Abstract

The study, conducted from October 2022 to March 2023, aimed to detect cutaneous leishmaniasis in 169 samples from visitors to Samarra General Hospital and dermatology outpatient clinics. Samples were confirmed via direct smear and PCR techniques. The study examined the spread of the parasite by various factors, revealing a higher infection rate in rural areas (64.5%) compared to urban areas (35.5%). The highest infection rate was observed in children aged 1-10 years (21.89%), followed by those aged 21-30 years (17.7%), while the lowest infection rate was in individuals aged 61-70 years (4.73%). Wet ulcers were more common (75.1%) than dry ulcers (24.9%). The infection rate was highest in January (27.22%), followed by February (21.89%), with March showing the lowest rate (10.65%). Facial infections were most common (42.6%), followed by lower extremities (40.23%), and the trunk had the lowest incidence (0.59%). Molecular analysis using PCR revealed that L. major was the predominant type (68.64%), with L. tropica accounting for 31.36%. Males showed a higher infection rate for both types, with L. major affecting 44.38% of males compared to 24.26% of females, and L. tropica infecting 21.3% of males versus 10.06% of females.

1. Study on cutaneous leishmaniasis in 169 samples, confirmed by smear and PCR.
2. Higher infection in rural areas, children, wet ulcers, and January-March period.
3. L.major prevalent (68.64%), higher male infection rates in both parasite types.

Keywords: leishmaniasis, pcr, diagnosis ,leishmania tropica, lishmania major

Introduction

Leishmaniasis is one of the nine largest parasitic diseases that affect humans and causes many health problems in addition to the rapid spread of the disease. Despite the seriousness and rapid spread of the disease, it is still neglected at the global level, as it is spread in 98 countries around the world, including Iraq and Iran. Brazil, Afghanistan, Syria, India, Bangladesh, and Sudan. Annual reports indicate that more than one million people are infected with leishmaniasis .(1)

The pathogen of leishmaniasis is a single-celled parasite of the genus Leishmania that is transmitted by the bite of a sandfly of the genus Phlebotomus in the Mediterranean and Middle East regions and the genus Lutzomyia in South and Central America. This disease represents 5 to 10% of diseases in tourists returning from Tropics (2)

Leishmaniasis is characterized by three clinical forms: Cutaneous Leishmaniasis, Mucocutanous Leishmaniasis, and Visceral Leishmaniasis (Cutaneous Leishmaniasis is given many names depending on the areas in which it occurs, including: Baghdad Boil, Aleppo Boil, and Oriental Sore, as well as Among other names there are two species that cause cutaneous leishmaniasis in Iraq, which are Leishmania tropica (L.tropica), are the cause of Anthroponotic Cutaneous Leishmaniasis (ACL) and Leishmania major (L.major) are the cause of Zoonotic Cutaneous Leishmaniasis ( ZCL), and the sand fly is the main carrier of the parasite, of which there are two species in Iraq: P.Sergenti and P.Papatasi(3)

Infection with the parasite occurs through the bite of a sand fly, which contains the promastigote stage, which forms inside the transmitting host in large numbers. Then, when the bite occurs, it enters through phagocytic cells in the skin and turns into the amastigote stage, which represents the infective stage of the parasite.

Also, the period between infection with the disease and the appearance of symptoms ranges from days to weeks. This discrepancy is due to reasons including the health condition of the infected person, environmental factors, the severity of the infection, and factors related to the parasite itself. (4)

The diagnosis of cutaneous leishmaniasis (CL) depends on the appearance of the parasite in smears or skin biopsy samples by direct microscopic examination and immunological examination, but these traditional methods of diagnosis lack high sensitivity and specificity and do not provide any evidence regarding the species causing the disease, so the polymerase chain reaction technique was used. Polymerase Chain Reaction (PCR) has been used in recent years to determine the species causing the disease. It is a technology that relies on amplifying part of the parasite's DNA and producing multiple copies to conduct molecular tests and determine the type of genes .(5

Methods

This was a descriptive laboratory-based study for isolation of Leishmania tropica and Leishmania major in Salah Al-Deen hospitals and Samarra hospital . The study conducted during the period from October 2022 - March 2023, Information was taken from patients according to a pre-prepared information form, Form No. (1). Cutaneous leishmaniasis patients who are participated in this study are attended the outpatient of the Dermatology Departments of the hospitals with cutaneous leishmaniasis and confirmed by direct smear done by the hospital laboratory staff.

Inclusion criteria

All positive cases for Leishmaniasis by direct microscopy.

Exclusion criteria

patients who received anti-leishmaniasis treatment before all patients were examined and then selected considering epidemiological risk factor for cutaneous leishmaniasis as well as signs of the disease. Lesion and the adjacent normal looking skin around them cleaned and disinfected. Skin biopsies of 4 mm in diameter was taken aseptically from the border of the ulcer using disposable scalpel blade. The blade was turned 90 degrees and scarped along the cut edge of the incision to remove and pick up skin tissue which was divided into two parts. One part was used for smear and one was stored at -20℃ for DNA extraction later used for PCR analysis [6]. The smear was prepared by smearing the biopsy on glass microscopic slide. After the smear was dried completed, it was fixed with absolute methanol, allowed to dry again and then stained with Giemsa stain. The amastigotes were found within macrophages. The presence of intra cytoplasmic, kinetoplast confirm the identification after staining, the cytoplasm appeared light blue, the nucleus and kinetoplast appeared red to purple. In very early and old lesion, very few organisms were presented. Biopsy tissue was collected directly in lysis buffer, DNA was isolated according to the user manual and kept at-20℃.

After confirming the presence of the parasite in the ulcers of the infected people through microscopic examination, 5 ml of blood was taken from the people infected with cutaneous leishmaniasis. After that, a portion of the blood was taken to conduct immune tests using serum to detect the presence of antibodies, especially IgM and IgG, which are formed as an initial response by the body against the disease. The presence and type of Leishmania parasites were confirmed through specific polymerase chain reaction (PCR) techniques for molecular diagnosis. After confirmation, DNA was extracted from samples and then sequenced with a view to analyzing the genetic diversity presented by various types of ulcers.

Results and Discussion

Leishmaniasis is classified as one of the six diseases on the World Health Organization's list. Statistics indicate that there are 12 million cases worldwide, including one million new cases annually (1). The epidemic of leishmaniasis is widespread and the disease is re-emerging and spreading in many regions, as it is spread in 98 countries around the world. The epidemiology and pathogenesis of leishmaniasis varies from one geographical area to another (10). The geographical spread of the disease also depends on the availability of the vector host and the environmental conditions suitable for the vector host, in addition to the factors that help the parasite grow in the host (1).

1 - Epidemiology :

1-Based on accommodation:

The results indicate a higher incidence of infection in rural areas compared to the number of infections in urban areas. Table (1-1) shows the results:

There are factors that have increased the incidence of infections in rural areas compared to urban areas. Among these reasons is that the majority of rural residents work in agriculture near their homes, where sand flies, which are the insect transmitting the disease, are present. The geographical nature of rural areas, in which fresh water must be available for agriculture, is an important factor in completing the life cycle of the insect transmitting the disease (11). In addition, poor health awareness and movement in areas infected with the disease are among the reasons for the spread of the disease, and the presence o-f reservoir hosts such as dogs and rodents, building houses of mud, and frequent exposure to stings from the vector insect during work are all factors that have increased the spread of the disease in rural areas (12).

2- Based on age:

The current results showed a high incidence in the age group (1-10), followed by the age group (21-30), and the lowest incidence rate in the age group (61-70).

Table (1-2): infection results based on age

The current and previous results on the disease show that it affects all age groups, but at different rates that depend on several factors, including the lack of development of the immune system of those infected at the age of ten years and under, as well as their inability to defend against the bite of the vector insect, as well as the children’s great desire to play outside the home for long periods of time, which increases their exposure to the vector insect (13). As for the older age groups, the low infection rate is due to the fact that these people were exposed to the infection during childhood, which provided them with long-term immunity against the parasite, as well as being more aware than children and able to avoid infection and leaving the house less than children.

3- Based on the type of ulcer:

Based on the type of ulcer, the results showed a higher incidence of infections in the wet type compared to the dry type. Table (1-3) shows the results:

The results of our study agree with the findings of researchers (14), who also indicated the prevalence of the wet type of infection, which is the most common. The reason for the prevalence of this type is the presence of a secondary infection that gives the appearance of an ulcer.

4- Based on the month:

The results of the current study revealed that the infection rate increased in January, followed by February, while the lowest infection rate was in March. Table (1-4) shows the results:

The researchers' study (3) indicated that the incubation period of the Leishmania parasite ranges between 2-6 months, as the sand fly bites the host in September or October, and cases begin to appear in January and February. The reasons for the difference in the monthly distribution of the disease epidemic are due to reasons including the association of the disease epidemic with sand fly activity associated with the climatic conditions during the study period in terms of temperature and humidity, and the development of female insects that need blood to complete the life cycle, in addition to repeated exposure to the vector during these months, which increases the emergence and spread of the disease. The World Health Organization (15) also indicated that the incidence of cutaneous leishmaniasis in Iraq reaches its highest levels during the winter and spring, as it increases in January and March, and the number begins to decrease with the beginning of summer.

5-Based on the site of injury:

The results of the study revealed a high incidence of infection in the face area, followed by the lower extremities area, while the lowest infection rate was in the torso area. Table (1-5) shows the results:

It can be said that the apparent distribution pattern is due to the areas being exposed and easily accessible to the sand fly, which is consistent with the study of (16). The results of the study also agreed with what was reached by (17) in the Arab Republic of Egypt, where the infection rate in the face and cheeks was the highest, and the infection rate in the face was 43.5% in a study conducted by (18).

2 - Using PCR technology to detect the genus of cutaneous Leishmania :

The parasite species responsible for cutaneous leishmaniasis were identified by collecting 169 samples from infected individuals. Leishmania parasite DNA was extracted from all samples and subjected to electrophoresis to check for its presence (Fig. 4). The DNA was then stored under refrigeration until needed. PCR reactions were performed using a thermal cycler to amplify a specific DNA fragment. For example, this fragment yielded a 560 bp band for L. major and a 750 bp band for L. tropica, which were found in the samples. PCR was used as a tool to amplify the DNA of the isolates in this research. All PCR products were run on a 2% agar gel showing bands of expected sizes. In L. tropica samples, 53 isolates had a 750 bp band while in L. major samples, 116 isolates had a 560 bp band.

Figure 1.Leishmania Skin Parasite DNA Extracted: Visual Confirmation through PCR"

Figure 2.PCR-Based Typing Reveals Electrophoretic Pattern of Leishmania major Isolate at 560 bp

Figure 3.Electrophoretic Pattern Analysis of Seven Isolates of Leishmania tropica at 750 bp: PCR Typing Results

Figure 4.Electrophoretic Pattern Analysis of Seven Isolates of Leishmania tropica at 750 bp: PCR Typing Results

Figure 5.Electrophoretic Pattern Analysis of Seven Isolates of Leishmania tropica at 750 bp: PCR Typing Results

Detection of cutaneous leishmaniasis by PCR :

PCR has proved to have great potential in the detection and diagnosis of various infection by different parasite species in number of clinical samples and in epidemiological studies, largely with application of PCR.

Approximately 20 Leishmania species are known to cause cutaneous, mucocutaneous, and visceral disorders in humans. Identification of the causative species in infected individuals is important for appropriate treatment and a favorable prognosis because infecting species are known to be the major determinant of clinical manifestations and may affect treatments for leishmaniasis.

Leishmania tropica was isolated from the clinical patients of cutaneous leishmaniasis in rural community of salah al-deen province and was identified through PCR, microscopy, and culture techniques. A total of 169 samples from the clinical patients were examined through PCR, microscopy, and culture which was L. major Males; 75(44.38%), L. major Females; 41(24.26%), L. tropica Males; 36(21.30%),L.tropica Female; 17(10.06%).Thus, the sensitivity of PCR is very high as compared to the conventional techniques(7) L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies [8].

Conclusion

In this study, L. major and L. tropica were firmly identified in suspected patients with a coinfection of L. major, L. tropica, According to DNA sequence of the parasite, Leishmania major is the predominant cause of leishmaniasis in Salah Al-Deen Province, The epidemiology of leishmaniasis was also determined according to several criteria, including sex, age, place of residence, site of infection, and months of the year. The sex of males most susceptible to infection was determined. Rural areas are also most susceptible to infection, and age groups (1-10) are most affected by cutaneous leishmaniasis. The study also revealed that the face area has the highest incidence of infection.

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