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Amebiasis caused by infection with Entamoeba histolytica is one of the most problematic parasitic diseases in developing and developed countries.1 E. histolytica infection is also related to diarrhea in patients with HIV infection and AIDS, as well as those infected with opportunistic protozoan parasites such as Cryptosporidium spp., Isospora belli, and microsporidia.2 Recently, a high prevalence of E. histolytica infection has been reported in HIV/AIDS patients in Japan and Taiwan.3–5 In China, it is estimated that 650,000 individuals are infected with HIV, and HIV/AIDS is becoming a major public health problem (http://data.unaids.org/Media/Press-Releases03/PR_china_060125_en.pdf). However, there is no information concerning the correlation between E. histolytica and HIV infections, and the prevalence of E. histolytica and E. dispar, which is morphologically indistinguishable from E. histolytica but is non-pathogenic, is not known in China. Because E. dispar is non-invasive, detection of high specific antibody titers indicates possible infection with E. histolytica.6 Therefore, this preliminary study was undertaken to estimate the seroprevalence of E. histolytica infection in HIV-infected persons in the Chinese population.
This study was approved by the Ethics Committees of Fudan University School of Medicine and Shanghai Public Health Center, which is affiliated with Fudan University. A total of 466 peripheral blood samples were collected from June to August 2005. Samples from 215 individuals with HIV/AIDS were obtained at Shanghai Public Health Center and at treatment centers for AIDS in Henan province and Anhui province. The subjects were selected randomly, but all were receiving anti-retroviral therapy. Symptomatic gastrointestinal complaints or disorders were not recorded at the time the blood samples were collected. For comparison, serum samples from 191 individuals with gastrointestinal symptoms but no HIV infection were obtained at Huashan Hospital, Shanghai. Gastrointestinal symptoms in these individuals were caused by acute gastritis, giant hypertrophic gastropathy, gastric ulcer, duodenal ulcer, acute hemorrhagic necrotizing enteritis, ulcerative colitis, and intestinal obstruction, but many cases did not have a final diagnosis. No specific pathogens have been detected in culture or microscopy of stool samples examined to date. Serology to HIV was examined using a Cambridge Biotech HIV1 Western blot kit (Cambridge Biotech Corp, Rockville, MD). Serum samples from 60 healthy individuals without a history of amebiasis were used as negative controls. All serum samples were inactivated at 56°C for 30 minutes and stored at –30°C or –80°C until use.
Anti-E. histolytica serology was examined by enzyme-linked immunosorbent assay (ELISA) using crude E. histolytica antigen and the recombinant fragment of C terminus of intermediate subunit of galactose and N-acetyl-D-galactosamine-inhibitable lectin of E. histolytica (C-Igl), as previously described.7 The cut-off for a positive result was defined as an ELISA value > 3 SD above the mean for healthy negative controls. Optical density (OD) values were plotted and analyzed using Prism ver. 4.0, and statistical analysis was performed with Stata ver. 7.0. A descriptive exploratory analysis of the data was performed to assess the distribution variables in the HIV-infected and HIV-uninfected groups. Categorical variables were compared using a Pearson 
2 test.
Quantification of CD4+ lymphocytes in individuals with HIV/AIDS was performed by cytofluorometry using a MultiTEST CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer’s instructions.
ELISA reactivities of sera from HIV-infected and HIV-uninfected patients are shown in Figure 1
. The cut-off OD values were 0.412 for crude antigen and 0.402 for C-Igl antigen. In HIV-infected individuals, seropositivity to these antigens was 12.1% and 7.9%, respectively (Table 1). These values were significantly higher than those for HIV-uninfected individuals, which were 3.1% and 0.5%, respectively (P < 0.005 and P < 0.001, respectively). There was no significant difference in seropositivity to E. histolytica between HIV-infected men and women. Positive serology to C-Igl antigen in HIV-infected individuals was similar (6.8%–9.1%) in every age category, except for patients < 20 years old, for whom there were a limited number of samples. Seropositivity to C-Igl in HIV-infected individuals from Shanghai was higher than those in samples from Anhui and Henan, but the differences were not significant (P = 0.140 and P = 0.122, respectively). Seroprevalence to E. histolytica was higher in patients with a CD4+ T-cell count of < 200/µL compared with those with a count of 
200/µL (P < 0.005). Stool samples from patients with positive serology to E. histolytica were obtained and examined microscopically using a direct smear and subsequent formalin-ether sedimentation. However, we were unable to detect E. histolytica/E. dispar, and no obvious symptoms of amebiasis were apparent at the time of serum collection.
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The prevalence of E. histolytica and E. dispar infections in China is not well understood. A survey in two villages in Shandong province from 1977 to 1984 showed an infection rate of E. histolytica/E. dispar of 6.4% using microscopy of direct smears of stool samples, with incidences of amebic dysentery and liver abscess of 2.8% and 0.9%, respectively.12 Another survey in a village in Hebei province in 1985 showed a positive serology rate of 13.2% and a positive rate for E. histolytica/E. dispar in stool of 9.4%.13 These reports suggest that E. histolytica infection may be endemic in rural areas of China. However, it has been reported that the prevalence of E. histolytica/E. dispar infection in Henan and Anhui provinces is 0.59% and 0.57%, respectively, using direct microscopy of stool samples.14 Furthermore, the prevalence of E. histolytica/E. dispar infection in Shanghai has recently been estimated to be < 0.1% (Xu and others, personal communication). Because microscopy has low sensitivity and specificity for detection of amebiasis, it is possible that these prevalence rates are underestimates. However, the low seroprevalence to E. histolytica in non-HIV patients from Shanghai in this study is in good agreement with these data. In addition, our previous observation that two of three E. histolytica/E. dispar isolates obtained from patients with diarrhea in Shanghai were not E. histolytica but E. dispar also supports the low seropositivity in the non-HIV group.15 Therefore, it is likely that the higher seroprevalence among HIV-infected persons is related to HIV infection and characteristics, rather than background seroprevalence because of inclusion of subjects from different districts. However, the reason why seropositivity in HIV patients from Shanghai was higher than in HIV patients from the other two provinces is unclear.
In China, HIV/AIDS patients with diarrhea are commonly treated with metronidazole, trimethoprim, and sulfamethoxazole, even if pathogenic protozoan parasites are not detected in the stool. Therefore, it is likely that the positive serology may be caused by past infection with E. histolytica.
It is unclear whether HIV infection is a risk factor for E. histolytica infections.10,11,16 However, the higher seropositivity in patients with a CD4+ cell count of < 200/µL in this study suggests that a deficiency in cellular immunity caused by HIV affects the chance of infection with E. histolytica. This observation is in accordance with a study in the United States, which estimated the risk ratios of E. histolytica/E. dispar infection in HIV-infected patients with CD4+ T cell counts of 0–99 and 100–199/µL as 1.7 and 1.5, respectively, compared with HIV patients with a CD4+ T cell count 200/µL.16 It remains unclear how immunodeficiency affects the chance of E. histolytica infection. However, it is likely that patients with lower CD4+ T cell counts may be subjected to more testing or that a lower CD4+ T cell count may reflect an increased accumulated risk for amebiasis caused by reactivation of latent infection or increased susceptibility to new infection.16
We have recently identified Igl on the surface of trophozoites of E. histolytica,17,18 and it has been shown that an ELISA using C-Igl is more specific than an ELISA using crude antigen.7 In this study, the seropositive rate in the ELISA using crude antigen was higher than that in the ELISA using C-Igl. All 17 samples that were positive for C-Igl were also positive with crude antigen. The higher seropositivity to crude antigen than to C-Igl antigen seems to be caused by its lower specificity, as previously reported.7 Therefore, the nine serum samples that were positive with crude antigen but negative with C-Igl may actually be false positives. Overall, our results show that the seroprevalence of E. histolytica infection in HIV/AIDS patients in China was 7.9%, which is a higher rate than in HIV-uninfected individuals. A further study will provide a better understanding of the correlation between these infectious diseases.
Received March 13, 2007. Accepted for publication July 2, 2007.
Acknowledgments: The authors thank Xiaozhang Pan for his encouragement of this study.
Financial support: This work was supported by a grant from SMHB (2001411), a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science, and a grant from the Ministry of Health, Labor and Welfare of Japan.
* Address correspondence to Xunjia Cheng, Department of Medical Microbiology and Parasitology, Fudan University School of Medicine, Shanghai 200032, China. E-mail: xjcheng{at}shmu.edu.cn 
Authors’ addresses: Yi Chen, Bin Yang, and Xunjia Cheng, Department of Medical Microbiology and Parasitology, Fudan University School of Medicine, Shanghai 200032, China, Telephone: 86-21-5423-7359, Fax: 86-21-5423-7122, E-mail: xjcheng{at}shmu.edu.cn. Yunzhi Zhang, Tangkai Qi, and Hongzhou Lu, Department of Infectious Diseases, Huashan Hospital Affiliated to Fudan University, Shanghai 200041, China, Telephone: 86-21-6248-9999. Hiroshi Tachibana, Department of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan, Telephone: 81-463-93-1121, Fax: 81-463-95-5450, E-mail: htachiba{at}iss.icc.u-tokai.ac.jp.
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