Detecting Cryptosporidium in Stool Samples Submitted to a Reference Laboratory

Stool sample selection and concentration.

Samples were received over a 5-month period from February to March; a total of 92 specimens were tested by all methods. Stool samples were concentrated using Fecal Parasite Concentrator Kits (Evergreen Scientific, Buffalo, NY). Briefly, 1–2 mL of homogenized stool in fixative or transport medium was poured into the provided test tube, and three drops of 20% Triton X-100 were added along with 1 mL of CitriSolv (Thermo Fisher Scientific, Hanover Park, IL). The tube was filled with 10% formalin, the centrifuge tube strainer was screwed in place, and the unit was rocked at 180° for 30 seconds. Once the liquid portion had all reached the centrifuge tube, then the unit was disassembled, capped, and centrifuged at 1,500 RPM for 10 minutes to pellet the debris and any parasites. The supernatant was poured off, and the pellet was resuspended in a small amount of 10% formalin. The stool was then applied to clean slides, in an area about the size of a dime, and left to dry overnight in preparation for modified acid-fast and DFA staining.

Staining and microscopy.

After fixing the dried specimens in methanol for 10 seconds and air-drying, modified acid-fast staining (Remel, San Diego, CA) was performed as per manufacturer’s instructions. Briefly, slides were placed in fuchsin stain for 5 minutes and in decolorizer for 1 second, and counterstained for 4 minutes. Between each step, the slides were rinsed briefly with deionized water. Slides were allowed to air-dry before viewing at 1000× using bright field microscopy. All of the sample on the slide was reviewed and determined positive if the staining, size, and morphology of organisms present matched with that of a typical Cryptosporidium oocyst.

Direct fluorescent antibody staining was performed on the supplied slides using a Cryptosporidium/Giardia detection kit (Meridian, Cincinnati, OH) according to the manufacturer’s instructions. One drop of the detection reagent and one drop of the counterstain were added to the dried stool specimen and allowed to incubate for 30 minutes in a humid chamber at room temperature, in the dark. Excess stain was removed from the slide with wash buffer, a drop of mounting medium was added to each sample, and a cover slip was applied to each slide. The entire sample applied to the slide was visualized under 200× magnification using the fluorescein isothiocyanate filter. Samples were determined positive if apple-green staining with the correct pattern and size for Cryptosporidium oocysts were visible.

Stock solution preparation.

To determine the limits of detection for the GI panel and the LDT, a stock of C. parvum at a concentration of 1 × 10⁶ oocysts/mL (Bunch Grass Farm, Drury, ID) was spiked into phosphate-buffered saline (PBS) and stool. The stool, which had previously been determined free of all parasites, was preserved in the alcohol-based fixative Total-Fix. One milliliter of the oocyst stock was spiked into 9 mL of PBS or stool and serially diluted to a concentration of one oocyst/mL, and then washed and extracted as described in the following text.

DNA extraction.

For clinical samples, 1 mL of homogenized stool in fixative or transport medium was centrifuged in a 1.5-mL tube for 5 minutes at full speed (20,500 × g), and the supernatant was discarded. The pellet was washed two times with 1 mL of PBS to remove any residual fixative and resuspended with 1 mL of NucliSENS easyMAG Lysis Buffer (Biomerieux, Durham, NC). The sample was transferred to Lysing Matrix E tubes (MP Biomedicals, Solon, OH), briefly vortexed, and placed on a 75°C heat block for 15 minutes. Samples were homogenized in a FastPrep instrument (Thermo Fisher Scientific, Hanover Park, IL) for 45 seconds at speed setting 6.0, and then centrifuged at 20,500 × g for 5 minutes to pellet debris. DNA was extracted using a standard stool extraction protocol on the easyMAG automated DNA extractor (Biomerieux). Briefly, in one vessel, 800 µL of stool supernatant, 1.2 mL NucliSENS Lysis Buffer, 140 µL magnetic silica (Biomerieux), and 10 µL of an internal extraction control were combined, extracted using Protocol Specific B 2.0.1, and eluted in 100 µL.

PCR amplification.

Sequences for primers and probes for each PCR reaction are provided in Table 1. Real-time reactions were performed using a QuantaBio 5X ToughMix (QuantaBio, Beverly, MA) containing hot start DNA polymerase, stabilizers, deoxyribonucleotide triphosphates (dNTPs), and MgCl₂. All cycling reactions were carried out on a ViiA7 RT-PCR system (Applied Biosystems, Foster City, CA) using the 96-well block under standard conditions beginning with a 10-minute 95° denaturation step followed by 45 cycles of 95° for 15 seconds and 60° for 1 minute. A volume of 10 µL of extracted DNA was tested in a final reaction volume of 25 µL; all specimens were tested in duplicate. A second set of duplicate reactions was tested using 1 µL of extracted DNA to test for inhibition.

Reactions for nested conventional PCR were performed in duplicate with a final volume of 50 µL containing 0.5 µM primer, dNTPs at 200µM each, 1 × Phire Reaction Buffer containing MgCl₂, 1 µL of Phire Hot Start II DNA Polymerase (Thermo Scientific), and 10 µL of DNA. When the18S rRNA gene was targeted, the conventional thermal cycling conditions were 98°C for 30 seconds followed by 35 rounds of 98°C for 5 seconds, 55.5°C (primary) and 61.6°C (secondary) for 5 seconds, 72°C for 15 seconds, and a final extension at 72°C for 1 minute. When the GP60 gene was targeted, the conventional thermal cycling conditions were 98°C for 30 seconds followed by 35 rounds of 98°C for 5 seconds, 65.1°C (primary) and 54.4°C (secondary) for 5 seconds, 72°C for 15 seconds, and a final extension at 72°C for 1 minute. Products were visualized on a 2% agarose gel stained with ethidium bromide. Products that were of the expected size (800 bp) were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions before being sequenced by the Wadsworth Center Applied Genomics Technology Cluster.

Positive amplification controls for the RT-PCR assay were designed using genus-level primers and amplifying 1) patient specimens previously determined to be positive for C. hominis and Cryptosporidium sp. and 2) C. parvum DNA extracted from oocysts isolated from cows (Bunch Grass Farm). Control plasmids were constructed using the TOPO TA Cloning Kit for Sequencing (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Briefly, ligation of appropriate PCR products into the pCR4-TOPO vector was followed by transformation into One Shot competent Escherichia coli cells (Invitrogen). The plasmid was then purified using the QIAGEN Plasmid Mini Kit (Qiagen) and stored at −20°C. To test for inhibition, an internal control plasmid¹⁹ was added to patient specimens after the heating and vortexing step, immediately before automated extraction. In addition, a no template control, which contained no added DNA, a negative extraction control (NEC), and a positive extraction control (PEC) were included in every RT-PCR assay. The PEC included the internal inhibition control plasmid, which was used both to monitor extraction efficiency and as a comparator for amplification from the internal control plasmid added to patient samples. The matrix of the NEC and PEC consisted of previously determined negative stool extracted in tandem with all patient samples to ensure no contamination occurred during handling or DNA extraction.

Commercial GI panel.

Samples were tested with a commercially available panel (FilmArray, BioFire, Salt Lake, UT) which includes bacterial, viral, and parasitic pathogens. As the GI panel is only intended for use with stool samples in Cary-Blair transport medium, samples preserved in fixatives were washed as described earlier and resuspended in 1 mL PBS followed by processing according to the manufacturer’s instructions. Briefly, hydration solution was first injected into the pouch, and then, sample buffer and 200 µL of sample were added to the sample/buffer injection tube, which was inverted three times and allowed to sit at room temperature for 5 seconds before being injected into the pouch. The pouch was loaded into the instrument where the sample undergoes extraction, nucleic acid purification, two rounds of PCR, DNA melt curve analysis, and result interpretation.

Rapid immunoassay assay.

All samples were tested using the antigen detection rapid immunoassay Giardia/Cryptosporidium Quik Chek (G/CQC) in accordance with the manufacturer’s instructions. Briefly, a preserved stool sample (100 µL) was added to a tube containing 400 µL of diluent and one drop of conjugate. Five hundred microliters of the diluted sample–conjugate mixture were transferred to the sample well of the cartridge and incubated at room temperature for 15 minutes. Wash buffer and two drops of substrate were added to the reaction window, and results were read after 10 minutes.

Preservative and demographics.

Samples were received in a variety of fixatives, as shown in Table 2. Preservation in formalin has been the standard for clinical parasitology, and most specimens were received in either 10% formalin (45.7%) or sodium acetate formalin (SAF) (23.9%). The age of patients ranged from 10 months to 91 years, although adults aged 22–65 years comprised 43.5% of the patient population (Table 3). For specimens where the gender of the patient was reported (86%), more specimens came from male patients (60.8%) than female patients.

Microscopy.

Microscopic examination consisted of both modified acid-fast staining and DFA analysis. A sample was considered positive by microscopy if either technique displayed organisms with the staining characteristics and morphological features of Cryptosporidium. Of the 92 samples, microscopy detected 20 positive samples; 19 were positive using both methods and one sample was positive only by modified acid-fast staining. Among the remaining 72 specimens, an additional 15 were positive by two or more methods, and five were positive by one method and confirmed by conventional PCR and sequencing. This resulted in 40 true-positive samples, 51 true-negative samples, and one specimen for which the status could not be resolved, leaving 91 samples (Figure 1, Supplemental Table 1).

Results obtained by each method compared with the composite reference.

Citation: The American Journal of Tropical Medicine and Hygiene 103, 1; 10.4269/ajtmh.19-0792

• Download Figure
• Download figure as PowerPoint slide

View Full Size

Results obtained by each method compared with the composite reference.

Citation: The American Journal of Tropical Medicine and Hygiene 103, 1; 10.4269/ajtmh.19-0792

• Download Figure
• Download figure as PowerPoint slide

Results obtained by each method compared with the composite reference.

Citation: The American Journal of Tropical Medicine and Hygiene 103, 1; 10.4269/ajtmh.19-0792

• Download Figure
• Download figure as PowerPoint slide

Laboratory-developed test.

Because molecular methods have been shown to be more sensitive than microscopy and antigen detection,²⁰ we developed an LDT for Cryptosporidium. The LDT was designed to specifically detect C. parvum and C. hominis, as these species are known to cause most cryptosporidiosis cases.²¹ The assay also was designed to detect Cryptosporidium at the genus level such that infections caused by other species could be detected. For the Cryptosporidium genus-level arm of the assay, the Cryptosporidium oocyst wall protein (COWP) gene was targeted, and primers and probes were designed with degeneracies in the sequence to detect as many species as possible (Supplemental Figure 1). The species-specific primers were designed based on previously published assays and target a gene of unknown function.^22,23 Forty specimens were positive in the LDT assay, and these were also determined to be positive by the composite reference. As expected, most specimens contained either C. parvum (n = 24, 60%) or C. hominis (n = 9, 22.5%). Seven specimens (17.5%) were detected only by the genus-level COWP portion of the assay. To rule out the possibility that the LDT was incorrectly amplifying DNA from other organisms in DNA extracted from stool samples, alternate primers were used to amplify the 18S and GP60 genes of Cryptosporidium for the seven samples detected at the genus level.^24,25 Sequencing of the 18S gene identified chipmunk genotype in three specimens, but four could not be identified further. These four samples, however, were confirmed as Cryptosporidium positive using other methods; one of the four samples was positive by all other methods, including microscopy, two were positive by the G/CQC immunoassay, and one was positive by the GI panel.

Giardia/Cryptosporidium Quick Chek.

The G/CQC antigen-based test was able to detect 21 of the 40 (52.5%) true-positive samples and was concordant with all other methods when true negatives were analyzed (n = 51). Thus, the sensitivity of the G/CQC compared with the composite reference was 52.5%, and the specificity was 100%. G/CQC detected Cryptosporidium in five samples that were missed by microscopy but failed to detect the parasite in four specimens that were positive by both modified acid-fast staining and DFA. All of the 19 false-negative specimens were positive when tested by the LDT. Twelve were identified as C. parvum, five as C. hominis, and two as other Cryptosporidium species. Thirteen of the samples were also positive in the GI panel, five were confirmed by sequencing, and one was positive via both microscopy methods. When tested using the LDT, samples that were positive by the antigen-based test had a lower average cycle threshold (Ct) value of 28.9 than the false negatives, where the average Ct value was 32.4. The G/CQC immunoassay provides optimal results when specimens are tested within 72 hours of collection. The specimens in this study were collected and tested before being submitted to the reference laboratory for confirmation or additional testing. Therefore, the test would not have been performed within that optimal window.

Gastrointestinal panel.

The GI panel originally identified 32 of the 40 specimens that were determined to be true positives and all 51 samples that were negative by the composite reference. Testing was repeated on the eight true positives that yielded negative results in the GI panel; of these samples, seven samples again yielded negative results on that platform. One sample gave a positive result on repeat testing. Taking the repeat testing into account, the GI panel had a sensitivity of 82.5% and a specificity of 100% compared with the composite reference.

Looking at the false-negative samples, one was positive by microscopy, by LDT, and by sequencing; and one was positive by both the G/CQC immunoassay and the LDT (Table 4). Five samples were positive by the LDT and confirmed by sequencing to verify the species. All of the specimens with false-negative results were preserved in 10% formalin, which is not a validated specimen type for the GI panel. Interestingly, 17 of the specimens that gave a true-positive result in the GI panel were also preserved in either 10% formalin or SAF. Seven specimens that gave positive results with the GI panel were preserved in Total-Fix, four were unpreserved, and for five specimens, the preservative was unknown.

Limits of detection and specificity.

The limit of detection (LOD) was determined for both the LDT and GI panel by spiking PBS and stool with known amounts of C. parvum oocysts and testing each concentration in duplicate. The overall LOD for the two assays was comparable. When spiked into PBS, both the GI panel and LDT were able to detect Cryptosporidium at a concentration of 100 oocysts/mL. When oocysts were spiked into stool, the GI panel detected oocysts at a concentration of 100 oocysts/mL and the LDT detected C. parvum oocysts in duplicate at 1,000 oocysts/mL and in one of two samples at concentrations of both 100 and 50 oocysts/mL.

Because C. hominis oocysts are not commercially available, the positive-control plasmid bearing the C. hominis target was used to test the LOD for this species in the LDT. When spiked into stool, the C. hominis plasmid was detected at a concentration of 4,000 copies/mL. As one oocyst contains four sporozoites, the LODs for C. parvum and C. hominis are comparable. Because the targets of the GI panel are not known, the LOD for C. hominis could not be assessed using that platform.

To evaluate the specificity of the LDT, DNA from a panel of 69 organisms including bacteria, viruses, and other parasites were evaluated for cross-reactivity (Table 5). Organisms tested included those causing similar gastrointestinal symptoms, pathogens likely to be found in stool specimens, and additional parasites. All samples were negative, except those containing Cryptosporidium.

Analysis of discordant samples.

To resolve results for the seven specimens that were positive by LDT and negative by the GI panel, each was retested by both molecular methods to confirm original results, and the RT-PCR products of the LDT were sequenced to confirm the presence of Cryptosporidium DNA. Six of the seven samples returned sequences confirming the presence of Cryptosporidium DNA. Although the seventh sample did not yield readable sequence, the presence of the target organism was confirmed by a positive result using the antigen-based G/CQC test (Table 4).