| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Leishmaniasis is a parasitic disease caused by hemoflagel-lates of the genus Leishmania and transmitted to humans by the bites of infected phlebotomine sandflies. Depending on the causative species, they can manifest as cutaneous, mucocutaneous, and visceral clinical forms.1 In Latin America, cutaneous, mucocutaneous and mucosal forms of leishmaniasis are referred to as American tegumentary leishmaniasis (ATL).2 This infection may be inapparent or may display a wide clinical spectrum, ranging from localized, sometimes self-healing cutaneous ulcers to severe mutilating mucosal lesions or diffuse cutaneous forms.3 American tegumentary leishmaniasis is found from Mexico to northern Argentina.4–7 In the province of Salta in northwestern Argentina, ATL is an endemic disease. In recent years, there has been a steady increase in the prevalence and/or recognition of the disease in this area, causing a negative socioeconomic impact.2,7–9
Diagnostic methods for ATL in this region are based on clinical evaluation, Montenegro skin test (MST), and demonstration of amastigotes in skin lesions by microscopic examination of cutaneous scrapings. Because the geographic distributions of leishmaniasis and Chagas disease overlap in many areas of northern Argentina, the MST may show cross-reactivity, which leads to misdiagnosis.2,8 Similarly, histoplasmosis, paracoccidioidomycosis, dermal tuberculosis, and other diseases may be misdiagnosed as early lesions of cutaneous leishmaniasis.1,10–12 Conventional smears display low sensitivity in detecting occult and subclinical infections.1 Moreover, this method is time-consuming in lesions with few parasites. To overcome these difficulties, new molecular approaches in the diagnosis of leishmaniasis have been developed. Among these, the polymerase chain reaction (PCR) is the most widely used. The PCR has been evaluated as a diagnostic tool in areas where leishmaniasis is endemic.13
Our group in collaboration with Hospital del Milagro (Salta, Argentina) has investigated the use of a low-cost, simplified PCR procedure that may be used in central reference laboratories for confirmation of results of conventional methods and/or more sensitive detection.14 Most of the patients attending this hospital were either residents or had spent some time in disease-endemic zones and had chronic lesions. Based on previous work, we developed a PCR procedure using generic primers.14–16 Because most Leishmania species, such as L. (Viannia) braziliensis, its variants, and L. (Leishmania) amazonensis, in Salta cause mucosal disease, systemic treatment is proposed for all species described.2,17,18 Primers 13A and 13B amplify a specific 120-basepair kinetoplast DNA sequence common to all Leishmania species.16 This sequence is highly repetitive and provides a basis for sensitive PCR detection. The use of these generic primers provides a sensitive technique for obtaining a rapid diagnosis and preventing development of mucosal disease.
The aim of the present study was to validate the PCR as diagnostic method in a field setting in which high sensitivity and specificity are required and other pathogens that produce clinically similar lesions are present. This is the first study using the PCR as a routine diagnostic tool for leishmaniasis in clinical practice in Argentina. Rather than screen for positive PCR results in large populations of patients, we tested the diagnostic usefulness of this method with selected bacterial and fungal diseases in the area. To optimize the PCR and measure its detection sensitivity for Leishmania sp., we used serial, calibrated dilutions of laboratory samples of Leishmania pifanoi promastigotes diluted in 200 µL of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) as described.19
This study included 82 patients, 20 with a parasitologically confirmed diagnosis of leishmaniasis (positive controls), 17 with a confirmed diagnosis of other ulcerative dieseases (negative controls), and 45 with suspected leishmaniasis. All patients were recruited from areas of vectorial transmission of L. (Viannia) braziliensis within three phytogeographically regions described previously in the province of Salta, Argentina.18 They attended the Instituto de Enfermedades Tropicales in Oran, Salta, or the Hospital del Milagro in Salta. All patients provided written informed consent. The study was reviewed and approved by the Ethical Committee of the Faculty of Health at the National University of Salta and the Ministry of Public Health.
Results in the 20 positive control patients had been confirmed by microscopy and clinical examination. Eighteen had cutaneous ulcers and 2 had mucocutaneous lesions. Among the 17 negative controls, 11 had lesions clinically compatible with ATL, but they were diagnosed as having other skin diseases such as myiasis (n = 2), leprosy (n = 2), varicose ulcer (n = 2), histoplasmosis (n = 2), paracoccidioidomycosis (n = 2), and traumatic ulcer (n = 1). The 45 patients with suspected leishmaniasis (Table 1
) underwent clinical examinations and laboratory evaluations. They were also tested by PCR for Leishmania and subclassified into groups with alternative diagnoses by additional parasitologic, bacteriologic, and mycologic tests. We also tested culture isolates of Histoplasma capsulatum, Paracoccidioides brasiliensis, Mycobacterium tuberculosis, Trichophyton sp., and Candida sp. obtained from six patients. A search for inhibitory factors was performed in all negative controls by adding to each PCR sample a potential inhibitor and positive sample in a proportion 10 to 1.
|
Dermal scrapings from the bottom and margin of lesions were obtained as described, stained, and examined by microscopy.20,21 All samples, including cultures, were collected with a sterile wooden toothpick, placed in an Eppendorf (Hamburg, Germany) tube containing 200 µL of TE buffer, heated for 10 minutes in a boiling water bath, and frozen at –20°C until use. After centrifugation at 14,000 rpm for 1 minute, 10µL of supernatant was used for the PCR.14,22,23 Triplicate samples from each lesion were usually collected for smears and the PCR. The MST was performed by injecting 0.1 mL of leishmanin (40 µg of protein nitrogen/mL) intradermally into the forearm of a person as described.2
The PCR with primers 13A (5'-GTGGGGGAGGGGCGT-TCT-3') and 13B (5'-ATTTTACACCAACCCCCAGTT-3') was performed according to the procedure of Belli et al;14 all positive samples amplified a 120-basepair product.14,16 Precautions were taken to avoid contamination of specimens and amplicons, as described.24
The detection limit of the PCR was 1 parasite/200 µL of TE (Figure 1). The 120-basepair amplification product was observed in all positive control patients, which indicates a PCR sensitivity of 100% (Figure 2). No product was observed from amplification of the negative control samples, which indicated a PCR specificity of 100% (Figure 3A and B). These results showed that PCR detects only Leishmania sp. and is useful for differential diagnosis. Lack of amplification was not associated with the presence of inhibitors (Figure 3C).
|
|
|
). Microscopic results were positive in 16 patients and negative in 29 patients The PCR results were positive in 25 patients and negative in 20 patients. All patients with positive microscopic results were also positive by PCR. Stained smears detected only 16 (64%) of 25 cases positive by PCR; this method detected an additional 9 of the 25 infected patients. The PCR protocol also showed higher sensitivity with mucocutaneous lesions, which confirmed results of previous studies in which PCR was the best method for diagnosis of mucosal disease.25 Assessment of the correlation between microscopic and PCR results and MST results indicated that most (22 of 32) patients positive by either by microscopy, PCR, or both methods were also positive by the MST; only 1 of 11 was negative by the MST. The 20 patients with negative PCR and microscopic results were sub-classified as follows. Ten had a positive result in the MST, 3 with bacteriologic infections, 1 with a mycologic infection, and 6 in which no specific pathogens were demonstrated. Ten had a negative result in the MST: 3 with bacteriologic infections, 3 with mycologic infections, and 4 in which no specific pathogens were demonstrated. On the asis of clinical and epidemiologic data, the 10 patients with a positive result in the MST, especially 6 with no other specific diagnosis, were most likely infected with Leishmania at low parasite loads.
Cure after treatment has been used as diagnostic evidence for leishmaniasis.26 However, we cannot compare this diagnostic evidence with PCR results; treatment is sporadic because most of our patients do not live in Salta and must travel considerable distances for treatment and hospital resources are insufficient. Moreover, these patients are often resistant to treatment because of reactivation of their lesions.
In the 9 patients who had positive results only by PCR, the diagnosis of Leishmania infection was also supported by clinical presentation (9 of 9), positive MST result (8 of 9), or epidemiology compatible with ATL (8 of 9). Histopathologic results showed a correlation with a diagnosis by PCR in some patients, which confirmed the results of a study conducted in an adjacent area endemic for leishmaniasis.27 Moreover, of the nine patients, four had the mucocutaneous form of the disease with a clinical presentation similar to mycosis, but results of mycologic diagnosis were negative in all nine patients. Of three 3 patients with cutaneous disease, one had a positive Leishmania culture, and the remaining two had a diagnosis by histopathologic results.
Although the PCR is an expensive method, it would be useful if it were available in developing countries such as Argentina where the areas of leishmaniasis transmission overlap with areas of other endemic ulcerative diseases. Use of this method would increase the number of positive diagnoses in patients in which smears are negative, which would enable them to receive adequate treatment and avoid development of mucocutaneous complications.
Received December 1, 2006. Accepted for publication June 20, 2007.
Acknowledgments: We thank Dr. Nestor Taranto and Pamela Cajal for providing positive control samples, and Drs. Paola Zago, Alejandra Falú, and Marta Urzagasti for their support during the clinical phase of the study.
Financial support: This study was supported by the Roemmers Foundation and Consejo de Investigación, Universidad Nacional de Salta, Argentina. Miguel A. Basombrío is an International Research Scholar of the Howard Hughes Medical Institute. The American Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses.
* Address correspondence to Alejandra Barrio, Avenida del Golf 159, B° Tres Cerritos, 4400, Salta, Argentina. E-mails: barrioa{at}unsa.edu.ar and aleba05{at}yahoo.com.ar 
Authors’ addresses: Alejandra Barrio, María C. Mora, Federico Ramos, and Miguel A. Basombrío, Instituto de Patología Experimental, Universidad Nacional de Salta, Avda. Bolivia 5150, 4400, Salta Argentina, Telephone: 54-38-7425-5333. Sonia Moreno and Ruth Samson, Hospital del Milagro, Servicio de Dermatología, Avda. Sarmiento 557, 4400, Salta Argentina, Telephone and Fax: 54-38-7431-7400.
Reprint requests: Alejandra Barrio, Avenida del Golf 159, B° Tres Cerritos, 4400, Salta, Argentina, E-mails: barrioa{at}unsa.edu.ar and aleba05{at}yahoo.com.ar.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
