The Journal of Molecular Diagnostics, Vol. 13, No. 3, May 2011

Copyright © 2011 American Society for Investigative Pathology

and the Association for Molecular Pathology.

Published by Elsevier Inc. All rights reserved.

DOI: 10.1016/j.jmoldx.2011.01.007

Consultations in Molecular DiagnosticsMolecular Diagnosis of Cystoisosporiasis Using

Extended-Range PCR Screening

Sean C. Murphy,* Daniel R. Hoogestraat,*Dhruba J. SenGupta,* Jennifer Prentice,*Andrea Chakrapani,† and Brad T. Cookson*‡

From the Departments of Laboratory Medicine * and

Microbiology,‡ University of Washington, Seattle, Washington,

and the Department of Pathology,† Oregon Health & Sciences

University, Portland, Oregon

The differential diagnosis of diarrhea in immunocom-promised patients encompasses many intestinal par-asites including the coccidian Cystoisospora belli.Gastrointestinal infection with C. belli leads tocystoisosporiasis with diarrhea and, depending onhost immune status, can cause extraintestinal disease.C. belli is usually diagnosed by examination of stoolor intestinal biopsy specimens; however, the organ-ism may be undetected using these test methods.Thus, more sensitive molecular tools for detection ofpathogenic parasites are desirable. Herein is de-scribed a patient with AIDS who had persistent diar-rhea of unknown cause. Microscopic examinations ofstool and ileal biopsy specimens were initially unre-markable for any specific pathogen. Screening ofDNA extracted from biopsy material using extended-range PCR primers recognizing conserved DNA se-quences found in many fungi and parasites revealedinfection with C. belli, which was confirmed at repeathistologic analysis. Extended-range PCR screeningwas used because the differential diagnosis was broadand other tools were not applied, yet this molecularapproach led to the appropriate diagnosis and treat-ment of the condition. Thus, this approach offers apromising test for diagnosis of parasitic diseases thatelude diagnosis using conventional methods. (J Mol

Diagn 2011, 13:359–362; DOI: 10.1016/j.jmoldx.2011.01.007)

Cystoisospora parasites are coccidia that cause diarrheain human beings and animals throughout the world.Cystoisospora belli, previously called Isospora belli beforereclassification, infects human beings exclusively.1 The

human diarrheal syndrome caused by C. belli is properly

termed “cystoisosporiasis.” Infection of immunocompe-tent persons usually results in self-limited nonbloody di-arrhea of 2- to 3-weeks duration.1 However, infection inimmunosuppressed patients can evolve into persistententeritis, with malabsorption and weight loss. Patientswith AIDS are particularly susceptible, and extraintestinalmanifestations such as invasion of mesenteric and tra-cheobronchial lymph nodes, liver, and spleen can occur.In addition to C. belli, other coccidia including Cryptospo-ridium spp. are also problematic in patients with AIDS.

The life cycle of C. belli has been reviewed in detail.1

Once shed in the stool, the single sporoblast within animmature oocyst divides to form two sporocysts, eachcontaining four sporozoites. The mature oocyst is theninfective, and can remain viable in the environment formonths. Human infection occurs when mature oocystsare ingested, releasing sporozoites into the small bowel,with subsequent invasion of gastrointestinal epithelium.Intracellular parasites undergo schizogony and releaseasexual-stage merozoites that invade additional epithelialcells. Infection propagates via asexual cycles of merozoiteinvasion, development of trophozoite and schizont stages,and release of daughter merozoites. Eventually, sexual-stage gametocytes emerge, and fertilization yields imma-ture oocysts, which are excreted.

The diagnosis of cystoisosporiasis is usually made byidentifying oocysts in stool wet mounts or stained fecalsmears.1 C. belli oocysts are characteristically ellipsoidand large (25 to 30 �m long), which makes them indis-tinguishable from other coccidia. Although they may bedetected using conventional methods, staining of director concentrated specimens using modified acid-fast2 orauramine-rhodamine or auramine O 3 procedures and UVmicroscopy1 can aid in detection and is standard prac-

Supported by the Department of Laboratory Medicine, University ofWashington Medical Center.

Accepted for publication January 4, 2011.

Address reprint requests to Brad T. Cookson, M.D., Ph.D., Departmentsof Laboratory Medicine and Microbiology, University of Washington Med-ical Center, 1959 NE Pacific Street, Box 357110, Seattle, WA 98195-7110; orSean C. Murphy, M.D., Ph.D., Department of Laboratory Medicine, University ofWashington Medical Centere, 1959 NE Pacific, NW120, Seattle, WA 98195.

E-mail: cookson@u.washington.edu or murphysc@u.washington.edu.

359

360 Murphy et alJMD May 2011, Vol. 13, No. 3

tice in several laboratories. Repeated stool examinationsmay be required for diagnosis because of low intermittentoocyst shedding. If stool examinations yield negativefindings, intestinal aspirate or biopsy specimens maydemonstrate intraepithelial C. belli parasites, or a duode-nal string test may be diagnostic.1 However, C. belli cancause disease with few parasites present and may bemissed at all of these tests,1 in particular if coccidiaare not included in the initial differential diagnosis.Cystoisosporiasis is treated using trimethoprim-sulfame-thoxazole. Widespread prophylactic use of this drugagainst Pneumocystis in HIV-positive patients has sub-stantially reduced the incidence of C. belli as an AIDS-defining illness.

DNA sequence-based screening using conserved ri-bosomal RNA (rRNA) genes in bacteria4 and the internaltranscribed spacer (ITS) regions between rRNA struc-tural genes in fungi5 enable diagnosis of a range ofpathogens, many of which elude diagnosis using con-ventional methods. Similar “global” screening tools arebeing developed for viruses.6 However, there are cur-rently no well-established screening methods for parasiteDNA. It was recently reported that DNA screening forfungi using ITS regions 1 and 2 and the 28S rRNA geneas DNA targets detected Taenia solium in patients withneurocysticercosis.7 Herein is reported the molecular di-agnosis of cystoisosporiasis in an HIV-positive patientwith diarrhea of unknown cause that initially eluded diag-nosis using conventional approaches.

Materials and Methods

Patient

A 44-year-old man with HIV-1 infection who was not re-ceiving antiretroviral therapy (CD4� T-cell count, 211/�L;HIV viral load, 227,000 copies/mL) had persistent non-bloody diarrhea. He had been living in rural Mexico for 2years. Diarrhea developed over the last 3 months, with aconcurrent 20-lb weight loss. An antiretroviral regimen(efavirenz-emtricitabine-tenofovir) was reinstituted, andloperamide was prescribed. Stool bacterial cultures werenegative for Salmonella, Shigella, Campylobacter, andEscherichia coli O157:H7, as were repeated stool exami-nations for ova and parasites, cryptosporidia, and mi-crosporidia. A Giardia enzyme immunoassay yieldednegative results. Endoscopic ileal biopsy specimensdemonstrated no inflammation, mucosal abnormalities, orevidence of infectious agents at hematoxylin and eosinstaining; additional staining procedures were not per-formed. PCR was performed on the ileal biopsy specimenusing extended-range bacterial and fungal primers.

DNA Analysis

Molecular diagnosis of biopsy material was performed asreported previously.7 Tissue was digested, DNA ex-tracted, and template DNA amplified by PCR using prim-ers for ITS region 1 (primers ITS1 and ITS2), ITS region 2(primers ITS3 and ITS4) and the 28S rRNA gene (primers

NL1 and NL4). PCR products were visualized on agarose

gels.7 Bidirectional sequencing was performed using theABI PRISM BigDye Terminator Cycle Sequencing Kit andthe ABI PRISM 3100 Analyzer (both from Applied Biosys-tems, Inc., Foster City, CA), and sequences were assem-bled into double-stranded contigs using Sequenchersoftware (Gene Codes Corp., Ann Arbor, MI) and sub-mitted to a GenBank Basic Local Alignment Search Tool(BLAST) search.

Figure 1. Molecular diagnosis of cystoisosporiasis using molecular methodsand retrospective confirmation by histologic examination. A: PCR-baseddetection of C. belli using primers directed against the 28S rRNA gene and ITSregions 1 and 2. PCR reactions were performed on extracted DNA, and PCRproducts were separated by electrophoresis using a 1% agarose gel andstained. Reaction products from primer sets are shown with the patienttemplate (Pt), positive control (�), and negative control (�), as shown. A100-bp DNA ladder is also shown. B: C. belli parasites demonstrated in theileal mucosa in a biopsy specimen from the described patient. Multinucleatedasexual schizonts (arrow), sexual macrogametocyte (wide arrowhead),and microgametocytes (narrow arrowheads) reside within enterocytes.The gastrointestinal epithelium demonstrated a normal-appearing epithelial

layer with intact lamina propria lacking significant inflammation. Hematox-ylin and eosin stain; original magnification �40.

Cystoisospora Diagnosis by PCR Screening 361JMD May 2011, Vol. 13, No. 3

Results

Universal bacterial primers (16S rRNA gene, heat shockprotein 65, and RNA polymerase � subunit) demon-strated no amplification products. This was somewhatsurprising because the patient was not receiving antibi-otic therapy and the sample was a gastrointestinal biopsyspecimen, in which normal bacterial microbiota were ex-pected. Nonetheless, all reactions were positive for ex-traction and amplification controls, and the presence ofPCR inhibitors was excluded. In contrast, all three sets ofuniversal fungal primers targeting ITS regions 1 and 2and the 28S rRNA gene produced PCR products onagarose gels (Figure 1). The ITS1 product (626 bp)yielded multiple unequivocal matches to C. belli in theITS1 region adjacent to the 18S rDNA (representativeGenBank accession DQ060683.2: 99% identity, 100%query coverage, BLAST E value 0.0). The 28S rDNAreaction product (629 bp) yielded matches to 28S rRNAgene sequences from Cystoisospora felis (GenBank ac-cession U85705.1: 93% identity, 100% query coverage,BLAST E value 0.0), Cystoisospora suis (GenBank acces-sion AF093428.1: 96% identity, 43% query coverage,BLAST E value 6�123), and C. belli (GenBank accessionAY063483.1: 94% identity, 36% query coverage, BLASTE value 6e�111). However, the 28S region is poorly rep-resented in the GenBank database (only four totalCystoisospora entries), making this region less reliablethan the ITS1 region for molecular speciation. The ITS2region amplification product was not sequenced. In lightof these findings, tissue biopsies were re-examined, andoccasional asexual forms of C. belli (schizonts and micro/macrogametes) were identified (Figure 1). Antiretroviraltherapy was resumed, and the patient’s diarrhea gradu-ally resolved.

Discussion

While coccidia-specific rRNA primers have been used todetect C. belli8, this is the first use of extended-rangemolecular screening with widely conserved primer-bind-ing sites to detect C. belli infection in a patient withcystoisosporiasis. Diagnosis initially eluded conventionaltesting because findings at stool examinations and bi-opsy specimen analysis were unremarkable. A molecularscreening approach included use of PCR primers capa-ble of detecting a broad range of bacterial, fungal, andsome parasitic pathogens. Using this approach, defini-tive diagnosis of C. belli was made by detection of its DNAin biopsy material and was confirmed upon retrospectivereview of tissue sections.

Eukaryotes possess four rRNA genes (18S, 5.8S, 28S,and 5S), which are highly conserved across differenttaxonomic groups. ITS sequences are noncoding regionsbetween 28S, 5.8S, and 18S rRNA genes. ITS1 (between18S and 5.8S rRNA genes) has been widely evaluated inmany eukaryotic organisms, and these sequences oftenprovide identification at the species level. Conservedprimer binding sites in the 18S and 5.8S rRNA genes

permit PCR amplification of the ITS1 sequence from both

fungal and parasitic DNA. Phylogenetic analysis ofITS1 sequences clearly delineated differences be-tween Cystoisospora and related parasites,9 and a PCRrestriction fragment length polymorphism-based assaywith ITS1 reliably identified and differentiated multiplespecies of Cystoisospora.10 Species-specific regionsof the adjacent 18S rRNA gene have been used as aspecific molecular target for C. belli,8 and were used toreclassify Cystoisospora into the family Sarcocystidaerather than Eimeriidae.11 In contrast, GenBank currentlycontains only four 28S rRNA sequences for Cysto-isospora; thus, the diagnosis using 28S rRNA primers isless definitive.

Primers for ITS region 1 (primers ITS1 and ITS2) havepreviously detected T. solium,7 and herein we report de-tection of C. belli using primers for all three regions (ITS1,ITS2, and 28S rRNA gene). In addition, in our laboratory,Toxoplasma gondii has been detected in clinical speci-mens using ITS1 region primers (oral communication onSeptember 19, 2009, D.T.S. and B.T.C.). ITS1 regionprimers are likely to detect other parasites as well, al-though the reverse primer of this pair is unlikely to detectPlasmodium, Giardia, or Trichomonas (personal commu-nication, D.T.S. and B.T.C.). Taenia was detected despitemultiple mismatches between PCR primers and the tar-get DNA sequences. Pathogen-specific molecular testsare available for parasites such as Cryptosporidium andare much more sensitive than microscopy.10,12 However,pathogen-specific molecular tests require that clinicianssuspect specific pathogens at the beginning of a di-agnostic workup. PCR screening with widely con-served primer binding sites, such as those used in thepresent study, is a valuable and efficient tool that canbe used to identify a range of suspected or unsus-pected etiologic agents including parasites. This ap-proach is useful in cases such as this in which thedifferential diagnosis is broad or in cases in whichparasites are not initially considered or pathogen-spe-cific testing is unrevealing or nonexistent.

References

1. Lindsay DS, Dubey JP, Blagburn BL: Biology of Isospora spp. from hu-mans, nonhuman primates, and domestic animals. Clin Microbiol Rev 1997,10:19–34

2. Ng E, Markell EK, Fleming RL, Fried M: Demonstration of Isosporabelli by acid-fast stain in a patient with acquired immune deficiencysyndrome. J Clin Microbiol 1984, 20:384–386

3. Hanscheid T, Cristino JM, Salgado MJ: Screening of auramine-stainedsmears of all fecal samples is a rapid and inexpensive way to increasethe detection of coccidial infections. Int J Infect Dis 2008, 12:47–50

4. Clarridge JE III: Impact of 16S rRNA gene sequence analysis foridentification of bacteria on clinical microbiology and infectious dis-eases. Clin Microbiol Rev 2004, 17: 840–862

5. Rakeman JL, Bui U, Lafe K, Chen YC, Honeycutt RJ, Cookson BT:Multilocus DNA sequence comparisons rapidly identify pathogenicmolds. J Clin Microbiol 2005, 43:3324–3333

6. Chiu CY, Urisman A, Greenhow TL, Rouskin S, Yagi S, Schnurr D,Wright C, Drew WL, Wang D, Weintrub PS, Derisi JL, Ganem D: Utilityof DNA microarrays for detection of viruses in acute respiratory tractinfections in children. J Pediatr 2008, 153:76–83

7. Harrington AT, Creutzfeldt CJ, SenGupta DJ, Hoogestraat DR, ZuntJR, Cookson BT: Diagnosis of neurocysticercosis by detection of

362 Murphy et alJMD May 2011, Vol. 13, No. 3

Taenia solium DNA using a global DNA screening platform. Clin InfectDis 2008, 48:86–90

8. Müller A, Bialek R, Fätkenheuer G, Salzberger B, Diehl V, Franzen C:Detection of Isospora belli by polymerase chain reaction using prim-ers based on small-subunit ribosomal RNA sequences. Eur J ClinMicrobiol Infect Dis 2000, 19:631–634

9. Samarasinghe B, Johnson J, Ryan U: Phylogenetic analysis ofCystoisospora species at the rRNA ITS1 locus and development of aPCR-RFLP assay. Exp Parasitol 2008, 118:592–595

10. Ryan UM, Bath C, Robertson I, Read C, Elliot A, McInnes L, Traub R,Besier B: Sheep may not be an important zoonotic reservoir for

Cryptosporidium and Giardia parasites. Appl Environ Microbiol 2005,71:4992–4997

11. Franzen C, Müller A, Bialek R, Diehl V, Salzberger B, Fätkenheuer G:Taxonomic position of the human intestinal protozoan parasiteIsospora belli as based on ribosomal RNA sequences. Parasitol Res2000, 86:669–676

12. Bruijnesteijn van Coppenraet LE, Wallinga JA, Ruijs GJ, Bruins MJ,Verweij JJ: Parasitological diagnosis combining an internally con-trolled real-time PCR assay for the detection of four protozoa in stool

samples with a testing algorithm for microscopy. Clin Microbiol Infect2009, 15:869–874