Cutaneous Leishmaniasis in El-Madinna Manowra region , Saudi Arabia in 2012

Objective: Leishmaniasis is a parasitic disease causing major public health problem in form of visceral and cutaneous types. The cutanoue leishmaniasis is caused by L. tropica, in low-land areas without reservoir; Arthroponatic leishmaniasis (ACL), Zoonotic Cutaneous Leishmaniasis ( ZCL), in high-land. This case report involved; 25 years old Egyptian active young single male adult, stayed in Utama (75 Km far from El-Madina Manowra on the road to Makkah). He presented with three skin lesions on his arms occurred within the last 1-3 months. on examination revealed; volcanolike indurated ulcers which clinically suspected as leishmania lesions. Materials and Methods: Laboratory investigations were involved; skin smear using Giemsa stain, Leishmanin test (LST), polymerase chain reaction (PCR), sequencing and phylogenitic analysis BLAST (NCBI). Results: Microscopy positive LDB (leishmanin donovani bodies), Leishmanin test (LST) was negative. PCR positive L. major. Sequence alignment were 100% with nine Iranian isolates and one Tunisian isolate. After one month of treatment with Pentostam (Sodium stibogluconate) local injections at the site of lesions the lesion progressed from ulcer to scar. Conclusion: L. major is a major species causing cutaneous leishmaniasis in Al-Medina Manowra region in Saudi Arabia. The usage of the polymerase chain reaction (PCR) is a useful diagnostic tool and help to identify the causative species.


Introduction:
Leishmaniasis is a parasitic disease existed in many countries . Presented in variable clinical patterns mainly visceral and cutaneous leishmaniasis which cause major health problem to communities. The cutaneous leishmaniasis is divided into two types; one type transmitted by arthropods (ACL) the other is transmitted by reservoir animals (ZCL) such as rodents. The disease is transmitted by a vector, sandfly such as Papattasi, Orientalis 1 .
Leishmaniasis are prevalent on four continents and considered endemic in 88 countries, 72 of which are developing countries. The worldwide prevalence of the disease is estimated at 12 million cases, with 400,000 to 600,000 new cases per year for visceral forms and of 1-1.5 million for the cutaneous forms _ population information.
There are about 1.5 million cases of Cutaneous Leishmaniasis each year worldwide. According to the World Health Organization leishmaniasis is endemic in 88 countries, with a total of 350 million people at risk. It is believed that worldwide 12 million people are affected by leishmaniasis so one person infected by cutaneous leishmaniasis every 20 seconds, and over 90% of cases occur in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil and Peru 2 .
The Leishmania species present with a similar clinical appearance, but with different prognosis during the course of the infection. It was conducted in Al-Utamah around seventy five kilometer north to Al-Madinah. It is a semi-cultivated desert area with houses and many farms containing sheep and poultry breeding houses, irrigated by underground water, which has created a rich habitat supporting endemic animal including desert rodents and sand flies populations. All the inhabitants are relatives with non indigenous workers, their main income depend on agriculture and live stock, i.e., working as farmers and shepherds. volcano-like indurated ulcers which clinically suspected as leishmania lesions. Laboratory investigations were performed which involved; skin smear using Giemsa stain, Leishmanin test (LST), polymerase chain reaction (PCR), sequencing and phylogenitic analysis BLAST (NCBI). The results were; Microscopy positive LDB (leishmanin donovani bodies), Leishmanin test (LST) was negative in day one with diameter less than 5mm and remained negative after repeated one month later. PCR positive L. major . Sequence alignment were 100% with nine Iranian isolates and one Tunisian isolate. After one month of treatment with Pentostam (Sodium stibogluconate) local injections at the site of lesions the lesion progressed from ulcer to scar. The patient compliance towards treatment and follow up was good and no side effects. The investigation procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (Research Center For Medical Colleges, King Khalid University) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from the patient after clear explanation of this study, after approval of the experimental protocol by the local human ethic committee (Research Center For Medical Colleges, King Khalid University). The clinical manifestation of CL can be misdiagnosed and confused with dermal diseases, however the disease in endemic areas can be easily diagnosed by the ulcers in the naked areas and the feature of the ulcers is very characteristic with prominent edged, volcanic appearance, started with papules and takes long term mostly one to six months without pain. The sample was taken from the edges of the lesion using insulin injection aspiration put in two clean microscopic slides with drop of normal saline, then the slides left to dry completely. Then Giemsa stain overlaid on the slides and fixed with 100% methanol, left to dry then covered with 10% Giemsa stain and left for 15 minutes then rinsed with distilled water by pouring water on the edge of the slide, then examined carefully for Leishmania amastigote microscopically at X100 power using the immersion oil lens. Neither culture nor serology was performed. Leishmanin Skin Test (LTS) was done. The Leishmanin antigen obtained from the Pasteur Institute of Iran L. major (reference strain MRHO/IR/75/ER) used for preparation of the leishmanin. The preparation contained a final concentration of L. major promastigotes of 6×10 6 in 1mL of phosphatebuffered saline (PBS). The LST was performed by intradermal injection of 0.1 mL skin test antigen on the volar surface of the left forearm using a 1.0 -mL sterile syringe and disposable needle. The result was read after 48-72 hours using the ballpoint pen technique. Induration size 5 mm was taken as positive reading. Molecular diagnosis was conducted using the filter paper to collect sample from secondarily infected ulcers The patients referred to the Dermatology Department of the AlMiqat Hospital, Al-Madinah Almonawarah, Saudi Arabia, with microscopic confirmed. Lesions and the adjacent normal-looking skin around them were cleaned and sterilized with disinfectant. Sterile saline (0.1 to 0.2 ml) was drawn into a syringe (1-ml, 25-gauge needle), and the needle was inserted into the nodule or ulcer's margin and rotated gently several times. A small amount of saline was expressed into the tissue and some tissue aspirate and freed tissue were withdrawn each was plotted in a sterile Whatman 3 MM filter paper 4 . Filter papers were stored with silica gel at 4°C until DNA extraction. DNA extracted from filter papers using; dried Blood Spot or ulcer aspirate Protocol (QIAGEN): placed 3 punched out circles from a dried blood spot into a 1.5ml Eppindorf tube and add 180ml of buffer ATL (cut 3mm by paper puncher), incubated at 85 ºC for 10mint, briefly centrifuge to remove drops from inside lid. 20ml protenase K (stock solution) was added and mixed by vortex and incubated at 56 ºC for 1 hour, briefly centrifuge to remove drops from inside lid. PCR was used by QIAGEN PCR Kit: dNTP mix (2.5 mM) containing all four dNTPs, 10x amplification buffer, Taq polymerase, Sterile distilled water, L. major specific set of primers: Primers: L5.8S: 5`TGATACCACTTATCG-CACTT 3`. LITSR: 5`CTGGATCATTTTC-CGATG 3`(320 pb).The PCR-Master Mix (MM) was prepared as indicated in Table 1. Vortex and centrifuge the MM shortly and dispense the MM in pre-chilled labeled PCR -tubes. To which 5 µl of template DNA was added. The total reaction volume will be 25 µl, vortex and centrifuge the mixture briefly. Overlay ( if necessary) the reaction mixtures with 1 drop (50 µl) mineral oil to prevent evaporation.
The PCR program was composed of; Hot start; 5 min 94 ºC. Denaturation: 30 sec94 ºC, annealing: 30 sec 65 ºC, extraction: 30 sec 72ºC for 35 cycles then extraction: 6 min 72ºC, finally stored at 4 ºC. PCR products was checked on agarose gels 1%. The collected data were statistically analyzed using the SPSS version. All these techniques were performed in the laboratory of Al-miquat Hospital in Al-Madinah Almonawarah and molecular analysis department of microbiology, college of medicine, King Khalid University, Abha. Saudi Arabia. The sequencing was performed abroad the sample was send to S. Korea. The total sequence is illustrated in (Fig. 1). The phylogenitic analysis by BLAST (Basic Local Alignment Sequence Tool) using NCBI (National Centre for Biological Information). The outcome showed 100% similar sequence with ten isolates of L. major from middle east region, nine from Iran which (unpublished data) and one isolate from Tunisia (published data).

Discussion:
Cutaneous leishmaniasis is an important health problem caused by the flagellated protozoa, Leishmania major and L. tropica. Leishmania is transmitted by female sandflies, Phlebotomus species. Cutaneous leishmaniasis occurs 1-12 weeks after exposure with a small inconspicuous papule on the exposed site which enlarges and finally ulcerates.
Direct detection of parasites is done by microscopic examination of clinical specimens or by cultiva-tion. However, all Leishmania are morphologically similar and species identi?cation is not possible using either of these techniques. Therefore, the ability to distinguish between Leishmania species is crucial in determining disease prognoses, as well as prescribing appropriate therapeutic regimens since some species are more refractory to treatment 5 .
As there is no vaccine, drug treatment is the only way to tackle leishmaniasis. The drugs of choices are sodium stibogluconate and meglumine anti-monite (both pentavallent antimony derivatives). Pentavalent antimony compounds have been in use for more than half a century, and they have shown response rates between 72% and 100% after intra lesion application. Their mechanism of action is not completely known. Development of resistance is of increasing concern; they require weeks of inttravenous administration and are frequently associated with malaise, anorexia, myalgia and arthralgia, electrocardiographic abnormalities, elevated aminotransferase levels, and chemical pancreatitis.