Mulkit Singh

Molecular Phylogenies of Blastocystis Isolates from Different Hosts: Implications for Genetic Diversity, Identification of Species, and Zoonosis

ABSTRACT Small-subunit (SSU) rRNA gene sequences were obtained by PCR from 12 Blastocystis isolates from humans, rats, and reptiles for which elongation factor 1α (EF-1α) gene sequences are already available. These new sequences were analyzed by the Bayesian method in a broad phylogeny including, for the first time, all Blastocystis sequences available in the databases. Phylogenetic trees identified seven well-resolved groups plus several discrete lineages that could represent newly defined clades. Comparative analysis of SSU rRNA- and EF-1α-based trees obtained by maximum-likelihood methods from a restricted sampling (13 isolates) revealed overall agreement between the two phylogenies. In spite of their morphological similarity, sequence divergence among Blastocystis isolates reflected considerable genetic diversity that could be correlated with the existence of potentially ≥12 different species within the genus. Based on this analysis and previous PCR-based genotype classification data, six of these major groups might consist of Blastocystis isolates from both humans and other animal hosts, confirming the low host specificity of Blastocystis. Our results also strongly suggest the existence of numerous zoonotic isolates with frequent animal-to-human and human-to-animal transmissions and of a large potential reservoir in animals for infections in humans.

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.

Real-Time Fluorescence-Based PCR for Detection of Malaria Parasites

ABSTRACT A fluorescence-based real-time 5′ nuclease PCR capable of detecting all four human malaria parasites was developed to screen large numbers of samples during an outbreak to prevent further transmission of malaria. The effectiveness of antimalarial therapy for malaria patients can be monitored by determining the reduction of parasitemia by this method.

Experimental Blastocystis hominis infection in laboratory mice

Abstract Young (less than 8 weeks old) immunocompetent BALB/c mice became infected with Blastocystis hominis after inoculation of fecal cysts orally and of in vitro axenic-culture forms intracecally. This study confirmed that the fecal cyst was the form responsible for external transmission and that the mode of transmission was by the fecal-oral route. The infection was self-limiting and the infected BALB/c mice appeared normal except that some of them showed weight loss and lethargy. Both vacuolar and granular forms were found in the cecum, but only cyst forms were observed in the colon. Histological examination of the cecum and colon showed intense inflammatory-cell infiltration, edematous lamina propria, and mucosal sloughing. It is apparent that although B. hominis is not invasive, it is capable of causing pathogenesis in BALB/c mice.

Elucidation of the life cycle of the intestinal protozoan Blastocystis hominis.

This paper elucidates the status of the different morphological forms ofBlastocystis and reports the existence of thin-and thick-walled cysts inB. hominis on the basis of current experimental evidence. It is suggested that the thin-walled cysts are autoinfectious, leading to multiplication of the organism in the intestinal tract. The thick-walled cysts are responsible for external transmission via the faecal-oral route. A life cycle forB. hominis is postulated on the basis of these findings.

DNA Polymorphism Revealed by Arbitrary Primers Polymerase Chain Reaction Among Blastocystis Strains Isolated from Humans, a Chicken, and a Reptile

DNA polymorphisms of different strains of Blastocystis isolated from humans, a chicken, and a reptile were examined by an arbitrary primer PCR method. Two strains of Blastocystis hominis isolated from humans in the USA and Japan yielded nearly identical PCR products. However, one strain of B. hominis (isolated from a human in Singapore) yielded quite different PCR products. Blastocystis sp. isolated from a chicken yielded PCR products similar to those of the former two strains, while Blastocystis lapemi, isolated from a reptile, shared no bands with any of the other isolates. These results indicate the possibility that our isolate from the chicken is a zoonotic strain, and that there is intraspecific variation of Blastocystis hominis.

Observations on the ultrastructure and viability of the cystic stage of Blastocystis hominis from human feces

This report describes the ultrastructure and viability of cysts of Blastocystis hominis from feces of infected patients. The cysts were round to ovoid, measured 2-5 μm in size, and contained a condensed cytoplasm that had vacuoles of varying sizes, four nuclei, and as many as six cristate mitochondria. The cell wall was rather electron-lucent. Surprisingly, chromatoid-like structures were found in the cytoplasm and nucleus of some of the cysts. These have not previously been reported in Blastocystis. The cysts can survive in water for up to 19 days at normal temperatures but are fragile at extreme temperatures and in common disinfectants.

Programmed cell death in a human intestinal parasite, Blastocystis hominis

Although programmed cell death (PCD) has been associated with multicellular organisms, there have been more reports of its presence in some protozoans. Our study shows the existence of PCD in an intestinal protozoan, Blastocystis hominis. Light and electron microscopy, biochemical and flow cytometry studies showed apoptosis-like death in B. hominis cells exposed to a cytotoxic monoclonal antibody (MAb 1D5). B. hominis cells displayed key morphological and biochemical features of apoptosis, namely, nuclear condensation and in situ fragmentation, reduced cytoplasmic volume, some externalization of phosphatidylserine and maintenance of plasma membrane integrity. No oligonucleosomal DNA laddering was observed in gel electrophoresis. This study supports earlier observations that the cellular machinery that is required to carry out PCD may have existed before the advent of multicellularity. Our study also ascribes a novel function for the B. hominis central vacuole in apoptosis; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space.

In vitro encystment and experimental infections of Blastocystis hominis

Cultures ofBlastocystis hominis were induced to encyst using three encystation media: (a) an encystation medium (EM) comprising yeast extract in buffered saline containing 50% horse serum, (b) an encystation medium (CEM) comprising EM conditioned with bacterial soluble products and (c) an encystation medium (TEM) containing 0.5% trypticase in EM. Two isolates ofB. hominis were studied, an axenized isolate C and a non-axenized isolate MS. In EM, isolate C did not encyst, whereas 6.1% of isolate MS had encysted by day 1. However, in CEM and TEM, 17.4% and 25.7% of isolate C, respectively, had encysted by day 5. In all three media, isolate MS encysted more readily than isolate C, with as much as 91.7% of the former encysting in TEM. As viewed by phase-contrast microscopy, cyst-like stages appeared highly refractile. Direct stool examination of juvenile Wistar rats infected with 10000 cyst-like stages of both C and MS isolates showedBlastocystis at day 2 post-infection. At autopsy on day 7, large numbers ofBlastocystis were seen in the cecum, with smaller numbers being observed in the large intestine. In contrast, rats fed with various inocula of the vacuolar stages of isolates C and MS did not become infected, indicating the importance of the encysted stages in the transmission of the parasite.

A survey of Blastocystis in reptiles.

A total of 28 species of reptiles were investigated forBlastocystis using light microscopy and in vitro culture in biphasic egg slant medium.Blastocystis species were detected in 8 (28.6%) of these 28 species in 3 tortoises (Geochelone elephantopus, G. elegans andG. carbonaria), 3 snakes (Boiga dendrophilla, Python reticulatus andElaphe radiata), 1 crocodile (Crocodylus porosus) and 1 iguana lizard (Cyclura cornuta). The reptilianBlastocystis appeared to be morphologically similar toB. hominis.