Cyclospora cayetanensis: review of an emerging intestinal pathogen

Cyclospora cayetanensis is an emerging pathogen. It is a new human coccidian agent of intestinal disease. Twenty years ago, the first known human cases of cyclosporiasis were reported in the medical literature. Cyclosporiasis occurs in persons of all ages and either in immunocompetent or immunocompromised hosts. The most characteristic feature of this infection is a syndrome of acute or chronic diarrhea. This parasite has a world-wide distribution. In previous reports, Cyclospora cayetanensis was associated with prolonged diarrhea in travellers, returning from developing countries. However, Cyclospora infection has recently been reported in non travellers in the United States and Canada. Cyclospora can be transmitted by ingestion of water or food contaminated with oocysts. The life cycle of Cyclospora cayetanensis is not fully known. Diagnosis of cyclosporiasis is made by direct examination of stool samples. To date, oral trimethoprim-sulfamethoxazole is the only effective treatment for Cyclospora infection.     

Evaluation of Streck tissue fixative, a nonformalin fixative for preservation of stool samples and subsequent parasitologic examination

We undertook a study to evaluate Streck tissue fixative (STF) as a substitute for formalin and polyvinyl alcohol (PVA) in fecal preservation. A comparison of formalin, PVA, (mercuric chloride based), and STF was done by aliquoting fecal samples into each fixative. Stool specimens were collected in Haiti, and parasites included Cyclospora cayetanensis, Giardia intestinalis, Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, and Necator americanus. Preserved stools were examined at various predetermined times (1 week, 1 month, and 3 months) to establish the quality of the initial preservation as well as the suitability of the fixative for long-term storage. At each time point, stool samples in fixatives were examined microscopically as follows: (i) in wet mounts (with bright-field and epifluorescence microscopy), (ii) in modified acid-fast-, trichrome-, and safranin-stained smears, and (iii) with two commercial test kits. At the time points examined, morphologic features remained comparable for samples fixed with 10% formalin and STF. For comparisons of STF- and 10% formalin-fixed samples, specific findings showed that Cyclospora oocysts retained full fluorescence, modified acid-fast- and safranin-stained smears of Cryptosporidium and Cyclospora oocysts were equal in staining quality, and results were comparable in the immunofluorescence assay and enzyme immunoassay commercial kits. Stool fixed in STF and stained with trichrome showed less-than-acceptable staining quality compared with stool fixed in PVA. STF provides an excellent substitute for formalin as a fixative in routine examination of stool samples for parasites. However, modifications to the trichrome staining procedures will be necessary to improve the staining quality for protozoal cysts fixed in STF to a level comparable to that with PVA.     

A case of human cyclosporiasis causing traveler's diarrhea after visiting Indonesia

This is the first case of human cyclosporiasis reported in Korea. We detected the oocyst of Cyclospora cayetanensis from a 14-yr old girl who complained of persistent diarrhea after traveling to Indonesia. Round oocysts sized about 8 to 9 m with wrinkle on the wall were found in modified acid fast stained stool specimen. Stainability was variable from red to pale. Oocyst wall showed typical autofluorescence under ultraviolet illumination. The exact diagnosis for the cause of diarrhea and treatment for this patient were not provided at the right moment from the hospital since the diagnostic system for the Cyclospora infection was not ready in the clinical laboratory of the hospital. More attention should be paid on Cyclospora as a cause of diarrhea especially for those returning from a trip to the tropics and an adequate diagnostic system for the Cyclospora infection should be implemented in clinical laboratories as soon as possible.     

Cyclospora cayetanensis infection in Lagos, Nigeria

Although reports of Cyclospora infection continue to increase globally, few cases have been reported from the African continent. We present 11 cases of cyclosporiasis detected from stool samples submitted to seven major hospital laboratories in Lagos, Nigeria between March 1999 and April 2000.     

Improved stool concentration procedure for detection of Cryptosporidium oocysts in fecal specimens.

Epidemiologic and laboratory data suggest that coprodiagnostic methods may fail to detect Cryptosporidium oocysts in stool specimens of infected patients. To improve the efficacy of stool concentration procedures, we modified different steps of the Formalin-ethyl acetate (FEA) stool concentration technique and evaluated these modifications by examining stool samples seeded with known numbers of Cryptosporidium oocysts. Because these modifications failed to improve oocyst detection, we developed a new stool concentration technique that includes FEA sedimentation followed by layering and flotation over hypertonic sodium chloride solution to separate parasites from stool debris. Compared with the standard FEA procedure, this technique improved Cryptosporidium oocyst detection. The sensitivities of the two concentration techniques were similar for diarrheal (watery) stool specimens (100% of watery stool specimens seeded with 5,000 oocysts per g of stool were identified as positive by the new technique, compared with 90% of stools processed by the standard FEA technique). However, the most significant improvement in diagnosis occurred with formed stool specimens that were not fatty; 70 to 90% of formed stool specimens seeded with 5,000 oocysts were identified as positive by the new technique, compared with 0% of specimens processed by the standard FEA technique. One hundred percent of formed specimens seeded with 10,000 oocysts were correctly diagnosed by using the new technique, while 0 to 60% of specimens processed by the standard FEA technique were found positive. Similarly, only 50 to 90% of stool specimens seeded with 50,000 oocysts were identified as positive by using the standard FEA technique, compared with a 100% positive rate by the new technique. The new stool concentration procedure provides enhanced detection of Cryptosporidium oocysts in all stool samples.     

Cyclosporiasis in an infant

This report describes cyclosporiasis in a seven month old infant who presented with incessant crying and refusal of feeds. The routine modified ZN stained smears showed the oocysts of Cyclospora when all other tests failed to reveal enteric pathogens. The need for the clinical laboratory to screen faeces samples for all possible pathogens in a given clinical situation needs to be emphasized.     

Cryptosporidium antigen detection in human feces by reverse passive hemagglutination assay.

A reverse passive hemagglutination (RPH) assay was developed for Cryptosporidium oocyst antigen with an antioocyst monoclonal antibody (MAb; MAb-C1) coupled to stabilized sheep erythrocytes. RPH was compared with microscopy of auramine-phenol-stained smears of 56 oocyst-positive fecal samples, each of which was tested blindly by RPH with two oocyst-negative samples received on the same day (a total of 112 controls). Thirty-nine additional fecal samples from human immunodeficiency virus type 1 antibody-positive patients with diarrhea (10 of which were positive in auramine-phenol-stained smears) were stored at -20 degrees C before testing. Thirty specimens with a variety of other fecal pathogens (all negative for oocysts) were also tested. Of the 237 samples tested, 69 were positive by one or both methods: 65 by RPH and 66 by microscopy. The kappa coefficient of agreement between the methods was very high at 0.926. The sensitivity of RPH was 93.9%, the specificity was 98.2%, the positive predictive value was 95.4%, and the negative predictive value was 97.7%. Visible oocyst numbers and RPH titers were measured after storage of fecal samples and oocyst concentrates for 8 days at 4 degrees C. Oocyst morphology was generally poor in specimens from the human immunodeficiency virus type 1 antibody-positive group, and it degenerated during the 8-day storage experiments. MAb-C1-reactive antigen eluted from oocysts to give progressively higher reciprocal titers during storage, and it was partially removed from the oocysts by concentration. RPH is a promising technique for the detection of Cryptosporidium antigen in human feces and may be useful when specimens are stored before testing. Studies of the sensitivity of Cryptosporidium immunoassays should take into account the possible release of antigen from oocysts.     

Detection of Cyclospora cayetanensis in travellers returning from the tropics and subtropics using microscopy and real-time PCR

We examined 100 stool specimens of returning travellers with diarrhoea for the presence of Cyclospora cayetanensis using fluorescence microscopy and real-time PCR. C. cayetanensis was found in four cases with microscopy and PCR. One additional sample was positive only by PCR, and could be confirmed by microscopic examination of several additional slides. C. cayetanensis was the most frequent parasitic cause of diarrhoea after Giardia duodenalis.     

Evaluation of an antigen capture enzyme-linked immunosorbent assay for detection of Cryptosporidium oocysts.

The diagnosis of the small (4- to 6-microns) Cryptosporidium oocysts is labor intensive and relies on stool concentration, with subsequent staining and microscopy. The primary purpose of this study was to evaluate the clinical utility of an antigen capture enzyme-linked immunosorbent assay (ELISA) (LMD Laboratories, Carlsbad, Calif.) in detecting Cryptosporidium oocysts in human stools. A total of 591 specimens (76 diarrheal, 515 control) obtained from 213 inhabitants of an urban slum in northeastern Brazil were examined by both ELISA and conventional microscopic examination (CME) of formalin-ethyl acetate-concentrated stool samples stained with modified acid-fast and auramine stains. Forty-eight diarrheal stools (63.2%) were positive for Cryptosporidium oocysts by CME, with 40 of these positive by ELISA. Thirty-five control stools (6.8%) had Cryptosporidium oocysts detected by CME, with 15 of these also positive by ELISA. All of the 480 nondiarrheal stools and all but one of the diarrheal stools negative by CME were negative by ELISA. The test had an overall sensitivity of 66.3% and a specificity of 99.8% (positive predictive value, 98.2%; negative predictive value, 94.8%). In the evaluation of human diarrheal stool samples, the test sensitivity increased to 83.3%, with a specificity of 96.4%, and, in analysis of samples from individual patients with diarrhea, the sensitivity was 87.9%, with a specificity of 100%. These results indicate that this stool ELISA is sensitive and specific for the detection of Cryptosporidium oocysts in human diarrheal stool specimens but has limited use in epidemiologic studies for the diagnosis of asymptomatic Cryptosporidium infection.     

Detection of astrovirus in pediatric stool samples by immunoassay and RNA probe.

Two new astrovirus assays, a rapid biotin-avidin enzyme immunoassay (EIA) and RNA probe hybridization, were developed and compared with an established astrovirus assay, an indirect EIA, and immune electron microscopy. Sensitivity and specificity were evaluated by using a screening panel of 22 astrovirus-positive and 305 astrovirus-negative fecal specimens. The biotin-avidin assay was equivalent in performance to the reference indirect assay, and both could detect about 10 ng of viral protein. Although the probe was more sensitive than either EIA and could detect higher dilutions of virus in tissue culture and stool specimens, it did not detect more astrovirus-positive fecal specimens. Of the 22 astrovirus-positive specimens detected by the EIAs, 20 were confirmed by immune electron microscopy with hyperimmune rabbit antiserum. To determine the usefulness of EIAs for large epidemiologic studies, EIAs were used to screen 1,289 stool specimens from three studies of children with and without diarrhea. Astrovirus was detected in 3.5% of specimens from children with diarrhea and 1.9% of specimens from those without diarrhea. Our results indicate that the biotin-avidin EIA is an efficient, sensitive, and specific method for routinely screening large numbers of fecal samples and that its application in epidemiologic studies may yield higher rates of astrovirus infection than have been found previously by other methods.     

Cyclospora cayetanensis presence in aquatic surroundings in Hanoi (Vietnam). Environmental study (well water, lakes and rivers)

Cyclospora cayetanensis is a pathogenic agent originating from the intertropical zone. It causes diarrhoeal diseases in local populations as well as in travellers visiting these zones. In the first part of this work, an epidemiological study on drinking water supply (reservoirs and consumers' taps) was conducted in Hanoi over 12 months; samples were daily collected and have revealed the presence of Cyclospora cayetanensis oocysts during the whole year in tanks and only during monsoon season. Molecular methods were used for species identification. In the second part, we tried to investigate different water sources in Hanoi city in order to detect Cyclospora cayetanensis environmental contamination: groundwaters, surface waters collected in lakes and rivers and also waters from treatment plants. Our results show that none Cyclospora cayetanensis oocyst was found in the groundwaters and in the desinfected finished waters after treatment. In contrast, in rivers and lakes samples, the level of positivity reached about 63.6% with significant differences between the districts regarding the rates of oocysts recovery: only 24% positive specimens in Hoan Kiem district, whilst respectively 80.4%, 78.3% and 65% positive samples in Hai Ba Trung, Dong Da and Ba Dinh districts. The results of this study seem to confirm that environmental water is contaminated by Cyclospora cayetanensis oocysts and points out the importance of water as a significant source of human transmission. It is quite obvious that observation could be probably extended to the other endemic areas.     

Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens.

Cryptosporidium sp. is a ubiquitous 4- to 6-micron protozoan parasite infecting the intestinal tract of humans. It causes mild to fulminant diarrhea in patients, especially immunocompromised persons, and it may be hard to detect by microscopic fecal examination. An indirect, double-antibody enzyme-linked immunosorbent assay (ELISA) was developed using specifically produced goat and rabbit antisera to detect Cryptosporidium antigens in human feces. Of 62 frozen stools from patients with cryptosporidiosis, as detected by at least two microscopic diagnostic techniques, 51 were positive by ELISA; all ELISA-negative specimens came from patients with fewer than five oocysts per 0.01 ml of concentrated fecal sample examined after modified acid-fast or fluorescent monoclonal antibody staining. A total of 182 specimens from persons without Cryptosporidium infection were negative by ELISA in 176 instances; 3 ELISA-positive specimens came from patients with cryptosporidiosis diagnosed earlier. The sensitivity of the assay was 82.3%, and specificity was 96.7%. The predictive value of a positive ELISA was 89.5%, and the predictive value of a negative ELISA was 94.2%. The ELISA was not affected by the presence of eight other intestinal parasites but was sometimes affected by repeated freezing and thawing of fecal specimens. All fecal specimens were heated to 100 degrees C for 2 min to reduce proteolytic enzyme activity, although the necessity of this step needs further evaluation. This first-generation ELISA is a simple, rapid, easily standardized test for Cryptosporidium antigens in stool samples which will be useful for diagnosis and for large-scale epidemiologic studies.     

Epidemiology of Cyclospora cayetanensis and other intestinal parasites in a community in Haiti

We conducted an exploratory investigation in a community in Haiti to determine the prevalence of Cyclospora cayetanensis infection and to identify potential risk factors for C. cayetanensis infection. In 2001, two cross-sectional stool surveys and a nested case-control study were conducted. In 2002, a follow-up cross-sectional stool survey was conducted among children < or =10 years of age. Stool specimens from study participants and water samples from their wells were examined for Cyclospora and other intestinal parasites. In stools, the prevalence of infection with Cyclospora in persons of all ages decreased from 12% (20 of 167 persons) in February 2001 to 1.1% (4 of 352 persons) in April 2001, a 90.8% decrease. For children < or =10 years of age, the prevalence rates were 22.5% (16 of 71 children) in February 2001, 3.0% (4 of 135 children) in April 2001, and 2.5% (2 of 81 children) in January 2002. Use of the water from the artesian well in the northern region of the community versus the one in the south was the only risk factor associated with Cyclospora infection in multivariate analyses (odds ratio, 18.5; 95% confidence interval, 2.4 to 143.1). The water sample from one of the nine wells or water sources tested (one sample per source) in January 2001, shortly before the investigation began, was positive for Cyclospora by UV fluorescence microscopy and PCR. None of the water samples from the 46 wells or water sources tested during the investigation (one sample per source per testing period, including the artesian wells) were positive for Cyclospora. Further studies are needed to assess the role of water as a possible risk factor for Cyclospora infection in Haiti and other developing countries.     

Equine<Emphasis Type="Italic"> Cryptosporidium parvum</Emphasis> infections in western Poland

A total of 564 fecal specimens from 318 horses used for recreational riding, child hippotherapy, and racing at ten commercial and government-run stables in western Poland were tested for Cryptosporidium parvum oocysts by microscopic examination of Ziehl–Neelsen stained smears, enzyme immunoassay, and combined direct immunofluorescent antibody and fluorescent in situ hybridization. Also, seven stool specimens from five personnel who had repeated contact with these horses were tested for C. parvum oocysts. Eleven horses that shed C. parvum oocysts were found in five of ten stables (50%). The prevalence of infection varied from 0% to 11.5%. The overall prevalence of equine C. parvum-associated cryptosporidiosis in the Wielkopolska region of western Poland was 3.5%. C. parvum oocysts were found only in fecal samples from mature horses, the number of oocysts was low, and infections were not associated with clinical signs. Oocysts were not found in human fecal specimens.     

Investigation of Cryptosporidium spp. antigen by ELISA method in stool specimens obtained from patients with diarrhea

Cryptosporidium spp. is an important parasitic protozoan causing diarrhea in developing and developed countries. The agent causes severe life-threatening diarrhea especially in immunocompromised hosts. Diagnosis of the Cryptosporidium oocyst in stool samples by conventional microscopy is labor-intensive and time-consuming. Thus, we aimed to evaluate the usefulness of a copro-antigen enzyme-linked immunosorbent assay (ELISA) test in detecting Cryptosporidium spp. from fecal specimens. For this aim, microscopy and specific antigen detection methods were compared to determine Cryptosporidium spp. In addition, specific antigen by ELISA method in stool was investigated in order to find out whether or not it contributes to the diagnosis of Cryptosporidium spp. One hundred and fifty-four stool specimens taken from patients whose ages ranged from 0 to 86 with diarrhea applied to Department of Parasitology, Balcali Hospital of Cukurova University in Adana, Turkey were used. All samples were examined for Cryptosporidium spp. antigen by ELISA and oocysts via gold standard modified acid-fast staining, between October 2008 and July 2009. Eight (5.19%) specimens were found to be positive by modified acid-fast staining method and 37 (24.03%) specimens by copro-antigen ELISA method were found to be positive. The sensitivity and specificity for copro-antigen ELISA were 100% and 80.1%, respectively. The results of copro-antigen ELISA indicate that the simple, rapid, reliable, and standardized immunoassay test is sensitive and specific for routine diagnosis and may be useful for large-scale epidemiological studies of cryptosporidiosis.     

Prevalence and molecular characterization of human and bovine <Emphasis Type="Italic">Cryptosporidium</Emphasis> isolates in Thailand

This study was performed to determine the prevalence and genotypes of Cryptosporidium species among HIV patients and cattle in Thailand. Stool specimens were collected from 46 HIV patients from Prabat Nampu Temple, Lop Buri Province in central Thailand. Two hundred fecal samples from dairy cattle were collected from seven farms in Chon Buri Province, the eastern part of Thailand. Each sample was concentrated by Sheather’s sucrose flotation technique and stained by acid fast stain (AFS) for the identification of oocysts by microscopy. All HIV stool samples and 83 fecal specimens from cattle were further tested using nested polymerase chain reaction (PCR) targeting the 18S SSUrRNA gene to characterize the detected species. In HIV patient samples, the detection rate was 28.7% by AFS and 4.35% by nested PCR. In cattle samples, the detection rate was 13% by AFS and 9.63% by nested PCR. After DNA sequencing results, we identified the genotypes of the Cryptosporidium from seven of the PCR positive samples. All were found to be C. parvum. The findings presented here represent the first genetic identification of Cryptosporidium species in cattle in Thailand.     

Direct appraisal of latex agglutination testing, a convenient alternative to enzyme immunoassay for the detection of rotavirus in childhood gastroenteritis, by comparison of two enzyme immunoassays and two latex tests.

During February and March 1984, 207 fecal samples from infants and children with gastroenteritis were tested for rotavirus with four techniques: two enzyme immunoassays (Rotazyme; Abbott Laboratories, North Chicago, Ill., and Enzygnost-Rotavirus; Calbiochem-Behring, La Jolla, Calif.) and two latex agglutination tests (Rotalex; Orion Research, Inc., Cambridge, Mass., and Slidex Rota-Kit; Biomérieux). All stool samples were also tested for yeasts and bacterial pathogens. Electron microscopy was used to investigate discrepant results. We found 47% positive samples with Enzygnost-Rotavirus, 38% with Rotazyme, 37% with Slidex Rota-Kit, and 34% with Rotalex. No specimen was found positive by Rotazyme only or Slidex Rota-Kit only. On the contrary, 12 samples which were positive with Enzygnost-Rotavirus only and 3 which were positive with Rotalex only were not confirmed as positive by electron microscopy. Both enzyme immunoassays gave 6% equivocal results; Slidex Rota-Kit gave significantly fewer equivocal results than did Rotalex: 2.9% versus 9.7% (P less than 0.01). The sensitivity and specificity of latex tests compared favorably with that of enzyme immunoassays. Latex agglutination tests can be performed by unskilled personnel and are rapid and relatively cheap. They appear to be very suitable for routine laboratory work and may prove useful for large-scale screening in developing countries.     

Comparison of sedimentation and flotation techniques for identification of Cryptosporidium sp. oocysts in a large outbreak of human diarrhea.

Cryptosporidiosis, previously seen mostly among immunocompromised patients, is now recognized among immunocompetent patients. During a large outbreak of cryptosporidiosis in two day-care centers, we compared two procedures for the demonstration of the organism in preserved stool specimens. Of 703 stool specimens tested by both techniques, Sheather sucrose flotation (SSF) identified 127 (18.1%) as positive for Cryptosporidium sp. oocysts. Ritchie Formalin-ethyl acetate sedimentation (F/EA) plus a modified cold Kinyoun acid-fast stain (MCK) of the sediment identified 129 (18.4%) as positive for Cryptosporidium sp. oocysts. The degree of agreement between the two tests was statistically highly significant (P less than 0.0001). A total of 161 (22.9%) were positive by one technique or the other; 95 (13.5%) were positive by both techniques. A total of 32 specimens were positive by SSF but negative by F/EA plus MCK, and 34 specimens were positive by F/EA plus MCK but negative by SSF. The discrepancies between the two techniques occurred in stool specimens that contained rare to a few oocysts. Other parasitic forms were found by both techniques. F/EA plus trichrome staining recovered 126 (17.9%) specimens with Giardia lamblia, whereas SSF recovered only 42 (6.0%) specimens with G. lamblia. No association (chi 2 = 0.02, P = 0.89) was observed between the presence of G. lamblia and Cryptosporidium sp. in these stool specimens. We concluded that F/EA plus MCK of the sediment was as effective in the concentration and identification of Cryptosporidium sp. oocysts as SSF. F/EA plus MCK may be advantageous as a single concentration method for general parasitology when Cryptosporidium sp. is also being sought.     

Infection and diarrhea caused by Cryptosporidium sp. among Guatemalan infants.

During July 1985 to June 1986, fecal excretion of Cryptosporidium oocysts was determined prospectively in a cohort of 130 infants, aged 0 to 11 months, living in a marginal urban area of Guatemala City, Guatemala. A total of 1,280 stool specimens were examined; 158 of them were collected during episodes of diarrhea, and 1,122 were collected during symptom-free periods, every 2 to 3 weeks, from every child. Of the children, 20 (15.4%) excreted Cryptosporidium oocysts during the observation period. Of the diarrheal episodes, 13 (8.3%) were associated with Cryptosporidium sp. Of the control specimens, seven (0.6%) were positive for oocysts. Most of the infections were documented during the months of February to May, at the end of the dry season. Cryptosporidium infections are very common among Guatemalan infants and are an important cause of diarrhea and weight loss. The introduction of liquid or solid foods in the diets of the children, the presence of domestic animals (dogs, cats, or poultry), and the absence of toilet facilities in the house seem to be important risk factors for infection; also, deficient nutritional status may predispose the infected child to Cryptosporidium-associated illness.