Sebastian Vernal

Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples To Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic.

ABSTRACT The precise diagnosis of American tegumentary leishmaniasis (ATL) is an essential task due to the diseases associated morbidity. A noninvasive, extremely sensitive, and highly specific exam is critical, particularly for mucosal leishmaniasis (ML), in which a low parasite quantity is expected. We aimed to compare the diagnostic accuracy of swab and biopsy sample analysis using SYBR Green- and TaqMan-based real-time PCR (qPCR) assays with that of a composite reference standard consisting of the Montenegro skin test, serology, histopathology, smears, culture, and conventional PCR. In total, 55 patients with ATL (ML, 18 patients; cutaneous leishmaniasis [CL], 37 patients) and 36 patients without ATL were studied. qPCR analysis of swabs was more accurate when using SYBR Green (87.88%; 95% confidence interval [CI], 77.86 to 93.73 patients) than when using TaqMan (78.79%; 95% CI, 67.49 to 86.92%) (P = 0.031). SYBR Green (84.72%; 95% CI, 74.68 to 91.25%) was also more accurate than TaqMan (73.61%; 95% CI, 62.42 to 82.41%) for biopsy samples (P = 0.008). All qPCR methods were 100% specific. Swabs and biopsy specimens had similar sensitivity when using the same chemistry (P = 0.125 for SYBR Green and P = 0.625 for TaqMan). Moreover, qPCR achieved better performance than most existing techniques used for the diagnosis of ATL and also detected the Leishmania parasite in a greater proportion of patients than the associated histopathology, smear, culture, and conventional PCR techniques did. Swabs therefore represent a useful diagnostic tool because they not only are noninvasive but also can achieve an accuracy similar to that of biopsy samples. The high accuracy of SYBR Green-based qPCR may also reduce the requirement for associated parasitological tests for ATL diagnosis.

Disseminated Leishmaniasis by Leishmania viannia Subgenus: A Series of 18 Cases in Southeastern Brazil

Background. Disseminated leishmaniasis (DL) is an emerging clinical form of American tegumentary leishmaniasis (ATL) that occurs mainly in Northeastern Brazil. This study describes 18 cases where DL has not yet been reported. Methods. Disseminated leishmaniasis cases were extracted from ATL recorded cases between 1987 and March 2015. Disseminated leishmaniasis identification was based on ≥10 mixed-type lesions, located in ≥2 body parts. Results. Eighteen (5.4%) patients were identified as DL. Polymerase chain reaction followed by enzymatic digestion confirmed Leishmania viannia subgenus in 17 patients; amastigotes forms were identified in another one. Conclusions. Considering that DL diagnosis and management is challenging, clinicians must be aware of this emerging clinical form of the disease.

Evidence of hidden leprosy in a supposedly low endemic area of Brazil

OBJECTIVES Show that hidden endemic leprosy exists in a municipality of inner São Paulo state (Brazil) with active surveillance actions based on clinical and immunological evaluations. METHODS The study sample was composed by people randomly selected by a dermatologist during medical care in the public emergency department and by active surveillance carried out during two days at a mobile clinic. All subjects received a dermato-neurological examination and blood sampling to determine anti-PGL-I antibody titers by enzyme-linked immunosorbent assay (ELISA). RESULTS From July to December 2015, 24 new cases of leprosy were diagnosed; all were classified as multibacillary (MB) leprosy, one with severe Lucios phenomenon. Seventeen (75%) were found with grade-1 or 2 disability at the moment of diagnosis. Anti-PGL-I titer was positive in 31/133 (23.3%) individuals, only 6/24 (25%) were positive in newly diagnosed leprosy cases. CONCLUSIONS During the last ten years before this study, the average new case detection rate (NCDR) in this town was 2.62/100,000 population. After our work, the NCDR was raised to 42.8/100,000. These results indicate a very high number of hidden leprosy cases in this supposedly low endemic area of Brazil.

Anti-phospholipid syndrome in seven leprosy patients with thrombotic events on corticosteroid and/or thalidomide regimen: insights on genetic and laboratory profiles

INTRODUCTION Corticosteroids and/or thalidomides have been associated with thromboembolism events (TBE) in multibacillary (MB) leprosy. This report aimed to determine genetic and laboratory profiles associated with leprosy and TBE. METHODS Antiphospholipid antibodies (aPL), coagulation-related exams, prothrombin and Leidens factor V mutations, and ß2-glycoprotein-I (ß2GPI) Val247Leu polymorphism were assessed. RESULTS Six out of seven patients with leprosy were treated with prednisone and/or thalidomide during TBE and presented at least one positive aPL. All patients presented ß2GPI polymorphism, and one showed prothrombin mutation. CONCLUSIONS Corticosteroid or thalidomide adverse effects and aPL and ß2GPI polymorphisms may cause TBE in patients with MB leprosy.

Maxadilan-simile expression in Nyssomyia neivai, a sandfly vector in an endemic region of Brazil, and its immunogenicity in patients with American tegumentary leishmaniasis

BACKGROUND Maxadilan (Max) is a salivary component in the sandfly Lutzomyia longipalpis (Lutz & Neiva 1912), a vector of visceral leishmaniasis. Max has a powerful vasodilatory effect and is a candidate vaccine that has been tested in experimental leishmaniasis. Nyssomyia neivai (Pinto 1926) is a vector of the pathogen responsible for American tegumentary leishmaniasis (ATL) in Brazil. OBJECTIVE We searched for Max expression in Ny. neivai and for antibodies against Max in ATL patients. METHODS cDNA and protein were extracted from the cephalic segment, including salivary glands, of Ny. neivai and analysed by polymerase chain reaction, DNA sequencing, and blotting assays. The results were compared with data obtained from Lu. longipalpis samples. We quantified antibodies against Max in serum samples from 41 patients with ATL (31 and 10 with the cutaneous and mucocutaneous forms, respectively) and 63 controls from the endemic northeastern region of São Paulo state, using enzyme-linked immunosorbent assay. FINDINGS Recognition of a Max-simile peptide by specific antibodies confirmed expression of a Max sequence in Ny. neivai (GenBank EF601123.1). Compared to controls, patients with ATL presented higher levels of antibodies against Max (p = 0.004); 24.4% of the patients with ATL and 3.2% of the controls presented anti-Max levels above the cutoff index (p = 0.014). The anti-Max levels were not associated with the specific clinical form of ATL, leishmanin skin test response, absence or presence of amastigotes in histopathologic exam, results of indirect immunofluorescence testing for leishmaniasis, or duration of cutaneous form disease. MAIN CONCLUSION High serum anti-Max levels did not protect patients against ATL, but confirmed previous natural exposure to Ny. neivai bites in this ATL endemic region.

Anti‐desmogleins autoantibodies detected by ELISA and blotting in bullous pemphigoid: what do they mean?

treat erythrodermic flares of MF/SS because it reduces bacterial colonization and debrides excess scale. It also decreases inflammation and promotes wound cleansing. Moreover, a pilot study showed that whirlpool therapy followed by a quick rinse eradicated more than four times as many bacteria compared with whirlpool therapy alone. In our protocol, the 0.25% acetic acid rinse further decreases the skin bacterial load and reduces skin pH, which inhibits S. aureus colonization. Because the severity of MF/SS is measured by the extent of skin erythema, clinicians must recognize staphylococcal colonization and sepsis in cases of erythroderma. Since the most common cause of death in MF/SS patients is infection, especially from the use of indwelling venous catheters causing line sepsis, treatment of S. aureus is critical. For patients who develop an erythrodermic flare of MF/SS secondary to S. aureus, we recommend the “Duvic regimen” consisting of IV antibiotics, whirlpool therapy, and steroid wet wraps.

Biological predictors shared by dementia and bullous pemphigoid patients point out a cross-antigenicity between BP180/BP230 brain and skin isoforms

Bullous pemphigoid (BP) following dementia diagnosis has been reported in the elderly. Skin and brain tissues express BP180 and BP230 isoforms. Dementia has been associated with rs6265 (Val66Met) polymorphism of the brain-derived neurotrophic factor (BDNF) gene and low serum BDNF. Here we investigated a possible cross-antigenicity between BP180/BP230 brain and skin isoforms. We assessed antibodies against BP180/BP230 and BDNF levels by ELISA and BDNF Val66Met SNP by PCR in three groups: 50 BP patients, 50 patients with dementia, and 50 elderly controls. Heatmap hierarchical clustering and data mining decision tree were used to analyze the patients’ demographic and laboratorial data as predictors of dementia-BP association. Sixteen percent of BP patients with the lowest serological BDNF presented dementia-BP clinical association. Anti-BP180/230 positivity was unexpected observed among dementia patients (10%, 10%) and controls (14%, 1%). Indirect immunofluorescence using healthy human skin showed a BP pattern in two of 10 samples containing antibodies against BP180/BP230 obtained from dementia group but not in the control samples. Neither allelic nor genotypic BDNF Val66Met SNP was associated with dementia or with BP (associated or not with clinical manifestation of dementia). Heatmap analysis was able to differentiate the three studied groups and confirmed the ELISA results. The comprehensive data mining analysis revealed that BP patients and dementia patients shared biological predictors that justified the dementia-BP association. Autoantibodies against the BP180/BP230 brain isoforms produced by dementia patients could cross-react with the BP180/BP230 skin isoforms, which could justify cases of dementia preceding the BP disease.

Relationship between pemphigus and American tegumentary leishmaniasis: insights from serological and genetic profiles

Background Antibodies against Leishmania peptides (Lbr-peps) and desmogleins (Dsgs) have been reported in pemphigus foliaceus (PF) and leishmaniasis patients, respectively. We aimed to compare serological and genetic features in a Brazilian region endemic for American tegumentary leishmaniasis (ATL) and pemphigus. Methods Commercial anti-Dsg ELISA and in-house ELISA with Lbr-peps were used to determine the serological profile, in addition to immunoblotting (IB) and indirect immunofluorescence (IIF) assays. HLA-DRB1 and -DQA1/DQB1 alleles were characterized by PCR combined with sequence-specific oligonucleotide probes (PCR-SSOP). The serological and genetic profiles were compared using 78 PF, 62 pemphigus vulgaris (PV) and 58 ATL patients against 163 and 1592 healthy controls, respectively. Results Some ATL patients showed positive results for anti-Dsg1 and/or anti-Dsg3 antibodies. They also revealed 130, 160 and/or 230 kDa epidermal peptides in IB. Moreover, some ATL samples exhibited pemphigus or a bullous pemphigoid pattern in IIF. ELISA and IB assays showed Lbr-peps in pemphigus patients. HLA-DQA1*01 and -DQA1*01:02 were protective and susceptibility alleles for ATL, respectively, but the opposite for pemphigus. Conclusions Anti-Dsgs in ATL may represent epiphenomena. Anti-Lbr-pep antibodies in pemphigus suggest a previous infection. A differential association of the HLA profile may contribute to the lack of co-association between pemphigus and ATL.

Geographical foci and epidemiological changes of pemphigus vulgaris in four decades in Southeastern Brazil

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Final diagnosis of 86 cases included in differential diagnosis of American tegumentary leishmaniasis in a Brazilian sample: a retrospective cross-sectional study

Background Cutaneous leishmaniasis is distributed worldwide, including Brazil. Its several clinical forms need to be distinguished from other dermatoses. Clinical similarities and lack of a gold standard diagnostic tool make leishmaniasis-like lesions a challenging diagnosis. Objectives. To report the final diagnosis of patients primarily suspected of having American tegumentary leishmaniasis (ATL). Methods. A retrospective cross-sectional study was conducted on the basis of medical records of 437 patients with clinical suspicion of ATL, registered in electronic hospital system between 1980 and 2013. Demographic, clinical, and laboratory data were compiled. Results. Analysis of 86 cases (19.7%) registered as ATL in one of the hypothesis revealed a different final diagnosis; 55 (63.9%) and 31 cases (36.1%) had skin and mucosal lesions, respectively. In 58 cases (67.4%), the requested PCR did not identify Leishmania sp. In 28 cases (32.5%), biopsies established the diagnosis and confirmed tumors, mycobacteriosis, and subcutaneous or systemic mycosis. Overall, 27% of the cases had inflammatory etiology, mainly nasal nonspecific inflammatory processes; 27% had infectious etiology, especially paracoccidioidomycosis and leprosy; 20% had neoplastic etiology, mainly basal and squamous cell carcinoma; 15% had miscellaneous etiology, including neuropathic ulcer, traumatic ulcers, idiopathic ulcer; 11% missed the follow-up. Study limitations Some cases had no final diagnosis due to loss of follow-up. Conclusion. ATL can be confused with several differential diagnoses, especially inflammatory and infectious granulomatous diseases as well as non-melanoma skin cancers. Clinicians working in tropical areas should be aware of the main differential diagnosis of leishmaniasis-like lesions.