Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction

Blastocystis hominis is a common enteric parasite of worldwide distribution. Its pathogenetic potential has not yet been established, although numerous case reports suggest that B. hominis may cause the development of various gastrointestinal symptoms and disorders. The detection of the parasite in stool specimens is conventionally done by microscopy of direct smears, fecal concentrates, or permanently stained smears; however, morphology-based diagnosis is problematic. The aim of this study was to develop and evaluate a polymerase chain reaction (PCR) technique for the direct detection of B. hominis in human stool samples. Primers were based on small subunit ribosomal DNA and able to detect > or =32 parasites/200 mg stool artificially spiked with cultured B. hominis. In the evaluation of 43 clinical specimens, the PCR was tested against the formol ethyl acetate concentration technique (FECT) and a culture technique, proving 100% test specificity and a significantly higher sensitivity than the FECT. The PCR method is recommended for screening clinical specimens for B. hominis infection and for use in prevalence studies.     

Ultrastructure of Blastocystis hominis in human stool samples.

A study of the ultrastructure of Blastocystis hominis in human stools found morphological differences between the organisms seen and those present in laboratory cultures. B. hominis found in stool samples showed little morphological variation with storage time before fixation, but were consistently smaller (approximately 5 microns in diameter), with a thicker surface coat than the cultured organisms. The large central vacuole, characteristic of the cultured organisms, and accepted as standard morphology of B. hominis, was rarely observed in organisms present in stool samples. Instead, a number of small vacuoles, or possibly a network of interconnected vacuoles, were noted. After short-term culture, organisms from these samples appeared with the typical vacuolated morphology. No large vacuoles were present in organisms obtained at colonoscopy. These results suggest that the vacuolated form as previously described may be an artefact of culture conditions, and that the form of B. hominis present in the gastrointestinal tract is avacuolar.     

Identification of Blastocystis hominis by colonic brush cytology. A case report.

Blastocystis hominis is a unicellular organism the pathogenic potential of which in humans remains unclear. It may be identified during a workup for gastrointestinal symptoms, usually in stool examined for ova and parasites. We describe a case in which B hominis was identified by cytologic examination in a patient with Crohn's disease who underwent colonoscopy and brushing of a transverse colon stricture. The morphologic features of this organism are described and contrasted with those of the uninucleate cyst form of Entamoeba histolytica.     

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.     

Infection status of intestinal parasites in children living in residential institutions in Metro Manila, the Philippines

A small scale survey was performed to know the infection status of intestinal parasite in children of the residential institutions and street communities in Metro Manila, Philippines. A total of 284 stool samples from 11 institutions and 3 street communities was examined by the formalin-ether concentration method. The scotch tape anal swab was adapted to 121 children to investigate the infection status of Enterovius vermicularis. It was found out that 62.0% of the children examined were positive for one or more intestinal parasites. Multiple infections were observed in 34.2% of the children. Among 172 children who gave detail information, the prevalence for Ascaris lumbricoides, Trichuris trichiura, and hookworm was 36.0%, 44.8%, and 7.0% respectively. Of the children examined, 47.7% were found to be harboring parasitic protozoans such as Entamoeba histolytica, Giardia lamblia, and Blastocystis hominis. The most prevalent of these protozoans was B. hominis with an infection rate of 40.7%. The prevalence of these infections among children living in institutions was relatively high. More efforts should be made to implement anthelminthic programs including bi-annual follow-up treatments.     

Blastocystis hominis in patients at the Ruiz y Paez University Hospital from Bolivar City, Venezuela]

Blastocystis hominis is a polymorphic protozoan of discussed taxonomic position, which is currently associated with human intestinal disease. In order to determine the prevalence of the microorganism in a sample of hospitalized patients, a study was carried out from november 1996 to april 1997 on 100 adult patients of both sexes aged 20 to 79 years at the "Ruíz y Páez" University Hospital of Bolivar city, Venezuela. A coproparasitological study was carried out using direct examination and Faust method. Infection by parasites and/or commensals was demonstrated in 48 patients. The most frequent agent was B. hominis with a prevalence of 42.0%. We did not find a statistically association between sex (P > 0.05) or age (X2 = 3.52; d.f; = 3) and B. hominis infection. B. hominis was most frequently identified as the single parasite (88.1%), and with a number of less than 5 cells per 400X microscopic field (73.8%). The infection was more common in patients with base chronic-immunosuppressive diseases, the major one being cancer. Diarrhea was observed in 27.0% of cases. Due to its high prevalence, especially as a single agent, together with the particular immunological characteristics of the patients studied, a potential pathogenic role of the opportunistic type is suggested for B. hominis.     

Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis

Diagnosis of amebiasis is usually performed on a clinical basis alone in most endemic countries having limited economic resources. This epidemiological study was conducted using modern diagnostic tests for amebiasis in the southeastern region of Turkey, an endemic area for amebiasis. The population of this study included patients with symptomatic diarrhea/dysentery attending both Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. A total of 380 stool specimens were collected and examined for Entamoeba by light microscopy (fresh, lugol, and trichrome staining) and stool antigen detection based- enzyme-linked immunosorbent assay (EIA) test (TechLab Entamoeba histolytica II). 24% (91/380) of stool specimens were positive for E. histolytica/Entamoeba dispar trophozoites/cysts microscopically using trichrome staining. 13% (51/380) of the stool specimens were found to be positive for E. histolytica by the EIA test, including 15% (14/91) of microscopy (+) stool specimens and 13% (37/289) of microscopy (-) stool specimens. Enteric parasites were common in these populations with 66% (251/380) of the study population harboring more than one parasite. In addition to the 13% (51/380) of patients determined to have E. histolytica by EIA, eighty-six patients (22.6%) had Blastocystis hominis, 54 (14.2%) Entamoeba coli, 44 (11.5%) Giardia lamblia, 16 (4.2%) Chilomastix mesnili, 15 (3.9%) Iodamoeba bütschlii, 12 (3.1%) Hymenolepis nana, 9 (2.3%) Endolimax nana, 9 (2.3%) Dientamoeba fragilis, and 8 (2.1%) had Ascaris lumbricoides. We concluded that E. histolytica infection was found in 13% of the patients presenting with diarrhea in Van and Sanliurfa Turkey.     

Blastocystis hominis: frequency of infection in ambulatory patients from the northern section of Santiago, Chile]

In July 1989-July 1990 period, a coproparasitological study of 6,162 ambulatory patients from the northern section of Santiago, was undertaken. Out of the total number of the studied individual, 88.6% were children, (51.4% females and 48.6% males). The global frequency of infection by B. hominis was 30.4%. In relation to age. Blastocystis hominis was found in: 13.1% of the group of children (1 month-2 years); 34.1% of the pre-school children, 45.4% in the school-children and 43.0% in the adults. B. hominis was frequently detected in association with other parasites and/or commensals, and observed alone in the 6.0% of the studied patients.     

Epidemiological survey of Giardia spp. and Blastocystis hominis in an Argentinian rural community

The aim of this study was to relate personal data, socio-cultural and environmental characteristics, and the presence of symptoms/signs with the frequencies of Giardia spp. and Blastocystis hominis among a rural population in Buenos Aires Province, Argentina. Of the surveyed population (350), 3.7% were infected with only Giardia spp. or 22.9% with B. hominis, and 2.3% were infected with both protozoa. The frequency of infection according to sex; 6.1% of males were infected and 1.6% of females by Giardia spp., 26.7% and 19.5% by B. hominis, and 2.4% and 2.2% by both parasites, respectively. Giardia spp. was detected in only three adults (over 14 years), but B. hominis was more frequent in adults than in children. The prevalences of these protozoa in this community are lower than those reported by other Argentinean studies, which is probably associated with the low density of the studied population (5.95 inhab/km2). Statistical analysis revealed that a male sex, flooding of the home, the use of a latrine, and an abdominal pain were correlated with the presence of these parasites, which indicate the importance of these factors in rural communities.     

Recent advances in Blastocystis hominis research: hot spots in terra incognita

Despite being discovered more than 80 years ago, progress in Blastocystis research has been gradual and challenging, due to the small number of laboratories currently working on this protozoan parasite. To date, the morphology of Blastocystis hominis has been extensively studied by light and electron microscopy but all other aspects of its biology remain little explored areas. However, the availability of numerous and varied molecular tools and their application to the study of Blastocystis has brought us closer to understanding its biology. The purpose of this review is to describe and discuss recent advances in B. hominis research, with particular focus on new, and sometimes controversial, information that has shed light on its genetic heterogeneity, taxonomic links, mode of transmission, in vitro culture and pathogenesis. We also discuss recent observations that B. hominis has the capacity to undergo programmed cell death; a phenomenon similarly reported for many other unicellular organisms. There are still many gaps in our knowledge of this parasite. Although there is a growing body of evidence suggesting that B. hominis can be pathogenic under specific conditions, there are also other studies that indicated otherwise. Indeed, more studies are warranted before this controversial issue can be resolved. There is an urgent need for the identification and/or development of an animal model so that questions on its pathogenesis can be better answered. Another area that requires attention is the development of methods for the transfection of foreign/altered genes into B. hominis in order to facilitate genetic experiments.     

Detection of Pentatrichomonas hominis DNA in biological specimens by PCR

AIM: To develop a sensitive and specific polymerase chain reaction for the detection of Pentatrichomonas hominis in biological specimens. METHODS: Three primers, associated in two primer pairs, were designed to amplify a sequence from the SSU rRNA gene of P. hominis. The specificity of both primer pairs was established by testing DNA extractions of different Trichomonad species, protozoa, bacteria, yeasts, and human leucocytes. The analytical sensitivity was determined through testing dilutions of P. hominis trophozoites. The clinical specificity and applicability of the assay was evaluated on stool samples and self-administered vaginal swabs. CONCLUSIONS: A highly specific and sensitive PCR assay was developed. Both primer pairs performed equally well. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of P. hominis in vaginal specimens has not been reported before.     

Intestinal parasites in Kampuchea, Takeo Province

Fresh stool samples obtained from 1407 adult patients who sought treatment in Takeo province hospital and 332 Takeo preschool and school-age children from 3 to 18 years of age were examined for the presence of intestinal parasites using the technique of native preparation and the flotation method of Faust with subsequent staining with Lugol solution to demonstrate cysts. In hospital patients, a total of 13 protozoan and 9 helminth species were diagnosed. The prevalence of Entamoeba histolytica (cysts and trophozoites) was highest in the age group 15-18 years (18.3%), the peak prevalence of Gairdia lamblia (27.6%) occurred in children of the age group 6-9 years. The highest frequency distribution of Pentatrichomonas hominis (20.1%) was recorded in 3 to 5 years old, that of Enteromonas hominis (12.8%) in 6 to 9 years old. The predominant helminth was Ancylostoma duodenale, with the peak prevalence (65.2%) in patients older than 18 years, followed by Ascaris lumbricoides and Strongyloides stercoralis. Almost half of children patients under 6 was infected with at least two species of parasites, patients over 6 were infected simultaneously with two or more intestinal parasites in an absolute majority of cases. In Takeo preschool and school children the spectrum of diagnosed protozoan and helminth species was somewhat narrower than seen in hospital patients, but their prevalence rates were higher, except for the flagellate Pentatrichomonas hominis. The highest prevalence rates recorded were for E. histolytica 29.5% (age category 10-14 years), for G. lamblia 34.8% (age category 6-9 years), for P. hominis 19.3% (age category 3-5 years), for E. hominis 10.5% (age category 3-5 years), for A. duodenale 85.9% (age category 15-18 years), for A. lumbricoides 26.1% (age category 6-9 years), and for S. stercoralis 18.8% (age category 6-9 years). As many as 70% of children at the age between 6 and 15 years were simultaneously infected with two or three species of intestinal parasites.     

Deoxyribonucleic Acid Homology and Relative Genome Size in Mycoplasma

The deoxyribonucleic acid homologies of Mycoplasma laidlawii type A and type B, M. pulmonis (#47 and #63), and M. hominis were determined by membrane methodology. The homology data revealed a difference in genome size between M. laidlawii type A and type B. This difference also held with stringent conditions of annealing (high temperature). Little or negligible homology was shown to exist between the M. laidlawii strains type A and type B and M. pulmonis strains 47 and 63 and M. hominis. M. hominis showed less than 10% homology to the M. pulmonis and M. laidlawii strains. Neither of the M. laidlawii strains showed more than 2% annealing to the M. pulmonis strains. Reaction rate studies are suggested as a means of demonstrating the phylogenetic relationship between the Mycoplasma and other microorganisms.     

Molecular characterizations of Cryptosporidium, Giardia, and Enterocytozoon in humans in Kaduna State, Nigeria

The use of molecular diagnostic tools in epidemiological investigations of Cryptosporidium, Giardia, and Enterocytozoon has provided new insights into their diversity and transmission pathways. In this study, 157 stool specimens from 2-month to 70-year-old patients were collected, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene was used to detect and differentiate Cryptosporidium species, and DNA sequence analysis of the 60 kDa glycoprotein (gp60) gene was used to subtype Cryptosporidium hominis and Cryptosporidium parvum. Giardia duodenalis, and Enterocytozoon bieneusi in the specimens were detected using PCR and sequence analysis of the triosephosphate isomerase (tpi) gene and internal transcribed spacer (ITS), respectively. C. hominis and C. parvum were found in two (1.3%) and one (0.6%) specimen respectively, comprising of Ia and IIe (with 8 nucleotide substitutions) subtype families. The G. duodenalis A2 subtype was detected in five (3.2%) specimens, while four genotypes of E. bieneusi, namely A, type IV, D and WL7 were found in 10 (6.4%) specimens. Children aged two years or younger had the highest occurrence of Cryptosporidium (4.4%) and Enterocytozoon (13.0%) while children of 6 to 17 years had the highest Giardia infection rate (40.0%). No Cryptosporidium, Giardia, and Enterocytozoon were detected in patients older than 60 years. Enterocytozoon had high infection rates in both HIV-positive (3.3%) and HIV-negative (8.3%) patients. Results of the study suggest that anthroponotic transmission may be important in the transmission of Cryptosporidium spp. and G. duodenalis while zoonotic transmissions may also play a role in the transmission of E. bieneusi in humans in Kaduna State, Nigeria.     

Opportunistic parasites among immunosuppressed children in Minia District, Egypt

A total of 450 stool samples were collected from inpatient and outpatient clinics of Pediatric Department, Minia University Hospital, Minia District, Egypt. Two groups of patients were studied, including 200 immunosuppressed and 250 immunocompetent children. Stool samples were subjected to wet saline and iodine mounts. A concentration technique (formol-ether sedimentation method) was carried out for stool samples diagnosed negative by wet saline and iodine mounts. Samples were stained by 2 different methods; acid fast stain (modified Ziehl-Neelsen stain) and Giemsa stain. Total 188 cases (94%) were diagnosed positive for parasitic infections among immunosuppressed children, whereas 150 cases (60%) were positive in immunocompetent children (P<0.0001). The most common protozoan infection in immunosuppressed group was Cryptosporidium parvum (60.2%), followed by Blastocystis hominis (12.1%), Isospora belli (9.7%), and Cyclospora caytenensis (7.8%). On the other hand, Entamoeba histolytica (24.6%) and Giardia lamblia (17.6%) were more common than other protozoans in immunocompetent children.     

Characterization of a Mycoplasma hominis gene encoding lysyl-tRNA synthetase (LysRS)

Abstract The gene encoding lysyl-tRNA synthetase ( lysS ) in Mycoplasma hominis was cloned and sequenced. The gene was found to have an open reading frame of 1466 bp encoding a polypeptide with a predicted molecular mass of 57 kDa. The amino acid sequence showed 44.3% and 43.7% identity to the Escherichia coli lysyl-tRNA synthetases, encoded by lysS and lysU . Only one lysyl-tRNA synthetase encoding gene was found in M. hominis . The G+C content of the gene was found to be 28.6%, which is significantly lower than in other prokaryotes. The gene was located 4 kb upstream of the M. hominis PG21 rRNA B operon.     

Programmed cell death in Blastocystis hominis occurs independently of caspase and mitochondrial pathways

We demonstrated previously that a cytotoxic monoclonal antibody (MAb) 1D5 elicits a programmed cell death (PCD) response in Blastocystis hominis and showed that caspase-3-like protease influences but is not essential for PCD in MAb 1D5-treated B. hominis. We also showed that mitochondrial dysregulation played a role in cell death. In the current study, we further analyzed the signaling pathways involved in PCD mediated by MAb 1D5. B. hominis cells were treated with MAb 1D5 or control MAb 5, either with or without pretreatment with a pan-caspase inhibitor, zVAD.fmk, and/or a mitochondrial transition pore blocker, cyclosporine A (CA). Flow cytometric examination of cell size, mitochondrial membrane potential (delta psi(m)), caspase activation and in situ DNA fragmentation showed that zVAD.fmk and CA, used independently or in combination, failed to inhibit MAb 1D5-mediated PCD. Interestingly, cell exposure to either inhibitor resulted in partial inhibition of DNA fragmentation while combined exposure of cells to inhibitors abolished DNA fragmentation completely. This study sheds new light on the conserved nature of PCD pathways in parasitic protozoa and is also the first report describing caspase- and mitochondria-independent cell death pathways in a protozoan parasite.     

Detection of antibodies to Herpesvirus simiae and Herpesvirus hominis in nonhuman primates

Sera from nonhuman primates, predominantly Macaca species, were assayed by a serum neutralization test for antibodies to antigenically related Herpesvirus simiae (B virus) and Herpesvirus hominis type 1. The data indicate that there would have been approximately 50% error in the diagnosis of Herpesvirus simiae infection if these sera had been tested only against Herpesvirus hominis antigen. The role of active guinea pig complement in the serum neutralization test was also evaluated and found to be required by many of the sera for reproducible and enhanced virus neutralization, particularly for B virus antibody determination. A plaque reduction assay was found to be highly sensitive, especially when complement (2.5-5.0 hemolytic units) was added, but impractical for large-scale serum surveys.     

Immunobiological properties of monoclonal antibodies to Mycoplasma hominis antigens

Approaches to obtaining stable mouse hybridomas synthesizing monoclonal antibodies (McAb) to M. hominis key antigens were developed. 4 clones capable of the stable synthesis of McAb of different IgG classes were obtained. Clones A3/2 and A5/D produced antibodies to the thermostable determinant to with a mol. wt. of 80-120 kD, sensitive to sodium periodate and resistant to potassium proteinase. Clone H9/B2 synthesized McAb which interacted with potassium proteinase-sensitive M. hominis thermolabile determinant with a mol. wt. of 80 kD. McAb of clone A3/2, labeled with fluorescein isothiocyanate and horse-radish peroxidase, specifically reacted with M. hominis antigens in the immunofluorescence test and the immunoenzyme assay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data may serve as prerequisites for the development of diagnostic test systems aimed at the detection of M. hominis antigens in different clinical substances.