Eduardo Henrique Gomes Rodrigues

Evaluation of PCR for Diagnosis of American Cutaneous Leishmaniasis in an Area of Endemicity in Northeastern Brazil

ABSTRACT PCR-based approaches targeting kinetoplast DNA were evaluated for the diagnosis of American cutaneous leishmaniasis (ACL) in regions of endemicity in northeastern Brazil. A total of 119 cutaneous biopsy specimens from patients with ACL and nonleishmaniasis cutaneous lesions were studied. Two PCR-based systems were used; one was specific for the subgenus Viannia, and the other was specific for the genus Leishmania. The PCR specific for the subgenus Viannia had a sensitivity of 95.4%, whereas the genus-specific PCR detected the target DNA in 88.2% of the samples tested. The specificities of the assays, determined with samples from a group with nonleishmaniasis cutaneous lesions, was 100%. The results of the conventional tests indicate that the sensitivities of the PCR-based methods were significantly higher than those of smear examination, histological staining, and isolation by culture (P < 0.05). Antibodies specific for Leishmania braziliensis were detected by indirect immunofluorescence in 82.9% of the patients tested. Parasites were isolated from 40 of 86 patients (46.5%). Sixty-seven percent of dermal scrapings and 66.2% of stained tissue sections were positive by microscopy. Amplified products from the subgenus-specific PCR hybridized with the Leishmania panamensis minicircle, confirming infection consistent with L. braziliensis. The evidence available at present incriminates L. braziliensis as the only causative agent of ACL in the state of Pernambuco in Brazil.

Cutaneous leishmaniasis in northeastern Brazil: a critical appraisal of studies conducted in State of Pernambuco

American cutaneous leishmaniasis (ACL) is a complex disease with clinical and epidemiological features that may vary from region to region. In fact, at least seven different Leishmania species, including Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) naiffi, Leishmania (Viannia) shawi, Leishmania (Viannia) lindenbergi, and Leishmania (Leishmania) amazonensis, have been implicated in the etiology of ACL in Brazil, and numerous phlebotomine sandfly species of the genus Lutzomyia have been regarded as putative or proven vectors. Because ACL is a focal disease, understanding the disease dynamics at the local level is essential for the implementation of more effective control measures. The present paper is a narrative review about the ACL epidemiology in Pernambuco, northeastern Brazil. Furthermore, the need for more effective diagnosis, treatment, control and prevention strategies for the affected populations is highlighted. This paper will provide researchers with a critical appraisal of ACL in Pernambuco. Hopefully, it will also be helpful for public health authorities to improve current control strategies against ACL at the state and country levels.

Sexually transmitted diseases in patients infected with HIV/AIDS in the state of Pernambuco, Brazil.

The data was obtained retrospectively from clinical records concerning 399 HIV infected patients. The HIV infected individuals predominated in the age group ranging from 20 to 40 years (73.4%) and 75% were male. The was no difference in the ratio of male and female patients regarding asymptomatic HIV infection or AIDS. The cases of HIV without AIDS concentrated in the age group ranging from 20-29 years while AIDS predominated in the age group ranging from 30-39 years. Only 0.8% were hemophilic, 3.5% injected drugs and 4.8% had hemotransfusions in the last 5 years. Regarding sexual behavior, 33% were heterosexuals, 11% bisexuals, 23% homosexuals and 33% did not disclose their sexual behavior. The presence of syphilis was the most frequent combination found (8.8%), followed by herpes (5.8%) and genital candidiasis (4.3%). Our results suggest an association between genital candidiasis and AIDS, although this was not demonstrated for the other STDs studied.

Phlebotomine sandflies (Diptera: Psychodidae) in São Vicente Férrer, a sympatric area to cutaneous and visceral leishmaniasis in the state of Pernambuco, Brazil.

INTRODUCTION In the last decades, a considerable geographic expansion of the leishmaniases in all regions of Brazil has been observed. The present study was carried out to identify the composition of the phlebotomine sandfly fauna and verify the seasonal variation of the main species after environmental changes occurred in São Vicente Férrer Municipality, State of Pernambuco, Brazil. METHODS Captures were carried out during four consecutive nights of each month using Centers for Disease Control and Prevention light traps from September 2009 to September 2010. The correlation between the number of phlebotomine sandflies captured and climatic factors (temperature and rainfall) was evaluated. RESULTS A total of 13,872 specimens belonging to 20 species were captured, of which, 6,247 (45%) were females, and 7,625 (55%) were males. Lutzomyia migonei was the most abundant species with 9,964 (71.8%) specimens, being predominant in the intradomicile and peridomicile areas with 108 (86.4%) and 9,746 (97%), respectively. In the forest remnants, Lutzomyia complexa 2,395 (65%) and Lutzomyia sordellii 770 (20.8%) predominated. The correlation analysis between the total number of sandflies captured and climatic factors did not show a significant influence on population density. CONCLUSIONS The high abundance of Lutzomyia migonei and Lutzomyia complexa indicates the possibility of new cases of cutaneous leishmaniasis (CL).

Discrimination of Leishmania braziliensis Variants by kDNA Signatures Produced by LSSP-PCR

The conventional methods for identification and typing of Leishmania species depend on previous culture isolation of the parasites. Not infrequently, culture is unsuccessful and may result in misrepresentation of the heterogeneity of the original isolate. Thus, more reliable and precise identification of genotypes of Leishmania spp. is important for a better clinical and epidemiological understanding of the disease. We evaluated the potential of LSSP-PCR targeting kDNA minicircles in discriminating different variants of the parasite with the use of clinical samples directly or cultivated parasites. The 1st step of this procedure consists of the amplification of the minicircles by conventional PCR; the 2nd step is low-stringency amplification of the minicircles previously amplified, with the use of 1 of the primers. Although LSSP-PCR produced complex and distinct kDNA signatures for isolates representing different species, further experiments demonstrated that the approach had the potential for discriminating intraspecific variants of L. braziliensis. Thus, the generated profiles were too variable to be useful as markers for species identification. Moreover, we demonstrated that the approach can be directly applied to clinical samples. In conclusion, LSSP-PCR targeting kDNA minicircles produces profiles that reflect polymorphisms of the predominant classes of minicircles, and can be useful for studies aimed at discriminating Leishmania braziliensis genotypes without the need for previous cultivation of the parasite.

Occupationally Acquired American Cutaneous Leishmaniasis

We report two occupationally acquired cases of American cutaneous leishmaniasis (ACL): one accidental laboratory autoinoculation by contaminated needlestick while handling an ACL lesion sample, and one acquired during field studies on bird biology. Polymerase chain reaction (PCR) assays of patient lesions were positive for Leishmania, subgenus Viannia. One isolate was obtained by culture (from patient 2 biopsy samples) and characterized as Leishmania (Viannia) naiffi through an indirect immunofluorescence assay (IFA) with species-specific monoclonal antibodies (mAbs) and by multilocus enzyme electrophoresis (MLEE). Patients were successfully treated with N-methyl-glucamine. These two cases highlight the potential risks of laboratory and field work and the need to comply with strict biosafety procedures in daily routines. The swab collection method, coupled with PCR detection, has greatly improved ACL laboratory diagnosis.

Molecular detection of Leishmania in phlebotomine sand flies in a cutaneous and visceral leishmaniasis endemic area in northeastern Brazil

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

Validação de abordagens moleculares para o diagnóstico da leishmaniose tegumentar americana em Pernambuco

The control of leishmaniasis depends on the availability of acurate and sensitive methods for the detection of Leishmania species. In the present study, two molecular approaches were evaluated for diagnosing American cutaneous leishmaniasis (ACL) in patients from endemic areas of Pernambuco State. One of the approaches is genus specific whereas the other is specific for the Viannia subgenus. The DNA detection limits were determined and the sensitivity and specificity calculated, for comparison with conventional diagnostic tests. The DNA detection limit of the PCR specific for the Viannia subgenus was 10 fg of total DNA, whereas this was 1 pg for the genus specific PCR. Diagnosis of ACL was performed on the basis of clinical-epidemiological criteria associated to positive reaction in at least one of the following tests: direct microscopy examination, histopathological examination and culture. Based upon these criteria, from the 98 patients initially diagnosed as ACL exclusivelly on clinicalepidemiological basis, 87 were considered ACL cases, and used for the sensitiviy calculation. Thirty one patients with cutaneous lesion caused by other agents were included as controls for the calculation of specificity. The genus specific PCR was positive in 60/67 biopsies from LTA, resulting in a sensitivity of 89.6%, whereas the PCR specific for the Viannia subgenus was positive in 82/87 cases of LTA, resulting in a sensitivity of 94.3%. However, these differences were not significant. Taken together with other observations, these results suggest that the Viannia subgenus is the only causative agent of ACL in Pernambuco State. Both PCRs showed 100% specificity. The diagnosis by direct observation of the parasite, histopathological examination, culture and indirect immunofluorescence was positive in 68.2%, 89.9%, 44.0% and 81.3% of the LTA cases, respectively. Since working with non radioactive material is clearly more convenient in endemic underdeveloped regions, a few preliminary experiments were carried out with PCR/hibridization with cold probes. In conclusion, the evaluated molecular methods showed high sensitivity and specificity, one of them being able to detect the subgenus Viannia. The simplification of sample collection and processing, and in-house preparation of reagents can transform the PCR into a low-cost technology appropriate for use in situations of limited resources, providing epidemiological information concerning the parasite identification that are relevant for the planning of control strategies.

Leishmania (Viannia) braziliensis isolated from the saliva of patients in a cutaneous leishmaniasis-endemic area of northeastern Brazil

Several studies have described the use of non-invasive collection methods, mostly based on the detection of parasite DNA, for diagnosis. However, no Leishmania specimens have been isolated from saliva. Here, we report the first isolation of Leishmania braziliensis from the saliva of humans with cutaneous leishmaniasis but without lesions on their mucosa. The isolates were obtained from salivary fluid inoculated in hamsters and were tested by multilocus enzyme electrophoresis. Seven samples from 43 patients suspected of having the disease were identified for in vivo culture. These findings suggest that saliva is a clinical sample that allows the isolation of Leishmania sp.