Parasitol Res (1999) 85: 833 ± 836
Ó Springer-Verlag 1999
ORIGINAL PAPER
Edith Valdez á Ma. del Carmen MartõÂ nez Alejandro GoÂmez á Roberto Cedillo á Jorge Arellano Marta E. PeÂrez á Fernando Ramos á Patricia MoraÂn Enrique GonzaÂlez á Olivia Valenzuela Emma I. Melendro á Manuel Ramiro Roberto Kretschmer á Onofre MunÄoz Cecilia XimeÂnez
HLA characterization in adult asymptomatic cyst passers of Entamoeba histolytica / E. dispar Received: 18 March 1999 / Accepted: 16 April 1999
Abstract The present work aimed at studying the possible association of HLA antigens with Entamoeba histolytica/E. dispar asymptomatic infection in a Mexican mestizo population. A case-control design was selected for evaluation of the role of genetic markers in parasite infection. For this purpose the HLA-A, HLAB, and HLA-DR pro®les of a population of asymptomatic E. histolytica/E. dispar adult cyst passers (cases) and a corresponding nonparasitized adult group (controls) followed for 12 months were identi®ed. Entamoeba species were identi®ed through zymodeme patterns and/ or ampli®cation of species-speci®c DNA sequences. A healthy, nonparasitized group of individuals was included as a control. Our results show that apparently, no speci®c HLA marker is associated with the asymptomatic cyst passers' condition. These ®ndings have to be added to previous results in which, in contrast to a demonstrated association between HLA-DR3 and amebic liver abscess in Mexican mestizo adults and infants, no signi®cant association with amebic rectocolitis was found.
E. Valdez á M. del Carmen MartõÂ nez á A. GoÂmez á R. Cedillo J. Arellano á M.E. PeÂrez á R. Kretschmer á O. MunÄoz Hospital de PediatrõÂ a, Centro MeÂdico Siglo XXI, IMSS, MeÂxico P. MoraÂn á M. Ramiro Hospital Regional lro, de Octubre del ISSTE, MeÂxico F. Ramos á E. GonzaÂlez á O. Valenzuela á E.I. Melendro C. XimeÂnez (&) Departamento de Medicina Experimental, Facultad de Medicina, UNAM, Dr. Balmis 148 Col. Doctores, C.P. 06726, MeÂxico D.F., A.P.70641 MeÂxico e-mail:
[email protected], Fax: (52)56-23-26-73
Introduction The recent establishment of two dierent Entamoeba species in terms of their pathogenicity (Clark and Diamond 1992; Diamond and Clark 1993) profoundly affected our understanding of previous epidemiology data with respect to the prevalence of infection. In 1986, Walsh reported that the prevalence of E. histolytica was 5 ´ 108 cases per year. This now has to be rephrased, since these ®gures apply to infection with E. histolytica and/or E. dispar and only 10% of infected individuals have invasive disease, which is believed always to be due to the E. histolytica species. On the other hand, recent studies in asymptomatic cyst passers on the characterization of isolates through ampli®cation of speci®c DNA sequences have demonstrated that asymptomatic infection is not the result of infection with E. dispar alone. Indeed, between 45% and 70% of asymptomatic cyst passers harbor both the E. histolytica and E. dispar species (AcunÄa-Soto et al. 1993; Newton-SaÂnchez et al. 1997). Invasive amebiasis occurs in dierent endemic areas that share some poorly understood risk factors associated with the host. However, only a few reports suggest an association with antigens of the major histocompatibility complex (MHC) (Arellano et al. 1991, 1992; PeÂrez-RodrõÂ guez et al. 1997). HLA-DR3 and its haplotype HLA-A2-DR3 as well as the complotype SC01/ haplotype HLA-DR4-SC01 were signi®cantly increased in Mexican mestizo patients with amebic liver abscess (ALA) as compared with a counterpart of healthy individuals of the same ethnic-socioeconomic group. Results of a similar analysis performed in children of the same group also showed increased expression of these HLA markers (PeÂrez-RodrõÂ guez et al. 1997). The present work evaluated the association of HLA markers in a group of asymptomatic cyst passers in Mexican mestizo, taking into account the recent data on mixed asymptomatic infection in Mexico.
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Subjects and methods Study design A case-control study was considered for determination of the association between a particular MHC haplotype or complotype and the risk for Entamoeba histolytica/E. dispar infection in a given population. Cases and controls were selected from cohorts of adult inhabitants of two geographic areas located in southeastern Mexico City. A two-step strategy was conducted for the selection of both groups. The ®rst step was scrutiny for the detection of asymptomatic cyst passers of E. histolytica/E. dispar and the nonparasitized group on the basis of microscopic examination of three consecutive stool samples. The second step consisted of the followup study (12 months) of both case and control groups to ensure the adequate classi®cation of each volunteer through a monthly clinical and stool-microscopic examination.
Fig. 1 Evolution of the Entamoeba histolytica/E. dispar cyst passers' condition
Treatment of stool samples The zinc sulfate ¯otation technique (Ash and Orihel 1987) was employed to detect E. histolytica/E. dispar cysts. In all samples a fresh stool aliquot was cultured in Robinson's medium and expanded to yield sucient numbers of trophozoites for zymodeme characterization (Sargeaunt and Williams 1979). Some of the expanded E. histolytica/E. dispar trophozoites were processed for DNA extraction and then subjected to polymerase chain reaction (PCR) evaluation using species-speci®c primers for E. histolytica or E. dispar characterization (Clark and Diamond 1992). The primers used for PCR ampli®cation were the 5¢ and 3¢ E. histolytica-speci®c primers (Psp5-Psp3) and the 5¢ and 3¢ E. disparspeci®c primers (NPsp5-NPsp3). PCR ampli®cation was performed as described elsewhere (Clark and Diamond 1992) using 30 cycles of 94 °C for 1 min, 55 °C for 1.5 min, and 72 °C for 2 min. The resulting ampli®cation products were visualized by electrophoresis in 0.8% agarose gels and ethidium bromide staining. For E. histolytica and E. dispar a speci®c product of 876 bp was obtained from Psp5 + 3 and Npsp5 + 3 primer ampli®cation, respectively. HLA characterization HLA-A-B and HLA-DR typing were performed in blood obtained from subjects through venous puncture and de®brinated through glass beads. The two-step microcytotoxicity method using Pel Freeze precharged plates (Brown Deer, Wis,) was used. This included 12 HLA-A, 21 HLA-B, and 12 HLA-DR speci®cities (Terasaki et al. 1978). Peripheral blood lymphocytes obtained in Ficoll-Hypaque gradient were harvested for HLA-A and HLA-B antigens. For HLADR antigen detection an enriched population of B-lymphocytes obtained with a nylon-wood column was used (Julius et al. 1973). Complotypes were studied in ethylenediaminetetraacetic acid (EDTA)-plasma by electrophoresis on agarose gel and immunoprecipitation (factor B), isolelectrofocusing and subsequent hemolytic assay (C2), and thin-layer agarose gel electrophoresis and immuno®xation (C4 A,B). Statistical analysis Results were analyzed by the chi-square test and Fisher's test using 2 ´ 2 contingency tables.
Results and discussion Our follow-up study assessed the evolution of the cyst passers' state (Fig. 1). More than 30% of the individuals
studied maintained the infection for 12 months after recruitment. The control group of healthy individuals, on the other hand, displayed no intestinal colonization during this observation period. We obtained a limited number of successfully positive stool cultures from infected individuals (
