OBJECTIVES: To determine the prevalence and risk factors associated with intestinal parasites in the population of San Juan Cosala, Jalisco, Mexico. METHODS: A total of 277 samples from 104 participants were analysed using direct smear, flotation, formaldehyde/ethyl acetate, and modified Kinyoun’s acid-fast stain methods. The Graham method was applied only for samples from children under 12 years of age for the diagnosis of Enterobius vermicularis. RESULTS: The prevalence of parasite infections in the study population was 77.9% including: Entamoeba histolytica/E. dispar/E. moshkovskii/E. bangladeshi (37.5%), Giardia intestinalis (11.5%); commensals: Endolimax nana (44.2%), Entamoeba coli (27.9%), Chilomastix mesnili (6.7%) and Iodamoeba bütschlii, (2.9%); emerging intestinal protozoans: Blastocystis spp. (49%), Cryptosporidium spp. (7.7%) and Cyclospora cayetanensis (2.9%); and helminths: Enterobius vermicularis (18.3%) and Ascaris lumbricoides (5.8%). The results also showed that 58.64% of the studied population presented polyparasitism. A significant association was found between protozoan infections and housewives, and houses that were not built with concrete ceilings, brick walls and cement floors (p < 0.05). CONCLUSION: Polyparasitism was observed in over half the study population. The most prevalent parasite was Blastocystis spp, whilst the prevalence of helminths was less than that of protozoans. The risk factors for infection to intestinal parasites were being a housewife and not having solid brick, cement and concrete materials for house construction.
Adult ; Ascaris lumbricoides ; Blastocystis ; Child ; Cryptosporidium ; Cyclospora ; Diagnosis ; Endolimax ; Entamoeba ; Enterobius ; Giardia lamblia ; Helminths ; Humans ; Methods ; Mexico ; Parasites ; Prevalence ; Protozoan Infections ; Retortamonadidae ; Risk Factors

A 3-month-old female Maltese puppy was hospitalized with persistent diarrhea in a local veterinary clinic. Blood chemistry and hematology profile were analyzed and fecal smear was examined. Diarrheal stools were examined in a diagnostic laboratory, using multiplex real-time polymerase chain reaction (PCR) against 23 diarrheal pathogens. Sequence analysis was performed using nested PCR amplicon of 18S ribosomal RNA. Coccidian oocysts were identified in the fecal smear. Although multiplex real-time PCR was positive for Cyclospora cayetanensis, the final diagnosis was Cystoisospora ohioensis infection, confirmed by phylogenetic analysis of 18S rRNA. To our knowledge, this the first case report of C. ohioensis in Korea, using microscopic examination and phylogenetic analysis.
Animals ; Chemistry ; Cyclospora ; Diagnosis ; Diarrhea ; Dogs ; Female ; Hematology ; Humans ; Infant ; Korea ; Oocysts ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; RNA, Ribosomal, 18S ; Sequence Analysis

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×10³ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.
Bacteriophage T4 ; Cryptosporidium parvum ; Cryptosporidium ; Cyclospora ; Diagnosis ; Diarrhea ; DNA ; Fluorescent Dyes ; Giardia lamblia ; Giardia ; Glutamate Dehydrogenase ; Humans ; Limit of Detection ; Methods ; Multiplex Polymerase Chain Reaction ; Oocysts ; Parasites ; Real-Time Polymerase Chain Reaction

Cyclospora cayetanensis is a foodborne and waterborne pathogen that causes endemic and epidemic human diarrhea worldwide. A few epidemiological studies regarding C. cayetanensis infections in China have been conducted. During 2013, a total of 291 stool specimens were collected from patients with diarrhea at a hospital in urban Shanghai. C. cayetanensis was not detected in any of the stool specimens by traditional microscopy, whereas five stool specimens (1.72%, 5/291) were positive by PCR. These positive cases confirmed by molecular technology were all in the adult group (mean age 27.8 years; 2.94%, 5/170) with watery diarrhea. Marked infection occurred in the rainy season of May and July. Sequence and phylogenetic analyses of the partial 18S rRNA genes of C. cayetanensis isolated showed intra-species diversity of this parasite. This study showed, for the first time, that C. cayetanensis is a pathogen in outpatients with diarrhea in Shanghai, albeit at a low level. However, the transmission dynamics of this parasite in these patients remain uncertain.
Adolescent ; Adult ; China ; epidemiology ; Cyclospora ; genetics ; isolation & purification ; Cyclosporiasis ; epidemiology ; Diarrhea ; etiology ; parasitology ; Feces ; parasitology ; Female ; Humans ; Male ; Middle Aged ; Outpatients ; Phylogeny ; Polymerase Chain Reaction ; RNA, Ribosomal, 18S ; analysis ; Retrospective Studies ; Young Adult

Cryptosporidium and Cyclospora are well-known coccidian protozoa that can cause waterborne and foodborne diarrheal illnesses. There have been a few reports regarding contamination in different vegetables with Cryptosporidium, but no data are available regarding the sources of Cyclospora infections in Korea. In the present study, we collected 6 kinds of vegetables (perilla leaves, winter-grown cabbages, chives, sprouts, blueberries, and cherry tomatoes) from July 2014 to June 2015, and investigated contamination by these 2 protozoa using multiplex quantitative real-time PCR. Among 404 vegetables, Cryptosporidium and Cyclospora were detected in 31 (7.7%) and 5 (1.2%) samples, respectively. In addition, Cryptosporidium was isolated from all 6 kinds of vegetables, whereas Cyclospora was detected in 4 kinds of vegetables (except perilla leaves and chives). Cryptosporidium (17.8%) and Cyclospora (2.9%) had the highest detection rates in chives and winter-grown cabbages, respectively. Cryptosporidium was detected all year long; however, Cyclospora was detected only from October to January. In 2 samples (sprout and blueberry), both Cryptosporidium and Cyclospora were detected. Further investigations using TaqI restriction enzyme fragmentation and nested PCR confirmed Cryptosporidium parvum and Cyclospora cayetanensis, respectively. In conclusion, we detected C. cayetanensis in vegetables for the first time in Korea. This suggests that screening should be employed to prevent these protozoal infections in Korea.
Blueberry Plant ; Brassica ; Chive ; Cryptosporidium parvum ; Cryptosporidium* ; Cyclospora* ; Korea* ; Mass Screening ; Perilla ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Vegetables*

A field survey studying intestinal parasites in humans and microbial pathogen contamination at environment was performed in a Laotian rural village to identify potential risks for disease outbreaks. A parasitological investigation was conducted in Ban Lak Sip village, Luang Prabang, Lao PDR involving fecal samples from 305 inhabitants as well as water samples taken from 3 sites of the local stream. Water analysis indicated the presence of several enteric pathogens, i.e., Aeromonas spp., Vibrio spp., E. coli H7, E. coli O157: H7, verocytotoxin-producing E. coli (VTEC), Shigella spp., and enteric adenovirus. The level of microbial pathogens contamination was associated with human activity, with greater levels of contamination found at the downstream site compared to the site at the village and upstream, respectively. Regarding intestinal parasites, the prevalence of helminth and protozoan infections were 68.9% and 27.2%, respectively. Eight helminth taxa were identified in fecal samples, i.e., 2 tapeworm species (Taenia sp. and Hymenolepis diminuta), 1 trematode (Opisthorchis sp.), and 5 nematodes (Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, trichostrongylids, and hookworms). Six species of intestinal protists were identified, i.e., Blastocystis hominis, Cyclospora spp., Endolimax nana, Entamoeba histolytica/E. dispar, Entamoeba coli, and Giardia lamblia. Questionnaires and interviews were also conducted to determine risk factors of infection. These analyses together with a prevailing infection level suggested that most of villagers were exposed to parasites in a similar degree due to limited socio-economic differences and sharing of similar practices. Limited access to effective public health facilities is also a significant contributing factor.
Adenoviridae ; Aeromonas ; Ancylostomatoidea ; Ascaris lumbricoides ; Blastocystis hominis ; Cestoda ; Cyclospora ; Disease Outbreaks ; Endolimax ; Entamoeba ; Entamoeba histolytica ; Giardia lamblia ; Helminths ; Human Activities ; Humans ; Hymenolepis ; Parasites ; Prevalence ; Protozoan Infections ; Public Health ; Risk Factors ; Rivers ; Shigella ; Strongyloides stercoralis ; Trichuris ; Vibrio ; Water*

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler’s diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×10³ oocysts for C. parvum, >1×10⁴ cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.
Cryptosporidium parvum* ; Cryptosporidium* ; Cyclospora* ; Diarrhea* ; Genes, rRNA ; Giardia lamblia* ; Giardia* ; Glutamate Dehydrogenase ; Humans ; Methods ; Multiplex Polymerase Chain Reaction ; Oocysts ; Parasites ; Polymerase Chain Reaction* ; RNA, Ribosomal, 18S

The genus Sarcocystis is not usually considered as an important enteric pathogen in immune compromised patients. It might be expected that species for which humans are the final host (Sarcocystis hominis and Sarcocystis suihominis as well as possibly others) would be encountered increasingly often in immunodeficient persons. This study aimed to address how to detect and differentiate Sarcocystis oocysts and/or sporocysts from enteric protozoans in the diarrheal samples of immunodeficient patients in Shiraz, Iran. Diarrheal samples of 741 immunodeficient patients with recurrent persistent or chronic diarrhea were examined by microscopy and molecular biological analysis. Oocysts-positive samples were 68 Cryptosporidium spp., 9 Cystoisospora belli (syn. Isospora belli), 2 Cyclospora cayetanensis, and 15 microsporidia (Enterocytozoon bieneusi). Sarcocystis-like sporocysts found from a woman were identified as Sarcocystis cruzi through 18S rDNA amplification and phylogenetic analysis. To the best of our knowledge, this is the first report of S. cruzi from a human.
Cryptosporidium ; Cyclospora ; Diarrhea ; DNA, Ribosomal ; Female ; Humans ; Iran* ; Isospora ; Microscopy ; Microsporidia ; Oocysts* ; Polymerase Chain Reaction ; Prevalence* ; Sarcocystis*

Graphis upretii is a new lichen species discovered in Vietnam. The species is characterized by a loosely corticate, rough, whitish grey to greyish green thallus, elongate and irregularly branched lirellae with an apically thin complete thalline margin (negrosina morph), laterally carbonized, entire proper exciple, clear hymenium, hyaline, 16~20 transversely locular ascospores, and about 50~95 x 10~15 microm in size. In addition, members of the taxon produce norstictic and stictic acids. Currently, the lichen flora of Vietnam include Arthonia radiata, Brigantiaea tricolor, Coenogonium implexum, Dirina paradoxa, Herpothallon sipmanii, Pertusaria pertusa, and Sarcographina cyclospora.
Carbon ; Classification ; Cyclospora* ; Hyalin ; Lichens* ; Vietnam*

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 101 to 102 oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.
Cryptosporidium/*isolation & purification ; Cyclospora/*isolation & purification ; DNA Primers/genetics ; DNA Restriction Enzymes/metabolism ; DNA, Protozoan/genetics/metabolism ; Humans ; Microsporidia/*isolation & purification ; Parasitology/*methods ; Polymerase Chain Reaction/*methods ; Polymorphism, Restriction Fragment Length ; Sensitivity and Specificity ; Water/*parasitology