Detection of Leishmania tropica Using Nested-PCR and Some of Their Virulence Factors in Thi-Qar Province, Iraq

Cutaneous leishmaniasis is one of endemic diseases in Iraq. It is considered as widely health problem and is an uncontrolled disease. The aim of the study is to identify of Leishmania species that cause skin lesions among patients in Thi-Qar Province, South of Iraq, also to detect some virulence factors of L. tropica. This study includes three local locations, Al-Hussein Teaching, Suq Al-Shyokh General and AlShatrah General Hospitals in Province for the period from the beginning of December 2018 to the end of September 2019. The samples were collected from 80 patients suffering from cutaneous leishmaniasis, both genders, different ages, various residence places and single and multiple lesions. Nested-PCR technique was used to amplify kinetoplast minicircle fragments DNA (kDNA). Conventional-PCR was performed for determination of some virulence factors (LPG1, GP63, CPA and PPG1 genes). The electrophoresis findings of kDNA gene showed two species of the parasite found in the study area, 65 samples were positive for cutaneous leishmaniasis, L. tropica at 750bp and L. major at 560bp. Generally, L. tropica (57.5%) was the most common specie and L. major (23.75%) appeared in a low level. There are no significant differences between the infections of males and females, while there are significant differences at the comparison between age groups. All virulence genes (LPG1, GP63, CPA and PPG1) appeared in all L. tropica isolates with high percentage (100%). L. tropica is the major specie which that caused CL in Thi-Qar province, while L. major appeared in low incidence. The virulence genes, which were reviewed, are necessary and important in pathogenesis of L. tropica.


Introduction:
Cutaneous leishmaniasis (CL) is considered as a global health problem, it is caused by the intracellular protozoa that belongs to the Leishmania genus, that is transmitted by the bite of the sand fly species (1). The most common leishmaniasis form is CL which infects skin epidermis to form papule usually painless and chronic, often at site of infected sand fly bite (2). Leishmania tropica and L. major cause CL and have same life cycle, but they are spread in different localities and have different intermediate and reservoir hosts (3). The disease transmits into human when human, sand fly and the reservoir share same the environment (4). L. tropica causes anthroponotic cutaneous leishmaniasis (ACL), while L. major causes the zoonotic cutaneous leishmaniasis (ZCL) (5). Leishmania must evade from the innate and adaptive immune responses. It is engulfed by macrophage but resist proteolysis and degradation in the phagolysosome (6). An infection of any type of Leishmania depends on complex, virulence factors and host immunologic response (7). Leishmania produces various virulence factors to facilitate transmission of parasite and infect mammalian host (8). Lipophosphoglycan (LPG) has an important role not only in invertebrate and vertebrate host, but too during early steps for the establishment of the infection (9). However, LPG plays an important role to avoid of the parasite lysis by the host complement system, either by preventing complement molecule attachment or inactivate the assembly of complement complex on the promastigote surface (10). It binds with complement receptor (CR3) and anchored molecules on macrophage surface to facilitate parasite engulfment (11). LPG1 is one of the key enzymes in the LPG biosynthesis, that are involved in the synthesis of the LPG glycan core. As well as, Leishmania that lack lpg1 gene express incomplete LPG, not contain PG domain (12). GP63 is as known leishmanolysin or is called Major Surface Protease (MSP) or promastigote surface protease (PSP), also it is called a zinc-dependent metalloprotease (13). GP63s vary in their sequence (especially in untranslated regions), array in the Leishmania genome (14,15). GP63 cleaves complement protein C3b and converts to C3bi, this leads to silent entry of parasite into macrophage (16). GP63 is responsible for a migration of the parasite through the extracellular matrix, avoid lysis by the complement system, evasion from macrophagic intracellular hydrolysis (12,17). Cysteine proteases (CPs) are degrading enzymes which cleave various proteins, as well play an essential role in many pathogenic protozoa and other microorganisms (18). CPs appear to have an essential role in pathogenicity, modulation or evasion of the host immune response, cell/ tissue degradation and damage, catalyze hydrolysis of various host proteins differentiation of promastigote and autophagy (19,20). There are several types of PPGs, including filamentous proteophosphoglycan and GPI-anchored membranous proteophosphoglycan (21). PPG is proved to bind to macrophage receptors in order to facilitate parasite invasion; this may explain that PPG plays an early role in infection and the prevalence (22,23). The PPG1 gene encodes a membrane-bound PPG (24). Lima et al. (25) confirmed a role of L. infantumderived LPG which it plays in the pro-inflammatory response in Leishmania infection. Mohammad and Hmood (26) recorded gp63 and GIPLs by a ratio of 100% in all L. major samples in AL-Qadisiyah Province in Iraq.
Despite of the importance of cutaneous leishmaniasis, and to our knowledge, there is limited information about Leishmania species prevalent in the area. This study aims to identify Leishmania species that cause human skin lesions in Thi-Qar Province, south of Iraq by kinetoplast minicircle DNA gene, as well as determine some virulence genes of L. tropica.

Study area and sample collection
The protocol of this study was approved by management of training and human development department/ Thi-Qar Health Office (32877 at 2/12/2018). The study includes three locations to the samples collected: Al-Hussein Teaching, Suq Al-Shyokh General and Al-Shatrah General Hospitals in Thi-Qar Province, South of Iraq for the period from the beginning of December 2018 to the end of September 2019. The hospitals have received patients suffering from cutaneous diseases. All medical information was taken from the hospital records. Written consent is obtained from all participants by the collector and the participation in the study is voluntary and they were free to withdraw at any time from the study. The samples were collected from lesion fluid of 80 patients with CL, both genders, different ages, various residence places in the province and from single and multiple lesions. Expected skin infection lesions were injected using normal saline (0.2 ml) in lesion edge, then pulled again, after that the fluid was kept in plain tube (27,28).

Genomic DNA extraction
DNA was extracted from lesion fluid using gSYAN DNA kit extraction kit Geneaid according to protocol of produced company (Geneaid/ Taiwan). DNA concentration was examined using a Nanodrop spectrophotometer and then stored at -20˚C until used in PCR amplification.

Nested-PCR amplification
The kDNA was amplified for identification of Leishmania isolates, using Nested-PCR, with some modification of PCR according to (29), which included two steps. Target DNA undergone the first run with external primers: CSB2XF and CSB1XR (Table 1), then the first run product undergone second run with internal specific primers:13Z and LiR.PCRmaster mix was prepared by (AccuPower® PCR PreMix kit. Bioneer, Korea). Nested PCR primers were provided by Macrogen Company, Korea. Nested PCR master mix prepared 5µL of genomic DNA, 10 pmol of each external primer and 13 µL of PCR water and placed in standard PCR tubes. Thermal condition of the PCR reaction included an initial denaturation at 95°C for 5 min followed by 30 cycles at 95˚C for 30 sec., 55˚C for 30 sec. and 72˚C for 1min and finally final extension 72˚C for 5min. A nested PCR master mix of second run included 3µL of first run product, 10 pmol of each internal primer and 15 µL of PCR water and placed in standard PCR tubes with thermal conditions for the PCR reaction. The PCR products passed electrophoresis in1% agarose gel with 3µL of ethidium bromide. PCR product (10 µl) was added into each comb well and 5 µl of (100bp ladder) in each well. Gel tray was fixed in electrophoresis chamber and filled by 1X TBE buffer. The electric current was connected at 100 volts and 80 mA for 1 hr. PCR products were visualized using an ultraviolet transilluminator.

Conventional-PCR amplification
The PCR technique was performed for detecting virulence factor genes in L. tropica. This method was carried out according to the method described by Al-Difaie (27). The primers were provided by Macrogen company, Korea. PCR master mix prepared 5µL of genomic DNA, 10 pmol of each R and F primers and 13 µL of PCR water and placed in standard PCR tubes. Thermal conditions and agarose electrophoresis are similar to the above.

Statistical Analysis
In the study, method of Chi-Square (χ 2 ) test was used in order to analyze the data using SPSS statistical package software V.17. Significant level was at P <0.05.

Results:
Agarose gel electrophoresis results for 80 samples of mitochondrial kinetoplast minicircle fragments DNA (kDNA) gene were amplified by Nested PCR, discovered 65 (81.25%) positive for cutaneous leishmaniasis (contain Leishmania parasite) and 15 (18.75%) negative samples, where observed significant difference (P>0.05) between them (Table 2). Furthermore, the results showed that two species of the parasite which that found in the study area. Statistically, there is a significant difference between L. tropica and L. major. L. tropica recorded the most common species, while L. major was a low incidence. 46 (57.5%) positive samples were L. tropica at 750bp and 19 (23.75%) positive samples were L. major at 560bp (Fig. 1). There are no significant differences between the infection of 24 (30%) males and 22 (27.5%) females. The age groups ≤ 20, 21-40 and >40 years old recorded 33 (41.25%), 10 (12.5%) and 3 (6.5%) respectively, and showed significant differences (Pvalue = 0.000) when the compared with other age groups. The electrophoresis results of the PCR product of LPG1, GP63, CPA and PPG1 genes observed in all of cutaneous Leishmania which were recorded as L. tropica isolates in the study (Figs. 2,3,4 and 5) at 311bp, 565 bp, 437 bp and 568 bp PCR product sizes, respectively.

Discussion:
Cutaneous leishmaniasis is an endemic disease in several countries. Foci of L. major in Egypt, Jordan, Libya, Sudan and Tunisia, while Afghanistan, Pakistan, Iran, Saudi Arabia, Syria, Iraq, Morocco, and Yemen are endemic for both L. tropica and L. major (30). In this study, Leishmania spp. was identified by kDNA. Amplification kDNA gene is a suitable coal for differentiating between CL species and numerous diagnostic tests (31,32). kDNA gene is the most sensitive since kinetoplast contains about 10,000 circular and convoluted kDNA minicircles per parasite (31,33).
Lieshmanial DNAs have been recorded in 65 (81.25%) cases and the rest were negative samples. The appearance of negative sample revealed not withdraw the parasite during the collection and may lack DNA in the sample or its crash. In addition, the results have shown that most CL patients in Thi-Qar Province were infected with L. tropica, while L. major was in a little number of patients. Geographical expansion and globally increase of leishmaniasis is still associated with vector population expansion (34). Also, increase in migration, rapid urbanization, deforestation and Leishmania adaptation with mammalian hosts and additional vectors (35). CL spread from endemic to non-endemic regions, if both sandfly vectors and reservoir hosts are present (36). The results of current study were no significant differences between infection of the males and the females, this may be due to the fact that Leishmania parasites infect both sexes at the same extent. Furthermore, there is no clear explanation for infection differences according to the gender. It might be individual and behavioral factors that play a role as mentioned in previous studies (37). This result is somewhat close to the results of Al-Difaie (27) (38) in Morocco. This result is inconsistent with Alsamarai and Alobaidi (39) Al-Haweja City and Rahi (40) in Wasit Province. The study recorded significant differences (p < 0.05) between other age groups. The highest rate of infection by L. tropica was among age group of ≤ 20 years old (41.25%), then followed by age group 21-40 years old. This can be explained as a result to previous exposure of the infection which give permanent immune for individuals (39). This is in consistent with the findings of Al-Difaie (27), El Hamouchi et al. (38) and Hassan et al. (41) in Kirkuk City. It is also inconsistent with Rahi (40) in Wasit Province, Khosravi et al. (37) and Ramezany et al.(32) in Iran.
The current study recorded the presence of Lipophosphoglycan (LPG1), leishmanolysin (GP63), Cysteine proteinase A (CPA) and Proteophosphoglycan (PPG1) genes in all of L. tropica isolates (100%). Al-Difaie (27). has found LPG, PPG and CP in all of L. major isolates in Al-Qadisiya Province. In Brazil, Lázaro-Souza et al. (12) have mentioned a growth of L. infantum delayed if deletion of LPG1 gene. L. major mutants which lack LPG1 and LPG2 genes are sensitive to host complement system, this leads to unable survival in both intermediate or definitive host cells (23). GP63 is not only to degradation and damage of transcription factors and various kinases, but also it modulates negative regulatory mechanisms of signaling pathways (15). Cysteine protease inhibition appears as an important strategy for elimination of disease. They are necessary for metabolism, intracellular survival and reproduction of parasite (42). CPA plays an essential key in the interactions of parasite with host cells, but is not important to replication promastigotes (43). Other studies have shown that inhibition of CPA and CPB or deletion their genes not only alters autophagy pathway, but for prevents transformation into amastigotes, thus support the hypothesis of autophagy are required for the differentiation (44,45). Samant et al. (45) have suggested that possibilities of PPG in drug resistance mechanisms and PPG abundance of L. donovani is as an evidence for resistant clinical isolates. PPG1 may play a direct important role in host-parasite interactions (46).

Conclusion:
The importance of Thi-Qar Province comes from frequent infections of CL. As all know, there is a very little data about the identification of Leishmania species responsible of human CL. The findings have showed two species co-existing in the study area that were L. tropica and L. major. The most cases of cutaneous lesions have been infected with L. tropica, that is the prevalent species. Some virulence genes have been investigated which confirmed their necessity and importance in pathogenesis of L. tropica. Therefore, it is essential to conduct more studies on the sandfly vectors and reservoirs of Leishmania species. However, nested-PCR is more suitable for direct diagnosis of Leishmania at species level.

Acknowledgements:
Out of gratitude, the authors thank the staff of dermatology and laboratory departments in Al-Hussein Teaching, Suq Al-Shyokh General and Al-Shatrah General Hospitals for their helping during the sample collection. Also, we would to say thanks for public health department-Thi-Qar health center for their kind assistance. This study did not support financially.