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On-line version ISSN 1678-9946
Rev. Inst. Med. trop. S. Paulo vol.51 no.6 São Paulo Oct./Dec. 2009
Caracterização molecular de Cryptosporidium spp. de pacientes de área urbana do Brasil infectados por HIV
Patrícia de LuccaI; Elizabeth Natal De GaspariII; Lígia M. BozzoliII; Mikaela Renata FunadaI; Sheila Oliveira de Souza SilvaI; Wilma IulianoIII; Rodrigo Martins SoaresI
IDepartamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, 05508-900, São Paulo, SP, Brazil
IIImmunology Section, Adolfo Lutz Institute, São Paulo, SP, Brazil
IIILaboratório de Parasitologia, Instituto Emílio Ribas, São Paulo, SP, Brazil
Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0%) isolates of C. hominis, four (14.8%) C. parvum, five (18.5%) C. felis and one (3.7%) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.
Keywords: Cryptosporidium spp.; Molecular characterization; Human; HIV; Zoonosis; Brazil.
Cryptosporidium spp. são importantes causas de doenças entéricas em humanos, mas podem também ser encontrados em animais. O presente estudo descreve a frequência relativa de diversas espécies de Cryptosporidium em amostras de humanos da cidade de São Paulo, Brasil, obtidas de janeiro a julho de 2007. Análises de sequências de produtos de nested PCR direcionadas ao genes codificadores da menor unidade ribosomal e da proteina de parede de oocistos revelaram 17 (63,0%) isolados de C. hominis, quatro (14,8%) C. parvum, cinco (18,5%) C. felis, e um (3,7%) C. canis. Estes resultados sugerem que, em ambientes urbanos no Brasil, o genótipo adaptado ao gato pode desempenhar potencial papel na transmissão zoonótica de criptosporidiose, enquanto a transmissão antroponótica da criptosporidiose causada pelo C. hominis parece predominar.
Cryptosporidium spp. are protozoan parasites of a wide range of vertebrates, including mammals, birds, reptiles, amphibians and fish14. Currently, there are at least 20 recognized species and 40 genotypes of Cryptosporidium13. Among them, five species, Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium meleagridis, Cryptosporidium felis and Cryptosporidium canis may infect immunocompetent and immunocompromised humans11,14. The use of small subunit rRNA-based genotyping tools revealed the presence of C. canis, C. felis and C. meleagridis in AIDS patients of several parts of the world in addition to the frequently found C. hominis and C. parvum15. To compare the differences in genetic diversity among Cryptosporidium species, DNA sequences of other markers such as, the Cryptosporidium oocyst wall protein (COWP) gene7,8,9, and the 70 kDa heat shock protein (HSP70) gene10 have also been used. In Brazil, few studies on the relative frequency of Cryptosporidium genotypes found in humans have been published, with C. parvum and C. hominis being incriminated as the most prevalent genotypes1,2,3,4,5. In this paper, we aimed to reveal and discuss the relative frequencies of Cryptosporidium in human patients from an infectious diseases hospital in the municipality of São Paulo, São Paulo State, Brazil.
MATERIAL AND METHODS
Oocysts of Cryptosporidium spp. were obtained from diarrheic feces of naturally infected human patients with HIV, irrespective of their age, sex or any other risk factor which could be associated with the infection. We analyzed all the Cryptosporidium isolates recovered from January to July 2007 from human patients from a hospital specialized in infectious diseases in the municipality of São Paulo, São Paulo State, Brazil. Twenty seven stool samples were examined from January to July 2007 for oocysts of Cryptosporidium spp. by a conventional sucrose flotation method. Floated material was transferred to a slide and examined by light microscopy. When 4-5 µm sized oocysts were observed, the slide was washed and the DNA was extracted from material as previously described6.
To amplify fragments of the small subunit rRNA and Cryptosporidium oocyst wall protein (COWP) coding genes, two nested-PCR protocols were used8,12. The nested-PCR products were sequenced using the secondary PCR primers and the Big Dye chemistry (Applied Biosystems, Foster City, California). Sequencing products were analyzed on a ABI377 automated sequencer. Both strands of each nested-PCR products were sequenced at least twice to increase the confidence of sequencing. The sequences were assembled and the contig formed with the phred-base calling and the phrap-assembly tool available in the suite Codoncode aligner v.1.5.2. (Codoncode Corp. Dedham, MA, USA).
Nucleotide sequences were deposited in Genbank under the accession numbers FJ232996 to FJ233040.
RESULTS AND DISCUSSION
From the 27 samples, 18 were positive by the nested-PCR based on 18S rRNA primers and by the nested PCR based on COWP primers. Nine samples were positive by only one PCR, being six positive by nested-PCR based on 18S rRNA and three positive by the nested PCR based on COWP (Table 1).
After analysis of the chromatograms, ~700 and 450 bp sequences were obtained from products of the 18S rRNA and COWP genes of each sample. The resulted sequences were aligned and compared with references sequences obtained from GenBank. From the results, 17 (63.0%) isolates were identified as C. hominis, four (14.8%) as C. parvum, five (18.5%) as C. felis, and one (3.7%) as C. canis. None of the sequences were discrepant from the well known species available in the GenBank and novel genotypes were not found (Table 1).
The isolates 1, 27, 28, 30 and 39 identified as C. felis were negative when tested by the COWP-based nested-PCR. DNA of the isolates 2, 17, 24, 25 was amplified by only one PCR assay because a small amount of material was available. Thus, the genotyping of these samples was accomplished by using only one molecular marker. The failure in amplifying fragments of COWP coding genes from C. felis might be caused by nucleotide diversity in the primer regions at this locus which prevented the anchorage of the primers for PCR amplification. In fact, C. felis is the most divergent species of Cryptosporidium when compared to C. canis, C. parvum and C. hominis.
Although the present study was not based on a statistically defined sampling, it showed that the relative frequencies of the genotypes of Cryptosporidium spp. in human patients of São Paulo city are somewhat similar to those found in other populations in developing countries. Developing and industrialized countries differ markedly in the proportion of infections caused by zoonotic species with the latter having more cases related to zoonotic transmissions15. In the present survey, a lower proportion of the isolates (37.0%) consisted of species potentially involved in zoonotic transmission.
However, it is noteworthy stressing that the source of infection is not possible to be inferred solely based on the description of the genotypic identification of the parasite. In fact, anthroponotic transmission of seemingly zoonotic Cryptosporidium species was already described in many parts of the world.
Two surveys for Cryptosporidium spp. infection in human population of South America reveal that C. hominis infections predominate over those caused by C. parvum, the two most common species found in humans15. In Peru, among 85 samples, 67 were identified as C. hominis, eight as C. parvum, one as C. felis and two as C. canis. In Venezuela, among 10 isolates, eight were revealed C. hominis, one C. parvum and one C. canis.
In Brazil, recent studies revealed that C. hominis predominate over C. parvum. BUSHEN et al. reported that among 42 isolates from children, 24 were C. hominis (57.1%) and 18 were C. parvum (42.9%)2. Multilocus genotyping of Cryptosporidium spp. associated with diarrhea outbreak in a day care unit by DNA sequencing analysis of fragments coding for 18S, COWP and a microssatelite locus (ML1) revealed the presence of C. hominis in all the 29 samples4. Cryptosporidium isolates identified in fourteen stool samples from five HIV patients and nine immunocompetent children revealed two C. parvum, eight C. hominis and two C. meleagridis1. In a larger survey5 involving 83 patients attended at a reference hospital in the São Paulo municipality, the majority of samples (88.5%) were C. hominis positive whereas zoonotic genotypes other than C. parvum were not identified. In this study, it is noteworthy mentioning that more than 60% of the isolates detected by microscopy remained unidentified.
The major difference of our results in comparison to those of other surveys in Brazil is the higher frequency of non-parvum and non-hominis (22.2%) Cryptosporidium spp. It is noteworthy mentioning that the occurrence of uncommon genotypes in humans may be due to patients who were HIV positive, hence more susceptible to infection than immunocompetent patients. In addition, the sampling methods used in each study may explain such a high difference in the relative frequencies of genotypes.
Among the species of Cryptosporidium spp. less frequently found in humans, three can be highlighted, C. meleagridis, C. felis and C. canis, the last one being the rarest15. In the present study, C. meleagridis was not found, C. canis was detected only once whereas C. felis was detected in a frequency very similar to C. parvum. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis.
To CNPq for the productivity scholarship for R. M. Soares and to Fapesp for a grant to P. Lucca (2007/50670-1).
This survey was approved by the ethical committee for scientific research of the Instituto Emílio Ribas, São Paulo, SP, Brazil.
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Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo
Av. Prof. Dr. Orlando Marques de Paiva 87
05508-900 São Paulo, SP, Brasil
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Received: 15 May 2009
Accepted: 13 October 2009