Dermatology Online Journal

Sensitivity of leishmanin skin test in patients of acute cutaneous leishmaniasis
A Manzur, Arfan ul Bari
Dermatology Online Journal 12 (4): 2

Department of Dermatology. PAF Hospital Sargodha, Pakistan. aamirderm@hotmail.com

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Abstract

Background: The leishmanin skin test (LST) is frequently used for clinical diagnosis of cutaneous leishmaniasis (CL). Although, LST is highly sensitive for CL, no definitive data exists as to how early in the disease does the test becomes positive. OBJECTIVE: The study was aimed to evaluate the sensitivity of LST in early cases of CL. PATIENTS AND METHODS: One hundred male patients with CL of not more than 2-weeks duration and having parasitologically proven diagnosis were enrolled in this study, carried out in a military hospital in Balochistan, Pakistan. LST was done by intradermal injection of 0.1 ml leishmanin on the volar surface of the forearm. The skin test reaction was read after 48 hours and then at 72 hours if the first reading was negative or marginal. If the initial test was negative, the LST was repeated after every 7 days until a positive reaction was produced. The total time from the onset of skin lesions until a positive LST was obtained was recorded for every patient. RESULTS: A positive LST was demonstrated in 78/100 patients with skin lesions of ≤ 2 weeks duration, thus showing a sensitivity of 78 percent for this short duration. The sensitivity of LST increased to 94 percent and 98 percent for CL lesions of 4 and 6 weeks duration, respectively. CONCLUSION: LST is sensitive even in those CL patients who present with lesions of very recent onset. Thus the test can be employed with confidence, for the diagnosis of CL, even in early disease.

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Introduction

Cutaneous leishmaniasis (CL) is endemic in Balochistan province of Pakistan [1]. It is one of the major health concerns for the public in general, and assumes a greater importance for the Army troops deployed there. Because most of the soldiers are nonresidents and thus not immune to CL, they are always at risk of acquiring the disease.

Delayed type hypersensitivity (DTH) is an important feature of all forms of CL. The leishmanin skin test (LST) or Montenegro test measures DTH reactions to an intradermal injection of a suspension of killed promastigotes. It is a useful and important tool for epidemiological, immunological, and diagnostic studies. The immunological procedure is similar to the tuberculin test [2, 3]. In corroborating the clinical diagnosis of CL with LST, it is important to know how long after infection or appearance of skin lesions the skin test becomes positive. Without this information the utility of this test as a diagnostic aid, particularly for acute CL lesions, becomes limited. Unavailability of any data on this issue prompted us to carry out this study. The present study was designed, therefore, to evaluate the sensitivity of LST in patients with acute CL lesions of ≤ 2 weeks duration.

Patients and methods

The study included 100 adult men with CL who had lesions of not more than 2-weeks duration, and who were attending the outpatient department at a military hospital in Quetta, Balochistan. These patients were selected from a total of 2560 CL patients during a 20-month period. The patients belonged to different areas of Pakistan, mostly Punjab and NWFP. The diagnosis of CL was made clinically and confirmed parasitologically. Patients were excluded from the study if they had taken drugs that could in any way influence the LST result (such as immunomodulators, corticosteroids, or antihistamines) or if they were diagnosed with a concurrent systemic illness.

L. major leishmanin, produced in the Pasteur Institute Tehran was used in the study. This Iran leishmanin contains killed L. major promastigotes at a concentration of 5 × 10⁶ promastigotes per ml, in phosphate buffered saline containing 0.01 percent thimerosal [2].

Leishmanin skin test was carried out on the first reporting day of clinically suspected cases of CL, fulfilling the above-mentioned criteria. The definitive parasitological diagnosis was made by smear microscopy, skin biopsy, or parasite culture. The smear was the initial test performed. Smears were obtained by both slit skin method and by needle aspiration method. Leishmania culture and skin biopsy were carried out in the smear-negative patients. Strain identification of the Leishmania species was not done. If the diagnosis was not confirmed parasitologically, the subject was dropped from the study regardless of the LST result. The skin over the volar surface of the forearm was cleansed with a 70 percent alcohol pad and 0.1 ml of the leishmanin preparation was injected intradermally. The injection was given by a 1-ml syringe with a 27g, 0.40 mm × 10 mm needle. During injection the beveled edge of the needle was kept upside. The skin test reaction was read after 48 hours and then at 72 hours if the first reading was negative or marginal. LST was measured using the ballpoint technique [4]. An induration of ≥ 5 mm was taken as a positive leishmanin test [5, 6]. If the initial test was negative, the LST was repeated after every 7 days until a positive reaction was obtained. Treatment was withheld, until a positive LST reaction, to avoid any effect of therapy on the test result. The total time from the start of skin lesions until a positive LST reaction developed was recorded for every patient and the total number of patients converting to positive was calculated at the end of 2nd, 3rd, 4th, 5th and 6th weeks. Sensitivity of LST at each time duration was calculated (sensitivity = true positives ÷ [true positives + false negatives]).

Results

A total of 100 parasitologically confirmed cases of CL with skin lesions of ≤ 14 days duration were leishmanin skin tested on their first visit. Below the upper limit of 14 days, the duration of skin lesions varied, the earliest being 5 days. A positive LST was obtained on the first or subsequent tests in 98 patients (98 %). Two patients (2 %) had a repeated negative LST reaction. In these two patients treatment was started after 2 months. They remained LST negative until the last test at 6 months. A total of 76 patients (76 %) had a positive reaction on the first LST, while 22 (22 %) converted to a positive LST on subsequent tests. Seventy-eight patients (78 %) had a positive reaction by the end of second week (the time duration is the total time from the appearance of skin lesions until a positive LST reaction). This gives a LST sensitivity of 78 percent for lesions up to 2 weeks old. Another 8 patients converted to a positive LST at the end of 3rd week and an additional 8 at 4th week; at the end of 5th week 3 more patients showed a positive LST. The last patient had a positive LST in the 6th week after a total time period of 41 days from the start of skin lesions. The earliest positive LST was recorded in a patient with disease duration of 7 days. The results have been summarized in Figure 1. Thus the sensitivity of LST increased from 78 percent at 2 weeks to 86 percent at 3 weeks, 94 percent at 4 weeks, 97 percent at 5 weeks and 98 percent at 6 weeks.

Figure 1: Graphic representation of sensitivity of LST after respective weeks

Discussion

Figure 2
A positive leishman skin test

The LST or Montenegro's test is an important tool for the diagnosis and epidemiological surveys of CL along with being an essential component of the vaccine trials. The first study of this test in humans was carried out by Montenegro in 1926 in Brazil [3]. Because of the high sensitivity of LST in cutaneous leishmaniasis, the test has been frequently applied in diagnosis [6, 7]. Its usefulness as a diagnostic aid in clinically suspicious lesions is high, because a positive result is a very strong support for the diagnosis of cutaneous leishmaniasis and is an indication for further investigations. The value of LST is especially supportive in the diagnosis of those lesions where parasites are scanty such as in healing sores and in leishmaniasis recidivans [6]. Although, LST cannot distinguish between present and past infection, the test retains its value for diagnosis in non-endemic areas or in visitors to an endemic area, as they have no background of leishmanin sensitivity [8]. Furthermore, a negative LST greatly reduces the possibility that the skin lesion is due to leishmaniasis. In addition, LST positivity is highly specific [2]. The safety and efficacy of Iran leishmanin has been previously documented [5]. Furthermore, the leishmanin produced in Iran, used in our study, has been selected by World Health Organization Tropical Disease Research center as the reference reagent for all epidemiologic and vaccine efficacy trials [9].

Most of the soldiers deployed in the endemic areas of Balochistan province are from non-endemic areas and thus lack any previous sensitization to Leishmania. In these subjects a positive LST strongly suggests a diagnosis of CL. In corroborating the clinical diagnosis with LST, understanding the timing of the onset of LST is of significant importance to avoid false negative results in those few cases that present with early disease. The available data gives a wide range for the time taken for LST to become positive. A duration ranging from a few days after infection to several months has been described in the literature [10, 11, 12, 13]. This wide range may be relevant to specific areas because the DTH response does depend on many factors relating to the parasite and the host [14]. In an earlier study, Dostrovsky and Sagher [2] suggested that the test becomes positive very soon after infection. Another study by Zvi and Sagher [15] conducted in healthy volunteers (who were injected intradermally with 2-16 million living promastigotes) also revealed a positive test in all cases after 72 hours. However, the authors admitted that this rapid conversion might not occur in a natural leishmania infection where a much smaller number of parasites are introduced via the sandfly bite.

Our study clearly demonstrated a high sensitivity of LST (78 %) for CL lesions of up to 2-weeks duration. This sensitivity increases to 94 percent and 98 percent at a disease duration of 4 and 6 weeks, respectively. Thus the test can be employed with confidence even in those cases that present with early lesions.

Conclusion

The leishmanin sensitivity test is highly sensitive for cutaneous leishmaniasis, even early in the disease. It should be included in the routine armamentarium of diagnostic tests for suspected CL lesions, even of recent onset.

References

1. Raja KM. Khan AA, Hameed A, Rahman SB. Unusual clinical variants of cutaneous leishmaniasis in Pakistan. Br J Dermatol 1998; 139: 111-13. PubMed

2. Dostrovsky A, Sagher F. The intracutaneous test in cutaneous leishmaniasis. Ann Trop Med Parasitol 1946; 40: 265-69.

3. Montenegro J. Cutaneous reaction in leishmaniasis. Arch Derm Syph 1926; 13: 187-94.

4. Sokal JE. Measurement of delayed type skin-test responses. N Engl J Med. 1975; 293 (10): 501-2. PubMed

5. Alimohammadian MH, Kivanjah M, Pak F. Evaluation of the efficacy of Iran leishmanin and comparison with leishmanins from Wellcome (UK) and Roma (Italy) in cured cutaneous leishmaniasis patients. Trans R Soc Trop Med Hyg 1993; 87: 550-1. PubMed

6. Pampiglione, Manson-Bahr PE, La Placa M, Borgatti MA, Micheloni F. Studies on Mediterranean leishmaniasis, IV. The leishmanin skin test in cutaneous leishmaniasis. Trans R Soc Trop Med Hyg 1976; 70: 62-5. PubMed

7. Faber WR, Oskam L, van Gool T, Kroon NC, Knegt-Junk KJ, Hofwegen H, van der Wal AC, Kager PA. Value of diagnostic techniques for cutaneous leishmaniasis. J Am Acad Dermatol 2003; 49: 70-4. PubMed

8. Manson-Bahr PEC. Diagnosis. In: The leishmaniasis in biology and medicine, clinical aspects and control. Peters W, Killick-Kendrick R, eds. London and New York: Academic Press. 1987; Vol. 2: 703-29.

9. Tropical disease research: progress 1975-94: highlights 1993-94: twelfth program report. UNDP/World Bank/WHO special programme for research and training in tropical diseases (TDR). Geneva, Switzerland: World Health Organization; 1995. p. 135-46.

10. Mauel J, Behin R. Leishmaniasis: Immunity immunopathology and immunodiagnosis. In: Cohen S, Warren KS, editors. Immunology of parasitic diseases. 2nd ed. Oxford: Blackwell Scientific Publications; 1982.p. 299-355.

11. Zijlstra EE, El-Hassan AM. Leishmanin and tuberculin sensitivity in leishmaniasis in the Sudan, with special reference to kala-azar. Trans R Soc Trop Med Hyg 1993; 87: 425-27. PubMed

12. Ali A, Ashford RW. Visceral leishmaniasis in Ethiopia. II. Annual leishmanin transformation in a population. Is positive leishmanin reaction a life long phenomenon? Ann Trop Med Parasitol 1993; 87: 163-7. PubMed

13. Saravia NG, Valderrama L, Labrada M, Holguin AF, Navas C, Palma G, Weigle KA. The relationship of Leishmania braziliensis subspecies and immune response to disease expression in New World leishmaniasis. J Infect Dis 1989; 159: 725-35. PubMed

14. Farah FS, Klaus SN, Frankenburg S, Klion AD, Nutman TB. Protozoan and helminth infections. In: Dermatology in General Medicine, Vol ll, 4th Ed. New York: McGraw-Hill; 1993; 2772-77.

15. Even-Paz Z, Sagher F. Some basic medical problems illustrated by experiments with cutaneous leishmaniasis. S Afr Med J 1961; 35: 576-81. PubMed

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