Emerging Infectious Diseases
[Volume 5 No.3 / May - June 1999]


Dispatches

New Cryptosporidium Genotypes in HIV-Infected Persons

Norman J. Pieniazek,* Fernando J. Bornay-Llinares,* Susan B. Slemenda,*
Alexandre J. da Silva,* Iaci N. S. Moura,* Michael J. Arrowood,* Oleg
Ditrich, and David G. Addiss*
Centers for Disease Control and Prevention, Atlanta, Georgia, USA; and
Institute of Parasitology AS CR, Cesk Budjovice, Czech Republic

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      Using DNA sequencing and phylogenetic analysis, we identified
      four distinct Cryptosporidium genotypes in HIV-infected
      patients: genotype 1 (human), genotype 2 (bovine)
      Cryptosporidium parvum, a genotype identical to C. felis, and
      one identical to a Cryptosporidium sp. isolate from a dog. This
      is the first identification of human infection with the latter
      two genotypes.

Protozoan apicomplexan parasites from the genus Cryptosporidium infect a
wide variety of hosts (1). The parasites are transmitted to humans through
contaminated drinking water (2), contact with infected animals, and contact
with infected persons (3). In the immunocompetent, cryptosporidiosis
manifests itself as self-limited diarrhea, sometimes accompanied by nausea,
abdominal cramps, fever, and vomiting. In the immunodeficient, however,
cryptosporidiosis may be severe, chronic, and life-threatening (4).

Cross-infection experiments, in which Cryptosporidium oocysts were obtained
from animals of one species and fed to animals of another species, have
investigated the host specificity of this parasite (5). The differences
observed in the host range of putative C. parvum isolates led to a proposal
to establish the Cryptosporidium isolate originating from guinea pigs,
morphologically indistinguishable from C. parvum, as a new speciesC.
wrairisolely on the basis of experimental infection (6). The possibility
of many Cryptosporidium species fostered the development of techniques
suitable for typing isolates. Commonly used techniques are isoenzyme
analysis (7), Western blotting (8,9), random amplified polymorphic DNA
analysis (10-12), polymerase chain reaction restriction fragment length
polymorphism (PCR-RFLP) analysis (13,14), and PCR followed by DNA
sequencing (11,15-19).

Early studies of the polymorphism of isolates classified as C. parvum found
significant geographic variation among isolates (20) in the region coding
for the small subunit ribosomal RNA (SSU-rRNA), commonly used for taxonomic
classification. Recently, it has been shown (21) that one of the sequences
used in this analysis (22) was erroneously identified as a C. parvum
sequence, while in fact it was C. muris. More recent work (e.g., Le Blancq
et al. [23] and GenBank entry AF040725) has shown that the SSU-rRNA region
of the C. parvum zoonotic (bovine) genotype does not show heterogeneity and
is practically identical to the sequence submitted to GenBank in 1993
(accession number L16996, [24,25]).

Recently, consistent results of typing bovine and human C. parvum isolates
led to unequivocal recognition of two genotypes of C. parvum. These two
genotypes were reproducibly differentiated by sequencing the SSU-rRNA
coding region (16), sequencing the Cryptosporidium thrombospondin-related
adhesion protein (TRAP-C2) gene (17), and PCR-RFLP analysis of the
Cryptosporidium oocyst wall protein (COWP) gene (15). Thus far, the
anthroponotic genotype (genotype 1) has been found only in infected humans,
while the zoonotic genotype (genotype 2) has been found both in infected
humans and in livestock, e.g., cows, lambs, goats, and horses. The
published partial SSU-rRNA sequence of the C. parvum genotype 1 (16) is
identical to the Cryptosporidium SSU-rRNA sequence that we observed in
PCR-amplified DNA from a patient with AIDS (GenBank accession number
L16997). That the data from different laboratories for these two genotypes
were reproducible calls into question the recent conclusion of Tzipori and
Griffiths (26) that "there are no clearly defined and fully characterized
reference `strains' of Cryptosporidium."

Other new Cryptosporidium genotypes have been identified in various animals
(pigs, mice, cats, and koalas) by sequencing the region coding for SSU-rRNA
(16,27,28). The cat genotype is thought to represent C. felis (28). When
morphologic and other data are available for these new genotypes, they may
be recognized as new Cryptosporidium species.

We present results of typing Cryptosporidium isolates by direct sequencing
the variable region of the SSU-rRNA amplified from fecal specimens with
Cryptosporidium genus-specific diagnostic PCR primers (25). Applying this
method to a set of specimens from a 3-year longitudinal study on the risk
for enteric parasitosis and chronic diarrhea in immunodeficient patients
with a low CD4+ count, we found the first human cases of infection by C.
felis and by a newly identified zoonotic Cryptosporidium species possibly
originating from a dog.

The Study

All analyzed specimens were collected from January 1991 through September
1994 in a study assessing the impact of enteric parasite-associated
diarrhea in persons infected with HIV (29). Study participants answered
comprehensive questionnaires concerning clinical and epidemiologic
information and provided stool specimens monthly. All stool specimens were
examined for C. parvum by Kinyoun carbol-fuchsin modified acid-fast stain
(30) and direct immunofluorescence (31). Stained slides were examined by
observing 200 oil-immersion fields. C. parvum was associated with 18 (5.1%)
of the 354 acute episodes and 36 (12.9%) of the chronic episodes of
diarrhea reported by the participants. All specimens from this study were
preserved in separate vials of 10% formalin and polyvinyl alcohol (Para-Pak
Stool System; Meridian Diagnostics, Inc., Cincinnati, OH) and thus were not
suitable for molecular analysis. Some specimens were originally aliquoted
and stored without preservation at -80C. Of this set, we selected 18
available specimens for 10 randomly chosen Cryptosporidium-positive
patients for this study.

An aliquot of approximately 300 l of each stool specimen was suspended in
1 ml of 0.01 M phosphate-buffered saline, pH 7.2, containing 0.01 M of EDTA
(PBS-EDTA), and the suspension was centrifuged at 14,000 x g, 4C for 5
minutes. The pellet from this centrifugation was washed two more times
under the same conditions. The pellet was resuspended in 300 l of PBS-EDTA
and used for DNA extraction, performed with the FastPrep disrupter and the
FastDNA kit (BIO 101, Inc., Vista, CA) (32). Extracted DNA was stored at
4C until PCR amplification.

Cryptosporidium genus-specific primers (CPBDIAGF and CPBDIAGR) were used to
amplify the Cryptosporidium SSU-rRNA variable region (24,25). The
conditions for PCR were 95C for 15 minutes; 45 cycles of denaturation at
94C for 30 seconds, annealing at 65C for 30 seconds, extension at 72C
for 1 minute, 30 seconds; followed by extension at 72C for 9 minutes; and
finished with a hold step at 4C.

PCR products were analyzed by electrophoresis on 2% SeaKem GTG agarose
(Cat. No. 50074, FMC Bioproducts, Rockland, ME), stained with ethidium
bromide, and visualized on UV transilluminator. The size of the diagnostic
fragment amplified with these primers for C. parvum genotype 1 (GenBank
accession number L16997) was 438 bp, for C. parvum genotype 2 (L16996) 435
bp, for C. baileyi (L19068) 428 bp, and for C. muris (L19069) 431 bp.

PCR products were purified by using the Wizard PCR Preps kit (Cat. No.
A7170, Promega, Madison, WI). Sequencing reactions were done with the
Perkin Elmer Big Dye kit (Cat. No. 4303149, PE Biosystems, Foster City, CA)
and analyzed on the Perkin Elmer ABI 377 automatic DNA sequencer. Sequences
were assembled by using the program SeqMan II (DNASTAR Inc., Madison, WI).
Sequences were aligned with the program MACAW (33) and analyzed by programs
from the PHYLIP (phylogeny inference program) package (34).

Findings

After the 18 fecal specimens were retrieved from storage and processed for
PCR with the CPBDIAGF/CPBDIAGR diagnostic primer set, visualization of PCR
products on 2% agarose gels found two different sizes of diagnostic bands.
The size of the Cryptosporidium diagnostic bands for patients 53, 75, 119,
124, 153, 278, and 554 did not differ significantly from the size of the
standard band (approximately 435 bp) obtained with cloned SSU-rRNA for the
C. parvum genotype 2 (Figure 1, lane 1). On the other hand, the
Cryptosporidium diagnostic bands in samples from patients 84, 184, and 485
were visibly larger, approximately 450 bp (Figure 1, lane 3 shows data for
patient 84).		

               [Fig]     
							    DNA sequence analysis showed
Figure 1. Agarose gel (2%) visualization      four types of sequences
of diagnostic polymerase chain reaction       (Table 1). The sequence of
products of four Cryptosporidium              the diagnostic band from
genotypes. Lane S, standard 100-bp ladder;    patient 53 was 435 bp long
lane 1, patient 53, zoonotic genotype 2;      and was identical to the
lane 2, patient 119, Cryptosporidium sp.      corresponding fragment of the
(zoonotic, canine genotype); lane 3,          C. parvum genotype 2 (GenBank
patient 84, C. felis (zoonotic, feline        accession number L16996)
genotype); lane 4, patient 75,                SSU-rRNA. The diagnostic
anthroponotic genotype 1.                     bands in patients 75, 124,
                                              153, 278, and 554 were 438 bp
                                              long and were identical to
the corresponding fragment of the C. parvum genotype 1 SSU-rRNA (GenBank
accession number L16997). The sequence from patient 119 was 429 bp long;
the diagnostic bands from patients 84, 184, and 485 were 455 bp long and
were identical to each other. Sequence similarity searches of GenBank
database and molecular phylogeny analysis of the two latter types of
sequences showed that, while they were not identical to any sequences in
GenBank, they clustered within other representative SSU-rRNA sequences from
the genus Cryptosporidium (results not shown). In addition, these sequences
were significantly different from recently reported partial SSU-rRNA
sequences (16,19,27,28). The 429-bp-long diagnostic fragment in patient 119
was identical to a recently identified canine C. parvum isolate SSU-rRNA
sequence (GenBank accession number AF112576). The longest sequence (455 bp)
from patients 84, 184, and 495 was identical in a 100-bp overlap to an
updated sequence (GenBank accession number AF097430) for C. felis (also
called feline C. parvum genotype) (28). Alignment of these sequences is
shown in Figure 2. In the hypervariable alignment region (from position 101
to 110), all genotypes display a variable number of thymidines. Genotype 1
has the longest stretch, 11 thymidines, while the Cryptosporidium sp.
(canine genotype) has only one. The sequence for C. felis has five
insertions at alignment positions 45, 86, 111, 187, and 218, as well as one
deletion of two thymidines at positions 197 and 198. Apart from the
difference in the hypervariable region, the Cryptosporidium sp. (canine
genotype) has a deletion of two bases at positions 49 and 50.

Results of genotyping the 18 specimens are in Table 2. For patients 53,
124, 153, 184, 278, and 485, only a single specimen was available. For
other patients (e.g., patient 554), four specimens collected during 12
months were available. The same Cryptosporidium genotype persisted
throughout a patient's infection.

Table 1. Cryptosporidium genotypes for 10 selected
patients in this study
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Patient no.    Band size(sup a)(bp) C. parvum genotype
-----------------------------------------------------------
53                    435           Zoonotic (genotype 2)
75, 124, 153          438           Anthroponotic
  278, 554                            (genotype 1)
119                   429           Cryptosporidium sp.
                                      (canine genotype)
84, 184, 485          455           C. felis
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(sup a)Size of the Cryptosporidium diagnostic band obtained 
with the CPBDIAGF/CPBDIAGR PCR primer pair

		
				[Fig]
     
Figure 2. Alignment of the Cryptosporidium small subunit ribosomal DNA
(SSU-rRNA) diagnostic fragments obtained with the CPBDIAGF/CPBDIAGR
polymerase chain reaction primer pair for the four genotypes. Only the first
300 columns of the alignment are shown, as the remaining columns were
identical for all genotypes. Gaps are shown with dashes (-), and bases
identical to the base in the first row (C. felis) are shown with dots (.).
Numbers to the right of the alignment show sequence positions for each
genotype. The sequences for the SSU-rRNA diagnostic fragment of all four
genotypes were submitted to GenBank and were assigned accession numbers
AF087574, AF087575, AF087576, and AF087577.


Table 2. Persistence of Cryptosporidium genotypes in
patients(sup a)
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                                 Month of follow-up
             No. of     -------------------------------------
Patient     available
no.         specimens    1  2  3  4  5 6  7  8 9  10  11  12
-------------------------------------------------------------
  53            1        B

  75            2        H  -  -  H

  84            4        F  -  F  -  F F

119             2        C  -  C

124             1        H

153             1        H

184             1        F

278             1        H

485             1        F

554             4        H  -  - -   - -  -  H -   H   -  H

-------------------------------------------------------------
(sup a)B, C. parvum genotype 2 (zoonotic, bovine); C,
Cryptosporidium sp. (zoonotic, canine); F, C. felis
(zoonotic, feline); H, C. parvum genotype 1 (anthroponotic);
-, specimen not available.

Conclusions

Using the sequence of a diagnostic fragment of SSU-rRNA, as well as two
well-established genotypes of C. parvum (anthroponotic genotype 1 and
zoonotic genotype 2), we detected two new Cryptosporidium genotypes. The
first, in patients 84, 184, and 485, was identical to the feline
Cryptosporidium genotype (28), also described as C. felis. The second, in
two specimens from patient 119, represents the newly identified
Cryptosporidium sp. found in a sequence originating from a dog (GenBank
accession number AF112576).

New taxons may be established within the genus Cryptosporidium on the basis
of the isolate's host range together with molecular data, even though
morphologic criteria are apparently lacking. We propose to use C. felis
Iseki, 1979, instead of feline C. parvum genotype; we believe that the
anthroponotic genotype 1 of C. parvum and the Cryptosporidium sp. (canine
genotype) described here should be named as distinct species. Although the
taxonomy of protists is morphology-based, the taxonomy of bacteria is being
reevaluated on the basis of molecular data (35). There is no reason to use
different approaches to these two kingdoms, nor is it necessary to
postulate that the basis for speciation of Cryptosporidium remains
ambiguous or that molecular data lend little support to separation of
Cryptosporidium into distinct, valid species (26). Further, we see no
evidence for the unconventional conclusions of Tzipori and Griffiths that
"...genetic markers in C. parvum can change upon passage to a different
host, possibly through a selective mechanism favoring different
populations" (26) nor for those of Widmer (21) that "...genetic studies and
experimental infections suggest that a selective mechanism triggered by a
change in the intestinal environment might be involved in shaping the
genetic make-up of C. parvum populations."

The finding of new Cryptosporidium genotypes in immunodeficient patients
might suggest a unique susceptibility to infections by divergent
Cryptosporidium species circulating in companion animals or livestock.
However, recent identification of C. felis in a cow (36) may indicate a
complex pattern of flow of different Cryptosporidium species in the
environment. When more data on the distribution of these species become
available, public health and preventive measures will need to be
reevaluated, and isolates may need to be renamed to reflect their natural
history. Finally, it is clear that Kinyoun carbol-fuchsin modified
acid-fast stain (30) and direct immunofluorescence (31) could detect all
genotypes, but this may not be true for diagnostic reagents that may be
affected directly (PCR) or indirectly (Ab) by the genetic composition of
the new Cryptosporidium isolates reported here. Thus, discovery of these
new Cryptosporidium genotypes in human cryptosporidiosis should cause
existing diagnostic tools to be reevaluated.

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Acknowledgment

      The authors thank Jane L. Carter for her expert help with running the
DNA sequencer.

      Dr. Pieniazek is a microbiologist with the National Center for
Infectious Diseases, CDC. His research interests include molecular
reference diagnosis of parasitic diseases and molecular systematics.

     Address for correspondence: Norman J. Pieniazek, Division of Parasitic
Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway
NE, Mail Stop F13, Atlanta, GA, 30341-3724, USA; fax: 770-488-4108; e-mail:
nxp3@cdc.gov.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

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