Bulovka Hospital, Prague, Czech Republic, provided 211 CSF samples that had been deep frozen at −80°C. CSF samples were collected from immunocompetent patients; the only other patient data reported were the year of birth and sex. We obtained total DNA from sediments obtained from thawed CSF together with extraction negative control in each series, as previously described (6). We used the same methods to isolate control DNA from purified E. intestinalis spores. The study was conducted beyond the routine screening of existing unused specimens and focused on potential detection of microsporidia in CSF recovered from immunocompetent patients hospitalized at 1 hospital. Because the study was performed using anonymized samples with no intervention tract, patient consent was not required.

We used an Encephalitozoon spp.–specific nested PCR to amplify the internal transcribed spacer region (9,10). We included DNA of E. intestinalis microsporidia as a PCR-positive control and ultrapure water as a negative control and evaluated PCR products by gel electrophoresis.

We quantified DNA from PCR-positive samples by using reverse transcription PCR to amplify a 268-bp region of the 16S rRNA gene of E. cuniculi (10). Each run included unspiked specimens and diluent blanks. We considered results positive when the fluorescence signal crossed the baseline at <43 cycles. We used a standard curve to calculate the total number of spores in 1 mL of each sample.

We used the QIAquick Gel Extraction Kit (QIAGEN, https://www.qiagen.com) to purify PCR amplicons of the internal transcribed spacer region and submitted amplicons to SEQme (https://www.seqme.eu) for sequencing in both directions. We manually edited nucleotide sequences by using the ChromasPro 2.1.4 program (Technelysium, https://technelysium.com.au) and used MAFFT version 7 (http://mafft.cbrc.jp) to align sequences with reference GenBank submissions. We also microscopically examined PCR-positive samples. We air dried a drop of CSF, fixed it with methanol, and stained with standard Calcofluor M2R (Sigma-Aldrich, https://www.sigmaaldrich.com) (11).

Of 211 CSF samples examined, 115 were from male patients and 96 from female patients. The median patient age was 34.0 (range 2–81) years (Table). Among all samples, 6 were positive for microsporidia DNA, 0.9% (1/115) of samples from male and 5.2% (5/96) of samples from female patients. The age of positive patients ranged from 13 to 75 years (median 45.5 years). The spore concentration in samples was 30–500 spores/mL.

Sequence analyses revealed 100% identity to E. cuniculi genotype II (GenBank accession no. MF062430) in all positive samples (Table; Figure 1). Microscopic analysis of Calcofluor M2R–stained smears confirmed the presence of spores (1–2 spores per slide) in samples obtained from 2 patients, nos. 139 and 185, who had the highest Encephalitozoon DNA burden (Figure 2). The other 4 patients were microscopically negative.

Although microsporidiosis is mainly detected in immunodeficient patients, data from the literature imply that otherwise healthy persons also are at risk (12,13). Those data indicate that apparently healthy persons could be infected without any clinical signs, and the risk increases with age (12). Whether microsporidial infection potentially leads to a deterioration in health that could be life-threatening in the event of a decline in immunity has not been determined (12,14,15).

The fecal–oral route is generally accepted as the most common transmission route because the spores are passed in the urine or feces of infected patients, then mostly contaminate water sources. Moreover, possible foodborne transmission, including through fresh vegetables and fruits, milk, cheese, and fermented meat products, has been reported (13). Besides those transmission routes, respiratory tract infection suggests airborne transmission by contaminated aerosols (13).

Microsporidia are small intracellular fungi capable of causing widespread infections within a few days, despite their lack of active motility and limited spreading possibilities (14). The exact spreading mechanism is still unknown; however, the possible connection between activation of proinflammatory cellular immune response and targeted transport of microsporidia toward inflammation site has been proposed on the basis of clinical and experimental data (6,7,11).

In this study, we detected microsporidia DNA in 3% of tested CSF samples from 211 patients of one hospital. The molecular data were supported by microscopy in 2 patients who had the highest spore loads. Although the other 4 PCR-positive patients tested microscopically negative, those results could be caused by limited sensitivity of microscopy in low burden samples, rather than laboratory contamination. Because we obtained uniform results from specific patients using both PCR and quantitative PCR, contamination is unlikely. Moreover, we can exclude laboratory contamination because the same trained personnel took the samples and ran PCRs under sterile conditions. In addition, PCR diagnostic laboratory is structurally divided into separate areas that adhere to the 1-direction workflow, and all negative controls used in sample processing were negative.

Our results for microsporidia detection indicate an increasing prevalence of latent microsporidiosis with patient age, which is consistent with the results of previous studies (12). Moreover, the presence of microsporidia in CSF represents a potentially serious condition; unfortunately, we cannot infer any association with the clinical condition of the patients because we did not have patient histories or reasons for collecting CSF samples. However, we can assume a possible link between the patients’ health issues and the presence of microsporidia in CSF, similar to those found in another study (15). That study reported a case of a paralyzed patient with a right frontal lobe abscess containing E. cuniculi genotype I; the patient was successfully treated following appropriate treatment regimen.

In conclusion, disseminated latent microsporidiosis can cause several serious diseases with nonspecific symptoms and ambiguous etiology that can be life-threating or fatal if misdiagnosed and left untreated. We encourage increased awareness of latent microsporidiosis and development of targeted screening that enables timely treatment.