Molecular study to diagnose and identify Leishmania spp. that causes Cutaneous leishmaniasis in humans in Maysan Governorate, Iraq.
Samer Hashim Alwan Al-Lamy* and Fadhil Abbas MunshedAl-Abady
Department of Biology, College of Education for Pure Sciences, University of Thi-Qar, Thi-Qar, Iraq.
*Email: [email protected] Abstract
The study aimed at molecular diagnosis of CL, Differentiate between two types of Leishmania spp., L. tropica and L. major, Determination of kDNA gene sequence, Recording of local isolates in NCBI-GenBank and its comparison with global isolates and Design of a phylogenetic tree to discover relationships between species. The study was conducted in the two main hospitals of Amarah city, Maysan as blood swabs were collected from skin lesions for 80 samples for the period from the beginning of March to the end of December 2020, Samples were examined in Central Health Laboratory in Maysan Governorate, DNA was extracted and then multiplied by a technique Nested-PCR and determine a genetic sequence of kDNA gene, after which Statistical analysis was done, Genetic sequence analysis was done, and the results were sent to the NCBI- GenBank to obtain accession numbers for local isolates and compare them with global isolates, and draw a phylogenetic tree. Results of Electrophoresis showed 50% for both positive and negative samples to CL, while the positive samples were 31.25% of Leishmania tropica and 18.75% of Leishmania major.
Keywords: Cutaneous leishmaniasis; Leishmania major; Leishmania tropica; kDNA gene;
Nested-PCR; Phylogenetic tree.
Introduction
The World Health Organization (WHO) has indicated that leishmaniasis is considered a tropical disease worthy of attention and a dangerous health problem. It has many clinical symptoms that can lead to death (1). Leishmaniasis is endemic in 98 countries and there are about 12 million or more infected and about 2 million new cases of leishmaniasis recorded annually (2).
Leishmaniasis appears in three different clinical forms: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL) (3). Leishmaniasis is a parasitic disease that results from infection with flagellated parasites of the genus Leishmania, which belong to the intracellular protozoan phylum and infect mammals, including humans (4).
Leishmaniasis is a transmissible disease that occurs through the bite of an infected female sand fly that transmits the parasite. Infection occurs widely in poor populations inhabiting tropical and subtropical countries (5).Vector insects are sandfly that belongs to the genus Phlebotomus and Lutzomyia which includes several species (1). Leishmania has two different forms, Promastigote is present in Invertebrate host, Amastigote is present in Vertebrate host (6). The most prevalent clinical manifestation of leishmaniasis is CL, where the symptoms appear on the skin in the form of a papule often at the site of the infected insect bite, and the papule is usually chronic and painless (7). It is worth noting that after An incubation period that may extend for several months, the vertebral host exhibits symptoms of a skin lesion, which usually gains healing spontaneously and gives permanent immunity but leaves a dejected scar (8). Generally, there are 2 million new cases of cutaneous leishmaniasis reported annually in Iraq, Saudi Arabia, Syria, Algeria, Iran, Afghanistan, Brazil and Peru (9). World Health Organization has identified endemic diseases in many countries of the world, including Iraq, as the infection occurs due to a bite of a female sand fly that carries the parasite of the type Leishmania tropica (dry or urban) or Leishmania major (Wet or Rural) (10). source of infection in first type is Anthroponotic (ACL), in second type is zoonotic (ZCL) (11). Diagnosis of CL is mainly based on clinical features, but laboratory confirmation of CL depends on microscopic examination of Giemsa stain lesion edge scraping or fine needle aspiration, but this procedure has a low
sensitivity (12). Therefore, molecular assays are widely used for epidemiological and diagnostic purposes to identify the parasite at a molecular level or species in hosts and vectors (13). Those methods of PCR technique are used to determine the genus of Leishmania, Species or subspecies that depend on different target genes such as Kinetoplast DNA gene (kDNA) (14). Aim of the study is a molecular diagnosis of CL, differentiate between L. tropica and L. major, Determination of kDNA gene sequence, Recording of local isolates in NCBI-GenBank and its comparison with global isolates and Design of a phylogenetic tree to discover relationships between species.
Materials and Methods
Geographical area and sample collection:
This study was conducted in Maysan Governorate, which is located in the southeastern part of Republic of Iraq, extends in a northeast-southwest direction, lies between latitudes 31.15o - 32.45o north and longitudes 46.30o - 47.30o east, bounded on the north and northwest by Wasit Governorate, from the south by Basra Governorate, from the west, Thi-Qar Governorate, while it is bordered on the east and northeast by Republic of Iran, the area of the Governorate is 16072 km2 and includes 6 districts and 9 sub-districts, thus it represents 3.7% of the area of Iraq amounting to 435025 km2 (15). The field of the study included two hospitals: Al-Sadr Teaching Hospital and Al-Zahrawi Surgical Hospital, Samples were collected from 80 people with cutaneous leishmaniasis in Maysan governorate, for the period from the beginning of March to the end of December 2020, Samples were examined in Central Health Laboratory in Maysan Governorate, to identify the type of leishmaniasis by the kDNA gene. Blood samples were collected from the patients by disinfecting the skin surrounding the lesion with 70% alcohol, then injecting 0.2 ml of normal saline solution by means of a 3 ml syringe and horizontally inside the red edge of the lesion, after which the injected liquid mixed with blood was withdrawn to obtain the parasite's DNA. The withdrawn liquid is placed in an Eppendrof tube 1.5 ml and stored at a temperature -20°C until it is used for DNA extraction, Nested-PCR and molecular diagnosis of the parasite (16).
Genomic DNA Extraction:
DNA was extracted from the skin lesion fluid using a gSYNC™ DNA Extraction Kit (Geneaid / Taiwan) according to the manufacturer's protocol, The extracted DNA was examined using Nanodrop spectrophotometer, which measures the concentration of DNA in units (ng/μl) and verifies a purity of the DNA by reading the absorbance at (260/280 nm), then stored at -20°C until used in Nested-PCR amplification.
Nested-PCR amplification:
Nested PCR includes use of two primers that go into two consecutive cycles, the output from the first cycle is the target of the second cycle in order to amplify it (17). Nested-PCR is used to identify Leishmania spp. which is achieved by amplifying kDNA gene in two steps (18), External primers for target DNA: CSB2XF 5'-CGA GTA GCA GAA ACT CCC GTT CA-3' and CSB1XR 5'-ATT TTT CGC GAT TTT CGC AGA ACG-3', Internal primers for first run output: 13Z 5'- ACT GGG GGT TGG TGT AAA ATA G-3' and LiR 5'-TCG CAG AAC GCC CCT-3'. Primers were provided by Macrogen Company, Korea. Nested-PCR-master mix was prepared according to GoTaq® G2 Green Master Mix kit by Promega, USA. Nested-PCR-master mix of first cycle was prepared from 5μL of genomic DNA, 1 μL (10 pmol) for each of external primers (forward and reverse), 12.5 μL of Green master mix and 5.5 μL of PCR water and put in Eppendorf tubes.
Thermocycler conditions of external Nested-PCR reaction included the following steps: Initial denaturation, 1 cycle at 95oC for 5 min , the second step included (denaturation, annealing and extension), 30 cycles at 95°C for 30 sec, 55°C for 30 sec and 72°C for 1 min, respectively, following step included final extension, 1 cycle at 72°C for 5 min. Nested-PCR master mix of
second cycle was prepared from 5 μL of first cycle output, 1 μL (10 pmol) for each of internal primers (forward and reverse), 12.5 μL of Green master mix and 5.5 μL of PCR water and put in Eppendorf tubes. PCR program was turend on. Products of PCR pushed through electrophoresis in agarose gel (1%) with ethidium bromide (3 μL). Gel tray was settled in electrophoresis chamber and loaded with 1X TBE buffer. Addition 10 μL of Product of PCR and 5 μl of 100bp ladder in to each well of comb. Then electric current passed at 100 volts and 80 mA for 1hr. Then electric current passed at 100 volts and 80 mA for 1hr and using ultraviolet transilluminator to visualize Products of PCR.
Sequencing, data analysis and Phylogenetic tree:
Ten samples belonging to the kDNA gene were seen on the gel, with an amount of 5 samples for each of L. tropica and L. major, then, Product of PCR purified and sent to Macrogene (Korean Company) for genetic sequence analysis. After obtaining the sequences of kDNA gene, It was sent to the genebank (NCBI-GenBank) in order to obtain accession numbers for recording and compared matches and mismatches between kDNA sequences in L.tropica and L. major isolates and the global L.tropica and L. major strains in Genebank using BLAST analysis software, (http://blast.ncbi.nlm.nih.gov/Blast.cgi) in order to regulate compatible sequences for each isolate.
kDNA gene sequences were cut by alignment analysis ClustalW using MEGA X software and multiple alignment analysis tool (MSG). Phylogenetic tree completed by analyzing a bioinformatics application, a multiple sequence alignment analysis (MSG) program, and using the UPGMA tree method in MEGA X version (19).
Statistical Analysis:
In this study, the experimental data analyzed by using the statistical program SPSS software V.25 (Inc., Chicago, IL, USA) to find the significant differences by using Chi-Square test (X2) at a significant level P=0.05 (20).
Results and Discussion
Molecular diagnosis of Cutaneous leishmaniasis showed 40 out of 80 (50%) for each of positive and negative samples. Statistical analysis showed that no significant differences between them at P>0.05. Also, molecular identification of positive samples showed two species of Leishmania in the study area: L. tropica recorded 25 out of 40 samples (62.5%), L. major recorded 15 out of 40 samples (37.5%). statistical analysis showed significant differences between them at P<0.05.
The agarose electrophoresis images showed product of the analysis of Nested-PCR reaction and the molecular sequencing of kDNA gene within DNA extracted from the skin lesion samples. As the path M: marker, its sequence (100bp - 2000bp) and the path (1-80) showed negative and positive samples which includes: L. major at 560 bp and L. tropica at 750 bp (Figure 1).
The present study aims to determine kDNA nucleotide sequence of 5 clinical isolates for each of L. tropica and L. major, which were selected from different places for the study area. Data of the nucleotide sequence was submitted to GenBank database, registered on the website, and provided with accession numbers, L. major isolates begin with MW550269 and end with MW550273 , L.
tropica isolates begin with MW550274 and end with MW550278. The results of the kDNA nitrogen bases sequence analysis for all studied samples were 99-100% consistent with those recorded by NCBI (Tables 1,2).
Phylogenetic tree designed to determine the extent of genetic convergence between local isolates and global strains. Local isolates of L. major (MW550269.1, MW550271.1( showed 100% match with Iranian isolate (KC869687.1), also (MW550270.1) showed 100% match with Iraqi isolate (MH511151.1) at total genetic change (0.10-0.30). Local isolates of L. tropica (MW550274.1, MW550276.1, MW550278.1) showed 100% match with Iranian isolate (AB678350.1) at total genetic change (0.006-0.020) (Figures 2,3).
PCR is a technique characterized by high sensitivity and reliability compared to conventional diagnostic methods (21). PCR-based diagnostic methods were faster and more sensitive to
diagnose leishmaniasis than conventional methods (22). Molecular methods based on PCR are necessary to diagnose leishmaniasis in clinical samples, in addition to distinguishing between parasites in terms of type, strains and genotypes (23). In the present study, Nested-PCR method was used to identify Leishmania spp. depending on the kDNA gene, due to the effectiveness of this technique for diagnosing and identifying leishmaniasis (24). Electrophoresis results for 80 skin lesions of 40 (50%) CL positive samples (containing Leishmanian DNA). The reason for the appearance of negative samples may be due to failure to withdraw the parasite during sample collection, lack of DNA in the sample, or a wrong clinical diagnosis of CL. However, the low sensitivity of PCR technique for detecting Amastigotes in samples that showed positive results on microscopy was often due to a lack of parasite loading in the original sample (25). This results is close to Abdolmajid et al. (11) recorded 66 (55%) positive cases by PCR. Ghasemloo et al. (26) recorded 98 (75.4%) and 70 (53.8%) positive cases by kDNA-PCR and ITS-PCR respectively after examined 130 CL lesions. Rahi et al. (27) showed that positive cases recorded 70% using kDNA of Leishmania parasites by Nested-PCR technique. The results of the study showed that there are two types of Leishmania in the local area. The highest percentage of CL patients in Maysan governorate were infected with L. tropica was 62.5% , while the percentage of L. major was lower in patients, 37.5%. Statistically, significant differences were observed between the two types. The geographical expansion of leishmaniasis and the global increase in those infected are associated with an overpopulation of the vector (28). In addition, there are other factors such as increased migration, expansion in urban areas at a high rate, elimination of forests, adaptation of Leishmania to host mammals and a number of additional vectors (29). In general, it is important to detect and diagnose the type of infection, whether L. major or L. tropica , Since the use of treatment is subject to different protocols (25), however, L. tropica is more common in the southern governorate, while L. major is more common in the northern and central governorate of Iraq. This results is close to Flaih (30) recorded 46 (57.5%) of L. tropica, while L. major was 19 (23.75%) out of 80 samples in Thi-Qar governorate. Seaad et al. (31) in Al-Qadissyia governorate, found 21 (33.87%) out of 62 samples, identified as L. major, whereas the rest believed L. tropica. Abdolmajid et al. (11) showed 66 (55%) of positive cases; included 46 L.
tropica and 20 L. major in Khorasan-Razavi governorate, Iran. Ghasemloo et al. (26) recorded in Kashan, Iran using kDNA-PCR; 71.4% L. tropica, 26.5% L. major and 2.1% for other types. The result was inconsistent with Abul-Doanej (32) in Maysan governorate , recorded 63.6% of L.
major and 36.4% of L. tropica. Al-Awadi (33) examined 95 samples in Thi-Qar governorate, and 70 samples were recorded positive for the disease, The results of the molecular examination showed 55 samples (57.9%) of L. major and 15 samples (15.8%) of L. tropica. Al-Hassani (16) recorded 52 (94.5%) positive samples in Eastern Al-Hamzah district, AlQadisiya governorate, the result was 49 (89.1%) samples were infected by L. major and 3 (5.4%) were infected by L.
tropica. Rahi et al. (27) identified L. major and L. tropica (38 and 4 samples respectively) in tested 42 positive samples in Kut City. Also, Izadi et al. (2) In Iran, diagnosed L. major as the cause of cutaneous leishmaniasis with 100% in Isfahan and 90.7% in Shiraz, while 9.3% of cases were detected in the city center of Shiraz due to L. tropica .
Conclusions
The most common cause of cutaneous leishmaniasis in Maysan governorate was L. tropica, In addition to L. major at a lower rate. The phylogenetic tree showed the genetic affinity between local Leishmania isolates and each of Iraqi and Iranian strains, whether L. tropica or L. major.
PCR technique is a sensitive and alternative method for traditional screening, diagnosis and differentiation of L. spp. Variation of kDNA nitrogen bases sequence in L. tropica and L. major isolates may cause different clinical symptoms of cutaneous lesion. Therefore, Diagnosis of genetic heterogeneity is important for determining appropriate treatment, control strategy and epidemiological studies.
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Table 1: Sequence matching between L. major isolates, local and registered in NCBI L.
major isolate number
GenBank accession number
Homology sequence identity L.
major country
GenBank accession number
Identity
%
No.1 MW550269.1 Iran KC869687.1 100
No.2 MW550270.1 Iraq MH511151.1 100
No.3 MW550271.1 Iran KC869687.1 100
No.4 MW550272.1 Iraq MH511151.1 99.90
No.5 MW550273.1 Iran KC869683.1 99.32
Table 2: Sequence matching between L. tropica isolates, local and registered in NCBI
L.tropica isolate number
Genbank accession number
Homology sequence identity L.tropica
country
Genbank accession number
Identity
%
No.1 MW550274.1 Iran AB678350.1 100
No.2 MW550275.1 Iran AB678350.1 99.71
No.3 MW550276.1 Iran AB678350.1 100
No.4 MW550277.1 Iran AB678350.1 99.85
No.5 MW550278.1 Iran AB678350.1 100
Figure 1: Gel electrophoresis images, L. major at 560 bp and L. tropica at 750 bp
Figure 2: Phylogenetic tree analysis of Leishmania major isolates
Figure 3: Phylogenetic tree analysis of Leishmania tropica isolates
