Optimization of Microculture and Evaluation of Miniculture for the Isolation of Leishmania Parasites from Cutaneous Lesions in Peru

Abstract

Traditional culture of Leishmania parasites is labor-intensive and shows poor sensitivity. We evaluated microculture and novel miniculture methods for diagnosis of cutaneous leishmaniasis (CL). Consecutive patients who came to the Leishmaniasis Clinic, Hospital Nacional Cayetano Heredia, Lima, Peru, were enrolled. Lesion aspirates were cultured in traditional tubes containing Novy-MacNeal-Nicolle medium and in miniculture tubes (Eppendorf, Hamburg, Germany) and capillary tubes (microculture) containing RPMI 1640 medium containing 20% fetal bovine serum. The reference standard was positive results in two of four tests (smear, culture, polymerase chain reaction, or leishmanin skin test). Outcome measures were sensitivity and time to positivity. Fifty-five patients with 74 lesions were enrolled. Of 59 lesions that fulfilled reference criteria for CL, 50 were positive by microculture (sensitivity=84.7%; P=0.001), 45 by miniculture (sensitivity=76.3%; P=0.042), and 35 by traditional culture (sensitivity=59.3%). Median time to positivity was three days by microculture and miniculture and five days by traditional culture (P<0.001). Microculture and miniculture are sensitive and efficient means of diagnosing CL.

An improved microculture method for diagnosis of cutaneous leishmaniasis

“…The aforementioned study (2004) also expressed that the high sensitivity of the MCM might be explained by the use of capillary tubes, which concentrate the sample material and provide microaerophilic conditions favorable for transformation of the amastigotes to promastigotes. The sensitivity and specificity of the IMC was found to be 98.4 and 100 %, respectively which is comparable with microculture results achieved by previous studies (Allahverdiyev et al 2004;Ihalamulla et al 2005Ihalamulla et al , 2006Boggild et al 2007Boggild et al , 2008Boggild et al , 2010.…”

“…While the present study used a new improved, noninvasive, safer (needle free), simpler, faster and more sensitive microculture method compared with studies carried out so far (Ihalamulla et al 2005(Ihalamulla et al , 2006Boggild et al 2007Boggild et al , 2008Boggild et al , 2010 as sampling was taken from the ulcer borders directly and without aspiration by needle.…”

“…Moreover, in each individual study, the composite reference standard against which each diagnostic test was compared was two of three or four tests with positive results, where tests refer to smears, culture by either method, or PCR. For evaluation of performance characteristics of each test, the lesion was the unit of analysis, as previously described by Boggild et al (2007Boggild et al ( , 2008.…”

“…To date, in all performed investigations for MCM evaluation, the samples have been obtained using the aspiration puncture by the syringe. In addition to, these studies had showed varied performance by sensitivity and saving incubation time (Boggild et al 2007(Boggild et al , 2008(Boggild et al , 2010. Therefore, further evaluation and also modification for MCM usefulness in the diagnosis of CL in other endemic countries is needed.…”

Comparison of Direct Microscopy, Culture and Polymerase Chain Reaction Methods for the Diagnosis of Cutaneous Leishmaniasis

“…Son yıllarda KL tanısında, ribozomal, "miniexon" veya "mini/maxicircle" kDNA gibi korunmuş veya değişken bölgelere ait sık tekrar eden değişik bölgeleri hedefleyen moleküler yöntemler kullanılmaktadır [12][13][14][15][16] . Moleküler bazlı yöntemlerin en önemli avantajının, 24 da duyarlılık oranlarını; PCR için %96.6, kültür için %84.7 ve DM için %66.1 olarak vermişlerdir. Bizim çalışmamızda ise DM, kültür ve PCR yöntemlerinin duyarlılıkları sırasıyla %78.4, %92 ve %81.1 olarak bulunmuştur.…”