Experimental Parasitology 110 (2005) 331–334

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Research brief

Entamoeba histolytica and/or Entamoeba dispar: Infection frequency in HIV+/AIDS patients in Mexico city

Patricia Moran a, Fernando Ramos a, Manuel Ramiro b, Octavio Curiel c, Enrique González a, Alicia Valadez a, Alejandro Gómez d, Gabriela García a,

Emma I. Melendro a, Cecilia Ximénez a,¤

a Departamento de Medicina Experimental, Facultad de Medicina, UNAM, Mexico Distrito Federal, Mexicob Clínica Lomas Altas, Mexico Distrito Federal, Mexico

c Hospital Regional 1ero. de Octubre, ISSSTE, Mexico Distrito Federal, Mexicod Coordinación de Investigación en Salud, Centro Médico Siglo XXI, IMSS, Mexico Distrito Federal, Mexico

Received 1 February 2005; received in revised form 22 March 2005; accepted 23 March 2005Available online 28 April 2005

Abstract

The objective of this work was to evaluate the frequency of Entamoeba histolytica/Entamoeba dispar intestinal infection in HIV+/AIDS subjects and their HIV¡ close relatives or sexual partners. Enteric parasites were investigated in stool samples by microscopicexamination and E. histolytica and E. dispar were identiWed by PCR. We found by microscopic analysis in HIV+/AIDS group thatthe E. histolytica/E. dispar complex was present in 5.9% of the members, while in the HIV¡ group was 2.9%. With PCR we found thatthe E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The diVerence in the results obtainedwith the microscopic and PCR is due to the diVerent sensibility of the procedures. Besides, we found patients who were infected withE. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studiedcommunity appear to be of low pathogenic potential. 2005 Elsevier Inc. All rights reserved.

Keywords: HIV+; AIDS; Parasitic infection

Intestinal parasites are some of the most important eti-ologic agents of diarrhea in patients with the acquiredimmunodeWciency syndrome (AIDS), both in developedand underdeveloped countries (Loughton et al., 1988;Smith et al., 1988). The most frequent opportunistic para-sites in HIV-infected individuals are intracellular protozoa(Isospora belli, Cryptosporidium parvum, and Cyclosporasp.), (Dupont and Marshall, 1995). However, infectionwith other extra-cellular parasites considered non-oppor-tunistic, but also pathogenic for humans, are as wellrelated to diarrheal disease in AIDS patients. Amongthese parasites Entamoeba histolytica, Giardia lamblia,

* Corresponding author. Fax: +5255 56 23 26 79.E-mail address: cximenez@servidor.unam.mx (C. Ximénez).

0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.exppara.2005.03.023

Strongyloides stercoralis, and Ascaris lumbricoides are themost important (Fontanet et al., 2000; Hung et al., 1999).

The adaptive cellular immune response, which is seri-ously damaged in AIDS patients, is a central mechanismof resistance against the parasites (Salata and Ravdin,1986). However, the eVect of HIV infection on suscepti-bility for amebic infection or invasive amebic disease isunknown. There are reports concerning the occurrenceof invasive amebiasis in endemic areas (Hung et al.,1999; Takeuchi et al., 1990; Reed et al., 1991). In thedeveloping world, including Mexico, parasite prevalenceis particularly high, and in many cases coincides with theHIV epidemic (Hung et al., 1999; Ramos et al., 2000).

Previous data showed a prevalence of 30% for E. his-tolytica/E. dispar infection in homosexual men; however,

332 P. Moran et al. / Experimental Parasitology 110 (2005) 331–334

invasive amebic diseases appeared to be uncommon(Quinn et al., 1983). In the present work, we evaluatedthe prevalence of amebic infection in groups of HIV+

patients who met the ‘Surveillance Criteria for AIDS’(Bartlett, 1999) of the Centers for Disease Control andPrevention. The HIV+ patients are seen at the AIDSClinic at the Hospital Regional 1ero. de Octubre, ISS-STE, in Mexico City. Simultaneously, HIV¡ close rela-tives or sexual partners of AIDS patients were includedas second population. During a 4 years period we incor-porated persons who wanted to participate in the study.Each individual was followed for a whole year.

This study was previously assessed and approved bythe Ethics Committee at the Hospital Regional 1ero. deOctubre, ISSSTE in Mexico City, in accordance with theMexican General Health Law for research in humanswhich is based on the Declaration of Helsinki (Investiga-ción en humanos, 2003). Three hundred and forty-threesubjects were included in the study after signing a writ-ten informed consent. The HIV+/AIDS patients were ona triple antiretroviral medication scheme. For the pur-pose of comparing two groups of individuals exposed tothe same E. histolytica/E. dispar infectious sources, weincluded 203 HIV+/AIDS patients in the experimentalgroup, and 140 healthy, non-HIV infected (HIV¡) closerelatives or sexual partners of HIV+/AIDS patients.

Detection of parasites was performed through micro-scopic examination of three consecutive days fresh stoolsamples previously stained with iodine solution (4%) at40£, and afterward cysts concentration using the Xota-tion technique in a zinc-sulfate gradient (d D 1.192°B)(Ash and Orihel, 1987).

The most common parasites we found were protozoa,some of them potential pathogens for humans (e.g.,G. lamblia and E. histolytica/E. dispar). Thirty-WveHIV+/AIDS patients (17.2%) excreted at least one intes-tinal parasite compared to 37 persons of the HIV¡ group(26.4%) who also were infected. The diVerence betweenboth groups was statistically signiWcant (p D 0.04, �2

test). However, with exception of Endolimax nana, whichwas more common in HIV¡ group (p < 0.01; �2 test), thefrequency of other protozoa was similar in both studiedgroups (Table 1). Furthermore, the E. histolytica/E. dis-par complex was detected in 5.9% of HIV+/AIDSpatients and in 2.9% of HIV¡group, which represents atwofold increase in frequency of E. histolytica/E. disparin HIV+/AIDS patients, nevertheless the diVerence wasnot statistically signiWcant. It is important to mentionthat Blastocystis hominis and Isospora belli were onlydetected in HIV+/AIDS patients.

Giardia lamblia was detected in 1.9% of HIV+/AIDSgroup in contrast with the 0.7% in non-HIV infectedindividuals. We should mention that Giardia and E. his-tolytica are etiological agents of diarrhea independent ofHIV infection. However, in HIV+/AIDS infected indi-viduals those agents are the cause of severe disease par-

ticularly when co-infection with Microsporidia, I. belli orC. parvum (Cimerman et al., 1999). In the studied groupswe did not Wnd Microsporidia or C. parvum, although wefound B. hominis and I. belli in some HIV+/AIDSpatients (B. hominis 4 cases and I. belli 1 case).

Parasitic infection is still a major problem in patientswith HIV+/AIDS patients all over the world (Savioliet al., 2002), although their prevalence has been docu-mented to be <5% (Reed, 2000). The identiWcation ofE. histolytica/E. dispar in HIV+/AIDS patients is adilemma for clinicians because it is considered thatE. dispar asymptomatic infections are more prevalentthan E. histolytica infection, which is the potentiallypathogenic specie. In this study, the identiWcation ofE. histolytica and E. dispar was made through PCR.With that purpose, DNA was extracted from sedimentobtained from stool samples placed in the zinc-sulfategradient (Acuña-Soto et al., 1993). Sediments were trans-ferred to 2 ml Eppendorf tubes, washed four times with0.15 M NaCl, and suspended in 300 �l of lysis buVer(100 mM EDTA, SDS 0.25%, pH 8). The tubes were fro-zen three times in ethanol-dry ice and thawed in a 37 °Cwater bath. Finally, 3 �l of 20 mg/ml proteinase K(Sigma Chemical, St. Louis, MO, USA) was added, andthe sample was incubated for 1 h at 55 °C. After diges-tion with proteinase K, lysates were adjusted to 0.7 MNaCl and 1% CTAB (Sigma Chemical). The mixturewas incubated at 65 °C for 20 min. DNA was extractedwith chloroform and phenol/chloroform, followed byprecipitation with ethanol. Precipitated DNA was sus-pended in water and passed through a Sephadex G-25spin column (Pharmacia-Biotech, Uppsala, Sweden).The DNA extracted was submitted to PCR ampliWca-tion assay under standard conditions (AmpliTaq Kit,Perkin Elmer Applied Biosystems, Foster City, CA,USA) for 35 cycles of one minute at 94 °C, 1.5 min at55 °C, and 2 min at 72 °C in a DNA thermocycler (PerkinElmer Applied Biosystems). For characterization ofE. histolytica or E. dispar species, species-speciWc primersfor the small ribosomal subunit rRNA gene were used.Psp5�–3� primers are speciWc for E. histolytica, and NPsp

Table 1Prevalence of parasite infection detected by microscopic examinationof stool samples

¤ Statistical analysis was performed with Pearson �2 square test.

Parasite Study groups p¤

HIV+/AIDS(n D 203)

HIV¡

(n D 140)

Blastocystis hominis 4 (1.9%) 0 (0.0%) NDEntamoeba coli 15 (7.3%) 14 (10.0%) 0.39Giardia lamblia 4 (1.9%) 1 (0.7%) 0.34E. histolytica/E. dispar 12 (5.9%) 4 (2.9%) 0.188Enteromona hominis 0 (0.0%) 3 (2.0%) NDEndolimax nana 13 (6.4%) 28 (20.0%) 0.0001Isospora belli 1 (0.4%) 0 (0.0%) NDIodamoeba butschlii 3 (1.4%) 1 (0.7%) 0.51

P. Moran et al. / Experimental Parasitology 110 (2005) 331–334 333

5�–3� are speciWc for E. dispar and both primers generatea fragment of 876 bp in size (Clark and Diamond, 1992).The samples were also ampliWed with RD primers thatare unspeciWc and let us know if individuals wereinfected with other protozoa. The ampliWes were visual-ized in 1.2% agarose gels in TBE buVer, stained withethidium bromide and photographed for later analysis.

One hundred and Wfty-eight samples were studied from203 HIV+/AIDS patients and 130 samples from 140 HIV¡

by PCR assays. In the HIV+/AIDS group E. histolyticaalone was detected in 19 samples (12%), E. dispar alone in14 (8.9%), and both Entamoeba species in 21 (13.3%)(Table 2). In HIV¡ group, 23 samples (17.7%) were posi-tive for E. histolytica; E. dispar was detected in 4 (3.1%)and both species were detected only in 1 (0.7%) (Table 2).Prevalence of E. histolytica (considering those personswho harbor only E. histolytica and those who have mixedinfections of E. histolytica and E. dispar) in HIV+/AIDSpatients was higher (40/158; 25.3%) than in HIV¡ group(24/130; 18.4%), but the diVerence was not statistically sig-niWcant (pD0.16; �2 test). Since E. histolytica and E. disparshare biological cycles, ecological niches, and transmissionmechanisms in humans, persons colonized with E. histoly-tica and/or E. dispar could be considered as a single group.In this way, Entamoeba infection prevalence [Eh+ (Eh +Ed)+ Ed] was signiWcantly greater in HIV+/AIDS patients(54/158; 34.1%) that in HIV¡ patients (28/130; 21.5%). Inthis case, the diVerence between both groups was statisti-cally signiWcant (pD0.019; �2 test). Further analysis of theseparate Entamoeba species showed that this diVerencewas due to a greater prevalence of E. dispar in HIV+/AIDS patients (35/158; 22.2%) versus HIV¡ group (5/130;3.8%) (pD 0.001, �2 test).

In our study, we explored the association of amebicinfection in two diVerent populations (HIV+/AIDS andHIV¡ individuals) exposed to the same environment andparasitic infectious sources. The E. histolytica specieprevalence was similar in HIV+/AIDS patients andHIV¡ group (p D 0.16; �2 test) (Table 2). However, as wepreviously mentioned, prevalence of E. dispar wasgreater in HIV+/AIDS patients than in HIV¡ group(p D 0.019; �2 test).

These data conWrm the high prevalence of E. histoly-tica strains in some regions of Mexico, particularly in

Table 2Prevalence of Entamoeba species as detected by PCR

¤ Statistical analysis was performed with Pearson �2 square test.

Parasite species Study groups p¤

HIV+/AIDS(n D 158)

HIV¡ (n D 130)

Entamoeba histolytica 19 (12.0%) 23 (17.7%) 0.17E. histolytica + E. dispar 21 (13.3%) 1 (0.7%) NDEntamoeba dispar 14 (8.9%) 4 (3.1%) NDOther protozoa 18 (11.4%) 44 (33.9%) 0.001Negative 86 (54.4%) 58 (44.6%) 0.097

Mexico City. However, during the follow up phase of thestudy, no E. histolytica-infected person developed clini-cal symptoms attributable to an invasion process. Thisfact suggests that the studied populations had been colo-nized with E. histolytica strains with low pathogenicpotential.

Acknowledgments

Authors especially thank Mrs. María Elena Ortiz andMr. Marco Gudiño for their secretarial and computerassistance. We thank María del Carmen García de Leónand José J. Castillo for their technical assistance. Thiswork includes a part of the doctoral dissertation in Bio-logical Sciences, UAM-X/I by Miss Patricia Morán.Financial support was provided by Grants DGAPAIN219599, DGAPA IN234202-2, and 2001 UCMEXUS-CONACYT collaborative grant.

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