The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction.

The present work focuses on the detection of SARS-CoV-2 in saliva, contributing to understanding the inhibition effect of the matrix and its influence on the results. Detection of viral genes ORF1ab, N, and E was performed by RT-PCR using saliva directly in the reaction without RNA extraction. Different amounts of saliva were spiked with increasing amounts of viral RNA from COVID-19 patients and subjected to RT-PCR detection. In parallel, 64 saliva samples from confirmed COVID-19 patients were used in two different amounts directly in the RT-PCR reaction and their results compared. The presence of saliva in the RT-PCR always causes a positive shift of the Ct values, but a very high between-person variability of its magnitude was obtained, with increases ranging from 0.93 to 11.36. Viral targets are also affected differently depending on the initial number of viral particles. Due to inhibitors present in saliva, the duplication of sample volume causes only 48 to 61% of the expected Ct value decrease depending on the viral target gene. The use of saliva has advantages, but also limitations, due to potential inhibitors present in the matrix. However, the choice of the target and the right amount of sample may significantly influence the results.

Impact of Thermal Pretreatment of Saliva on the RT-PCR Detection of SARS-CoV-2.

The use of saliva directly as a specimen to detect viral RNA by RT-PCR has been tested for a long time as its advantages are relevant in terms of convenience and costs. However, as other body fluids, its proven inhibition effect on the amplification reaction can be troublesome and compromise its use in the detection of viral particles. The aim of the present work is to demonstrate that saliva pretreatment may influence the RT-PCR amplification of three gene targets of SARS-CoV-2 significantly. A pool of RNA from confirmed COVID-19 patients was used to test the influence of heat pretreatment of saliva samples at 95°C for 5, 10, 15 and 20 min on the amplification performance of ORF1ab, E, and N SARS-CoV-2 genes. Prolonged heating at 95°C significantly improves the Ct value shift, usually observed in the presence of saliva, increasing the limit of detection of viral genes ORF1ab, E, and N. When tested using a cohort of COVID-19 patients' saliva, the increased time of heat pretreatment resulted in a significant increase in the detection sensitivity.

Performance of saliva as a specimen to detect SARS-CoV-2.

Massive testing to detect SARS-CoV-2 is an imperious need in times of epidemic but also presents challenges in terms of its concretization. The use of saliva as an alternative to nasopharyngeal swabs (NPS) has advantages, being more friendly to the patient and not requiring trained health workers, so much needed in other functions. This study used a total of 452 dual samples (saliva and NPS) of patients suspected of having COVID-19 to compare results obtained for the different specimens when using RT-PCR of RNA extracted from NPS and saliva, as well as saliva directly without RNA extraction. SARS-CoV-2 was not detected in 13 saliva (direct) of the 80 positive NPS samples and in 16 saliva (RNA) of a total of 76 NPS positive samples. Sensitivity of detection of viral genes ORF1ab, E and N in saliva is affected differently and detection of these genes in saliva samples presents great variability when NPS samples present Ct-values above approximately 20, with sensitivities ranging from 76.3% to 86.3%. On average an increase in 7.3 Ct-values (average standard deviation of 4.78) is observed in saliva samples when compared to NPS. The use of this specimen should be carefully considered due to the false negative rate and the system used for detection may be also very relevant since the different viral genes are affected differently in terms of detection sensitivity using saliva.

Complementary exams in the diagnosis of American tegumentary leishmaniasis.

The diagnosis of American Tegumentary Leishmaniasis is a difficult but essential task when considering the high toxicity profile of the drugs available. Since the discovery of its etiologic agent, numerous diagnostic tests have been developed. None of the tests available today can be considered as the gold standard, since they do not add enough accuracy for the disease detection. Good epidemiological and clinical knowledge of the disease are fundamental precepts of the dermatology practice and precede the rational use of existing diagnostic tests. In this article we aim, through extensive literature review, to recall fundamental concepts of any diagnostic test. Subsequently, based on this information, we will weave important comments about the characteristics of existing diagnostic tests, including immunological tests such as Montenegro's skin test, serology and detection of parasites by direct examination, culture or histopathology. Finally we will discuss the new technologies and options for the diagnosis of Cutaneous Leishmaniasis. The molecular biology technique is considered a promising tool, promoting the rapid identification of the species involved. We also aim to educate dermatologists about a disease with high morbidity and assist in its difficult recognition.

Complementary exams in the diagnosis of american tegumentary leishmaniasis

The diagnosis of American Tegumentary Leishmaniasis is a difficult but essential task when considering the high toxicity profile of the drugs available. Since the discovery of its etiologic agent, numerous diagnostic tests have been developed. None of the tests available today can be considered as the gold standard, since they do not add enough accuracy for the disease detection. Good epidemiological and clinical knowledge of the disease are fundamental precepts of the dermatology practice and precede the rational use of existing diagnostic tests. In this article we aim, through extensive literature review, to recall fundamental concepts of any diagnostic test. Subsequently, based on this information, we will weave important comments about the characteristics of existing diagnostic tests, including immunological tests such as Montenegro's skin test, serology and detection of parasites by direct examination, culture or histopathology. Finally we will discuss the new technologies and options for the diagnosis of Cutaneous Leishmaniasis. The molecular biology technique is considered a promising tool, promoting the rapid identification of the species involved. We also aim to educate dermatologists about a disease with high morbidity and assist in its difficult recognition.

Pyoderma gangrenosum leading to bilateral involvement of ears.

Pyoderma gangrenosum is a destructive inflammatory disease that commonly occurs in an idiopathic way. Its occurrence in the auricular area is very rare, although this fact does not seem to determine a different behavior of the disease with regard to ulcer aspects and response to treatment. The authors report the case of a patient with pyoderma gangrenosum affecting both earlobes. The patient responded well to treatment with oral prednisone and has not shown relapses after a six-month follow-up.

The use of the Er:YAG 2940nm laser associated with amorolfine lacquer in the treatment of onychomycosis.

Onychomycosis is a common disease, accounting for up to 50% of all ungual pathologies. We have been developing a clinical trial (ClinicalTrials.gov: NCT01528813) using a 2940nm Er:YAG laser to fractionally ablate human nails in vivo, aiming to increase topical amorolfine lacquer delivery to the nail unit, increasing the efficacy of topical treatment of distal and lateral subungual onychomycosis. Partial results have shown an increase in areas of nail plate free of disease. We believe that ablative lasers can increase the efficacy of topical onychomycosis treatment.

The use of the Er:YAG 2940nm laser associated with amorolfine lacquer in the treatment of onychomycosis / Uso do laser de Er:YAG 2940nm associado ao esmalte de amorolfina no tratamento da onicomicose

Onychomycosis is a common disease, accounting for up to 50% of all ungual pathologies. We have been developing a clinical trial (ClinicalTrials.gov: NCT01528813) using a 2940nm Er:YAG laser to fractionally ablate human nails in vivo, aiming to increase topical amorolfine lacquer delivery to the nail unit, increasing the efficacy of topical treatment of distal and lateral subungual onychomycosis. Partial results have shown an increase in areas of nail plate free of disease. We believe that ablative lasers can increase the efficacy of topical onychomycosis treatment.

A onicomicose é afecção frequente, representando até 50% do total das doenças ungueais. Um ensaio clinico (ClinicalTrials.gov: NCT01528813) em atual desenvolvimento usa o laser de Er:YAG 2940nm para realizar ablação fracionada in vivo de unhas humanas visando aumentar a permeabilidade ungueal ao esmalte de amorolfina, visando aumentar a eficácia do tratamento tópico da onicomicose subungueal distal lateral. Resultados parciais tem demonstrado um aumento na área ungueal livre de doença nas unhas tratadas com o laser, em comparação ao uso isolado do esmalte. Acreditamos que lasers ablativos possam aumentar a eficácia do tratamento tópico da onicomicose.

Recurrent cutaneous leishmaniasis.

We present a case of an 18-year-old male patient who, after two years of inappropriate treatment for cutaneous leishmaniasis, began to show nodules arising at the edges of the former healing scar. He was immune competent and denied any trauma. The diagnosis of recurrent cutaneous leishmaniasis was made following positive culture of aspirate samples. The patient was treated with N-methylglucamine associated with pentoxifylline for 30 days. Similar cases require special attention mainly because of the challenges imposed by treatment.

Phacomatosis pigmentokeratotica--a patient with hypophosphatemic rickets.

The epidermal naevus syndrome (ENS) is a sporadic condition characterized by congenital epidermal naevi associated with anomalies in other organ systems, most commonly the central nervous system and skeleton. We report a case of ENS presenting hypophosphataemic rickets resistant to traditional therapeutic agents.

Active search for leprosy cases in Midwestern Brazil: a serological evaluation of asymptomatic household contacts before and after prophylaxis with bacillus Calmette-Guérin.

Leprosy is a disease caused by Mycobacterium leprae that carries a high risk of disability, making early diagnosis mandatory. This study aimed to determine the applicability of anti-PGL-1 IgM antibody detection, using the ML FLOW technique, as an assistant tool for the detection of leprosy infection in asymptomatic household contacts (AHHC) of multibacillary leprosy index cases from Midwest Brazil. Serological changes induced by the prophylaxis of these household contacts with Bacillus Calmette-Guérin (BCG) were also verified. A total of 91 AHHC were assessed, among which, 18.68% (n = 17) presented both positive bacilloscopy and positive anti-PGL-1 IgM serology. Positivity concordance between these two laboratorial exams (Kappa Index = 1; p < 0.001) was indicated, however, one case did not demonstrate concordance between the semiquantitative assessment of anti-PGL-1 IgM and the bacilloscopy index (Kappa Index = 0.96; p < 0.001). Among the 17 AHHC with positive bacilloscopy, eight were reassessed after prophylaxis with BCG and two of them presented negative anti-PGL-1 IgM serology, being these patients who had presented a bacilloscopy index of < 2[+] in the initial assessment. This study shows that anti-PGL-1 IgM detection may be used as a tool to determine the bacillary load in AHHC and to detect immune changes related to prophylaxis by nonspecific vaccination.

The use of ablative lasers in the treatment of facial melasma.

Melasma represents a pigmentary disorder that is difficult to treat. This study aims to broadly review the use of ablative lasers (Er:YAG and CO2) in the treatment of melasma, presenting the level of evidence of studies published to date. A total of 75 patients were enrolled in four case series studies (n=39), one controlled clinical trial (n=6) and one randomized controlled clinical trial (n=30). Studies on the Er:YAG laser showed better results with the use of short square-shaped pulses, which determined low rates of post-inflammatory hyperpigmentation and long-lasting maintenance of results. Likewise, studies on the CO2 laser proved the benefits of short pulse duration along with low-density energy. Post-treatment maintenance with the use of antipigmenting creams was necessary and effective to sustain long-term results. Ablative lasers may represent another useful and effective tool against melasma. Postinflammatory hyperpigmentation and difficulty in sustaining long-term results still represent the main limitations to a broader use of ablative lasers. Based on actual evidence, the use of this technology should be restricted to patients with recalcitrant disease. Further studies will help establish optimal laser parameters and treatment regimens.

Recurrent cutaneous leishmaniasis / Leishmaniose recidiva cútis

We present a case of an 18-year-old male patient who, after two years of inappropriate treatment for cutaneous leishmaniasis, began to show nodules arising at the edges of the former healing scar. He was immune competent and denied any trauma. The diagnosis of recurrent cutaneous leishmaniasis was made following positive culture of aspirate samples. The patient was treated with N-methylglucamine associated with pentoxifylline for 30 days. Similar cases require special attention mainly because of the challenges imposed by treatment.

Paciente do sexo masculino, 18 anos. Dois anos após tratamento insuficiente para leishmaniose tegumentar americana, apresentou, na mesma localização, lesão formada por cicatriz atrófica central e nódulos verrucosos na periferia. Era imunocompetente, hígido e negava qualquer trauma local. O diagnóstico de leishmaniose recidiva cutis foi feito através de cultura do aspirado da lesão. Realizou tratamento com N-metilglucamina (20mgSbV/kg/dia) associado à pentoxifilina (1200mg/dia) durante 30 dias alcançando cura clínica. Os casos semelhantes requerem atenção diferenciada pela dificuldade ao tratamento.

Active search for leprosy cases in Midwestern Brazil: a serological evaluation of asymptomatic household contacts before and after prophylaxis with bacillus Calmette-Guérin / Busca ativa por casos de hanseníase no Centro-Oeste do Brasil: avaliação sorológica dos contatos domiciliares assintomáticos antes e após a profilaxia com Bacillus Calmette-Guérin

Leprosy is a disease caused by Mycobacterium leprae that carries a high risk of disability, making early diagnosis mandatory. This study aimed to determine the applicability of anti-PGL-1 IgM antibody detection, using the ML FLOW technique, as an assistant tool for the detection of leprosy infection in asymptomatic household contacts (AHHC) of multibacillary leprosy index cases from Midwest Brazil. Serological changes induced by the prophylaxis of these household contacts with Bacillus Calmette-Guérin (BCG) were also verified. A total of 91 AHHC were assessed, among which, 18.68% (n = 17) presented both positive bacilloscopy and positive anti-PGL-1 IgM serology. Positivity concordance between these two laboratorial exams (Kappa Index = 1; p < 0.001) was indicated, however, one case did not demonstrate concordance between the semiquantitative assessment of anti-PGL-1 IgM and the bacilloscopy index (Kappa Index = 0.96; p < 0.001). Among the 17 AHHC with positive bacilloscopy, eight were reassessed after prophylaxis with BCG and two of them presented negative anti-PGL-1 IgM serology, being these patients who had presented a bacilloscopy index of < 2[+] in the initial assessment. This study shows that anti-PGL-1 IgM detection may be used as a tool to determine the bacillary load in AHHC and to detect immune changes related to prophylaxis by nonspecific vaccination.

A hanseníase é doença causada pelo Mycobacterium leprae, apresentando elevado potencial incapacitante, o que torna indispensável seu diagnóstico precoce. O estudo visa determinar a aplicabilidade da detecção de anticorpos anti-PGL1-IgM por meio da técnica do ML FLOW como ferramenta adjuvante ao diagnóstico de hanseníase em contatos domiciliares assintomáticos (AHHC) de pacientes multibacilares procedentes da região Centro-Oeste do Brasil, bem como, documentar o comportamento sorológico após a profilaxia com a vacina Bacillus Calmette-Guérin (BCG). Foram avaliados 91 AHHC atendidos no Hospital Universitário de Brasília - Brasil, dos quais 18,68% (n = 17) apresentaram positividade para baciloscopia e anti-PGL1-IgM, totalizando uma concordância completa entre os dois grupos (Índice Kappa = 1; p < 0,001). Em apenas um dos casos não observou-se concordância entre a avaliação semi-quantitativa do anti-PGL1-IgM e índice baciloscópico (Índice Kappa = 0,96; p < 0,001). Oito dos 17 AHHC com baciloscopia positiva foram reavaliados após profilaxia com BCG e apenas dois apresentaram negativação dos títulos anti-PGL1-IgM, sendo tais casos correspondentes aos que haviam apresentado índice baciloscópico menor do que 2[+] na avaliação inicial. O estudo corrobora o potencial do anti-PGL1-IgM como ferramenta de predição da carga bacilar em AHHC da região Centro-Oeste do Brasil, e surpreende alterações imunes relacionadas à profilaxia obtida pela vacinação não específica com BCG.

The use of ablative lasers in the treatment of facial melasma / O uso de lasers ablativos no tratamento do melasma facial

Melasma represents a pigmentary disorder that is difficult to treat. This study aims to broadly review the use of ablative lasers (Er:YAG and CO2) in the treatment of melasma, presenting the level of evidence of studies published to date. A total of 75 patients were enrolled in four case series studies (n=39), one controlled clinical trial (n=6) and one randomized controlled clinical trial (n=30). Studies on the Er:YAG laser showed better results with the use of short square-shaped pulses, which determined low rates of post-inflammatory hyperpigmentation and long-lasting maintenance of results. Likewise, studies on the CO2 laser proved the benefits of short pulse duration along with low-density energy. Post-treatment maintenance with the use of antipigmenting creams was necessary and effective to sustain long-term results. Ablative lasers may represent another useful and effective tool against melasma. Postinflammatory hyperpigmentation and difficulty in sustaining long-term results still represent the main limitations to a broader use of ablative lasers. Based on actual evidence, the use of this technology should be restricted to patients with recalcitrant disease. Further studies will help establish optimal laser parameters and treatment regimens.

O melasma representa desordem pigmentar de difícil tratamento. O presente estudo tem como propósito apresentar ampla revisão da literatura acerca do uso de laser ablativos (Er:YAG e CO2) no tratamento do melasma, estabelecendo o nível de evidência dos estudos publicados até o instante. Um total de 75 pacientes foram envolvidos entre quatro séries de casos (n=39), um ensaio clínico controlado (n=6) e um ensaio clínico controlado e randomizado (n=30). Os estudos acerca do laser de Er:YAG demonstraram melhores resultados com o uso de pulsos de forma quadrada, os quais determinaram menores taxas de hiperpigmentação pós-inflamatória. Ademais, os estudos com laser de CO2 também demonstraram benefício no uso de pulsos curtos com baixa densidade de energia. O uso de cremes despigmentantes no período pós-tratamento se mostrou necessária e efetiva na manutenção de resultados à longo prazo. Os lasers ablativos, por conseguinte, podem representar ferramenta efetiva e de grande utilidade no manejo do melasma. Entretanto, hiperpigmentação pós-inflamatória e dificuldade na manutenção de resultados à longo prazo parecem representar as principais limitações atuais ao seu amplo uso. Por conseguinte, com base nas atuais evidências, o uso de tais tecnologias ainda deve ser restrita à casos de doença recalcitrante. Novos estudos ainda são necessários para o estabelecimento de parâmetros e regimes ideais de tratamento.

Periungual leishmaniasis.

The vast majority of cases of cutaneous leishmaniasis are represented by limb injuries. A female patient, white, presented an ulcer with infiltrated borders located on the fourth finger of the left hand following occupational exposure in an area of native forest. Diagnosis of cutaneous leishmaniasis caused by Leishmania of the subgenus Viannia was confirmed. The patient failed to respond to treatment with antimony, but achieved clinical cure after this was associated with pentoxifylline. The case highlights the rarity of the periungual location of the leishmanial lesion and the difficulties encountered in therapy.

Periungual leishmaniasis / Leishmaniose periungueal

The vast majority of cases of cutaneous leishmaniasis are represented by limb injuries. A female patient, white, presented an ulcer with infiltrated borders located on the fourth finger of the left hand following occupational exposure in an area of native forest. Diagnosis of cutaneous leishmaniasis caused by Leishmania of the subgenus Viannia was confirmed. The patient failed to respond to treatment with antimony, but achieved clinical cure after this was associated with pentoxifylline. The case highlights the rarity of the periungual location of the leishmanial lesion and the difficulties encountered in therapy.

A grande maioria dos casos de leishmaniose tegumentar é representada por lesões nos membros. Paciente feminina, branca, diabética, apresentou úlcera com bordas infiltradas, localizada no quarto quirodáctilo esquerdo, após exposição ocupacional em área de mata nativa. Foi confirmado o diagnóstico de leishmaniose tegumentar por Leishmania do subgênero Viannia. Não respondeu ao tratamento com antimonial, mas obteve cura clínica após associação com a pentoxifilina. O caso destaca-se pela raridade da localização periungueal da lesão leishmaniótica e pela dificuldade terapêutica.

Comparison of four botulinum neurotoxin type A preparations in the treatment of hyperdynamic forehead lines in men: a pilot study.

BACKGROUND: Botulinum neurotoxin type A (BTXA) offers a minimally invasive approach for the treatment of facial rhytides and has been well accepted by male patients. However, few comparative studies about the efficacy of the different BTXA preparations in this emerging group of patients are available. OBJECTIVE: To access the efficacy of four BTXA preparations in the treatment of hyperdynamic forehead lines in men. METHODS: Twelve male patients with a mean age of 30.5±5.26 years, with moderate or severe hyperdynamic forehead lines on a facial wrinkle scale (FWS), were involved in the study. Using an intra-individual controlled study design, either 17 mouse units (m.U) of Botox, Prosigne, and Xeomin or 51 Speywood units (s.U) of Dysport were applied to each side of a patient's forehead. Subjects with absent or mild forehead lines were considered responders. Study evaluations were performed monthly up to day 150. RESULTS: All patients responded in a side-to-side evaluation and no asymmetries or differences in the degree of reduction of hyperdynamic forehead lines were noted at day 30 and day 60 (P=0.340). Analysis of all combined forehead sides treated with a given BTXA did not show statistically significant differences in terms of maintenance of improvement between different BTXA preparations at day 150 (P=0.528). CONCLUSION: The data obtained in this study revealed a similar effectiveness of the BTXA preparations evaluated in the treatment of hyperdynamic forehead lines in men, when considering a dose-ratio of 1 m.U of Botox, Xeomin or Prosigne to 3 s.U of Dysport.

Congenital dermatofibrosarcoma protuberans.

Congenital dermatofibrosarcoma protuberans (DFSP) is a rare dermal and subcutaneous neoplasm of low-grade malignant behavior that is characterized by a low frequency of metastases with locally invasive growth. Its occurrence at birth and during childhood is rare. We present a case of a patient who was born with a light brown macule on his right buttock that was misdiagnosed as localized scleroderma. The lesion progressed into reddish atrophic plaques and nodules extending to the iliac region and the gluteal fold. At 5 years of age, a diagnosis of congenital DFSP was made based on clinical and immunohistochemical characteristics (CD34 positivity and spindle cell proliferation). Although there was a delay in diagnosis, a 3-step excision was proposed with a final step of Mohs micrographic surgery (MMS).