Phlebotomine sand flies and Leishmania species in a focus of cutaneous leishmaniasis in Algeria

Cutaneous leishmaniasis is a disease caused by various Leishmania spp., which are transmitted by phlebotomine sand flies. Algeria is one of the most affected countries, with thousands of cutaneous leishmaniasis cases registered every year. From March to November of 2016 and 2017, sand flies were collected in 12 municipalities in Setif province, North-Eastern Algeria. Sand flies were identified and females were tested by PCR for detecting Leishmania DNA. Additionally, cutaneous leishmaniasis cases notified during the study period were analysed. Out of 1804 sand flies collected, 1737 were identified as belonging to seven species, with Phlebotomus perniciosus (76.2%), Ph. papatasi (16.7%) and Ph. sergenti (5.0%) being the most common species, representing together 97.9% of the collected specimens. The remaining specimens were identified as Sergentomyia minuta, Se. fallax, Ph. longicuspis and Ph. perfiliewi. The number of sand flies collected monthly was positively correlated with temperature. Out of 804 females tested, nine Ph. perniciosus (1.1%) scored positive for Leishmania infantum (n = 5), L. major (n = 3) and L. tropica (n = 1), respectively. During the study period, 34 cutaneous leishmaniasis cases were notified in Setif, of which 58.8% were patients residing in two urban and peri-urban municipalities and 41.2% in rural areas. The finding of Ph. perniciosus as the most abundant species in Setif suggests that this sand fly may be adapted to different biotopes in the North-East region of Algeria. The detection of different Leishmania spp. in Ph. perniciosus suggests a complex epidemiological picture of cutaneous leishmaniasis in Setif, with the involvement of different etiological agents and possibly with different reservoir hosts and vectors.

The geographical distribution of CL and VL is closely associated to the sand fly population structure and distribution [9,10]. In Algeria, 24 sand fly species of the genera Phlebotomus and Sergentomyia have been identified, with Phlebotomus perniciosus, Ph. perfiliewi, Ph. longicuspis, Ph. papatasi and Ph. sergenti being proven or suspected vectors of Leishmania spp. to humans [11,12]. Nonetheless, data about the environmental factors that may affect the dispersion and population dynamics of sand flies, are still poor understood in some areas of this country. This is the case of Setif province, where 1288 cases of human CL and 501 of VL have been reported to the public health authorities from 1993 to 2017. The present study focused on: i) the evaluation of the diversity and seasonality of sand flies in rural and urban areas and ii) detection of Leishmania spp. DNA in sand fly of Setif province. We also analysed data on CL cases notified in Setif during the study period.

Study area
Sand flies were collected in Setif (36˚11'29"N, 5˚24'34"E), a semi-arid region belonging to the highland of North-Eastern Algeria ( Table 1, Fig 1), with an extension of 6550 km 2 and 1,489,979 inhabitants. This area is characterized by hot and dry summer (from the end of May until the beginning of October) with an average annual precipitation of 402 mm. The monthly average temperature reaches 5.8˚C in January and 24.8˚C in July, with monthly average relative humidity of 37% in July and of 79.1% in December. The monthly average speed of the wind is 2.9 m/s, with highest values (7.4 m/s) being recorded during April and May.

Collection and identification of sand flies
Sand flies were collected from March to November 2016 and 2017, in 12 municipalities of Setif (Table 1, Fig 1). Twenty collection sites were selected, including animal stables (n = 17), an old building (n = 1), a forest (n = 1), a discharge of domestic waste (n = 1), a discharge of water waste (n = 1), and a water treatment station (n = 1) ( Table 1, Fig 2). Sand flies were collected using 651 sticky paper (15 cm × 27 cm, covering up to 52.76 m 2 ) and CDC lights traps as described [13,14]. CDC light traps were installed monthly, for 41 nights during the two collection seasons from 7:30 pm to 5:00 am. Collections were carried out once a month using sticky paper traps until the appearance of the first sand fly and then every 5 ± 2 days until their total disappearance. All specimens were preserved in labelled glass vials containing 70% ethanol and subsequently processed for morphological identification, using taxonomic keys and descriptions [15][16][17][18].

Molecular detection of Leishmania spp. in female sand flies
Genomic DNA was extracted from thorax and abdomen (heads and last segments were removed for morphological identification) of female sand flies (n = 804) using a commercial kit (DNeasy Blood & Tissue Kit, Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. Detection of Leishmania spp. DNA was performed by conventional PCR using primers LGITSF2 (5'-GCATGCCATATTCTCAGTGTC-3') and LGITSR2 (5'-GGCCAACGCGAAGTTGAATTC-3') targeting partial region of the rRNA internal transcribed spacer 2 (ITS2, from 372 to 450 bp) [20]. Each PCR reaction consisted of 4 μl of sand fly genomic DNA and 46 μl of PCR mix containing 2.5 mM MgCl2, 10 mM Tris-HCl (pH 8.3),   Amplified products were examined on 2% agarose gels stained with GelRed (VWR International PBI, Milan, Italy) and visualized on a GelLogic 100 gel documentation system (Kodak, New York, USA). Amplicons were purified and sequenced in both directions using the same primers as for PCR, employing the Big Dye Terminator v.3.1 chemistry in an automated sequencer (3130 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA). The ITS2 Leishmania sequences were aligned using the ClustalW program [21] and compared with those available in GenBank using the BLASTn tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
The phylogenetic relationships of Leishmania spp. were inferred by Maximum Likelihood (ML) method based on the Tamura 3-parameter model [22] with Invariant sites (I), selected by best-fit model [23]. Evolutionary analysis was tested with 8000 bootstrap replications, using MEGA6 software [24]. The phylogenetic analysis was run using ITS2 reference sequences of Leishamania species available in GenBank. The sequence from Trypanosoma evansi (Accession number LC199491) was used as outgroup.

Human cases
To assess the relationship occurring between the diversity of vector populations and the risk of human infection, CL cases notified in Setif province were included in the study. From 2016 to 2017, data regarding patients (i.e., sex, age and place of residence) were obtained from the Direction of Health and Population (DHP) of Setif. Data were obtained and processed anonymously, with the authorization of the head of the prevention department from the same direction (Ref. 1173/HPD/PD/2019).

Meteorological data
From March to November 2016 and 2017, temperature (C˚), relative humidity (%), rainfall (mm) and wind speed (m/s) data were obtained from National Office of Meteorology (ONM) of Setif. These data are firstly registered in the auxiliary station located in Ain Oulmen (site G), then reported to the central station situated in the Setif International Airport (World Meteorological Organization code: 60445). Temperature, relative humidity and wind speed were registered daily every 3 hours, whilst precipitation data were recorded twice per day. The ombrothermic diagram [25] has been established according to monthly mean temperature and relative humidity, based on average data recorded during 2016 and 2017 (Fig 3).
Sand fly species dominance was estimated by the relative abundance (RA %), expressed by the ratio between number of specimens for each species and the total number of specimens caught ×100 [27]. Sex ratio was calculated by dividing the number of females by the number of males. Lilliefors test was used to test the normality of the data. The total number of specimens for each species collected monthly and the number of females and males according to species were compared using non-parametric tests (Kruskal-Wallis H-test, Mann-Whitney U-test) respectively. Spearman's (rs) correlation coefficients were used to assess the relationship between the number of sand flies and meteorological variables (i.e., average monthly temperature, relative humidity, rainfall and wind speed). Statistical analyses were performed using IBM SPSS statistics 23 rd version for Windows 10. Differences were considered statistically significant when P<0.05.   Table 2). Out of 20 collection sites, eight (i.e., A1, A2, A3, B1, B2, C1, C2, E) and three (i.e., A1, A2, B1) sites located in rural areas were positive for sand flies in 2016 and 2017, respectively ( Table 2). No urban and peri-urban area scored positive for sand flies during the study period. All sand fly-positive sites were located in mountainous areas in the extreme south of Setif, neighbouring north Saharan regions. The highest number of taxa and RA of specimens were registered in sites with animal shelters for the first year of collection, with a slight increasing of RA in second year for Ph. papatasi (98.1%) and Ph. perfiliewi (100%) from site A and for Ph. perniciosus (28.28%), Ph. sergenti (47.8%) and Se. minuta (60%) from site B.
The number of specimens for each species collected monthly vary significantly in 2016 (Kruskal-Wallis H-test, H = 13.78, df = 6, P = 0.03) or in 2017 (Kruskal-Wallis H-test, H = 13.24, df = 6, P = 0.03). Sand flies were present during the summertime period (April-October), but not during the winter period (November-March) (Fig 4). In particular, sand flies were collected from the middle of June and until the first days of November in 2016, peaking in August, when average monthly temperature and humidity of 24.4˚C and 42.6% were registered. In 2017, sand flies occurred from the middle of July to the end of November with two peaks in August (average monthly temperature and relative humidity of 27.3˚C and 38.8%, respectively) and in September (average monthly temperature and relative humidity 20.2˚C and 50.5%, respectively). Number of sand flies collected in 2016 and 2017, mean daily temperature and relative humidity recorded at each day of collection are reported in supplementary tables (S1 and S2 Tables).
The number of females was lower than males with an overall female:male ratio of 0.89, with the exclusion of Ph. longicuspis, Se. minuta, Se. fallax, for which a higher number of females specimens were collected ( Table 2). The number of females and males did not vary significantly according to species (Mann-Whitney U-test, U = 22.5, n 1 = 7, n 2 = 7, P = 0.80).
Out of 801 females tested, nine Ph. perniciosus (1.1%) collected from two municipalities (A1, A2) scored positive for Leishmania spp. DNA. In particular, five specimens were positive for L. infantum, three for L. major and one for L. tropica (Table 3).
Reflecting the genetic identification, the phylogram of ITS2 sequences grouped each Leishmania species examined in three paraphyletic clades including those of each corresponding reference Leishmania spp. (Fig 5).

Discussion
A high degree of sand fly species richness was recorded in Setif, with a higher relative abundance registered in rural areas of two municipalities (i.e., Boutaleb and Hamma), where sheep, goat and horses were the most common animals found. Even if no significant variations in terms of meteorological variables (i.e., temperature, relative humidity, rainfall and wind speed) were recorded between 2016 and 2017 in Setif, the spatio-temporal dynamics and the abundance of sand flies changed between the both seasons. Indeed, a decrease in number of positive collection sites and of sand fly species and abundance was recorded during the second year. This may be related to the relatively low abundance of sand flies collected during the whole study period. On the other hand, the absence of sand flies recorded for urban and peri-urban municipalities could also be due to the chemical treatment (deltamethrin) applied in these sites (i.e., A2, A3, B2 and C) during May and June 2017. However further studies, perhaps using local data loggers, would be valuable to confirm these analyses, which were done using data from meteorological stations. Overall, of 24 sand fly species present in Algeria [15,28], seven (i.e., Ph. perniciosus, Ph. papatasi, Ph. sergenti, Ph. longicuspis, Ph. perfiliewi, Se. minuta and Se. fallax) were identified in the present study, with most of them being found in sympatry in different municipalities. A similar sand fly population structure has been observed also in the northern/north-eastern and middle-eastern regions of Algeria, where up to ten sand fly species were identified (i.e., Biskra region) [27,29,30].
The occurrence of Ph. perniciosus as the most abundant species may be partly explained by the capacity of this sand fly to adapt to different environments such as stables and human houses. In addition, the high abundance of Ph. perniciosus was probably due to its endo-exophilic and anthropozoophilic behaviour [11,27,31] and because it is well adapted to the zones having from sub-humid to semi-arid climate [15,29]. A similar abundance (up to 77%) for this sand fly species was also recorded in other provinces in Northern and North-Eastern Algeria [11,28,31]. Phlebotomus papatasi was the second most common species collected. This finding is in contrast with those obtained from other studies carried out in North and North-Eastern Algeria where frequencies of 0.2% and 1.2% for Ph. papatasi were registered in Constantine, Tipaza and Tizi-Ouzou provinces, respectively [11,31,32]. The abundance of Ph. papatasi in Setif may be explain by its ability to adapt to semi-arid climate. Indeed, it is well known that Ph. papatasi, an anthropophilic and endophilic species, is typical of Saharan and arid areas and rarely collected in semi-arid regions [8,15,31].
The finding of Ph. sergenti with a frequency of 5% is in line with data from previous studies conducted in north and north-east regions of Algeria, where a relative frequency of 4.8% was registered [8,29,32,33]. The relatively low frequency of this species in Setif may be due to the semi-arid climate of the region, as Ph. sergenti is a species which typically occurs in Saharan and arid zones and lives in wall crevices, rodent burrows and rocky areas [15]. On the other hand, the low numbers of Ph. longicuspis and Ph. perfiliewi, which occurred during the whole study period, may be due to the unsuitable environmental conditions and/or host presence [15,28,29].
The finding of Se. minuta and Se. fallax with relative abundances of up 62.5% and 100%, respectively (see Table 2), agrees with previous data revealing that Sergentomyia species are abundant in all the Algerian territory and have adapted in all the bioclimatic areas, from semiarid and Saharan to sub-humid and humid bioclimatic stages [27,34,35].
Considering that the presence of a competent vector is a determinant factor for the local circulation of a given vector-borne pathogen, the retrieval of proven vectors in different areas of Setif may explain the local circulation in this province of different Leishmania spp., whose genetic identification was supported by the phylogenetic analysis. Furthermore, the presence of Ph. sergenti and Ph. papatasi, the proven vectors of L. tropica and L. major [36,37], suggest the circulation of these parasites in this region. This was reinforced by the detection of L. tropica and L. major DNA in Ph. perniciosus. Noteworthy, the mere detection of L. tropica and L. major DNA does not imply that Ph. perniciosus is a vector. The detection of different Leishmania spp. in non-competent vectors has been previously reported [38][39][40].
Even if no sand flies were collected in urban and peri-urban areas of Setif, CL cases have been registered in these areas. However, the possibility that these CL patients may have acquired the infection in rural environments cannot be ruled out. Indeed, most of the 34 cases of CL herein analysed were young males between 10 and 30-years old, employed in agricultural activities, especially during the summer, and consequently exposed at high risk of sand fly bites.
The high number of CL cases registered in Algeria may be explained by the extension of classical foci and by the emergence of new foci across the country [26,41,42]. The occurrence of different bioclimatic zones (e.g., Mediterranean climate in the north and humid and semiarid zones in the southern Sahara), the desertification in the steppe of the northern Sahara, and population movements from endemic to non-endemic areas (and vice versa) have likely contributed to the increase in the number of CL cases in Algeria in the past decades [6,29,32,33,43,44].
This study confirms the presence of different proven vectors of Leishmania spp. (e.g., Ph. perniciosus, Ph. papatasi and Ph. sergenti) in several municipalities of Setif, highlighting the risk of infection in humans and animals by three different Leishmania species (i.e., L. infantum, L. major and L. tropica). Sand flies were positively correlated with temperature and public health authorities should benefit from this information to establish optimized vector control strategies in Setif and surrounding risk areas in Algeria.