Uniform staining of Cyclospora oocysts in fecal smears by a modified safranin technique with microwave heating.

Cyclospora, a coccidian protist, is increasingly being identified as an important, newly emerging parasite that causes diarrhea, flatulence, fatigue, and abdominal pain leading to weight loss in immunocompetent persons with or without a recent travel history as well as in patients with AIDS. Modified Kinyoun's acid-fast stain is the most commonly used stain to identify the oocyst of this parasite in fecal smears. Oocysts of Cyclospora stain variably by the modified acid-fast procedure, resulting in the possible misidentification of this parasite. We examined fecal smears stained by six different procedures that included Giemsa, trichrome, chromotrope, Gram-chromotrope, acid-fast, and safranin stains. We report on safranin-based stain that uniformly stains oocysts of Cyclospora a brilliant reddish orange, provided that the fecal smears are heated in a microwave oven prior to staining. This staining procedure, besides being superior to acid-fast staining, is fast, reliable, and easy to perform in most clinical laboratories.     

Detection of Cyclospora cayetanensis oocysts in human fecal specimens by flow cytometry

A diagnosis of cyclosporiasis typically involves stool examinations for the presence of Cyclospora oocysts by means of microscopy. In recent years, flow cytometry has been gaining in popularity as a novel method of detecting pathogens in environmental and clinical samples. The present study is an evaluation of a flow cytometric method for the detection and enumeration of Cyclospora oocysts in human fecal specimens associated with food-borne outbreaks of cyclosporiasis in Ontario, Canada. Flow cytometry results were generally very comparable to the original microscopy results for these specimens, in terms of both presence or absence of oocysts and relative oocyst concentrations. Of the 34 fecal specimens confirmed positive for Cyclospora by microscopy, 32 were also found positive by flow cytometry, and 2 others were considered equivocal. Of the eight fecal specimens reported to be negative by microscopy, two were found positive by flow cytometry and five others were considered equivocal. These two flow cytometry-positive samples and one of the equivocal samples were confirmed by microscopic reexamination, suggesting that flow cytometry may be more sensitive than microscopy. While the sample preparation time for flow cytometry is similar to or slightly longer than that for microscopy, the actual analysis time is much shorter. Further, because flow cytometry is largely automated, an analyst's levels of fatigue and expertise will not influence results. Flow cytometry appears to be a useful alternative to microscopy for the screening of large numbers of stool specimens for Cyclospora oocysts, such as in an outbreak situation.     

Modified detergent Ziehl-Neelsen technique for the staining of Cyclospora cayetanensis.

Cyclospora cayetanensis is a cause of prolonged diarrhoea, mainly in travellers. Laboratory diagnosis may be achieved by a number of methods such as the staining of faecal smears by the modified Ziehl-Neelsen (ZN) technique. Safer methods using this technique have been described for the staining of acid fast bacilli and cryptosporidia by replacing the phenol content of the carbol fuschin stain with various concentrated detergents. In this report the technique was modified slightly using a non-concentrated detergent and applied to the staining of oocysts of C cayetanensis. It was found that oocysts of C cayetanensis do not stain using the modified detergent ZN method when compared with similar preparations containing oocysts of Cryptosporidium spp.     

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.     

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.     

Molecular characterization of clinical isolates of Cyclospora cayetanensis from patients with diarrhea in India

Purpose: Cyclospora cayetanensis is an intestinal coccidian protozoan that has emerged as an important cause of both epidemic and endemic protracted diarrhea worldwide. Though humans appear to be the only natural hosts; the role of animals as natural reservoir is uncertain but of increasing concern. The present study aimed to study the prevalence of coccidian in different groups such as immunocompromised, clinically apparent immunocompetent and healthy individuals. Also, the study isolates were assessed for heterogeneity among the sequences. Materials and Methods: Stool samples from different groups of patients were collected. The parasite was detected in stool by different diagnostic tools such as light microscopy and nested PCR-restriction fragment length polymorphism using 18S ribosomal RNA as the target gene. Results: The prevalence of C. cayetanensis was 2.4% (19/800) in the present study. The PCR assay amplified Cyclospora cayetanensis DNA in only 89% (17/19) isolates. Further, sequencing revealed no significant difference among the study isolates and the non-primates. Phylogenetic analysis of the study isolates however, formed two clusters. While one cluster showed close evolutionary association with the C. cayetanensis strains, the other cluster showed evolutionary association with the two non-primate species. Conclusion: The methods described here for detection of C. cayetanensis oocysts are simple, efficient, specific, and sensitive and therefore can be effectively applied for laboratory diagnosis and environmental assessment of fresh produce and water sources. Clinicians should include Cyclospora infection in the differential diagnosis of prolonged or relapsing diarrheal illness even in clinically apparent immunocompetent individuals.     

Immunohistochemical staining of plastic embedded bone marrow trephine biopsy specimens after microwave heating.

AIMS--To investigate (1) whether adequate immunohistochemical staining can be achieved on sections cut from plastic embedded bone marrow trephine biopsy specimens after microwave heating in citrate buffer; and (2) whether this immunohistochemical staining is comparable with that achieved on routine sections cut from paraffin wax embedded trephine biopsy specimens after decalcification procedures. METHODS--Sixty five consecutive bone marrow trephine biopsy specimens of more than 1 cm in length were divided transversely into two equal parts. One part was processed in paraffin wax followed by decalcification. The other part was embedded in the epoxyresin Polarbed 812 followed by the cutting of 1 micron sections. Both parts underwent immunohistochemical staining by an identical panel of antibodies. With Polarbed 812 plastic embedded sections, microwave heating in citrate buffer was undertaken before the application of antisera. RESULTS--On sections cut from plastic embedded material, immunohistochemical staining was generally satisfactory, easy to interpret and comparable with that achieved with paraffin wax embedded material. Exceptions were antibodies to neutrophil elastase and CD61 where immunostaining was consistently negative on plastic embedded sections. Immunohistochemical staining for CD20 was consistently more reliable on plastic embedded sections. CONCLUSIONS--The results provide evidence that, with few exceptions, satisfactory immunohistochemical staining is possible on plastic embedded bone marrow trephine biopsy specimens after microwave heating in citrate buffer. This, combined with the advantage of superior cellular morphology with semi-thin (1 micron) sections of plastic embedded material, make such embedding procedures the preferred method for the processing of bone marrow trephine biopsy specimens.     

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.     

Electron microscopy identification of microsporidia (Enterocytozoon bieneusi) and Cyclospora cayetanensis from stool samples of HIV infected patients

Microsporidia (Enterocytozoon bieneusi) and Cyclospora cayetanensis have been reported worldwide causing diarrhoea in AIDS patients. Stool samples from HIV infected patients were subjected to routine examination for parasites, followed by special staining techniques to detect microsporidia and Cyclospora cayetanensis. Confirmed positive cases of these parasites were further processed for electron microscopy identity of the parasites and characteristic details. Scanning and transmission electron microscopy showed better morphological and structural details of the parasites.     

Recognition of whole Cryptosporidium oocysts in feces by negative staining and electron microscopy

Crytosporidium oocysts in feces were recognizable in the electron microscope when prepared by the negative staining technique used by virologists. Their size, shape, and surface markings were sufficiently characteristic for cryptosporidiosis to be diagnosed by this method if more suitable methods are not available.     

Rapid immunoperoxidase staining of lymphocyte antigens using microwave irradiation

The innovative application of microwave irradiation in the immunoperoxidase staining of a wide range of labile lymphocyte antigens is described. Fifteen second irradiation at 320 watts produced excellent adherence of the frozen sections to the glass slides without loss of surface antigen staining. This brief procedure eliminated the need for much longer periods of freeze drying or drying at 4 degrees C. Microwave irradiation was also used to accelerate the incubation of the primary antibody. Thirty seconds of irradiation at 320 watts produced specific antigen staining of an intensity which was as good as that obtained with much longer durations of incubations at room temperature. Cytomorphologic preservation was also good and background staining was minimal.     

Immunohistochemical analysis of Hodgkin's disease using microwave heating.

AIMS--To assess the effect of microwave heating on immunohistochemical staining of CD15 and CD30 antigens in Hodgkin''s disease tissue samples. METHODS--Formalin fixed, paraffin wax embedded sections from 20 cases of Hodgkin''s disease (six mixed cellularity, 14 nodular sclerosis) were immunostained for CD15, using two antibodies (DAKO-M1 and Leu-M1) and for CD30 using the antibody Ber-H2. The staining was carried out by conventional techniques which included pretreatment of sections with trypsin and on untreated sections following heating with microwaves. With antibody Leu-M1 an additional method, using a specific antimouse IgM bridge both with and without microwave heating, was also included. The results for each method were compared by counting positively stained Reed-Sternberg cells and estimating the staining intensity. RESULTS--Microwave heating resulted in a substantial increase in the number of cells stained with antibodies to CD15 and also in the staining intensity. The best results were obtained using Leu-M1 with specific rabbit anti-mouse IgM bridge and microwave heating. Dramatic enhancement of the staining of Reed-Sternberg cells for CD30 was achieved following microwave heating, together with disappearance of the non-specific staining of plasma cells. CONCLUSION--Microwave heating is strongly recommended for the immunohistochemical staining of CD15 and CD30 expressed by Reed-Sternberg cells in Hodgkin''s disease.     

Rapid fixation and immunofluorescent staining of cultured cells using microwave irradiation

Abstract

Microwave irradiation during tissue fixation and immunostaining reduces the sample preparation time. Microwave irradiation also facilitates the penetration of fixatives and antibody solutions into the tissues, resulting in efficient fixation and reduction of non-specific antibody binding, respectively. Experimental procedures involving immunofluorescence microscopy are time-consuming as this method relies on antigenantibody reaction. Here, we utilized a technique involving exposure of cultured cells and tissues to intermittent microwave irradiation and immunostaining of fixed samples. Intermittent microwave irradiation during fixation reduces the required incubation time with blocking and antibody solutions, and results in good preservation of the immunoreactivity of fixed cells. Microwave irradiation also reduces the non-specific binding of fluorescein-labeled antibodies. These microwave-assisted rapid immunofluorescence techniques are useful for analysis of molecular compositions in cultured cell systems.     

Use of microwave oven improves morphology and staining of cryostat sections.

The quality of microscopic image of cryostat sections that had been subjected to microwave assisted fixation was compared with that resulting from conventional air drying of the sections. The role of microwaves in producing rapid special stains on cryostat sections was also assessed. Methods used permitted stains such as periodic acid Schiff, alcian blue, Gordon and Sweets's reticulin, Masson Fontana, Elastica, Prussian blue and Van Gieson to be performed within three minutes of cutting a cryostat section. The cytological detail of nuclei was much clearer using the microwave technique, allowing more accurate determination of cell type. The microwave oven seems to have major potential in improving the diagnostic accuracy of surgical frozen sections.