Indigenous Development and Evaluation of Leishmanin Skin Test for Diagnosis of

Cutaneous Leishmaniasis Nasrin Abdul Hai, Muhammad Atiq–ur–Rehman, Fazal Haq

Centre of Excellence Science and Technology (CESAT), Islamabad, Pakistan nasreenhayee@gmail.com

Abstract: - Leishmaniasis is caused by a protozoanparasites belong to genus Leishmania and this disease isendemic in certain areas of Pakistan. The clinicaldiagnosis of disease can be confirmed by variouslaboratory investigations like Direct Agglutination Test(DAT), Enzyme Linked Immunoabsorbent Assay(ELISA), Immunofluorescence Assay (IFA), PolymeraseChain Reaction (PCR), direct microscopy, culture andLeishmanin.In this research study the total sample size was 95 male patients with suspected CL of not more than 2 weekduration. Leishmanin test, Direct Microscopy andLeishmania culture was performed on all these patients.These patients were serving soldiers referred by PNSShifa, deployed in different regions of Baluchistanwhere Cutaneous Leishmaniasis is prevalent. A fewcivilians as private patients also reported and wereincluded in the study. All patients of CutaneousLeishmaniasis who reported for the first time wereincluded in our study. Those patients who wereinhabitants of Baluchistan were suspicious of havingdeveloped active immunity by virtue of previous sandfly exposure was excluded from the study. Similarlypatients were also excluded from the study if they hadtaken drugs that could in any way influence the LST result. LST was done by intradermal injection of 0.1mlLeishmanin suspension on the volar surface of forearm.The site of injection was examined for nodule formation/indurations after 48-72 hours and the size of indurationwas recorded and the response was graded. SalineAspirates were collected from the lesions by an expertperson. The Saline Aspirate was processed and examined microscopically for the presence ofpromastigotes and the results of the two diagnostic toolswere compared. All the information was recorded andcompiled.Leishmanin skin test (LST) was positive in 87 patients out of 95(92%), and saline aspirate was positive in 36 patients (38%), 02 patients did not reported, back forobservation. LST test is sensitive and effective even inthose Cutaneous Leishmaniasis patients with lesions ofvery recent onset.

I. INTRODUCTION Leishmaniasis is worldwide in distribution and one

of the major parasitic diseases recognized by World Health Organization (WHO). It is endemic in 88 countries with an estimated yearly incidence of 1 – 1.5 million cases of Cutaneous Leishmaniasis & 0.5

Million cases of Visceral Leishmaniasis. The population at risk is estimated at 350 million people, with an overall prevalence of 12 million [1], [2]. In Pakistan the most common manifestation of infection is Cutaneous Leishmaniasis (CL). It is endemic in Baluchistan province of Pakistan while sporadic cases are seen all over the country [3]. In Pakistan Cutaneous Leishmaniasis is responsible for important public health and economical problems in affected regions. Non-Immune army personals are more susceptible and badly affected with this disease when deployed in the endemic areas.

In endemic areas the disease is adequately diagnosed by its clinical appearance. However, for the effective treatment, the definitive diagnosis of CL in laboratory requires either the demonstration of Leishmania parasites in smears, in culture media, in biopsy or in experimental animals. Anti Leishmania antibodies can be detected in the serum of patient by several serological tests like Direct Agglutination Test (DAT), Enzyme Linked Immunoabsorbent Assay (ELISA) or Immuno Fluorescent Assay (IFA) [9], [10], [11] but the titers are usually low and for these tests need sophisticated equipments & specialized skills. Whereas the diagnostic methods like Leishmanin Skin Test (LST), Direct Microscopy and Leishmania Culture are more useful & cost effective techniques.

Delayed Hypersensitivity is an important feature of all forms of human Leishmaniasis and can be measured by LST also known as Montenegro test. LST is based on cell-mediated immunity [3], [18]. In present study Leishmanin reagent was prepared indigenously from local strain as per WHO guide lines [20]. The purpose of this study was to determine the effectiveness of indigenously prepared Leishmanin Skin Test and Leishmania culture in the diagnosis of Cutaneous Leishmaniasis in Pakistan.

II. Materials & METHODS

The study and laboratory investigations were conducted at CESAT Islamabad from January to December 2008. The study population was mostly serving army personnel, deployed in different regions

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of Baluchistan where Cutaneous Leishmaniasis is endemic. These patients were referred by PNH Shifa. The total number of patients included in this study was 95 having no more than 02 weeks duration of illness.

The inclusion criteria were mainly clinical. They were all males with age ranging from 19 – 50 years. They had one or more nodules / lesions mainly on exposed areas of the body. History of visiting endemic area was one of the inclusion criteria.

Too young, old and those patients, having lesions not suggestive of Leishmaniasis or were suggestive of some other pathology like syphilis, were excluded from the study. Similarly those patients who were inhabitant of Baluchistan and were suspicious of having developed active immunity by virtue of previous exposure were excluded from the study. Patients were also excluded from the study if they had taken drugs that could in any way influence the LST result. A total of 02 patients did not report back for observation after 48 hours and were excluded from the study. A. Parasite Isolation And Cultivation

Parasites were isolated from human cutaneous lesions by needle aspiration or from biopsy samples from the edges of the lesions using fine needle 1 ml insulin syringe. The biopsy material was homogenized in sterile normal saline solution. Needle aspirate and tissue homogenates were inoculated onto Novy, McNeil and Nicolle (NNN) Medium. The isolates were incubated at 22 – 25 oC and examined for the presence of promastigotes form of the parasite every 48 hours during first 02 weeks (fortnight) Negative Cultures were examined again at the end of 3rd and 4th week, being then discarded if still negative. Once primary isolates had been established, sub cultures were made into same medium until sufficient quantity had been built up. Mass cultivation of the organisms was carried out in Tobies Diphasic Modified Medium covered with Locke’s Solution as an overlay and harvested during the log phase of growth by centrifugation. B. Antigen Preparation

Leishmanin skin test was indigenously developed with local identified Leishmania strain i.e. Leishmania major Zymodeme LON1 according to the safety procedures described in World Health Organization Guidelines [20] characterized by isoenzyme technique.

For antigen preparation, promastigotes of Leishmania major strain identified as Zymodeme LON1 were mass cultured. The mass culture of promastigotes was centrifuged & the pellet obtained was re-suspended and washed four times by centrifugation using PBS pH 7.2 in pyrogen free

saline / physiological saline at 35000 rpm at 4oC for 15 minutes. After final wash promastigotes were suspended in 0.5% Phenol w/v in physiological Saline @ 1 x 106 promastigotes per ml Error!Reference source not found.(Rab et al., 1997) C. Performance Of LST & Saline Aspirate (SA)

A single dose of 0.1 ml of indigenously prepared Leishmanin reagent was injected intradermally on the volar surface of the forearms of all patients along with the same quantity of Control, to identify false positive results. Skin Reaction was read by Ball Point Pen Method described by [13]. All reactions with an induration size of 5 mm or more were recorded as positive and the reaction was graded. Grading was done according to the diameter of the indurations: 5-6 mm induration was graded as Grade I; 6-8 mm as Grade II and 8 mm as Grade III [7].

The samples for direct microscopy and culture isolates were obtained through Saline Aspirate from all patients. These were inoculated into Novy, McNeil and Nicolle (NNN) Culture Medium and incubated at 22-25 °C. The solid phase of the NNN media comprised fresh, aseptically collected, defebrinated rabbit blood, mixed with agar and gentamicin [12]. The liquid phase comprised of 0.85% saline. Microscopy was carried out after 48 hours, repeated after 72 hours then after every 48 hours till 02 week.

A questionnaire, containing important clinical & social details including age, sex, occupation, total service, reason to enter into endemic area, place of posting, onset of disease and lesion(s) (number, location & type), was filled in.

III. RESULTS The LST was positive in 87 patients (92%) out of

total 95 referred cases and 84 (88.4%) were found true positive (induration 5 mm), 2 (2.1%) were false-positive (reaction at sides, test & control) and 6 (6.3%) were true negative i.e. no any reaction. The grading of LST was mostly I and II.

Figure 1: A positive Leishmanin Skin Test Reaction

Proceedings of 2014 11th International Bhurban Conference on Applied Sciences & Technology (IBCAST)Islamabad, Pakistan, 14th – 18th January, 2014 90

Figure 2: %age of positive cases by LST & SA

The Saline Aspirate (SA) was obtained from 91 patients and in 4 patients the SA could not be taken due to biopsy already done at the infected site and secondary infection. Out of 91 patients, 36 (38%) were culture positive and 55 (58%) were culture negative. In 36 patients SA & LST both were positive. So the result of positive LST is much higher than SA. Culture isolates depend on parasites load on the site from which material is obtained and experience of observer, While LST is sensitive and effective even in those Cutaneous Leishmaniasis patients with lesions of very recent onset. Our study clearly demonstrated a high sensitivity of LST (92%) for CL of up to 02 week duration. Thus the LST can be confidentially employed for early diagnosis of CL.

ACKNOWLEDGEMENTS The Team is extremely indebted to the Honourable

Dr. Rakhshanda Bilal for her continuous encouragement; motivation and support that resulted in the outcome of a research paper. Contributions of Mr. Mazhar Ali Shahid, Rana Pervaiz Akhtar, Mr. Riaz Hussain Qamar are highly appreciated for guidance and facilitating the team to conduct this work. Contributions of PNH Shifa are also need to be appreciated at all levels for referring suspected patients of Cutaneous Leishmaniasis and confidence building. The study was financially supported by CESAT.

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Comparison b/w LST and SA results

Figure 3: Graphic representation of LST sensitivity vs. SA

Proceedings of 2014 11th International Bhurban Conference on Applied Sciences & Technology (IBCAST)Islamabad, Pakistan, 14th – 18th January, 2014 91