Impact of two rounds of mass drug administration using diethylcarbamazine combined with albendazole on the prevalence of Brugia timori and of intestinal helminths on Alor Island, Indonesia.

BACKGROUND
Annual mass drug administration (MDA) using diethylcarbamizine (DEC, 6 mg/kg) combined with albendazole (alb, 400 mg) is recommended by the Global Programme to Eliminate Lymphatic Filariasis (GPELF). This strategy has been shown to be efficient in the of control bancroftian filariasis, but data on brugian filariasis as well as on the positive side effects on intestinal helminths are lacking.


METHODS
The effect of one selective treatment and two rounds of MDA using DEC and alb on the prevalence and intensity of Brugia timori infection were studied on Alor island using a cross-sectional and a cohort approach. Before the campaign and ten months after each treatment cycle microfilariae (mf) were assessed by filtration of night blood. Before and ten months after MDA, stool samples were collected and the prevalence of intestinal helminths were determined.


RESULTS
In all, the mf-rate dropped from 26.8% before any treatment to 3.8% following the second MDA. Almost all mf-positive, treated individuals showed very low mf densities. The crude prevalence of hookworm dropped from 25.3% to 5.9%. The reduction of prevalence of Ascaris lumbricoides (32.3% to 27.6%) and Trichuris trichiura (9.4% to 8.9%) was less pronounced. Within a cohort of 226 individuals, which was examined annually, the prevalence of A. lumbricoides dropped from 43.8% to 26.5% and of T. trichiura from 12.8% to 6.6%. The results indicate that this MDA approach reduces not only the mf prevalence of B. timori but also the prevalence of hookworm and to a lesser extent also of A. lumbricoides and T. trichiura.


CONCLUSION
The MDA using DEC and alb as recommended by GPELF is extremely effective for areas with brugian filariasis. The beneficial effect of MDA on intestinal helminths may strengthen the national programme to eliminate lymphatic filariasis in Indonesia and may set resources free which are otherwise used for deworming campaigns of schoolchildren.


Background
Lymphatic filariasis (LF) has been targeted by the World Health Organization for elimination as a public health problem by the year 2020 [1,2]. It is caused by three species of filarial parasites: Wuchereria bancrofti infects about 115 million people in Africa, India and other tropical and subtropical areas, whereas Brugia malayi infects about 13 million people in south India and south-east Asia and it is replaced by its sibling species Brugia timori in eastern Indonesia and Timor-Leste [3]. In Asia, the key strategy of the Global Programme to Eliminate LF (GPELF) is an annual mass drug adminstration (MDA) of all individuals at risk of infection with a single annual dose of diethylcarbamazine (DEC) combined with albendazole (alb) for at least four to five subsequent years [2,4]. This approach has been shown to reduce microfilaraemia of W. bancrofti and B. malayi efficiently [5][6][7]. Studies in areas endemic for W. bancrofti also indicate that the treatment with DEC combined with alb has the additional long-term beneficial effect of reducing prevalence and intensity of infection with intestinal helminths such as Ascaris lumbricoides, hookworms and Trichuris trichiura [8][9][10][11].
In 2001, The Department of Health of the Indonesian Government decided to participate in the GPELF. Although Indonesia has a long history in filariasis control programmes [12], filariasis is still in many areas a large public health problem and the new strategy recommended by GPELF was never evaluated. Therefore, studies were initiated to investigate the efficacy of a single dose DEC combined with alb to control W. bancrofti and B. timori infections on Alor island [13]. The treatment was judged to be efficient and safe enough to be employed in an MDA approach and an area endemic for B. timori was selected for annual follow-up studies [13][14][15].
In the present study we examined the prevalence of B. timori microfilaraemia following one selective treatment and two annual rounds of MDA using DEC combined with alb in a highland village of Alor island. We assessed the prevalence of intestinal helminths before MDA and its impact on the prevalence of the most common intestinal helminths, A. lumbricoides, hookworms and T. trichiura. We provide data on the effectiveness of two annual single doses of DEC and alb treatment to control B. timori and intestinal helminths.

Study area
The study was performed in Mainang village on Alor island (East Nusa Tenggara Timor, Indonesia). This village was first characterized in April 2001, and it was found to have a prevalence of B. timori microfilaraemia of about 25%. [16]. About 40% of the inhabitants showed signs of infection and 80% presented IgG4 antibodies reactive with a recombinant B. malayi antigen, BmR1 [16,17]. Anopheles barbirostris mosquitoes were identified as vectors for B. timori [18]. An animal reservoir for this Brugia species is not known and wild-life as well as domestic animals were rare in the study village. Before treatment intestinal helminths were common, especially A. lumbricoides (prevalence 2002, 32.3%) and hookworms (prevalence 2002, 25.3%) as well as T. trichuria (prevalence 2002, 9.4%). In the study area a few individuals received diethylcarbamazine (DEC) before 1990 and no anthelminthics were commercially available on the island. However, a deworming campaign was performed in schoolchildren at irregular intervals using generic benzimidazole derivates before the year 2000. In the village about 60% of eligible children attended school.

Sample collection
Originally, the study was planned as a cohort-study with annual re-examinations. Since the most appropriate travel period to Alor is also harvesting season, many farmers were absent from their home during the re-examinations. Therefore, in 2001Therefore, in , 2002Therefore, in , 2003 and 2004 between 37 and 51% of the total resident population (about 1,500 individuals) were examined and data were analysed using a cross-sectional approach. years) in the three surveys which included stool collection. Data of these individuals were also analysed separately as a cohort.
In May 2001 about 200 selected individuals with microfilariae (mf) and/or lymphoedema received a single dose of DEC (6 mg/kg) combined with alb (400 mg) [13]. This number represented about 40% of the total number of individuals with mf and/or lymphoedema in the study area. A second survey was performed in April/May 2002 to assess the long-term efficacy of the filariasis treatment in selected individuals. The treatment results of these mfpositive individuals were reported previously [13,14]. For comparison their data were also included in this report. In addition, during the 2002 survey, baseline data on the prevalence of intestinal helminths were collected. Reexamination following MDA was carried out from April to May 2003 and from March to April 2004. Inhabitants of the endemic villages were called by their local health workers to a central place, usually the Puskesmas (primary health center). For each volunteer, sex, age and name were noted and after a brief clinical examination venous blood Filaria Journal 2005, 4:5 http://www.filariajournal.com/content/4/1/5 was collected between 7.00 p.m. and 11 p.m. One labeled stool container was provided to each individual participating in the study and collected one day later by local health workers.
The participants came from three different residential quarters of Mainang, Welai Selatan, Malaipea and Tominuku, but no significant differences with regard to the prevalence of B. timori or intestinal helminths were found [16]. All individuals over the age of two years were asked to participate in the study. Informed consent was obtained from all adults or, in the case of children, from their parents. The study was approved by the ethical board of the University of Indonesia, Jakarta. Following the registration, the individuals were examined by experienced physicians for clinical signs of lymphatic filariasis. During the survey patients with lymphoedma were introduced to hygiene of affected legs and other procedures which may help to stop the progression or alleviate symptoms of their disease.

Mass drug administration
In May and June 2002 and 2003, after each survey, a community based MDA using a single dose of DEC and alb for the entire eligible population was performed. Pregnant or breastfeeding women, children younger than two years or persons suffering from acute illness were not treated. A medication regimen based on the classification of age rather than on weight was used (Table 1), since this was found to facilitate treatment.
The MDA of the rest of the island was conducted by the District Health Authority, which claims a coverage rate (number of distributed doses per number of residents) of 78%. Staff from the GTZ performed MDA in 6 randomly selected B. timori and/or W. bancrofti endemic villages with a total population of about 6,000 inhabitants and introduced a developed educational programme [19]. For MDA a mixed approach was used, involving both, the primary health care system and the community members directly. Health staff and volunteers were trained for correct drug distribution and documentation as well as side effect recognition and its treatment. The impact of this campaign was tested by using KAP (knowledge, attitudes, practice) surveys before and after the intervention [19]. In this project a MDA coverage rate of 75% was achieved, whereas 15% of the population, according to WHO guidelines, were not eligible for treatment. Besides the effect of treatment the educational programme has increased the knowledge about filariasis. In our study village Mainang in April 2003 a compliance rate (number of persons who reported to have taken the drug per number of residents) for the 2002 MDA of 67% was determined (n = 772).

Assessment of microfilariae
For the identification of mf-positive individuals, 1 ml of anticoagulated blood (EDTA) was filtered through a polycarbonate membrane with a 5 µm pore size (Millipore, Eschborn, Germany). Subsequently mineral water was passed through the membrane for blood cell lysis. The membrane was placed on a slide, air-dried and fixed with methanol. Following Giemsa staining, slides were airdried, examined microscopically using 100-fold magnification and mf were counted.

Assessment of helminth eggs
In the field, the Harada-Mori hatching test was used to detect living hookworm larvae. Briefly about 0.5 g fresh stool was spread on a wet filter paper and placed with 1 ml water in a specially designed plastic bag. The bags were placed upright in a window, covered by paper and incubated for 6 to 10 days. The samples were examined microscopically for the presence of hookworm larvae at a 63fold magnification. The rest of the stool samples were preserved in the field using 4% formaldehyde. In the laboratory in Jakarta, 1-2 g of stool was examined by the formalin/ether enrichment method for the presence of helminth eggs. The most prevalent geohelminths, A. lumbricoides, hookworm and T. trichiura were analysed in this study, since other species such as Hymenolepis spp. and Strongyloides stercoralis were only found in a few cases.
In the baseline survey in 2002 the Kato-Katz smear was used to assess the helminth eggs quantitatively. Compared to the enrichment method and the Harada-Mori test the Kato Katz technique had a poor sensitivity and this method was not applied in the following years. In 2002 the number of eggs per gram (epg) in infected individuals as determined by the Kato Katz smear was usually relatively low with medians for A. lumbricoides, hookworms and T. trichiura of 3,500 epg, 50 epg and 75 epg, respectively. For the enrichment method and the Harada Mori test, the numbers of helminth eggs or hookworm larvae were scored as follows: low density (1-10 eggs per slide or 1-50 hookworm larvae per plastic bag), moderate density (11-100 eggs or 51-500 larvae) or high density (more than 100 eggs or 500 larvae). Usually, scoring data are in good agreement with results obtained by the Kato Katz Smear [20].

Statistical analysis
EpiInfo 2002 Revision2 was used for documentation and analysis of the data. As index for the mf density within a study group the geometric mean was used. For the estimation of the community mf load (CMFL) a log (x+1) transformation was used. Data on distribution and density of mf were compared using the chi-square test or the Mann-Whitney U test.

Brugia timori
In the years 2001 to 2004 a total of 586, 769, 768, and 704 individuals, respectively, were examined for the presence of mf ( In order to determine the dynamics of the mf-status during the investigation period, the mf prevalence in a cohort of 145 individuals, who participated in all four annual surveys, was determined (      (Fig. 3A).
In contrast to these crude data, a significant decrease of A. lumbricoides prevalence was observed in a cohort of 226 individuals, who were examined in 2002, 2003 and 2004 (Fig. 4A). In these three years the prevalence dropped in this cohort from 43.8% to 28.3% and to 26.5%. This is a reduction of 39.7% after two rounds of MDA with a compliance rate of 86% and 95%. http://www.filariajournal.com/content/4/1/5 g stool, whereas before treatment 50% of the examined individuals had 3,500 eggs /g or more.

Hookworms
The crude prevalence of hookworm infection decreased from 25.3% in 2002 to 8.2% in 2003 and the 5.9% in 2004 (Table 3). This is an average reduction of prevalence of 76.7%. In children the rate of infection dropped from 19.3 to 7.2% in 2003 and remained low with 8.2% in 2004, while in the other age groups the decrease of prevalence continued also in 2004 (Fig. 3B). No difference in hookworm reduction was observed in male and female individuals. In the cohort of 226 individuals with full annual data sets, the prevalence dropped from 2002 to 2004 from 34.5% to 17.3% and 14.2%, respectively. This equals following two rounds of MDA within this time course to a reduction of 59% (Fig. 4B). In 2003, 20 new infections were observed and 59 individuals became hookworm negative. In 2004, 25 new infections were detected, while 32 persons turned negative. This corresponds to an average rate of new infections of 13.5% and an average clearance rate of about 80%. The mean egg count before MDA was already relatively low, with about 65% of individuals with light infections of less than 2,000 eggs /g. In 2003 and 2004 the intensity of infection was even lower with more than 90% having light infections. The results from the formol/ether enrichment methods were in principal confirmed by the data obtained from Harada Mori culture, although in a few cases the hatching test was more sensitive.

Trichuris trichiura
The crude prevalence of T. trichiura was almost stable from 2002 to 2004 with only a non-significant decrease from 9.4% to 8.7% and to 8.9%, respectively ( Table 3). The prevalence of infected children under the age ten years increased only slightly from 9.6% in 2002 to 11.8% in 2003, but dropped to 7.6% in 2004 (Fig. 3C). A more pronounced reduction of prevalence was observed in the cohort of 226 individuals, who where examined in all three years. In this group the prevalence was in 2002, 12.8% and a year later it was 10.2% and in 2004 it was only 6.6%. This is a significant reduction over two years of 48.4%.

Discussion
This study shows the effect of one selective treatment and two rounds of MDA using DEC combined with alb on the most prevalent helminths, B. timori, A. lumbricoides, hookworms and T. trichiura in a community on Alor island. The results confirm and show for the first time at the com-   http://www.filariajournal.com/content/4/1/5 munity level, that the MDA approach as recommended by the GPELF is highly effective at reducing the prevalence and the intensity of infection of B. timori. Furthermore, a positive impact on the prevalence, especially of hookworm infection, but also of A. lumbricoides and of T. trichiura, was observed. The data support the hypothesis that B. timori is an excellent candidate for elimination, while the campaign will also have a large impact on the reduction of intestinal helminths [15]. B. timori occurs only east of the Wallace line in Indonesia and Timor-Leste. This restricted distribution makes this species not only a good candidate for local elimination, but B. timori may also be a prime candidate for eradication of lymphatic filariae [15,21].
B. timori is closely related to B. malayi and tools and strategies developed to support the elimination of B. malayi infection may apply for both species [13][14][15]17,18]. It has been shown that the combination of a single annual dose of DEC combined with alb is very efficient in the control of brugian filariasis and a higher efficacy of this regimen has been suggested as compared to bancroftian filariasis [6,13,14]. The successful control of B. timori on parts of Flores island using multiple doses of DEC has been reported already some decades ago [12]. However, this strategy has caused many logistical problems and was never extended to the remaining parts of Flores and other islands. The present study proved the principle that a single annual dose of DEC combined with alb is highly suitable to control and most probably to eliminate B. timori infection. Before treatment, among a cohort of 145 individuals, 45 were mf-positive with a geometric mean mf density of almost 150 mf/ml. Three years later, following one selective treatment and two rounds of MDA in this group only 3 individuals were found to be mf-positive, who had mf densities of 1-2 mf/ml.
Our data show that a larger percentage of mf-positive individuals claimed not to have participated in MDA, compared to the average compliance rate. It can be concluded that a high compliance rate is necessary for reducing the mf prevalence to levels under which transmission cannot be sustained. In addition, a few individuals who received no treatment still had high mf densities. If these individuals participate in the next rounds of MDA, side effects may occur and affected individuals may spread their problems in the community which eventually reduce compliance. However, extensive health information campaigns can help to ensure high compliance rates [19].
On the community level not much data exists about the control of brugian filariasis by annual MDA using DEC combined with alb. For the control of W. bancrofti infection in Asia a number of extensive field studies were published using annual MDA with DEC alone or in combination with alb. Results from Papua New Guinea indicate promising prospects for elimination [22,23]. In India, after six rounds of a single dose treatment with DEC the prevalence of microfilaraemics was reduced by 86% and the mf density by 91% [7]. Computer models predict that in this area a further decline of mf prevalence will occur even after the cessation of MDA [24]. In this study (B.timori), similar reductions of prevalence and mf density were observed after only one selective treatment and two rounds of MDA. It is possible that models would also predict a further decline of mf prevalence for our study, but epidemiological parameters differ largely between W. bancrofti infections in Pondicherry (India) and B. timori infections on Alor island and further studies on the dynamics of B. timori control are needed. In addition, it has been discussed for W. bancrofti in the pacific area that MDA should be accompanied by local vector control [25].
Our results showed that MDA using a combination of DEC and alb also has an impact on the reduction of intestinal nematode infections. In the highland village examined the original prevalence and intensity of infections with A. lumbricoides, hookworms and T. trichiura was relatively low. This is in agreement with previous surveys on Alor and on other islands of volcanic origin in east Nusa Tenggara Timur [26,27]. Although, both hookworm species occur in eastern Indonesia, data from Flores indicate that Necator americanus, may be the prominent species [27].
The strongest reduction of prevalence following two rounds of MDA among the intestinal helminths was observed in hookworm infections, in both, the cross-sectional group of an average of 600 individuals and a cohort of 226 individuals. Although a large number of re-infections occurred, the crude prevalence dropped from 25.3% to 5.9% and in the cohort from 34.5% to 14.2%, ten months after the second round of MDA. This equals a reduction of 76.7% and 58.8%, respectively. The hookworm prevalence is usually reduced by about 80% shortly after treatment with alb [28,29]. Although DEC alone may reduce the output of hookworm eggs, it is assumed that it has no influence on its prevalence [30]. In our study we observed a large number of hookworm re-or new infections. In 13.5% (2003) and 14.0% (2004) of the cohort new infections were observed. From Java, an even higher re-infection rate with N. americanus of about 50% one year after anthelminthic treatment was reported [31]. Despite of the occurrence of re-and new infections, the drop in hookworm prevalence can be explained by the relatively short survival time of hookworm larvae in the environment as compared to the mean survival time of eggs of A. lumbricoides and of T. trichiura.
Following the first round of MDA the crude prevalence of A. lumbricoides dropped from 32.2% to 22.1%, but after the second round it was 27.6%. More consistently were the results in the cohort. Before MDA the prevalence was 43.3%, following the first round it was 28.3% and following the second round it was 26.5%. Albendazole is very effective against A. lumbricoides and median cure rates are over 95% [28,29,32]. DEC alone has a minor therapeutic effect on Ascaris [10,33]. Although some treated individuals may expel adult worms after DEC, the overall prevalence of infection may be not affected [30]. These observations are confirmed by other studies, which show that DEC alone has no significant impact on A. lumbricoides, but the combination of DEC with alb has relevant cure and egg reduction rates [34]. As in other intestinal helminths, re-and new infections occur regularly, and the time point of re-examination is critical. In our cohort we observed an annual rate of new infections of 25%. Children have an especially high risk for re-or new-infections and show a lower decrease in worm burden compared to adults [35]. In another study it was observed that eight months after treatment 55% of children were re-infected [36]. Re-infection with A. lumbricoides may return six months after treatment to almost 90% of the pre-treatment prevalence and worm density may drop to about 75% [35].
The crude prevalence of T. trichiura in the community before and after MDA was almost identical, ranging between 9.4% and 8.7%. However, following two rounds of MDA the prevalence dropped in the cohort from 12.8% to 6.6%. Although re-and new infections occurred, it is important to note that in 2004 most new infections were observed in those individuals which were negative for T. trichiura for the previous two years. Trichuris trichiura is known to be only poorly sensitive to albendazole and the reported reduction rates for alb range between 38% and 47.7% [28,29,32]. From Sri Lanka a cure rate of T. trichiura of 43.6% and an egg reduction rate of 70.3% was reported [37]. The combination of DEC with alb showed different results, ranging from no significant impact on the prevalence but with significant egg reduction of 79.4% one week after treatment, to a cure rate of 81.6% and an egg reduction of 84% [10,34]. The study from Sri Lanka reported that this drug combination has a cure rate of 30% and an egg reduction rate of 70% [36].
For areas endemic for W. bancrofti there are an increasing number of studies which show the positive effect of filariasis control using MDA with DEC combined with alb on the reduction of intestinal helminths [8][9][10][11]38]. The results of the present study can extend this observation to areas endemic for Brugia infections. Although it is unlikely that MDA as used for filariasis elimination will eliminate intestinal helminths from most areas, a reduction may be achieved to levels which may cause no significant morbidity. Other intervention strategies, such as for example the development of a hookworm vaccine [39], may take advantage of reduced prevalences in order to achieve a long-lasting elimination of intestinal helminths, as it has been accomplished in most industrialised countries. In areas with filariasis control by the MDA using DEC combined with alb, separate de-worming campaigns for school-age children may become superfluous. This could set available resources free which can then be used to support MDA. Co-ordination is needed within the local health administration to use the limited funds more efficiently. The present study showed that MDA using DEC combined with alb is effective to control B. timori and that this has also impact on the reduction of geohelminths.

Conclusion
Annual MDA using DEC in combination with alb is highly effective in the control of B. timori infection and has a positive impact on the reduction of intestinal helminths. Given a high compliance rate the strategy can lead to elimination of B. timori on Alor and on other islands in Indonesia.