The Use of the Internal Transcribed Spacer Region for Phylogenetic Analysis of the Microsporidian Parasite Enterocytozoon hepatopenaei Infecting Whiteleg Shrimp (Penaeus vannamei) and for the Development of a Nested PCR as Its Diagnostic Tool.

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2% nucleotide sequence identity) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

Case Report: Diagnosing Dermatophytoses in Children.

Dermatophytoses of the skin and scalp are common disorders in the pediatric population. The resemblance of the clinical presentation to other dermatoses can make fungal infections challenging to diagnose. We present three cases of dermatophytoses in children. The presence of fungi within skin lesions was confirmed in all cases. The diagnoses were "id" reaction in response to Trichophyton tonsurans infection, Kerion celsi because of Microsporum canis infection, and hair loss during microsporosis. Based on our review and clinical experience, we suggest diagnostic paths and treatments for dermatophytoses in children.

Opportunistic microsporidiosis unveiled by fine-needle aspiration cytology of cervical lymph node with literature review.

Microsporidia are highly specialized obligate intracellular organisms closely related to fungi, traditionally linked to diarrheal diseases in acquired immunodeficiency syndrome patients. Over the past two decades, an increasing incidence of extraintestinal infections affecting various organ systems, especially in immunocompromised individuals, has been observed. The report presents a unique case of lymph node microsporidiosis in a 38-year-old male, positive for human immunodeficiency virus, with coinfections of hepatitis B and C. Fine-needle aspiration cytology (FNAC) from cervical lymph node yielded pus-like, necrotic material with periodic acid-Schiff stained smear uncovering small round to oval spores on microscopy suspicious for microsporidia. Based on polymerase chain reaction and sequencing done with aspiration material, the causative agent was identified as Vittaforma corneae. This rare encounter highlights the significance of recognizing unique morphological characteristics of infectious organisms and employing appropriate ancillary techniques for precise identification. The case underscores the crucial role of FNAC in diagnosing opportunistic infections involving the lymph nodes and the growing significance of molecular tests for specific pathogen confirmation.

The global epidemiology of Microsporidia infection in birds: A systematic review and meta-analysis.

This study aimed to assess the global status and genetic diversity of Microsporidia infection in different birds. An online search was conducted in international databases from 1 January 1990 to 30 June 2022. A total of 34 articles (including 37 datasets) were included for the final meta-analysis. The pooled global prevalence of Microsporidia infection in birds was 14.6% (95% CI: 11.6-18.1). The highest prevalence of Microsporidia was found in wild waterfowl which was 54.5% (28.1-78.6). In terms of detection methods, the pooled prevalence was estimated to be 21.2% (95% CI: 12.1-34.4) and 13.4% (95% CI: 10.3-17.3) for using microscopic and molecular detection methods, respectively. Enterocytozoon bieneusi was the most common pathogen (24/31; 77.42% of the studies) according to PCR-based methods, and genotype D was the highest reported genotype (nine studies). In conclusion, designing strategies for the control and prevention of Microsporidia infection in birds should be recommended.

Microscopic and Molecular Investigation of Intestinal Microsporidia in HIV + /AIDS and Cancer Patients Undergoing Chemotherapy in Mazandaran Province, North of Iran.

BACKGROUND: The aim of this study was to identify Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples of HIV + /AIDS and cancer patients undergoing chemotherapy, and comparing the results to healthy individuals in Mazandaran province, north of Iran. METHODS: Stool samples were collected from 50 HIV + /AIDS patients, 50 cancer patients, and 50 healthy samples referred to medical centers in north of Iran. Stool samples were kept in 2.5% potassium dichromate at 4 °C, and stained by modified trichrome for light microscopy examination. The multiplex/nested-PCR targeted the small subunit ribosomal RNA (SSU rRNA) gene. To characterize genotypes, the nested PCR products sequenced by Bioneer Company and was subjected to phylogenetic analyses. RESULTS: Ten of 50 samples (20%) of HIV + /AIDS patients, 5 of 50 samples (10%) of cancer patients, and 1 of healthy individuals (2%) were microscopically positive. From 50 HIV + / AIDS patients, E. bieneusi and Encephalitozoon spp. were detected in 10 (20%) and 6 (12%) cases, respectively. Furthermore, among cancer patients, 7 (14%) and 2 (4%) cases were E. bieneusi and Encephalitozoon spp., respectively. Out of 50 samples of healthy individuals, only 3 (6%) cases of E. bieneusi were observed. The genotypes D and M were detected among positive samples of E. bieneusi. CONCLUSIONS: E. bieneusi and then Encephalitozoon spp. are common intestinal microsporidia in HIV + /AIDS patients and cancer patients undergoing chemotherapy in Mazandaran province. E. bieneusi genotype D seems to be the predominant genotype in Mazandaran province. Due to the considerable prevalence of intestinal microsporidia, physicians are advised to pay more attention to this opportunistic infection in high-risk groups.

Characterization of Enterocytozoon hepatopenaei causing hepatopancreatic microsporidiosis in L. vannamei and a new molecular method for its detection in shrimps, and other environmental samples.

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is a disease of utmost concern in almost all shrimp growing countries. The pathogen was characterized by ultramicrography, histopathology and phylogenetic analysis of 18srDNA. A total of 183 biological samples were collected from all major shrimp growing states of the country.The histology technique could be used very well in identifying the site of infection and can aid in diagnosis of EHP. Wet mount and Ultramicrography were employed to observe the structure of spores. A single step PCR based method was developed for detecting the pathogen from variety of DNA samples including shrimp and non-shrimp sources.The developed PCR assay proved to be a robust and reliable technique to detect EHP in shrimps and environmental samples and for assessing the distribution of pathogen within geographical zones, thus aid in mitigating the disease. The PCR primers was also used to generate DIG labelled probe which was successful in binding to the EHP infected cells in HP of shrimp. The presence of pathogen was confirmed from many non-shrimp environmental samples suggests that they could act as reservoirs for recurrent infection in shrimp ponds. Proper control of these reservoirs will be the first step in recovering an EHP affected pond back to normal.

Investigation of the presence of Enterocytozoon bieneusi and Encephalitozoon intestinalis in immunosuppressed patients with diarrhea by IFA and real time PCR methods.

PURPOSE: This study aimed to investigate the frequency of Enterocytozoon bieneusi and Encephalitozoon intestinalis in patients with diarrhea in the immunosuppressed. METHODS: Patients between the ages of 18-85 who applied to different clinics of Mus Bulanik and Bitlis State Hospitals and were referred to the microbiology or parasitology laboratory were selected for this study. A total of 200 individuals, including 88 immunosuppressed with diarrhea patients, 38 immunocompetent with diarrhea patients, 38 immunosuppressed without diarrhea patients, and 36 immunocompetent without diarrhea individuals, were included. Collected stool samples were evaluated using IFA-MAbs and real-time PCR methods to determine the frequency of E.intestinalis and E.bieneusi. RESULTS: E. intestinalis was detected in 59 (29.5%) of 200 samples and E. bieneusi was detected in 46 (23.0%) of them. Mixed infection was detected in 16 (8%) of the positive samples. While there was no statistically significant difference between E. intestinalis positivity and gender, age, diarrhea status and immune system status, a statistically significant relationship was determined between E. bieneusi positivity and diarrhea. When the real-time PCR method was accepted as the gold standard, the sensitivity of the IFA-MAbs method in the diagnosis of E. intestinalis was 94.54%, the specificity was 97.24, the sensitivity in the diagnosis of E. bieneusi was 95.45%, and the specificity was 98.72%. The overall accuracy of the IFA-MAbs method was 96.5% for the diagnosis of E. intestinalis and 98% for the diagnosis of E. bieneusi. CONCLUSIONS: The findings suggest that E. intestinalis and E. bieneusi should be considered in both immunosuppressed and healthy individuals with diarrhea. IFA-MAbs method can be used in addition to the real-time PCR method to diagnose E. intestinalis and E. bieneusi.

Trachipleistophora hominis: molecular characterization of a rare pathogen causing microsporidial stromal keratitis.

The authors report the clinical and microbiological findings of a unique case of stromal keratitis caused by a rare microsporidium, Trachipleistophora hominis. This case of stromal keratitis was in a 49-year-old male with a history of COVID-19 infection and diabetes mellitus. Corneal scraping specimens revealed numerous microsporidia spores upon microscopic examination. PCR of the corneal button revealed the presence of T. hominis infection, which could be controlled by penetrating keratoplasty surgery. The graft was clear with no recurrence of infection until the last follow-up 6 weeks postsurgery. This is the first case of human stromal keratitis caused by this organism in a post-COVID infection, confirmed by molecular diagnosis.

Molecular Prevalence of Microsporidia Infection in Patients with Lung Cancer.

Infections are still among the most important causes of morbidity and mortality in patients with lung cancer, which has the highest rate of cancer-related deaths in the world. Microsporidia, which are opportunistic parasitic fungi, primarily localize to the intestine by ingestion but can disseminate to the respiratory tract or can be acquired by spore inhalation. Cancer patients are at higher risk for microsporidia, a life-threatening infection, than the normal population is. We aimed to characterize the prevalence of microsporidia infection for the first time by evaluating the intestinal and respiratory tracts of patients with lung cancer. In this study, we investigated 98 patients with lung cancer and 103 healthy individuals for microsporidia infection and evaluated the clinical findings of patients who were found to be positive. Sputum and stool samples were tested by microscopic examination, in addition to pan-microsporidia and genus-specific polymerase chain reactions. Nine patients with lung cancer had positive results for microsporidia (9.2%), which was significantly higher than the rate in healthy individuals (P = 0.008), and most of them had clinical findings. Among these positive patients, polymerase chain reaction revealed microsporidia in the sputum samples of seven patients, the stool sample of one patient, and both the sputum and stool samples of one patient. Encephalitozoon cuniculi was identified as the predominant pathogen in 87.5% (7/8) of positive sputum samples. Microsporidia infection was significantly associated with advanced stages of cancer. However, in the control group, Encephalitozoon intestinalis was detected in the stool sample of an individual without clinical symptoms. Microsporidia, especially E. cuniculi, should be considered as a cause of respiratory tract infection as well as intestinal infection in cancer patients and should be screened in respiratory samples of these patients when they have pulmonary symptoms.

Microsporidia-Associated Anterior Uveitis After Keratoconjunctivitis.

OBJECTIVE: The aim of this study was to describe the clinical features and management of uveitis associated with microsporidial keratoconjunctivitis (MKC). METHODS: The medical records of clinically diagnosed or microbiologically proven patients with MKC between July 2016 and August 2021 were reviewed. Patients with documented evidence of keratic precipitates (KPs) or anterior chamber cells were analyzed for their demography, clinical features, and treatment. Patients with microsporidial stromal keratitis and herpes simplex virus keratouveitis were excluded from the study. RESULTS: Of the 2212 patients reviewed within the study period 171 of 172 eyes (7.7%) had documented evidence of KPs and/or anterior chamber cells. The patients' mean age was 43.8 ± 13.8 years, and there were more men (n = 120). The mean duration of appearance of KPs was 6.9 ± 5.5 days, and 28% (n = 48 of 171) appeared on the day of presentation. Superficial punctate keratitis was central and diffuse in 48 and 49 patients, respectively. The treatment was either lubricant alone (45.3%; 78 eyes) or combined with topical steroids (54.7%; 94 eyes). The mean duration of the resolution was longer in the "corticosteroid" than "no corticosteroid" group: KPs: 15.3 ± 6.5 days versus 12.3 ± 5.8 days ( P = 0.007) and superficial punctate keratitises: 15.4 ± 9.4 days versus 11.7 ± 6.2 days ( P = 0.01). The presenting visual acuity with a pinhole was 0.26 ± 0.26 (logMAR) and it improved to 0.03 ± 0.07 on resolution ( P < 0.0001, paired t test). CONCLUSIONS: Uveitis after MKC is a self-limiting entity that often resolves without corticosteroid. One must exercise caution in using steroids in the presence of active corneal lesions.

Microsporidia-induced stromal keratitis: a new cause of presumed immune stromal (interstitial) keratitis.

BACKGROUND AND OBJECTIVE: To describe the clinical features, diagnosis and management of immune stromal keratitis/interstitial keratitis (IK) associated with microsporidial epithelial keratitis. METHODS: Between October 2020 and January 2021, medical records of IK patients microbiologically proven as microsporidia from samples collected from corneal epithelium on smear examination, and/ or molecular analysis were reviewed. Demography, clinical profile and treatment were analysed. Real-time PCR (RT-PCR) for adenovirus (ADV), Epstein-Barr virus (EBV), herpes simplex virus (HSV) and varicella-zoster virus (VZV) was done. RESULTS: Twenty of 152 (13%) microbiologically proven cases of microsporidial keratitis were diagnosed as IK during the study period, the mean age and duration of symptoms were 35.7±11.4 years and 46.3±27.7 days, respectively. Half had predisposing risk factors, like trauma; and 30% had prior recurrences. One-fourth of patients were using antivirals on presentation. Characteristic presentations included disciform keratitis(n=12), incomplete/complete ring(n=5), and combination(n=3), along with variable subepithelial infiltrates (n=14). All cases had stromal oedema, with an intact epithelium and fine pigment dusting on endothelium. Corneal epithelial scrapings had scanty microsporidia spores in smears of 17/20 (85%), and pan-microsporidial DNA was identified in 14/20 (70%), with Vittaforma corneae by sequencing in 11/20 (55%). Other viruses detected were ADV (14,70%), VZV (2,10%), EBV (1,5%) and HSV (1,5%). Rapid resolution of inflammation and oedema within 2 weeks of starting steroids was seen in all cases. CONCLUSION: Microsporidia epithelial keratitis induced stromal inflammatory keratitis; is distinguished from microsporidial keratoconjunctivitis and stromal keratitis, by characteristic clinical features, and response to topical steroids.

Past, present, and prospects in microsporidial keratoconjunctivitis- A review.

Ocular microsporidiosis comprises two entirely different spectra of disease as keratoconjunctivitis and stromal keratitis. Microsporidial keratoconjunctivitis (MKC) has been increasingly reported in the past two decades, probably due to raised awareness, simpler diagnostic procedures, and a better understanding of the clinical presentation. It is characterized by the presence of raised, coarse, punctate, multifocal, round to oval, greyish-white corneal epithelial lesions which usually evolve into nummular scars before resolution. Conjunctivitis seen is non-purulent and of mild-moderate intensity, with mixed papillary-follicular reaction. The mode of transmission and pathogenesis is poorly understood. Despite lack of inflammatory response, uncommon associations reported were- endotheliitis, corneal edema, limbitis, uveitis, and sub-epithelial infiltrates. There has been no consensus on the management of MKC. It varies from the use of multiple antimicrobial agents to simple lubricants. The majority of the disease goes underdiagnosed or misdiagnosed and treated as adenoviral keratoconjunctivitis, with topical steroids or anti-virals empirically. Changing trends have been noticed in the pattern of infection, possibly with increasing evidence of Vittaforma corneae as causative organisms, previously reported to cause stromal keratitis. An elaborate review of the past and present literature on MKC is provided in this review article, along with gaps in knowledge, and future directions of research.

Identification of Enterocytozoon bieneusi in an HIV-negative bronchiectasis patient with respiratory infection based on metagenomic next-generation sequencing: A case report.

Bronchiectasis is often caused by serious infections. Infections caused by Enterocytozoon bieneusi (E. bieneusi) is most common in the immunocompromised host, such as HIV-positive patients. Herein, we reported an HIV-negative patient with bronchiectasis infected with E. bieneusi, which diagnosed by mNGS and validated by Sanger sequencing. During the treatment of albendazole, the patient gradually recovered. This is the first report of a case of respiratory E. bieneusi infection in a bronchiectasis patient. This finding highlights the efficacy of mNGS for pathogen diagnosis in bronchiectasis patients and the potential treatment option of albendazole for bronchiectasis patients with E. bieneusi infection.

Evaluation of the Bio-Evolution Microsporidia generic and typing real-time PCR assays for the diagnosis of intestinal microsporidiosis.

Cases of intestinal microsporidiosis infection are underestimated and affect both immunocompromized and immunocompetent patients. Real-time PCR is superseding microscopic examination for its diagnosis in medical analysis laboratories. However, few manufacturers include microsporidia in their PCR panel for the diagnosis of infectious gastroenteritis. Here, we evaluated the performances of the real-time PCR assays microsporidia generic and microsporidia typing (Bio-Evolution, France) on the Rotor-Gene Q real-time PCR cycler (Qiagen, France). We included 45 negative and 44 positive stool samples for Enterocytozoon bieneusi (n = 34, with various genotypes), Encephalitozoon intestinalis (n = 4), Encephalitozoon hellem (n = 4), and Encephalitozoon cuniculi (n = 2). We also studied a four-year survey of an inter-laboratory quality control program including 9 centers that used this commercial assay. Sensitivity and specificity of the microsporidia generic assay were 86.4% and 93.3%, respectively. Encephalitozoon hellem and Encephalitozoon cuniculi were detected by the microsporidia generic PCR assay but not by the microsporidia typing PCR assay. These results were consistent with the results of the inter-laboratory quality control program. In conclusion, Bio-Evolution Real-time PCR assays are useful tools for intestinal microsporidiosis, but negative results for microsporidia typing assays require supplementary analyses to confirm E. hellem or E. cuniculi infections.

Title: Évaluation des tests de PCR en temps réel Bio-Evolution Microsporidia generic et typing pour le diagnostic de la microsporidiose intestinale. Abstract: Les microsporidioses intestinales sont des infections sous-estimées affectant à la fois les patients immunodéprimés et immunocompétents. Le diagnostic microscopique en laboratoire médical est aujourd'hui supplanté par la PCR en temps réel. Cependant, peu de fabricants incluent les microsporidies dans leurs panels PCR pour le diagnostic des gastro-entérites infectieuses. Ici, nous avons évalué les performances des tests PCR en temps réel microsporidia generic et microsporidia typing (Bio-Evolution, France) sur le thermocycleur PCR en temps réel Rotor-Gene Q (Qiagen, France). Nous avons inclus 45 échantillons de selles négatifs et 44 échantillons positifs pour Enterocytozoon bieneusi (n = 34, avec divers génotypes), Encephalitozoon intestinalis (n = 4), Encephalitozoon hellem (n = 4) et Encephalitozoon cuniculi (n = 2). Nous avons également analysé les résultats sur 4 ans d'un programme de contrôle qualité inter-laboratoires dont 9 centres ont utilisé ces kits commerciaux. La sensibilité et la spécificité du kit microsporidia generic étaient respectivement de 86,4 % et 93,3 %. Encephalitozoon hellem et E. cuniculi ont été détectés par le kit microsporidia generic mais pas par le kit microsporidia typing. Ces résultats étaient cohérents avec ceux du programme de contrôle de qualité inter-laboratoires. En conclusion, les tests de PCR en temps réel Bio-Evolution sont des outils intéressants pour la microsporidiose intestinale, mais un résultat négatif pour le test de typage microsporidia nécessite une analyse supplémentaire pour confirmer les infections à E. hellem ou E. cuniculi.

MULTI-STATE OCCUPANCY MODEL ESTIMATES PROBABILITY OF DETECTION OF AN AQUATIC PARASITE USING ENVIRONMENTAL DNA: PSEUDOLOMA NEUROPHILIA IN ZEBRAFISH AQUARIA.

Detecting the presence of important parasites within a host and its environment is critical to understanding the dynamics that influence a pathogen's ability to persist, while accurate detection is also essential for the implementation of effective control strategies. Pseudoloma neurophilia is the most common pathogen reported in zebrafish (Danio rerio) research facilities. The only assays currently available for P. neurophilia are through lethal sampling, often requiring euthanasia of the entire population for accurate estimates of prevalence in small populations. We present a non-lethal screening method to detect P. neurophilia in tank water based on the detection of environmental DNA (eDNA) from this microsporidium, using a previously developed qPCR assay that was adapted to the digital PCR (dPCR) platform to complement current surveillance protocols. Using the generated dPCR data, a multi-state occupancy model was also implemented to predict the probability of detecting the microsporidium in tank water under different flow regimes and pathogen prevalence. The occupancy model revealed that samples collected in static conditions were more informative than samples collected from flow-through conditions, with a probability of detection at 80% and 47%, respectively. There was also a positive correlation between the frequency of detection in water and prevalence in fish based on qPCR.

[A case of microsporidial keratitis].

A case of keratitis caused by microsporidia infection was reported. A 57-year-old female patient, without any obvious predisposing cause, presented with eye redness, eye abrasion and vision loss for one year in the left eye. The patient was diagnosed with viral keratitis based on laboratory examinations and clinical symptoms two months ago in our hospital. He was given outpatient treatment for antivirus. Two months later, he was admitted to our hospital with worsened condition that presented with corneal ulcer. After admission, corneal scraping examination was performed for the detection of microsporidia with calcofluor white (CFW) and Ziehl-Neelsen staining, the smear revealed multiple oval spore-like structures, with acid-fast positive and showed blue fluorescence on potassium hydroxide with CFW stain, confirming a diagnosis of microsporidial keratitis in the left eye. Treatment: topical use of ofloxacin eye ointment and voriconazole eye drops was not effective, and then penetrating keratoplasty was performed, and the patient's condition was stable after surgery. At present, they are still in treatment and follow-up.

Progression of infection and detection of Pseudoloma neurophilia in zebrafish Danio rerio Hamilton by PCR and histology.

Pseudoloma neurophilia is a critical threat to the zebrafish (Danio rerio) model, as it is the most common infectious agent found in research facilities. In this study, our objectives were two-fold: (1) compare the application of diagnostic tools for P. neurophilia and (2) track the progression of infection using PCR and histology. The first experiment showed that whole-body analysis by qPCR (WB-qPCR) can be a standardized process, providing a streamlined diagnostic protocol, without the need for extraction of specific tissues. Evaluating the course of infection in experimentally infected fish, we showed key dynamics in infection. Starting with a low dose exposure of 8000 spores/fish, the prevalence remained low until 92 days post-exposure (dpe), followed by a 30%-40% prevalence by histology or 40%-90% by PCR until the end of the experiment at 334 dpe. WB-qPCR positively detected infection in more fish than histology throughout the study, as WB-qPCR detected the parasite as early as 4 dpe, whereas it was undetected by histology until 92 dpe. We also added a second slide for histologic analyses, showing an increase in detection rate from 24% to 26% when we combined all data from our experiments, but this increase was not statistically significant.

Anncaliia algerae Microsporidiosis Diagnosed by Metagenomic Next-Generation Sequencing, China.

We report a case of Anncaliia algerae microsporidia infection in an immunosuppressed kidney transplant recipient in China. Light microscopy and transmission electron microscopy initially failed to identify A. algerae, which eventually was detected by metagenomic next-generation sequencing. Our case highlights the supporting role of metagenomic sequencing in early identification of uncommon pathogens.

Successful use of albendazole and fenbendazole therapy in a cat with persistent diarrhea due Enterocytozoon bieneusi.

Enterocytozoon bieneusi, also known as microsporidia, is an obligate, opportunistic, and neglected intracellular pathogen that causes diarrhea in humans. Although identified in the cat feces by epidemiological studies, no association with diarrhea has been demonstrated. We demonstrated a case of chronic enteritis by E. bieneusi in a 1-year-old male Maine Coon cat, neutered with diarrhea for nine months, by histopathological analysis of gastrointestinal biopsies and PCR of feces. The treatment with albendazole (10 days) followed by fenbendazole (5 days) proved to be effective and safe after diagnosis. This description highlights the need to investigate these pathogens in cases of diarrhea due to their importance in public health since they are zoonotic agents.

Molecular diagnosis of intestinal microsporidia infection in HIV/AIDS-patients in Zahedan city, Southeast of Iran.

Microsporidia belong to the intracellular spore-like pathogen, that can cause infection in invertebrates and vertebrates, including humans. Encephalitozoon spp. and Enterocytozoon bieneusi, are important causes of chronic diarrhea, especially in patients with HIV/AIDS. Therefore, in this study, modified trichrome staining (MTS) and nested polymerase chain reaction (nested PCR) methods were used for the diagnosis of common intestinal microsporidia in faecal samples of patients with HIV/AIDS in Zahedan, southeastern Iran, for the first time. Stool samples were collected from 50 HIV/AIDS-infected patients with gastrointestinal symptoms whose infections were confirmed by serology test. Prepared smears from each stool sample were stained using the MTS method. Nested PCR was used to amplify 440 bp and 629 bp fragments of 16S rRNA genes in E. bieneusi and Encephalitozoon spp., respectively. Based on the MTS method and the nested PCR, 8 (16%) and 12 (24%) stool samples were positive, respectively. According to the results of nested PCR, eight, three, and one case were infected with E. bieneusi, Encephalitozoon spp., and both of them, respectively. Findings indicated microsporidiosis in HIV/AIDS-infected patients in Zahedan is an important health problem. Therefore, this opportunistic microorganism in HIV/AIDS-infected patients should be diagnosed using sensitive and accurate methods.