Mixed Cryptosporidium infections and HIV.Mixed Cryptosporidium cryptosporidium (krĭp'tōspərĭd`ēəm), genus of protozoans having at least four species; they are waterborne parasites that cause the disease cryptosporidiosis. infections were detected in 7 of 21 patients with a diagnosis of rare Cryptosporidium canis or C. fells infections; 6 patients were infected with 2 Cryptosporidium spp. and 1 patient with 3 species, Mixed infections may occur more frequently than previously believed and should be considered when assessing cryptosporidiosis Cryptosporidiosis Definition Cryptosporidiosis refers to infection by the sporeforming protozoan known as Cryptosporidia. Protozoa are a group of parasites that infect the human intestine, and include the better known Giardia. . ********** Cryptosporidium spp. infect humans and other vertebrate animals. Persons with compromised immune systems can suffer life-threatening chronic diarrhea, especially when their CD4+ lymphocyte lymphocyte: see blood; immunity. lymphocyte Type of leukocyte fundamental to the immune system, regulating and participating in acquired immunity. Each has receptor molecules on its surface that bind to a specific antigen. counts fall <200 cells/[micro]L. At least 7 Cryptosporidium spp. have been detected in immunocompromised immunocompromised /im·mu·no·com·pro·mised/ (-kom´pro-mizd) having the immune response attenuated by administration of immunosuppressive drugs, by irradiation, by malnutrition, or by certain disease processes (e.g., cancer). patients (1). Nonetheless, the role of concurrent or mixed infections in the pathogenesis and transmission of Cryptosporidium spp. is unclear. Mixed infections of Cryptosporidium hominis and C. parvum have been reported in several patients from Switzerland and England (2,3). Additional studies from the United Kingdom reported simultaneous infections with these 2 species: 4 cases in 2 waterborne outbreaks and 2 cases of sporadic infections from 1995 to 1999 (4). In a more recent study, 12% of 135 clinical specimens from Aberdeenshire, Scotland, had concurrent C Concurrent C - 1. An extension of C with rendezvous-based concurrency. Versions for most Unix systems are available commercially from AT&T. ["Concurrent C", N.H. Gehani et al, Soft Prac & Exp 16(9):821-844 (1986)]. ["The Concurrent C Programming Language", N. . parvum and C. hominis infections (5). Mixed C. hominis--C, parvum infections were also seen in 2 of 38 archived human specimens in a study conducted in the United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. (6). These observations suggest that mixed Cryptosporidium infections are not uncommon. Mixed infections may not be readily identified by commonly used molecular diagnostic tools because of preferential polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) ) amplification of the predominant genotypes or the specificity of molecular tools (6). For example, PCR--restriction fragment length polymorphism polymorphism, of minerals, property of crystallizing in two or more distinct forms. Calcium carbonate is dimorphous (two forms), crystallizing as calcite or aragonite. Titanium dioxide is trimorphous; its three forms are brookite, anatase (or octahedrite), and rutile. (RFLP RFLP abbr. restriction fragment length polymorphism RFLP restriction fragment length polymorphism. RFLP ) tools based on the small subunit (SSU SSU Small Subunit SSU Sonoma State University SSU Savannah State University (Savannah, Georgia) SSU Shawnee State University (Ohio) SSU Salisbury State University ) rRNA gene are frequently used in genotyping Cryptosporidium spp. because they have higher sensitivity and detect more species than PCR-RFLP PCR-RFLP Polymerase Chain Reaction–Restriction Fragment Length Polymorphism tools based on other genes (7). Two previous studies in Peru used an SSU-rRNA--based PCR-RFLP tool to genotype Cryptosporidium specimens from children (8) and AIDS patients (1). A variety of Cryptosporidium spp. were found in both patient populations; C. hominis was the predominant species, followed by C. parvum, C. meleagridis, C. canis, and C. felis, but mixed infections were rarely detected (1,8). However, a recent study of some of the specimens that used PCR tools that selectively amplify DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. of C. parvum and closely related species identified concurrent infections of C. hominis in specimens previously diagnosed as having only C. canis, C. muris, or C. suis (7). Another recent study has shown that an SSU rRNA--based PCR-RFLP tool had only a 31%-74% success rate in detecting concurrent infections with C. parvum and C. hominis (9). The Study We addressed the question of whether Peruvian HIV-positive patients infected with the usual C. canis or C. felis parasites were co-infected with C. hominis, C. parvum, or C. meleagridis (7). The study protocol was approved by the participating institutional review boards. All participants gave written informed consent. Mixed infections were identified by using 2 PCR-RFLP tools that only amplify C. hominis, C. parvum, or C. meleagridis (7). One tool was based on the dihydrofolate reductase dihydrofolate reductase enzyme catalyzing the conversion of folate to 5,6,7,8-tetrahydrofolate, which is the key carrier of one-carbon units in purine and pyridime synthesis, the pathway for the breakdown of histidine and the synthesis of S-adenosylmethionine from S (DHFR DHFR Dihydrofolate reductase, see there ) gene (10) and the other on the Cryptosporidium oocyst oocyst /oo·cyst/ (-sist) the encysted or encapsulated ookinete in the wall of a mosquito's stomach; also, the analogous stage in the development of any sporozoan. o·o·cyst n. wall protein (COWP COWP Cowpens National Battlefield (US National Park Service) CoWP Cobalt Tungsten Phosphide ) gene (11). Fifty-six stool specimens from 21 HIV-infected persons with previous diagnoses of C. canis or C.felis with an SSU rRNA--based PCR-RFLP tool were re-analyzed with these 2 molecular tools. DNA was extracted by using the QIAamp stool DNA extraction DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis. Outline of a DNA extraction There are three basic steps in a DNA extraction, the details of which may vary depending on the type of sample and any substances that may kit (Qiagen Inc., Valencia, CA, USA), and 1 [micro]L DNA was used in nested PCR analyses of the DHFR and COWP genes. Secondary PCR products positive for Cryptosporidium were digested with restriction enzymes BpuA I for the DHFR tool or Rsa I for the COWP tool (10,11). Results of RFLP diagnosis were confirmed by DNA sequence DNA sequence Genetics The precise order of bases–A,T,G,C–in a segment of DNA, gene, chromosome, or an entire genome. See Base pair, Base sequence analysis, Chromosome, Gene, Genome. analysis. All secondary PCR products were sequenced with a 3100 ABIPrism Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences obtained were aligned with reference sequences from GenBank by using BioEdit version 7.0.5 (Isis Pharmaceuticals, Carlsbad, CA, USA). The PCR analysis of both DHFR and COWP genes showed that 17 specimens from 7 patients yielded products of the expected size for Cryptosporidium spp. (Figure, panel A, and Table). Restriction analysis of DHFR products with BpuA I showed that 4 patients had banding patterns indicative of C. hominis, 1 patient had the pattern of C. parvum, 1 patient had the pattern of C. meleagridis, and 1 patient had the patterns of C. hominis and C. meleagridis (Figure panel B). Likewise, RFLP analysis of the COWP PCR products digested with Rsa I showed 3 banding patterns that were in complete agreement with the results obtained for the DHFR PCR-RFLP tool (Figure panel C). Therefore, 2 of the 12 C. canis--infected patients had C. hominis, 1 had C. parvum and 1 had both C. hominis and C. meleagridis; of the 9 C. felis-infected patients, 2 had C. hominis and 1 had C. meleagridis (Table). All DHFR and COWP PCR products were sequenced, which confirmed the results of the RFLP diagnosis. Altogether, 8, 2, and 3 DHFR sequences were obtained for C. hominis, C. parvum, and C. meleagridis, respectively. The C. hominis and C. meleagridis DHFR sequences were identical to XM_660774 and AY391725, respectively. The C. parvum DHFR sequences were homologous homologous /ho·mol·o·gous/ (ho-mol´ah-gus) 1. corresponding in structure, position, origin, etc. 2. allogeneic. ho·mol·o·gous adj. 1. to XM_625460, with an insertion at position 37 and 4 bp substitutions at positions 66, 69, 364, and 367. Likewise, 10, 2, and 3 COWP sequences were obtained for C. hominis, C. parvum, and C. meleagridis, respectively, and were identical to AF481960, AF266273, and AY 166840, respectively, in GenBank. The C. parvum DHFR nucleotide sequence obtained from this study is deposited in GenBank under accession no. DQ352814. To confirm the original diagnosis of C. canis and C. felis infection, we reanalyzed all DNA preparations of these specimens with the SSU rRNA genotyping tool (7). Results were in complete agreement with those obtained previously (7): 19 specimens from 12 patients had C. canis, 15 specimens from 9 patients had C. felis, and no specimens had mixed Cryptosporidium spp., as indicated by RFLP patterns (Table and Figure panel D). Data on diarrhea at study enrollment were available for 4 of the 7 patients with mixed infections and all 14 patients without mixed infections. Among persons with mixed infections, 1 did not have diarrhea, 2 had diarrhea lasting [less than or equal to] 30 days, and 1 had diarrhea [greater than or equal to] 5 months. Seven of 14 patients without mixed infections had diarrhea: 5 had acute diarrhea lasting [less than or equal to] 30 days, and 2 had chronic diarrhea lasting >5 months (difference in prevalence of diarrhea for mixed versus single infections was not significant by the Fisher exact test). The average CD4+ lymphocyte count among the patients with mixed infections was 130 cells/[micro]L. Of the 7 patients with mixed infections, 3 had specimens collected >30 days after the first detection, and mixed infections with the same species were still identified. The persistence of 2 species for >1 month is in contrast to a report that 1 Cryptosporidium genotype rapidly displaces the other during experimental infections of animals (6). Conclusions Concurrent infection with multiple Cryptosporidium spp. may affect clinical manifestations since C. hominis and C. parvum induce different sequelae sequelae Clinical medicine The consequences of a particular condition or therapeutic intervention in humans (12). The frequent finding of C. hominis in C. canis- and C. felis-infected persons also raises the question of infection sources. Although these 2 species are traditionally associated with animals, anthroponotic transmission may play a role in their acquisition in humans. Recent analyses demonstrate that a large proportion of human infections with C. parvum, another traditional zoonotic Zoonotic A disease which can be spread from animals to humans. Mentioned in: Zoonosis species, are actually due to anthroponotic transmission (13,14). Our results also suggest that although the SSU rRNA-based PCR-RFLP tool or similar PCR techniques can detect and differentiate a wide range of Cryptosporidium species or genotypes, their usefulness in detecting mixed infections was compromised by preferential PCR amplification of the dominant species or genotype in specimens. This problem is likely inherited with most PCR tools. Thus, the use of PCR tools with broad specificity in combination with species-specific tools is needed to address the issue of mixed Cryptosporidium infections. Our findings demonstrate that mixed infections are more frequent and persist longer in HIV-infected patients than previously believed. The clinical importance of these findings is not clear because of the study's cross-sectional nature. Future studies should employ tools that can detect mixed Cryptosporidium infections in longitudinal studies longitudinal studies, n.pl the epidemiologic studies that record data from a respresentative sample at repeated intervals over an extended span of time rather than at a single or limited number over a short period. , evaluate the frequency of mixed infections of C. hominis and C. parvum, and assess their clinical and epidemiologic implications in both immunocompetent im·mu·no·com·pe·tent adj. Having the normal bodily capacity to develop an immune response following exposure to an antigen. im and immunocompromised persons. This work was supported in part by funds from the Opportunistic Infections Opportunistic infections Infections that cause a disease only when the host's immune system is impaired. The classic opportunistic infection never leads to disease in the normal host. Working Group of the Centers for Disease Control and Prevention Centers for Disease Control and Prevention (CDC), agency of the U.S. Public Health Service since 1973, with headquarters in Atlanta; it was established in 1946 as the Communicable Disease Center. . Dr Gilman is supported by National Institutes of Health-National Institute of Allergy and Infectious Diseases grants Peru-TMRC 5P01AI051976-04 and R21 AI 059661-01. Dr Cama is a researcher at the Centers for Disease Control and Prevention and an associate at Johns Hopkins University Johns Hopkins University, mainly at Baltimore, Md. Johns Hopkins in 1867 had a group of his associates incorporated as the trustees of a university and a hospital, endowing each with $3.5 million. Daniel C. , Bloomberg School of Public Health. His current research interests include studies on the molecular epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases, of enteric enteric /en·ter·ic/ (en-ter´ik) within or pertaining to the small intestine. en·ter·ic adj. 1. Of, relating to, or within the intestine. 2. parasites that affect humans and have zoonotic potential zoonotic potential n. The potential for animal infections to be transmissible to humans. . References (1.) Cama VA, Bern C, Sulaiman IM, Gilman RH, Ticona E, Vivar A, et al. Cryptosporidium species and genotypes in HIV-positive patients in Lima, Peru. J Eukaryot Microbiol. 2003;50(Suppl):531-3. (2.) Fretz R, Svoboda P, Ryan UM, Thompson RC, Tanners M, Baumgartner A. Genotyping of Cryptosporidium spp. isolated from human stool specimens in Switzerland. Epidemiol Infect. 2003;131:663-7. (3.) Gile M, Warhurst DC, Webster KA, West DM, Marshall JA. A multiplex allele allele (əlēl`): see genetics. allele Any one of two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome. specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of Cryptosporidium parvum genotypes 1 and 2. Parasitology Parasitology The scientific study of parasites and of parasitism. Parasitism is a subdivision of symbiosis and is defined as an intimate association between an organism (parasite) and another, larger species of organism (host) upon which the parasite is . 2002;125:35-44. (4.) McLauchlin J, Amar C, Pedraza-Diaz S, Nichols GL. Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: results of genotyping Cryptosporidium spp. in 1,705 fecal specimens from humans and 105 fecal specimens from livestock animals. J Clin Microbiol. 2000;38:3984-90. (5.) Mallon M, MacLeod A, Wastling J, Smith H, Reilly B, Tait A. Population structures and the role of genetic exchange in the zoonotic pathogen Cryptosporidium parvum. J Mol Evol. 2003;56:407-17. (6.) Tanriverdi S, Arslan MO, Akiyoshi DE, Tzipori S, Widmer G. Identification of genotypically mixed Cryptosporidium parvum populations in humans and calves. Mol Biochem Parasitol. 2003;130:13-22. (7.) Jiang J, Xiao L. An evaluation of molecular diagnostic tools for the detection and differentiation of human-pathogenic Cryptosporidium spp. J Eukaryot Microbiol. 2003;50(Suppl):542-7. (8.) Xiao L, Bern C, Limor J, Sulaiman I, Roberts J, Checkley W, et al. Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. J Infect Dis. 2001;183:492-7. (9.) Reed C, Sturbaum GD, Hoover PJ, Sterling CR. Cryptosporidium parvum mixed genotypes detected by PCR-restriction fragment length polymorphism analysis. Appi Environ Microbiol. 2002;68:427-9. (10.) Gibbons Famous people named Gibbons include:
(11.) Pedraza-Diaz S, Amar C, Nichols GL, McLauchlin J. Nested polymerase chain reaction Nested polymerase chain reaction is a modification of polymerase chain reaction intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites. for amplification of the Cryptosporidium oocyst wall protein gene. Emerg Infect Dis. 2001;7:49-56. (12.) Hunter PR, Hughes S, Woodhouse S, Raj N, Syed Q, Chalmers RM, et al. Health sequelae of human cryptosporidiosis in immunocompetent patients. Clin Infect Dis. 2004;39:504-10. (13.) Alves M, Xiao L, Sulaiman I, Lal AA, Matos O, Antunes F. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol. 2003;41:2744-7. (14.) Mallon ME, MacLeod A, Wastling JM, Smith H, Tait A. Multilocus genotyping of Cryptosporidium parvum type 2: population genetics Population genetics The study of both experimental and theoretical consequences of mendelian heredity on the population level, in contradistinction to classical genetics which deals with the offspring of specified parents on the familial level. and sub-structuring. Infect Genet genet: see civet. Evol. 2003;3:207-18. Address for correspondence: Lihua Xiao, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, Mailstop F12, Atlanta, Georgia 30333, USA; email: lxiao@cdc.gov The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated. * Tropical Medicine tropical medicine, study, diagnosis, treatment, and prevention of certain diseases prevalent in the tropics. The warmth and humidity of the tropics and the often unsanitary conditions under which so many people in those areas live contribute to the development and Institute "Pedro Kouri," Havana, Cuba; ([dagger]) Ministry of Public Health, Havana, Cuba; ([double dagger]) Instituto de Ecologia y Sistematica, Havana, Cuba; ([section]) Instituto de Medicina Veterinaria, Havana, Cuba; and ([paragraph]) National Microbiology Laboratory The National Microbiology Laboratory (NML) is located in the Canadian Science Centre for Human and Animal Health in Winnipeg, Manitoba. This modern state-of-the-art facility houses the NML's Biological Safety Level 4 (BSL-4) containment laboratory, currently Canada's only BSL-4 , Winnipeg, Manitoba, Canada Vitaliano Cama,* ([dagger]) Robert H. Gilman, ([dagger] [double dagger]) Aldo Vivar, ([section]) Eduardo Ticona, ([section]) Ynes Ortega, (#) Caryn Bern, * and Lihua Xiao * * Centers for Disease Control and Prevention, Atlanta, Georgia, USA; ([dagger]) Johns Hopkins University, Baltimore, Maryland, USA; ([double dagger]) Asociacion Benefica Proyectos en Informatica, Salud, Medicina y Agricultura, Lima, Peru; ([section]) Hospital Arzobispo Loayza, Lima, Peru; ([paragraph]) Hospital Dos de Mayo, Lima, Peru; and (#) University of Georgia Organization The President of the University of Georgia (as of 2007, Michael F. Adams) is the head administrator and is appointed and overseen by the Georgia Board of Regents. , Griffin, Georgia, USA
Table. Results of multilocus genotyping of Cryptosporidium
specimens originally diagnosed as Cryptosporidium canis and
C. felis by an SSU rRNA-based PCR-RFLP tool *
Cryptosporidium
genotype by
locus (no.
No. No. days between specimens)
specimens first and last
Participant tested specimen SSU rRNA
0043D 7 29 C. canis
0214D 2 5 C. canis
0448D 4 45 C. canis
1083D 1 -- C. canis
1322D 2 2 C. canis
0002D 1 -- C. canis
0034D 7 56 C. canis
0482D 1 -- C. canis
0500D 1 -- C. canis
0533D 3 3 C. canis
0670D 4 414 ([dagger]) C. canis
0725D 1 -- C. canis
0044A 1 -- C. felis
0076A 4 31 C. felis
0668A 3 3 C. felis
0673A 5 31 C. felis
0817A 2 2 C. felis
0891A 1 -- C. felis
1344A 3 3 C. felis
0569D 2 2 C. felis
0776D 1 -- C. felis
Cryptosporidium genotype by
locus (no. specimens)
Mixed
Participant COWP DHFR infection
0043D -- -- No
0214D -- -- No
0448D C. hominis (1) C. hominis (2) Yes
and C. and C.
meleagridis (2) meleagridis (2)
1083D -- -- No
1322D -- -- No
0002D -- -- No
0034D C. parvum (2) C. parvum (2) Yes
0482D C. hominis (1) C. hominis (1) Yes
0500D -- -- No
0533D -- -- No
0670D C. hominis (4) C. hominis (2) Yes
0725D -- -- No
0044A C. meleagridis C. meleagridis Yes
(1) (1)
0076A C. hominis (3) C. hominis (1) Yes
0668A C. hominis (1) C. hominis (2) Yes
0673A -- -- No
0817A -- -- No
0891A -- -- No
1344A -- -- No
0569D -- -- No
0776D -- -- No
* PCR-RFLP, polymerase chain reaction-restriction fragment length
polymorphism; SSU, small subunit; COWP, Cryptosporidium oocyst wall
protein; DHFR, dihydrofolate reductase gene.
([dagger]) Specimens correspond to 2 visits 14 months apart.
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