Molecular detection of Leishmania infantum DNA and host blood meal identification in Phlebotomus in a hypoendemic focus of human leishmaniasis in northern Algeria

Abstract

BackgroundLeishmania parasites are transmitted by female phlebotomine sand flies that maintain the enzootic cycle by circulating between sylvatic and domestic mammals. Humans are part of this cycle as accidental hosts due to the vector’s search for a source of blood. In Algeria, Human Leishmaniases (HL) are endemic and represent a serious public health problem because of their high annual incidence and their spread across the country. The aim of this study is to identify sand fly species fauna (vectors of Leishmania), determine their infection rate and identify their feeding preferences using molecular tools in a hypoendemic focus of HL located in the province of Tipaza, northern Algeria.Methodology/Principal findingsAn entomological survey using CDC light traps was conducted between July and October of 2015 in four HL affected peri-urban locations in the province of Tipaza, northern Algeria. Sand flies were identified using the morphological criteria of the genitalia for the males and spermathecae for the females. Leishmania DNA was detected in pooled female sand flies (N = 81 pools with 8–10 specimens per pool) using quantitative real-time polymerase chain reaction (qPCR) targeting two different genes: kDNA-PCR and 18S rRNA. To identify their blood meal sources, blood-fed female sand flies were analyzed by PCR-sequencing targeting the vertebrate cytochrome c oxidase I (COI) gene. A total of 4,045 sand flies were caught, of which 3,727 specimens were morphologically identified. Seven species were recorded: P. (L.) perniciosus (50.28%), P. (L.) perfiliewi (26.13%), P. (L.) longicuspis (21.92%), Sergentomyia (S.) minuta (0.85%), P. (P.) papatasi (0.42%), P. (L.) langeroni (0.32%) and P. (L.) ariasi (0.05%). Afterwards, 740 female specimens were randomly selected and divided into 81 pools and were then screened to investigate the presence of Leishmania spp. L. infantum DNA was detected in three pools, corresponding to three sand fly specimens (one each). The infection rate was 0.33% (2/600) for P. (L.) perniciosus and 2.56% (1/39) for P. (L.) perfiliewi. Analysis of the blood feeding sources (N = 88 specimens) revealed that sand flies belonging to Larroussius subgenera, mainly (71.5%) feed on small ruminants. Human blood is the second feeding source (17%), eight specimens (9%) were found to feed on equines and no domestic reservoir (dog) blood was found.Conclusions/SignificanceThe presence of human leishmaniasis cases, the high abundance of Phlebotomus (Larroussius) species which are proven or suspected vectors of L. infantum, and the detection of L. infantum DNA from its natural vectors (P. (L.) perniciosus, P. (L.) perfiliewi), in addition to the blood-feeding of positive females for L. infantum on humans blood, prove that the major elements of the epidemiological transmission cycle of L. infantum are present and indicate risk factors for an outbreak of the disease in the province of Tipaza.

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

“…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.…”

“…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.…”

Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies

“…Several methodologies to identify blood meal in sand flies have been used, including enzyme-linked immunosorbent assay (ELISA), mass spectrometry, precipitin test and polymerase chain reaction (PCR) [6][7][8][9][10][11]. More recently, quantitative real-time PCR [12,13] and PCR followed by amplicon sequencing [14] demonstrated promising results, with high-level sensitivity. Although all these methods generated important information about the feeding behaviour of sand flies, they may present several drawbacks, such as low sensitivity and specificity (e.g.…”

Molecular xenomonitoring and host identification of Leishmania sand fly vectors in a Mediterranean periurban wildlife park

“…Selecting appropriate methods for accurate diagnosis has been the focus of much attention. Several genes have been successfully used including mitochondrial cytochrome b and cytochrome c oxidase subunit I (COI), 12S and 16S rRNA genes (Alcaide et al, ; Bennai et al, ; Branco et al, ; Bravo‐Barriga et al, ; Cotteaux‐Lautard et al, ; González et al, ; González, Jiménez, Hernández, Martín‐Martín, & Molina, ; Jiménez et al, ; Maia et al, ; Valinsky, Ettinger, Bar‐Gal, & Orshan, ) and the nuclear prepronociceptin (PNOC) gene (Haouas et al, ; Jaouadi et al, ). Moreover, molecular diagnosis of L. infantum infection indicates that many of vertebrate species may become infected with the parasite, but their ability to transmit infection back to the vector and therefore their potential contribution to the epidemiology of infection has not been proven in most cases (Quinnell & Courtenay, ; Tomassone et al, ).…”