2.10.1.1.3. Intergenic spacer region (IGS) :
The IGS is a genomic element varying in size, number and sequence across Leishmania species, and isolates. The Leishmania IGS contains a 60 to 64 bp-long repeat element of 16 to 275 copies, causing length variations of 4 to 12 Kbp (113 ). The size of the IGS region is roughly the same in Sauroleishmania and Leishmania species, demonstrating its conservation at the subgenus level. The IGS is less conserved than the rRNA genes and thus is more suitable to map the evolutionary relationships between closely related Leishmania species. (114, 115).

2.10.1.1.4 Mini-exon or Spliced leader RNA (SL RNA) gene:
It is of kinetoplastid protists is present in 100 to 200 tandemly repeated copies per nuclear genome and has been used for Leishmania phylogenomics (114,115). Each repeat consists of three major parts:
A transcribed 39 bp-long exon (or mini-exon) that is highly conserved between positions 1 to 9 and 21 to 39 (116); A moderately conserved intron, ranging in size from 55 to 101 bp; and a non-transcribed highly variable spacer (51 to 341 bp) used for Leishmania genotyping.
Its variation in size allows preliminary discrimination between major Old and New World Leishmania complexes (117,118,119,120). The 39 bp mini-exon can be used to quantify gene expression because it is present at the 50 ends of mRNAs (121).

2.10.2. Non-chromosomal DNA :
2.10.2.1. Kinetoplast DNA (mitochondrial DNA) :
All Kinetoplastid ?agellates possess a single mitochondrial genome known as the kinetoplast DNA (kDNA), which consists of several thousand circular DNA molecules linked together in a concatenated network (103).
It is a mass of circular DNA that consists of thousands of mini-circles (~1Kb each) and several dozen maxicircles (~23 Kb each) (121).

2.10.2.1.1. kDNA maxicircle :
encode genes homologous to those present in the mitochondrial DNA of other eukaryotes (121).

2.10.2.1.2. kDNA minicircle :
Minicircles makeup approximately 95% of kDNA and encode small RNA molecules termed guide RNAs, which provide information for RNA-editing of the maxi-circle encoded transcripts (122). kDNA is traditionally the most frequently used target for detection and typing of Leishmania because of its multicopy nature and through high sensitivity (123).

2.11. Diagnosis :
The broad clinical spectrum of cutaneous leishmaniasis makes a diagnosis of resent and Previous cases difficult. Differential diagnosis is important because diseases of other causes but with a similar clinical spectrum to leishmaniasis e.g. leprosy, skin cancers, tuberculosis, cutaneous mycoses, are common in leishmaniasis-endemic areas (124), Parasitological diagnosis remains the gold standard in cutaneous leishmaniasis diagnosis, because of its high specificity.

2.11.1.Direct examination :
Microscopic examination is probably the most common diagnostic approach used, because more sophisticated techniques are expensive and rarely available at primary, secondary, and tertiary health-care levels in endemic areas, using Giemsa, Wright’s, Leishman’s or other stains(13). (125) said that during stain the Leishmania amastigotes appear round to oval parasites, with a round basophilic nucleus and a small rod-like kinetoplast in macrophages or freed from ruptured cells (126). The use of an Electron microscope gives the best result for identification both the size and disposition of nucleus and kinetoplast (127).

2.11.2. Parasitic isolation and identification :
Leishmania spp was isolated from skin lesion by taking aspirate from the lesion, which converted motile promastigotes after seven days by using the culture (128).
There are different culture media which used for culturing the Leishmania spp. Novy-MacNeil-Nicole medium, RPMI 1640, peptone- yeast extract, brain heart infusion and nutrient broth, Evan’s modified Tobie’s medium (EMTM), Grace’s medium and Schneider’s Drosophila medium (129).
The animal inoculation into hamsters may also be valuable, especially for adaptation of the parasite (130).

2.11.3. Histopathology :
Tissues biopsy were processed according to (131), briefly, the tissue from the skin collected and placed in 10% formalin for histopathological studies The histopathology lesion can increase the likelihood of detecting the organism when few parasites are present, the morphological differences at light microscope level which it is a rapid examination.
2.11.4. Leishmanin Skin Test (LST) :
Delayed hypersensitivity is an important feature of cutaneous forms of human leishmaniasis and can be measured by the leishmanin test, also known as the Montenegro reaction. Leishmanin is a killed suspension of whole (0.5-1 x 10/ml) or disrupted (250 µg protein/ml) promastigotes in pyrogen-free phenol saline (132). No cross-reactions occur with Chagas’ disease, but some cross-reactions are found with cases of glandular tuberculosis and lepromatous leprosy. Leishmanin Skin Test is usually used as an indicator of the prevalence of cutaneous and mucocutaneous Leishmaniasis in human and animal populations and successful cure of visceral leishmaniasis (133,134), During active kala-azar disease, there will be no or negligible cell-mediated immune response. However, the leishmanin antigen is not commercially available and no field study has been carried out in India (135).

2.11.5. Serological tests :
Also called immunological test, the elevated antibody titers against promastigote or amastigote antigens, their fractions or recombinant antigens have been extensively exploited for specific serodiagnosis in last two decades in the form of complement fixation test, direct agglutination test (DAT), ELISA, dot-ELISA, immunoblot, strip test, indirect immunofluorescent antibody test (IFAT), indirect haemagglutination antibody (IHA), latex agglutination, immunodiffusion or immunoblotting may also be available (136). However, diagnosis of leishmaniasis remains problematic and the problem occurred due to cross-reaction not only between different Leishmania spp. but also between different flagellates and unrelated organism particularly Trypanosoma cruzi (74).
2.11.6. Rapid Test For Cutaneous Leishmaniasis CL Detect :
The CL Detect Rapid Test is a qualitative, in vitro immunochromatographic assay for the rapid detection of Leishmania species antigen in ulcerative skin lesions. The test is intended for use with dental broach samples from less than four-month-old ulcerative skin lesions that are obtained from patients with suspected cutaneous leishmaniasis (CL). The test targets the peroxiredoxin antigen of Leishmania species that may cause CL. The CL DetectTM Rapid Test is intended in the diagnosis of CL and must be interpreted within the context of all relevant clinical and laboratory findings(137).

2.11.7. Molecular tests :
There are many molecular tests to indicate the species, subspecies and/or strain of Leishmania which is identified by specialized techniques including isoenzyme analysis, PCR, DNA hybridization kinetoplast DNA and restriction endonuclease analysis (138).
Polymerase Chain Reaction is particularly sensitive and more specific also can be used to detect Leishmania spp. in blood, skin biopsies, lymph nodes, bone marrow and conjunctival swabs, the target sequences for characterization include either nuclear DNA such as the small subunit rRNA (SSU rRNA) gene, a repetitive genomic sequence and the miniexon (spliced leader) gene repeat (139), also Real-Time PCR are very important techniques to detect the parasite quantitatively and qualitatively (82).

2.12. PCR-restriction fragment length polymorphism (RFLP) analysis :
PCR-restriction fragment length polymorphism (RFLP)-based analysis is a popular technique for genotyping. The technique exploits that SNPs, MNPs and microindels often are associated with the creation or abolishment of a restriction enzyme recognition site (140). The first step in a PCR-RFLP analysis is the amplification of a fragment containing the variation. This is followed by treatment of the amplified fragment with an appropriate restriction enzyme. Since the presence or absence of the restriction enzyme recognition site results in the formation of restriction fragments of different sizes, allele identification can be done by electrophoretic resolvement of the fragments (141).

Table 2.3. Advantages and disadvantages of PCR-RFLP (141).

Advantages Inexpensive
Easy to design
Applicable to the analysis of single nucleotide polymorphisms as well as microindels
No requirement for expensive instruments
No requirement for extensive training of laboratory staff
Miniaturisable
Disadvantages Requires that a variation generates or abolishes a restriction enzyme recognition site
Some restriction enzymes are expensive
Exact genotyping cannot be achieved in the event that there is more than one nucleotide variation in a restriction enzyme recognition site
Requires relatively large amounts of hand-on-time Long time from start to completion of the analysis Not suitable for high-throughput analysis

2.13. Treatment :
Uncomplicated CL is usually self-limiting. Lesions resolve over months leaving a scar. Different Leishmania species have different self-resolution times, L. aethiopica and L. tropica being characteristically taken much longer than L major.
Treatment choice is determined mainly by disease severity: the size, number, location, and likely chronicity of lesions; the potential of the Leishmania species to disseminate; established mucocutaneous or diffuse disease; and the presence of co-morbidities and immunosuppressant states such as HIV co-infection (142).
The first line of treatment for all of the types of leishmaniasis is the pentavalent antimonials- meglumine antimoniate (Glucantime) and sodium stibogluconate (Pentostam), it has remained the standard therapy for more than 60 years (143). This agent has multiple toxicities and is increasing unsuccessful due to the expansion of parasite resistance, and there are many defects such as painful administration and a long period of treatment ( 144, 44). The WHO explore that antimonials dosages should not surpass 20 mg/kg/day, and due to its elevated toxicity, the dosage of antimony ingested per day should not be uttermost than 850 mg (145). The other forms of drugs are amphotericin B, miltefosine, paromomycin. The basic pharmacologic therapies below, Table (2.4) (146,147).

Table (2.4): The basic Pharmacologic Therapies and their
Applications (146,147)
Drug Application
Pentavalent antimony (pentostam)
(sodium stibogluconate ) Used in cutaneous leishmaniasis; not marketed in the United States, but obtainable through the CDC under an Investigational New Drug (IND) protocol
Liposomal amphotericin B (AmBisome) Operative versus pentavalent antimony
resistant mucocutaneous disease and VL.
Oral miltefosine (Impavido) Confirmed by the Fluorescein diacetate
(FDA) in March 2014 for VL due to L. donovani; CL due to L. panamensis, L. guyanensis, and L. braziliensis; and mucosal leishmaniasis due to L. braziliensis
Intramuscular pentamidine Operative versus VL but related with
constant diabetes mellitus and disease recurrence.
Orally administered ketoconazole,
itraconazole, fluconazole and dapsone They may be beneficial in hastening
healing in patients with CL that does not develop to mucosal disease and tends toward self-resolve.
Topical paromomycin Shown to be operative versus CL caused by L. mexicana and L. major.
Sitamaquine Subjecting phase three trials
local therapies for some forms of CL include local heat therapy at 40-42°C and cryotherapy.

(148) revealed that the Therapeutic failure in leishmaniasis is a common problem in endemic areas. This may be associated with multiple factors that depend both on the parasite and on the mammalian host. Regarding the mammalian host, therapeutic failure can be attributed to altered drug pharmacokinetics, reinfection or immunologic compromise. In most cases where chemotherapy fails to cure the patient, the natural susceptibility of parasites to drugs happens to be low, or alternatively, the infecting parasite has developed chemo-resistance.
(13) revealed that new topical treatment Gentian violet with Arabic Gum was the best drug for CL gave(93%) with highly significant when compared with other drugs Miltefosine(67%) Pentostam (46%), Arabic Gum alone (33%).

2.14. Disease control (Vector and reservoir control) :
Because the strategies available are expensive and labour intensive, and cutaneous leishmaniasis is a nonfatal disease, prevention and control strategies have mainly focused on the treatment of the human disease, rather than on the elimination of reservoirs or reduction of human-vector contact (44). Hence, most approaches have been limited to pilot research studies and only a few have been brought up to operational scale (149).
Measures involving the participation of the at-risk human population focus on personal protection from cutaneous leishmaniasis, including insecticide-impregnated materials like bed nets curtains clothes, or bed sheets and repellents which may offer an alternative in places with poor health-service infrastructure and per domestic Leishmania transmission. Several studies have shown that pyrethroid-treated bed nets provide 50–65% protection against infection or disease (150). However, similar to house spraying, the long-term feasibility of insecticide-treated materials is debatable, because of logistical constraints (e.g. re-impregnation of materials) and the intervention’s economic cost.
In forested environments (e.g. in most endemic areas of South and Central America) health authorities are usually limited to treating human cutaneous leishmaniasis cases. Although prevention and control strategies (e.g. environmental management, spraying of a sand fly resting sites) have been explored, targeting the sand fly vector effectively in these habitats is difficult (6).
Sand flies are highly susceptible to insecticides. Although they possess the necessary biochemical mechanisms (151), reports of resistance are few. Anecdotal evidence from Peruvian and Iranian malaria eradication campaigns in the 1950s suggested that residual spraying of houses is effective against endophilic and endophagic sand fly vectors, which was subsequently shown in controlled studies (152). The measure taken against malaria in Iraq reduce the intensity of sand fly in recent year (153).

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