Canine ringworm caused by Trichophyton mentagrophytes - detection by SYBR-Green real-time PCR

Background: Dermatophytes, fungi of universal distribution, invade semi or fully keratinized structures, such as skin, fur/ hair and nails. The various species of dermatophytes are classified into three genera anamorphic: Microsporum, Trichophyton and Epidermophyton. The genus Epidermophyton includes only E. floccosum, that rarely affects animals. The main species responsible for the disease in dogs and cats are Microsporum canis, M. gypseum and Trichophyton mentagrophytes, which were characterized through conventional mycological methodology (microscopic examination with KOH and culture). Molecular methodologies, such as real-time PCR, can contribute to a rapid laboratory diagnosis, helping clinicians to initiate an early antifungal treatment. This case report describes a case of canine dermatophytosis due to Trichophyton mentagrophytes detected from a clinical sample by SYBR-Green real-time PCR. Case: A 8-year-old dog, rescued from the street, was referred to a private veterinary clinic in the city of Canoas, RS, Brazil, presenting generalized lymphadenomegaly, crusted lesions all over the body, generalized alopecia, signs of excoriation and epistaxis. Initially, were administered prednisone [1 mg/kg every 48 h, BID] and cephalexin [30 mg/kg, BID]. Weekly baths with benzoyl peroxide were also given. The therapy was not clinically successful. Wood's Lamp Test was negative. As a differential diagnosis, PCR for detection of Leishmania was negative. Complete blood count and serum biochemical assay were also performed. For mycological diagnosis, hair specimen was clarified and examined microscopically using 10% potassium hydroxide (KOH) for the visualization of chains of arthroconidia (ectothrix invasion of hair). The infected hair was plated onto MycoselTM Agar, incubated at 28°C for 15 days. Microscopy of hyphae/ conidia and macroscopic colony characteristics (colors and texture) were conducted for the differentiation of the species within the genus Microsporum and Trichophyton. In addition, real-time PCR was applied for direct analysis of the fungal DNA obtained from the hair sample. Microscopic examination was negative. The dermatophyte present in the hair sample was confirmed as Trichophyton mentagrophytes by culture and qPCR (melting-point analysis). The patient was treated with systemic itraconazole [10 mg/ kg SID - 90 days]. Twice-weekly application of 2.5 % miconazole and 2% chlorhexidine shampoo until complete cure. Discussion: Dermatophytosis is often listed as self-limiting infection; however, animal dermatophytosis can spread between pets, as well as a zoonotic transmission to humans. The literature on dermatophytosis indicates that Microsporum canis is the predominant etiological agent, followed by M. gypseum. Trichophyon mentagrophytes that appear in a lower percentage of isolation. The culture of hair, even with specific medium containing chloramphenicol and cyclohexamide, may present contaminating fungi, not related to dermatophytosis, which can inhibit or override the growth of dermatophytes. The use of real-time PCR provided a faster and specific diagnosis of dermatophytosis when compared to the conventional mycological methodology for detection and identification of T. mentagrophytes, which takes around 10 to 15 days for culture. It is possible to use this technique as an alternative diagnosis for dermatophytes associated to clinical hair samples of dogs.

DETERMINAÇÃO DE CETAMINA E NORCETAMINA EM CABELO, COMO MODELOS DE DROGAS BÁSICAS PARA A INVESTIGAÇÃO TOXICOLÓGICA SOBRE O GOLPE BOA NOITE CINDERELA

A cetamina é um anestésico amplamente difundido, principalmente em medicina veterinária. Devido a seu efeito dissociativo, indução à amnésia e a efeitos psicodélicos é frequentemente utilizada como droga de abuso e como droga facilitadora para práticas criminosas, dentre as quais o chamado golpe boa noite Cinderela. Entretanto, esse tipo de crime é de difícil comprovação, pois, necessita de perícia imediata e metodologias analíticas sensíveis, validadas de acordo com o tipo de matriz biológica em exame. O cabelo tem se mostrado uma matriz importante na investigação criminal por conta da ampla janela de detecção, maior que a de outras matrizes biológicas; além disso, é possível realizar a análise multissecional, que distingue o uso recreacional frequente do uso criminoso, usualmente realizado com dose única, capaz de incapacitar uma vítima. A cromatografia líquida acoplada à espectrometria de massas sequencial (CLEM/ EM) vem sendo muito empregada na área forense devido a sua especificidade e sensibilidade. No presente estudo foi desenvolvida uma metodologia para determinação da cetamina e norcetamina em cabelo por CL-EM/EM em amostras de cabelo pulverizado, empregando a extração das amostras por tampão e purificação por extração em fase sólida com fase mista polimérica/troca catiônica. O desenvolvimento incluiu uma etapa de produção de amostras fortificadas por incorporação do analito no cabelo, onde diferentes métodos de incorporação foram testados. A escolha do meio, do processo mecânico de extração e testes com diferentes tipos de pré-tratamento do cabelo - picotamento e dois graus de moagem foram feitos com as amostras fortificadas por incorporação. A validação realizada mostrou que o método possui linearidade na faixa de 0,02 10 ng mg-1 para cetamina e 0,04 4 ng mg-1 para norcetamina. Os parâmetros de exatidão, precisão, efeito matriz e recuperação ficaram dentro dos limites de aceitação principalmente segundo a ANVISA e SWGTOX. A metodologia validada foi aplicada a amostras autênticas (n=3) de indivíduos que receberam dose única de cetamina como medicação anestésica. A análise do segmento de cabelo que corresponde à época em que o medicamento foi aplicado demonstrou a presença do fármaco nas amostras.

Ketamine is a widespread anesthetic, especially in veterinary medicine. Due to its dissociative effect, inducing amnesia and psychedelic effects, it is often used as a drug of abuse and as a facilitator for drug facilitated crimes, among which the best known is the so-called in Brazil "Ketamine is a widespread anesthetic, especially in veterinary medicine. Due to its dissociative effect, inducing amnesia and psychedelic effects, it is often used as a drug of abuse and as a facilitator for drug facilitated crimes, among which the best known is the so-called in Brazil "good night Cinderella blow, also known as date rape crime. However, this kind of crime is difficult to prove because it requires immediate forensic investigation, by different sensitive, validated analytical methodologies, according to the type of biological matrix in examination. Hair is an important matrix in criminal investigation because of its wide detection window, larger than that of other biological matrices. Furthermore, with hair it is possible to perform multi-sectional analysis, which distinguishes frequent recreational from criminal use, normally done with a single dose capable of incapacitating a victim. Liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS) has been widely used in forensics because of its specificity and sensitivity. In this study we developed a methodology for determination of ketamine and norketamine in hair by LC-MS/MS in pulverized hair samples, using a buffer for extraction and solid phase extraction (SPE) as a clean-up step. The development of the method included the production of fortified samples, by incorporation of the analyte into hair. The choice of extraction medium and of mechanical assistance (vortex or ultrassonic bath) was performed with samples fortified by incorporation. The effect of sample pretreatment hair cutting or pulverization at two different levels on the amount of analyte extracted was also investigated. The validation performed showed that the method was linear in the range of 0.02 to 10 ng-mg-1 for ketamine and 0.04 to 4 ng mg-1 for norketamine. Accuracy, precision, and matrix effects were within the acceptance limits mainly according to ANVISA and SWGTOX. The validated methodology was applied to authentic samples (n = 3) from subjects who received a single dose of ketamine as an anesthetic medication. The analysis of the hair segment that corresponds to the time when the drug was applied demonstrated the drug presence in the samples. blow, also known as date rape crime. However, this kind of crime is difficult to prove because it requires immediate forensic investigation, by different sensitive, validated analytical methodologies, according to the type of biological matrix in examination. Hair is an important matrix in criminal investigation because of its wide detection window, larger than that of other biological matrices. Furthermore, with hair it is possible to perform multi-sectional analysis, which distinguishes frequent recreational from criminal use, normally done with a single dose capable of incapacitating a victim. Liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS) has been widely used in forensics because of its specificity and sensitivity. In this study we developed a methodology for determination of ketamine and norketamine in hair by LC-MS/MS in pulverized hair samples, using a buffer for extraction and solid phase extraction (SPE) as a clean-up step. The development of the method included the production of fortified samples, by incorporation of the analyte into hair. The choice of extraction medium and of mechanical assistance (vortex or ultrassonic bath) was performed with samples fortified by incorporation. The effect of sample pretreatment hair cutting or pulverization at two different levels on the amount of analyte extracted was also investigated. The validation performed showed that the method was linear in the range of 0.02 to 10 ng-mg-1 for ketamine and 0.04 to 4 ng mg-1 for norketamine. Accuracy, precision, and matrix effects were within the acceptance limits mainly according to ANVISA and SWGTOX. The validated methodology was applied to authentic samples (n = 3) from subjects who received a single dose of ketamine as an anesthetic medication. The analysis of the hair segment that corresponds to the time when the drug was applied demonstrated the drug presence in the samples.