PCR-Based Diagnosis of Acute and Chronic Cutaneous Leishmaniasis Caused by Leishmania ( Viannia )

Abstract

We evaluated PCR methods for diagnosis of acute and chronic cutaneous leishmaniasis (CL) in an area of Colombia where Leishmania (Viannia) is endemic. The PCR method specifically amplified whole linearized minicircle kinetoplast DNA (kDNA) of the Leishmania subgenus Viannia from biopsy lysates. PCR products were detected in agarose gels. For 255 acute cases, this PCR method had greater sensitivity (75.7%) than each conventional method, i.e., microscopic examination of Giemsa-stained lesion scraping (46.7%), biopsy culture (55.3%), aspirate culture (46.3%), and the conventional methods combined (70.2%). Among 44 cases of chronic CL, amplification of biopsy DNA was more sensitive (45.5%) than the individual (4.5 to 27.7%) and combined (27.3%) conventional methods. The detection of kDNA in biopsies from chronic lesions was enhanced by a chemiluminescent dot blot hybridization, which produced a sensitivity of 65.8% when alone and 90.9% when in combination with DNA extraction of biopsy lysates (P < 0.001). Three biopsies from 84 skin lesions of other etiologies were falsely positive by PCR (specificity, 96.4%). PCR detected kDNA more frequently in biopsies (detection level, 83.9%) than in aspirates (74.7%) from 103 cases of acute CL. Among aspirates from 53 chronic cases of CL, the alternative methods, DNA extraction and hybridization, increased sensitivity from 41.5 to 56.6% (P > 0.05). This enhanced PCR method in chronic biopsies was so much more sensitive than conventional methods that it should be considered the preferred diagnostic method for chronic CL. These findings support the appropriate incorporation of PCR into diagnostic strategies for cutaneous leishmaniasis.The leishmaniases are a group of illnesses of the skin, oral and respiratory mucosae and the reticuloendothelium caused by protozoa of the genus Leishmania. Of these, the cutaneous form is the most widespread, afflicting primarily rural and periurban populations exposed to the infected sand fly vector. Cutaneous leishmaniasis (CL) is most frequently diagnosed by clinical evaluation, either alone or in combination with the leishmanin skin test. Clinical evaluation usually suffers from lack of standardization (13,38) and is hampered by the fact that even fairly typical acute lesions can be confused with other dermatological problems, such as sporotrichosis (9). The leishmanin skin test is highly sensitive but lacks specificity when employed in areas of endemicity because it cannot distinguish acute lesions from past infection. A definitive, laboratory diagnosis of mucosal or cutaneous leishmaniasis traditionally requires either the visualization of amastigotes or the isolation of replicative Leishmania from lesions (24). The most widely employed laboratory methods for CL are microscopic examination of lesion scrapings, biopsy impression smears, and histopathology. The most sensitive conventional diagnostic methods, culture of lesion biopsies and aspirates, are available only in reference laboratories. Even these less available, more sensitive methods ...

Polymerase chain reaction with lesion scrapping for the diagnosis of human American tegumentary leishmaniasis

“…Isaza et al (2002) have obtained 90.6% of PCR positivity in biopsies and 80.5% in lesion scrapping. Among the ATL (positive PD and/or MST), the results showed 91.1% of positivity, similar to the ones found by Lopez et al (1993), Rodriguez et al (1994, Pirmez et al (1999), Marques et al (2001), and Weigle et al (2002). In all cases where one of the conventional exams was positive, and the lesion PCR negative, another lesion material sample was extracted and amplified.…”

“…nia antigens, but do not distinguish between current infection and past infection (Belli et al 1998, Weigle et al 2002. The serological techniques have some inconveniences as cross reaction between Leishmania antigens and other parasites as Trypanosoma cruzi (Vexenat et al 1996), as well as low sensitivity due to low antibody titers, a characteristic of tegumentary leishmaniasis (Badaró & Reed 2001).…”

Biosensors for Efficient Diagnosis of Leishmaniasis: Innovations in Bioanalytics for a Neglected Disease

“…9,10 Culturing of replicative Leishmania specimens from the skin lesion border may also be performed. 4,10,11 These methods, however, often lead to misdiagnosis; the culture assays are positive in only ca. 70% when the patient has a severe disease.…”

“…70% when the patient has a severe disease. 11 Moreover, contaminations may affect culture procedures, whereas the his-* To whom correspondence should be addressed. E-mail: zuco@if.sc.usp.br.…”

Utilização do alvo hsp70 em técnicas de biologia molecular para diferenciação de subgêneros de Leishmania spp

“…(Ashford, 2000;Weigle et al , 2002) A sensibilidade da cultura varia entre 60% a 70% devido a pele estar mais exposta ao ambiente, o que facilita a contaminação por fungos e bactérias (Silva, 2013) e o tempo de leitura pode ser de até 28 dias, dessa forma, tornando o diagnóstico mais demorado, além de não diferenciar as espécies (Ashford, 2000;Weigle et al, 2002).…”