Title Field study on the distribution of Entamoeba histolytica and Entamoeba dispar in the northern Philippines as detected by the polymerase chain reaction

We used the polymerase chain reaction (PCR) to study the distribution of Entamoeba histolytica and E. dispar in 1,872 individuals in 14 communities in the northern Philippines. Here we report a field study using a DNA extraction protocol from formalin-fixed stool specimens as previously reported. This assay detected 137 stools (7.318%) containing E. dispar and 18 stools (0.961%) containing E. histolytica. The most affected age group for E. histolytica/E. dispar infections were those 5–14 years of age. There was no significant difference in the sex distribution of E. histolytica, while in the case of E. dispar, a higher prevalence was observed in females (9.186%) than in males (5.731%) (P , 0.01). An apparent clustering of stool-positive cases of E. histolytica and E. dispar was also observed in the northern part of the study area. The results of this survey demonstrate that E. dispar is highly prevalent in the communities studied. Moreover, it offers promise for the PCR using DNA extracted from formalin-fixed stools as a sensitive epidemiologic tool for detecting E. histolytica and E. dispar infections. The intestinal protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. Entamoeba histolytica is responsible for up to 100,000 deaths per year, placing it second only to malaria in mortality due to protozoan parasites.1 The infection is common in developing countries and predominantly affects individuals with poor socioeconomic conditions, nonhygienic practices, and malnutrition. A number of epidemiologic studies of E. histolytica infection were performed before the clear distinction of two separate species, E. dispar and E. histolytica, was established.2 Because they are morphologically indistinguishable, studies based on stool surveys have raised the question on the validity of most of these studies. Moreover, seroepidemiologic studies that were carried out in a number of endemic countries3–8 usually reflected the seroprevalence of the disease even in the near past. Also, the major problem with current serologic test results is that they remain positive for years after an episode of amebiasis. To date, the standardization of antigen preparation and the relative measurement of positive cut-off antibody titers for use in serodiagnosis still pose a problem. In recent years, a number of methods such as isoenzyme typing,9 DNA probes for the polymerase chain reaction (PCR) and hybridization,10–13 restriction pattern analysis,14–19 and monoclonal antibodies20–24 have been developed for the clear distinction of the two species. Clearly, accurate diagnostic tools are required for the clinical and public health management of the disease. The use of a stool ELISA has recently been shown to be useful in routine diagnostic procedure and epidemiologic studies.25,26 However, a comparative study on the use of the ELISA and PCR for the detection of E. histolytica and E. dispar indicated that the PCR was more advantageous than the ELISA in epidemiologic studies.27 It is therefore important to assess the usefulness of the PCR in accumulating data on the prevalence of E. histolytica and E. dispar in the field that are more accurate. We previously reported that a successful extraction of DNA from formalin-fixed stool samples for the PCR is a useful tool for rapid diagnosis of both E. histolytica and E. dispar infections.28 Here we report the application of this method to document the prevalence of the two species in a number of communities in the northern Philippines. Our preliminary hospital-based studies showed that amebic diarrheal diseases in Baguio City and nearby communities in the northern Philippines are frequent; however, their prevalence and distribution are not well-characterized. We therefore conducted a large-scale field study to determine the distribution of E. histolytica and E. dispar in these communities. MATERIALS AND METHODS Study sites. The study was conducted during the months of December 1995, January and December 1996, and January 1997 in the communities (locally called barangays) within and nearby Baguio City, The Philippines (Figure 1). This study area belongs to the Cordillera Autonomous Region and is the highest city in The Philippines, with an average altitude of 1,500 meters above sea level. Three major land forms characterize the relief features of the city: valley, plateau, and steep slopes. Throughout the year, the weather is cool but is coolest during the months of December to February with an average temperature of 188C. Housing is inadequate in the city, given a rapid population increase and inadequate area for settlement expansion aside from poor economic conditions. Ninety-nine percent of the total number of households in the city have access to potable water. Only 0.95% of the total population have doubtful water sources, most of which are places where water pipes are inaccessible in view of the high elevation of the city. Collection of samples. The day before the scheduled sample collection, the research team visited the randomly selected households, explained the purpose of the study and the requirements for sample collection, and distributed specimen cups. Particular care was taken in explaining the need to label the specimen cups with name, age, and sex and to avoid sample contact with soil and with samples from other individuals. Informed consent was obtained from all individuals. This protocol was approved by the Ethical Committees for Human Studies of the Institute of Tropical Medicine at Nagasaki University and Baguio General Hospital and Med917 E. HISTOLYTICA AND E. DISPAR IN THE NORTHERN PHILIPPINES FIGURE 1. Map of Baguio City, The Philippines, showing the study areas. ical Center (Baguio City, The Philippines). The samples were then collected the next morning and immediately transported to the laboratory. Each sample was macroscopically inspected for its consistency and for the presence of soil contamination, blood, or mucus. Routine microscopic diagnosis of parasites was then performed. Microscopic examination of parasites. The presence of parasites was determined by microscopic examination of fresh stools and formalin-ether concentrated specimens. Genomic DNA extraction and the PCR. Genomic DNA was extracted from cysts present in formalin-fixed stool specimens as previously described28 with slight modifications. Briefly, the pellet resulting from the sedimentation procedure was washed four times with phosphate-buffered saline. The pellet from the last wash was resuspended in TE buffer (100 mM Tris, pH 8.0, and 25mM EDTA) and subjected to freezing (using dry ice and ethanol for 5 min) and thawing (at 378C in a water bath for 2 min) six times. After the last treatment, the solution was mixed with 200 ml of 0.2% Triton-X 100 and then heated in 988C water bath for 10 min. The mixture was then incubated with 200 mg/ml of proteinase K in lysis buffer (100 mM Tris, pH 8.0, 1% sodium dodecyl sulfate, 25 mM EDTA) at 808C for 2 hr, and the DNA was extracted using the routine phenol-chloroform extraction procedure.29 The PCR was then carried out using primers specific for E. histolytica and E. dispar (p11 plus 918 RIVERA AND OTHERS