Acid Fast Trichrome Research Articles

Occurrence of microsporidia spp. infection in children with diarrhea in southern Iran.

Microsporidia spp. are recognized emerging zoonotic microorganisms in immunocompromised and immunocompetent populations. Therefore, we conducted a study to investigate the occurrence, sociodemographic and risk factors of microsporidia spp. infection using microscopic and molecular techniques in children with diarrhea in Jahrom city (Fars province, southern Iran). Stool samples were gathered from 395 children aged ≤ 14 years between January 2017 and January 2018. Next, a questionnaire includes the age, sex, living area, immune system status, breast milk consumption, contact with animals, and type of water source was completed for all children. For microscopic assessment, modified acid-fast trichrome staining was applied for detection of microsporidia spores. Furthermore, DNA extraction and PCR were accomplished for all samples. A total of 15 (3.79%; 95% confidence interval [CI] 2.14-6.19) and 8 (2.02%; 95% CI 0.88-3.95) samples were positive for microsporidia infection by microscopic and molecular techniques, respectively. Although none of the sociodemographic and risk factors were significant (pvalue > 0.05), the prevalence of this infection was higher in some variables (e.g., rurality, contact with animals and well water consumption). Three random samples were sequenced that all isolates belonged to Enterocytozoon bieneusi. Cognizance of microsporidia occurrence, risk factors, and disease disorders for the health specialists, physicians, children's families are important for appropriately control the infection.

Evaluation of different laboratory techniques for diagnosis of intestinal microsporidiosis in diarrheic children

Background: Microsporidia are a group of obligate intracellular organisms that can infect all animals including man causing disease in both immunocompetent individuals and immunosuppressed patients. The most common microsporidia causing gastrointestinal infection worldwide are Enterocytozoonbieneusi and Encephalitozoonintestinalis. Objectives: The present study was designed with the aim of estimating the rate of microsporidia in diarrheic children and the evaluation of different staining techniques and nested polymerase chain reaction (PCR) for the clinical diagnosis of intestinal microsporidiosis. Subjects and Methods: One hundred and fifty diarrheic stool samples were collected from Abou El-Rich children hospital laboratory. Samples were examined by “Ryan-Blue” modified trichrome stain (MTS), modified ZiehlNeelsen (MZN), acid fast trichrome (AFT) stain, and nested polymerase chain reaction (nPCR). Results:Microsporidium spp. was detected in 12 (8%) of samples using MTS. Compared with the other detection techniques the respective sensitivities and specificities of MZN stain was 100% and 98.57%; nPCR was 80% and 100%; and AFT was 85.71% and 97.87%. Conclusion: Using specific staining techniques as MZN and AFT for diagnosis of intestinal microsporidiosis in diarrheal stool samples are nearly as efficacious as PCR, but even better in some cases with the added advantage of being an inexpensive diagnostic method compared to PCR. Another benefit is the detection of other oocyst forming parasites such as Cryptosporidium, which makes staining techniques very suitable for developing countries

Incidence of Parasitic Diarrhea in Patients with Common Variable Immune Deficiency.

Parasites might cause atypical and severe infections in immunocompromised hosts. The prevalence of diarrhea among common variable immune deficiency (CVID) syndrome patients varies between 20% and 94%, which indicates that diarrhea and gastrointestinal system (GIS) complaints could be the second leading cause of morbidity in CVID patients after respiratory tract infections. This study aimed to assess the prevalence of intestinal parasites in CVID patients with GIS complaints and diarrhea. In this study, all cases followed up in the Immunology and Allergy Clinic of Ege University School of Medicine from July 2008 to August 2015 with the diagnosis of CVID were reviewed retrospectively. The stool samples of patients with diarrhea were identified using direct microscopy of native (0.09% NaCl) and Lugol's iodine preparations followed by formol-ethyl acetate concentration to apply modified Kinyoun, trichrome, acid-fast trichrome, and modified trichrome stains for the presence of intestinal parasites. Overall, 26 of 37 CVID patients had diarrhea; white and red blood cells (WBCs and RBCs, respectively) were identified in 11 and 10 of these 26 samples, respectively. Intestinal parasites were found to be present in 7 of the 11 patients with WBCs and 3 of the 10 patients with RBCs. With the addition of patients who neither had WBCs nor RBCs in their stool, a parasitic agent was detected in 13 (50%) of the 26 patients with diarrhea. There was no significant difference between the diarrheic patients with or without intestinal parasites with respect to cramps, fever, nausea and vomiting, tenesmus, bloody feces, and presence of mucus in the stool. Only one patient had malabsorption, which was not associated with intestinal parasites. The most common parasites detected in this study were Cryptosporidium spp. (n=9; 69.2%), Giardia spp. (n=7; 53.8%), and Blastocystis spp. (n=3; 23.1%). We also identified that parasitic diarrhea in CVID patients tended to last longer (M (mean): 16.2 days) than other causes of infectious diarrhea; this is in accordance with previous studies. Cryptosporidium spp. was found be the major cause of parasitic intestinal infection in this patient population. It was concluded that parasitic infections may cause chronic diarrhea, which are major causes of morbidity in CVID patients. Therefore, special attention is necessary for the identification of intestinal parasites in CVID patients with diarrhea.

Prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi in cancer patients under chemotherapy

Microsporidia species are obligate intracellular parasites and constitute one of the most important opportunistic pathogens that can cause severe infections especially in immunocompromised patients. Enterocytozoon bieneusi and Encephalitozoon intestinalis are the most common species among 14 microsporidia species identified as human pathogens. The aim of this study was to investigate the prevalence of E.intestinalis and E.bieneusi in cancer patients under chemotherapy by immunofluorescent antibody and conventional staining methods. A total of 123 stool samples obtained from 93 patients (58 male, 35 female) with cancer who were followed in oncology and hematology clinics of our hospital and 30 healthy volunteers (13 male, 17 female) were included in the study. Fifty-one (55%) of the patients had complain of diarrhea. The presence of E.intestinalis and E.bieneusi were investigated by a commercial immunofluorescence antibody test using monoclonal antibodies (IFA-MAbs; Bordier Affinity Products, Switzerland) in all of the samples, and 50 of the samples were also investigated by modified trichrome, acid-fast trichrome and calcofluor staining methods. A total of 65 (69.9%) patients were found positive with IFA-MAbs method, including 43 (46.2%) E.intestinalis, 9 (9.7%) E.bieneusi and 13 (14%) mixed infections. In the control group, 5 (16.7%) subjects were positive with IFA-MAbs method, including 2 (6.7%) E.intestinalis, 1 (3.3%) E.bieneusi and 2 (6.7%) mixed infections. The difference between the positivity rate of the patient and control groups was statistically significant (p< 0.05). Of the patients with diarrhea, 68.6% (35/51) were infected with microsporidia, and the difference between cases with and without (48.6%) diarrhea was statistically significant (p< 0.05). When 50 samples in which all of the methods could be performed were evaluated, the frequency of microsporidia were detected as follows; 66% (n= 33) with IFA-MAbs, 34% (n= 17) with modified trichrome staining, 24% (n= 12) with acid-fast trichrome staining and 42% (n= 21) with calcofluor staining methods. Our data indicated that the use of IFA-MAbs method along with the conventional staining methods in diagnosis of microsporidia will increase the sensitivity. As a conclusion, the prevalence of E.intestinalis and E.bieneusi in cancer patients under chemotherapy was detected quite high (69.9%) in our study, it would be appropriate to screen these patients regularly in terms of microsporidian pathogens.

Bacterial and parasitic etiological agents in HIV seropositive diarrhoea cases.

Introduction: Studies in India indicated that diarrhoea has been the third common clinical presentation in AIDS patients. It is a second leading cause of hospital visit in developing nations and makes its place top ten worldwide. A diagnostic spectrum of enteric pathogen causing diarrhoea includes bacteria, parasites, fungi & viruses. Some pathogen tend to occur more frequently to cause more severe disease in person infected with HIV than in HIV uninfected persons with diarrhoea. These include Salmonella spp., Isospora spp., Crytptosporidium spp., Microsporidia spp., Cytomegalovirus. Aims & Objectives: Present study was undertaken to find out which etiological agents are most commonly responsible for causing diarrhoea in HIV seropositive patients. Material Methods: Total 181 cases were included in study out which 150 were HIV seropositive and 31 were HIV seronegative. Diagnosis of HIV infection was done as per NACO guidelines and CD4 count estimation was done. Isolation of bacterial pathogen was done using standard bacteriological procedures and parasite detection was done by direct examination after formal ether concentration technique, using staining as: modified Kinyouns acid fast staining and modified acid fast trichrome staining. Results: In this study, most common bacterial pathogen detected was Escherichia coli ( 57.6%), followed by Proteus spp (9.4%), % klebsiella spp ( 7.7%). Most common protozoal parasite was Cryptosporidium (53.6%), & Microsporidium (2.9%) and most common helminthic parasite was S. stercoralis (1.6%). All the enteric pathogens were predominantly detected with CD4+ count < 200 cells/ul. Conclusion: Cryptosporidium was the most common 53.6% parasitic enteropathogen detected while diarrheogenic E. coli 18.2% was the most common bacterial enteropathogens isolated. Majority of cases were with <200 CD4+ count had more than one pathogen.

HIV/AIDS-Associated Opportunistic Protozoal Diarrhea

Human immunodeficiency virus (HIV) infection has altered both the epidemiology and outcome of enteric opportunistic parasitic infections. This study was done to determine the prevalence and species/genotypes of intestinal coccidian and microsporidial infections among HIV/AIDS patients with diarrhea and/or a history of diarrhea alternately with an asymptomatic interval, and their association with CD4 T cell count. This cross-sectional study was done from May 2010 to May 2011 in Shiraz University of Medical Sciences, South of Iran. A blood sample was obtained from HIV-positive patients for a CD4 T cell count upon enrollment. Sociodemographic data and a history of diarrhea were collected by interviewing 356 consecutive participants (273 males and 83 females). Whenever possible more than a fecal sample was collected from all the participants and examined for parasites using direct, physiological saline solution ethyl acetate, an acid-fast trichrome stain, nested polymerase chain reaction, and sequencing techniques for the detection, confirmation, and genotyping of Cryptosporidium spp., Cyclospora cayetanensis, Isospora belli, and intestinal microsporidia (Enterocytozoon bieneusi). The most common opportunistic and nonopportunistic pathogens were Cryptosporidium spp. (C. parvum and C. andersoni), E. bieneusi, Giardia lamblia, Sarcocystis spp., and Blastocystis homonis affecting 34, 8, 23, 1, and 14 patients, respectively. C. cayetanensis, I. belli, Enterobius vermicularis, and Hymenolepis nana were observed in few patients. A CD4 count <200 cells/μl was significantly associated with the presence of opportunistic parasites and diarrhea (p<0.05). Opportunistic intestinal parasites should be suspected in any HIV/AIDS patient with chronic diarrhea. Tropical epidemic nonopportunistic enteric parasitic infections among such patients should not be neglected in Iran.

PP-171 Detection of Microsporidia and Cryptosporidium in stool specimens from AIDS patients by Modified Trichrome-Blue and Acid-Fast Trichrome staining methods

PP-171 Detection of Microsporidia and Cryptosporidium in stool specimens from AIDS patients by Modified Trichrome-Blue and Acid-Fast Trichrome staining methods

Diagnosis of intestinal microsporidiosis in pediatric oncology patients in Egypt using modified acid fast trichrome staining versus PCR

AbstractMicrosporidia are intracellular opportunistic parasites that cause chronic diarrhea in AIDS patients. Little is known about the prevalence of these pathogens in pediatric cases with cancer and diarrhea. Unidentified causes of chronic diarrhea were previously encountered in pediatric cancer patients at the National Cancer Institute in Egypt. Therefore, this study tried to search for the contribution of microsporidia as a causative agent of diarrhea in this population using acid-fast trichrome stain as a specific staining and the PCR in order to evaluate the staining technique in clinical diagnosis of microsporidia. Between January 2008 and June 2009, 271 diarrheic samples from pediatric patients with cancer were studied. Microsporidia were confirmed in 13 (4.8%) cases by both PCR and staining, and additional 2 samples were positive only by staining. As a negative control, stool samples from 60 diarrheic children without malignant cancer and no microsporidia detection were examined by these two methods. So, it can be concluded that adding a diagnostic test for microsporidia to the clinical laboratory work in hospitals concerned with cancer is essential. Acid fast trichrome staining technique as being nearly efficacious as the PCR, but simpler and less expensive, can replace the molecular techniques for the diagnosis of microsporidia.

Diagnostic markers for encephalitozoonosis in pet rabbits

Encephalitozoonosis is a common disease in pet rabbits, routinely diagnosed in vivo by serological examination or post mortem by histopathology. Recently molecular techniques have become increasingly important as diagnostic tools. The application of different diagnostic markers for in vivo and post mortem determination of E. cuniculi in naturally infected pet rabbits were compared. The examined population was divided into 33 rabbits with symptoms of encephalitozoonosis (group I) and 38 animals without symptoms (group II) which were all tested by serological analysis (Indirect Immunofluorescence Test), histological examination including special spore staining (Ziehl-Neelsen, acid-fast trichrome) and conventional and nested PCR (organs, body fluids). Additionally, in group III lens material (n=10) of animals (n=9) with phacoclastic uveitis were examined by conventional PCR. Infections with E. cuniculi could be determined post mortem in 78.8% of the rabbits of group I and in 57.9% of group II by histological examination combined with spore staining. In group I 69.7% and in group II 50.0% showed seroconversion. Conventional PCR was only sufficiently sensitive in samples of eyes with phacoclastic uveitis (n=10; 100%). Therefore nested PCR was performed in tissue samples, urine and cerebrospinal fluid (CSF) with positive results in 63.6% of group I and 42.1% of group II. Positive results in serology, pathohistology (spore detection and histological changes in the brain and/or kidneys) and nested PCR were obtained in 52.1% of the rabbits (group I and II, n=71), whereas 31.0% showed negative results in all three diagnostic techniques. 5.6% of the rabbits were positive in two methods and 11.3% in one method. In 37 rabbits (group I and II) with positive results in nested PCR, E. cuniculi DNA could be detected more frequently in the brain (91.9%) than in the kidney (54.1%). Furthermore nested PCR of urine revealed positive results in 29.7% of the rabbits (n=37) with seroconversion and/or confirmed E. cuniculi infection by spore detection. All 25 samples of CSF tested negative in nested PCR. Conventional PCR of eyes with phacoclastic uveitis was an excellent diagnostic marker in living rabbits, while nested PCR of urine or CSF was not reliable. Histological examination combined with special staining was the most sensitive method in post mortem diagnostics. Nested PCR appears to be a good post mortem method to investigate organs, especially brains, of chronically infected animals.

Encephalitozoonosis in pet rabbits (Oryctolagus cuniculus): pathohistological findings in animals with latent infection versus clinical manifestation

Encephalitozoon cuniculi is a common infectious agent of rabbits. The aim of this study was to determine the distribution and extent of histological lesions in the brain and in the kidney of naturally infected pet rabbits with or without clinical encephalitozoonosis. In 71 animals (33 with symptoms) which died or were euthanised, histopathological examination including staining of spores (Ziehl-Neelsen, acid-fast trichrome) was performed and changes were described quantitatively. The cerebrum was the most frequently affected brain region (97.5%), whilst the cerebellum (55%) and the vestibular cores (37.5%) were less commonly concerned. Granulomas were found in 77.5% of animals with encephalitis and in 12.5% of rabbits with interstitial nephritis. Although cerebral granulomas were found irrespective of the grade of histological changes, they were significantly correlated with changes at higher grades. There was no correlation between the severity of encephalitis and neurological symptoms. Since severe lesions were also found in clinically inconspicuous animals, histological findings of inflammatory lesions are not indicative of overt encephalitozoonosis as the causative agent for neurological signs. Other diseases causing neurological symptoms, such as suppurative encephalitis, otitis media as well as malignant lymphoma were also detected in the rabbit population that was examined in the present study.

Assessment of Cryptosporidium Removal from Domestic Wastewater Via Constructed Wetland Systems

Constructed wetlands have been recognized as offering a removal treatment option for high concentrations removal of chemical and biological contaminants in domestic wastewater. The enteric protozoan parasite Cryptosporidium is considered one of the highly resistant to treatment and highly infectious organisms to humans and animals. Moreover, some species of Cryptosporidium are known to have a zoonotic nature. In this investigation a pilot scale for domestic wastewater treatment system was used, consisting of the following steps in series: (1) up-flow anaerobic sludge blanket (UASB) reactor, (2) free water surface (FWS) wetland unit, and (3) sub-surface flow (SSF) wetland unit. This treatment system was fed with domestic wastewater to assess its efficiency in removing Cryptosporidium oocysts. The obtained Cryptosporidium oocysts were detected and enumerated by two different staining techniques ‘acid fast trichrome (AFT) and modified Ziehl Neelsen (MZN) stains’. Polymerase chain reaction (PCR) technique was also used to detect Cryptosporidium DNA in wastewater samples. Results revealed that anaerobic treatment (using UASB reactor) could remove about 53.1% of Cryptosporidium oocysts present in raw wastewater. The in-series connection between the two wetland units allowed complete elimination of Cryptosporidium oocysts as the first (FWS) wetland unit removed 95.9% of the oocysts present in anaerobically treated wastewater and the remaining portion of oocysts was completely removed by the second (SSF) wetland unit. Cryptosporidium oocysts were detected in 95.8% of raw wastewater samples with a mean count of 43.8 oocysts/l when AFT stain was used while they were detected in only 87.5% of raw wastewater samples with a mean count of 35.6 oocysts/l when MZN stain was used. Polymerase chain reaction (PCR) technique was able to detect Cryptosporidium DNA in only 45.8% of raw wastewater samples. Positive PCR results were only achieved in wastewater samples containing 52 oocysts or more per liter.

Fate of Cryptosporidium during wastewater treatment via constructed wetland systems

Constructed wetlands have been recognized as offering a removal treatment option for high concentrations of chemical and biological contaminants in domestic wastewater. The enteric protozoan parasite Cryptosporidium is considered to be one of the most highly resistant and most infectious organisms for humans, animals and birds. Moreover, some species of Cryptosporidium are known to have a zoonotic nature. In this investigation a pilot scale for a domestic wastewater treatment system is used, consisting of the following steps: (1) up‐flow anaerobic sludge blanket (UASB) reactor; (2) free water surface (FWS) unit; and (3) sub‐surface flow (SSF) unit. This treatment system was fed with domestic wastewater to assess its efficiency in removing Cryptosporidium oocysts. The obtained Cryptosporidium oocysts were detected by two different staining techniques (acid fast trichrome ‘AFT’ and modified Ziehl Neelsen ‘MZN’ stains). Results revealed that the AFT stain was more sensitive and reliable for the detection of Cryptosporidium oocysts than MZN stain. Anaerobic treatment (using UASB reactor) could remove about 47.49% of Cryptosporidium oocysts present in raw wastewater. The FWS unit could remove about 94.35% of the oocysts that escaped from UASB treatment unit. No oocysts were detected after SSF treatment. The physicochemical properties of the final effluent were within the limits for wastewater reuse.

Evaluation of a combined acid-fast-trichrome stain for detection of microsporidia and Cryptosporidium parvum.

M and Cryptosporidium parvum are important agents of enteritis, capable of causing severe chronic disease in immunocompromised individuals.1,2 Currently, detection of these organisms in stool specimens requires at least 2 special stains or procedures; a chemofluorescent agent or a modified trichrome stain is commonly used to detect microsporidia,3–5 and an acid-fast stain, direct immunofluorescent assay (DFA), or enzyme immunoassay is used to detect C parvum.6–8 Recently, Ignatius et al9 described a combination acid-fast–trichrome (AFT) stain, which allows detection of these organisms, as well as Cyclospora cayetanensis and Isospora belli, with a single procedure. We modified this AFT procedure, incorporating the use of commercially available reagents, and compared the results of the modified AFT stain with those of our standard procedures for stool specimens received in the laboratory for a 15-month period.

Evaluation of a combined acid-fast-trichrome stain for detection of microsporidia and Cryptosporidium parvum.

M and Cryptosporidium parvum are important agents of enteritis, capable of causing severe chronic disease in immunocompromised individuals.1,2 Currently, detection of these organisms in stool specimens requires at least 2 special stains or procedures; a chemofluorescent agent or a modified trichrome stain is commonly used to detect microsporidia,3–5 and an acid-fast stain, direct immunofluorescent assay (DFA), or enzyme immunoassay is used to detect C parvum.6–8 Recently, Ignatius et al9 described a combination acid-fast–trichrome (AFT) stain, which allows detection of these organisms, as well as Cyclospora cayetanensis and Isospora belli, with a single procedure. We modified this AFT procedure, incorporating the use of commercially available reagents, and compared the results of the modified AFT stain with those of our standard procedures for stool specimens received in the laboratory for a 15-month period.

A new acid-fast trichrome stain for simultaneous detection of Cryptosporidium parvum and microsporidial species in stool specimens.

The detection in stool specimens of Cryptosporidium parvum and microsporidia, the most frequent parasitic pathogens causing diarrhea in AIDS patients, until now has depended on two different staining methods. However, since double infections occur and minimization of laboratory costs is mandatory, development of a method for simultaneous detection of these parasites appeared desirable. We report on a new, inexpensive, and easy-to-perform staining procedure to demonstrate both acid-fast oocysts of C. parvum and other coccidia, as well as microsporidial spores. This acid-fast trichrome stain yields results comparable to those obtained by the Kinyoun and modified trichrome methods and considerably reduces the time necessary for microscopic examination.