Molecular evidence supports the expansion of visceral leishmaniasis towards non-program districts of Nepal

Leishmaniasis, one of the neglected tropical diseases, is caused by a protozoan parasite, Leishmania spp. and transmitted by bite of infected female sandfly (Phlebotomus spp. and Lutzomyia spp.) [1]. Globally, 350 million people from over 98 countries in the tropical and sub-tropical regions are at risk of this disease [2]. Leishmaniasis is categorized as cutaneous, muco-cutaneous and visceral leishmaniasis (VL) based on the parasite species specific clinical manifestations [3, 4]. VL is caused by Leishmania donovani and is the most common clinical presentation in the Indian sub-continent including Nepal [5, 6]. Continuous high grade fever, weight-loss and an enlarged liver and spleen are the most common features of VL patients, and if not treated, case fatality rate may reach as high as 95%, though it can be reduced to < 3% with proper clinical management on time [7]. The disease is now expanding to the newer areas due to rapid urbanization, sharp increase in migration and adaptation of the Leishmania parasite to additional vectors and mammalian hosts [8].

VL has been identified by WHO as a public health problem and target for its elimination (< 1 case/10,000 people/year) from the Indian sub-continent including Nepal has been set for 2020 [9, 10]. As of now, VL is thought to be endemic in 12 districts of the low land Terai region of Nepal. Therefore these 12 districts have been considered as program districts and attain higher priorities for public health support programs in Nepal while the remaining 63 districts are regarded as non-program districts [9, 10]. The recent surge in the number of cases reported from non-program districts has become a serious issue in achieving VL elimination from this region [11‐14]. The main aim of the present study is identification and confirmation of Leishmania spp. by employing a molecular approach in VL suspected patients’ samples from non-program districts of Nepal.

To understand the trend of VL in past 10 years in Nepal, we plotted the number of VL cases from both program and non-program districts (national totals) including the cases from STIDH during 2013 to 2017 (Fig. 3a). The trends of VL cases during this period were clearly different between program and non-program districts of Nepal. In the past 10 years, there was significantly decreasing trend of VL cases in the program districts of Nepal (p = 0.001), while a significantly increasing trend of VL cases was observed in non-program districts (p = 0.002) (Fig. 3b). The national trend for overall incidence of VL also significantly decreased over this time period (p = 0.001). Although the number of cases attended at the STIDH is smaller than the national totals (2013–2017 and study year 2016), the trend observed in this hospital was also quite similar to that of the country’s national scenario. Furthermore, STIDH is the only central specialized hospital for treatment of tropical diseases including leishmaniasis in the country and these 14 cases represent all of the cases at the central hospital (STIDH) over the study period (2016). National reports consist of all the VL cases reported throughout the country (peripheral hospitals, health facilities) including the cases from STIDH.

Out of 14 patients, initial screening was performed by rK39 dipstick test and two patients were required bone marrow aspiration for confirmation (Fig. 1). Two bone marrow aspiration positive cases were also positive by PCR. Out of 14 VL suspected cases, six were positive by rK39, six were negative by rK39 and two cases remained inconclusive for rK39. Only 2 (33.3%) rK39 positive cases were positive by PCR. Sociodemographic, clinical and molecular results are presnted in Table 1. Of the 14 clinically suspected VL cases, 4 (28.6%) samples had the expected band size (approx. 720 bp); the same as the positive control (L. donovani) (Fig. 4). These PCR positive cases were from Chitwan, Dang, Dolpa and Sindhuli districts of Nepal (Fig. 2). A case of VL from Nepal’s mountain region (Dolpa) reported previously [11] was also included in the present study. PCR positive samples were sequenced to determine the identity of the PCR product. The sequences were deposited in GenBank (Accession number: MK803363–65). Using the nucleotide BLAST database search through the NCBI, we found that the PCR positive samples possessed high homology with the L. donovani complex, the causative agent of VL.

Together, our data suggest that - 1) the number of VL cases was found to be increasing in non-program districts of Nepal despite the successful reduction in program districts, and 2) VL cases from these non-program districts were confirmed by a molecular approach (sequencing). It is high time to focus the VL control program in non-program districts to achieve the elimination goals.

All four districts (Chitwan, Dang, Sindhuli and Dolpa) with cases positive by molecular tools are located in the VL non-program districts. Chitwan and Dang districts are located in the lowland areas (250–2500 asl) of Nepal while Sindhuli is in the hilly region (300–3000 asl) and Dolpa is in the mountain region (1500–7500 asl) (Fig. 2). Chitwan district is located along the bank of Narayani river (one of the 3 largest rivers in the country) while Dang is located in the valley. Chitwan and Sindhuli are in an immediate geographical proximity to endemic districts, there will be higher chance to transmit the diseases in those districts due to the movement of people. Although the Government of Nepal, Ministry of Health and Population is committed to eliminate VL by 2020, the rise in incidence of cases from non-program districts could be a major obstacle in achieving the set target [12‐14]. The serial reports of such incidences may be attributed to the possibility of spread from program districts or the presence of VL parasites in non-program districts, and it was largely overlooked in the past. The study on vector dynamics was conducted at two high altitude districts of Nepal [14] and found local VL transmission. But extensive vector and parasite infection studies are yet to be conducted so as to ascertain the presence of parasite in other non-endemic areas with reported VL cases.

Ten years data of VL patients from both program and non-program districts presented in Fig. 3b clearly demonstrates an increasing trend in non-program districts. Increasing developmental projects/improved connectivity and trade/tourism in recent years have posed high risk of VL through the introduction/transmission of vector and Leshmania infected individuals into the non-program districts. And, the outcome is the presence of VL in areas previously thought to be free. VL prevalence is strongly associated with poverty and access to health care facilities in developing countries including Nepal [16]. In Nepal’s rural settings, houses with damp earthen floors are common, an ideal breeding site for sandflies, however data on actual distribution of the vector is lacking. Awareness raising programs and government support reduced the overall VL cases in Nepal [10] though largely overlooked in non-program districts. Global rise in temperature could play a vital role in increased adaptability of the vectors which subsequently increases the habitat area for the vector borne diseases pathogens worldwide [17‐20]. Rising socioeconomic status of local people and their enhanced accessibility to patient diagnosis and parasites detection could also have impacted this incidence. Previously inaccessible places or places without proper health facilities have now improved situation [19‐21] both in private and government sectors. Nevertheless, the increasing case reports is a fact, though the reason behind it might not be explained by a single attribute instead it could be a cumulative outcome of multidimensional circumstances and needs to be properly investigated and addressed.

The VL control program has greatly benefited from rK39 test, however it is inadequate when it comes to elimination phase due to declining positive predictive values. Despite the claimed sensitivity and specificity [22], the rK39 test (antibody detection) has several shortcomings, such as need of expert clinical interpretation, false positive results due to post-recovery antibody retention for years and among residents of VL endemic areas [23], and time required for detectable antibody production delaying the diagnosis. To address this gap, a more specific test is required [24, 25] and PCR is a better alternative due to the following advantages: applicable to varieties of samples at early acute phase helping transmission reduction by timely diagnosis [24, 25], less invasive than bone-marrow aspiration [26, 27], prompt species identification (L. donovani complex) [26, 27], and improved sensitivity/ specificity using buffy coat [28], Although we did not compare the diagnostic performance in our setting due to small sample size, PCR is certainly useful in a country embarking elimination like Nepal where malaria, typhoid, tuberculosis, brucellosis and leptospirosis are co-endemic.

Our study provides important insights into the VL expansion to non-program districts which is strongly supported by use of PCR/sequence homology-based method in VL confirmation. However, due to the limited resources in our exploratory study, we could not collect larger number of samples and carry out necessary diagnostic tests for more conclusive results. Similarly, distribution of vector and parasites prevalence in all non-program districts was not studied. Not all the patients underwent bone marrow aspiration for diagnosis since the rK39 had discordant results with PCR for some patients. A small amount of blood was collected on filter paper and used for DNA extraction which might compromise the DNA concentration although we did not confirm it.

With the rising number of VL cases reported from non-program districts of Nepal, it has become imperative for formulating new strategies that can overcome the present hassle of elimination obstacles as well as prevent an impending epidemic in low to high mountainous areas. Therefore, these findings should be considered as a signal for inception of a robust strategic plan for accommodation of non-program districts in the elimination program. Moreover, innovative strategies including molecular investigation should also be institutionalized to understand the origin and spread of these strains especially at this pre-elimination phase of VL.