- Email: [email protected]
AIDS patients in Mexico city
Experimental Parasitology 110 (2005) 331–334 www.elsevier.com/locate/yexpr
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, Mexico b Clínica Lomas Altas, Mexico Distrito Federal, Mexico c Hospital Regional 1ero. de Octubre, ISSSTE, Mexico Distrito Federal, Mexico d 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 2005 Available 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 microscopic examination and E. histolytica and E. dispar were identiWed by PCR. We found by microscopic analysis in HIV+/AIDS group that the 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 that the E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The diVerence in the results obtained with the microscopic and PCR is due to the diVerent sensibility of the procedures. Besides, we found patients who were infected with E. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community 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 etiologic agents of diarrhea in patients with the acquired immunodeWciency syndrome (AIDS), both in developed and underdeveloped countries (Loughton et al., 1988; Smith et al., 1988). The most frequent opportunistic parasites in HIV-infected individuals are intracellular protozoa (Isospora belli, Cryptosporidium parvum, and Cyclospora sp.), (Dupont and Marshall, 1995). However, infection with other extra-cellular parasites considered non-opportunistic, but also pathogenic for humans, are as well related to diarrheal disease in AIDS patients. Among these parasites Entamoeba histolytica, Giardia lamblia, *
Corresponding author. Fax: +5255 56 23 26 79. E-mail address: [email protected] (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 the most important (Fontanet et al., 2000; Hung et al., 1999). The adaptive cellular immune response, which is seriously damaged in AIDS patients, is a central mechanism of resistance against the parasites (Salata and Ravdin, 1986). However, the eVect of HIV infection on susceptibility for amebic infection or invasive amebic disease is unknown. There are reports concerning the occurrence of invasive amebiasis in endemic areas (Hung et al., 1999; Takeuchi et al., 1990; Reed et al., 1991). In the developing world, including Mexico, parasite prevalence is particularly high, and in many cases coincides with the HIV epidemic (Hung et al., 1999; Ramos et al., 2000). Previous data showed a prevalence of 30% for E. histolytica/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 evaluated the prevalence of amebic infection in groups of HIV+ patients who met the ‘Surveillance Criteria for AIDS’ (Bartlett, 1999) of the Centers for Disease Control and Prevention. The HIV+ patients are seen at the AIDS Clinic at the Hospital Regional 1ero. de Octubre, ISSSTE, in Mexico City. Simultaneously, HIV¡ close relatives or sexual partners of AIDS patients were included as second population. During a 4 years period we incorporated persons who wanted to participate in the study. Each individual was followed for a whole year. This study was previously assessed and approved by the Ethics Committee at the Hospital Regional 1ero. de Octubre, ISSSTE in Mexico City, in accordance with the Mexican General Health Law for research in humans which is based on the Declaration of Helsinki (Investigación en humanos, 2003). Three hundred and forty-three subjects were included in the study after signing a written informed consent. The HIV+/AIDS patients were on a triple antiretroviral medication scheme. For the purpose of comparing two groups of individuals exposed to the same E. histolytica/E. dispar infectious sources, we included 203 HIV+/AIDS patients in the experimental group, and 140 healthy, non-HIV infected (HIV¡) close relatives or sexual partners of HIV+/AIDS patients. Detection of parasites was performed through microscopic examination of three consecutive days fresh stool samples previously stained with iodine solution (4%) at 40£, and afterward cysts concentration using the Xotation 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-Wve HIV+/AIDS patients (17.2%) excreted at least one intestinal parasite compared to 37 persons of the HIV¡ group (26.4%) who also were infected. The diVerence between both groups was statistically signiWcant (p D 0.04, 2 test). However, with exception of Endolimax nana, which was more common in HIV¡ group (p < 0.01; 2 test), the frequency of other protozoa was similar in both studied groups (Table 1). Furthermore, the E. histolytica/E. dispar complex was detected in 5.9% of HIV+/AIDS patients and in 2.9% of HIV¡group, which represents a twofold increase in frequency of E. histolytica/E. dispar in HIV+/AIDS patients, nevertheless the diVerence was not statistically signiWcant. It is important to mention that Blastocystis hominis and Isospora belli were only detected in HIV+/AIDS patients. Giardia lamblia was detected in 1.9% of HIV+/AIDS group in contrast with the 0.7% in non-HIV infected individuals. We should mention that Giardia and E. histolytica are etiological agents of diarrhea independent of HIV infection. However, in HIV+/AIDS infected individuals those agents are the cause of severe disease par-
Table 1 Prevalence of parasite infection detected by microscopic examination of stool samples Parasite
Blastocystis hominis Entamoeba coli Giardia lamblia E. histolytica/E. dispar Enteromona hominis Endolimax nana Isospora belli Iodamoeba butschlii ¤
p¤
Study groups HIV+/AIDS (n D 203)
HIV¡ (n D 140)
4 (1.9%) 15 (7.3%) 4 (1.9%) 12 (5.9%) 0 (0.0%) 13 (6.4%) 1 (0.4%) 3 (1.4%)
0 (0.0%) 14 (10.0%) 1 (0.7%) 4 (2.9%) 3 (2.0%) 28 (20.0%) 0 (0.0%) 1 (0.7%)
ND 0.39 0.34 0.188 ND 0.0001 ND 0.51
Statistical analysis was performed with Pearson 2 square test.
ticularly when co-infection with Microsporidia, I. belli or C. parvum (Cimerman et al., 1999). In the studied groups we did not Wnd Microsporidia or C. parvum, although we found B. hominis and I. belli in some HIV+/AIDS patients (B. hominis 4 cases and I. belli 1 case). Parasitic infection is still a major problem in patients with HIV+/AIDS patients all over the world (Savioli et al., 2002), although their prevalence has been documented to be <5% (Reed, 2000). The identiWcation of E. histolytica/E. dispar in HIV+/AIDS patients is a dilemma for clinicians because it is considered that E. dispar asymptomatic infections are more prevalent than E. histolytica infection, which is the potentially pathogenic specie. In this study, the identiWcation of E. histolytica and E. dispar was made through PCR. With that purpose, DNA was extracted from sediment obtained from stool samples placed in the zinc-sulfate gradient (Acuña-Soto et al., 1993). Sediments were transferred to 2 ml Eppendorf tubes, washed four times with 0.15 M NaCl, and suspended in 300 l of lysis buVer (100 mM EDTA, SDS 0.25%, pH 8). The tubes were frozen three times in ethanol-dry ice and thawed in a 37 °C water bath. Finally, 3 l of 20 mg/ml proteinase K (Sigma Chemical, St. Louis, MO, USA) was added, and the sample was incubated for 1 h at 55 °C. After digestion with proteinase K, lysates were adjusted to 0.7 M NaCl and 1% CTAB (Sigma Chemical). The mixture was incubated at 65 °C for 20 min. DNA was extracted with chloroform and phenol/chloroform, followed by precipitation with ethanol. Precipitated DNA was suspended in water and passed through a Sephadex G-25 spin column (Pharmacia-Biotech, Uppsala, Sweden). The DNA extracted was submitted to PCR ampliWcation 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 at 55 °C, and 2 min at 72 °C in a DNA thermocycler (Perkin Elmer Applied Biosystems). For characterization of E. histolytica or E. dispar species, species-speciWc primers for the small ribosomal subunit rRNA gene were used. Psp5⬘–3⬘ primers are speciWc for E. histolytica, and NPsp
P. Moran et al. / Experimental Parasitology 110 (2005) 331–334 Table 2 Prevalence of Entamoeba species as detected by PCR Parasite species
+
Entamoeba histolytica E. histolytica + E. dispar Entamoeba dispar Other protozoa Negative ¤
p¤
Study groups ¡
HIV /AIDS (n D 158)
HIV (n D 130)
19 (12.0%) 21 (13.3%) 14 (8.9%) 18 (11.4%) 86 (54.4%)
23 (17.7%) 1 (0.7%) 4 (3.1%) 44 (33.9%) 58 (44.6%)
0.17 ND ND 0.001 0.097
Statistical analysis was performed with Pearson square test.
333
Mexico City. However, during the follow up phase of the study, no E. histolytica-infected person developed clinical symptoms attributable to an invasion process. This fact suggests that the studied populations had been colonized with E. histolytica strains with low pathogenic potential.
Acknowledgments
2
5⬘–3⬘ are speciWc for E. dispar and both primers generate a fragment of 876 bp in size (Clark and Diamond, 1992). The samples were also ampliWed with RD primers that are unspeciWc and let us know if individuals were infected with other protozoa. The ampliWes were visualized in 1.2% agarose gels in TBE buVer, stained with ethidium bromide and photographed for later analysis. One hundred and Wfty-eight samples were studied from 203 HIV+/AIDS patients and 130 samples from 140 HIV¡ by PCR assays. In the HIV+/AIDS group E. histolytica alone was detected in 19 samples (12%), E. dispar alone in 14 (8.9%), and both Entamoeba species in 21 (13.3%) (Table 2). In HIV¡ group, 23 samples (17.7%) were positive 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 persons who harbor only E. histolytica and those who have mixed infections of E. histolytica and E. dispar) in HIV+/AIDS patients was higher (40/158; 25.3%) than in HIV¡ group (24/130; 18.4%), but the diVerence was not statistically signiWcant (p D 0.16; 2 test). Since E. histolytica and E. dispar share biological cycles, ecological niches, and transmission mechanisms in humans, persons colonized with E. histolytica 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%). In this case, the diVerence between both groups was statistically signiWcant (p D 0.019; 2 test). Further analysis of the separate Entamoeba species showed that this diVerence was due to a greater prevalence of E. dispar in HIV+/ AIDS patients (35/158; 22.2%) versus HIV¡ group (5/130; 3.8%) (p D 0.001, 2 test). In our study, we explored the association of amebic infection in two diVerent populations (HIV+/AIDS and HIV¡ individuals) exposed to the same environment and parasitic infectious sources. The E. histolytica specie prevalence was similar in HIV+/AIDS patients and HIV¡ group (p D 0.16; 2 test) (Table 2). However, as we previously mentioned, prevalence of E. dispar was greater in HIV+/AIDS patients than in HIV¡ group (p D 0.019; 2 test). These data conWrm the high prevalence of E. histolytica strains in some regions of Mexico, particularly in
Authors especially thank Mrs. María Elena Ortiz and Mr. Marco Gudiño for their secretarial and computer assistance. We thank María del Carmen García de León and José J. Castillo for their technical assistance. This work includes a part of the doctoral dissertation in Biological Sciences, UAM-X/I by Miss Patricia Morán. Financial support was provided by Grants DGAPA IN219599, DGAPA IN234202-2, and 2001 UC MEXUS-CONACYT collaborative grant.
References Acuña-Soto, R., Samuelson, J., De Girolami, P., Zárate, L., MillanVelasco, F., Schoolmick, G., Wirth, D., 1993. Application of the polymerase chain reaction to the epidemiology of pathogenic and non-pathogenic Entamoeba histolytica. American Journal of Tropical Medicine and Hygiene 48, 58–70. Ash, R.L., Orihel, T.C., 1987. Collection and preservation of faeces. Parasites: A Guide to Laboratory Procedures and IdentiWcation. American Society of Clinical Pathologists, Chicago, IL, ASCP Press, pp. 5–14. Bartlett, J.G., 1999. The Johns Hopkins Hospital 1998–1999 Guide to Medical Care of Patients with HIV Infection. Williams and Wilkins, Baltimore, MD. Cimerman, S., Cimerman, B., Salomao, L.D., 1999. Enteric parasites and AIDS. Sao Paulo Medical Journal 117, 266–273. Clark, G.C., Diamond, L.S., 1992. DiVerentiation of pathogenic Entamoeba histolytica from other intestinal protozoa by riboprinting. Archives Medical Research 23, 15–18. Dupont, H.L., Marshall, G.D., 1995. HIV-associated diarrhea and wasting. Lancet ii, 352–356. Fontanet, A.L., Sahlu, T., Rinkedewit, T., Messele, T., Masho, W., Woldemichael, T., Yeneneh, H., Coutinho, R.A., 2000. Epidemiology of infections with intestinal parasites and human immunodeWciency virus (HIV) among Sugar-State residents in Ethiopia. Annals of Tropical Medicine and Parasitology 94, 269–278. Hung, C.C., Chen, P.S., Hsieh, S.M., Wong, J.M., Fang, C.T., Chang, S.C., Chen, M.Y., 1999. Invasive amoebiasis: an emerging parasitic disease in patients infected with HIV in an area endemic for amoebic infection. Acquired ImmunodeWciency Syndrome 13, 2421– 2428. Investigación en humanos. In: Ley General de Salud, Gobierno Federal de los Estados Unidos Mexicanos. SISTA editor; 2003. pp. 32– 34. Loughton, B.E., Druckman, D.A., Vernon, A., Quinn, T.C., Pol, B.P., 1988. Prevalence of enteric pathogens in homosexual men with and without acquired immunodeWciency syndrome. Gastroenterology 94, 948–993. Quinn, T.C., Stamm, W.E., Goodell, S.E., 1983. The polymicrobial origin of intestinal infection in homosexual men. New England Journal of Medicine 309, 576–582.
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Ramos, F., Valdez, E., Morán, P., González, E., Padilla, G., Gómez, A., Ramiro, M., Melendro, E.I., Muñoz, O., Clark, G., Ximénez, C., 2000. Prevalence of Entamoeba histolytica and Entamoeba dispar in highly endemic rural population. Archives of Medical Research 31, 34–35. Reed, S.L., 2000. Editorial response: Entamoeba infection in human immunodeWciency virus–infected patients: not just a tropical problem. Clinical Infectious Disease 30, 959–961. Reed, S.L., Wessed, D.W., David, C.E., 1991. Entamoeba histolytica infection and AIDS. American Journal of Medicine 90, 269– 271. Salata, R.A., Ravdin, J.L., 1986. Review of human immune mechanisms directed against Entamoeba histolytica. Review Infection Disease 8, 261–272.
Savioli, L., StausWeld, S., Bundy, D.A.P., Mtchel, L.A., Bhatia, R., Engels, D., Montresor, A., Neira, M., Shein, Am., 2002. Schitosomiasis and soil transmitted helminthics infections: forgoing control eVorts. Transactions of the Royal Society of Tropical Medicine and Hygiene 96, 577–579. Smith, P.D., Lane, H.C., Gill, V.J., Manischewitz, J.F., Quinnan, G.N., Fauci, A.S., Masur, H., 1988. Intestinal infections in patients with acquired immunodeWciency syndrome (AIDS). Annals of Internal Medicine 108, 328–333. Takeuchi, T., Miyahira, Y., Kobayashi, S., Nozaki, T., Mottha, S., Matsuda, J., 1990. High seropositivity for Entamoeba histolytica in Japanese homosexual men: further evidence for the occurrence of pathogenic strains. American Journal of Tropical Medicine and Hygiene 84, 250–251.
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, Mexico b Clínica Lomas Altas, Mexico Distrito Federal, Mexico c Hospital Regional 1ero. de Octubre, ISSSTE, Mexico Distrito Federal, Mexico d 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 2005 Available 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 microscopic examination and E. histolytica and E. dispar were identiWed by PCR. We found by microscopic analysis in HIV+/AIDS group that the 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 that the E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The diVerence in the results obtained with the microscopic and PCR is due to the diVerent sensibility of the procedures. Besides, we found patients who were infected with E. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community 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 etiologic agents of diarrhea in patients with the acquired immunodeWciency syndrome (AIDS), both in developed and underdeveloped countries (Loughton et al., 1988; Smith et al., 1988). The most frequent opportunistic parasites in HIV-infected individuals are intracellular protozoa (Isospora belli, Cryptosporidium parvum, and Cyclospora sp.), (Dupont and Marshall, 1995). However, infection with other extra-cellular parasites considered non-opportunistic, but also pathogenic for humans, are as well related to diarrheal disease in AIDS patients. Among these parasites Entamoeba histolytica, Giardia lamblia, *
Corresponding author. Fax: +5255 56 23 26 79. E-mail address: [email protected] (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 the most important (Fontanet et al., 2000; Hung et al., 1999). The adaptive cellular immune response, which is seriously damaged in AIDS patients, is a central mechanism of resistance against the parasites (Salata and Ravdin, 1986). However, the eVect of HIV infection on susceptibility for amebic infection or invasive amebic disease is unknown. There are reports concerning the occurrence of invasive amebiasis in endemic areas (Hung et al., 1999; Takeuchi et al., 1990; Reed et al., 1991). In the developing world, including Mexico, parasite prevalence is particularly high, and in many cases coincides with the HIV epidemic (Hung et al., 1999; Ramos et al., 2000). Previous data showed a prevalence of 30% for E. histolytica/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 evaluated the prevalence of amebic infection in groups of HIV+ patients who met the ‘Surveillance Criteria for AIDS’ (Bartlett, 1999) of the Centers for Disease Control and Prevention. The HIV+ patients are seen at the AIDS Clinic at the Hospital Regional 1ero. de Octubre, ISSSTE, in Mexico City. Simultaneously, HIV¡ close relatives or sexual partners of AIDS patients were included as second population. During a 4 years period we incorporated persons who wanted to participate in the study. Each individual was followed for a whole year. This study was previously assessed and approved by the Ethics Committee at the Hospital Regional 1ero. de Octubre, ISSSTE in Mexico City, in accordance with the Mexican General Health Law for research in humans which is based on the Declaration of Helsinki (Investigación en humanos, 2003). Three hundred and forty-three subjects were included in the study after signing a written informed consent. The HIV+/AIDS patients were on a triple antiretroviral medication scheme. For the purpose of comparing two groups of individuals exposed to the same E. histolytica/E. dispar infectious sources, we included 203 HIV+/AIDS patients in the experimental group, and 140 healthy, non-HIV infected (HIV¡) close relatives or sexual partners of HIV+/AIDS patients. Detection of parasites was performed through microscopic examination of three consecutive days fresh stool samples previously stained with iodine solution (4%) at 40£, and afterward cysts concentration using the Xotation 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-Wve HIV+/AIDS patients (17.2%) excreted at least one intestinal parasite compared to 37 persons of the HIV¡ group (26.4%) who also were infected. The diVerence between both groups was statistically signiWcant (p D 0.04, 2 test). However, with exception of Endolimax nana, which was more common in HIV¡ group (p < 0.01; 2 test), the frequency of other protozoa was similar in both studied groups (Table 1). Furthermore, the E. histolytica/E. dispar complex was detected in 5.9% of HIV+/AIDS patients and in 2.9% of HIV¡group, which represents a twofold increase in frequency of E. histolytica/E. dispar in HIV+/AIDS patients, nevertheless the diVerence was not statistically signiWcant. It is important to mention that Blastocystis hominis and Isospora belli were only detected in HIV+/AIDS patients. Giardia lamblia was detected in 1.9% of HIV+/AIDS group in contrast with the 0.7% in non-HIV infected individuals. We should mention that Giardia and E. histolytica are etiological agents of diarrhea independent of HIV infection. However, in HIV+/AIDS infected individuals those agents are the cause of severe disease par-
Table 1 Prevalence of parasite infection detected by microscopic examination of stool samples Parasite
Blastocystis hominis Entamoeba coli Giardia lamblia E. histolytica/E. dispar Enteromona hominis Endolimax nana Isospora belli Iodamoeba butschlii ¤
p¤
Study groups HIV+/AIDS (n D 203)
HIV¡ (n D 140)
4 (1.9%) 15 (7.3%) 4 (1.9%) 12 (5.9%) 0 (0.0%) 13 (6.4%) 1 (0.4%) 3 (1.4%)
0 (0.0%) 14 (10.0%) 1 (0.7%) 4 (2.9%) 3 (2.0%) 28 (20.0%) 0 (0.0%) 1 (0.7%)
ND 0.39 0.34 0.188 ND 0.0001 ND 0.51
Statistical analysis was performed with Pearson 2 square test.
ticularly when co-infection with Microsporidia, I. belli or C. parvum (Cimerman et al., 1999). In the studied groups we did not Wnd Microsporidia or C. parvum, although we found B. hominis and I. belli in some HIV+/AIDS patients (B. hominis 4 cases and I. belli 1 case). Parasitic infection is still a major problem in patients with HIV+/AIDS patients all over the world (Savioli et al., 2002), although their prevalence has been documented to be <5% (Reed, 2000). The identiWcation of E. histolytica/E. dispar in HIV+/AIDS patients is a dilemma for clinicians because it is considered that E. dispar asymptomatic infections are more prevalent than E. histolytica infection, which is the potentially pathogenic specie. In this study, the identiWcation of E. histolytica and E. dispar was made through PCR. With that purpose, DNA was extracted from sediment obtained from stool samples placed in the zinc-sulfate gradient (Acuña-Soto et al., 1993). Sediments were transferred to 2 ml Eppendorf tubes, washed four times with 0.15 M NaCl, and suspended in 300 l of lysis buVer (100 mM EDTA, SDS 0.25%, pH 8). The tubes were frozen three times in ethanol-dry ice and thawed in a 37 °C water bath. Finally, 3 l of 20 mg/ml proteinase K (Sigma Chemical, St. Louis, MO, USA) was added, and the sample was incubated for 1 h at 55 °C. After digestion with proteinase K, lysates were adjusted to 0.7 M NaCl and 1% CTAB (Sigma Chemical). The mixture was incubated at 65 °C for 20 min. DNA was extracted with chloroform and phenol/chloroform, followed by precipitation with ethanol. Precipitated DNA was suspended in water and passed through a Sephadex G-25 spin column (Pharmacia-Biotech, Uppsala, Sweden). The DNA extracted was submitted to PCR ampliWcation 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 at 55 °C, and 2 min at 72 °C in a DNA thermocycler (Perkin Elmer Applied Biosystems). For characterization of E. histolytica or E. dispar species, species-speciWc primers for the small ribosomal subunit rRNA gene were used. Psp5⬘–3⬘ primers are speciWc for E. histolytica, and NPsp
P. Moran et al. / Experimental Parasitology 110 (2005) 331–334 Table 2 Prevalence of Entamoeba species as detected by PCR Parasite species
+
Entamoeba histolytica E. histolytica + E. dispar Entamoeba dispar Other protozoa Negative ¤
p¤
Study groups ¡
HIV /AIDS (n D 158)
HIV (n D 130)
19 (12.0%) 21 (13.3%) 14 (8.9%) 18 (11.4%) 86 (54.4%)
23 (17.7%) 1 (0.7%) 4 (3.1%) 44 (33.9%) 58 (44.6%)
0.17 ND ND 0.001 0.097
Statistical analysis was performed with Pearson square test.
333
Mexico City. However, during the follow up phase of the study, no E. histolytica-infected person developed clinical symptoms attributable to an invasion process. This fact suggests that the studied populations had been colonized with E. histolytica strains with low pathogenic potential.
Acknowledgments
2
5⬘–3⬘ are speciWc for E. dispar and both primers generate a fragment of 876 bp in size (Clark and Diamond, 1992). The samples were also ampliWed with RD primers that are unspeciWc and let us know if individuals were infected with other protozoa. The ampliWes were visualized in 1.2% agarose gels in TBE buVer, stained with ethidium bromide and photographed for later analysis. One hundred and Wfty-eight samples were studied from 203 HIV+/AIDS patients and 130 samples from 140 HIV¡ by PCR assays. In the HIV+/AIDS group E. histolytica alone was detected in 19 samples (12%), E. dispar alone in 14 (8.9%), and both Entamoeba species in 21 (13.3%) (Table 2). In HIV¡ group, 23 samples (17.7%) were positive 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 persons who harbor only E. histolytica and those who have mixed infections of E. histolytica and E. dispar) in HIV+/AIDS patients was higher (40/158; 25.3%) than in HIV¡ group (24/130; 18.4%), but the diVerence was not statistically signiWcant (p D 0.16; 2 test). Since E. histolytica and E. dispar share biological cycles, ecological niches, and transmission mechanisms in humans, persons colonized with E. histolytica 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%). In this case, the diVerence between both groups was statistically signiWcant (p D 0.019; 2 test). Further analysis of the separate Entamoeba species showed that this diVerence was due to a greater prevalence of E. dispar in HIV+/ AIDS patients (35/158; 22.2%) versus HIV¡ group (5/130; 3.8%) (p D 0.001, 2 test). In our study, we explored the association of amebic infection in two diVerent populations (HIV+/AIDS and HIV¡ individuals) exposed to the same environment and parasitic infectious sources. The E. histolytica specie prevalence was similar in HIV+/AIDS patients and HIV¡ group (p D 0.16; 2 test) (Table 2). However, as we previously mentioned, prevalence of E. dispar was greater in HIV+/AIDS patients than in HIV¡ group (p D 0.019; 2 test). These data conWrm the high prevalence of E. histolytica strains in some regions of Mexico, particularly in
Authors especially thank Mrs. María Elena Ortiz and Mr. Marco Gudiño for their secretarial and computer assistance. We thank María del Carmen García de León and José J. Castillo for their technical assistance. This work includes a part of the doctoral dissertation in Biological Sciences, UAM-X/I by Miss Patricia Morán. Financial support was provided by Grants DGAPA IN219599, DGAPA IN234202-2, and 2001 UC MEXUS-CONACYT collaborative grant.
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