Plasmodium Falciparum and Schistosoma Mansoni Coinfection and the Side Benefit of Artemether-lumefantrine in Malaria Patients

Introduction: The distribution of both malaria and schistosomiasis exhibits a large geographical overlap in tropical environments, particularly in sub-Saharan Africa. This part of the world currently harbours more than 85% of the estimated global burden of these diseases. Studies showed that artemisinin derivatives used for the treatment of malaria also have an antischistosomal effect. This study aimed to investigate the extent of malaria-schistosomiasis co-infection and the antischistosomal effect of artemether-lumefantrine when administered to treat falciparum malaria in Kemise, Northeast Ethiopia. Methodology: Stool samples were collected from 152 microscopically confirmed malaria patients and diagnosed for schistosomiasis using the Kato-Katz technique before treatment. The schistosomiasis cure rate and egg reduction were determined in co-infected patients, who were treated with artemether-lumefantrine,. Results: Twenty-eight out of 152 malaria patients were co-infected (18.4%, n = 152) with schistosomiasis. All 28 co-infected patients were found stool-negative for Schistosoma mansoni eggs four weeks after treatment. The extent of co-infection was associated with age, sex and educational level. Cure rate and egg reduction rate following the treatment of artemether-lumefantrine were 100%. Conclusion: The co-infection rate was associated with patient characteristics. Artemether-lumefantrine was effective against S. mansoni in co-infected patient. Multicenter and randomized trials, however, are needed for a better understanding of the efficacy of artemether-lumefantrine against schistosome infection with ranges of intensity.


Introduction
Malaria remains a challenge to both public health and socio-economic development, particularly in sub-Saharan African countries [1].In Ethiopia, an estimated 65% of the population is reported to be at risk of acquiring malaria infection [2].In malaria endemic areas, co-infection with multiple parasites, including Schistosoma species, is common [3,4].
In Kemise, surveys have been conducted on intestinal parasite and malaria coinfection [5,6].Even though malaria and schistosomiasis have been geographically co-endemic in Kemise as they are in many places in sub-Saharan Africa, studies on the prevalence of schistosome and P. falciparum coinfection is still limited.The present study therefore attempts to report the level of malaria and schistosomiasis co-infection.
Praziquantel is the drug of choice for the treatment of schistosomiasis.Although no resistance to praziquantel has been described, low cure rates of schistosomiasis have been reported [7].Isolates from the resistant infections in Egypt are less susceptible to praziquantel-induced tegumental damage in vitro, suggesting that the worms are in some way less responsive to the drug [8,9].Studies controversially indicated that resistance to praziquantel may be emerging.Higher treatment coverage in the framework of the roll-out of the neglected tropical diseases control program are expended to increase evolutionary pressure and hasten the emergence of resistance in Schistosoma species populations [10].
Efforts have been made by drug researchers to obtain alternatives to praziquantel, and artemisinin derivatives have found to be active against S. mansoni using in vivo and in vitro tests [11,12].In an explorative study in Sudan investigating the antischistosomal effect of artemether-lumefantrine in patients taking the drug for the management of malaria, all the patients were found to be stoolnegative for schistosome eggs after treatment [13].
In Ethiopia, artemisinin based combination therapy is the drug of choice for the treatment of uncomplicated falciparum malaria.Particularly, artemether-lumefantrine has been used in Ethiopia since 2006.In addition to malaria-causing plasmodium species, artemether has been proved to have activity against several species of schistosomes [14].In areas where malaria and schistosomiasis are co-endemic, the artemisinins taken by malaria patients may also exhibit antischistosomal properties though little is known about the impact of the co-infection state on the drug response [15].
The aim of the present work was to investigate the extent of co-infection of P. falciparum malaria and schistosomiasis, and investigate the effect of artemether-lumefantrine against schistosomiasis in the co-infected patients attending Kemise Health Center.This study aimed to determine the side benefits of artemether-lumefantrine based therapy in falciparum malaria patients co-infected with S. mansoni.

Study area and population
The study was conducted at Kemise Health Center in Kemise town, northeast Ethiopia.The town is in the Oromia Zone of the Amhara Region.Kemise is located at 325 km northeast of Addis Ababa, the capital.It has an altitude of 1424 m above sea level.The town has an estimated total population of 18,897, of whom 10,151 are males and 8,746 are females [15].
In Kemise, malaria transmission is seasonal and unstable [16].The town is situated in an area where water collections are potential sites for the breading of anopheline mosquito larvae and infected snails, resulting in a high level of both malaria and schistosomiasis burden in the community.

Study subjects
Study participants, who fulfilled the inclusion criteria, were enrolled in the study for a follow-up period of one month.Patients above 5 years old, coinfected with P. falciparum and S. mansoni, were included in the study.

Study design
A prospective longitudinal study was conducted at Kemise Health Center, in Northeast Ethiopia during the high malaria transmission season, i.e. from September to November, 2009.The study was designed to determine the extent of P. falciparum and schistosomiasis confection and the effect of artemether-lumfantrine against S. mansoni in coinfected patients at a dosage regimen used for the treatment of P. falciparum malaria.As the study was non-comparative, randomization was not performed.

Sample size determination
The required sample size for the study was calculated using a formula for a single population proportion.We assumed 0.9% malaria prevalence [5].
Taking critical value at 95% confidence level (Zα/ 2 = 1.96) and degree of precision 0.05, the minimum sample size was 138.Ten percent was added to account for patients who were likely to be lost during follow-up, withdraw, or have incomplete records.The minimum desired sample size for the study was 152 patients with uncomplicated P. falciparum malaria.

Inclusion and exclusion criteria
The inclusion criteria were patients above five years old with uncomplicated P. falciparum malaria, diagnosed to have patent schistosome infection, mild P. falciparum, absence of danger signs or signs of severe and complicated falciparum malaria according to the definition given by World Health Organization, and absence of febrile conditions caused by diseases other than malaria.The exclusion criteria were pregnancy, infection with species other than P. falciparum, age younger than five years, complicated/severe P. falciparum malaria on screening/at study entry, praziquantel treatment before entry into the study, or severe clinical signs/symptom of the disease such as ascites and hepatosplenomegaly.

Detection of malaria parasite
Blood smears were prepared from each clinical malaria case to detect malaria parasites.These smears were then stained using the Giemsa staining method [17].Each stained smear was then examined for detection of P. falciparum using light microscope.

Detection of schistosome infection
As soon as a malaria case was confirmed microscopically, a stool sample was collected from the patient for S. mansoni examination.Stool samples were examined for the presence of schistosome eggs using the standard Kato-Katz technique for three consecutive days [17].After treatment with artemether-lumefantrine, stool samples of the coinfected cases were examined using the Kato-Katz method at days 28, 29 and 30 [18].In an attempt to improve the sensitivity of Kato-Katz technique, duplicate Kato-Katz thick smears were prepared from each sample collected for three consecutive days [19,20].The World Health Organization cut of value [light 1 to 99 eggs per gram (epg), moderate 100 to 399 epg and heavy > 400 epg] was used for classification of intensity of S. mansoni infection.

Treatment of malaria and schistosomiasis co-infected patients
Microscopically confirmed patients with P. falciparum and S. mansoni co-infection as well as uncomplicated P. falciparum malaria patients were treated with artemether-lumefantrine per national guidelines.

Study variables
The independent variables for this study were socio-demographic characteristics (age, weight, and educational status) and the artemether-lumefantrine intervention.The dependent variables were coinfection rate, intensity of schistosome infection, and treatment outcome.

Data processing and management
The laboratory results and clinical examinations were recorded for each patient.Intensity of infection of schistosomiasis was expressed as epg.Cure rate was expressed as the percentage of S. mansoni and P. falciparum co-infected patients who became S. mansoni ova negative after treatment.

Data quality control
Patients were recruited by trained clinical research assistants, including health officers and nurses.Both clinical and laboratory examinations were conducted on patients presenting with signs and symptoms of malaria.

Data analysis
SPSS version 15 software (SPSS IBM, Chicago, Ill, USA) was used to analyze data.Pearson's  2 test and paired t-test were used for statistical analysis of categorical and continuous data, respectively.The a priori statistical significance level was 0.05.Egg reduction rate was assessed by the change in geometric mean egg count per gram (GMEC) and calculated as [1-(GMEC after treatment/GMEC before treatment)] x 100.Treatment outcome was expressed as cure "for patients whose status changed from schistosome eggpositive to negative after treatment," and non-cure "for those whose status remained schistosome egg-positive after treatment."

Ethical considerations
The study protocol was submitted to the Institutional Review Board of the Faculty of Medicine, Addis Ababa University, and obtained ethical clearance to conduct this study.Before enrollment, written and informed consent was obtained from each adult patient and from the guardians/parents of each child.Illiterate participants had the written and informed consent after they chose a literate witness to read the consent document; agreement of illiterate participants was indicated by their thumb prints on the written consent form.
The cure rate after treatment was calculated as the percentages of individuals who had tested as schistosome egg free after treatment.The results showed that both the cure rate and the percentage of egg reduction were 100% at the end of the follow-up period (Table 3).Though the findings for the malarial infection response are being prepared for publication, all the 28 co-infected patients were cured from their uncomplicated P. falciparum malaria.

Discussion
The current study revealed that Kemise town is coendemic for S. mansoni and falciparum malaria with a co-infection rate of 18.4%, which is similar to the rate reported in an investigation from Tanzania (22.6%) [21].Males have been shown to have higher coinfection rates compared with females.The observed differences among males and females might be exposure related.
School-aged children are one of the high-risk groups for the overlap of two infections [22].The current findings also revealed that the 6-to 14-yearold group (school-aged children) had the highest coinfection rate.In the current study, the co-infection rate decreased as age and educational level increased.This inverse relationship might be due to awareness in avoiding any exposure to the two disease-causing agents, Plasmodium and Schistosoma species, through minimizing contact with infected water bodies and proper use of bed nets.The other possible age-specific reason could be that children have incompetent immunity against malaria and schistosomiasis.The association between age and co-infection rate has also been reported somewhere else [21,23].
The demographic variables (age, gender and educational level) considered in the study were not significantly associated with the intensity of S. mansoni infection.These findings, except the association between age and intensity of infection in one report, are not unusual and have been previously described in other settings [24,25].
Artemether-lumefantrine was introduced in Ethiopia for the treatment of P. falciparum infection in 2006.From experimental studies, artemisinin    derivatives act against other parasites, as well as against tumor cells [26].Hence it is cogent to investigate the clinical side benefits of artemisinins when used in the treatment of malaria.The present study revealed that all co-infected patients became S. mansoni egg negative following artemetherlumefantrine therapy at doses used for the treatment of malaria.This is comparable to the results of a study conducted in Sudan, in which malaria patients treated with artemether-lumefantrine were negative for S. mansoni eggs after a month check-up [13].In addition, artemether alone showed significant prophylactic action against S. mansoni in the Côte d'Ivoire study [27].
The cure rate of schistosomiasis in the present study was 100%, unlike the treatment outcome of the Kenya study that was based on patients with a high intensity of S. mansoni infection [28].The results of the current study may be due to the light schistosome infection of the participants, as our findings and those reported elsewhere have shown that artemisinins act better against light infections [13,29,30].

Conclusion
Demographic characteristics (gender, educational level, and age) influence the P. falciparum and S. mansoni co-infection rate in Kemise.Artemetherlumefantrine is highly effective against schistosomiasis in uncomplicated falciparum malaria co-infected patients at a dose used for the treatment of falciparum malaria.Artemether-lumefantrine may make possible the use of a single regimen to treat patients with S. mansoni and falciparum malaria coinfection.However, before the use of artemetherlumefantrine alone in the treatment of schistosomiasis in malaria co-infected patients, there is a need for multicenter and randomized trial evidence on the range of S. mansoni infection level.

aP
= 0.010 for the comparison of co-infection in males and females b P = 0.008 for the comparison of co-infection in age groups c P = 0.024 for the comparison of co-infection in educational level d P = 0.177 for the comparison of co-infection in rural residents and urban

Table 1 .
Extent of Schistosoma and falciparum malaria co-infection among patients Kemise Health Center, Oromia Zone of Amhara Region, December 2009

Table 2 .
Schistosoma mansoni egg load of malaria patients attending in Kemise Health Center, Oromia Zone of Amhara Region, December 2009

Table 3 .
Effect of artemether-lumfantrine against S. mansoni in falciparum malaria co-infected patients