In order to assess the applicability of multiplexed fluorescence in situ

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 computer virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R staining; 85.5% versus 72.7 and 70.9% respectively. The study exhibited 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 (25 38 39 40 Until recently microsporidian species have rarely been considered in the differential diagnosis of opportunistic infections in human immunodeficiency computer virus (HIV)/AIDS patients (13 37 Identification of human-virulent microsporidian spores represents a challenge because microsporidia ABT-492 can infect a variety of nonhuman hosts and spore morphology is usually insufficient for species identification (12 25 However species-specific identification of microsporidian spores is essential for advising HIV/AIDS patients on how to avoid exposure since the epidemiology of microsporidia varies considerably (11 12 25 Identification of microsporidian spore species is essential for prompt and proper pharmacological therapy in order to reduce the risk of progression to disseminated contamination with fatal end result since different treatments may be indicated depending on the species recognized (5 9 12 13 33 The global spread of microsporidia and increased HIV/AIDS frequency illustrates the need for a rapid sensitive and reliable spore identification and differentiation method (37). The fluorescence in situ hybridization (FISH) assay uses fluorescently labeled 19-bp oligonucleotide probes targeted to microsporidium species-specific sequences of 16S rRNA; therefore spore identification ABT-492 is species specific (18 19 21 31 The FISH assay was originally developed for (21); however alignment of the ABT-492 respective 16S rRNA regions of 22 other species of microsporidia (21) allowed the design of oligonucleotide probes specific to (18) and to and (19). Through the use of numerous fluorochromes for probe labeling spores are stained in yellow reddish green or blue and orange respectively (18 19 21 31 which facilitates the simultaneous use of all four probes i.e. multiplexing. The multiplexed approach has been successfully applied to screening freshly collected environmental samples (19) and animal i.e. bird fecal samples (31). The RNA and DNA of pathogens in preserved samples is stable for years (1 20 35 which can potentially allow retrospective analyses. The multiplexed FISH assay (19 31 ABT-492 has not previously been utilized for archival long-term formalin-preserved fecal samples although FISH assay using a single can be applied to archival formalin-preserved fecal samples from HIV/AIDS patients with verified intestinal microsporidiosis. MATERIALS AND METHODS A total of 110 diarrheic fecal samples collected from 110 HIV/AIDS patients (Johns Hopkins Hospital Baltimore MD) with intestinal microsporidiosis between 1992 and 2003 (Fig. ?(Fig.1)1) were analyzed. The samples were stored in 15-ml plastic tubes at 4°C in 10% buffered formalin at 1:1 milliliter ratio (stool to preservative). At the time of collection the samples were verified to be positive for microsporidian spores by examination of Chromotrope-2R (2)-stained direct wet smears under a 100× immersion oil objective lens. FIG. 1. Diarrhetic fecal samples from HIV/AIDS patients with intestinal microsporidiosis positive for microsporidian spores at the time Mouse Monoclonal to V5 tag. of collection as determined by Chromotrope-2R-stained direct wet smears examined under a 100× immersion oil objective … During initial processing of the fecal specimens in 2005 the tubes were vortexed (for 3 min) and 3 ml of each specimen were transferred to a new tube. The tubes were centrifuged (5 0 × spores were assayed by PCR. DNA was extracted from concentrated and purified spores resuspended in approximately 500 μl of sterile PBS using reagents of the FastDNA-kit (Q-Biogene Carlsbad CA) (7). The samples were disrupted in the FP 120 cell disruptor for 10 s at a velocity of 5.5. Purified DNA was stored at 4°C for later analysis by PCR..