Dermal Scrapings Research Articles

Bacilloscopy and polymerase chain reaction of slit-skin smears and anti-phenolic glycolipid-I serology for Hansen's disease diagnosis.

The bacilloscopy of the slit-skin smear (SSS) is the exclusive laboratory test associated with dermato-neurological evaluation for Hansen’s disease (HD) diagnosis; however, it is negative in the majority of PB or primary neural forms. Thus, a PCR technique involving different sequences and target genes has been performed with an aim to increase the sensitivity and specificity of M. leprae identification, especially in patients with low bacillary loads. Additionally, serological assays based on antibody response reflect infection levels and indicate that this could be a simpler, less invasive technique for estimating M. leprae exposure. Serological tests and PCR have been shown to be more sensitive and accurate than the SSS. Our study aimed to measure accuracy and performance among the SSS and PCR of dermal scrapings stored on filter paper and APGL-I serology for diagnosis in HD. A cross-sectional study analyzing the medical records (n = 345) of an HD outpatient-dermatology clinic from 2014 to 2021 was conducted. Accuracy performance parameters, correlation, and concordance were used to assess the value among the SSS, PCR, and APGL-I exams in HD. The SSS presented 24.5% sensitivity, 100% specificity, 37.4% accuracy, and the lowest negative predictive value (21.5%). The PCR assay had 41, 100, and 51% sensitivity, specificity, and accuracy, respectively. PCR and APGL-I serology increased the detection of HD cases by 16 and 20.6%, respectively. PCR was positive in 51.3% of patients when the SSS was negative. The SSS obtained moderate concordance with PCR [k-value: 0.43 (CI: 0.33–0.55)] and APGL-I [k-value: 0.41 (CI: 0.31–0.53)]. A moderate positive correlation was found between the APGL-I index and the bacillary index (r = 0.53; P < 0.0001). Thus, the use of the SSS is a low sensitivity and accuracy method due to its low performance in HD detection. The use of PCR and serological tests allows for a more sensitive and accurate diagnosis of patients.

Imaging Leishmania major Antigens in Experimentally Infected Macrophages and Dermal Scrapings from Cutaneous Leishmaniasis Lesions in Tunisia.

Leishmania major cutaneous leishmaniasis (CL) lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection. The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens and access its reliability in the routine diagnosis of CL. The developed DIF assay used IgG polyclonal antibodies produced in rabbits by intravenous injections of live L. major metacyclic promastigotes chemically coupled to fluorescein isothiocyanate. Applied to L. major infected RAW macrophages, corresponding macrophage-derived amastigotes and dermal scrapings from CL lesions, the immunofluorescence assay stained specifically Leishmania amastigotes and showed a diffuse Leishmania antigen deposit into cytoplasm of phagocytic cells. Reliability of DIF in CL diagnosis was assessed on 101 methanol-fixed dermal smears from 59 positive and 42 negative CL lesions diagnosed by direct microscopy and/or kDNA real-time PCR. Sensitivity and specificity of DIF was 98.3% and 100%, respectively, being more sensitive than microscopy (p < 0.001) and as sensitive as ITS1-PCR. ITS1-PCR-RFLP allowed Leishmania species identification in 56 out of the 58 DIF-positive smears, identifying 52 L. major, two L. infantum and two L. tropica cases, which indicates antigenic cross-reactivity between Leishmania species.

Diagnostic Efficacy of Recombinase-Polymerase-Amplification Coupled with Lateral Flow Strip Reading in Patients with Cutaneous Leishmaniasis from the Amazonas Rainforest of Perú.

Cutaneous leishmaniasis (CL) is highly prevalent in rural and sylvatic regions of Latin America, with an estimated 55,000 annual cases. Diagnosis in resource-limited areas still relies on microscopy of dermal scrapings, while more sensitive methods like PCR are not attainable due to costs and lack of adequate health infrastructure. Isothermal amplification of Leishmania DNA can be performed without sophisticated equipment and training and may become a point of care (POC) test for health care centers with scarce resources. We evaluated the efficacy of recombinase-polymerase-amplification (RPA-LF) to diagnose CL in 226 patients attending a clinic in Puerto Maldonado within the Peruvian Amazon basin. Conventional PCR targeting kinetoplast DNA (kDNA-PCR) was used as the gold standard. Eight of 226 patients were considered true negatives (microscopy, kDNA-PCR, and RPA-LF negative), while RPA-LF resulted positive in 186 of 204 kDNA-PCR positive patients, yielding 91.2% (confidence interval [CI] = 86.5-94.4%) sensitivity and 93% (CI 88.6-95.8%) positive predictive value. There were 14% (32/226) discrepant samples alternating positive and negative results in similar proportions between both tests. Quantitative PCR used to resolve the discrepancies suggested that they occurred in samples with scarce parasite numbers as determined by high cycle threshold (Ct) values (≥32; cutoff 35.5). Microscopy had the lowest sensitivity of all methods (45.4%). Nested real-time PCR performed in 71 samples determined that Leishmania (Viannia) braziliensis was highly prevalent (69/71), and Leishmania (Viannia) lainsoni was present in only two isolates. Results indicated that RPA-LF has POC potential for CL endemic areas, yet further simplification and optimization coupled with field validation will be necessary to confirm its broad applicability.

Cutaneous leishmaniasis as an increasing threat for Iranian travellers attending religious ceremonies.

The Islamic Republic of Iran is one of the most important endemic foci of cutaneous leishmaniasis in the world. Annually, a large number of Iranian Shia pilgrims travel to Iraq from this area in order to participate in one of their most important religious ceremonies. This trip has coincided with the seasonal activity of sand flies in recent years. So, cutaneous leishmaniasis could be a serious threat for pilgrims on these trips. To report cases of cutaneous leishmaniasis among Iranian Shia pilgrims attending a religious ceremony in Iraq during 2017. Sixteen patients were referred to our laboratory in the Department of Parasitology and Mycology at Qazvin University of Medical Sciences. Dermal scrapings and stained slides prepared of skin lesions were used to morphological diagnosis. DNA extraction and PCR amplification were optimized to identification of Leishmania species. All of the patients were infected with cutaneous leishmaniasis in microscopic survey. L. major was detected by molecular approach. The number of lesions observed in patients were 1 (31%), 2 (25%), and ≥ 3 (44%). Since a large number of Shia Muslims participate in the annual religious ceremonies, serious measures must be taken to prevent the disease.

Virulence Factors and Antifungal Susceptibility in Candida Species Isolated from Dermatomycosis Patients.

Dermatomycoses caused by Candida spp. are increasingly common, however there are few reports in the literature regarding their epidemiology, pathogenesis and antifungal susceptibility of these fungal pathogens. This study aimed to identify, characterize virulence and determine the antifungal susceptibility of Candida species isolated from patients with dermatomycoses. We studied a total of 45 yeast samples isolated from dermal scrapings from patients treated at a public hospital, Bauru, São Paulo, Brazil. After identification, the samples were analyzed for protease activity, phospholipase, biofilm formation and antifungal susceptibility. The most common species was Candida albicans (40%), followed by C. krusei (22.22%), C. parapsilosis complex (22.22%) and C. famata (6.67%). Regarding virulence mechanisms, 57.78% and 28.89% of the samples showed enzymatic activity for protease and phospholipase, respectively. A total of 13.33% of the samples showed secretion of both enzymes. All isolates were biofilm producers, and a higher production was observed in C. tropicalis isolates. Regarding the antifungal susceptibility test, 97.78% of the samples had the highest levels of the minimal inhibitory concentration (MIC) for terbinafine, and one C. krusei sample showed intermediate susceptibility to fluconazole. The correct identification of dermatomycosis-related microorganisms, as well as a better knowledge of their pathogenicity and susceptibility against the antifungals used in the clinic, is extremely important for an efficient preventive and therapeutic action.

Comparison of Diagnostic Methods in Cutaneous Leishmaniasis in Iraq

Sixty -four specimens collected passively from patients with cutaneous ulcers suggestive of leishmaniasis attending hospital and health centres in all parts of Iraq for diagnosis were included in the study. The present study was conducted from June to December 2012 in Al-Karamah Teaching Hospital in Wasit Province, Iraq. Dermal scrapings were analysed both by parasitological (examination of Giemsa-stained smears and in vitro cultivation) methods and by a genus-specific PCR detection. Microscopy revealed amastigotes in 38 samples (59.4%) and in vitro cultivation gave positive results in 19 cases (29.7%), whereas PCR detected Leishmania in 44 samples (68.75 %). There are two forms of Cutaneous leishmaniasis (CL) in Iraq are caused by two species of Leishmania . Of 44 well-documented cases in our study ; 28 (63.6 %) were caused byLeishmania major and 16 (36.4 %) by Leishmania tropica.

Active surveillance of American tegumentary leishmaniasis in endemic areas in rural Bolivia

American tegumentary leishmaniasis (ATL), including mucocutaneous leishmaniasis (MCL) and localized cutaneous leishmaniasis (LCL), is endemic in Bolivia. We describe the results of active surveillance of ATL from 2001 to 2006 and assess demographic data related to ATL epidemiology in the Yungas valleys. Community-based active ATL surveillance was performed by the institutions SERVIR, CÁRITAS, and the Health Services Department of La Paz, whose files were reviewed retrospectively. A cross-sectional survey was carried out to assess demographic data in two communities. Two thousand nine hundred nine cases of ATL were detected from 2001 to 2006: 2,488 (85.5%) corresponded to LCL and 421 (14.5%) to MCL. A reduction in the proportion of mucosal cases was observed between 2001 and 2006. The proportion of MCL cases increased with age and was higher among males (15.5% versus 12.1%, p=0.018). The rate of positivity via direct observation of the parasite in dermal scrapings and in parasite cultivation was significantly higher for LCL than for MCL (p<0.001 and p=0.009, respectively). The rate of reactivity in the leishmanin skin test was higher in the group with mucosal lesions (p=0.012). The cross-sectional survey showed that 40% of the families had emigrated from the Altiplano. It is necessary to undertake continuous case detection of ATL in the area, where the disease presents a high rate of mucosal cases. Increasing incidence seems to be associated with immigration and continuous deforestation to expand the crop-growing areas.

Epidemiologic and Clinical Features of Cutaneous Leishmaniasis in Southeastern Tunisia

Species-specific diagnosis was performed in 66 patients with cutaneous leishmaniasis (CL) living in Tataouine focus in southeastern Tunisia. Leishmania DNA was extracted directly from dermal scrapings (n = 66) and from parasites obtained in culture (n = 12). Species were identified by using polymerase chain reaction-restriction fragment length polymorphism analysis for internal transcribed spacer region 1 and isoenzyme analysis. Leishmania tropica and L. major were identified in 31 (47%) and 35 (53%) cases respectively. Leishmania tropica CL cases were geographically scattered, and L. major CL cases were clustered. Lesions caused by L. tropica were mostly single (83.8%) and face-localized (55.8%), and lesions caused by L. major were multiple (57.1%; P < 0.001) and situated on limbs (83.7%; P < 0.001). For both species, most lesion onsets were reported during June-January. However, lesions that emerged during February-May were mainly caused by L. tropica (83.3%; P < 0.01). Moreover, the delay before seeking medical advice was higher for L. tropica infections than for L. major infections (P < 0.05).

Evaluation of blood oxidant/antioxidant balance in dogs with sarcoptic mange

The aim of this study was to investigate of oxidant/antioxidant balance in dogs with sarcoptic mange. The study materials consisted of totally 30 cross-breed male dogs; 15 with sarcoptic mange (study group) and 15 healthy as control. Blood samples for analyses were taken from control and study group. In study group, microscopic examination of dermal scrapings of 15 dogs revealed S. scabies. Lipid hydroperoxide level, total oxidant status and oxidative stress index in dogs with sarcoptic mange were higher ( P < 0.01, P < 0.01 and P < 0.05, respectively) than the control. Otherwise; sulphydril levels in dogs with sarcoptic mange were lower ( P < 0.05) than that of control. No significant differences were observed in total antioxidant capacity between groups. Our results suggest a possible relationship between oxidant/antioxidant imbalance and sarcoptic mange infestation in dogs.

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

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. 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. 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. 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.

Leishmaniasis among Gold Miners, French Guiana

To the Editor: In 2004, the Cayenne General Hospital and public health centers recorded 348 new cases of cutaneous leishmaniasis (CL) in French Guiana (1). A case of CL was considered confirmed if cutaneous lesions were present for >2 weeks; the patient had a compatible epidemiologic history; and microscopic examination of dermal scrapings, parasite cultivation, or both showed positive results for Leishmania. According to the population estimate given by the French National Institute for Statistics and Economical Studies (INSEE, Cayenne), the incidence of CL in 2004 was 0.2%–0.4% and has been relatively stable since 1979 (2,3). However, when the annual number of cases per village were examined, new CL cases were heterogeneously distributed. Saint Elie, a gold-mining village in the inland neotropical forest, had an apparent incidence rate of 25.9% in 2004 and 28.9% in 2005 (Figure); risk for infection in this village was, on average, 65× higher than anywhere else in French Guiana. We tested samples from 12 random CL patients with a Leishmania-specific polymerase chain reaction–restriction fragment length polymorphism test that targeted the internal transcribed spacer 1 of ribosomal RNA genes with primers SSU-12103-D (5´-GGGAATATCCTCAGCACGT-3´) and 5.8S-13333-R (5´-CGACACTGAGAATATGGCATG-3´) (4). All these patients were infected with Leishmania guyanensis. Figure Number of new cutaneous leishmaniasis (CL) cases registered in health centers of 4 villages of French Guiana (Iracoubo, Regina, Saul, and Saint Elie) from 2000 to 2005. For each village, the 1999 population estimate (French National Institute ... Isolated in dense rainforest (no road or airport) and with 239 inhabitants (INSEE, Cayenne), Saint Elie is situated on a gold seam; miners illegally create trails from the village to deposits in a 10-km circumference in the dense forest around the village. Compared to other French Guianan villages, such as Saul and Regina, which are similarly isolated in the rainforest and have 160 and 765 inhabitants (INSEE, Cayenne), respectively, and Iracoubo, the village closest to Saint Elie with 1,430 inhabitants (INSEE, Cayenne), substantially more new CL cases have been observed in Saint Elie since 2003. Since 2000, medical rounds have been undertaken every 15 days in the villages of Saint Elie and Saul, whereas people from Regina and Iracoubo have doctors at their disposal every day. Official records indicate that the population of Saint Elie has doubled in the past 10 years, reaching 239 inhabitants in 1999 (INSEE, Cayenne). However, 860 new medical files have been registered in the Saint Elie Health Centre since 2000. This finding could be explained by the high number of illegal workers in this area. Patient interviews showed that most of these workers (≈90%) originated from the poorest northern Brazilian states (Para, Amapa, Roraima, and especially Maranhao). Thus, the incidence rate of 25.9%, calculated on the basis of 239 inhabitants, was likely overestimated. Taking into account a substantial turnover in migrant populations, the denominator could be 500–1,000 inhabitants, and the incidence rate would be 6.2%–12.4%. All patients worked in the small-scale gold mines surrounding Saint Elie, and CL cases were recorded without seasonal fluctuations. Imported cases are possible, but reports are likely to be anecdotal because clinical observations, estimated dates of infection, and duration of patient stay in Saint Elie were congruent and because all genotyped strains were Guianan L. guyanensis (1). Several infection risk factors exist simultaneously in this situation. In a CL-endemic area, immigrant populations, who are mostly nonimmune, exert pressure on the environment (deforestation) that directly increases their risk for exposure to infected vectors, in the absence of prophylactic measures. The initial short-term effect of deforestation is the mobilization of aggressive adult sandflies, which have been disturbed while resting. However, the ability of zoophilic vectors to adapt to peridomestic environments has also already greatly influenced the distribution of leishmaniases in South America (5–7). Considering the uncertainty of the population estimate, turnover, and immunity status, we assume that incidence rates should be considered cautiously. Nevertheless, we found that gold mining in forested areas constitutes a risk factor for CL, at least in French Guiana and probably in all Amazonian rainforests. This risk could be a public health concern. Larger studies in other gold-mining areas are required to quantify the incidence of CL among workers to effectively focus prophylactic and preventive campaigns.

Usefulness of PCR in the diagnosis of cutaneous leishmaniasis in Tunisia

We assessed the efficiency of a PCR method in establishing the diagnosis of cutaneous leishmaniasis (CL) in Tunisian patients. Four hundred and thirty specimens collected passively from patients with cutaneous ulcers suggestive of leishmaniasis attending health centres for diagnosis were included in the study. Dermal scrapings were analysed both by parasitological (examination of Giemsa-stained smears and in vitro cultivation) methods and by a genus-specific PCR detecting a fragment of the 18S rRNA gene. Microscopy revealed amastigotes in 245 samples (57.0%) and in vitro cultivation gave positive results in 88 cases (20.5%), whereas PCR detected Leishmania in 301 samples (70%). The sensitivities inferred from our results were 99.3%, 80.8% and 29% for PCR, microscopic examination and in vitro cultivation, respectively. The different forms of CL in this country are caused by three species of Leishmania and are treated with the same protocol. Of 303 well-documented cases in our study, 99% were probably caused by Leishmania major and 1% by Leishmania infantum. The lack of species-specific diagnosis is not known to affect treatment or prognosis in Tunisia. These data support the incorporation of PCR into diagnostic strategies for CL, particularly in Tunisia.

Single-step multiplex PCR assay for characterization of New World Leishmania complexes.

We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested. The assay detected L. braziliensis in dermal scrapings from cutaneous leishmanial lesions, Leishmania chagasi in dermal scrapings of atypical cutaneous leishmaniasis, and L. mexicana from lesion aspirates from infected hamsters. We have minimized the material requirements and maximized the simplicity, rapidity, and informative content of this assay to render it suitable for use in laboratories in countries where leishmaniasis is endemic. This assay should be useful for rapid in-country identification of Leishmania parasites, particularly where different Leishmania complexes are found in the same geographical area.