Molecular detection of Leishmania species in Sand Flies by PCR-RFLP technique in refugee camps

[Detecção molecular de espécies de Leishmania em flebotomíneos por meio da técnica PCR-RFLP em campos de refugiados]

ABSTRACT

Leishmaniasis is one of the most important health dilemmas facing the World Health Organization (WHO), due to it being widespread and the great diversity of sand flies that transmit it. This study aimed to detect the presence of Leishmania parasites in the sand flies spread in Refugee camps by PCR- RLFP technique. A total of 437 sandflies were collected and classified into two species Phlebotomus papatasi and Phlebotomus sergenti. DNA was extracted from the female fly species, then the PCR reaction was amplified by two primers (LITSR, L5.8S) that transcribed a partial internal transcribed spacer (ITS)-1 gene for Leishmania parasite with a length of 320 bp. PCR showed the presence of Leishmania DNA in females of both P. papatasi (10%) and P. sergenti (20%). To determine Leishmania species transmitted by the two previous fly species, the RFLP-PCR technique was performed by the HaeIII enzyme for Leishmania DNA extracted from them. RFLP-PCR showed that P. papatasi females transmitted Leishmania major and P. sergenti females transmitted Leishmania tropica in Refugee camps. It could be concluded that leishmaniasis is widely distributed in Refugee camps due to the presence of its vector.

Keywords:
HaeIII enzyme; P. papatasi; P. sergentii; RFLP-PCR; Leishmania

RESUMO

A leishmaniose é um dos mais importantes dilemas de saúde enfrentados pela Organização Mundial da Saúde (OMS) devido à sua ampla disseminação e à grande diversidade de flebotomíneos que a transmitem. Este estudo teve como objetivo detectar a presença de parasitas de Leishmania nos flebotomíneos espalhados em campos de refugiados por meio da técnica PCR-RLFP. Um total de 437 flebotomíneos foi coletado e classificado em duas espécies: Phlebotomus papatasi e Phlebotomus sergenti. O DNA foi extraído das espécies de flebotomíneos das fêmeas e, em seguida, a reação de PCR foi amplificada por dois primers (LITSR, L5.8S) que transcreveram um gene parcial do espaçador transcrito interno (ITS)-1 para o parasita Leishmania com um comprimento de 320 pb. A PCR mostrou a presença de DNA de Leishmania em fêmeas de P. papatasi (10%) e P. sergenti (20%). Para determinar as espécies de Leishmania transmitidas pelas duas espécies de moscas anteriores, a técnica de RFLP-PCR foi realizada pela enzima HaeIII para o DNA de Leishmania extraído delas. A RFLP-PCR mostrou que as fêmeas de P. papatasi transmitiam Leishmania major e as fêmeas de P. sergenti transmitiam Leishmania tropica nos campos de refugiados. Pode-se concluir que a leishmaniose está amplamente distribuída nos campos de refugiados devido à presença de seu vetor.

Palavras-chave:
enzima HaeIII; P. papatasi; P. sergentii; RFLP-PCR; Leishmania

INTRODUCTION

Phlebotomy belonging to family (Psychodidae) is of the order Diptera. It is one of the most important medical insects, as its females form a group of vectors of many pathogens such as protozoa, bacteria, and viruses (Defuentes et al., 2005; Mariwan et al., 2021; Manseur et al., 2022). Leishmania parasites that cause leishmaniasis are transmitted by phlebotomy (Cecílio et al., 2022; Usman et al., 2023). Leishmaniasis is one of the most common infectious diseases globally and exists in multiple forms, including cutaneous leishmaniasis (Al-Rashed et al., 2022; Vries and Schallig, 2022), visceral leishmaniasis (Saini et al., 2020; Scarpini et al., 2022) and mucocutaneous leishmaniasis (Handler et al., 2015; Severino et al., 2022). This disease spreads in four continents Asia, Africa, Europe, and America. The World Health Organization (WHO) estimated that the number of new infections annually ranges between 700,000 to one million infections globally (Leishmaniose, 2022) due to the great diversity of vectors that transmit its parasites. Many methods have been used to detect the presence of Leishmania in living organisms, whether hosts or vectors. The most accurate techniques are polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) (Monroy-Ostria et al., 2014; Echchakery et al., 2017). So, this study aimed to classify Phlebotomus species that spread in refugee camps in Raqqa (Syria). Molecular identification of Leishmania species was also detected in female Phlebotomus species by PCR-RFLP technique.

MATERIALS AND METHODS

Insect collection. Sand flies were collected in the refugee camps in Raqqa (geographical location E º35.9399, Nº38.5887) using sticky paper traps (white papers A4 size greased with castor oil). Flies were identified at both morphological and morphometric levels according to the local taxonomic key of Lewis (1982).

DNA extraction. Total DNA (Phlebotomus DNA and DNA organisms inside them) was extracted from 100 female insects. The 50 females of P. sergenti were divided into 10 groups, each group included 5 females, and similarly, 50 females of P. papatasi were divided into 10 groups, each group included 5 females. Each group was placed in an Ependorf tube, then frozen, and extracted using the QIAamp DNA Mini Kit (Qiagen, Germany).

Polymerase chain reaction (PCR). The PCR mixture was prepared within a volume of 25μL, including 12.5μL reaction mixture (0.2mm dNTPs and 1.5Mm MgCl₂ 1.25U Taq Polymerase, 1× PCR Buffer), 1μL of the forward primer (L5.8S: 5ˊ-TGA TAC CAC TTA TCG CAC TT-3ˊ), 1μL of the reverse primer (LITSR: 5ˊ-CTG GAT CAT TTT TCC GAT G-3ˊ), 4μL of the DNA sample, and then complete the mixture with 6.5.μL distilled water for the final volume of 25μL. The amplification reaction was carried out in the PEQ Lab thermal circulator according to the following program: initial denaturation at 94°C for 5 min, then 30 cycles consisting of 94 °C for 30 sec, primers coalesced at 53°C for 40 sec and copied at 72 °C for 1 min. Finally, a copy was performed at 72°C for 10 min (Schonian et al., 2003).

Gel electrophoresis. The PCR products were separated by electrophoresis on the Agarose gel at a concentration of 2% by a Mupid-One electrophoresis device (Takara company). Gel was prepared by dissolving 2gm of Agarose powder in 100mL electrophoresis solution 1×TBE (Tris-Borate-EDTA buffer) and warmed using a microwave, then cooled to about 50°C and added 5μL of ethidium bromide at a concentration of 0.5mg/mL, after that poured into gel composition mold until solidification, where placed in an electrophoresis tank containing 1000mL of 1×TBE electrophoresis solution. Loading the samples: First, 3μL of Loading buffer solution was mixed with 7 μL of PCR products, and then loaded into the gel wells. Similarly, 5μL of the marker was mixed with Loading buffer solution and loaded in the 1^st well. Finally, electrophoresis was performed at a potential difference of 100 volts for 30 minutes. Gel was examined by UV in a gel documentation device, a digital image was taken for the gel to analyze the results.

Restriction fragment length polymorphism (RFLP). Enzymatic restriction of PCR products was carried out according to the following steps: (1) 15μL of DNA amplified by PCR technique was placed in the Eppendorf tube. (2) 10 units of the restriction enzyme (HaeIII) were added. (3) 5μL of the restriction enzyme protective solution. (4) Incubate previous mixture at a temperature of 37°C for 2 hr. (5) Transfer the mixture to the electrophoresis device to show the results.

RESULTS

Identification of sand flies collected from refugee camps. A total of 437 sand flies were classified according to local taxonomic key in two species, Phlebotomus papatasi and Phlebotomus sergenti. P. papatasi was more prevalent than P. sergenti (Table 1).

Phlebotomus were characterized by their small size, as length of its members ranged between (2-4) mm and their yellow-brown color. Its body was covered with dense hairs. The wings are placed during rest diagonally at an angle of 45 degrees upwards, thus taking the form of a butterfly, and the head is located perpendicular to the axis of the body, elongated in shape, and covered with dense hairs (Figure 1 A, B). The most important taxonomic characteristic of males P. papatasi is back end, which was characterized by the presence of the distinctive cylindrical shape and equipped with five spoon-shaped spines, three of which are final (located at the end of the pen) and two basal (Figure 1 C). The most important feature that distinguishes females of P. papatasi is the sperm preservative, which consisted of ten rings, the last of which was the largest and carried many poems (Figure 1 D).

Table 1
Species of sand flies collected from refugee camps

Species	Sandflies		Total	Percentage
	Males	Females
P. papatasi	218	61	279	63.84 %
P. sergenti	103	55	158	36.16 %

The most important taxonomic characteristic of males P. sergenti is the posterior end, which contains an oval-shaped reproductive pen, with two long spines at the end, one thick and another thin near its base (Figure 2 A). Most important feature of females P. sergenti is the presence of a sperm capsule: it consists of (4-6) rings, and the last ring is larger than the others (Figure 2 B).

Detection of Leishmania DNA in P. papatasi by PCR. About 50 females of P. papatasi were divided into 10 groups (each group included 5 females) and then were examined. The DNA was amplified by PCR with the primers (L5.8S and LITSR) for the partial ITS-1 gene region. PCR showed that the results of 10% (1/10 groups) were positive only for Leishmania DNA and the length of the amplified fragment was 320 bp (Figure 3).

Detection of Leishmania DNA in P. sergenti by PCR. About 50 females of P. sergenti were divided into 10 groups (each group included 5 females) then were examined. The DNA was amplified by PCR with the two primers (L5.8S, LITSR) for the partial ITS-1 gene region. PCR showed that the results of 20% (2/10 groups) were positive only for Leishmania DNA and the length of the amplified fragment was 320 bp (Figure 4).

PCR-RFLP. It was performed using HaeIII enzyme and the results of electrophoresis were as follows: The results of PCR-RFLP on DNA extracted from Leishmania present in P. papatasi showed two bands, the first with a length of approximately 200 bp, and the second with a length of approximately 120 bp. This corresponds to the DNA of Leishmania major parasite (Figure 5). The results of PCR-RFLP on DNA extracted from Leishmania that is present in P. sergenti showed two bands, the first with a length of 190 bp, and the second with a length of 60 bp, and this corresponds to the DNA of the Leishmania tropica parasite.

Figure 1
Sand fly of P. papatasi. (A) male, (B) female, (C) pen in male, (D) sperm preservative in female.

Figure 2
Sand fly of P. sergenti. (A) male reproductive pen, (B) sperm preservative in females.

Figure 3
Electrophoresis of PCR products by two Primers (L5.8S, LITSR). M: marker, Numbers groups were detected of Leishmania DNA in P. papatasi

Figure 4
Electrophoresis of PCR products by two Primers (L5.8S, LITSR). M, marker; L1, L2 two groups contain Leishmania DNA in P. sergenti

Figure 5
PCR-RFLP products by HaeIII (M, marker; L1, DNA Leishmania major in female P. papatasi; L2, L3, DNA Leishmania tropica in P. sergenti.

DISCUSSION

In this study, the PCR-RFLP technique of the partial ITS-1 gene region was used to identify species of Leishmania transmitted by (sand flies) Phlebotomus that spread in refugee camps. PCR-RFLP is an important and widely used technique to identify Leishmania species (Gadisa et al., 2007; Mouttaki et al., 2014; Mosleh et al., 2015) and compare those species.

In recent years, cases of cutaneous leishmaniasis in refugee camps have increased. In the past, it was widely believed that all Phlebotomus species are transmitted Leishmania species without specific, but according to reference studies, there is a variation between Phlebotomus species in ability to transport Leishmania species, due to geographically related and varied from one country to another, so this research came to detect Leishmania in Phlebotomus species that spread in refugee camps and to identify species of Leishmania transmitted by each Phlebotomus species.

A total of 437 sand flies were classified into two species P. papatasi and P. sergenti. P. papatasi (63.84%) was more prevalent than P. sergenti (36.16%). A possible hypothesis for this issue is that females P. papatasi feed on rodent blood in addition to human blood, while females of P. sergenti prefer to feed on human blood only (Akhoundi et al., 2013) and this is consistent with taxonomic studies of Phlebotomus in the world that confirm the dominance of the spread P. papatasi on the rest of the species of sand flies (Sofizadeh et al., 2017; Zivdari et al., 2018; Karmaoui, 2020).

The results of the PCR showed the presence of Leishmania DNA in females P. papatasi (10%) and P. sergenti (20%). This is consistent with other studies that reported P. papatasi (Parvizi et al., 2005; Parvizi and Ready, 2006) and P. sergenti (Es-Sette et al., 2014; Ajaoud et al., 2013) could transmit Leishmania parasites.

PCR-RFLP with HaeIII restriction enzyme for PCR products on the DNA of Leishmania parasites extracted from females P. papatasi showed the presence of two bands, 200 bp and 120 bp. When comparing this result with the relevant global studies, it was found that the DNA extracted from P. papatasi belongs to Leishmania major, and this proves that P. papatasi is a vector of the Leishmania major parasites in refugee camps. This corresponds to previous studies (Parvizi and Ready, 2008; Dabirzadeh et al., 2016; Kykalová et al., 2021) reported that the main vector of Leishmania major is P. papatasi in the Middle East (Knight et al., 2023) and North Africa (Karmaoui et al., 2022) and several countries as Sudan (Soltani et al., 2015).

The results of PCR-RFLP performed on the DNA of Leishmania parasites extracted from female P. sergenti showed the presence of two bands, 190 bp and 60 bp, it belongs to Leishmania topica parasites and this is confirmed by global studies (Dweik et al., 2013; Mosleh et al., 2015; Es-Sette et al., 2014; Koohsar et al., 2020), and this proves that P. sergenti is a vector of the Leishmania tropica parasite in refugee camps, and this is consistent with global studies (Mosleh et al., 2015; Karmaoui et al., 2022) reported that in Jordan, P. sergenti is the primary vector of Leishmania tropica (Janini et al., 1995), as well as, in Saudi Arabia (Karmaoui et al., 2022) and Turkey (Demir and Karakuş, 2015).

By comparing the percentage of prevalent L. major and L. tropica in our study (10% to 20%) with another study (Paiva et al., 2006), it was the highest in refugee camps, and due to the poor living, the proximity of refugee tents to each other and low level of health. L. tropica is more prevalent than L. major because the former which is transmitted by females of P. sergenti does not require a stored host and is transmitted between humans only, while the latter, which is transmitted by P. papatasi females, requires a stored host from rodents (Mirzaei et al., 2014; Karakuş et al., 2019).

CONCLUSION

It could be concluded that leishmaniasis is widely distributed in refugee camps (Syria) because of the natural occurrence of its vector. Further studies should be done to control sand flies to reduce the spread of leishmaniasis.

ACKNOWLEDGMENTS

Authors extend their appreciation to Researchers Supporting Project (RSP2024R99) King Saud University, Riyadh, Saudi Arabia.

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Publication Dates

• Publication in this collection
  24 June 2024
• Date of issue
  Jul-Aug 2024

History

• Received
  06 Nov 2023
• Accepted
  02 Jan 2024