Usefulness of sampling with cotton swab for PCR-diagnosis of cutaneous leishmaniasis in the New World

In this study, we tested the polymerase chain reaction (PCR)-method to diagnose cutaneous leishmaniasis (CL) by taking exudate materials from lesions with cotton swabs, using our previously tested (PCR) panel comprised of Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) mexicana and L. (L.) amazonensis. The objectives of the present study were to improve the sampling method convenient for the patients and to test the usefulness of samples taken with cotton swabs. Sixteen patients were clinically diagnosed to have CL including one case of diffuse cutaneous leishmaniasis (DCL) in Ecuador and the causative Leishmania parasites were identified by PCR. All the 12 samples from CL patients of La Mana, positive for Leishmania DNA, were identified as L. (V.) panamensis, while two from CL of Huigra and one from DCL of San Ignacio were L. (L.) mexicana. In the field condition, taking biopsy material is not only painful but sometimes causes iatrogenic bacterial infections. Considering the sensitivity of the test, and convenient sampling procedure, it may be suggested that collection of exudates using cotton swabs may be a better alternative to biopsy sample for PCR-diagnosis of CL.     

Leishmania (Viannia) infection in the domestic dog in Chaparral, Colombia

Peridomestic transmission of American cutaneous leishmaniasis is increasingly reported and dogs may be a reservoir of Leishmania (Viannia) in this setting. We investigated the prevalence of infection in dogs in Chaparral County, Colombia, the focus of an epidemic of human cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis. Two (0.72%) of 279 dogs had lesions typical of cutaneous leishmaniasis that were biopsy positive by kinetoplast DNA polymerase chain reaction-Southern blotting. Seroprevalence was 2.2% (6 of 279) by enzyme-linked immunosorbent assay. Buffy coat and ear skin biopsy specimens were positive by polymerase chain reaction-Southern blotting in 7.3% (10 of 137) and 11.4% (12 of 105) of dogs, respectively. Overall 20% of dogs (21 of 105) showed positive results for one or more tests. Amplification and sequencing of the Leishmania 7SL RNA gene identified L. guyanensis in one dog and L. braziliensis in two dogs. No association was identified between the risk factors evaluated and canine infection. Dogs may contribute to transmission but their role in this focus appears to be limited.     

The rarity of infection with Leishmania (Viannia) braziliensis among patients from the Manaus region of Amazonas state,Brazil, who have cutaneous leishmaniasis

The frequency of Leishmania ( Viannia) braziliensis infection was assessed in 79 of the 138 patients with cutaneous leishmaniasis who attended a reference outpatient unit in Manaus, Amazonas state, between the August and December of 1997. The disease was characterized by one or more cutaneous ulcers, the skin lesions being frequently associated with satellite lymph-node enlargement. All parasite isolates were identified using monoclonal antibodies and enzyme electrophoresis. Only two (2.8%) of the 71 patients from whom parasites were successfully isolated were found to be infected with L. ( V.) braziliensis, the other 69 isolates being identified, from their isoenzyme profiles, as L. ( V.) guyanensis. In the Manaus region, therefore, almost all human cutaneous leishmaniasis is the result of infection with L. (V.) guyanensis, and L. ( V.) braziliensis is a relatively rare cause of the disease.     

The sensitivity of clinical isolates of Leishmania from Peru and Nepal to miltefosine

Clinical isolates of Leishmania, from visceral leishmaniasis (VL) cases in Nepal and from cutaneous leishmaniasis (CL) cases in Peru, were cultured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to type species and strain. Promastigotes from 38 isolates, within eight passages from isolation, were used to infect mouse peritoneal macrophage cultures in vitro, and the amastigote sensitivity to miltefosine was determined. The concentration required to kill 50% of intracellular amastigotes from Nepalese VL isolates, all typed as Leishmania (L.) donovani (N = 24) from both Sbv responders and nonresponders, ranged from 8.7 to 0.04 microg/mL. In contrast, the concentration required to kill 50% intracellular amastigotes from isolates from Peru, typed as L.(V.) braziliensis (N = 8), was > 30 to 8.4 microg/mL, L.(V.) guyanensis (N = 2) > 30 to 1.9 microg/mL, L.(L.) mexicana (N = 1) > 30 microg/mL, and L. (V.) lainsoni (N = 4) was 3.4 to 1.9 microg/mL. This demonstrates a notable difference in the intrinsic sensitivity of Leishmania species to miltefosine in vitro. If this model can be correlated to therapeutic outcome, it may have implications for the interpretation of clinical trials.     

Failure of both azithromycin and antimony to treat cutaneous leishmaniasis in Manaus, AM, Brazil

A non-randomized controlled clinical trial was carried out in order to evaluate both azithromycin and antimony efficacy in cutaneous leishmaniasis in Manaus, AM, Brazil. Forty nine patients from both genders, aged 14 to 70, with cutaneous ulcers for less than three months and a positive imprint for Leishmania spp. amastigotes were recruited into two groups. Group I (26 patients) received a daily-single oral dose of 500 mg of azithromycin for 20 days and Group II (23 patients) received a daily-single intramuscular dose of 20 mg/kg of meglumine antimony, also for 20 days. Azithromycin cured three of 24 (12.5%) patients on days 60, 90 and 120 respectively whereas therapeutic failure was considered in 21 of 24 (87.5%) cases. In group II, antimony cured eight of 19 (42.1%) cases as follows: three on day 30, one each on day 60 and day 90, and three on day 120. Therapeutic failure occurred in 11 of 19 (57.9%) individuals. The efficacy of antimony for leishmaniasis was better than azithromycin but analysis for the intention-to-treat response rate did not show statistical difference between them. Although azithromycin was better tolerated, it showed a very low efficacy to treat cutaneous leishmaniasis in Manaus.     

Species diversity causing human cutaneous leishmaniasis in Rio Branco, state of Acre, Brazil

OBJECTIVE: Information on Leishmania species diversity in western Brazilian Amazon and the clinical picture of human cutaneous leishmaniasis it causes is scarce. We describe clinical findings, diagnostic procedures and identification of Leishmania species in patients from that region. METHODS: The sample consisted of 50 patients, prospectively evaluated for epidemiological and clinical characteristics by means of a structured questionnaire. Conventional and molecular tools were applied to confirm the parasitological diagnosis and identify the species responsible for the disease. RESULTS: Patients were predominantly male (76.5%) and living in rural areas. Median average age was 18 years and median average disease evolution was 8 weeks. For the diagnostic procedures of leishmanin skin test, direct visualization of amastigotes in dermal scrapings and parasite culture of aspirates of the ulcer border were positive for 98%, 52% and 34%, respectively. Molecular methods applied to DNA extracted from skin biopsies of the 50 patients yielded 100%, 82% and 44% positivity by PCR minicircle kDNA, PCR-RFLP ITS1rDNA and PCR-glucose-6-phosphate (G6P), respectively. Fourteen samples from 13 patients were successfully isolated and identified. Multilocus enzyme electrophoresis, PCR-RFLP ITS1rDNA and PCR-G6P permitted identification of the Leishmania species responsible for the aetiology of American tegumentary leishmaniasis in 60% of the examined patients: 16 Leishmania (Viannia) braziliensis, 12 Leishmania (Viannia) lainsoni, 1 Leishmania (Viannia) guyanensis and 1 putative hybrid of Leishmania (Viannia) naiffi and L. (V.) lainsoni. CONCLUSION: The clinical and epidemiological behaviour of cutaneous leishmaniasis in Acre, Brazil, is similar to other Amazon scenarios previously described; however Acre's complex parasite diversity may be contributed to the concomitant circulation of at least three distinct Leishmania species. The implementation of control interventions in the studied area must take into consideration the possibility of various expected phlebotomine vectors and reservoirs.     

Clinical features and diagnosis of 42 travellers with cutaneous leishmaniasis

BACKGROUND: Leishmania species that occur within different geographical areas may cause different clinical manifestations, virulence and drug sensitivity. Patients/Methods. All patients with a clinical diagnosis of cutaneous leishmaniasis seen at the Hospital for Tropical Diseases from 1997 to 2000 were identified and clinical details recorded onto a database, with emphasis on clinical presentation, risk factors, travel history and laboratory diagnosis. RESULTS: Forty-two patients were identified, 23 of whom had travelled to New World and 19 to Old World countries. Clinical presentation typically consisted of a single nodule with ulceration. In 50% infection was caused by L. (Viannia) braziliensis. PCR was performed in specimens from 34 patients and species identification was possible in 32 cases (sensitivity 94%), the two PCR negative patients had amastigotes demonstrated by histology and culture. Patients were treated with established therapies. Seventy one percent were cured by treatment, 12% had a spontaneous cure, 7% were lost to follow-up and the remaining 10% required a second-line therapy. No relapses were reported during a mean follow-up period of 27 months. CONCLUSIONS: Our study highlights the need for comprehensive investigations and the advantages of PCR in the diagnosis of patients with suspected leishmaniasis in non-endemic regions of the world.     

Etiologic agent of an epidemic of cutaneous leishmaniasis in Tolima, Colombia

American cutaneous leishmaniasis (ACL) has been characterized as a zoonotic disease. However, peridomestic and domestic transmission have been recorded in at least nine countries in Central and South America. The present study was undertaken to identify the etiologic agent of a peridomestic epidemic of ACL in the Department of Tolima, Colombia. Leishmania isolates were obtained during the diagnosis of 56 patients with ACL who consulted the local leishmaniasis control program in three municipalities in Tolima. Species were identified using monoclonal antibodies and isoenzyme electrophoresis. A total of 53 (94.6%) of 56 isolates were identified as Leishmania (Viannia) guyanensis. Three isolates (5.4%) were identified as L. (V.) panamensis. Leishmania (V.) guyanensis is the probable etiologic agent of the largest epidemic of cutaneous leishmaniasis recorded in Colombia. This species has not previously been reported outside the Amazon and southeastern regions of Colombia, and has not been described in the peridomestic setting or linked with an epidemic.     

The persistence, dissemination, and visceralization tendency of Leishmania major in Syrian hamsters

I monitored the persistence, dissemination, and the possible visceralization tendency of Leishmania major, the causative parasite of cutaneous leishmaniasis in North Africa and the Middle East in Syrian hamsters (Mesocricetus auratus). Hamsters were inoculated subcutaneously in the hind footpad, with 1 x 10(6)L. major metacyclic promastigotes and were sacrificed at months 1, 3, 6 and 10 post-infection (pi). Skin lesions, blood, spleens, livers and kidneys were screened by both Giemsa-stained smears and a polymerase chain reaction (PCR) for detection of L. major amastigotes. A few weeks pi, 61.7% of the inoculated hamsters developed a cutaneous lesion only at the inoculation site, while 38.3% of them developed non-self-healed lesions at sites distant from the inoculation site. PCR identified all the positive stained smears as well as false-negative ones, indicating that PCR was more sensitive than stained smears. The results confirmed the dissemination and persistence of L. major amastigotes in all tissues examined, except the kidneys, for a period extending to 10 months, only in those hamsters suffering from disseminated cutaneous lesions. Parasite DNA was detected in the bloods starting from the first month pi and starting from month 3 pi in the spleens and livers. Some, but not all, the animals with disseminated infections proved to be positive for parasite DNA in their organs. Persistence of the L. major amastigotes in the tissues differed from those of other species causing visceral diseases. These findings demonstrate a possible visceralization tendency for L. major previously recorded for L. tropica and L. mexicana.     

Polymerase chain reaction detection of Leishmania kDNA from the urine of Peruvian patients with cutaneous and mucocutaneous leishmaniasis

We hypothesized that Leishmania kDNA may be present in urine of patients with cutaneous leishmaniasis (CL). Urine samples and standard diagnostic specimens were collected from patients with skin lesions. kDNA polymerase chain reaction (PCR) was performed on samples from patients and 10 healthy volunteers from non-endemic areas. Eighty-six of 108 patients were diagnosed with CL and 18 (21%) had detectable Leishmania Viannia kDNA in the urine. Sensitivity and specificity were 20.9% (95% confidence interval [CI] 12.3-29.5%) and 100%. Six of 8 patients with mucocutaneous involvement had detectable kDNA in urine versus 12 of 78 patients with isolated cutaneous disease (P < 0.001). L. (V.) braziliensis (N = 3), L. (V.) guyanensis (N = 6), and L. (V.) peruviana (N = 3) were identified from urine. No healthy volunteer or patient with an alternate diagnosis had detectable kDNA in urine. Sensitivity of urine PCR is sub-optimal for diagnosis. On the basis of these preliminary data in a small number of patients, detectable kDNA in urine may identify less localized forms of infection and inform treatment decisions.     

Radical cure of experimental cutaneous leishmaniasis by the bisphosphonate pamidronate

The effects in vivo of the bisphosphonate drug pamidronate, used in bone resorption therapy, were investigated in an experimental model of cutaneous leishmaniasis. Pamidronate at an intraperitoneal dose of 10 mg/kg/day for 5 days effects a radical cure of cutaneous leishmaniasis in Balb/c mice, as evidenced by long-term disappearance of lesions; disappearance of amastigotes in lesion sites, as determined by histopathological analysis and cultivation of material obtained from lesions; and polymerase chain reaction analysis of necropsy material, using probes specific for kinetoplast DNA. Pamidronate is, therefore, a new lead compound for the synthesis of drugs effective against cutaneous leishmaniasis.     

Cutaneous leishmaniasis treatment

The causative species of cutaneous leishmaniasis determines the clinical features and courses, and treatments. Intralesional or systemic antimonials are the gold standard for the treatment of these diseases. However, as for visceral leishmaniasis, other therapeutic options appear promising. Paromomycin ointments are effective in Leishmania major, L. tropica, L. mexicana, and L. panamensis lesions. In L. braziliensis localized leishmaniasis, both paromomycin and imiquimod may be topically applied. Oral fluconazole and zinc sulfate are useful in L. major. Oral azithromycin, effective in vitro and in mice, needs further investigation in human leishmaniasis. On the contrary, data with oral itraconazole are disappointing. Oral miltefosine, which is very effective in visceral leishmaniasis caused by L. donovani, appears ineffective in L. major and L. braziliensis infections. Intramuscular pentamidine is required for L. guyanensis cutaneous leishmaniasis, for which systemic antimony is not effective. Liposomal amphotericin B could be an alternative to antimony in south American cutaneous leishmaniasis with mucosal involvement (especially L. braziliensis and L. guyanensis infections).     

Leishmania braziliensis from the Pacific coast region of Colombia: foci of transmission, clinical spectrum and isoenzyme phenotypes

Tegumentary leishmaniasis is highly prevalent in the Pacific coast region of Colombia. We have identified 90 foci of transmission in this region based on 179 parasitologically diagnosed patients. Human transmission occurred in mangrove forests, secondary growth and intervened tropical rain forest. A parasitological diagnosis, that is, either isolation or visualization of Leishmania was made in 68.6% of suspected cases. Three phenotypically distinguishable groups of L. braziliensis were encountered based on isoenzymes: L. b. panamensis variants (82%), variants of L. b. braziliensis (14.5%), and stocks intermediate between L. b. panamensis and L. b. guyanensis reference strains (3.5%). The L. b. braziliensis variants produced cutaneous disease alone relatively infrequently (12% of classified cutaneous stocks) but were more frequently (38% of all mucosal stocks) isolated from mucosal lesions. Leishmania infection of the mucous membranes caused a wide spectrum of disease, severity being closely related to time of evolution. Both contiguous and metastatic spread to the mucous membranes was supported by the clinical course of 19 mucosal cases.     

Imported cutaneous leishmaniasis in a short-term traveler returning from Central Mali - The role of PCR

Leishmaniasis is a parasitic infection caused by the obligate intracellular protazoa leishmania. The most commonly encountered form is cutaneous leishmaniasis (CL), which generally manifests as a chronic, painless ulcer. Recent increases in the incidence of CL worldwide due in large part to increased immigration and international travel, combined often with the lack of familiarity with the disease in non-endemic settings, pose the continued problems of delayed diagnosis and inappropriate treatment. A case is described of imported cutaneous leishmaniasis occurring in a 48 year-old male who presented with multiple painless, progressively ulcerating lesions after returning from a one week trip to Bandiagara, Mali, West Africa. After four months of misdiagnoses and ineffective treatments, he was referred to a tropical disease specialist where the diagnosis was made with a skin biopsy followed by a tissue impression smear, culture and PCR. Appropriate treatment was initiated and the lesions resolved with minimal scarring. The goals of this case report are threefold: first, to stress the importance of associating chronic ulcers in a traveler with potential cutaneous leishmaniasis; second, to emphasize the clinical utility of PCR for the diagnosis; and third, to discuss the clinical approach to treatment.     

Sensitivity of an immunoenzymatic test for detection of anti-L. brasiliensis antibodies compared to other tests used for the diagnosis of American cutaneous leishmaniasis

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.     

Use of kDNA-based polymerase chain reaction as a sensitive and differentially diagnostic method of American Tegumentary Leishmaniasis in disease-endemic areas of northern Argentina

We evaluated the polymerase chain reaction (PCR) for the diagnosis of endemic American Tegumentary Leishmaniasis in Salta, Argentina. Diverse Leishmania species, coexistence of mycotic and varicose ulcers, and high endemicity T. cruzi, represent diagnostic challenges in the region. We performed a simplified PCR using sensitive, generic primers on samples obtained by a non-invasive method. We tested different culture types and clinical specimens with other microorganisms that induce leishmaniasis-like lesions. The PCR had a sensitivity and specificity of 100%. Forty-five patients with presumptive leishmaniasis were compared to the PCR, smears, and the Montenegro skin test (MST). In the same population, the PCR had an increased sensitivity, detecting 25 of 45 cases compared with 16 of 45 for smears and had a higher sensitivity in detecting mucocutaneous lesions. Diagnosis by PCR was supported by clinical presentation, positive MST results, compatible epidemiology, and in some cases histopathologic results or isolation of parasites by culture. These findings indicate the convenience of incorporating this PCR into diagnostic strategies for detecting leishmaniasis in northern Argentina.     

First case of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Suriname

The main causative agent of cutaneous leishmaniasis (CL) in Suriname is Leishmania (Viannia) guyanensis. This case report presents a patient infected with Leishmania (Viannia) braziliensis, a species never reported before in Suriname. This finding has clinical implications, because L. braziliensis has a distinct clinical phenotype characterized by mucocutaneous leishmaniasis, a more extensive and destructive form of CL that requires different treatment. Clinicians should be aware that chronic cutaneous ulcers in patients from the Guyana region could be caused by L. braziliensis.     

"Localized" leishmania lymphadenitis: a light and electron microscopic study

Nineteen cases of "localized" leishmania lymphadenitis without any evidence of visceral leishmaniasis are reported. Fifteen males and 4 females aged 5 to 30 years have presented with localized lymphadenopathy of up to 3 months duration. The disease has a seasonal incidence of late summer to mid-winter. Cutaneous leishmaniasis even when present, usually was overlooked. Lesions of cutaneous leishmaniasis were absent in 3; healed in 1; small, similar to insect bites in 5; classic in 3; lupoid in 1 and unknown in 6 patients. Serum leishmanial antibody determination by IFAT performed on 12 cases were positive. Toxoplasma serology was negative. Histological picture of one lymph node biopsy showed an (anergic) intact histiocytic response characterized by thousands of intracellular amastigotes, no necrosis and inconspicuous plasma cells. In 17 biopsies the picture was that of histiocytic granulomata with varying degrees of necrosis, moderate numbers of amastigotes in several foci, fibrosis and varying numbers of plasma cells. One biopsy from a lupoid case shows numerous epitheloid granulomata, no organisms, no necrosis and inconspicuous plasma cells. Electron microscopy has been performed on 8 biopsies to confirm leishmania amastigotes. Differential diagnosis from toxoplasmosis and cat-scratch disease is discussed. Histological types of responses in the lymph nodes are comparable to those described in cutaneous leishmaniasis: an anergic response with intact macrophage granuloma, a histiocytic response with necrosis, and a lupoid type of response with epithelioid granulomas.