Access to clean and safe drinking water that is free from pathogenic protozoan parasites, especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans, is still an issue in Southeast Asia (SEA). This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA, using real-time polymerase chain reaction (qPCR) assays.
The influx of low skilled workers from socioeconomically deprived neighbouring countries to Malaysia has raised concerns about the transmission of communicable gastrointestinal diseases such as giardiasis and cryptosporidiosis to the local population. Therefore, a cross sectional study was conducted to investigate the prevalence of both diseases and the genetic diversity of these pathogens in the migrant population. Microscopic examination of faecal samples from 388 migrant workers involved in five working sectors were screened and 10.8% (n = 42) were found to be positive with Giardia spp. and 3.1% (n = 12) with Cryptosporidium spp. infections. PCR amplicons at the triosephosphate isomerase (tpi) gene were successfully obtained for Giardia duodenalis from 30 (30/388; 7.73%) samples with assemblages AII and B in 13 (13/30; 43.3%) and 17 (17/30; 56.7%) positive samples, respectively. Nine samples (9/388; 2.3%) were identified as Cryptosporidium parvum using PCR-RFLP analysis. Country of origin, duration of residence in Malaysia and working sectors significantly influenced G. duodenalis assemblage AII infections amongst the targeted population. Meanwhile, C. parvum infection was significantly associated with those working in the food service sector. Despite the low presence of pathogenic G. duodenalis and C. parvum in the study population, the results highlight the risk of anthroponotic foodborne and waterborne transmission and therefore call for implementation of control strategies through improvements in personal hygiene and sanitation standards.
This cross-sectional study determined the prevalence of cryptosporidiosis in HIV-infected patients using polymerase chain reaction (PCR). Stool specimens were collected from HIV infected patients who were admitted to Hospital Raja Perempuan Zainab II, Kota Bharu, Malaysia, for various indications from December 2004 to December 2005. A modified acid-fast stain was performed on the direct stool smears, then the stool specimens were further tested using nested PCR targeting the 18S rRNA gene of Cryptosporidium parvum, with a built-in internal control (IC). Out of 59 samples, 11 were positives. Nested PCR identified a total of nine samples (16%) compared to microscopy, which identified only three samples. All PCR negative results showed IC amplicons, suggesting that these samples were true negatives and were not due to inhibition of PCR. This study highlights the importance of molecular diagnosis in determining the true prevalence and epidemiology of C. parvum.
Cryptosporidium species are protozoan parasites that infect humans and a wide variety of animals. This study was aimed at identifying Cryptosporidium species and genotypes isolated from avian hosts. A total of 90 samples from 37 different species of birds were collected throughout a 3-month period from April 2008 to June 2008 in the National Zoo of Kuala Lumpur, Malaysia. Prior to molecular characterization, all samples were screened for Cryptosporidium using a modified Ziehl-Neelsen staining technique. Subsequently samples were analysed with nested-PCR targeting the partial SSU rRNA gene. Amplicons were sequenced in both directions and used for phylogenetic analysis using Neighbour-Joining and Maximum Parsimony methods. Although 9 (10%) samples were positive for Cryptosporidium via microscopy, 8 (8.9%) produced amplicons using nested PCR. Phylogenetic trees identified all the isolates as Cryptosporidium parvum. Although C. parvum has not been reported to cause infection in birds, and the role of birds in this study was postulated mainly as mechanical transporters, these present findings highlight the significant public health risk posed by birds that harbour the zoonotic species of Cryptosporidium.
Cryptosporidium is a protozoan parasite of humans and animals and has a worldwide distribution. The parasite has a unique epidemiology in Middle Eastern countries where the IId subtype family of Cryptosporidium parvum dominates. However, there has been no information on Cryptosporidium species in Yemen. Thus, this study was conducted in Yemen to examine the distribution of Cryptosporidium species and subtype families. Fecal samples were collected from 335 patients who attended hospitals in Sana'a city. Cryptosporidium species were determined by PCR and sequence analysis of the 18 s rRNA gene. Cryptosporidium parvum and C. hominis subtypes were identified based on sequence analysis of the 60 kDa glycoprotein (gp60) gene. Out of 335 samples, 33 (9.9%) were positive for Cryptosporidium. Of them, 97% were identified as C. parvum whilst 1 case (3%) was caused by C. hominis. All 7 C. parvum isolates subtyped belonged to the IIaA15G2R1 subtype. The common occurrence of the zoonotic IIa subtype family of C. parvum highlights the potential occurrence of zoonotic transmission of cryptosporidiosis in Yemen. However, this postulation needs confirmation with future molecular epidemiological studies of cryptosporidiosis in both humans and animals in Yemen.
The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.
Cryptosporidium is an intestinal protozoan parasite which causes diarrhoea in animals and has recently been reported to cause similar symptoms in man. Cryptosporidiosis is a zoonotic infection and the first human case was reported in 1976.2 Since then the number of cases has increased by the hundreds especially among patients with acquired immuno-deficiency syndrome (AIDS), as a result of the severe symptoms that they cause in the im-munosuppressed patients and also due to the better screening techniques which have been developed resulting in the detection of cases.3 To date 20 species of Cryptosporidium have been reported but these cannot be differentiated morphologically. The differences are based mainly on the host from which the parasites were recovered. The species responsible for causing infection in man is Cryptosporidium parvum.4 Of the 20 species reported several are invalid because the oocysts of some of them were found to be the same as the sporocyst of Sarcocystis. It is now concluded that there are only two species that infect mammals (C. Muris and C. parvum). (Copied from article).
The aim of this prospective study was to determine the prevalence of Cryptosporidium parvum in hospitalized children in Kota Bharu, Malaysia. Over a 19 month study period, 258 stool samples were examined from 159 children; 109 with diarrhea and 50 controls without diarrhea. Modified Ziehl-Neelsen staining method and a polymerase chain reaction (PCR) assay were used to detect C. parvum and the samples were also examined for the presence of other intestinal parasites. Only 1 of the 109 (0.9%) children with acute diarrhea was positive for C. parvum by microscopy and PCR. Thirty-one percent of children were infested with other intestinal parasites, the most common being Ascaris lumbricoides and Trichuris trichiura. In conclusion, we found C. parvum to be an uncommon infective agent in hospitalized children with or without diarrhea in Kota Bharu, Malaysia.
A total of 1,885 blood and stool samples of four main protozoan parasitic infections were retrospectively reviewed from January, 2000 to April, 2004. Eleven of the 1,350 stool samples were shown positive for Cryptosporidium and Giardia infections; one of the 5 cases was clinically diagnosed as gastrointestinal cryptosporidiosis, while 6 cases were giardiasis. In patients with giardiasis, children were among the high-risk groups, making up 66.7% of these patients. The common presenting signs and symptoms were: diarrhea (83.3%), loss of appetite (83.3%), lethargy (83.3%), fever (66.7%), nausea/vomiting (50.0%), abdominal pain (16.7%), dehydration (16.7%) and rigor and chills (16.7%). Metronidazole was the drug of choice and was given to all symptomatic patients (83.3%). For the blood samples, 28 of the 92 peripheral smears for Plasmodium spp infection were diagnosed as malaria. The age range was from 4 to 57, with a median of 32.5 years. The sex ratio (M:F) was 3.6:1, while the age group of 30-44 years was the most commonly affected in both sexes. The majority of patients were foreigners (60.7%) and non-professional (39%). Plasmodium vivax (71%) infection was the most common pathogen found in these patients, along with a history of traveling to an endemic area of malaria (31%). The predominant presenting signs and symptoms were: fever (27%), rigor and chills (24%), nausea/vomiting (15%) and headache (8%). Chloroquine and primaquine was the most common anti-malarial regimen used (78.6%) in these patients. The seroprevalence of toxoplasmosis in different groups was 258/443 (58%): seropositive for IgG 143 (32.3%); IgM 67 (15%); and IgG + IgM 48 (10.8%). The age range was from 1 to 85, with a mean of 34 (+/- SD 16.6) years. The predominant age group was 21 to 40 years (126; 28.4%). The sex ratio (M:F) was 1.2:1. Subjects were predominantly male (142; 32%) and the Malay (117; 26.4%). Of these, 32 cases were clinically diagnosed with ocular toxoplasmosis. The range of age was from 10 to 56 years with a mean of 30.5 (+/- SD 12.05) years. The sex ratio (M:F) was 1:1.7. The majority were in the age group of 21 to 40 years, female (20; 62.5%), and Malay (17; 53%). They were also single (16; 50%), unemployed (12; 37%), and resided outside Kuala Lumpur (21; 65.6%). The more common clinical presentations were blurring of vision (25; 78%), floaters (10; 31%) and pain in the eye (7; 22%). We found that funduscopic examination (100%) and seropositivity for anti-Toxoplasma antibodies (93.7%) were the main reasons for investigation. Choroidoretinitis was the most common clinical diagnosis (69%), while clindamycin was the most frequently used antimicrobial in all cases. Among HIV-infected patients, 10 cases were diagnosed as AIDS-related toxoplasmic encephalitis (TE) (9 were active and 1 had relapse TE). In addition, 1 case was confirmed as congenital toxoplasmosis.
Intestinal protozoa are increasingly being studied because of their association with acute and chronic diarrhoea in immunocompromised as well as immunocompetent patients. Various community outbreaks due to contamination of water or food with these protozoa have further highlighted their importance in public health. Among these important pathogens are Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli, and microsporidia. Except for the cyst-forming G. duodenalis and E. histolytica, the others are intracellular and form spores which are passed out with the faeces. These organisms are also found in various animals and birds and zoonotic transmission is thought to occur. These infections are distributed worldwide, with a higher prevalence in developing compared to developed countries. However, the relative importance of zoonotic infections especially in developing countries has not been studied in detail. The prevalence rates are generally higher in immunodeficient compared to immunocompetent patients. Higher prevalence rates are also seen in rural compared to urban communities. Most studies on prevalence have been carried out in developed countries where the laboratory and other health infrastructure are more accessible than those in developing countries. This relative inadequacy of laboratory diagnosis can affect accurate estimates of the prevalence of these infections in developing countries. However, reports of these infections in travellers and workers returning from developing countries can provide some indication of the extent of these problems. Most studies on prevalence of amoebiasis in developing countries were based on morphological identification of the parasite in faecal smears. As the pathogenic E. histolytica is morphologically indistinguishable from that of non-pathogenic E. dispar, estimates of amoebiasis may not be accurate. The epidemiology of human microsporidia infections is not completely understood. Two species, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans and it is believed that human to human as well as animal to human infections occur. However, the importance of zoonotic infections has not been fully characterised. G. duodenalis cysts, microsporidia and Cryptosporidium oocysts have been detected in various ground water resources, but their role in community outbreaks and maintenance of the infection has not been fully characterised. The taxonomic classification and pathogenic potential of B. hominis are still controversial. While considered by many as yeast, fungi or protozoon, recent sequence analysis of the complete SSUrRNA gene has placed it within an informal group, the stramenopiles. This review covers recent published data on these zoonotic infections and examines their public health importance in Asian countries.