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.

Awareness of Environmental Change, Climate Variability, and Their Role in Prevalence of Mosquitoes among Urban Dwellers in Southern Ghana.

This study evaluates the extent to which urban residents of Accra and Sekondi-Takoradi (all of southern Ghana) were aware of environment and climate-related variability. A total of 150 questionnaires were given to adults of 40 years and above who lived in the cities for at least 35 years. SPSS version 16 was used to process the data. Results show that all respondents in Accra Metropolitan Area (AMA) and 96% of them in Sekondi-Takoradi Metropolitan Area (STMA) noted the deteriorating vegetation cover over the cities. Similarly, 93% and 83% of them in AMA and STMA, respectively, observed that land use pattern of the two cities has changed considerably. There was positive and statistically significant correlation between respondents' educational attainment and their awareness of changes in land use/land cover (r = 0.18, n = 140, p = 0.03). Also, 71% in AMA and 89% in STMA stated that temperature of the cities had been increasing over the years. In all, 82.9% of the respondents mentioned that they had problem with the increasing population of mosquitoes. Respondents demonstrated ample knowledge of environment and climate variability and should be engaged in preventing further environmental degradation. The top-down approach to environmental issues has failed; the bottom-up approach to environmental maintenance is needed.

Prevalence of Plasmodium falciparum delayed clearance associated polymorphisms in adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1) genes in Ghanaian isolates.

BACKGROUND: Plasmodium falciparum delayed clearance with the use of artemisinin-based combination therapy (ACTs) has been reported in some African countries. Single nucleotide polymorphisms (SNPs) in two genes, P. falciparum adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1), have been linked to delayed clearance with ACT use in Kenya and recurrent imported malaria in Britain. With over 12 years of ACT use in Ghana, this study investigated the prevalence of SNPs in the pfap2mu and pfubp1 in Ghanaian clinical P. falciparum isolates to provide baseline data for antimalarial drug resistance surveillance in the country. METHODS: Filter paper blood blots collected in 2015-2016 from children aged below 9 years presenting with uncomplicated malaria at hospitals in three sentinel sites Begoro, Cape Coast and Navrongo were used. Parasite DNA was extracted from 120 samples followed by nested polymerase chain reaction (nPCR). Sanger sequencing was performed to detect and identify SNPs in pfap2mu and pfubp1 genes. RESULTS: In all, 11.1% (9/81) of the isolates carried the wildtype genotypes for both genes. A total of 164 pfap2mu mutations were detected in 67 isolates whilst 271 pfubp1 mutations were observed in 72 isolates. The majority of the mutations were non-synonymous (NS): 78% (128/164) for pfap2mu and 92.3% (250/271) for pfubp1. Five unique samples had a total of 215 pfap2mu SNPs, ranging between 15 and 63 SNPs per sample. Genotypes reportedly associated with ART resistance detected in this study included pfap2mu S160N (7.4%, 6/81) and pfubp1 E1528D (7.4%, 6/81) as well as D1525E (4.9%, 4/81). There was no significant difference in the prevalence of the SNPs between the three ecologically distinct study sites (pfap2mu: χ2 = 6.905, df = 2, P = 0.546; pfubp1: χ2 = 4.883, df = 2, P = 0.769). CONCLUSIONS: The detection of pfap2mu and pfubp1 genotypes associated with ACT delayed parasite clearance is evidence of gradual nascent emergence of resistance in Ghana. The results will serve as baseline data for surveillance and the selection of the genotypes with drug pressure over time. The pfap2mu S160N, pfubp1 E1528D and D1525E must be monitored in Ghanaian isolates in ACT susceptibility studies, especially when cure rates of ACTs, particularly AL, is less than 100%.

Diversity in breeding sites and distribution of Anopheles mosquitoes in selected urban areas of southern Ghana.

BACKGROUND: Anopheles vectors of malaria are supposedly less common in urban areas as a result of pollution, but there is increasing evidence of their adaptation to organically polluted water bodies. This study characterized the breeding habitats of Anopheles mosquitoes in the two major urban areas in southern Ghana; Accra (AMA) and Sekondi-Takoradi (STMA) Metropolitan Areas, during dry and wet seasons. METHODS: Anopheles mosquito larvae were sampled using standard dipping methods to determine larval densities. The origin, nature and stability of 21 randomly selected sites were observed and recorded. Mosquito larvae were reared to adults and Anopheles species identified by both morphological and molecular means. RESULTS: Sixty-six percent of Anopheles habitats were permanent and 34% temporal, and 74.5% man-made while 25.5% were natural. Puddles and urban farm sites accounted for over 51% of all Anopheles mosquitoes sampled. The mean larval densities among the habitat types was highest of 13.7/dip for puddles and lowest of 2.3/dip for stream/river, and the variation between densities were significant (P = 0.002). The mean larval densities were significantly higher in the wet season than in the dry season for the two study areas combined (P = 0.0191) and AMA (P = 0.0228). Over 99% of the 5,802 morphologically identified Anopheles species were An. gambiae (s.l.) of which more than 99% of the studied 898 were An. coluzzii (62%) and An. gambiae (s.s.) (34%). Urban farms, puddles, swamps and ditches/ dugouts accounted for approximately 70% of all An. coluzzii identified. Conversely, drains, construction sites, streams/rivers and "others" contributed 80% of all An. gambiae (s.s.) sampled. The wet season had significantly higher proportion of Anopheles larvae compared to the dry season (Z = 8.3683, P < 0.0001). Also, the proportion of Anopheles mosquitoes produced by permanent breeding sites was 61.3% and that of temporary sites was 38.7%. CONCLUSION: Taken together, the data suggest that man-made and/ or permanent habitats were the main contributors to Anopheles larval populations in the cities and that regulation of the anthropogenic processes that lead to development of breeding places and proper environmental management can drastically reduce mosquito breeding sites in urban areas of Ghana.

A STAT6 Intronic Single-Nucleotide Polymorphism is Associated with Clinical Malaria in Ghanaian Children.

Malaria pathogenesis may be influenced by IgE responses and cytokine cross-regulation. Several mutations in the IL-4/STAT6 signaling pathway can alter cytokine cross-regulation and IgE responses during a Plasmodium falciparum malarial infection. This study investigated the relationship between a STAT6 intronic single-nucleotide polymorphism (rs3024974), total IgE, cytokines, and malaria severity in 238 Ghanaian children aged between 0.5 and 13 years. Total IgE and cytokine levels were measured by ELISA, while genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Compared with healthy controls, heterozygosity protected against clinical malaria: uncomplicated malaria (odds ratios [OR] = 0.13, P < 0.001), severe malarial anemia (OR = 0.18, P < 0.001), and cerebral malaria (OR = 0.39, P = 0.022). Levels of total IgE significantly differed among malaria phenotypes (P = 0.044) and rs3024974 genotypes (P = 0.037). Neither cytokine levels nor IL-6/IL-10 ratios were associated with malaria phenotypes or rs3024974 genotypes. This study suggests a role for rs3024974 in malaria pathogenesis and offers further insights into an IL-4/STAT6 pathway mutation in malaria pathogenesis.

Pesticide application among farmers in the catchment of Ashaiman irrigation scheme of Ghana: health implications.

Pesticide use in modern day agriculture has increased tremendously. Pesticides are used to control pests and weeds, as well as protect crops from postharvest losses; however, their effects on humans and the environment cannot be overstated. This study examined pesticide acquisition, handling, and use among 120 farmers within the catchment of a small urban irrigation scheme. Also, in-depth interviews and focus group discussions were conducted among selected farmers through which further data was collected to augment that of the survey. Twelve types of pesticides, including herbicides, insecticides, and fungicides, were found in use in the study areas. Three main sources of information about pesticides were identified, 43.3% from extension officers, 39.2% from agrochemical dealers, and 10% from colleague farmers. Seventy-five percent (75%) of the respondents purchased the pesticides from agrochemical shops. Out of 74 farmers who were observed spraying pesticides on their farms, only 25.7% wore dresses that covered their whole body but without goggles. About sixty-seven percent (66.7%) of the farmers whose chemical got finished left the containers on their farms or threw them into the bushes around. The frequency of application was influenced by affordability and size of farm, among others. The study recommended that training of farmers on pesticide handling and use should be intensified.

High Levels of IL-10 and CD4+CD25hi+ Treg Cells in Endemic Burkitt's Lymphoma Patients.

BACKGROUND: The interplay between Epstein-Barr virus infection, malaria, and endemic Burkitt's Lymphoma is not well understood. Reports show diminished EBV-specific Th1 responses in children living in malaria endemic areas and deficiency of EBNA1-specific IFN-γ T cell responses in children with endemic Burkitt's Lymphoma (eBL). This study, therefore, examined some factors involved in the loss of EBNA-1-specific T cell responses in eBL. METHODS: T-cell subset frequencies, activation, and IFN-γ- or IL-4-specific responses were analyzed by flow-cytometry. Plasma cytokine levels were measured by ELISA. RESULTS: CD4+ and CD8+ cells in age- and sex-matched healthy controls (n = 3) expressed more IFN-γ in response to all immunostimulants than in pediatric endemic BL (eBL) patients (n = 4). In healthy controls, IFN-γ expression was higher than IL-4 expression, whereas in eBL patients the expression of IL-4 by CD4+ cells to EBNA-1 was slightly higher than IFN-γ. Moreover, the blood levels of TNF-α was significantly lower (p = 0.004) while IL-10 was significantly higher (p = 0.038), in eBL patients (n = 21) compared to controls (n = 16). Additionally, the frequency of CD4+CD25hi+ T cells was higher in both age-matched acute uncomplicated malaria (n = 26) and eBL (n = 14) patients compared to healthy controls (n = 19; p = 0.000 and p = 0.027, respectively). CONCLUSION: The data suggest that reduced Th1 response in eBL might be due to increased levels of IL-10 and T reg cells.

Selective activation of TCR-gammadelta+ cells in endemic Burkitt's lymphoma.

BACKGROUND: The overlap in geographical distribution of Plasmodium falciparum malaria and endemic Burkitt's lymphoma (eBL)--an aggressive Epstein-Barr virus (EBV)-associated B-cell tumour occurring almost exclusively in the tropics--strongly suggests a link between the two diseases. It is suspected that the polyclonal B-cell activation in P. falciparum malaria may precipitate a breakdown in homeostatic T-cell control of EBV-immortalized B-cell proliferation. Previous studies have suggested that a particular T-cell subset, characterized by expression of Vdelta1+ gammadelta T-cell receptors, is important for maintaining B-cell homeostasis, both in P. falciparum- exposed populations and in individuals subject to polyclonal B-cell activation of other aetiology. The objective of the present study was, therefore, to characterize lymphocyte phenotypes and to investigate possible differences in T-cell subset composition and activation status in P. falciparum-exposed Ghanaian children with and without eBL. METHODS: Venous blood samples in heparin from 21 eBL patients (mean age: 7.0 years; range: 3-11 years), referred to the Burkitt's Tumour Centre at Korle-Bu Teaching Hospital, Accra and 15 healthy, age and sex matched children, were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, R-phycoerythrin (RPE)- and RPE-Cy5-conjugated antibodies (CD3, CD4, CD8, CD25, CD69, CD95, HLA-DR, TCR-gammadelta, Vdelta1, Vdelta3, Vgamma9 and B-cells) and acquired on a flow cytometer. RESULTS: A reduction in the proportion of CD3+ cells in eBL patients, due mainly to perturbations among TCR-gammadelta+ cells was observed. In contrast, the proportions of CD4+ or CD8+ cells were relatively unaffected, as were the mean numbers of peripheral blood mononuclear cells. CONCLUSION: Selective changes in numbers and activation status of TCR-gammadelta+ cells occurs in Ghanaian children with eBL, a pattern which is similar to P. falciparum-induced changes. The data supports the hypothesis of a regulatory role for Vdelta1+ TcR-gammadelta T-cells in maintaining B-cell homeostasis and provides insights into the pathogenesis of eBL.