The diagnosis of CL classically relies on microscopic examination. These classical methods require the presence of a relatively high number of viable or morphologically intact parasites; this may pose a problem particularly in the chronic phase of CL where parasite levels in skin lesions are very low. In contrast, the molecular approach is both sensitive and specific (Laskay et al.,1995). In this study, we set up a well-documented, genus-specific PCR to detect Leishmania species in clinical cutaneous samples and compared this method with the classical method.
The present study revealed a significantly high prevalence of cutaneous leishmaniasis in the age group between 1-10 years (24.6%)and (28.7%)by using microscopic examination and conventional PCR assay, This result is agreement with other Iraqi studies as (Al-Mayale 2004;Al-Difaie and Jassem 2014) in Al-Qadisiya governorate. Also (Rahi et al.,2014) observed in another study higher infection appeared in age group 10 years and less. the present result agrees with that in neighboring countries as in Syria (Al-Nahhas and Kaldas 2013), in Jordan (Al-Athamneh et al.,2014) decided that age group (2-19) years was also more susceptible to the infection by CL than another age group, in Iran (Mehdi et al.,2016) and Turkey (Fahriye et al.,2017).
Whereas (Al-Obaidi et al.,2016) obtained the highest infections of CL atin the age group (5-14yr.), While the lowest infection of CL was observed in the age group (>1yr.). also (Al-Warid et al.,2017) who confirmed that majority of cases were recorded among age groups 15–45 years old. The reason for the low rate of elderly patients may be related to the fact that they were infected during their early ages and acquired long-term immunity during childhood ,another factor is that older people do not admit to the treatment of CL while they know this disease and disfiguring scars are not as important for them as for youngsters (Akçal? et al.,2007).
Also, this differences could be due to this age playing outdoors for a long time and more exposure to the infected sand flies, many investigators postulated that the decrease in incidence with age was due to development of immunity by previous infections (Al-Samarai and AlObaidi 2009).
The result of present and other studies pointed to, this diseases can infect the individuals at any age. Also, the (WHO 2014) reported that people of all ages are at risk for infection if they live or travel where Leishmania spp is found.
Depending on genders, The present study revealed the rate of cutaneous leishmaniasis which is higher in males than in females by using microscopic examination and PCR assay. In Iraq (Al-Samarai and AlObaidi 2009) also found that males were (57%) and females were (31.7%). Another study by (Abdulwahab 2013) recorded that the infection in males was 65% than females (35%). In Saudi Arabia (Amin et al.,2013) documented that the incidence rate of CL was higher in males than in females from 2000 to 2010. In Syria (Shanehsaz and Ishkhanian 2013) found that Syrian males are more infected with the parasite than females, In Jordan (Al-Athamneh et al.,2014), The same result in Turkey (Mustafa et al.,2017), This is probably happened due to the cultural habits of most areas.
Otherwise, the results of the present work appeared to disagree with the other previous Iraqi studies by (Rahi et al.,2014) and (Al-Qadhi et al.,2013). These results may be attributed to the fact that males are more exposed to the insect biting more than females due to working outdoors and also due to men are less covering than women then exposed (Al-Samarai et al.,2016). Although it is believed that sex hormones may influence the establishment and the course of parasitic diseases, behavioural factors, making male individuals more likely to be exposed to vectors in fields and other transmission environments, are probably equally or more important (Rahi et al.,2013). The males are more exposed to the environment where the sand flies present by walking near rivers or swimming beside males work in the farms, while the females mostly staying in the houses (Kharfi et al.,2004). Asmaa et al., (2017) found the highest percent of infection related to the geographical site which was near water stream flow all year and abundance of fresh water holes which provide sand flies a suitable environment to complete its life cycle and increase agriculture activities.
The distribution of cutaneous leishmaniasis in rural area was higher than the urban area, this result is agreement with other Iraqi studies as (Rahi et al.,2013) and ( Al-Samarai et al.,2016), in Libya (Sabra et al.,2013), in Syria (Al-Nahhas and Kaldas 2013), in Jordan (Al-Athamneh et al.,2014), in Iran (Mehdi et al.,2016), and in Turkey (Fahriye et al.,2017). However, a different result had been recorded by (AL-Hucheimi 2014; AL-Atabi 2014).
Leishmaniasis usually is more common in rural than in urban areas because there are many factors that play an important role in the presence and distribution of CL in this district, including the presence of animal reservoirs such as rodents, dogs, etc.; the presence of marshes; and the use of clay to build some of the houses in villages that belong to this district area. Furthermore, as an agricultural area, attracts and harbors many kinds of insects; therefore, its population works long hours in the farms where they are more exposed to insects bites (Al-Samarai and AlObaidi 2009). but it is found in the outskirts of some cities (Mustafa et al.,2017).
Different parts of the patient’s body were observed with infection of Baghdad boil including face, arms, legs and feet, but the number of patients infected in arms (upper limbs) had the highest percentage (48%) when compared to other sites of infection,
This result agrees with (Al-Difaie and Jassem 2014) and (Hassan 2017) in Iraq suggested that the highest rate of the lesion was on upper limbs but (Khalifa et al.,2004),( Rahi et al.,2013) and (Al-Obaidi et al.,2016) found the face and hand the highest infection. Also, This result agrees with another study in Iran as (Talari et al.,2006 and Hojat et al.,2012).
In general, the presence and distribution of lesions depend on which parts of the body are exposed and on the susceptibility of the host) Al-Samarai and AlObaidi 2009). CDC(2014)directed that, to minimize the amount of exposed (uncovered) skin, to the extent that is tolerable in the climate, wear long- sleeved shirts, long pants, and socks; and tuck your shirt into your pants.
The number of lesions in CL patients ranged between one to 10 in different body parts. One ulcerated lesion was documented in 67 (55%) of patients, while two lesions were observed in 30(25 %) patient, 3-5 ulcerated lesions were found in 16(13%) patient and multiple lesions (5-10) were found in 9 (7%),
This result agreement with (Al-Mayale 2004) in ALQadisiya, (Al-Hucheimi 2005) in Al- Kufa, (Musa 2011) in Baghdad and (Rahi et al.,2013; Rahi 2015) in Kut city were show the incidence rate of single lesions in CL patients was higher (67.2%) than of the multiple lesions 32.8 % in Iraq, and (Khalifa et al.,2004) in Saudi Arabia.
In contrast with (Al-Difaie and Jassem 2014) reported that the incidence rate of multiple lesions in CL patients was higher than of the single lesions, this can be due to the fact that some ulcers do not necessarily lead to the appearance of scars for several possible reasons, i.e. immune system interference or early healing of the ulcers, spontaneously or due to treatment. Also which is in agreement with previous reports indicating more exposure as a result of educational and occupational situations (Lafi et al.,2007).
The direct staining smear considers good first examination to CL, need a small amount of material from the edge of the lesion which stained easily by Giemsa stain (CDC 2011).
The main reason that cells are stained is to enhance visualization of the cell or the cellular components under a microscope, make them easier to see, also it can highlight (Khademvatan et al.,2012). Staining helps in the identification of the sample by smear colour change without getting into the complete analysis of the sample and easy to observe the morphology, size and shape. The Giemsa stain provides a better stain intensity, show some details that may be unclear otherwise, especially in cells, but some smears of Giemsa stain gave negative results and the parasite doesn’t see or disappeared (Younis et al.,2017), that attributed to many reasons, patient take treatment, mistake in time staining, smear thickness and sometimes distortion of the cell wall may occur (Mustafa et al.,2017). These classical methods require the presence of a relatively high number of viable or morphologically intact parasites; this may pose a problem particularly in the chronic phase of CL where parasite levels in skin lesions are very low (Laskay et al.,1995; Rahi 2015).
Most of the slides that were high scored amastigote numbers as microscopy – positive were also positive by PCR-RFLP. Although the costs for PCR-RFLP diagnosis are higher and its concordance is lower than microscopic examination, but this method can identify Leishmania species without the need for cultivatin them (Schönian et al.,2003;Al-Jawabreh et al.,2006).
The ITS1 gene was chosen to detect 18 ribosomal RNA (SSU)RNA in Leishmania, it is found on chromosome 27 and exon 1 in the parasite. This gene consider as an important virulent factor for the parasite to make infection as reported by (Mauricio et al.,2004). In the current study, the amplification of ITS1 gene fragment with 350 bp showed clear band when electrophoresis on 2% agarose gel. Belal et al.,(2012) established that PCR and Genotyping analysis of Leishmania spp. PCR based methods have proven to be highly sensitive and specific compared to the standard methods and are considered exceedingly valuable for diagnosis. Identification of the Leishmania type is important, because different species may require distinct treatment regimens. Furthermore, such data are also valuable in epidemiologic studies where the distribution of Leishmania species in human and animal hosts, is a prerequisite for designing appropriate control measures (Maraghi et al.,2013).
The results of restriction amplified ITS1 gene product of Leishmania by the endonuclease Hae III gave 42 L.tropica and 20 L.major were diagnosed with RFLP methods. These results were confirmed by (WHO 2014) an agreement with many studies, in Iraq (Ali et al.,2015; Hassan 2017), in Afghanistan (Faulde et al.,2008) and Iran (Talari et al.,2006) who found the dry lesions more than wet lesions, in contrast with other studies done in Iraq (Al-Difaie and Jassem 2014), Pakistan (Ul Bari and ber Rahman 2006), India by (Sharma and Mahajan 2015) and in America (Amalia et al.,2014) reported that dry skin lesions less than wet skin lesion.
The presence of incidence of L. major in this and other studies in Iraq may be due to the presence of reservoir animals in large numbers in some areas in Iraq, especially rodents and dogs. Obviously, dense populations of natural hosts of L.major, together with abundant vector sand flies are the key elements responsible for the high rate of human infection (CDC 2012). also, it must know vector sand flies responsible for human infection by L. topica only (Craig et al.,2013).
The application of PCR and RFLP benefit to characterize the Leishmania species causing cutaneous leishmaniasis in Iraq. two types of Leishmania spp. , L.major as mentioned previously by (Al-Saqur and Al-Obaidi 2013) and L. tropica as mentioned previously by (Sharma and Mahajan 2015) and that confirmed another Iraqi study (Rahi et al.,2013) and another study in nearby countries such as Saudi Arabia (Amin et al.,2013) and Iran (Azizi et al.,2012).
According to the results of this study, we concluded the demographic study investigation revealed that the High infection rate was noticed in the age group of 1 to 10 years. and males were more exposure to infection than females, upper limbs had the highest percentage when compared to other sites of infection, and single lesion appeared in patients more than multiple lesions.
Characterization of Leishmania isolates collected from different parts of Al Ramadi city showed that L.tropica and L.major are the agents of cutaneous leishmaniasis.