Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization

Abstract

In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 ؋ 10 3 to 4.4 ؋ 10 5 for E. bieneusi (mean, 8.8 ؋ 10 4 /ml), 2.3 ؋ 10 2 to 7.8 ؋ 10 4 (mean, 1.5 ؋ 10 4 /ml) for E. intestinalis, and 1.8 ؋ 10 2 to 3.6 ؋ 10 2 for E. hellem (mean, 2.7 ؋ 10 2 /ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings.

Specific Detection and Localization of Microsporidian Parasites in Invertebrate Hosts by Using In Situ Hybridization

“…So far, most of the few studies that have applied FISH to microsporidia have focused on the detection of spores of microsporidia infecting vertebrates, especially humans (18,19,36). Our study shows that not only the spores but all of the stages of the microsporidian life cycle can be detected by FISH (see Fig.…”

“…S1 and S2 in the supplemental material). Moreover, previous studies applied this method to stool samples, intestinal biopsy samples, or environmental samples (18,36). Our study shows that this method is also suitable for use with whole-mount tissues, allowing the study of the dynamics of cell invasion of microsporidia within tissues.…”

“…FISH probes target particular regions of the rRNA and can be designed to detect a broad range of microorganisms (if the targeted rRNA region is well conserved across these microorganisms) or to be very specific (if the targeted region is unique); they are thus considered to be phylogenetic stains (17). While this method is widely used to detect and localize bacteria, only a few studies have applied this method to microsporidia (18)(19)(20). This is partly due to the fact that microsporidia are eukaryotes with unusual rRNAs (21,22) and many of the tools developed for bacteria to design FISH probes are therefore unsuitable (23)(24)(25).…”

The effect of a taste-enhancement process for cold-stored raw shell-stock oysters (Crassostrea virginica) on the spillage of human enteropathogens

“…The eluting fluid was processed by the CAM-filter dissolution method (Graczyk et al 1997). The resulting pellets from oyster and eluting fluid samples were processed by combined fluorescence in situ hybridization (FISH) and immunofluorescent antibody (IFA) method for C. parvum and G. lamblia (Graczyk et al 2006;2007a) and by the multiplexed FISH assay for microsporidian spores (Slodkowicz-Kowalska et al 2006;Graczyk et al 2007b;Jedrzejewski et al 2007). …”

Mikrosporidialar ve Mikrosporidiyozis

“…Mikrosporidiaların dört türüne (E. bieneusi, E. intestinalis,, E. cuniculi, ve E. hellem) özgü hazırlanmış oligonük-leotit problarla yapılan bir multipleks fluoresan in situ hibridizasyon çalışmasında, sporları boyamaya yönelik kromotrop-2R ve kalkoflor beyazı M2R boyalarının kullanıldığı mikroskopik tanıya göre daha duyarlı olduğu gösterilmiştir (43). Formalinle sabitlenmiş, parafine gömülmüş klinik örneklerden mikrosporidiaların teşhisinde rutinde kullanılan histokimyasal boyalara göre daha başarılı olduğu gösterilmesine rağmen birçok aşamasının olması, prob hibridizasyonu için bir gece beklenmesi, epi-fluoresan bir mikroskop gerektirmesi, duyarlılığının PCR'dan daha düşük olması bu tekniğin rutin kullanımını kısıtlamaktadır (40).…”