Open Access

Cutaneous leishmaniasis in northwestern Saudi Arabia: identification of sand fly fauna and parasites

  • Najoua Haouas1Email author,
  • Omar Amer2,
  • Fawwaz Freih Alshammri3,
  • Shorooq Al-Shammari2,
  • Latifa Remadi1 and
  • Ibrahim Ashankyty2
Parasites & Vectors201710:544

https://doi.org/10.1186/s13071-017-2497-6

Received: 4 June 2017

Accepted: 25 October 2017

Published: 2 November 2017

Abstract

Background

Cutaneous leishmaniasis (CL) is a vector-borne disease transmitted by the bite of an infected sand fly. This disease is highly prevalent in Saudi Arabia where Leishmania major and L. tropica are the etiological agents. In the region of Hail, northwestern of Saudi Arabia, the incidence is about 183 cases/year. However, the epidemiology of the disease in this area is not well understood. Thus, an epidemiological survey was conducted in 2015–2016 to identify the circulating parasite and the sand fly fauna in the region of Hail. Skin lesion scrapings were collected from suspected patients with CL.

Methods

The diagnosis was made by microscopic examination of Giemsa-stained smear and PCR. The parasite was identified by PCR and sequencing of the single copy putative translation initiation factor alpha subunit gene. Sand fly specimens were collected and identified morphologically. Total DNA was extracted from the abdomen of female specimens and Leishmania DNA was detected by PCR.

Results

Among the 57 examined patients, 37 were positive for CL. The identification of the parasite has revealed the single species Leishmania major. The 384 sand flies were collected belonged to two genera (Phlebotomus and Sergentomyia), six sub-genera and six species. Phlebotomus papatasi, Ph. kazeruni and Sergentomyia clydei were the dominant species. Leishmania DNA was detected in two females of Ph. papatasi two of Ph. kazeruni and one specimen of Sergentomyia clydei.

Conclusions

Leishmania major is confirmed to be the etiological agent of cutaneous leishmaniasis in northwestern Saudi Arabia. The molecular detection of Leishmania DNA in Ph. papatasi and Ph. kazeruni supports the potential role of these two species in the transmission of Leishmania. Further epidemiological studies are needed to prove their role and to evaluate the burden of CL in the study region.

Keywords

Cutaneous leishmaniasis Leishmania major Sand flyHailNorthwestern Saudi Arabia

Background

Cutaneous leishmaniasis is a parasitic disease caused by a flagellated protozoan belonging to the genus Leishmania. It is known to be the ninth largest disease burden among the 13 parasitic and bacterial neglected tropical diseases worldwide [1, 2]. Although it is self-healing, CL causes skin ulcers and disfiguring scars that can result in serious social and psychological stigma [35]. Because of the devastating consequences to the patient, CL is recognized as a special public health problem. In the Middle East and across to central Asia, CL is endemic with an average estimated annual incidence of 321,300 cases [6]. Among countries of this region, Saudi Arabia was reported as the fourth most endemic focus of zoonotic CL after Afghanistan, Iran and Pakistan with an estimated incidence ranging from 9600 to 15,800 cases/year [6]. In this region, the first real documented cases date from 1973 [7]. Many Saudi provinces are endemic for CL including the Al-Hassa oasis, Al-Madinah Al-Munawarah and Al Qassim provinces where desert rodents (Psammomys obesus and Meriones libycus) are the main reservoir hosts and the vector Ph. papatasi is prevalent [811]. In most endemic areas in Saudi Arabia, the causative organism was identified as L. major. Cutaneous leishmaniasis due to L. tropica is less prevalent compared to zoonotic CL caused by L. major. It occurs within small endemic foci in the west (Al Madina Al-Munawarah and Al Qassim) and southwest (high plateau of Aseer) provinces [9, 10, 1215].

Despite the large distribution of this parasitic disease, studies focusing on the identification of the Leishmania species and the sand fly fauna are rare. Indeed, the characterization of the parasite circulating in this geographical area only started at the beginning of the 1990s. Since then and up to now only about 224 Leishmania isolates have been identified to the species level either by isoenzymatic methods (n = 38 isolates) or using a molecular approach (from culture and human clinical samples n = 102 and sand flies n = 84) [9, 10, 13, 1618].

Several sand fly investigations have described phlebotomine species composition, their geographical distribution and their role in disease dynamics in Saudi Arabia. These surveys have revealed the presence of 25 species with a predominance of Ph. papatasi in all investigated areas of the kingdom [1922]. Moreover, it was demonstrated that the geographical distribution of CL coincides with the distribution of the sand fly vectors Ph. papatasi and Ph. sergenti [20, 23].

According to the Saudi Ministry of Heath seven-year reports (2006–2012), Hail Province, Northwestern Saudi Arabia, is reported as the fifth most infected region among the 20 Saudi provinces after El Qassim, Al-Madinah Al-Munawarah, El Hassa and Riyadh [24]. Indeed, the average reported incidence of CL in Hail was 183 cases/year. Despite the endemic state of Hail province to CL, no published report is available describing the epidemiological profile of this disease including the identification of the parasite and sand fly species and their geographical distribution.

Here, we aimed to identify both the Leishmania species and the sand fly species composition in the Hail region in northwestern Saudi Arabia including the monthly abundance of circulating phlebotomine species. Such data are used to understand the structure of this CL focus and to clarify the role of the sand fly species in the transmission of the disease and thereby to establish effective control and preventive measures against this parasitic disease.

Methods

Description of the study area

Hail is located in the northwestern region of the Kingdom of Saudi Arabia (between 25°35′–29°00′N, 39°01′–44°45′E). It is bordered by Al Jouf to the North, Northern borders to the northeast, Al Qassim and Riyadh to the south and Tabuk and Al Madina Al Munawara to the west. Hail covers an area of almost 118,322 km2 and has a population of 527,000 (Ministry of the Interior, 2013 estimate) (Fig. 1). It is located at 914 m above mean sea level and has an annual rainfall of 100.6 mm. The principal part of Hail is composed of the Nafud Desert, covering about 64,000 km2. The weather system of Hail Region is arid to extra arid. Summer temperatures typically rise as high as 50 °C during daytime with a diurnal variation of about 25 °C. Winter temperatures hover around freezing at night especially at higher altitudes although the ground occasionally freezes and daytime temperatures nearly always reach 25 °C in the sun.
Fig. 1

Location of sand fly collection sites (stars in red) in the Hail Province, northwestern KSA

Sample collection

Human CL cases were collected from dermatology services in different hospitals of the Hail region. For each patient, socio-demographic (gender, age, geographical residency of patients and nationality) and clinical data (number of lesions, precise location, size, clinical aspect and evolution duration of each lesion) were recorded on a pre-printed sheet. For each patient, two samples were collected with the first used to prepare two smears for Giemsa staining and microscopic examination. The second sample was stored in phosphate buffered saline (PBS) at -20 °C for further molecular studies.

Sand flies were captured twice a week from September 2015 to October 2016 in nine geographical locations within Hail Province (Fig. 1) from where the regional health authorities reported human CL cases. On each occasion, thirty sticky traps (A4 white paper sheets coated with crude Castor oil) were placed in different biotopes. Indoor (animal shelters) traps were placed at 1.5 m above ground sheltered from air streams. Outdoor (at the entrance of rodent burrows and in sandy areas and valleys) traps were put on the ground with a stick as shown in Fig. 2. Traps were placed at sunset and collected the next day before sunrise.
Fig. 2

Examples of collection sites: a near rodent burrow; b in valleys; c sandy area; d inside animal stables

Molecular detection and identification of Leishmania parasites

DNA extraction was performed on clinical samples and female sand flies using the ReliaPrep™ gDNA Tissue Miniprep System Kit (Promega, Madison, United States), following the manufacturer’s instructions.

PCR amplification was carried out using the genus-specific primers of the conserved sequences of the small subunit ribosomal DNA (SSU-rDNA) gene (PCR-Lei) for the detection of Leishmania spp. infection according to the protocol of Spanakos et al. [25]. This PCR is genus-specific and does not allow the identification of the species. The forward primer Lei70L was 5′-CGC AAC CTC GGT TCG GTG TG-3′ and the reverse primer Lei70R was 5′-CGC GGT GCT GGA CAC AGG GTA-3′. PCR was performed in a final volume of 50 μl containing 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM of each deoxynucleotide, 3 units of Taq DNA Polymerase (all obtained from Promega) and 100 pM of each primer. PCR was performed in a PTC 100 Thermal Cycler, with the following conditions: initial denaturation at 94 °C for 5 min, 40 cycles at 94 °C for 30 s, 65 °C for 30 s and 72 °C for 30 s and a final elongation step at 72 °C for 10 min. For each experiment, positive (DNA extracted from Leishmania culture) and negative (Human DNA extracted from a healthy donor) controls were used. Products were separated by 2% agarose gel electrophoresis containing 0.5 mg/ml ethidium bromide and visualized on a UV transilluminator.

Thereafter, DNA from the positive sample was re-analyzed using a second set of primers targeting a coding DNA sequence (CDS) of the putative translation initiation factor alpha subunit gene according to the protocol of El Baidouri et al. [26]. Genomic DNA was amplified by real-time PCR using SYBR Green method (Light Cycler 480 II, Roche, Rotkreuz, Switzerland). The amplified products were sequenced on both strands (Eurofins MWG Operon, Ebersberg, Germany) and the obtained sequences were blasted using Blastn algorithm against the “non-redundant” GenBank sequence database for the identification of Leishmania species.

Phlebotomine sand flies identification

Sand flies were firstly washed with 90% ethanol to remove excess oil and then kept in labelled vials containing 70% ethanol. The head and posterior part of the abdomen of both male and female sand flies were dissected, cleared in Marc-Andrée solution [27] and then mounted between slide and coverslip in chloral gum medium [28]. Their abdomens and thorax were preserved in ethanol for further molecular analysis. Female and male specimens were morphologically identified by observing head and genital structures under the microscope using the morphological keys and characters of identification proposed by Croset et al. [29] and Leger et al. [30]. For each female specimen, the presence of eggs (gravid), blood (total or partial engorged) or unfed (no visible blood in the abdomen) status was recorded.

Leishmania DNA detection and identification from female sand flies

After removal of the head and posterior extremity from each female specimen, the remainder of the body was transferred to individual sterilized 1.5 ml vials, from which genomic DNA was isolated using ReliaPrep™ gDNA Tissue Miniprep System kit (Promega, Madison, United States), following the manufacturer’s instructions. The maceration of the insect’s tissues was carried out with a piston pellet, and the final elution volume was 100 μl. DNA extracts were stored at -20 °C until use.

Five μl of the extracted DNA was used as a template for the PCR reaction for the detection of a Leishmania SSU rDNA sequence as described by Spanakos et al. [25] and detailed above. Identification of Leishmania species detected from female sand flies was performed by the amplification and sequencing of the putative translation initiation factor alpha subunit locus according to the protocol of El Baidouri et al. [26].

Results

Number of recruited human cases

From September 2015 to October 2016, 57 patients with suspected human CL were included in our study. The patients ranged from one to 65 years of age, with a median of 30 years. The sex ratio of men to women was 2.8 (men: n = 42; women: n = 15). Among them, 29 (50.87%) were Saudi and 28 (49.12%) were non Saudi (Egyptian: n = 10; Indian: n = 8; Pakistani: n = 4; Sudanese: n = 4; Bangladeshi: n = 1; Filipino: n = 1).

Diagnosis and epidemiological characteristics of cutaneous leishmaniasis

Thirty-seven patients (64.91% of the diagnosed cases) were diagnosed as cases of CL. Among the 57 diagnosed patients, 18 (31.57%) had a positive direct examination. The diagnosis of the 39 remaining negative cases was confirmed by the detection of Leishmania DNA using PCR-Lei. Thus, among these 39 negative cases, 19 cases (48.71%) had shown a positive PCR result confirming their infection with the Leishmania parasite.

Among the 37 confirmed CL cases, 29 were males (78.37%), and 8 were females (21.62%). Their age ranged from three to 65 years old with a median age of 34 years. The majority (72.97%) of cases occurred in the age group 15–45 years. CL cases declined for the two other age groups (i.e. 8.10% in patients < 15 and 18.91% in patients > 45 years of age).

By comparing the infection rate between Saudi and non-Saudi patients, 17 (45.94%) were indigenous, and 20 (54.05%) were expatriate workers (Egyptian: n = 6; Indian: n = 6; Pakistani: n = 4; Sudanese: n = 3; and Bangladeshi: n = 1). All patients (Saudi and non-Saudi) confirmed that they had not left the Hail region in the previous five months before the onset of lesions. The positive cases were unequally distributed in the different regions of the Province of Hail. In fact, the majority of CL cases were from Hail city (n = 22, 59.45%), followed by Chinan (n = 7, 18.91%). Sporadic cases were from Al Rodha (n = 2, 5.4%), Baqaa (n = 2, 5.4%), Qina (n = 2, 5.4%), Samirah (n = 1, 2.7%) and Guefar (n = 1, 2.7%).

Clinical features of cutaneous leishmaniasis

Among the positive CL cases, 94.6% had lesions over exposed parts of the body. The most commonly affected sites were the lower limbs (n = 21, 56.75%) (leg: n = 13; foot: n = 8) followed by the upper limbs (n = 10, 27.02%) and face (n = 4, 10.81%). Nevertheless, two CL lesions (5.40%) were found on the back (n = 1, 2.7%) and the groin (n = 1, 2.7%).

Among positive CL cases, the size of the lesion varied between 0.3 and 9 cm; about 40% of positive lesions were 3–5 cm (n = 15), whereas 27.02% were 1–3 cm. The lesions of 1 cm and > 5 cm represented 16.21% each. Among the same group, 22 patients (59.45%) had a single lesion, 10 patients (27.02%) with 2–5 lesions and 5 patients (13.5%) had 5–20.

The duration of the disease varied between three weeks to four months. Most CL cases (75.67%) presented for diagnosis one to two months after the onset of their skin lesions. However, 5.40% of the patients diagnosed their disease within three weeks and 18.91% after more than two months. The detailed data are summarized in Table 1.
Table 1

The clinical features of cutaneous leishmaniasis in the Hail region, the Kingdom of Saudi Arabia

Parameter

n (%)

Location of the lesion

 Lower limbs

21 (56.75)

 Upper limbs

10 (27.02)

 Face

4 (10.81)

 Other

2 (5.40)

Size of the lesion (cm)

  < 1

6 (16.21)

 1–3

10 (27.02)

 3–5

15 (40.54)

  > 5

6 (16.21)

Number of lesions

 1

22 (59.45)

 2–5

10 (27.02)

 5–10

3 (8.10)

  > 10

2 (5.40)

Duration of the disease (months)

  < 1

2 (5.40)

 1–2

28 (75.67)

 2–3

4 (10.81)

  > 3

3 (8.10)

An important clinical polymorphism of the CL lesions was noticed among the studied series. Among the 37 positive cases of CL, 6 different clinical forms were noted. The ulcero-crusted form was the most common (n = 19, 51.35%) followed by the ulcerated form (n = 8, 21.62%), the nodular (n = 4, 10.81%), the pseudotumoral (n = 3, 8.10%), the vegetant (n = 2, 5.40%) and the nodulo-papular (n = 1, 2.70%).

Leishmania species identification in clinical samples

Among the 37 positive CL cases, 25 (including 18 positive cases by direct examination and 7 positive cases by PCR-Lei) were positive for the amplification of the single copy gene. These PCR products were sequenced and identified by comparison with the nucleotide-nucleotide Basic Local Alignment Search Tool (BLAST) (GenBank DNA sequence database, National Centre for Biotechnology Information) (www.ncbi.nlm.nih.gov/blast/). All of the 25 CL cases were identified as L. major. No case of L. tropica was identified.

Sand fly species composition and abundance

During the entomological survey, a total of 46 weekly collections were performed from September 2015 to October 2016. In total, 384 wild-caught phlebotomine sand flies were captured including 212 males and 172 females. Among these females, four specimens were blood fed, and the abdomen of 18 others were filled with eggs. Regarding sand fly individuals, the highest number of flies were recorded in Guefar (n = 187), followed by Mrifag (n = 119) and Al Swifla (n = 27). However, only eight specimens were collected in Al Hmayria, seven in Mogag and a single fly in Baqaa.

The collected sand fly specimens were distributed among two genera [Phlebotomus (n = 292, 76.04%) and Sergentomyia (n = 92, 23.95%)], six sub-genera [Phlebotomus (n = 242, 63.02%), Paraphlebotomus (n = 49, 12.76%), Larroussius (n = 1, 0.26%), Sintonius (n = 81, 21.09%), Sergentomyia (n = 9, 2.34%), and Grassomyia (n = 2, 0.52%)] and six species (Ph. papatasi, Ph. kazeruni, Ph. syriacus, Se. clydei, Se. antennata and Se. dreyfussi) (Tables 2 and 3).
Table 2

Relative abundance and sex ratios of Phlebotomus spp. identified from nine collecting sites in the Hail Province

 

Collection locality

Geographical coordinate

Ph. papatasi

Ph. kazeruni

Ph. syriacus

Total (%)

M (%)

F (%)

M:F

M (%)

F (%)

M:F

M (%)

F (%)

M:F

M (%)

F (%)

M:F

S1

Al Swifla

27°34′N, 41°45′E

7 (2.39)

8 (2.73)

0.875

1 (0.34)

 

 

7 (2.39)

9 (3.08)

0.77

S2

Guefar

27°24′N, 41°33′E

64 (21.91)

62 (21.23)

1.032

13 (4.45)

 

1 (0.34)

 

78 (26.71)

62 (21.23)

1.25

S3

Mrifag

27°22′N, 41°38′E

46 (15.75)

32 (10.95)

1.437

6 (2.05)

12 (4.10)

0.5

 

52 (17.80)

44 (15.06)

1.18

S4

Baqaa

27°53′N, 42°24′E

 

1 (0.34)

 

 

1 (0.34)

 

S5

Al Hafir

27°38′N, 41°16′E

2 (0.68)

 

4 (1.36)

2 (0.68)

2

 

4 (1.36)

4 (1.36)

1

S6

Mogag

27°22′N, 41°10′E

1 (0.34)

2 (0.68)

0.5

4 (1.36)

 

 

5 (1.71)

2 (0.68)

2.5

S7

G. Ashrawat

27°20′N, 41°27′E

5 (1.71)

1 (0.34)

5

3 (1.02)

 

 

8 (2.73)

1 (0.34)

8

S8

Qina

27°46′N, 41°25′E

3 (1.02)

2 (0.68)

1.5

3 (1.02)

 

 

3 (1.02)

5 (1.71)

0.6

S9

Al Hmayria

26°55′N, 41°38′E

7 (2.39)

 

 

 

7 (2.39)

 
 

Total

 

133 (45.54)

109 (37.32)

 

31 (10.61)

18 (6.16)

 

1 (0.34)

  

165 (56.5)

127 (43.5)

1.3

  

242 (82.87)

 

49 (16.78)

 

1 (0.34)

 

292 (100)

 
Table 3

Relative abundance and sex ratios of Sergentomyia spp. identified from nine collecting sites in the Hail Province

 

Collection locality

Geographic coordinate

Se. clydei

Se. antennata

Se. dreyfussi

Total (%)

 

M (%)

F (%)

M:F

M (%)

F (%)

M:F

M (%)

F (%)

M:F

M (%)

F (%)

M:F

S1

Al Swifla

27°34′N, 41°45′E

4 (4.34)

2 (2.17)

2

2 (2.17)

3 (3.26)

0.66

 

6 (6.52)

5 (5.43)

1.2

S2

Guefar

27°24′N, 41°33′E

24 (26.08)

21 (22.82)

1.143

1 (1.08)

 

1 (1.08)

 

25 (27.17

22 (23.91)

1.13

S3

Mrifag

27°22′N, 41°38′E

13 (14.13)

9 (9.78)

1.444

1 (1.08)

 

 

14 (15.21)

9 (9.78)

1.55

S4

Baqaa

27°53′N, 42°24′E

 

 

 

 

S5

Al Hafir

27°38′N, 41°16′E

1 (1.08)

4 (4.34)

0.25

 

 

1 (1.08)

4 (4.34)

0.25

S6

Mogag

27°22′N, 41°10′E

 

 

 

 

S7

G. Ashrawat

27°20′N, 41°27′E

 

 

 

 

S8

Qina

27°46′N, 41°25′E

2 (2.17)

 

2 (2.17)

 

1 (1.08)

 

1 (1.08)

4 (4.34)

0.25

S9

Al Hmayria

26°55′N, 41°38′E

1 (1.08)

 

 

 

1 (1.08)

 
 

Total

 

42 (45.65)

39 (42.39)

 

3 (3.26)

6 (6.52)

 

2 (2.17)

  

47 (51.08)

45 (48.91)

1.04

  

81 (88.04)

 

9 (9.78)

 

2 (2.17)

 

92 (100)

 

Sergentomyia dreyfussi was exclusively found at the station of Guefar and Qina (Table 3). Among the Phlebotomus species caught, Ph. papatasi was the most predominant (82.87%) species, followed by Ph. kazeruni (16.78%) and Ph. syriacus (0.34%). For the genus Sergentomyia, Se. clydei represents 88.04% of the collected specimens, followed by Se. antennata (9.78%). Se. dreyfussi was rare and represented just 2.17% of the collected Sergentomyia flies. All blood-fed female sand flies were identified as Ph. papatasi.

Overall, the male to female sand fly ratio was 6:5 (212/172). This sex ratio (M/F) varied according to the sand fly species and the station (Tables 2 and 3). For all of the three identified Phlebotomus species, the males outnumbered the females. However, in case of Se. antennata the ratio of male/female is 0.5, whereas Ph. syriacus and Se. dreyfussi were only represented by males.

The monthly abundance (fly/month) was examined for the three common sand fly species Ph. papatasi, Ph. kazeruni and Se. clydei (Fig. 3). In general Ph. papatasi and Se. clydei sand fly species were active from April to November with increased activity between July and August indicated by prominent peaks during these two months. However, Ph. kazeruni species had shown a peak of activity in May which markedly decreased in June and July, and the species disappeared from August when the temperature exceeded 40 °C.
Fig. 3

The monthly abundance of the most common sand fly species (Ph. papatasi, Ph. kazeruni and Se. clydei) in the Hail Province, northwestern KSA

Leishmania DNA detection in female sand flies

Molecular detection of Leishmania was carried out using individual DNA extract prepared from 75 sandflies female specimens (44 Ph. papatasi, 15 Ph. kazeruni, 14 Se. clydei and 2 Se. antennata). Leishmania DNA was detected using SSU rDNA sequence as described by Spanakos et al. [25]. The infection rate was calculated as 6.66% (5/75). Among these five infected specimens, two were Ph. papatasi, two Ph. kazeruni and one Se. clydei. All of them were non-blood-fed females. The presence of Leishmania DNA was further confirmed by sequencing of the PCR products using the same primers as the amplification step.

The DNA extract of the five infected specimens was used for a second PCR amplifying a single copy housekeeping gene to identify the Leishmania species. Although the PCR was repeated on five separate occasions, no specimen was found positive for Leishmania DNA. This is the consequence of the low sensitivity of this second PCR compared to the first one which amplifies multi-copy DNA sequence.

Discussion

Cutaneous leishmaniasis is endemic in Hail. According to the Ministry of Health of the Kingdom of Saudi Arabia (2006–2012) reports, about 183 CL cases are reported each year in this region [24]. Despite its endemic state, this disease is rarely studied in the Hail region. A single retrospective study was carried out in 2014 emphasising the extension of this disease in different parts of Hail [31]. No data are available concerning the causative species of this disease or the vector. For this reason, the purpose of our study was to identify the Leishmania species causing the disease and the sand flies species circulating in the Hail region.

To our knowledge, this is the first time that Leishmania species are identified in this study area. All positive human CL cases were caused by L. major indicating the presence of a homogeneous focus of zoonotic cutaneous leishmaniasis (ZCL) in this study area. Nevertheless, the absence of human CL cases caused by L. tropica in our study did not exclude the presence of small micro-foci of anthroponotic CL (ACL) in Hail region. Further studies are needed to investigate all Hail regions to affirm or deny the presence of ACL since this nosogeographical CL form is less prevalent than ZCL. This last form is endemic in many regions of Saudi Arabia including Al-Hassa Oasis, Al-Madinah Al-Munawarah and Al Qassim provinces where desert rodents (Psammomys obesus and Meriones libycus) are the main reservoir hosts [811, 19]. However, ACL caused by L. tropica was reported only in small foci such Aseer in the southwestern region [9, 10, 13, 14].

Variation in the clinical presentation of CL was noticed among the positive cases. Six different clinical forms were noticed. The ulcero-crusted form was the most common clinical form followed by the ulcerated form then the nodular form. The same result was reported in Tunisia by Masmoudi et al. [32] who noticed 54.9% of the diagnosed patients with this clinical form [32]. Also, the same study had reported the presence of nine clinical forms among a studied population of 102 cases of CL. Douba et al. [33] have reported five types of CL in the region of Aleppo, Syria. Among them, the two most common types were the papulonodular and plaque forms.

It is important to highlight that the size of the lesion is directly linked to the duration of infection. Here, small nodular lesions were noted among patients who have diagnosed their disease within one month or less since their onset. It is known that a single Leishmania species can elicit a range of clinical patterns [3436]. The specific factors that influence the clinical outcome of these infections remain to be completely elucidated but likely is influenced by host genetics or immune responses [37], potentially different sand fly vector species or populations [38, 39] and the presence of Leishmania spp. Hybrids [40, 41]. Clinical polymorphism could also be explained by the degree of virulence of the Leishmania strain causing the lesion. Indeed, recent studies have reported that the degree of virulence of Leishmania decreases in the presence of a mutation in the gene encoding a protein BBSome complex or the gene encoding the enzyme Sphingosine kinase [42]. Further, Zangger et al. [43] showed that the virulence of L. guyanensis is due to its co-infection by a virus LRV (Leishmania RNA virus). Major efforts are still needed to understand the real causes of the observed clinical polymorphism better.

The second part of our study was an entomological survey of sand flies in the Hail Province of Saudi Arabia. The goal of this investigation was to identify the species of sand fly and assess the infection rate in female specimens. Thus, six species were identified including three of the genus Phlebotomus (Ph. papatasi, Ph. kazeruni and Ph. syriacus) and three of the genus Sergentomyia (Se. antennata, Se. clydei and Se. dreyfussi). Phlebotomus papatasi was the most abundant species identified in the current study. This result is consistent with the epidemiological state of leishmaniasis in Hail where ZCL is the extended noso-geographical form in this study. It is known that in Saudi Arabia, ZCL is transmitted to humans from infected rodent reservoir hosts (Psammomys obesus and Meriones libycus) through the bites of the sand fly vector Ph. papatasi [44]. The same species was also reported as the predominant sand fly species in many parts of the Kingdom of Saudi Arabia, such as Al-Madinah Al-Munawarah [10, 23], Jeddah [45], Riyadh [46] and Al-Qassim [47]. Since 1985, Killicki-Kendrick et al. [44] have isolated L. major MON-26 from a specimen of Ph. papatasi is proving its role as a vector of ZCL. A study on the seasonal abundance of Ph. papatasi in Riyadh had demonstrated that the greatest number of sand flies occurred most commonly in the summer with two peaks in June and September [46].

It is important to highlight that a complete survey carried in Saudi Arabia to study its phlebotomine sand fly fauna was that of Lewis & Buttiker [21] in 1982. During this survey, 25 phlebotomine species were identified in the whole of Saudi Arabia [21]. Inside this study area, sand fly fauna was identified in the Hail region. Between 1975 and 1980 only 14 specimens were collected in Hail, and only two species were identified: Se. clydei (79%) and Se. antennata (21%). During the same investigation, the second survey in Hail was carried in 1981, which identified Ph. kazeruni, Ph. syriacus, Ph. naqbenius, Se. christophersi, Se. clydei, Se. fallax and Se. tiberiadis. Since 1981, no study has been carried out in this region. Here, we identify for the first time Se. dreyfussi from two specimens. More entomological investigations are crucial to study the abundance of this species in the different parts of Hail.

Assessment of the Leishmania infection rate in sand flies is of paramount importance, and PCR-based approaches have been successfully used for detection of parasite DNA in sand flies [4850]. The detection of Leishmania DNA in two unfed females of Ph. papatasi caught in a rural area of Hail provides additional evidence in favour of its role as a potential vector for L. major in this study area. Also, the detection of Leishmania DNA in two unfed Ph. kazeruni specimens (a close species to Ph. sergenti) suggests a potential role of Paraphlebotomus spp. in the transmission of L. tropica. It should be pointed out that the molecular detection of Leishmania DNA in sand flies is not enough to incriminate these insects as vector conclusively. Such finding could be explained by recent feedings on reservoirs resulting in parasite DNA remnants after blood meal digestion. Thus, isolation of the parasite from the midgut of these sand flies and its isoenzymatic identification remain mandatory to confirm this hypothesis. Finally, one specimen of Se. clydei was also found to be positive for the detection of Leishmania. In the Old World, phlebotomine species of the genus Sergentomyia are known as reptile-biting sand flies transmitting Sauroleishmania or the “lizard Leishmania” parasite [51]. Nevertheless, some recent investigations in Tunisia and Portugal have reported the detection of L. major and L. infantum DNA in Se. minuta [4852, 53] suggesting its potential role in the transmission of mammal-infecting Leishmania. Also, Senghor et al. and Berdjane-Brouk et al. suggested that Sergentomyia species could be involved in the transmission of L. major and L. infantum in Africa [5455]. To confirm this hypothesis, it will be crucial to demonstrate that Sergentomyia species feed on humans and can support the complete development of the parasite in natural conditions after the digestion of an infectious blood meal.

Conclusion

In conclusion, L. major is the parasitic agent responsible for CL in Hail, Saudi Arabia. It exhibits a large clinical polymorphism and consequently should be included in the differential diagnosis of many common and uncommon dermatological diseases. The life-cycle of L. major is still not elucidated in this area but the detection of Leishmania DNA in two specimens of Ph. papatasi is evidence for the role of this sand fly species in the transmission of L. major. Further studies including the isolation and identification of the parasite from sand fly are needed to prove this hypothesis. Entomological data showed that Ph. papatasi, Se. clydei and Ph. kazeruni are the most dominant species in our collection. The detection for the first time of Leishmania DNA in two specimens of Ph. kazeruni supports the vector status of this species. Our investigation has provided a valuable set of data about the epidemiological features of CL in Northwestern Saudi Arabia. Nevertheless, further studies in humans, vectors and potential reservoirs are needed to determine the burden of cutaneous leishmaniasis in the whole of Hail.

Abbreviations

CL: 

cutaneous leishmaniasis

ZCL: 

zoonotic cutaneous leishmaniasis

Declarations

Acknowledgments

We thank all the staff of the Control of Communicable Diseases and Vectors Administration in Hail region (Mr Hichem Al Tamimi, Marzouk Al-Shammary, Abdul Rahman Al-Salah and Salem Al-Nozha) for their contribution in field work and sand fly collection. Also, we are grateful to Dr Christophe Ravel for identifying Leishmania species and Dr Jérôme Depaquit for helping in sand fly morphological identification. Finally, we sincerely acknowledge Mr Slaheddine Amor for his technical help.

Ethical approval and consent to participate

This study was embedded in activities of the KACST research project, which has received ethical approval from the CAMS Medical Research Ethics Committee in University of Hail, Hail, KSA (reference no. 10/Apr/015).

Funding

This study received financial support from the King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, ID number SG-35-181. The funders had no role in study design, data collection and analysis, interpretation of the data, or writing the manuscript.

Availability of data and materials

The data sets supporting the conclusions of this article are included within the article. The sequences are submitted in the GenBank database under the accession numbers MG018949–MG018957.

Authors’ contributions

This work was accomplished by the contribution of all authors. NH has been involved in the study design, sand fly identification, results analysis and the manuscript drafting. OA has provided local contacts, contributed to the study design and manuscript reviewing. FFA has contributed to the collection of human samples and the annotation of the human clinical cases. SA was involved in the collection and identification of sand flies. LR has performed the molecular study. IA has contributed in data analysis. All authors read and approved the final manuscript.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Laboratoire de Parasitologie-Mycologie Médicale et Moléculaire (LR12ES08), Département de Biologie Clinique B, Faculté de Pharmacie, Université de Monastir
(2)
College of Applied Medical Sciences, Clinical Laboratory Sciences Department, University of Hail
(3)
Dermatology service, King Khaled Hospital

References

  1. Hotez PJ, Molyneux DH, Fenwick A, Ottesen E, Sachs SE, Sachs JD. Incorporating a rapid-impact package for neglected tropical diseases with programs for HIV/AIDS, tuberculosis and malaria. PLoS Med. 2006;3(Suppl 5):576–84.Google Scholar
  2. Molyneux DH, Hotez PJ, Fenwick A. Rapid-impact interventions: how a policy of integrated control for Africa’s neglected tropical diseases could benefit the poor. PLoS Med. 2005;2(Suppl 11):e336.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Kassi M, Kassi M, Afghan AK, Rehman R, Kasi PM. Marring leishmaniasis: the stigmatization and the impact of cutaneous leishmaniasis in Pakistan and Afghanistan. PLoS Negl Trop Dis. 2008;2(Suppl 10):e259.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Vares B, Mohseni M, Heshmatkhah A, Farjzadeh S, Safizadeh H, Shamsi-Meymandi S, et al. Quality of life in patients with cutaneous leishmaniasis. Arch Iran Med. 2013;16(Suppl 8):474–7.PubMedGoogle Scholar
  5. WHO. Control of the leishmaniases: report of a meeting of the WHO expert committee on the control of leishmaniases. World Health Organ Tech Rep Ser 949.  Geneva: WHO; 2010.Google Scholar
  6. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. The WHO leishmaniasis control team. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 2012;7(Suppl 5):e35671.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Morsy TA. Shoura MI. Some aspects of cutaneous leishmaniasis in Riyadh, S.A. Proc 9th Int Cong Trop Med Malar. 1973;1:138.Google Scholar
  8. Al-Tawfiqa JA, AbuKhamsin A. Cutaneous leishmaniasis: a 46-year study of the epidemiology and clinical features in Saudi Arabia (1956–2002). Int J Infect Dis. 2004;8(Suppl 4):244–50.View ArticleGoogle Scholar
  9. El-Beshbishy HA, Al-li KH, El-Badry AA. Molecular characterization of cutaneous leishmaniasis in Al-Madinah Al-Munawarah province, western Saudi Arabia. Int J Infect Dis. 2013a;17(Suppl 5):e334–8.Google Scholar
  10. El-Beshbishy HA, Al-Ali KH, El-Badry AA. Molecular characterization of Leishmania infection in sand flies from Al-Madinah. Al-Munawarah province, western Saudi Arabia. Exp Parasitol. 2013b;134(Suppl 2):211–5.Google Scholar
  11. Peters W, Al-Zahrani MA. The leishmaniases as a public health problem in Saudi Arabia. Saudi Med J. 1987;8(Suppl 4):333–43.Google Scholar
  12. Al-Qurashi AR, Ghandour AM, Osman M, Al-Juma M. Dissemination in cutaneous leishmaniasis due to Leishmania major in different ethnic groups in Saudi Arabia. Int J Dermatol. 2000;39(Suppl 11):832–6.View ArticlePubMedGoogle Scholar
  13. Shalaby I, Gherbawy Y, Jamjoom M, Banaja AE. Genotypic characterization of cutaneous leishmaniasis at al Baha and Al Qasim provinces (Saudi Arabia). Vector Borne Zoonotic Dis. 2011;11(Suppl 7):807–13.View ArticlePubMedGoogle Scholar
  14. Yehia L, Adib-Houreih M, Raslan WF, Kibbi AG, Loya A, Firooz A, et al. Molecular diagnosis of cutaneous leishmaniasis and species identification: analysis of 122 biopsies with varied parasite index. J Cutan Pathol. 2012;39(Suppl 3):347–55.View ArticlePubMedGoogle Scholar
  15. SalamN, Al-Shaqha WM, Azzi A. Leishmaniasis in the Middle East: incidence and epidemiology. PLoS Neg Trop Dis. 2014;8(Suppl 10):e3208.Google Scholar
  16. Bin DS, Mostafa OM, Abdoon A, Al-Quraishy SA, Alqahtani AA. Isoenzyme electrophetic characterization of Leishmania major, the causative agent of zoonotic cutaneous leishmaniasis in North and West Saudi Arabia. J Egypt Soc Parasitol. 2010;40(Suppl 2):465–78.Google Scholar
  17. Karram S, Loya A, Hamam H, Habib RH, Khalifa I. Transepidermal elimination in cutaneous leishmaniasis: a multiregional study. J Cutan Pathol. 2012;39(Suppl 4):406–12.View ArticlePubMedGoogle Scholar
  18. Morsy TA, Sarwat MA, al Shaiby AZ, Fawzy AF, Arafa MA. Isoenzyme characterization of Leishmania isolates from patients in el nour specialized hospital, Saudi Arabia. J Egypt Soc Parasitol. 1993;23(Suppl 3):871–6.PubMedGoogle Scholar
  19. Al-Zahrani MA, Peters W, Evans DA, Chin C, Smith V, Lane RP. Phlebotomus sergenti, a vector of Leishmania tropica in Saudi Arabia. Trans R Soc Trop Med Hyg. 1988;82(Suppl 3):416.View ArticlePubMedGoogle Scholar
  20. Ibrahim AA, Abdoon AM. Distribution and population dynamics of Phlebotomus sand flies (Diptera: Psychodidae) in an endemic area of cutaneous leishmaniasis in Asir region, southwestern Saudi Arabia. J Entomol. 2005;2(Suppl 1):102–8.Google Scholar
  21. Lewis DJ, Buttiker W. Insects of Saudi Arabia: the taxonomy and distribution of Saudi Arabian phlebotomine sandflies (Diptera: Psychodidae). Fauna of Saudi Arabia. 1982;4:353–97.Google Scholar
  22. Mustafa MB, Hussein SM, Ibrahim EA. Al-Seghayer SM, al Amri SA, Gradoni L. Phlebotomus papatasi (Scopoli), vector of zoonotic cutaneous leishmaniasis in Riyadh province, Saudi Arabia. Trans R Soc Trop Med Hyg. 1994;88(Suppl 1):40.View ArticlePubMedGoogle Scholar
  23. El-Badry A, Al-Juhani A, el-K I, Al-Zubiany S. Distribution of sand flies in El-Nekheil province, in Al-Madinah Al-Munawwarah region, western of Saudi Arabia. Parasitol Res. 2008;103(Suppl 1):151–6.Google Scholar
  24. MOHSA, 2006-2012. Statistical books for the years 2006–2012. Ministry of health of Saudi Arabia. Available at : http://www.moh.gov.sa/en/Ministry/Statistics/book/Pages/defa ult.aspx (accessed on March 2014).
  25. Spanakos G, Patsoula E, Kremastinou T, Saroglou G, Vakalis N. Development of a PCR-based method for diagnosis of Leishmania in blood samples. Mol Cell Probes. 2002;16(Suppl 6):415–20.View ArticlePubMedGoogle Scholar
  26. El Baidouri F, Diancourt L, Berry V, Chevenet F, Pratlong F, Marty P, Ravel C. Genetic structure and evolution of the Leishmania genus in Africa and Eurasia: what does MLSA tell us. PLoS Negl Trop Dis. 2013;7(Suppl 6):e2255.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Abonnenc E. Les phlébotomes de la région éthiopienne (Diptera: Psychodidae). Sér Ent Méd Parasitol: Cah ORSTOM. 1972;55:239.Google Scholar
  28. Madulo-Leblond G. Les phlébotomes (Diptera: Phlebotomidae) des Iles Ioniennes. PhD Thesis, Champagne-Ardenne University, Faculty of Pharmacy, Reims, France; 1983.Google Scholar
  29. Croset H, Rioux JA, Maistre M, Bayar N. Les phlébotomes de Tunisie (Diptera, phlebotomidae) Mise au point systématique, chronologique et éthologique. Ann Parasitol Hum Comp. 1978;53(Suppl 6):711–49.View ArticlePubMedGoogle Scholar
  30. Léger N, Pesson B. Madulo-Leblond G, Abonnenc E. Differentiation of females of the subgenus Larroussius Nitzulescu 1931 (Diptera: Phlebotomidae) of the Mediterranean region. Ann Parasitol Hum Comp. 1983;58(Suppl 6):611–23.Google Scholar
  31. Haouas N, Amer O, Ishankyty A, Alazmi A, Ishankyty I. Profile and geographical distribution of reported cutaneous leishmaniasis cases in northwestern Saudi Arabia, from 2010 to 2013. Asian Pac J Trop Med 2015;8(Suppl 4):287–91.View ArticlePubMedGoogle Scholar
  32. Masmoudi A, Ayadi N, Boudaya S, Meziou TJ, Mseddi M, Marrekchi S, et al. Polymorphisme clinique de la leishmaniose cutanée du centre et sud tunisien. Bull Soc Pathol Exot. 2007;100(Suppl 1):36–40.PubMedGoogle Scholar
  33. Douba MD, Abbas O, Wali A, Nassany J, Aouf A, Tibbi MS, et al. Chronic cutaneous leishmaniasis, a great mimicker with various clinical presentations: 12 years’ experience from Aleppo. J Eur Acad Dermatol Venereol. 2012;26(Suppl 10):1224–9.View ArticlePubMedGoogle Scholar
  34. Abbas K, Musatafa MA, Abass S, Kheir MM, Mukhtar M, Elamin EM, Elhassan AM. Mucosal leishmaniasis in a Sudanese patient. Am J Trop Med Hyg. 2009;80(Suppl 6):935–8.PubMedGoogle Scholar
  35. Elamin EM, Guizani I, Guerbouj S, Gramiccia M, El Hassan AM, Di Muccio T, et al. Identification of Leishmania donovani as a cause of cutaneous leishmaniasis in Sudan. Trans R Soc Trop Med Hyg. 2008;102(Suppl 1):54–7.View ArticlePubMedGoogle Scholar
  36. Khalil EA, Musa AM, Elgawi SH, Meshasha A, Gamar Eldawla I, Elhassan MO, et al. Revival of a focus of visceral leishmaniasis in central Sudan. Ann Trop Med Parasitol. 2008;102(Suppl 1):79–80.View ArticlePubMedGoogle Scholar
  37. Sakthianandeswaren A, Foote SJ, Handman E. The role of host genetics in leishmaniasis. Trends Parasitol. 2009;25(Suppl 8):383–91.View ArticlePubMedGoogle Scholar
  38. Ben Hadj Ahmed S, Chelbi I, Kaabi B, Cherni S, Derbali M, Zhioua E. Differences in the salivary effects of wild-caught versus colonized Phlebotomus papatasi (Diptera: Psychodidae) on the development of zoonotic cutaneous leishmaniasis in BALB/c mice. J Med Entomol. 2010;47(Suppl 1):74–9.View ArticlePubMedGoogle Scholar
  39. Laurenti MD, Silveira VM, Secundino NF, Corbett CE, Pimenta PP. Saliva of laboratory-reared Lutzomyia longipalpis exacerbates Leishmania (Leishmania) amazonensis infection more potently than saliva of wild caught Lutzomyia longipalpis. Parasitol Int. 2009;58(Suppl 3):220–6.View ArticlePubMedGoogle Scholar
  40. Ravel C, Cortes S, Pratlong F, Morio F, Dedet JP, Campino L. First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major. Int J Parasitol. 2006;36(Suppl 13):1383–8.View ArticlePubMedGoogle Scholar
  41. Volf P, Benkova I, Myskova J, Sadlova J, Campino L, Ravel C. Increased transmission potential of Leishmania major/Leishmania infantum hybrids. Int J Parasitol. 2007;37(Suppl 6):589–93.View ArticlePubMedPubMed CentralGoogle Scholar
  42. Price HP, Paape D, Hodgkinson MR, Farrant K, Doehl J, Stark M, Smith DF. The Leishmania major BBSome subunit BBS1 is essential for parasite virulence in the mammalian host. Mol Microbiol. 2013;90(Suppl 3):597–611.View ArticlePubMedPubMed CentralGoogle Scholar
  43. Zangger H, Ronet C, Desponds C, Kuhlmann FM, Robinson J, Hartley MA, et al. Detection of Leishmania RNA virus in Leishmania parasites. PLoS Negl Trop Dis. 2013;7(Suppl 1):1–11.Google Scholar
  44. Killick-Kendrick R, Leaney AJ, Peters W, Rioux JA, Bray RS. Zoonotic cutaneous leishmaniasis in Saudi Arabia: the incrimination of Phlebotomus papatasi as the vector in the Al-Hassa oasis. Trans R Soc Trop Med Hyg. 1985;79(Suppl 2):252–5.View ArticlePubMedGoogle Scholar
  45. Abu-Zinada NY. A spotlight survey of sandflies of the genus Phlebotomus in Jeddah, Saudi Arabia. J Egypt Soc Parasitol 1999;29(Suppl 1):85–9.Google Scholar
  46. Morsy TA, Abou el-Ela RG, Rifaat MM, al Dakhil MA. The seasonal and daily activities of Phlebotomus papatasi in Riyadh. J Egypt Soc Parasitol. 1995;25(Suppl 3):699–711.Google Scholar
  47. El-Sibae MM, Eesa NM. A study on Phlebotomus species, the vectors of leishmaniasis in Gassim, Saudi Arabia. J Egypt Soc Parasitol. 1993;23(Suppl 1):231–8.PubMedGoogle Scholar
  48. Maia C, Parreira R, Cristóvão JM, Freitas FB, Afonso MO, Campino L. Molecular detection of Leishmania DNA and identification of blood meals inwild caught phlebotomine sand flies (Diptera: Psychodidae) from southern Portugal. Parasit Vectors. 2015;8:173.View ArticlePubMedPubMed CentralGoogle Scholar
  49. Rossi E, Bongiorno G, Ciolli E, Di Muccio T, Scalone A, Gramiccia M, et al. Seasonal phenology, host-blood feeding preferences and natural Leishmania infection of Phlebotomus perniciosus (Diptera Psychodidae) in a high-endemic focus of canine leishmaniasis in Rome province, Italy. Acta Trop. 2008;105(Suppl 2):158–65.View ArticlePubMedGoogle Scholar
  50. Yang BB, Chen DL, Chen JP, Liao L, XS H, Analysis XJN. Of kinetoplast cytochrome b gene of 16 Leishmania isolates from different foci of China: different species of Leishmania in China and their phylogenetic inference. Parasit Vectors. 2013;6:32.View ArticlePubMedPubMed CentralGoogle Scholar
  51. Bates PA. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int J Parasitol. 2007;37(Suppl 10):1097–106.View ArticlePubMedPubMed CentralGoogle Scholar
  52. Barhoumi W, Fares W, Cherni S, Derbali M, Dachraoui K, Chelbi I, et al. Changes of sand fly populations and Leishmania infantum infection rates in an irrigated village located in arid central Tunisia. Int J Environ Res Public Health. 2016;13(Suppl 3):E329.Google Scholar
  53. Jaouadi K, Ghawar W, Salem S, Gharbi M, Bettaieb J, Yazidi R, et al. First report of naturally infected Sergentomyia minuta with Leishmania major in Tunisia. Parasit Vectors. 2015;8:649.View ArticlePubMedPubMed CentralGoogle Scholar
  54. Senghor MW, Niang AA, Depaquit J, Ferté H, Faye MN, Elguero E, et al. Transmission of Leishmania infantum in the canine leishmaniasis focus of Mont-Rolland, Senegal: Ecological, parasitological and molecular evidence for a possible role of Sergentomyia sand flies. PLoS Negl Trop Dis. 20016;10(Suppl 11):e0004940.Google Scholar
  55. Berdjane-Brouk Z, Koné AK, Djimdé AA, Charrel RN, Ravel C, Delaunay P, et al. First detection of Leishmania major DNA in Sergentomyia (Spelaeomyia) darlingi from cutaneous leishmaniasis foci in Mali. PLoS One. 2012;7(Suppl 1):e28266.View ArticlePubMedPubMed CentralGoogle Scholar

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