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Detection of Enterocytozoon hepatopenaei (EHP) (microsporidia) in several species of potential macrofauna-carriers from shrimp (Penaeus vannamei) ponds in Malaysia

Wan Sajiri WMH, Kua BC, Borkhanuddin MH

J Invertebr Pathol, 2023 Jun;198:107910.
PMID: 36889458 DOI: 10.1016/j.jip.2023.107910

Infection by the microsporidian parasite Enterocytozoon hepatopenaei (EHP) has become a significant problem in the shrimp cultivation industry in Asian countries like Thailand, China, India, Vietnam, Indonesia, and Malaysia. The outbreak of this microsporidian parasite is predominantly related to the existence of macrofauna-carriers of EHP. However, information about potential macrofauna-carriers of EHP in rearing ponds is still limited. In this study, the screening of EHP in potential macrofauna-carriers was conducted in farming ponds of Penaeus vannamei in three states in Malaysia, namely Penang, Kedah, and Johor. A total of 82 macrofauna specimens (phyla: Arthropoda, Mollusca, and Chordata) were amplified through a polymerase chain reaction (PCR) assay targeting genes encoding spore wall proteins (SWP) of EHP. The PCR results showed an average prevalence of EHP (82.93%) from three phyla (Arthropoda, Mollusca and Chordata). The phylogenetic tree generated from the macrofauna sequences was revealed to be identical to the EHP-infected shrimp specimens from Malaysia (MW000458, MW000459, and MW000460), as well as those from India (KY674537), Thailand (MG015710), Vietnam (KY593132), and Indonesia (KY593133). These findings suggest that certain macrofauna species in shrimp ponds of P. vannamei are carriers of EHP spores and could be potential transmission vectors. This study provides preliminary information for the prevention of EHP infections that can be initiated at the pond stage by eradicating macrofauna species identified as potential vectors.

Matched MeSH terms: Microsporidia*
Fulltext Pathogenicity of a microsporidium isolate from the diamondback moth against Noctuid moths: characterization and implications for microbiological pest management

Ghani IA, Dieng H, Abu Hassan ZA, Ramli N, Kermani N, Satho T, et al.

PLoS One, 2013;8(12):e81642.
PMID: 24349104 DOI: 10.1371/journal.pone.0081642

Due to problems with chemical control, there is increasing interest in the use of microsporidia for control of lepidopteran pests. However, there have been few studies to evaluate the susceptibility of exotic species to microsporidia from indigenous Lepidoptera.

Matched MeSH terms: Microsporidia, Unclassified/classification; Microsporidia, Unclassified/genetics; Microsporidia, Unclassified/pathogenicity*
Fulltext Gastrointestinal polyposis and microsporidia infection in a HIV-infected child

Chan KJ, Ong SY, Rohela BM, Lee WS

Pediatr Neonatol, 2018 12;59(6):636-637.
PMID: 29804938 DOI: 10.1016/j.pedneo.2018.05.003
Matched MeSH terms: Microsporidia
Incidence of microsporidia in cancer patients

Lono AR, Kumar S, Chye TT

J Gastrointest Cancer, 2008;39(1-4):124-9.
PMID: 19459072 DOI: 10.1007/s12029-009-9065-z

INTRODUCTION: Microsporidia are considered opportunistic pathogens as evidenced by the significant detection in immunocompromised HIV/AIDS population. Cancer patients receiving chemotherapy are considered to be immunosuppressed.
MATERIALS AND METHODS: Stool samples were collected from 311 cancer patients in the Klang Valley. Each sample underwent water-ether concentration and staining with modified trichrome stain.
RESULTS AND DISCUSSION: Sixty-eight samples were positive by oil immersion examination. Polymerase chain reaction amplification with specific primers on those samples amplified Encephalitozoon intestinalis from two of the samples and Encephalitozoon hellem from one sample.

Matched MeSH terms: Microsporidia/isolation & purification*; Microsporidia/ultrastructure
Intestinal microsporidial infections among Orang Asli (aborigine) children from Malaysia

Norhayati M, Al-Mekhlafi HM, Azlin M, Nor Aini U, Shaik A, Sa'iah A, et al.

Ann Trop Med Parasitol, 2007 Sep;101(6):547-50.
PMID: 17716439
Matched MeSH terms: Microsporidia/classification*; Microsporidia/isolation & purification
Fulltext Human microsporidiosis in Malaysia: review of literatures

Nurul Shazalina Zainudin, Siti Nur Su’aidah Nasarudin, Norhayati Moktar, Aishah Hani Azil, Emelia Osman

Malaysian Journal of Public Health Medicine, 2017;17(2):9-18.

The purpose of this study is to review the literature on microsporidiosis in various high-risk groups among the Malaysian population, i.e., HIV/AIDS, cancer, hospitalised patients and Orang Asli, and to update information with regards to microsporidia prevalence, diagnosis and association of the disease with gastrointestinal symptoms in Malaysia. Hospitalised patients showed the highest prevalence (28.3%) of microsporidiosis compared to other risk groups. This review did not find any direct correlations between gastrointestinal symptoms and microsporidiosis. Since microsporidiosis is an emerging threat to the high-risk groups, greater awareness should be instilled among clinicians to consider microsporidiosis in their differential diagnosis if no other causes can be defined.

Matched MeSH terms: Microsporidia
Fulltext New insights of Microsporidial infection among asymptomatic aboriginal population in Malaysia

Shahrul Anuar T, M Al-Mekhlafi H, Md Salleh F, Moktar N

PLoS One, 2013;8(8):e71870.
PMID: 24014078 DOI: 10.1371/journal.pone.0071870

Studies on microsporidial infection mostly focus on immunodeficiency or immunosuppressive individuals. Therefore, this cross-sectional study describes the prevalence and risk factors of microsporidiosis among asymptomatic individuals in Malaysia.

Matched MeSH terms: Microsporidia/isolation & purification
Evaluation of gram-chromotrope kinyoun staining technique: its effectiveness in detecting microsporidial spores in fecal specimens

Salleh FM, Al-Mekhlafi AM, Nordin A, Yasin 'M, Al-Mekhlafi HM, Moktar N

Diagn Microbiol Infect Dis, 2011 Jan;69(1):82-5.
PMID: 21146718 DOI: 10.1016/j.diagmicrobio.2010.08.028

This study was conducted to evaluate the modification of the usual Gram-chromotrope staining technique developed in-house known as Gram-chromotrope Kinyoun (GCK) in comparison with the Weber Modified Trichrome (WMT) staining technique; as the reference technique. Two hundred and ninety fecal specimens received by the Microbiology Diagnostic Laboratory of Hospital Universiti Kebangsaan Malaysia were examined for the presence of microsporidial spores. The sensitivity and specificity of GCK compared to the reference technique were 98% and 98.3%, respectively. The positive and negative predictive values were 92.5% and 99.6%, respectively. The agreement between the reference technique and the GCK staining technique was statistically significant by Kappa statistics (K = 0.941, P < 0.001). It is concluded that the GCK staining technique has high sensitivity and specificity in the detection of microsporidial spores in fecal specimens. Hence, it is recommended to be used in the diagnosis of intestinal microsporidiosis.

Matched MeSH terms: Microsporidia, Unclassified/cytology; Microsporidia, Unclassified/isolation & purification*
Disseminated microsporidiosis: An underdiagnosed and emerging opportunistic disease

Mohamed Yusoff PS, Osman E, Raja Sabudin RZA

Malays J Pathol, 2021 Apr;43(1):9-18.
PMID: 33903300

Disseminated microsporidiosis is a life-threatening disease resulting from the haematogenous spread of microsporidia species. The diagnosis is challenging owing to its subtle nonspecific clinical presentation, which usually reflects the underlying organ involved. Therefore, a high index of suspicion is required for early diagnosis. Besides, tools for confirmatory laboratory diagnosis are limited. Currently, there is no direct diagnostic method that can detect the infection without involving invasive procedures. Clinical confirmation of disseminated microsporidiosis is usually based on light and transmission electron microscopy of infected tissue specimens. These are then followed by species detection using polymerase chain reaction (PCR). Disseminated microsporidiosis shows the potential to be cleared up by albendazole or fumagillin if they are detected and treated early. Based on a series of case reports, this review aims to present a current update on disseminated microsporidiosis with emphasis on the clinical manifestations based on the organ system infected, diagnostic approach and treatment of this devastating condition.

Matched MeSH terms: Microsporidia
Prevalence of microsporidia in an indigenous Orang Asli community in Pahang, Malaysia

Lono A, Kumar GS, Chye TT

Trans R Soc Trop Med Hyg, 2010 Mar;104(3):214-8.
PMID: 19716577 DOI: 10.1016/j.trstmh.2009.07.006

Microsporidia are ubiquitous parasites thought to be closely related to fungi. Their presence in the environment means that humans are frequently exposed to infection. Stool samples were collected from 151 indigenous villagers from the eastern state of Pahang in 2005. The samples were concentrated with water-ether sedimentation, stained with modified trichrome stain and examined under oil-immersion microscopy. Thirty-two specimens (21.2%) were positive for microsporidia. Microsporidia were observed as ovoid or rounded ovoid shapes measuring approximately 1mum, with a bright pink outline containing a central or posterior vacuole. PCR amplification with specific primers on microscopy-positive specimens amplified Encephalitozoon intestinalis DNA from five of the ten specimens used.

Matched MeSH terms: Microsporidia/isolation & purification
A preliminary study on the prevalence of intestinal microsporidiosis in patients with and without gastrointestinal symptoms in Malaysia

Norhayati M, Azlin M, Al-Mekhlafi MH, Anisah N, Nor Aini U, Fatmah MS, et al.

Trans R Soc Trop Med Hyg, 2008 Dec;102(12):1274-8.
PMID: 18602128 DOI: 10.1016/j.trstmh.2008.05.019

An observational study was carried out to establish the existence of intestinal microsporidiosis among patients with and without gastrointestinal symptoms in Hospital Universiti Kebangsaan Malaysia, Malaysia. A total of 893 faecal specimens from hospitalized patients were examined for microsporidia using a modification of the usual Gram-chromotrope stain technique. One hundred and sixteen (13.0%) patients were positive for microsporidia: 84 (72.4%), 27 (23.3%) and 5 (4.3%) were low, moderate and high excreters of microsporidia spores, respectively. Of the 91 patients with available medical records, microsporidiosis was commonly observed in children aged 0-6 years (26.4%) and adults aged >or=31 years (57.2%). About one-third of this infection was observed in immunocompetent individuals. Among the immunosuppressive group, microsporidia were observed to be more prevalent in patients with haematological malignancy or a combination of malignancy and diabetes mellitus. About 74% of the patients who had microsporidia in their faeces had gastrointestinal symptoms, which could be related to infections or induced by immunosuppressive therapy. The role of microsporidia in causing gastrointestinal symptoms in this population is as yet unclear.

Matched MeSH terms: Microsporidia/isolation & purification
Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp

Tang KF, Pantoja CR, Redman RM, Han JE, Tran LH, Lightner DV

J Invertebr Pathol, 2015 Sep;130:37-41.
PMID: 26146228 DOI: 10.1016/j.jip.2015.06.009

A microsporidian parasite, Enterocytozoon hepatopenaei (abbreviated as EHP), is an emerging pathogen for penaeid shrimp. EHP has been found in several shrimp farming countries in Asia including Vietnam, Thailand, Malaysia, Indonesia and China, and is reported to be associated with growth retardation in farmed shrimp. We examined the histological features from infected shrimp collected from Vietnam and Brunei, these include the presence of basophilic inclusions in the hepatopancreas tubule epithelial cells, in which EHP is found at various developmental stages, ranging from plasmodia to mature spores. By a PCR targeting the 18S rRNA gene, a 1.1kb 18S rRNA gene fragment of EHP was amplified, and this sequence showed a 100% identity to EHP found in Thailand and China. This fragment was cloned and labeled with digoxigenin-11-dUTP, and in situ hybridized to tissue sections of infected Penaeus vannamei (from Vietnam) and P. stylirostris (Brunei). The results of in situ hybridization were specific, the probe only reacted to the EHP within the cytoplasmic inclusions, not to a Pleistophora-like microsporidium that is associated with cotton shrimp disease. Subsequently, we developed a PCR assay from this 18S rRNA gene region, this PCR is shown to be specific to EHP, did not react to 2 other parasitic pathogens, an amoeba and the cotton shrimp disease microsporidium, nor to genomic DNA of various crustaceans including polychaetes, squids, crabs and krill. EHP was detected, through PCR, in hepatopancreatic tissue, feces and water sampled from infected shrimp tanks, and in some samples of Artemia biomass.

Matched MeSH terms: Microsporidia; Microsporidia, Unclassified
Infections of Blastocystis hominis and microsporidia in cancer patients: are they opportunistic?

Chandramathi S, Suresh K, Anita ZB, Kuppusamy UR

Trans R Soc Trop Med Hyg, 2012 Apr;106(4):267-9.
PMID: 22340948 DOI: 10.1016/j.trstmh.2011.12.008

Chemotherapy can cause immunosuppression, which may trigger latent intestinal parasitic infections in stools to emerge. This study investigated whether intestinal parasites can emerge as opportunistic infections in breast and colorectal cancer patients (n=46 and n=15, respectively) undergoing chemotherapy treatment. Breast cancer patients were receiving a 5-fluorouracil/epirubicin/cyclophosphamide (FEC) regimen (6 chemotherapy cycles), and colorectal cancer patients were receiving either an oxaliplatin/5-fluorouracil/folinic acid (FOLFOX) regimen (12 cycles) or a 5-fluorouracil/folinic acid (Mayo) regimen (6 cycles). Patients had Blastocystis hominis and microsporidia infections that were only present during the intermediate chemotherapy cycles. Thus, cancer patients undergoing chemotherapy should be screened repeatedly for intestinal parasites, namely B. hominis and microsporidia, as they may reduce the efficacy of chemotherapy treatments.

Matched MeSH terms: Microsporidia/immunology; Microsporidia/pathogenicity*
Detection of microsporidia in local HIV-positive population in Malaysia

Lono A, Kumar S, Chye TT

Trans R Soc Trop Med Hyg, 2011 Jul;105(7):409-13.
PMID: 21596411 DOI: 10.1016/j.trstmh.2011.03.006

The HIV-positive population, due to their immuno-compromised nature, is considered more susceptible to parasitic infections than other populations. However despite the reports of other opportunistic pathogens such as Cryptosporidium and tuberculosis reported in vulnerable communities, microsporidia have not been highlighted in the local HIV-positive population in Malaysia. This study aimed to provide preliminary information on the prevalence of microsporidia in the local HIV-population. Microsporidia were detected in 21/247 (8.5%) stool samples from the HIV-infected individuals, a significantly higher (P-value <0.05) prevalence than in the control group, in which 5/173 (2.9%) were positive. HIV patients were 3x more at risk for acquiring microspordium (OR: 3.12; 95% CI 1.15-8.44). Spores were ellipsoid in shape with outlines that stained dark pink with the interior a lighter shade. Approximately 21% of the positive specimens were from individuals in the 40-49 years age group. Ten individuals who were positive for microsporidia were also positive for other enteric parasites such as Blastocystis hominis and Giardia lamblia. We detected Encephalitozoon intestinalis DNA following nested PCR from three of 10 samples analysed, as demonstrated by an amplicon of 370bp. From the findings reported, it appears that microsporidial infection in humans may actually be more common than reported. We strongly advocate greater emphasis on personal hygiene through public education on personal hygiene and the consumption of boiled or filtered water.

Matched MeSH terms: Microsporidia/isolation & purification*
Dense populations of the microsporidian Enterocytozoon hepatopenaei (EHP) in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp

Tang KFJ, Han JE, Aranguren LF, White-Noble B, Schmidt MM, Piamsomboon P, et al.

J Invertebr Pathol, 2016 Oct;140:1-7.
PMID: 27530403 DOI: 10.1016/j.jip.2016.08.004

White feces syndrome (WFS) is an emerging problem for penaeid shrimp farming industries in SE Asia countries, Thailand, Malaysia, Vietnam, Indonesia, China, and in India. This occurrence of this syndrome is usually first evidenced by the appearance of white fecal strings floating on surface of the shrimp ponds. The gross signs of affected shrimp include the appearance of a whitish hindgut and loose carapace, and it is associated with reduced feeding and growth retardation. To investigate the nature of the white feces syndrome, samples of white feces and shrimp hepatopancreas tissue were collected from Penaeus vannamei in affected farms in Indonesia, and these were examined histologically. Within the white feces, we found densely packed spores of the microsporidian Enterocytozoon hepatopenaei (abbreviated as EHP) and relatively fewer numbers of rod-shaped bacteria. From WFS ponds, hepatopancreas samples form 30 individual shrimp were analyzed by histology and in situ hybridization. The results showed that all of the shrimp examined were infected with EHP accompanied by septic hepatopancreatic necrosis (SHPN). Midgut epithelial cells were also infected and this increased the number of tissue types being affected by EHP. By PCR, EHP was detected in all the samples analyzed from WFS-affected ponds, but not in those sampled from healthy shrimp ponds. To determine the modes of transmission for this parasite, we performed feeding and cohabitation bioassays, the results showed that EHP can be transmitted through per os feeding of EHP-infected hepatopancreas tissue to healthy shrimp and through cohabitation ofinfected and healthy shrimp. In addition, we found the use of Fumagillin-B, an antimicrobial agent, was ineffective in either reducing or eliminating EHP in infected shrimp.

Matched MeSH terms: Microsporidia
Fulltext Important zoonotic intestinal protozoan parasites in Asia

Mak JW

Trop Biomed, 2004 Dec;21(2):39-50.
PMID: 16493397

Intestinal protozoa are increasingly being studied because of their association with acute and chronic diarrhoea in immunocompromised as well as immunocompetent patients. Various community outbreaks due to contamination of water or food with these protozoa have further highlighted their importance in public health. Among these important pathogens are Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli, and microsporidia. Except for the cyst-forming G. duodenalis and E. histolytica, the others are intracellular and form spores which are passed out with the faeces. These organisms are also found in various animals and birds and zoonotic transmission is thought to occur. These infections are distributed worldwide, with a higher prevalence in developing compared to developed countries. However, the relative importance of zoonotic infections especially in developing countries has not been studied in detail. The prevalence rates are generally higher in immunodeficient compared to immunocompetent patients. Higher prevalence rates are also seen in rural compared to urban communities. Most studies on prevalence have been carried out in developed countries where the laboratory and other health infrastructure are more accessible than those in developing countries. This relative inadequacy of laboratory diagnosis can affect accurate estimates of the prevalence of these infections in developing countries. However, reports of these infections in travellers and workers returning from developing countries can provide some indication of the extent of these problems. Most studies on prevalence of amoebiasis in developing countries were based on morphological identification of the parasite in faecal smears. As the pathogenic E. histolytica is morphologically indistinguishable from that of non-pathogenic E. dispar, estimates of amoebiasis may not be accurate. The epidemiology of human microsporidia infections is not completely understood. Two species, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans and it is believed that human to human as well as animal to human infections occur. However, the importance of zoonotic infections has not been fully characterised. G. duodenalis cysts, microsporidia and Cryptosporidium oocysts have been detected in various ground water resources, but their role in community outbreaks and maintenance of the infection has not been fully characterised. The taxonomic classification and pathogenic potential of B. hominis are still controversial. While considered by many as yeast, fungi or protozoon, recent sequence analysis of the complete SSUrRNA gene has placed it within an informal group, the stramenopiles. This review covers recent published data on these zoonotic infections and examines their public health importance in Asian countries.

Matched MeSH terms: Microsporidia

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