Detecting Cryptosporidium in Stool Samples Submitted to a Reference Laboratory

Kimberly Mergen Parasitology Laboratory, Wadsworth Center, NYSDOH, Albany, New York;

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Noel Espina Parasitology Laboratory, Wadsworth Center, NYSDOH, Albany, New York;

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Allen Teal Parasitology Laboratory, Wadsworth Center, NYSDOH, Albany, New York;

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Susan Madison-Antenucci Parasitology Laboratory, Wadsworth Center, NYSDOH, Albany, New York;
School of Public Health, Biomedical Sciences, University at Albany, Albany, New York

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When considering methods of detecting Cryptosporidium in patient samples, clinical and public health laboratories have historically relied primarily on microscopy. However, microscopy is time intensive and requires trained personnel to accurately identify pathogens that are present. Even with skilled analysts, the parasitemia level has the potential to fall below the level of detection. In addition, public health laboratories do not always receive specimens in fixatives that are compatible with the desired microscopic method. Antigen-based and molecular methods have proven to be effective at identifying Cryptosporidium at low levels and require less training and hands-on time. Here, we have developed and validated a real-time polymerase chain reaction (RT-PCR) laboratory-developed test (LDT) that identifies Cryptosporidium hominis and Cryptosporidium parvum, and also includes detection at the genus level to identify additional species that occasionally cause disease in humans. Results of the molecular test were compared with those obtained from modified acid-fast microscopy, immunofluorescent microscopy, an antigen-based detection rapid test, and a commercial gastrointestinal panel (GI panel). Of 40 positive samples, microscopy and antigen-based methods were able to detect Cryptosporidium in only 20 and 21 samples, respectively. The GI panel detected 33 of the 40 positive samples, even though not all specimens were received in the recommended preservative. The LDT detected Cryptosporidium in all 40 positive samples. When comparing each method for the detection of Cryptosporidium, our results indicate the LDT is an accurate, reliable, and cost-effective method for a clinical public health reference laboratory.

INTRODUCTION

The protozoon parasite Cryptosporidium causes the intestinal disease cryptosporidiosis in humans. Cryptosporidiosis symptoms typically include watery diarrhea, dehydration, and weight loss, and may last for up to 2 weeks. Infections are spread by consuming or having contact with contaminated water, consuming contaminated food, or close contact with people or animals that are infected. In North America, swimming pools and other recreational waters are common sources of infection for multiple reasons; the oocysts are passed in large numbers by infected individuals, are immediately infectious, hardy in the environment, insensitive to chlorine levels maintained in pools, too small (4–6 µm diameter) to be removed by typical filtration systems, and are able to cause an infection when as few as 10 organisms are ingested.1,2

In 2014, 8,682 cases of cryptosporidiosis were reported to the CDC. However, diagnosis is likely to be significantly underreported, and estimates of the number of actual episodes of illness due to Cryptosporidium in the United States are as high as 748,000.3,4 From 1995 to 2004, the average incidence was slightly more than one case per 100,000 people, with a range of 0.9–1.4. In 2005, however, this number started to increase following a jump in the number of reported cases, leading to a new higher steady-state incidence rate of almost three cases per 100,000 people.5 Examples of contact with contaminated water that contributed to the increase in reported cases include an outbreak in a New York spray park in 2005,6 contaminated swimming pools in Utah,7 a man-made lake in Texas in 2008,8 and contaminated drinking water in a municipality in Oregon in 2013,9 which sickened 28% of the community. Increased awareness due to outbreaks such as these, as well as advances in testing methods, could have also contributed to the higher reported incidence rates. Given that only a small percentage of cryptosporidiosis cases are believed to be reported, efforts to detect outbreaks earlier are likely to benefit from greater awareness of and more sensitive testing for this intestinal parasite.

Diagnosis by microscopic examination of acid-fast stained slides has been the gold standard, and although it is inexpensive, it is also labor intensive and lacks sensitivity compared with other methods. Immunofluorescent microscopy is more sensitive but still labor intensive. Antigen detection is simple to perform using rapid diagnostic tests, but their performance has been variable.1013 More recently, FDA-approved multiplex panels that detect a variety of gastrointestinal pathogens, including Cryptosporidium, have become available, and these are now widely used.14,15 The commercial assays are easy to perform but can be costly. Laboratory-developed tests (LDTs), including real-time PCR (RT-PCR) assays,16 are less expensive and easily designed, as the genomes for Cryptosporidium hominis17 and Cryptosporidium parvum18 have been sequenced. However, not all laboratories have the capacity to develop or validate LDTs, and the required instrumentation is expensive. A complicating factor for references laboratories is that samples are received in a variety of fixatives. Thus, the choice of testing method is likely to depend on several factors including available resources, workload, and the population being tested.

As a provider of clinical public health reference testing in New York State, the Wadsworth Center Parasitology Laboratory confirms the presence of Cryptosporidium in patient specimens submitted from hospitals and commercial laboratories. All specimens are evaluated by modified acid-fast staining and direct fluorescent antibody (DFA) immunomicroscopy; however, for approximately 25% of submitted specimens, Cryptosporidium cannot be identified by microscopy. In this study, we developed a LDT that specifically targets C. parvum and C. hominis and includes degenerate genus-level primers and a probe to detect other species of Cryptosporidium, which occasionally cause disease in humans. Because microscopy was anticipated to be the least sensitive method, the focus was on specimens that were negative when tested by acid-fast staining and immunofluorescent microscopy. After routine analysis, specimens were tested by the LDT as well as a rapid immunoassay for antigen detection and a gastrointestinal panel (GI panel). In addition to the microscopy-negative samples, 20 microscopy-positive specimens were included in the study to verify the specificity of the classical methods. For the purposes of comparison, a composite reference was used such that a specimen was considered a true positive if two or more methods yielded a positive result or the presence of Cryptosporidium could be confirmed by DNA sequencing.

MATERIALS AND METHODS

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.

Table 1

Primers and probes

OrganismTypePrimers and probeTargetReference
C. parvum and C. hominisReal-time primersF—TTA ATG TAA CTC CAG CTG AAT TCT TTT TC R—GGA GTT CAG ATT CTT TAA TTT AAT CTA TCA TTT AATGene of unknown functionRef. 22 23
C. hominisProbeP—Cy5/ATT TAT CTC TTA CTT CGT GGC GGC G/IBRQ
C. parvumProbeP—TexRd/ATT TAT CTC TTC GTA GCG GCG/BHQ2
Crypto. sppReal-time primersF—TGY CCN CCN GGN TTY GTN GAY R—YGG NGG RCA YTC BGG RTT NGG WGG RGCCryptosporidium oocyst wall proteinThis study
P—FAM/CAG GCA TWG/ZEN/TRA AYG CAA CAC AAT CTC T/IBFQ
Crypto. spp.Conventional (nested)F1—TTC TAG AGC TAA TAC ATG CG18SRef. 25
R1—CCC ATT TCC TTC GAA ACA GGA
F2—GGA AGG GTT GTA TTT ATT AGA TAA AG
R2—CTC ATA AGG TGC TGA AGG AGT A
Crypto. parvum/hominisConventional (nested)F1—ATG AGA TTG TCG CTC ATT ATCGP60Ref. 24 25
R1—TTA CAA CAC GAA TAA GGC TGC
F2—TCC GCT GTA TTC TCA GCC
R2—GGA AGG AAC GAT GTA TCT

C. hominis = Cryptosporidium hominis; C. parvum = Cryptosporidium parvum.

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.

RESULTS

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.

Table 2

Fixative type for submitted patient samples

FixativeNumber of samples (%)*
10 % formalin42 (45.7)
Sodium acetate formalin22 (23.9)
Total Fix9 (9.8)
Cary-Blair4 (4.3)
Polyvinyl alcohol2 (2.2)
ProtoFix1 (1.1)
ParaPak clean vial1 (1.1)
Fixative type not available11 (12.0)

Percentage of 92 samples.

Table 3

Age distribution

Age (years)Number of specimens (%)*
0–11 (1.1)
1–516 (17.4)
6–2115 (16.3)
22–6540 (43.5)
> 658 (8.7)
Unknown12 (13.0)

Percentage of 92 samples.

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).

Figure 1.
Figure 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

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.

Table 4

Sequence analysis of discrepant samples

SampleGastrointestinal panel resultLaboratory-developed test real-time PCR result (average Ct value)Sequence matchOther test method
2(−)C. parvum (34.3)C. parvumMicroscopy (+)
65(−)C. parvum (40.2)C. parvumNA
66(−)C. parvum (34.8)C. parvumNA
77(−)C. parvum (36.0)C. parvumNA
83(−)C. parvum (29.7)C. parvumNA
86(−)C. hominis (38.8)C. hominisNA
87(−)Cryptosporidium sp. (33.9)NAG/CQC

C. hominis = Cryptosporidium hominis; C. parvum = Cryptosporidium parvum.

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.

Table 5

Specificity of the laboratory-developed test

OrganismNumber of species testedResult
Areomonas spp.11Negative
Adenovirus1Negative
Babesia microti1Negative
Blastocystis hominis1Negative
Campylobacter spp.3Negative
Clostridium difficile1Negative
Clostridium sordellii1Negative
Coxsackievirus3Negative
Cryptosporidium hominis1Positive
Cryptosporidium parvum1Positive
Cryptosporidium sp.1Positive
Cyclospora cayetanensis1Negative
Entamoeba histolytica1Negative
Enterovirus4Negative
Escherichia coli8Negative
Giardia lamblia1Negative
HIV2Negative
Leishmania spp.2Negative
Microsporidia1Negative
Norovirus2Negative
Plasmodium spp.4Negative
Plesiomonas shigelloides1Negative
Salmonella enteritidis1Negative
Shigella spp.2Negative
Staphylococcus aureus (Ent A-E)5Negative
Trypanosoma brucei1Negative
Vibrio spp.7Negative
Yersinia enterocolitica1Negative

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).

DISCUSSION

Although microscopy has long been the gold standard for the detection of Cryptosporidium, more sensitive molecular-based methods are now available. We tested 91 stool specimens that were submitted to the public health laboratory to confirm the presence of Cryptosporidium. Of those, 20 were positive based on microscopy, another 20 were positive when tested by other methods, and 51 were negative in all assays. Of the 40 samples determined positive by the composite reference, most were found positive by a molecular method and in some cases, by the LDT alone (Figure 2). The LDT described here identified 20 specimens that were negative when tested by microscopy but determined to be positive by the composite reference. The average Ct value for those specimens when tested by the LDT was 31.7, compared with an average Ct value of 29.2 for the 20 samples that were positive by microscopy. Similarly, as noted earlier, specimens that yielded a false-negative result by G/CQC had a higher Ct value than specimens that were positive by G/CQC. Although the number of samples in each group was low, the results suggest that specimens with a low parasite burden may be missed by the G/CQC immunoassay. These results are consistent with previous studies26,27 which indicate microscopy and G/CQC lack sensitivity compared with nucleic acid amplification–based testing.

Figure 2.
Figure 2.

Distribution of positive samples by method.

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

Although amplification-based testing has better sensitivity, inhibition can be a problem when performing PCR on stool samples, which are inherently variable. In the LDT described here, inhibition was evaluated in two ways. First, the inhibition of sample amplification was monitored by adding an internal inhibition control plasmid to the patient sample, and PEC and Ct values were compared after amplification. Second, for each patient sample, a 1:10 dilution of the extracted DNA was tested to dilute the effect of any possible inhibitors. In two cases, a sample tested at the 1:10 dilution gave duplicate positive results, whereas the undiluted sample gave one positive and one negative result. This indicates that in a small percentage of samples, amplification was inhibited and inhibition could be overcome by diluting the DNA extract.

We also compared the results of the LDT to a commercially available GI panel. All of the samples that yielded discordant results had been preserved in 10% formalin, which is not approved for this assay. Interestingly, the GI panel was able to successfully detect Cryptosporidium in 17 samples that had been preserved in a formalin-based fixative. Although the LDT performed well with formalin-fixed specimens in this study, we have observed (data not shown) that the Ct values for formalin-fixed stool increased with longer storage times. The results suggest an interference with extraction, inhibition of DNA amplification, or both. Although each formalin-preserved sample processed in this study was washed two times with PBS to remove as much fixative as possible, it is unknown how long each sample was stored in formalin before being washed. Cary-Blair transport medium and preservatives that are alcohol-based are preferred when performing molecular analysis.18 The compatibility of these fixatives with molecular analysis was also evident in this study. Both the LDT and the GI panel produced a positive result for all samples that were submitted in Total-Fix or Cary-Blair transport medium.

For samples that gave discordant results when comparing the LDT and the GI panel, conventional PCR and sequencing were performed to confirm the presence of Cryptosporidium DNA. For six of the seven samples, sequencing verified the results of the LDT. For one sample, an interpretable sequence could not be obtained; however, the G/CQC antigen-based test was consistent with the presence of Cryptosporidium (Table 4). Of note, this sample had been stored in formalin, potentially contributing to a negative result on the GI panel and the inability to obtain readable DNA sequence.

Another consideration for reference or clinical laboratories is the cost of performing an assay. In Table 6, we show the cost of each assay, excluding labor. Microscopy and G/CQC are the least expensive, although personnel costs for microscopy can be high as it is labor intensive. Additionally, microscopy and G/CQC were not as sensitive. The LDT is slightly more expensive than microscopy or G/CQC at approximately $15 per sample, including DNA extraction and PCR. The LDT is affordable even when performed in duplicate, as there is only a modest increase in cost for the additional PCR reagents. The cost of a single GI panel is approximately eight-fold higher at $130 per sample, at the time of study. When comparing personnel costs for the LDT and GI panel, although the sample preparation for the LDT can be time consuming, DNA extraction and amplification can be completed for approximately 45 samples in an 8-hour workday. By comparison, the GI panel requires less hands-on specimen preparation time. However, a single instrument has the maximum capacity of processing one sample at a time and is limited to roughly 7–8 samples in a day.

Table 6

Cost of an assay

Detection methodApproximate cost ($)
Microscopy7.50
Giardia/Cryptosporidium Quik Chek11.50
Laboratory-developed test15.00
Gastrointestinal panel130.00

Some limitations of this study were due solely to the study being performed by a public health reference laboratory. For example, the optimal time to perform an antigen-based test is within 72 hours of collection. At a reference laboratory, this is not always feasible, as some submitters may hold and batch samples to send multiple specimens to the laboratory at once. Also, the fixative used to collect a sample may not be compatible with the testing algorithm the laboratory has established. Molecular methods are most compatible with a fixative that is ethanol-based or with a specimen that is unpreserved. However, most stool samples for parasitology testing are still collected in a formalin-based fixative, which does not allow for optimal performance of the LDT or the GI panel. In addition, to properly perform DFA microscopy analysis, a polyvinyl alcohol fixative is not recommended because of the high levels of background that can be generated. As a reference laboratory, we must adapt and perform each test to the best of our capabilities given the specimens we receive.

The LDT described here is a method that laboratories can use to accurately and reliably test for Cryptosporidium in patient stool samples. It has proven to be a sensitive, specific, and cost-efficient option that allows for fast and easy result interpretation. In addition, the LDT can be used in a high-throughput situation, such as an outbreak. The assay specifically detects C. parvum and C. hominis. Other species can be detected by the genus-level primers, although the sensitivity is decreased because of degeneracy in the primers and probes. An added benefit of the LDT is that the extracted DNA can be genotyped to aid in source tracking and epidemiological investigations.

Supplemental figure and table

Acknowledgment:

We thank the Wadsworth Center Applied Genomics Technology Cluster for performing all sequencing.

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    Adams DA, Thomas KR, Jajosky RA, Foster L, Sharp P, Onweh DH, Schley AW, Anderson WJ; Nationally Notifiable Infectious Conditions G, 2016. Summary of notifiable infectious diseases and conditions - United States, 2014. MMWR Morb Mortal Wkly Rep 63: 1152.

    • Search Google Scholar
    • Export Citation
  • 4.

    Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM, 2011. Foodborne illness acquired in the United States–major pathogens. Emerg Infect Dis 17: 715.

    • Search Google Scholar
    • Export Citation
  • 5.

    Painter JE, Hlavsa MC, Collier SA, Xiao L, Yoder JS; CDC, 2015. Cryptosporidiosis surveillance–United States, 2011–2012. MMWR 64: 114.

  • 6.

    Yoder JS et al. CDC, 2008. Surveillance for waterborne disease and outbreaks associated with recreational water use and other aquatic facility-associated health events–United States, 2005–2006. MMWR Surveill Summ 57: 129.

    • Search Google Scholar
    • Export Citation
  • 7.

    CDC, 2012. Promotion of healthy swimming after a statewide outbreak of cryptosporidiosis associated with recreational water venues--Utah, 2008-2009. MMWR Morb Mortal Wkly Rep 61: 348352.

    • Search Google Scholar
    • Export Citation
  • 8.

    Cantey PT et al. 2012. Outbreak of cryptosporidiosis associated with a man-made chlorinated lake–Tarrant county, Texas, 2008. J Environ Health 75: 1419.

    • Search Google Scholar
    • Export Citation
  • 9.

    DeSilva MB et al. 2016. Communitywide cryptosporidiosis outbreak associated with a surface water-supplied municipal water system–Baker city, Oregon, 2013. Epidemiol Infect 144: 274284.

    • Search Google Scholar
    • Export Citation
  • 10.

    Robinson TJ, Cebelinski EA, Taylor C, Smith KE, 2010. Evaluation of the positive predictive value of rapid assays used by clinical laboratories in Minnesota for the diagnosis of cryptosporidiosis. Clin Infect Dis 50: e53e55.

    • Search Google Scholar
    • Export Citation
  • 11.

    Minak J, Kabir M, Mahmud I, Liu Y, Liu L, Haque R, Petri WA Jr., 2012. Evaluation of rapid antigen point-of-care tests for detection of giardia and Cryptosporidium species in human fecal specimens. J Clin Microbiol 50: 154156.

    • Search Google Scholar
    • Export Citation
  • 12.

    Alexander CL, Niebel M, Jones B, 2013. The rapid detection of Cryptosporidium and Giardia species in clinical stools using the quik chek immunoassay. Parasitol Int 62: 552553.

    • Search Google Scholar
    • Export Citation
  • 13.

    Roellig DM et al. 2017. Community laboratory testing for Cryptosporidium: multicenter study retesting public health surveillance stool samples positive for Cryptosporidium by rapid cartridge assay with direct fluorescent antibody testing. PLoS One 12: e0169915.

    • Search Google Scholar
    • Export Citation
  • 14.

    Buss SN, Leber A, Chapin K, Fey PD, Bankowski MJ, Jones MK, Rogatcheva M, Kanack KJ, Bourzac KM, 2015. Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis. J Clin Microbiol 53: 915925.

    • Search Google Scholar
    • Export Citation
  • 15.

    Madison-Antenucci S et al. 2016. Multicenter evaluation of BD max enteric parasite real-time PCR assay for detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica. J Clin Microbiol 54: 26812688.

    • Search Google Scholar
    • Export Citation
  • 16.

    Mary C et al. ANOFEL Cryptosporidium National Network. 2013. Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis. J Clin Microbiol 51: 25562563.

    • Search Google Scholar
    • Export Citation
  • 17.

    Xu P et al. 2004. The genome of Cryptosporidium hominis. Nature 431: 11071112.

  • 18.

    Abrahamsen MS et al. 2004. Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304: 441445.

  • 19.

    Teal AE, Habura A, Ennis J, Keithly JS, Madison-Antenucci S, 2012. A new real-time PCR assay for improved detection of the parasite Babesia microti. J Clin Microbiol 50: 903908.

    • Search Google Scholar
    • Export Citation
  • 20.

    Elsafi SH, Al-Maqati TN, Hussein MI, Adam AA, Hassan MM, Al Zahrani EM, 2013. Comparison of microscopy, rapid immunoassay, and molecular techniques for the detection of Giardia lamblia and Cryptosporidium parvum. Parasitol Res 112: 16411646.

    • Search Google Scholar
    • Export Citation
  • 21.

    Ryan U, Fayer R, Xiao L, 2014. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology 141: 16671685.

    • Search Google Scholar
    • Export Citation
  • 22.

    Hadfield SJ, Robinson G, Elwin K, Chalmers RM, 2011. Detection and differentiation of Cryptosporidium spp. in human clinical samples by use of real-time PCR. J Clin Microbiol 49: 918924.

    • Search Google Scholar
    • Export Citation
  • 23.

    Jothikumar N, da Silva AJ, Moura I, Qvarnstrom Y, Hill VR, 2008. Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays. J Med Microbiol 57: 10991105.

    • Search Google Scholar
    • Export Citation
  • 24.

    Alves M, Xiao L, Sulaiman I, Lal AA, Matos O, Antunes F, 2003. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol 41: 27442747.

    • Search Google Scholar
    • Export Citation
  • 25.

    Fayer R, Xiao L, 2008. Cryptosporidium and Cryptosporidiosis. Boca Raton, FL: CRC Press; IWA Publishing.

  • 26.

    Laude A et al. 2016. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review. Clin Microbiol Infect 22: 190 e1190 e8.

    • Search Google Scholar
    • Export Citation
  • 27.

    ten Hove RJ, van Esbroeck M, Vervoort T, van den Ende J, van Lieshout L, Verweij JJ, 2009. Molecular diagnostics of intestinal parasites in returning travellers. Eur J Clin Microbiol Infect Dis 28: 10451053.

    • Search Google Scholar
    • Export Citation
  • 28.

    CDC, DPDX, 2016. Stool Specimens - Molecular Diagnosis. Available at: https://www.cdc.gov/dpdx/diagnosticprocedures/stool/moleculardx.html. Accessed 2019.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Susan Madison-Antenucci, Parasitology Laboratory, Wadsworth Center, NYSDOH, 120 New Scotland Ave., Rm. 3112, Albany, NY 12201-2002. E-mail: s.antenucci@health.ny.gov

Authors’ addresses: Kimberly Mergen, Noel Espina, and Allen Teal, Parasitology Laboratory, Wadsworth Center, NYSDOH, Albany, NY, E-mails: kimberly.mergen@health.ny.gov, noel.espina@health.ny.gov, and allen.teal@health.ny.gov. Susan Madison-Antenucci, Parasitology Laboratory, Wadsworth Center, NYSDOH, Albany, NY, and School of Public Health, Biomedical Sciences, University at Albany, Albany, NY, E-mail: s.antenucci@health.ny.gov.

  • 1.

    Katsumata T, Hosea D, Ranuh IG, Uga S, Yanagi T, Kohno S, 2000. Short report: possible Cryptosporidium muris infection in humans. Am J Trop Med Hyg 62: 7072.

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  • 2.

    Chappell CL, Okhuysen PC, Langer-Curry R, Widmer G, Akiyoshi DE, Tanriverdi S, Tzipori S, 2006. Cryptosporidium hominis: experimental challenge of healthy adults. Am J Trop Med Hyg 75: 851857.

    • Search Google Scholar
    • Export Citation
  • 3.

    Adams DA, Thomas KR, Jajosky RA, Foster L, Sharp P, Onweh DH, Schley AW, Anderson WJ; Nationally Notifiable Infectious Conditions G, 2016. Summary of notifiable infectious diseases and conditions - United States, 2014. MMWR Morb Mortal Wkly Rep 63: 1152.

    • Search Google Scholar
    • Export Citation
  • 4.

    Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM, 2011. Foodborne illness acquired in the United States–major pathogens. Emerg Infect Dis 17: 715.

    • Search Google Scholar
    • Export Citation
  • 5.

    Painter JE, Hlavsa MC, Collier SA, Xiao L, Yoder JS; CDC, 2015. Cryptosporidiosis surveillance–United States, 2011–2012. MMWR 64: 114.

  • 6.

    Yoder JS et al. CDC, 2008. Surveillance for waterborne disease and outbreaks associated with recreational water use and other aquatic facility-associated health events–United States, 2005–2006. MMWR Surveill Summ 57: 129.

    • Search Google Scholar
    • Export Citation
  • 7.

    CDC, 2012. Promotion of healthy swimming after a statewide outbreak of cryptosporidiosis associated with recreational water venues--Utah, 2008-2009. MMWR Morb Mortal Wkly Rep 61: 348352.

    • Search Google Scholar
    • Export Citation
  • 8.

    Cantey PT et al. 2012. Outbreak of cryptosporidiosis associated with a man-made chlorinated lake–Tarrant county, Texas, 2008. J Environ Health 75: 1419.

    • Search Google Scholar
    • Export Citation
  • 9.

    DeSilva MB et al. 2016. Communitywide cryptosporidiosis outbreak associated with a surface water-supplied municipal water system–Baker city, Oregon, 2013. Epidemiol Infect 144: 274284.

    • Search Google Scholar
    • Export Citation
  • 10.

    Robinson TJ, Cebelinski EA, Taylor C, Smith KE, 2010. Evaluation of the positive predictive value of rapid assays used by clinical laboratories in Minnesota for the diagnosis of cryptosporidiosis. Clin Infect Dis 50: e53e55.

    • Search Google Scholar
    • Export Citation
  • 11.

    Minak J, Kabir M, Mahmud I, Liu Y, Liu L, Haque R, Petri WA Jr., 2012. Evaluation of rapid antigen point-of-care tests for detection of giardia and Cryptosporidium species in human fecal specimens. J Clin Microbiol 50: 154156.

    • Search Google Scholar
    • Export Citation
  • 12.

    Alexander CL, Niebel M, Jones B, 2013. The rapid detection of Cryptosporidium and Giardia species in clinical stools using the quik chek immunoassay. Parasitol Int 62: 552553.

    • Search Google Scholar
    • Export Citation
  • 13.

    Roellig DM et al. 2017. Community laboratory testing for Cryptosporidium: multicenter study retesting public health surveillance stool samples positive for Cryptosporidium by rapid cartridge assay with direct fluorescent antibody testing. PLoS One 12: e0169915.

    • Search Google Scholar
    • Export Citation
  • 14.

    Buss SN, Leber A, Chapin K, Fey PD, Bankowski MJ, Jones MK, Rogatcheva M, Kanack KJ, Bourzac KM, 2015. Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis. J Clin Microbiol 53: 915925.

    • Search Google Scholar
    • Export Citation
  • 15.

    Madison-Antenucci S et al. 2016. Multicenter evaluation of BD max enteric parasite real-time PCR assay for detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica. J Clin Microbiol 54: 26812688.

    • Search Google Scholar
    • Export Citation
  • 16.

    Mary C et al. ANOFEL Cryptosporidium National Network. 2013. Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis. J Clin Microbiol 51: 25562563.

    • Search Google Scholar
    • Export Citation
  • 17.

    Xu P et al. 2004. The genome of Cryptosporidium hominis. Nature 431: 11071112.

  • 18.

    Abrahamsen MS et al. 2004. Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304: 441445.

  • 19.

    Teal AE, Habura A, Ennis J, Keithly JS, Madison-Antenucci S, 2012. A new real-time PCR assay for improved detection of the parasite Babesia microti. J Clin Microbiol 50: 903908.

    • Search Google Scholar
    • Export Citation
  • 20.

    Elsafi SH, Al-Maqati TN, Hussein MI, Adam AA, Hassan MM, Al Zahrani EM, 2013. Comparison of microscopy, rapid immunoassay, and molecular techniques for the detection of Giardia lamblia and Cryptosporidium parvum. Parasitol Res 112: 16411646.

    • Search Google Scholar
    • Export Citation
  • 21.

    Ryan U, Fayer R, Xiao L, 2014. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology 141: 16671685.

    • Search Google Scholar
    • Export Citation
  • 22.

    Hadfield SJ, Robinson G, Elwin K, Chalmers RM, 2011. Detection and differentiation of Cryptosporidium spp. in human clinical samples by use of real-time PCR. J Clin Microbiol 49: 918924.

    • Search Google Scholar
    • Export Citation
  • 23.

    Jothikumar N, da Silva AJ, Moura I, Qvarnstrom Y, Hill VR, 2008. Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays. J Med Microbiol 57: 10991105.

    • Search Google Scholar
    • Export Citation
  • 24.

    Alves M, Xiao L, Sulaiman I, Lal AA, Matos O, Antunes F, 2003. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol 41: 27442747.

    • Search Google Scholar
    • Export Citation
  • 25.

    Fayer R, Xiao L, 2008. Cryptosporidium and Cryptosporidiosis. Boca Raton, FL: CRC Press; IWA Publishing.

  • 26.

    Laude A et al. 2016. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review. Clin Microbiol Infect 22: 190 e1190 e8.

    • Search Google Scholar
    • Export Citation
  • 27.

    ten Hove RJ, van Esbroeck M, Vervoort T, van den Ende J, van Lieshout L, Verweij JJ, 2009. Molecular diagnostics of intestinal parasites in returning travellers. Eur J Clin Microbiol Infect Dis 28: 10451053.

    • Search Google Scholar
    • Export Citation
  • 28.

    CDC, DPDX, 2016. Stool Specimens - Molecular Diagnosis. Available at: https://www.cdc.gov/dpdx/diagnosticprocedures/stool/moleculardx.html. Accessed 2019.

    • Search Google Scholar
    • Export Citation
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