based study in the Central Region, Ghana.

About 20-90% of the world's population has had contact with Toxoplasma gondii parasites. The aim of this study was to determine the seroprevalence and risk factors associated with T. gondii infection in the Central Region, Ghana. A community-based cross-sectional study was conducted in three selected communities. Serum samples were tested for the presence of anti-T. gondii IgG and IgM antibodies by ELISA. A serological criterion for seropositivity was a positive test result for any of the two anti-Toxoplasma IgG or IgM antibodies or a combination of both. In all, 390 participants of mean age 47.0 years consisting of 118 (30.%) males and 272 (69.7%) females were tested. The overall seroprevalence of T. gondii was 85% (333/390) where fishermen, farmers and fishmongers, respectively, had the highest seropositivity. IgG and IgM antibodies were detected in 329 (84%) and 25 (6%), respectively, while both IgG and IgM antibodies were detected in 21 (5%) of the participants. Respectively, 1% (4/390) and 79% (308/390) of participants tested positive for IgM-only and IgG-only antibodies. There was a significant relationship between Toxoplasma seropositivity and contact with soil, presence of a cat in the surrounding area, age, sources of drinking water, level of formal education, and socioeconomic status. The results suggest that the seashore may serve as a good ground for sporulation and survival of Toxoplasma oocysts.

Mouse monoclonal antibodies against human urinary protein Sm-UP(2)IP of Schistosoma mansoni.

There are innumerable clinical and pathological problems associated with schistosomiasis that have necessitated various control programs. Successful control would naturally depend on effective rapid diagnosis in the field. However, the overlapping distribution of urinary and intestinal schistosomiasis in hyperendemic areas calls for differential diagnosis. This study was aimed at producing anti-Schistosoma mansoni monoclonal antibodies (MAbs) for possible utilization in assays to detect antigens in the urine of infected persons. In order to raise antibodies to less immunogenic urinary parasite antigens, BALB/c mice were immunized with Schistosoma mansoni soluble worm antigens (Sm-SWA) while urinary proteins (Sm-UP(2)IP), isolated from infected human urine samples, was used as a final booster before cell fusion. Hybridoma cells were obtained by the fusion of mouse myeloma and spleen cells from the immunized mice, which were screened by microplate ELISA and then studied further to obtain anti-S. mansoni specific MAbs. The MAbs analyzed presented IgM isotypes. The reactivity of anti-S. mansoni MAbs with Sm-UP(2)IP, 13/43 (30.2%), MAbs showed stronger reactivity. It was observed that one of the MAbs cross-reacted with antigen associated with S. haematobium urinary antigen (Sh-UP(2)IP). Nine (9/13, 69.2%) MAbs recognized glycoprotein antigenic epitopes of Sm-UP(2)IP and Sm-SWA. On the other hand, 4/13 (30.8%) MAbs recognized carbohydrate antigenic epitopes. Band size of 8.9 kDa associated with Sm-UP(2)IP was detected by the 13 MAbs. With Sm-SWA, all the MAbs detected band sizes of 177.8 and 158.5 kDa. In addition, three MAbs recognized a 38.9 kDa band. The generation of anti-S. mansoni species-specific MAbs offers opportunities to develop a specific MAb-based diagnostic tool for use in the field to detect Schistosoma mansoni infection in Ghana.

Sero-epidemiology of toxoplasmosis amongst pregnant women in the greater accra region of ghana.

OBJECTIVES: To investigate Toxoplasma infection among pregnant women in relation to exposure to infection risk, age and pregnancy-related risk factors. DESIGN AND METHODS: This cross-sectional study involved 294 pregnant women attending ante-natal clinic in Accra who consented to participate. Personal and Toxoplasma infection risk related data were obtained by questionnaire interviews. Venous blood was safely drawn from each participant and spun to obtain sera. Each of the 159 randomly selected serum samples was tested for specific anti-Toxoplasma (anti-T. gondii) antibodies IgG, IgA and IgM using a commercial ELISA kit (Calbiotech Inc., CA). ELISA results were correlated with exposure to possible infection risk factors as well as age and pregnancy-related risk factors. RESULTS: The 159 women aged 15-40 years in their first, second and third trimesters, numbered 29, 70 and 60, respectively. An overall anti-T. gondii antibodies IgG, IgA and IgM seroprevalence of 92.5% (147/159) was recorded, with 4.1% (6/147) of them having anti-IgG only. The remaining 88.7% (141/159) had anti-Toxoplasma antibodies IgG, IgA and IgM in various combinations and consisted of 17.7% (25/141) in their first, 44.0% (62/141) in their second, and 38.3% (54/141) in their third, trimesters. Twelve women (7.6%) were seronegative for all 3 antibodies CONCLUSIONS: Seroprevalence was high among the women and exposure to contact with cats' faeces was found to be the major T. gondii infection risk factor. Age and pregnancy-related risk factors did not have association with T. gondii infection within the limitations of this study.

A monoclonal antibody-based dipstick assay for diagnosis of urinary schistosomiasis.

A Schistosoma haematobium species-specific mouse immunoglobulin (Ig) G1 monoclonal antibody (mab) Sh2/15.F that bound a 29 kDa peptide was utilized to develop a membrane-based dipstick enzyme-linked immunosorbent assay for specific diagnosis of urinary schistosomiasis. Strips of polyvinylidene difluoride membrane were wetted with methanol and stored in distilled water. The strips were used to capture urinary antigens which were then revealed by incubation in a mixture of specific mab and peroxidase-conjugated goat anti-mouse IgG. The assay correctly identified 26/30 (87%) of egg-negative control individuals and 53/54 (98%) of parasitologically confirmed cases including all of 6 individuals treated with praziquantel (40 mg/kg) but not cured. Also, the assay detected S. haematobium antigens in the urine of 3 individuals from whom 2 specimens had to be examined microscopically to confirm infection, thus suggesting that the mab detection method may have greater sensitivity than microscopy.

Limited field evaluation of a rapid monoclonal antibody-based dipstick assay for urinary schistosomiasis.

A rapid, visually read monoclonal antibody (MoAb)-based dipstick assay for specific diagnosis of urinary schistosomiasis was field tested with microscopy and the use of hematuria and proteinuria in a schistosomiasis hematobia endemic area in Southern Ghana. The study group consisted of 229 individuals (114 males and 115 females) aged 1 to 86 years; 145/229 (63.3%) of the subjects submitted stool samples from which no S. mansoni eggs were detected. However, infections with Necator americanus (hookworms) 33.1%, Ascaris lumbricoides 2.8%, Trichuris trichiura (whipworm) 2.8%, and Strongyloides stercoralis 0.7% were detected but did not appear to influence the results of the MoAb-dipstick assay. Urinary schistosomiasis prevalence was estimated as 47.6% by microscopy, 48% by MoAb-dipstick, 39.7% by microhematuria, and 23.6% by proteinuria. The MoAb-dipstick correctly identified 108/109 (99.1%) of microscopically confirmed cases and 118/120 (98.3%) of egg-negative individuals, thereby giving a sensitivity of 99.1% and a specificity of 98.3%. On the other hand, microhematuria and proteinuria were, respectively, 76.1% and 40.4% sensitive, and 94.2% and 92.5% specific when compared to microscopy. Microhematuria and proteinuria had significantly lower sensitivity (P < 0.001) than either microscopy or dipstick.

Extraction of Schistosoma haematobium antigens from infected human urine and generation of potential diagnostic monoclonal antibodies to urinary antigens.

Proteins in Schistosoma haematobium infected human urine were concentrated by precipitation with saturated ammonium sulphate 50% (v/v) and various fractions obtained at different stages of precipitation tested for presence of schistosome antigens (ShAgs) by dot-ELISA. The protein fraction (UP2S) obtained following two-times precipitation was found to contain high concentrations of ShAg. Fraction UP2S was dialysed against phosphate-buffered saline (pH 7.4) and further purified by Sephadex G-200 column chromatography. Two protein peaks were eluted of which the first peak UP2S(pkI) was found to contain high concentrations of ShAgs as determined by microplate-ELISA. The second peak UP2S(pkII) consisted of human urine proteins. Further analysis of UP2S(pkI) revealed that ShAgs were mainly in the form of immune complexes with human IgG, IgM, IgA, IgE and complement C3. The ShAgs in both UP2S and UP2S(pkI) were found to be active as they induced immune responses in mice which produced antibodies reactive with S. haematobium worm as well as soluble egg antigens (SEA). Pure ShAgs were obtained from UP2S following dissociation of immune complexes with a carbonate buffer (pH 11.42) and further purification on Sephadex G-200. Immunizations with UP2S led to the generation of MoAbs which could bind both SEA and UP2S.