Rev. Inst. Med. trop. S. Paulo
50(3):139-143, May-June, 2008
(1) Instituto Básico de Biociências, Universidade de Taubaté, Taubaté, SP, Brasil.
(2) Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil.
(3) Instituto Adolfo Lutz, Sorocaba, SP, Brasil.
(4) Hospital Albert Einsten, São Paulo, SP, Brasil.
(5) Centers for Disease Control and Prevention, Atlanta, GA, USA.
Correspondence to: Herminia Yohko Kanamura, Instituto Básico de Biociências, Universidade de Taubaté, Av. Tiradentes 500, 12030-180 Taubaté, SP, Brasil. E-mail: email@example.com.
GENOTYPIC IDENTIFICATION OF Cryptosporidium SPP. ISOLATED FROM HIV-INFECTED
PATIENTS AND IMMUNOCOMPETENT CHILDREN OF SÃO PAULO, BRAZIL
Ana Julia Urias dos Santos ARAÚJO(1,2), Herminia Yohko KANAMURA(1,2), Marcos Eduardo de ALMEIDA(1), Aparecida Helena de Souza GOMES(3),
Thais Helena Lemos PINTO(4) & Alexandre Januário DA SILVA(5)
Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine
immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR
followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion
with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on
this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a
profile that corresponded to Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular
methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.
KEYWORDS: Cryptosporidiosis; PCR; Genotyping; Cryptosporidium parvum; Cryptosporidium hominis; Cryptosporidium
Nearly 100 years after description of the first Cryptosporidium
species, in mice by Tyzzer, it is known that Cryptosporidium spp. can
occur in distinct classes of vertebrates, such as fishes, reptiles,
amphibians, birds and mammals12,20,27,28.
In humans, infection by Cryptosporidium sp. was first registered
in 1976, and Cryptosporidium parvum (previously known as cattle
genotype or genotype 2) and Cryptosporidium hominis (previously
known as C. parvum - human genotype or genotype 1) have been
recorded as the two species most frequent cause of human
cryptosporidiosis, either associated with sporadic cases of infections
or outbreaks, some of them being very large as the one that occurred
in Milwaukee WI, USA, which affected approximately 400,000
persons12,17. To date, all the cryptosporidiosis outbreaks occurring
worldwide have been caused by C. hominis and C. parvum, with several
being associated with consumption of drinking water or exposure to
recreational water contaminated with Cryptosporidium oocysts of
zoonotic and anthroponotic origins15,27,30. Due to the size and frequency
of these outbreaks, cryptosporidiosis became a serious public health
issue worldwide and prompted reevaluation of the microbiological
standards for drinking water by health authorities in developed and
developing countries. In countries like Brazil, it is mandatory that
potable water be also free of Cryptosporidium sp. and Giardia sp.3.
Although all outbreaks studied to date have been associated with the
two species of Cryptosporidium listed above, it is also known that at least
some zoonotic species, such as Cryptosporidium canis, Cryptosporidium
felis, and Cryptosporidium meleagridis, can indeed infect both
immunocompromised and immunocompetent persons4,14,18,21,22,24.
In different regions of Brazil, including São Paulo, studies involving
patients with compromised immune systems either HIV-positive or
not, and children, with or without diarrhea, have shown different rates
for presence of Cryptosporidium spp. in their stools5,7,8,15,19,25.
Serological surveys conducted in Brazil showed the presence of anti-
Cryptosporidium antibodies in a large number of children living in
slums of the northeastern region of Fortaleza, state of Ceara1, and also
in individuals with no intestinal symptoms living in the state of São
Paulo. In this last study the frequencies for anti-Cryptosporidium
antibodies varied from 10% to 80%, according to the age group9.
Environmental contamination with Cryptosporidium was also reported
in rivers and other public water sources11,13. Despite the great number
of reports about occurrence of Cryptosporidium infection in Brazil,
only few studies addressed the molecular characterization of the isolates
found in the studied clinical specimens. In one of the studies carried
out in the northeast region, two isolates were identified as C. parvum,
one as C. hominis, but the authors were not able to genetically
characterized one of the isolates2. Other study identified seven Brazilian
children infected with C. hominis14 and another revealed the presence
of a single genotype of C. hominis based on analysis of three genes in
29 samples from a diarrhea outbreak investigated in a day care in São