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Journal of Clinical Microbiology, April 2007, p. 1255-1260, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.01975-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Thaddeus K. Graczyk,^1,2* Michael A. Johansson,² Leena Tamang,¹ Govinda S. Visvesvara,³ Laci S. Moura,³ Alexandre J. DaSilva,³ Autumn S. Girouard,² and Olga Matos⁴

Department of Environmental Health Sciences, Division of Environmental Health Engineering, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, Maryland 21205,¹ Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, Maryland 21205,² Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Public Services, Atlanta, Georgia 30341,³ Unidade de Protozoarios Oportunistas/VIH e Outras Protozooses, Unidade de Parasitologia e Microbiologia Medicas, Instituto de Higiene e Medicina Tropical, 1495-233 Lisbon, Portugal⁴

Received 22 September 2006/ Returned for modification 4 December 2006/ Accepted 25 January 2007

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 x 10³ to 4.4 x 10⁵ for E. bieneusi (mean, 8.8 x 10⁴/ml), 2.3 x 10² to 7.8 x 10⁴ (mean, 1.5 x 10⁴/ml) for E. intestinalis, and 1.8 x 10² to 3.6 x 10² for E. hellem (mean, 2.7 x 10²/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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Microsporidia are obligate intracellular parasites, which are considered to be emerging opportunistic pathogens among both immunocompromised and immunocompetent people. The most common species identified are Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi (25, 38, 39, 40). Until recently, microsporidian species have rarely been considered in the differential diagnosis of opportunistic infections in human immunodeficiency virus (HIV)/AIDS patients (13, 37). Identification of human-virulent microsporidian spores represents a challenge because microsporidia can infect a variety of nonhuman hosts, and spore morphology is 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 infection with fatal outcome since different treatments may be indicated depending on the species identified (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 is species specific (18, 19, 21, 31). The FISH assay was originally developed for E. hellem (21); however, alignment of the respective 16S rRNA regions of 22 other species of microsporidia (21) allowed the design of oligonucleotide probes specific to E. intestinalis (18) and to E. bieneusi and E. cuniculi (19). Through the use of various fluorochromes for probe labeling, E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi spores are stained in yellow, red, 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 testing 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) has not previously been used for archival, long-term formalin-preserved fecal samples, although FISH assay using a single E. bieneusi-specific probe has been used for formalin-fixed, paraffin-embedded duodenal biopsy (3, 34). However, recently, it has been demonstrated that long-term formalin preservation of fecal samples did not affect species-specific oligonucleotide microarray reactions (i.e., FISH and PCR) for microsporidia (37). The purpose of the present study was to determine whether multiplexed FISH assay for the identification of microsporidian spores of E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi can be applied to archival, formalin-preserved fecal samples from HIV/AIDS patients with verified intestinal microsporidiosis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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. 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 100x immersion oil objective lens.

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FIG. 1. Diarrhetic fecal samples from HIV/AIDS patients with intestinal microsporidiosis positive for microsporidian spores at the time of collection as determined by Chromotrope-2R-stained direct wet smears examined under a 100x immersion oil objective lens (A) and samples positive for microsporidian spores by multiplexed FISH tested in 2005 (B).

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 (5,000 x g, 5 min), and the pellet resuspended in 1.5 ml of 75% ethanol. Smears were prepared from all samples in duplicate using 20 µl per smear. The smears were air dried, fixed with methanol, and stained with Chromotrope-2R and CalcoFluor White M2R (2). The smears were examined by light microscopy by using an x100 immersion oil objective lens. Approximately 1.0 ml of each fecal specimen was washed off to remove the ethanol by centrifugation (5,000 x g, 5 min), and the pellet was resuspended and emulsified with 3 ml of sterile phosphate-buffered saline (PBS; pH 7.4), and processed by sugar-phenol flotation (2). Then, 1 ml of the top layer was collected, centrifuged (8,000 x g, 10 min), and the supernatant was discarded. The pellet was resuspended in 200 µl of 75% ethanol and stored at 4°C. A total of 10 µl of the suspension was placed on each of two 5-mm-diameter lysine-coated wells on a Teflon-coated glass slide (Carlson Scientific, Inc., Peotone, IL). Wells were air dried and fixed with methanol. The wells were stained with Chromotrope-2R and CalcoFluor White M2R (2), respectively. The entire area of a well was examined under a x100 immersion oil objective lens. The remaining volume of the suspension was used for multiplexed FISH analysis.

The samples were coded, and the multiplexed FISH assay was carried out in Eppendorf tubes in a total volume of 100 µl of hybridization buffer at 57°C for 3 h, using a 1 mM concentration of each oligonucleotide probe (all probes in the same Eppendorf tube) (18, 19, 31). The spores were permeabilized by incubation for 15 min in acetone (21). E. bieneusi-, E. intestinalis-, E. hellem-, and E. cuniculi-specific oligonucleotide probes (18, 19, 21, 31) were synthesized by the DNA Analysis Facility of the Johns Hopkins University, Baltimore, MD, in a 1.0 µM scale, purified by high-pressure liquid chromatography, and 5' labeled with a single molecule of a fluorochrome, Hex, 6-Fam, Tet, and Cy3 (19). Positive control spores for the multiplexed FISH assay were obtained from in vitro cell line infections (i.e., E. intestinalis, E. hellem, and E. cuniculi) or purified from a human fecal sample (i.e., E. bieneusi). After hybridization, the tubes were centrifuged twice at 4°C (2,000 x g, 5 min), and the pellets were resuspended in 100 µl of sterile PBS. Five 20-µl samples were transferred onto five lysine-coated wells (5-mm in diameter) on a Teflon-coated glass slide and air dried. The entire area of a well was examined with the aid of an Olympus BH2-RFL epifluorescence microscope, a dry x60 objective lens, and a BP450-490 exciter filter; the spores were enumerated, and the samples were uncoded.

For confirmation purposes, diarrhetic fecal samples previously determined by multiplexed FISH assay to be positive for E. hellem 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 speed of 5.5. Purified DNA was stored at 4°C for later analysis by PCR. PCRs were performed with microsporidian species-specific primers based on the small-subunit rRNA gene of E. bieneusi, E. hellem, E. intestinalis, and E. cuniculi (6, 8, 36). For the PCR, 0.3 µM concentrations of each primer and AmpliTaq Gold PCR Master Mix (Applied Biosystems, Foster City, CA) were mixed in a 50-µl final volume. The cycling parameters were 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 1 min, with a final extension of 72°C for 7 min. Negative controls and the spores of other microsporidian species were included in the PCR testing. All PCR products were analyzed on a 2% agarose gel (Agarose GTG/LE; American Bioanalytical, Natick, MA) and stained with ethidium bromide for visualization.

Statistical analysis was carried out with Statistix 7.0 (Analytical Software, St. Paul, MN). Variables were tested by Wilk-Shapiro ranking plots to determine whether their distribution conformed to a normal distribution. The results are presented as the mean for continuous variables and as the percentage for categorical data. The statistical significance of association between the sample status (i.e., spore negative or spore positive) and the sample age was assessed by the Kolmogorov-Smirnov test. Statistical significance between fractions was assessed by chi-square test. Statistical significance was considered to be at a P value <0.05.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Retrospective analysis of the 110 specimens by Chromotrope-2R and CalcoFluor White M2R stains found 77 of 110 (70.0%) dried fecal smears positive for microsporidian spores. This detection percentage represents a significant loss of sensitivity compared to the time of collection (chi-square test; G = 3.93, P < 0.05). Conversion to a spore-negative status was observed among the oldest samples, i.e., from 14 to 10 years old (i.e., collected between 1992 and 1996; Fig. 1), and there was a significant association of the sample status with the age of the sample (Kolmogorov-Smirnov test; t = 0.19, P < 0.03). Chromotrope-2R-stained spores were pinkish and had a characteristic morphology of microsporidia with a clear vacuole-like polar end. The spores were ovoid in shape and measured from 0.9 to 2.4 µm in length.

In comparison, when Chromotrope-2R and CalcoFluor White M2R stains were applied to concentrated material, microsporidian spores were identified in 80 of 110 (72.7%) and 78 of 110 (70.9%) samples, respectively, whereas the multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples (Fig. 1). Again, the negative samples represented the oldest samples in the collection, i.e., from 14 to 10 years old (Fig. 1). The multiplexed FISH assay identified spores of E. bieneusi in 49 of 94 (52.1%) samples and E. intestinalis in 43 of 94 (45.8%) samples, and 2 of 94 samples (2.1%) were positive for E. hellem spores (Fig. 2). Of 94 samples, 9 (9.6%) were positive for both E. bieneusi and E. intestinalis spores. FISH-processed E. bieneusi spores stained bright yellow, E. hellem spores bright green or blue (Fig. 2), and E. intestinalis bright red; all species displayed typical microsporidian morphology with more intense fluorescent staining in the polar half of the spore. The intensity and brightness of fluorescent staining were similar to the intensity and brightness observed for positive control spores of the respective species.

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FIG. 2. Spores of Encephalitozoon hellem identified by the FISH in diarrheic fecal samples from HIV/AIDS patients and species identification confirmed by the PCR. Note the typical microsporidian morphology with more intense fluorescent stain in the polar half of the spore. Scale bar, 2 µm.

PCR analysis confirmed that the spores in the two fecal samples identified as E. hellem by the multiplexed FISH assay represented this species.

Quantitative spore counts per milliliter of diarrhetic stool based on the multiplexed FISH assay yielded concentration values from 3.5 x 10³ to 4.4 x 10⁵ for E. bieneusi (mean, 8.8 x 10⁴/ml), from 2.3 x 10² to 7.8 x 10⁴ (mean, 1.5 x 10⁴/ml) for E. intestinalis, and from 1.8 x 10² to 3.6 x 10² for E. hellem (mean, 2.7 x 10²/ml). The concentration of E. bieneusi spores in positive samples was significantly higher than that of either E. intestinalis or E. hellem spores (two-sample t test; t = 4.03, P < 0.001), and the concentration of E. hellem spores was significantly lower than that of the other two species (two-sample t test; t = 2.29, P < 0.02).

Top Abstract Introduction Materials and Methods Results Discussion References

 
E. bieneusi is most frequently recognized in humans with various immunodeficiencies (38, 39) and was first described in 1985 as an intestinal pathogen of a HIV-infected patient (10). Since then, more than a thousand cases have been identified in immunocompromised patients in whom the pathogens produce life-threatening infections (25, 38). E. bieneusi-associated microsporidiosis is most frequently confined to the small intestine and results in diarrhea followed by weight loss; however, infections could also be asymptomatic (7, 13, 27). Intestinal microsporidiosis is a serious, insidious disease of HIV/AIDS patients and frequently progresses to extraintestinal infections, i.e., urinary, respiratory, disseminated systemic infections, sinusitis, otitis, and keratoconjunctivitis (9, 14, 22, 30, 32). Microsporidia are on the Contaminant Candidate List of the U.S. Environmental Protection Agency because their transmission routes are not fully known, spore removal and inactivation in drinking water and parasite identification in clinical samples is technologically challenging (29), and infections in humans are difficult to treat (5, 13).

It is thought that intestinal microsporidiosis is predominantly caused by E. bieneusi and E. intestinalis (10, 11, 38). However, E. hellem has also been identified by the oligonucleotide microarray method and PCR in diarrhetic fecal samples from HIV/AIDS patients (15, 26, 37). It is possible that the presence of E. hellem spores in fecal samples of HIV/AIDS patients is a result of the ingestion of spores involved with the respiratory infection rather than evidence of actual intestinal infection. The significantly lower concentration of E. hellem spores observed in the present study compared to the concentrations observed for E. bieneusi and E. intestinalis supports this plausible scenario.

The present study, together other studies (16, 37, 41), provides evidence that coinfection of microsporidia in HIV/AIDS patients with intestinal microsporidiosis is not an uncommon event (26). Coinfection prevalence can be quite high; 91.7% of all positive samples contained spores of both E. bieneusi and E. intestinalis (37). The prevalence of E. bieneusi and E. intestinalis coinfection observed in the present study was much lower, i.e., 9.6%.

The reasons why a multiplexed FISH assay did not identify microsporidian spores in 20 of 110 (18.2%) fecal samples verified to have spores at the time of collection are not clear. The spores could be present in low concentrations and may have been lost during the purification and concentration steps or could have disintegrated over time during storage. Alternatively, their rRNA could have been altered by the long-term formalin storage (i.e., 10 to 14 years) or may have been damaged by the permeabilization procedure and then disintegrated during the hybridization step. However, the lack of spore visualization in the same samples by FISH and the use of Chromotrope-2R and CalcoFluor White M2R applied to purified and concentrated material and the fact that this phenomenon was observed only in the oldest samples indicate that microsporidian spores spontaneously disintegrated over the long-term formalin storage period. Thus, the present study demonstrates that fecal samples stored in formalin for longer than 10 years may not be suitable for retrospective analysis by techniques targeting nucleic acids.

In many clinical laboratories worldwide, routine diagnosis of intestinal microsporidiosis in HIV/AIDS patients still relies on histochemical staining and light microscopy, techniques that are laborious, are insensitive, require skilled microscopists, and generally result in an underestimation of disease prevalence (37). Light microscope techniques are effective as a screening test when the spore concentration is high but do not provide the species-specific identification necessary for proper clinical management and therapy (23). The clinical application of PCR has several drawbacks: DNA extraction requires harsh and lengthy treatment of spores, species differentiation requires subsequent PCRs and sometimes restriction enzyme digests, and PCR is hampered by stool-derived inhibitors (37). Clinical application of immunological techniques is limited because specific antibodies against infectious microsporidia in humans are not widely available (23). Early FISH assays used for identification of E. bieneusi in duodenal histological sections (3, 34), although received favorably, have been criticized for requiring expertise and a long hybridization time (37). This method is appropriate for testing fecal samples preserved in formalin, the type of storage regarded as safe by personnel dealing with HIV/AIDS patients (23).

HIV/AIDS-associated intestinal microsporidiosis has been reported from all over the world, indicating that microsporidia are common opportunistic pathogens and that improved diagnostics would benefit a great number of patients. The recently developed oligonucleotide microarray is an excellent method for the simultaneous identification of E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi spores in fecal samples (37). However, method complexity makes its use in clinical laboratory unrealistic since microarray production, operation, result interpretation, and final diagnosis require very expensive equipment and would have to be outsourced to a highly specialized genetic facility or center (37). Oligonucleotide microarray analysis (37) is not a quantitative method and cannot assess the intensity of spore shedding. Also, oligonucleotide microarray reactions are affected by inhibitors present in fecal samples that decrease the efficiency of PCR and primer extension labeling and can cause false-negative readings (37). Multiplexed FISH utilizes, in part, the same technology as the oligonucleotide microarray (i.e., oligonucleotide probe hybridization); however, it does not include PCR and is not vulnerable to inhibitors present in fecal and environmental samples (18, 19, 31). For clinical applications, only FISH probe production has to be outsourced to a genetic facility; however, the probes are prepared in large quantities and can be conveniently stored for long-term laboratory use. For clinical diagnosis and research purposes, it is better to examine FISH-processed material without immersion oil and coverslips since otherwise the fluorescence deteriorates rapidly and the slides are not suitable for long-term storage.

The epidemiology of microsporidiosis in immunocompetent and immunocompromised people is still unclear in many aspects (24). Studies on the intensity of intestinal microsporidiosis showed that there are significant temporal variations in the concentration of E. bieneusi spores shed by HIV/AIDS patients (4, 17, 28). The mean spore concentration varied in HIV-positive children from 2.4 x 10² to 1.2 x 10⁵/g, and the maximum concentration reached 5.7 x 10⁵/g in a HIV-negative child (28). The concentrations of spores (of unspecified species) in HIV/AIDS adults varied from 4.5 x 10⁵ to 4.4 x 10⁸/ml (17). Thus, the concentration of E. bieneusi spores observed in the present study (range, 3.5 x 10³ to 4.4 x 10⁵/ml; mean, 8.8 x 10⁴/ml) falls within the reported ranges (17, 28). However, even in specimens with high spore counts, clumping of spores as reported previously (17) was not observed in the present study, which most likely is related to the long-term storage in formalin.

We have demonstrated that multiplexed FISH assay is a reliable, quantitative, molecular fluorescence microscopy method for the simultaneous detection of E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi spores in fecal samples of HIV/AIDS patients and is a useful tool for quantifying spore shedding intensity in intestinal microsporidiosis. This method could aid in the timely and appropriate treatment of both immunocompetent and immunocompromised people and thus prevent progression to disseminated microsporidiosis in HIV/AIDS patients.

 
We thank Thom Spahr, Johns Hopkins School of Medicine, Baltimore, MD, for access to diarrhetic samples from HIV/AIDS patients.

This study was supported by the Johns Hopkins Center in Urban Environmental Health (grant P30 ES03819), Alternatives Research and Development Foundation, the NOAA Chesapeake Bay Office (grant no. NA04NMF4570426), the Procter and Gamble Foundation, and the Johns Hopkins Center for a Livable Future.

 
* Corresponding author. Mailing address: Department of Environmental Health Sciences, Division of Environmental Health Engineering, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205. Phone: (410) 614-4984. Fax: (410) 955-0105. E-mail: tgraczyk{at}jhsph.edu

Published ahead of print on 7 February 2007.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, April 2007, p. 1255-1260, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.01975-06
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