Report on the Schistosomiasis Control Program in Cambodia
SUMMARY OF ACTIVITY
1) Epidemiological survey in Kratie Province
We surveyed the prevalence of schistosomiasis mekongi in Kratie Province, Cambodia, between 25 April and 8 May 2003. We performed seroepidemiological studies, examined stools, performed ultrasonographic examinations, and collected host snails.
Seroepidemiological studies were conducted in six villages along the Mekong River. We used rapid-ELISA with Schistosoma mekongi egg antigen. Positive rates in children were 25.7% at Achen, 45.1% at Kompong Krabei, 59.4% at Kbal Chuor, 31.1% at Sambok, 8.8% at Talous, and 13.5% at Hanchey Leu.
We examined stools by the Kato-Katz method in two low endemic villages, Talous (n = 149) and Hanchey Leu (n = 101). No positive cases were found. Next we examined stools by a formalin-detergent technique in Talous (n = 143), Hanchey Leu (n = 22), and Achen (n = 3). The 22 individuals from Hanchey Leu were chosen because they had high values in the rapid-ELISA. S. mekongi eggs were found in one individual at Talous and in two at Hanchey Leu. This result indicates the usefulness of using rapid-ELISA as a first screening test in schistosomiasis control.
In ultrasonographic examinations we found differences in the frequency and severity of symptoms between villages showing high and low endemicity. This result indicates that ultrasonography is useful for morbidity studies of schistosomiasis mekongi.
We collected around 1000 intermediate host snails, Neotricula aperta, at Klokor in Kratie Province. The snails were brought back to Japan and maintained in the Department of Tropical Medicine and Parasitology, Dokkyo University School of Medicine. They have been used for maintaining S. mekongi in the laboratory.
2) Use of SMP (sodium metaperiodate)-ELISA for serodiagnosis of schistosomiasis mekongi
We tried the SMP-ELISA technique for serodiagnosis of schistosomiasis mekongi. SMP-ELISA is an improved ELISA technique designed to reduce cross-reaction in serodiagnosis of schistosomiasis mansoni (Alarcon de Noya, et al., 2000). We found that SMP-ELISA reduces cross-reaction in serodiagnosis of schistosomiasis mekongi also.
3) Technology transfer of serodiagnostic technique to National Malaria Centre (CNM) in Phnom Penh
From 12 to 26 January 2004, four CNM staff learned our ELISA technique for serodiagnosis for schistosomiasis mekongi under our guidance.
Schistosomiasis mekongi, caused by the blood-living parasite Schistosoma mekongi, is a serious health problem in the lower Mekong River basin. Until recently, little has been known about the prevalence of the disease in Cambodia, although more than thirty years has passed since the discovery of the disease in 1968 at Kratie town, in the northeast of the country. We have been conducting a series of surveys of schistosomiasis mekongi in Cambodia since 1997 in collaboration with the National Malaria Centre (CNM), Ministry of Health, Cambodia, for the purpose of clarifying its epidemiological status. Our results have revealed high endemicity mainly in the upstream regions of the Mekong River and in Kratie Province, and decreasing endemicity in the downstream direction. Schistosomiasis control in Cambodia has been based on large-scale mass treatment operation since 1995. National Malaria Centre (CNM), covering all the disease endemic areas and resulted in a dramatic fall in the prevalence of the infection from 70% to 5%. In this report, stool examinations were also applied to the same study subjects as those seen in the seroepidemiological studies (Table). Only a few schoolchildren were found to be schistosome egg-positive in spite of the seroepidemiological results showing rather high positivity in each study area. This operation may have been responsible for the remarkable decrease in the number of egg-positive patients.
1) Epidemiological survey in Kratie Province: Seroepidemiological studies and stool examination were conducted to determine the disease endemicity in villages along the Mekong River and to identify individuals suspected of having schistosomiasis. Abdominal ultrasonographic examination was performed to establish a standard for diagnosis of the disease from ultrasonographic images.
2) Use of SMP-ELISA for serodiagnosis of schistosomiasis mekongi: The object of the study was the reduction of false-positive results by serodiagnosis. SMP-ELISA was designed to reduce non-specific cross-reactions by oxidization of polysaccharide residues in antigen molecules with sodium metaperiodate (SMP).
3) Technology transfer of serodiagnostic technique to National Malaria Centre (CNM) in Phnom Penh: The object of the mission was to establish a laboratory system at the CNM and to teach the staff to perform serodiagnosis of schistosomiasis mekongi.
The epidemiological survey team comprised Dr. Muth Sinuon, Dr. Suon Seila, and two medical technicians from the CNM; Professor Hajime Matsuda and Dr. Masashi Kirinoki from Dokkyo University School of Medicine; and Dr. Hiroshi Ohmae from Institute of Basic Medical Science, University of Tsukuba, Japan. The team received technical support from local staff of the Provincial Health Department of Kratie Province.
Dr. Masashi Kirinoki taught the ELISA technique to staff of the CNM.
The epidemiological survey was conducted from 25 April to 5 May 2003 as follows:
25 April: Briefing at CNM, Phnom Penh
28 April: Briefing at Kratie Provincial Health Department
28 April-3 May: Epidemiological survey in Kratie Province
4 May: Snail collection
5 May: Briefing at CNM, Phnom Penh.
The serodiagnostic method was taught at CNM from 12 to 26 January 2004.
ACTIVITIES AND RESULTS
1. Epidemiological survey in Kratie Province
The seroepidemiological survey was conducted in six villages. We have used the rapid-ELISA method for field surveys since 2002. This method was developed in our laboratory to produce results from a simple protocol in a short time in the field. This method has three advantages: 1) finger pricking produces enough blood; 2) it uses whole blood samples; 3) and results are available in around 1 hour in either the village or the laboratory. The results are shown in Table 1 . Positive rates in children in each village were 13.5% (12/89) at Hanchey Leu, 8.8% (12/136) at Talous, 31.1% (32/103) at Sambok, 59.4% (60/101) at Kbal Chuor, 45.1% (23/51) at Kompong Krabei, and 25.7% (27/105) at Achen. The distribution of ELISA values in each village are shown by box plots in Figure 1, and the locations of target villages and positive rates are indicated in Figure 2. In comparison with a previous study, serological positive rates in Kratie Province are tending to decrease. The results of rapid-ELISA were significantly correlated with those of conventional ELISA.
We examined stools by the Kato-Katz method in Hanchey Leu (n = 101) and Talous (n = 149), but found no positive cases. We also examined stools by the formalin-detergent technique in three villages. The results are shown in Table 2. The 22 individuals from Hanchey Leu were chosen because they had high values in the rapid-ELISA. Positive rates were 9.1% (2/22) at Hanchey Leu, 0.7% (1/143) at Talous, and 0% (0/3) at Achen. The fact that 2 positives were found in the 22 selected individuals at Hanchey Leu indicates the usefulness of using rapid-ELISA as a first screening test for schistosomiasis in low endemic areas or in endemic areas after mass treatment with drugs.
Around 1000 intermediate host snails, Neotricula aperta, were collected at Klokor in Kratie Province. The snails were brought back to Japan and maintained in the Department of Tropical Medicine and Parasitology, Dokkyo University School of Medicine. They have been used for maintaining S. mekongi in the laboratory.
2. Use of SMP-ELISA for serodiagnosis of schistosomiasis mekongi
Serodiagnosis of infectious diseases by ELISA has some limitations because of false-positive results due to immunological cross-reaction with other factors. Since polysaccharide molecules in glycoproteins might be main target antigens for cross-reaction, Alarcon de Noya et al. (2000)* pretreated the antigens with sodium metaperiodate (SMP) to oxidize the polysaccharide molecules. SMP-ELISA reduced cross-reaction in immunodiagnosis of schistosomiasis mansoni. In the present study, we tried the SMP-ELISA technique for serodiagnosis of schistosomiasis mekongi. Sera from 34 individuals with schistosomiasis (positive for S. mekongi egg antigen), sera from 100 children in Phnom Penh, where no schistosome infection has been reported, and sera from 25 individuals in Japan were subjected to ELISA using S. mekongi soluble egg antigen with or without SMP treatment. Distributions of ELISA values are shown in Figure 3. The ranges of ELISA values of the patients and the children in Phnom Penh overlapped in ELISA without SMP treatment, but they were separated in SMP-ELISA. This result indicates an advantage of SMP-ELISA in specificity of immunodiagnosis.
In a previous epidemiological study in Cambodia, we used ELISA with S. japonicum egg antigen and found a decrease of disease endemicity. To confirm the result, we examined serum samples from four villages, including previously collected sera, by SMP-ELISA with S. mekongi egg antigen. Sera collected from 1998 to 2003 had been kept at -80 ℃ and were thawed just before use. The distribution of ELISA values in each village is shown by box plots in Figure 4.
3. Technology transfer of serodiagnostic technique to CNM
Practice classes in our serodiagnosis technique were conducted from 12 to 26 January 2004. Equipment and chemicals were purchased by WHO, and some equipment was donated by Dokkyo University School of Medicine. Four CNM staff (one nurse and three laboratory technicians) learned the ELISA protocol, SMP treatment, and preparation of blood samples. Although they are now competent, more practice and more laboratory equipments are required.
4. Morbidity study using ultrasonography in schistosomiasis mekongi endemic area of Cambodia
4-1. Study area and methods
To compare morbidity due to schistosomiasis mekongi, we conducted ultrasonographic (US) examinations in two village groups in a schistosomiasis mekongi endemic area in Kratie Province. The high endemic village group consists of Achen, Kompong Krabei, and Ampilteuk; the low endemic village is Chhlong (Table 3). The 483 inhabitants (366 in high endemic villages, 117 in Chhlong) had an age range of 6-64 years. Yokogawa medical (RT-220) ultrasonographic equipment was used in this study. All US images were analyzed and classified according to international criteria (Ultrasound in schistosomiasis: A practical guide to standardized use of ultrasonography for the assessment of schistosomiasis morbidity). The severity of portal hypertension was classified as mild, moderate, or severe.
4-2. Results−Evaluation of morbidity due to schistosomiasis mekongi by ultrasound
In the high endemic village group, out of 366 inhabitants who underwent US examinations, 139 (38%) showed dilatation of the portal vein (Table 4). Five inhabitants showed ascites and 107 (29%) showed collateral vessels. In Chhlong, out of 117 inhabitants, 10 showed dilatation of the portal vein, none showed ascites, and two showed collateral vessels.
In the high endemic village group, the frequency and severity of portal hypertension were high in the 15 to 34 age group (Table 5). They were low in people younger than 15 years and in people older than 45 years. This relationship between portal hypertension and age was not found in the low endemic village (Table 6).
The clinical manifestations of S. mekongi infection are similar to those of S. japonicum and S. mansoni infections. The primary lesion in S. mekongi infection is a granulomatous reaction to the deposited ova. This lesion leads to the development of periportal fibrosis and portal hypertension. As the disease develops, the liver becomes hard and shrunken. In the advanced stage, patients present with hematemesis and/or signs or symptoms of splenomegaly, ascites, edema, and spider angiomas.
In S. japonicum infection, the fibrotic lesions are detected in both the periportal area and the liver parenchyma. The parenchymal lesions can be detected as a characteristic septum-formation-like network or "fishscales" by US. Most US changes due to S. mekongi infection are similar to those of S. mansoni infection. In S. mekongi infection, however, the typical network pattern of S. japonicum infection has not been found. To perform further morbidity studies of S. mekongi infection, we need specific standardization of US diagnosis.
In the 1990s, splenomegaly due to S. mekongi infection was reported to be more severe than that due to S. japonicum infection; and among young adults, larger splenomegaly with collateral vessels was frequently detected by US. In our study, however, we did not find severe portal hypertension with collateral vessels and/or ascites among young people in either high or low endemic villages.
As the prevalence of S. mekongi infection has decreased with mass treatment, the intensity and morbidity due to the infection have changed. Especially in young people, US images show improvements. In low endemic areas, the sensitivity of the Kato-Katz stool examination is not high, and morbidity due to schistosomiasis cannot be easily detected by clinical symptoms. The results of our seroimmunological studies using ELISA and morbidity studies using US in endemic areas in Cambodia since 1997 have been incorporated into the schistosomiasis control activities. As the next step, adequate indicators such as seroimmunological or US examination will be necessary to monitor changes of endemicity and morbidity.
(* Alarcon de Noya et al., Exp. Parasitol., 95, 106-112, 2000)