Parasitic infection among HIV/AIDS patients at Bela-Bela clinic, Limpopo province, South Africa with special reference to Cryptosporidium.

Intestinal parasitic organisms are common pathogens among HIV patients worldwide and have been known to cause severe and life-threatening diarrhea in such subjects. In the present study, the prevalence of Cryptosporidium spp and other intestinal parasites in stool samples from 151 HIV/AIDS patients attending a HIV treatment center in South Africa was determined using' standard parasitological methods, as well as molecular methods including PCR and quantitative PCR for confirmation of Cryptosporidium spp. In addition, the loop-mediated isothermal amplification (LAMP) method was evaluated for detection of Cryptosporidium spp in 24 stool samples. Standard parasitological methods indicated that Cryptospo- ridium spp (26.5%), Entamoeba spp (26.5%) and Giardia lamblia (13%) were the most common protozoan parasites, while Ascaris lumbricoides (8%), Schistosoma mansoni (6%) and Trichuris trichiura (4.6%) were the most commonly found helminths. PCR, quantitative PCR and LAMP methods identified Cryptosporidium spp in 28% (30/106), 35% (53/151) and 58% (14/24) of the stool samples, respectively. Multiple infections (34%) were commonly found in the study population. Females above 45 years had the highest Cryptosporidium prevalence (58%). Prevention measures must be implemented in order to curb the negative impact of Cryptosporidium-causing diarrhea among HIV/AIDS patients in this region as well as other parasitic infections identified in this study.

Prevalence and distribution of Entamoeba species in a rural community in northern South Africa.

Amoebiasis occurs worldwide and affects about 20-50 million people annually. Stool samples were collected from patients attending different rural clinics in Northern South Africa in the present study. Microscopic examination was performed for the initial detection of Entamoeba parasites. A multiplex PCR protocol based on the small subunit rRNA gene of E. moshkovskii, E. dispar, and E. histolytica, was used for the differential detection of the three Entamoeba species (collectively referred to as Entamoeba complex). A total of 170 participants were recruited in the study, with a mean age of 35.9 ±â€¯17.8 years and a median of 37.0 years. The prevalence of Entamoeba species was found to be 34.7% and 33% by PCR and microscopy, respectively. E. histolytica had a prevalence of 4.1%, E. dispar 14.7% and E. moshkovskii 15.9%. Of the three species, only E. histolytica was significantly associated with diarrhoea and was more prevalent among HIV patients even in the absence of diarrhoea while the other two were not, although the difference was not significant (p > 0.05). This is the first study in South Africa to describe the prevalence of E. moshkovskii. E. dispar was significantly associated with abdominal pains (p = 0.003). Further studies are needed to clarify the role of E. moshkovskii and E. dispar in abdominal pain and diarrhoea.

Prevalence and genetic characterization of Giardia lamblia in relation to diarrhea in Limpopo and Gauteng provinces, South Africa.

BACKGROUND: Very few studies have determined the prevalence and assemblage distribution of Giardia lamblia in South Africa. The present study aimed to ascertain the prevalence of G. lamblia infection and the spread of the various assemblages in two communities in South Africa - Giyani, Limpopo province (rural community) and Pretoria Guateng province (urban community). METHODS: Prevalence was determined by immunological and molecular methods analyzing a total of 516 stool samples collected from patients visiting different health centres in Giyani and Pretoria. For immunological assays, samples were screened by ELISA to detect G. lamblia antigen. Furthermore, a semi nested PCR amplifying the triose phosphate isomerase (tpi) gene was used to differentiate between the two most common human assemblages (A and B). FINDINGS: Of the 516 participants, 40 (7.75%) were identified as positive by ELISA. A statistically significant correlation was observed between the stool texture and Giardia infection (ᵡ2 = 10.533; p = .005). G. lamblia was significantly associated with watery stool types in females p = .008. Furthermore, a significant association was also noticed between the origin of samples (ᵡ2 = 9.725; p = .002). No significant correlation between age and gender was noted. Regarding the age groups, most people who were infected were between 3 and 20 years. A statistically significant association was seen (p = .001) with the distribution of the pathogen with the stool type. The prevalence of Giardia infection was higher in watery stool samples (71.4%) in Giyani region (rural) whereas in Pretoria, high prevalence was found in loose stool samples (6.2%). Generally, the distribution was statistically significant in the stool type collected for the study (p = .005). Genotyping revealed more G. lamblia assemblage B (17.8%) than assemblage A (1.7%). Furthermore, 21.0% of the samples exhibited single infection while 4.2% had mixed infections. Assemblage B was more common in Giyani than in urban Pretoria. CONCLUSIONS: The study confirms Giardia as an important cause of diarrhea in the concerned communities with people in rural areas more at risk compared to those in urban areas with higher prevalence among younger patients. Therefore, health education campaigns should target young age groups.

Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing.

Three LAMP (loop-mediated isothermal DNA amplification) assays were applied to detect Cryptosporidium species DNA in a total number of 270 fecal samples originating from cattle, sheep and horses in South Africa. DNA was extracted from 0.5 g of fecal material. Results of LAMP detection were compared to those obtained by nested PCR targeting the Cryptosporidium 18 small subunit rRNA (18S) gene. All samples were negative by nested PCR, while up to one-third of samples were positive by LAMP assays. The SAM-1 LAMP assay, shown to detect C. parvum, C. hominis and C. meleagridis, amplified Cryptosporidium DNA in 36 of 107 cattle (33.64%), in 26 of 85 sheep (30.5%) and in 17 of 78 horses (21.79%). The HSP LAMP specific to C. muris and C. andersoni, amplified Cryptosporidium DNA in one cow (0.9%), five sheep (5.8%) and seven horses (8.9%). The gp60 LAMP assay, shown to detect C. parvum produced no amplified Cryptosporidium DNA, likely due to low sample DNA concentrations. The specificity of LAMP assays was confirmed by sequencing of the LAMP products generated in positive samples. Sequence products from the three LAMP assays showed high identity to the target gene sequences confirming the specificity of LAMP. In this study, the LAMP procedure was clearly superior to nested PCR in the detection of Cryptosporidium species DNA. Use of LAMP is proposed as an efficient and effective tool for epidemiologic survey studies including screening of healthy animals in which Cryptosporidium oocyst shedding is characteristically low and likely below the detection limit of PCR in conventional sample concentrates.

Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences.

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. MSP1a is involved in the adhesion and transmission of A. marginale by ticks and varies among geographic strains in the number and sequence of amino-terminal tandem repeats. The aim of this study was to characterize the genetic diversity of A. marginale strains collected from countries in North and South America, Europe, Asia, Africa and Australia, inclusive of all continents. In this study, we characterized 131 strains of A. marginale using 79 MSP1a repeat sequences. These results corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The phylogenetic analyses of MSP1a repeat sequences did not result in clusters according to the geographic origin of A. marginale strains but provided phylogeographic information. Seventy-eight percent of the MSP1a repeat sequences were present in strains from a single geographic region. Strong (> or =80%) support was found for clusters containing sequences from Italian, Spanish, Chinese, Argentinean and South American strains. The phylogenetic analyses of MSP1a repeat sequences suggested tick-pathogen co-evolution and provided evidence of multiple introductions of A. marginale strains from various geographic locations worldwide. These results contribute to the understanding of the genetic diversity and evolution of A. marginale and tick-pathogen interactions.

Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10(-6) dilution of 10-fold serially diluted samples. Furthermore, DNA extracted from blood collected from a horse experimentally infected with T. equi was amplified by a T. equi LAMP primer set from days 2 to 35 post-infection, demonstrating the high sensitivity of these primers. Of 55 samples collected from China, 81.8% and 56.3% were positively detected by LAMP for T. equi and B. caballi infections, respectively. In contrast, 91.8% and 45.9% of the 37 samples collected from South Africa were LAMP positive for T. equi and B. caballi, respectively. These results suggest that LAMP could be a potential diagnostic tool for epidemiological studies of equine piroplasmosis.

Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes.

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries.

Ticks and tick-borne diseases of livestock belonging to resource-poor farmers in the eastern Free State of South Africa.

The paper provides a summary of three studies conducted in the eastern Free State of South Africa between 1998 and 2000. In a questionnaire-based study approximately 21% of interviewed resource-poor farmers (n = 150) indicated that they experienced problems with ticks and tick-borne diseases. About 56% of farmers indicated that tick-related problems were most severe in summer, while 32% indicated that the most problems were encountered in winter. About 12% indicated that the tick problems were experienced throughout the year. Farmers also indicated that the highest tick burdens were experienced between spring and early winter. The principal ticks infesting cattle (n = 30) were found to be Boophilus decoloratus (53.1%). Rhipicephalus evertsi evertsi (44.7%), Rhipicephalus follis (1.0%), Rhipicephalus gertrudae (0.7%) and Rhipicephalus warburtoni (0.4%). On small stock (n = 188), R. evertsi evertsi (68%) and B. decoloratus (32%) were recorded as the main ticks in the study area. A sero-epidemiological survey of cattle (n = 386) showed that 94% of cattle were seropositive for Babesia bigentina by IFAT, while 87% were sero-positive for Anaplasma by indirect ELISA. All the animals were sero-negative for Babesia bovis and this is probably because the tick vector, Boophilus microplus, is not present in the study area. All sheep and goats were sero-positive for Theileria species by IFAT while 85% of sheep and 100% of goats tested positive for Anaplasma species by competition inhibition ELISA. The high incidence of positive serological results for B. bigemina and Anaplasma in cattle, and Theileria and Anaplasma in sheep and goats and the absence of clinical cases would indicate that this area is endemically stable for these diseases.

Ethyleneglycol-Bis-((-aminoethyl ether) N,N,N(1),N(1), -Tetraacetic acid (EGTA) inhibits Leishmania donovani in vitro.

Exposure of Leishmania donovani culture promastigotes to ethyleneglycol-bis-((-aminoethyl ether) N,N,N(1),N(1),-tetraacetic acid (EGTA) concentrations of between 0.2 to 1.6 mg/ml significantly inhibited their growth, though the different concentrations did not significantly differ between themselves on their effect on promastigotes in cell free media. EGTA concentrations of between 0.05 and 0.1 mg/ml were non-toxic to mouse peritoneal macrophages in vitro. Treatment of L. donovani-infected macrophages with EGTA at concentrations of 0.05, 0.1 and 0.2 mg/ml contributed significantly to a decline in amastigote parasite-loads in the macrophages. The higher the chelator concentration within the acceptable toxic levels for macrophages, the greater was the rate at which parasites were cleared from the macrophages. It is speculated that EGTA chelates manganese from phosphoenol pyruvate (PEP) carboxykinase, a TCA-rate limiting enzyme in the metabolism of Leishmania parasites. A manganese-complex is also probably used by these parasites as a defense mechanism against oxygen-derived radicals. Chelation of manganese would destabilise PEP carboxykinase, and therefore severely interfere with the parasite metabolism. All these factors would render the Leishmania parasite susceptible to digestion in the lysosomal vacuoles of the macrophage, hence the observed significant reduction in parasite-loads of L. donovani-infected EGTA-treated macrophages in vitro. These results suggest an exciting future potential use of chelators in the experimental chemotherapy of visceral leishmaniasis.

Pristane (2,6,10,14-Tetramethyl-pentadecane) inhibits disease progression in Leishmania-infected Balb/c mice.

The course of Leishmania infection in pristane-primed BALB/c mice infected with either Leishmania major or Leishmania donovani was examined. Pristane-primed L. donovani infected mice had spleen parasite-loads that were 13 times less than controls. Likewise pristane-primed L. major infected animals had significantly smaller footpad lesion areas than controls. Pristane-primed mice had an atypical haematology compared to controls. To the best of our knowledge, this is the first report to demonstrate that pristane inhibits progression of disease in Leishmania-infected BALB/c mice.

Establishment of an appropriate inoculum dose of Leishmania donovani promastigotes required to establish a visceral infection in laboratory animal rodent models.

Syrian hamsters and BALB/c mice were inoculated intraperitoneally with various doses of stationary phase Leishmania donovani promastigotes derived from primary, secondary and tertiary cultures. Axenic derived amastigotes from a tertiary culture and mass-culture derived promastigotes from primary, secondary, and tertiary cultures were also used. Animals were sacrificed after 30 days incubation period and parasite-loads quantified from Giemsa stained spleen smears. A primary inoculum dose of 1 x10(8) was found to be the most appropriate in effecting a visceral infection. This parasite dose resulted in a spleen parasite-load of 10-20 amastigotes per field of microscope view at x1,000 magnification. Those involved in candidate vaccine molecules or experimental drugs against kala-azar will find these results useful.

Screening of metal ion chelators against Leishmania donovani-infected Syrian hamsters.

Leishmania donovani-infected Syrian hamsters were treated intraperitoneally with 0.23 mmoles/kg/day of EDTA, EGTA, HEEDTA and 100 mg/kg/day of Pentostam R. The control group received 0.1 ml of phosphate buffered saline. After 30 days of treatment, the animals were sacrificed. Of the Pentostam-treated animals, 5 out 6 had negative spleen cultures, while all the chelator and PBS-treated ones yielded parasites. While all the Pentostam-treated animals had negative bone marrow cultures, only 1 out of 6 HEEDTA-treated hamsters yielded parasites. Spleen, liver and bone marrow parasite- loads calculated from chelator-treated animals were consistently significantly higher than for Pentostam-treated animals. These results suggest that although metal ion chelators have some antileishmanial potential, their in vivo activity against L. donovani is low compared to Pentostam.

Comparison of Giemsa and acridine orange stains for the diagnosis of Leishmania donovani in biopsies of infected hamsters.

Identical impression smears of spleen, liver and bone marrow biopsy materials from Leishmania donovani-infected hamsters were stained using either acridine orange or Giemsa. Spleen parasite-loads calculated from the two stains for identical biopsy material were significantly different from each other. However, liver and bone marrow parasite- loads calculated from either Giemsa-stained or acridine orange-stained biopsies were not significantly different from each other. This study has shown that acridine orange, which is a quick and simple technique, has great potential in the diagnosis of kala-azar when liver and bone marrow biopsies are used.