Visceral Leishmaniasis-Malaria Coinfection and Their Associated Factors in Patients Attending Metema Hospital, Northwest Ethiopia: Suggestion for Integrated Vector Management

Background Despite high prevalence of visceral leishmaniasis and malaria in the study area, their coinfection remains unknown. Therefore, this study was aimed to document VL-malaria coinfections and their associated factors. Methods A cross-sectional study was conducted among clinical suspected VL patients attending Metema hospital, Northwest Ethiopia, from January 2014 to June 2014. Blood sample was tested by rk39 antigen-based DiaMed IT-Leish dipstick and Giemsa stain microscopic examination of thick and thin blood smears for malaria detection was performed. Result A total of 384 VL suspected patients were included in the study. Out of these, the prevalence of VL was 83 (21.6%) while the prevalence of malaria was 45 (11.7%). Of malaria cases, 40 (89%) were positive for P. falciparum and 5 (11%) positive for P. vivax. The overall prevalence of VL-malaria coinfection was 16 (4.2%). One-hundred eighty (46.9%) study participants have history of travel. Of these, 10 (5.6%) have VL-malaria coinfections. Age less than 5 years was associated with VL-malaria coinfection. Conclusion This study highlights the importance of performing malaria screening amongst VL patients living in malaria-endemic areas, particularly in patients under five years.


Background
Leishmaniasis and malaria are among the most important six diseases on the World Health Organization (WHO) or Tropical Disease Research list. There are 2 million new cases of Leishmaniasis diagnosed every year [1]. As for malaria, 300-660 million people become infected yearly with the malignant Plasmodium falciparum, and 200-300 children are dying every hour from this disease [2,3]. According to the world malaria report 2016, there were about 212 million cases of malaria and 429,000 deaths [4].
Visceral leishmaniasis (VL) is the phlebotomine sand flies borne disease caused by an intracellular protozoan parasite of the Leishmania donovani complex [5], while malaria is Anopheles mosquito borne disease caused by an intracellular protozoan parasite of Plasmodium species [6]. Usually, the transmission of both parasites occurs when the female insect takes a blood meal. Measures to combat these two diseases usually aimed at interrupting the life-cycle of the parasite by destroying the parasite or its vector. Destroying the parasite could be achieved by vaccination or treatment. Destroying the vector could be achieved by many ways such as introducing an enemy of the vector to the environment, using the sterile male technique, destroying the habitat of the vector, or spraying pesticides [7].
Visceral leishmaniasis and malaria are the two major lifethreatening parasitic diseases that still remain a serious public health problem, particularly in endemic areas [8,9]. Differential diagnosis of VL often includes malaria among other febrile splenomegalies, due to its clinical overlap. Malaria, in fact, is widespread in tropical and subtropical regions of the world, where it accounts for more than 250 million cases annually, the vast majority of which occurs among children under 5 years old [10]. Transmission can occur throughout the year or be seasonal, depending on the region [11]. In the latter case, transmission seasons for VL and malaria may not coincide, but the two diseases still overlap, due to the longer incubation period of VL. The overlap in disease distribution suggests the two diseases could cooccur in the same host.
Visceral leishmaniasis and malaria coinfections have been reported across various African and Asian countries, with the prevalence among VL patients ranging from 20.8% and 6.4% in Uganda [12,13] to 10.7% in Sudan [14] and 1.2% in Bangladesh [15] and a rate of 5.9% among Indian patients with fever and splenomegaly [16], with the exception of the case-reports [17][18][19][20], whose evidence remains anecdotal. Despite high prevalence of the VL [21] and malaria [22] in the study area, their coinfection is unknown. Therefore, this study was designed to document the prevalence of VL-malaria coinfection and their associated factors among patients attending Metema hospital.

Study Area and Period.
This study was conducted at Metema hospital from January 2014 to June 2014. Metema is a town in Northwestern Ethiopia, on the border with Sudan. This town is located in the North Gondar Administrative Zone, Amhara region, 897 km North of Addis Ababa and 197 km from the ancient city of Gondar, and it has a latitude and longitude of 12 ∘ 58 N 36 ∘ 12 E with an elevation of 685 meters above sea level. This area is malarious and has cases of VL.

Study Design, Study Population, and Sampling Technique.
A cross-sectional study was carried out at Metema hospital to determine VL-malaria coinfection among all clinically suspected VL patients. Visceral leishmaniasis suspected patients who have taken treatment for malaria and/or VL for the last two weeks were excluded from the study. Visceral leishmaniasis suspects are defined as those individuals who develop clinical evidence of infection usually have fever, loss of appetite (anorexia), weight loss, fatigue, anemia, enlargement (swelling) of the spleen and liver, and abnormal blood tests [23].

Data Collection and Processing.
Well-structured questionnaire was prepared in English version by the research team. It was translated later into the local language, Amharic. The questionnaire addresses patients' sociodemographic information and associated factors. A pretest was conducted among five percent of the total sample size by trained data collectors and any ambiguous questions and repetitive ideas were corrected. Additional response categories were added based on the pretest findings.

Parasitological Techniques.
After interviewing the patients, fresh peripheral whole blood sample was tested by the rk39 ICT with sensitivity of 100% and specificity of 98% [24] for diagnosis of VL, and Giemsa stain microscopic examination of thick and thin blood smears for malaria detection and species differentiation was performed systematically.

Data
Analysis. Data were entered and analyzed using SPSS version 20 software. The Pearson Chi-square test was employed to examine associated factors with the coinfection.
value < 0.05 was considered statistically significant.

Ethical Consideration.
Ethical clearance was obtained from Institutional Review Board of University of Gondar. Written informed consent was obtained from each of the volunteer study subjects or guardian of children. Patient information was anonymized and deidentified prior to analysis. Positive results were given for physicians working in the hospital for treatment according to the national treatment guideline.

Sociodemographic Characteristics.
A total of 384 VL suspected participants were included in this study. Out of these, 334 (87%) were males. The mean (standard deviation) age was 28.1 (11.8) years (ranging from 2 to 78 years). Majority 227 (59.1%) of the study participants were in the age group of 15-29 years old. Among the study groups, 230 (59.9%) were illiterate and 267 (69.5%) were farmers followed by 54 (14.1%) daily laborers. The majority of study participants 234 (60.9%) were from rural residents (Table 1).

Associated Factors for VL-Malaria
Coinfection. From the total study participants, 292 (76%) were used bed nets. Of these, 13 (4.5%) patients were VL-malaria coinfected. Most of the study participants, 217 (56.5%) sleep inside home. One

Discussion
Visceral leishmaniasis and malaria coinfection may exist in endemic areas due to similarity in clinical manifestations of the two diseases but their coinfection has been poorly investigated. In the present study, the overall prevalence of VL-malaria coinfection was 4.2%. This is in agreement with the study which is conducted in India 5.9% among Indian patients with fever and splenomegaly [16]. However, this prevalence was lower as compared to study reported in Uganda 19% [25]. This variation might be due to difference in the VL and malaria prevalence which is 21.6% and 11.7% in this study and 57% and 31% in Uganda, respectively [25]. In this study, prevalence of VL-malaria coinfection was nonsignificantly higher in males than females. A similar finding was also reported in other studies [25][26][27]. The higher prevalence rate might be due to the fact that males engage in activities which make them more prone to infective mosquito and sand fly bites as compared to females who are mostly at home and protected from such infective bites.
Visceral leishmaniasis has been a major health problem in North Gondar, especially in lowlands of Metema and Humera plains, which are important endemic foci in Northwest of Ethiopia [28]. In the present study, its prevalence was 21.6%, which was consistent with the other study done in the same study area, 22.6% [21], however, lower than the study in other areas of Ethiopia which was reported as 39.1% [29]. The variation might be due to heterogeneity of the vectors. Following agricultural development in the region, a large number of labor migrants from the different areas were moved to the endemic areas for crop harvesting. This led to outbreaks of VL, in many areas in the country which resulted in high morbidity and mortality [30].
Malaria is also one of the most public health problems in Ethiopia. The total land mass of the country is regarded as malarious and about 68% of the total population is at risk of malaria infection [31]. In this study, the prevalence of malaria was 11.7%, which is relatively lower than previous study done in the same study area (17%) [22]. The variation might be due to difference in study participants. The predominant Plasmodium species detected was P. falciparum. This was in agreement with other previous studies [22,32,33].
Visceral leishmaniasis-malaria coinfection was significantly higher in patients with the age groups of less than five years ( = 0.004) followed by patients in the age groups of fifteen to twenty-nine years. This indicates that children less than five years old are prone to acquire VLmalaria coinfections which might be associated with their low immune system. On the other hand, high prevalence of VL-malaria coinfection within fifteen to twenty-nine years old might be associated with their daily activities. Farming is extensive in Metema due to the fact that young daily laborers move to Metema from different areas for application of herbicide and for gathering of crops. Because of high temperature in this area, daily activities are accomplished especially during night. This may expose them to the bite of mosquitoes and sand fly which means a more concentrated effort should be given to educate these age groups about this disease and its vector.
In this study, majority of study subjects use their bed nets. However, the prevalence of VL-malaria coinfection was high in these subjects. This might be due to improper and infrequent use of their bed net or it might be due to lack of impregnating their bed nets regularly. In the study area most of study participants who were coinfected with VL-malaria were daily laborers followed by merchants. This might be associated with their daily activities which may expose them for vector bite.
This study is limited to the data obtained from the VL suspected patients, which may reduce the prevalence of malaria. Moreover, this study for VL diagnosis depends only on serological tests. There is no microscopically confirmed parasitological data for VL. To fill all these gaps, there is a need for further study including all febrile patients with different study design.
In conclusion, this study tries to see VL-malaria coinfection for the first time and found 4.2% coinfection rate. A significantly higher number of VL-malaria coinfections were showed in study subjects whose age was less than five years old. Based on these findings, we recommend that malaria screening be implemented for all VL patients who live in malaria-endemic areas in order to promptly initiate antimalarial drug treatment. Moreover, to minimize the disease burden, health planners and administrators need to give intensive health education to increase the community awareness about the two diseases' prevention and control strategies.

Conflicts of Interest
The authors declared that they have no conflicts of interest.

Authors' Contributions
Getachew Ferede participated in conception and design of the study, data collection and analysis, interpretation of the findings, drafting the paper, and the write-up. Ermias Diro, Sisay Getie, Gebeyaw Getnet, and Yegnasew Takele participated in conception and design of the study and interpretations of the findings and reviewed the manuscript. Yitayih Wondimeneh and Anteneh Amsalu participated in conception and design of the study, data analysis, and interpretations of the findings and reviewed manuscript. All authors reviewed and approved the final paper.