OBJECTIVETo simultaneously detect viable Cryptosporidium parvum oocysts and Giardia duodenalis cysts for the purpose of reducing time and cost spent.

METHODSA duplex reverse transcription polymerase chain reaction (RT-PCR) method was newly developed.

RESULTSUsing duplex RT-PCR method for the hsp70 gene, viable (oo)cyst concentrations of 10(1) and 10(3) (oo)cysts/100 microL could be detected for C. parvum and G duodenalis, respectively. However, after heat-shock stimulation the expression of hsp70 mRNAs was detectable at 10(0) and 10(1) (oo)cysts/100 microL concentrations of C. parvum and G duodenalis, respectively. Thus, the detection sensitivity was significantly increased when the viable (oo)cysts were exposed to heat shock.

CONCLUSIONThis study describes a new duplex RT-PCR method for hsp70 gene to detect the viable (oo)cysts of the C. parvum and G duodenalis with less time consumed and at a lower cost. This newly developed duplex RT-PCR method may be used to detect these parasites not only in aquatic environments but also in clinical samples.

The status of intestinal parasitic infections and seroprevalence of cystic echinococcosis in the inhabitants in Dornod and Selenge, Mongolia, was observed with stool and serum samples from 165 and 683 residents from August 9 to August 15, 2003. A total of 10 inhabitants (6.1%) were found to be infected with protozoan cysts or oocysts by stool examinations; 7, 1 and 2 cases with Entamoeba coli, Giardia lamblia and Cryptosporidium parvum, respectively. A total of 62 people (9.1%) revealed significantly high levels of specific antibodies against Echinococcus granulosus by enzyme-linked immunosorbent assay (ELISA). The surveyed areas are rural areas and the sanitized tab water is not available. There is a possibility of endemic transmission of water-borne protozoan diseases in these areas. The clinical echinococcosis is needed to be searched from the seropositive inhabitants.
Protozoan Infections/*epidemiology/parasitology ; Mongolia/epidemiology ; Humans ; Giardia lamblia/isolation & purification ; Gastrointestinal Diseases/*epidemiology/*parasitology ; Entamoeba/isolation & purification ; Echinococcosis/*epidemiology ; Cryptosporidium parvum/isolation & purification ; Animals

In order to determine the distribution and prevalence of human cryptosporidiosis on western and southern coastal islands of Jeollanam-do (Province), fecal samples were collected from 2,541 people residing on 25 islands, 13 in the western coasts and 12 in the southern coasts, during July and August 2000. Fecal smears were prepared following formalin-ether sedimentation of the samples and stained by a modified acid-fast procedure. The presence of Cryptosporidium oocysts was determined by light microscopy. Cryptosporidium oocysts were detected in 38 specimens (1.5%). The oocyst positive rate varied (0-6.0%) according to island; the highest was detected on Oenarodo (6.0%), followed by Naenarodo (5.6%) and Nakwoldo (5.4%). The majority (35 persons, 94.6%) of Cryptosporidium-infected individuals were older than 50 years of age. Men (22/1,159; 1.9%) were infected at a higher rate than women (16/1,382; 1.2%). The results of the present survey indicate that human Cryptosporidium infections (due to Cryptosporidium hominis and/or C. parvum) are maintained at a relatively low prevalence on coastal islands of Jeollanam-do, Republic of Korea.
Sex Distribution ; Prevalence ; Oocysts ; Middle Aged ; Male ; Korea/epidemiology ; Infant, Newborn ; Infant ; Humans ; Female ; Feces/parasitology ; Cryptosporidium parvum/growth & development/*isolation & purification ; Cryptosporidium/growth & development/*isolation & purification ; Cryptosporidiosis/*epidemiology/parasitology ; Child, Preschool ; Child ; Animals ; Aged ; Age Distribution ; Adult ; Adolescent

The present study was undertaken to know the infection status of Cryptosporidium parvum among the residents of Chorwon-gun, Kangwon-do in 1993. Total 461 fecal samples were collected from the inhabitants residing in Chorwon-gun during the period of August 12 to September 14, 1993. Fecal smears were prepared by formalin-ether sedimentation, and examined after modified acid fast staining. Of the 461 fecal samples, 9 (1.9%) were positive for C. parvum oocysts. The positive cases were limited to thirties (4) patients, forties (3), and sixties (2), and no oocyst was detected in other age groups. The oocyst positive rate for male was 1.4% and that of female was 2.6%.
Adolescent ; Adult ; Age Factors ; Aged ; Animals ; Child ; Cryptosporidiosis/*epidemiology/parasitology ; Cryptosporidium parvum/isolation & purification ; Feces/parasitology ; Female ; Human ; Korea/epidemiology ; Male ; Middle Aged ; Parasite Egg Count ; Sex Factors

To investigate the infection status of pigs with Cryptosporidium parvum, 589 fecal samples were collected from pigs raised at farm in Chungcheongbuk-do and Chungcheongnam-do. Of the 589 pig fecal samples, 62 (10.5%) were positive for C. parvum. The area showing the highest positive rate was Dangjin-gun, Chungcheongnam-do (14.0%), and the lowest (0%) Salmi-myon, Chungcheongbuk-do. The positive rate of C. parvum in Judok-eup increased from 12.7% in the winter to 22.1% in the summer. The results of this study suggest that the pigs may be a source of human C. parvum infection.
Animal Husbandry ; Animals ; Cryptosporidiosis/epidemiology/parasitology/*veterinary ; Cryptosporidium parvum/*isolation & purification ; Feces/parasitology ; Korea/epidemiology ; Parasite Egg Count ; Swine ; Swine Diseases/*epidemiology/*parasitology

Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from 10(3) to 10(4) oocysts, and the nested PCR method was able to detect 10(0) to 10(2) oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.
Animals ; Cryptosporidiosis/diagnosis/*parasitology ; Cryptosporidium parvum/genetics/*isolation & purification ; DNA Primers/*genetics ; DNA, Protozoan/diagnostic use/genetics ; Humans ; Polymerase Chain Reaction/*methods ; Sensitivity and Specificity

Cryptosporidium parvum is a zoonotic protozoan parasite that causes cryptosporidial enteritis. Numerous outbreaks of cryptosporidiosis have been reported worldwide. Cryptosporidium is transmitted to hosts via consumption of contaminated water and food but also by direct contact with contaminated soil or infected hosts. The present study investigated farm soil collected from 34 locations along the western Korean peninsula and 24 vegetables purchased from local grocery markets in Seoul. The soil and vegetable samples were examined by real-time polymerase chain reaction (qPCR) to estimate the risk of infection. Eleven of 34 locations (32.4%) and 3 of 24 vegetable samples (12.5%) were contaminated with Cryptosporidium parvum, as confirmed by TaqI enzyme digestion of qPCR products and DNA sequencing. It is suggested that Cryptosporidium infection can be mediated via farm soil and vegetables. Therefore, it is necessary to reduce contamination of this organism in view of public health.
Base Sequence ; Cryptosporidiosis/parasitology ; Cryptosporidium parvum/*genetics/*isolation & purification ; DNA, Protozoan/analysis/genetics ; Enteritis/parasitology ; Foodborne Diseases/*parasitology ; Humans ; Sequence Alignment ; Sequence Analysis, DNA ; Soil/*parasitology ; Vegetables/*parasitology

From May to June 2012, a waterborne outbreak of 124 cases of cryptosporidiosis occurred in the plumbing systems of an older high-rise apartment complex in Seoul, Republic of Korea. The residents of this apartment complex had symptoms of watery diarrhea and vomiting. Tap water samples in the apartment complex and its adjacent buildings were collected and tested for 57 parameters under the Korean Drinking Water Standards and for additional 11 microbiological parameters. The microbiological parameters included total colony counts, Clostridium perfringens, Enterococcus, fecal streptococcus, Salmonella, Shigella, Pseudomonas aeruginosa, Cryptosporidium oocysts, Giardia cysts, total culturable viruses, and Norovirus. While the tap water samples of the adjacent buildings complied with the Korean Drinking Water Standards for all parameters, fecal bacteria and Cryptosporidium oocysts were detected in the tap water samples of the outbreak apartment complex. It turned out that the agent of the disease was Cryptosporidium parvum. The drinking water was polluted with sewage from a septic tank in the apartment complex. To remove C. parvum oocysts, we conducted physical processes of cleaning the water storage tanks, flushing the indoor pipes, and replacing old pipes with new ones. Finally we restored the clean drinking water to the apartment complex after identification of no oocysts.
Cryptosporidiosis/*epidemiology/*parasitology ; Cryptosporidium parvum/genetics/growth & development/*isolation & purification ; Disease Outbreaks ; Drinking Water/*parasitology ; Housing ; Humans ; Oocysts/growth & development ; Republic of Korea/epidemiology ; Water Supply/analysis

The present study investigated the prevalence rate of Cryptosporidium parvum as a cause of diarrhea. We examined 942 stools of unidentified reasons occurring in patients in whom no immunosuppression had been detected. We examined the stools for Cryptosporidium parvum via modified acid-fast staining. The clinical records of all of the positive patients were then analyzed. Nine (1%) of the stools among the 942 diarrheal patients were positive for C. parvum. The positive rate in the males was 1.1% (6/522) and the positive rate of the females was 0.7% (3/420). Age distribution revealed that the highest positive rates were in patients in their sixties, with a positive rate of 2.5% (4/158). In the clinical tests, levels of c-reactive protein, erythrocyte sedimentation rates, and neutrophil proportions were normally increased in the peripheral blood, whereas the lymphocyte proportion exhibited a tendency towards decrease. The pathological findings were compatible with an inflammatory reaction in the host.
Adult ; Aged ; Animals ; Child, Preschool ; Cryptosporidiosis/*epidemiology/immunology ; *Cryptosporidium/isolation & purification ; Cryptosporidium parvum/isolation & purification ; Diarrhea/*epidemiology/immunology/parasitology ; Feces/parasitology ; Female ; HIV Seronegativity ; Humans ; Immunocompetence ; Korea/epidemiology ; Lymphocyte Count ; Male ; Middle Aged ; Prevalence ; Research Support, Non-U.S. Gov't ; Staining and Labeling

Two species of Cryptosporidium are known to infect man; C. hominis which shows anthroponotic transmission between humans, and C. parvum which shows zoonotic transmission between animals or between animals and man. In this study, we focused on identifying genotypes of Cryptosporidium prevalent among inhabitants and domestic animals (cattle and goats), to elucidate transmittal routes in a known endemic area in Hwasun-gun, Jeollanam-do, Republic of Korea. The existence of Cryptosporidium oocysts was confirmed using a modified Ziehl- Neelsen stain. Human infections were found in 7 (25.9%) of 27 people examined. Cattle cryptosporidiosis cases constituted 7 (41.2%) of 17 examined, and goat cases 3 (42.9%) of 7 examined. Species characterizations were performed on the small subunit of the rRNA gene using both PCR-RFLP and sequence analysis. Most of the human isolates were mixtures of C. hominis and C. parvum genotypes and similar PCR-RFLP patterns were observed in cattle and goat isolates. However, sequence analyses identified only C. hominis in all isolates examined. The natural infection of cattle and goats with C. hominis is a new and unique finding in the present study. It is suggested that human cryptosporidiosis in the studied area is caused by mixtures of C. hominis and C. parvum oocysts originating from both inhabitants and domestic animals.
Rural Health ; Prevalence ; Polymorphism, Restriction Fragment Length ; Polymerase Chain Reaction/methods ; Mutation/genetics ; Molecular Sequence Data ; Korea/epidemiology ; Humans ; Goats ; Goat Diseases/epidemiology/*parasitology ; Genotype ; Genes, rRNA/genetics ; DNA, Protozoan/chemistry ; DNA Primers/chemistry ; Cryptosporidium parvum/genetics/isolation & purification ; Cryptosporidium/classification/*genetics ; Cryptosporidiosis/*epidemiology/*parasitology ; Cattle Diseases/epidemiology/*parasitology ; Cattle ; Base Sequence ; Animals