Diagnóstico micológico de histoplasmosis en Uruguay, reporte de una serie de casos de la última década / Mycological diagnosis of histoplasmosis in Uruguay, report of a series of cases of the last decade

Introducción: La histoplasmosis es una micosis sistémica que afecta a humanos, su agente Histoplasma capsulatum, hongo dimorfo, es ubicuo en la naturaleza. Frecuentemente se presenta como reactivación en personas con infección por VIH/SIDA, con manifestaciones polimórficas y diseminadas. Las lesiones mucocutáneas son una importante llave diagnóstica. Objetivo: Contribuir al conocimiento de esta patología a través del reporte de los diagnósticos de laboratorio de histoplasmosis realizados en Uruguay en los últimos 10 años. Materiales y Métodos: Se realizó un estudio observacional, retrospectivo de las histoplasmosis diagnosticadas en el laboratorio de referencia de Micología de Facultad de Medicina y dos laboratorios clínicos. Se enrolaron los registros clínicos y analíticos asociados. Resultados: Fueron 69 los diagnósticos de histoplasmosis. Más de 80% correspondió a personas con infección por VIH/SIDA. El 62,3% del total presentó lesiones de piel y/o mucosas y en 58% el diagnóstico se realizó mediante el estudio de estas. El 62,3% de los diagnósticos se realizaron mediante la visualización al microscopio óptico de frotis coloreados. Conclusiones: La mayoría de las histoplasmosis se vinculan a la infección por VIH/SIDA. El estudio micológico de las lesiones de piel y/o de mucosas, es accesible, mínimamente invasivo, rápido y presenta una excelente performance diagnóstica.

Background: Histoplasmosis is a systemic mycosis that affects humans, its agent Histoplasma capsulatum, a dimorphic fungus, is ubiquitous in nature. It frequently presents as reactivation in people with HIV/AIDS infection, with polymorphic and disseminated manifestations. Mucocutaneous lesions are characteristic and an important diagnostic key. Aim: To contribute to the knowledge of this pathology through the report of histoplasmosis laboratory diagnosis made in Uruguay in the last 10 years. Methods: We conducted an observational, retrospective study of diagnosed histoplasmosis in the Mycology reference laboratory of the Faculty of Medicine and two clinical laboratories. Associated clinical and analytical records were obtained. Results: There were 69 histoplasmosis diagnoses. More than 80% corresponded to people with HIV/AIDS infection. 62.3% of the total presented skin and/or mucosal lesions and in 58% the diagnosis was made by studying them. 62.3% of the diagnoses were initially made by viewing colored smears under an optical microscope. Conclusions: Most histoplasmosis is linked to HIV/AIDS infection. Exposure to a high fungal load is a constant in cases of immunocompetent individuals. The mycological study of skin and/or mucosal lesions is accessible, minimally invasive, fast and has excellent diagnostic performance.

Otomycosis caused by the cryptic and emerging species Aspergillus sydowii: two case reports.

Two cases of otomycosis have been reported in patients undergoing tympanomastoidectomy. The first one had chronic otitis media, hypertrophic concha and nasal septum deviation, tympanic perforation and otorrhea. The second had otalgia, pruritus, chronic otitis media and cholesteatoma. Direct examination showed mycelial septate filaments with a branch at an angle close to 45°, later identified as Aspergillus sydowii by sequencing the BenA and CaM genes. Susceptibility testing showed low MIC of amphotericin B, itraconazole, ketoconazole and ciclopirox olamine. In both cases, ketoconazole was instituted for 10 days. Otomycosis is a challenge as it is primarily recurrent in patients undergoing surgery. The clinical implication, the identification of the emerging pathogen and the determination of MIC were necessary for the knowledge of the epidemiological profile and establishment of the treatment.

Aspergillus are fungi that can cause ear disease. In severe infections, these fungi can be present for long periods inside the ear, and commonly belong to the species Aspergillus section Nigri and Aspergillus flavus. In this work, we present two cases of ear infections by a different species, Aspergillus sydowii. Patients had obstructed nasal cavities, crooked internal separation of the nose and complaints of secretion in the ear. The efficient diagnosis allowed a treatment that resulted in the death of the fungus and the cure of the patient.

Epidemiology and Diagnostic Perspectives of Dermatophytoses.

Dermatophytoses affect about 25% of the world population, and the filamentous fungus Trichophyton rubrum is the main causative agent of this group of diseases. Dermatomycoses are caused by pathogenic fungi that generally trigger superficial infections and that feed on keratinized substrates such as skin, hair, and nails. However, there are an increasing number of reports describing dermatophytes that invade deep layers such as the dermis and hypodermis and that can cause deep infections in diabetic and immunocompromised patients, as well as in individuals with immunodeficiency. Despite the high incidence and importance of dermatophytes in clinical mycology, the diagnosis of this type of infection is not always accurate. The conventional methods most commonly used for mycological diagnosis are based on the identification of microbiological and biochemical features. However, in view of the limitations of these conventional methods, molecular diagnostic techniques are increasingly being used because of their higher sensitivity, specificity and rapidity and have become more accessible. The most widely used molecular techniques are conventional PCR, quantitative PCR, multiplex PCR, nested, PCR, PCR-RFLP, and PCR-ELISA. Another promising technique for the identification of microorganisms is the analysis of protein profiles by MALDI-TOF MS. Molecular techniques are promising but it is necessary to improve the quality and availability of the information in genomic and proteomic databases in order to streamline the use of bioinformatics in the identification of dermatophytes of clinical interest.

Método convencional para o diagnóstico de micoses: um estudo comparativo / Conventional method for the diagnosis of mycoses: a comparative study

Introdução: O método clássico para o diagnóstico de micoses é realizado pelo Exame Micológico Direto (EMD) e cultural, que possibilita a visualização de estruturas fúngicas vegetativas e estruturas reprodutivas, respectivamente. Essa combinação é fundamental para reduzir possíveis erros analíticos e aumentar a precisão do diagnóstico. Métodos: Com a finalidade de verificar a frequência do EMD e cultural, e comparar seus parâmetros de sensibilidade e especificidade, realizamos uma análise retrospectiva entre janeiro de 2018 e maio de 2020, de 1603 laudos micológicos oriundos de um laboratório de análises clínicas, localizado em Porto Alegre. Resultados: Após a análise dos laudos observamos que a maioria dos casos apresentaram o EMD negativo com cultura positiva (36,24%). Na sequência, 30,87% dos casos foram de amostras negativas e 25,57% dos laudos foram positivos para ambos os exames. A minoria dos casos (7,29%) apresentou o EMD positivo com cultura negativa. Conclusão: Esta análise revelou que o exame cultural é mais sensível e específico, demonstrando uma maior confiabilidade no diagnóstico. Entretanto, vale ressaltar que a realização dos exames em conjunto, além de reduzir possíveis erros analíticos, proporciona um diagnóstico melhor fundamentado. (AU)

Introduction: The classic method for the diagnosis of mycoses is performed by both direct mycological examination (DME) and culture, which allow the visualization of vegetative and reproductive fungal structures, respectively. This combination is essential to reduce possible analytical errors and increase the accuracy of the diagnosis. Methods: To assess the frequency of DME and culture, and compare their parameters of sensitivity and specificity, we performed a retrospective analysis of 1603 mycological reports produced between January 2018 and May 2020 in a clinical analysis laboratory in Porto Alegre, southern Brazil. Results: After analyzing the reports, we observed that most cases presented a negative DME and a positive culture (36.24%). Subsequently, 30.87% of the cases were negative for both tests, and 25.57% were positive for both tests. The minority of cases (7.29%) presented a positive DME and a negative culture. Conclusion: Our analysis revealed that cultural examination is more sensitive and specific, showing greater reliability in the diagnosis. However, it is noteworthy that performing the tests together, in addition to reducing possible analytical errors, provides a more consistent diagnosis. (AU)

[Microbiological diagnosis of invasive mycosis]. / Diagnóstico microbiológico de las micosis invasoras.

The prognosis of invasive fungal infections (IFI) depends on the speed of diagnosis and treatment. Conventional diagnostic methods are of low sensitivity, laborious and too slow, leading to the need for new, faster, and more efficient diagnostic strategies. There are several techniques for diagnosing a candidemia that are faster than the conventional blood culture (BC). Once yeast growth in BC is detected, species identification can be speeded up by mass spectrometry (30minutes), commercialised molecular techniques (60-80minutes) or fluorescent in situ hybridization (90minutes). The combined detection of biomarkers (antimicellium, mannan and anti-mannan or ß-glucan) has shown to be of greater use than their individual use. Commercialised nucleic acid amplification techniques (Septifast®, T2Candida®) are very reliable alternatives to BC. The detection of the capsular antigen of Cryptococcus, by means of latex agglutination or immuno-chromatography, is a valuable technique for cryptococcosis diagnosis. Direct microscopic examination and culture of representative specimens is used for the conventional diagnosis of IFI by filamentous fungi. Detection of galactomannan and ß-glucan are considered diagnostic criteria for probable invasive aspergillosis and probable IFI, respectively, despite the lack of specificity of the latter. The detection of fungal volatile organic compounds in breath is an interesting diagnostic strategy in pulmonary infections. Although widely used, nucleic acid detection techniques are not considered diagnostic criteria for IFIs caused by moulds in consensus documents, due to their lack of standardisation. However, they are the only alternative to culture methods in invasive infections by Scedosporium/Lomentospora, Fusarium, zygomycetes, or dematiaceous fungi.

Examen mycologique en dermatologie.

Dermatomycoses are dermatological infections very commonly encountered in private dermatological practice since they affect up to one third of the population. However, the symptoms are very often shared by other skin infections and disorders and may be highly atypical. It is thus impossible to make a diagnosis with any certainty on clinical grounds alone. For this reason, mycological diagnosis is essential to either confirm or rule out dermatomycosis, and is unavoidable when antifungal therapy is required for the treatment of ringworm of the scalp or beard, or for onychomycosis. It is also vital where therapy guided by the clinical appearance of lesions has failed or in the event of recurring skin lesions. Confirmation of mycosis enables antifungals to be initiated and a negative test warrants investigation for other underlying causes for the lesions seen. However, regardless of the mycological diagnostic technique employed, the quality of the results depends chiefly on the quality of sampling of the infected site, but also on the expertise of the microbiologist. Standard mycological testing remains the most informative, the least expensive and the sole examination capable of isolating the causative fungus irrespective of the type of mycosis, such as dermatophytosis, scytalidiosis, mould-induced ungual infection, candidiasis, or infections due to Malassezia sp. This is the only examination able to identify epidemiological variations. All other more recent techniques are either based upon simple demonstration of the fungal elements involved, without identification of the fungal species in question, or else they are reliant upon a fungal database that is generally highly incomplete.

Early Diagnosis of Cutaneous Mucormycosis Due to Lichtheimia corymbifera After a Traffic Accident.

A case report of cutaneous mucormycosis and obstacles to early diagnosis is presented. A 38-year-old male was involved in a car accident that led to amputation of both lower limbs. Subsequently, he developed fungal wound infection of the left lower limb stump. The infection was detected very early, although the diagnosis was difficult because only a small area was affected and histopathological examination was initially negative. The infection was proven by microscopy, culture and histopathology. The isolate was identified by sequencing of the rDNA ITS region gene (internal transcribed spacer region of ribosomal DNA) as Lichtheimia corymbifera. Liposomal amphotericin B and surgery were successful in management of the disease.

Diagnosis of dermatophytosis: an evaluation of direct examination using MycetColor® and MycetFluo®.

Dermatophytes are an important cause of superficial fungal infection. Direct examination of skin, nail, or hair samples remains essential in diagnosis, as it provides a quick response to the clinician. However, mycological analysis, including direct examination and culture, often lacks sensitivity. The use of stains or fluorochromes may enhance the performance of direct examination. We analyzed 102 samples from patients with suspected dermatophytosis in 4 different diagnostic mycology laboratories. Two reagents, MycetColor® and MycetFluo®, which use Congo red and calcofluor dye, respectively, were evaluated for the direct microscopic examination of skin, hair, and nail specimens. The results were compared to those of culture and conventional direct examination. Both reagents were able to clarify the specimens and also to specifically stain fungal elements. Microscopic examination of the specimens was greatly facilitated with MycetFluo®, which allowed a higher number of positive cases to be detected compared to the other methods.

Disseminated histoplasmosis caused by Histoplasma capsulatum var. duboisii in a non-HIV patient in Burkina Faso: Case report.

Histoplasmosis is a fungal infection due to Histoplasma capsulatum. The African form of this mycosis, caused by Histoplasma capsulatum var. duboisii, remains rare. We report a case of disseminated African histoplasmosis with skin, lymph nodes, bones and viscera localizations. The 22-year-old patient was HIV-seronegative and was considered immunocompetent. The presence of Histoplasma capsulatum var duboisii in ulcerations and a nodule pus aspiration was confirmed by direct microscopic examination and by culture. The medical treatment was based on fluconazole. Even though a regression of the symptoms was observed, the patient died. In disseminated African histoplasmosis, an early laboratory diagnosis must be carried out for accurate treatment.

[Onychomycoses due to molds]. / Les onychomycoses à moisissures.

Onychomycoses represent about 30% of superficial mycosis that are encountered in Dermatology consults. Fungi such as dermatophytes, which are mainly found on the feet nails, cause nearly 50% of these onychopathies. Yeasts are predominantly present on hands, whereas non-dermatophytic moulds are very seldom involved in both foot and hand nails infections. According to literature, these moulds are responsible for 2 to 17% of onychomycoses. Nevertheless, we have to differentiate between onychomycoses due to pseudodermatophytes such as Neoscytalidium (ex-Scytalidium) and Onychocola canadensis, which present a high affinity for keratin, and onychomycoses due to filamentous fungi such as Aspergillus, Fusarium, Scopulariopsis, Acremonium... These saprophytic moulds are indeed most of the time considered as colonizers rather than real pathogens agents. Mycology and histopathology laboratories play an important role. They allow to identify the species that is involved in nail infection, but also to confirm parasitism by the fungus in the infected nails. Indeed, before attributing any pathogenic role to non-dermatophytic moulds, it is essential to precisely evaluate their pathogenicity through samples and accurate mycological and/or histological analysis. The treatment of onychomycoses due to non-dermatophytic moulds is difficult, as there is today no consensus. The choice of an antifungal agent will first depend on the species that is involved in the infection, but also on the severity of nail lesions and on the patient himself. In most cases, the onychomycosis will be cured with chemical or mechanical removing of the infected tissues, followed by a local antifungal treatment. In some cases, a systemic therapy will be discussed.

Concordancia entre los métodos del diagnóstico micológico de la paracoccidioidomicosis / Concordance between methods of mycological diagnosis of paracoccidioidomycosis

La paracoccidioidomicosis (PCM) es una enfermedad crónica, sistémica, granulomatosa, endémica en nuestro país, producida por un hongo dimorfo denominado Paracoccidioides brasiliensis. Existen numerosas técnicas para realizar el diagnóstico de esta entidad. Nos planteamos la posibilidad de realizar un estudio para determinar la concordancia que pudiese existir entre las diferentes técnicas que se utilizan para el diagnóstico de la PCM. Se realizó un registro de historias clínicas. Se evaluaron 251 historias clínicas de pacientes con diagnóstico de PCM, de la consulta externa de la Sección de Micología Médica “Dr. Dante Borelli” del IMT-UCV, entre los años 2000 y 2010. Se determinó la concordancia entre los métodos diagnósticos por medio del análisis de concordancia de atributos para datos binarios. Entre el examen directo y el cultivo, no hubo acuerdo. Entre la serología y el examen directo, se encontró que hubo equivalencia, así como entre el cultivo y la serología. No se pudo calcular correlación alguna con la histopatología, ya que no hubo datos negativos, en vista de que todas las muestras procesadas fueron positivas. El diagnóstico de la PCM se basa en la identificación y el aislamiento del hongo. Es obligatoria la realización del examen directo en fresco de toda muestra clínica. Nuestro estudio de muestra que deben realizarse todos los métodos que estén al alcance (examen directo en fresco, cultivo, serología, histopatología) a fin de aumentar la probabilidad de llegar a un diagnóstico certero de esta patología

Paracoccidioidomycosis (PCM) is a chronic, granulomatous disease, endemic in our country, produced by a dimorphic fungus, Paracoccidioides brasiliensis. Several techniques are used for the diagnosis. A study was performed to determine the agreement that could exist between the different techniques, used for the diagnosis of PCM. Clinical records of patients with diagnosis of PCM was made. 251 clinical records were reviewed, from the Sección de Micología Médica “Dr. Dante Borelli”, IMT- UCV, between 2000 and 2010. The agreement between the methods was determined by means of the analysis of agreement of attributes for binary data. Between direct examination and culture, there was no agreement. Between serology and direct examination, there was equivalence, as well as between culture and serology. Correlation could not be calculated with histopathology, since there were no negative data, due to the fact that all the processed samples were positive. The diagnosis of the PCM is based on the identification and isolation of the fungus. Direct examination is mandatory in all clinical samples. Our study demonstrates that all the methods must be performed (direct examination, culture, serology, histopathology) in order to increase the probability of reaching an accurate diagnosis

Diagnóstico de la histoplasmosis diseminada asociada al sida / AIDS-related disseminated histoplasmosis diagnosis

En un paciente con histoplasmosis asociada al sida y lesiones cutáneas, se procedió a realizar el diagnóstico de la micosis mediante la metodología habitualmente empleada, la cual se describe. En estos pacientes, el empleo de la escarificación de las lesiones cutáneas ofrece un método rápido, sencillo y económico para el diagnóstico de esta micosis endémica. La severa inmunodeficiencia de estos pacientes eleva la carga microbiana presente en los tejidos y materiales clínicos, lo cual facilita la aplicación de los métodos directos de diagnóstico micológico como microscopia y cultivo. La observación en las preparaciones microscópicas de levaduras intracelulares, que se tiñen de manera defectuosa con la técnica de Giemsa, orienta a un diagnóstico micológico rápido, confirmado luego a través por los cultivos y permite la administración inmediata del tratamiento antifúngico, lo cual mejora las posibilidades de éxito terapéutico.

In a patient with AIDS-related histoplasmosis, and skin lesions, the diagnosis of mycosis through usual methodology was made, which was described in this paper. The use of skin lesion scarification in these patients offers an economic, fast and simple method for diagnosis of endemic mycosis. The severe immunodeficiency of these patients raises the microbial charge present in tissues and clinical materials, which facilitates the application of direct mycological diagnosis methods such as microscopic analysis and culturing. The observation of intracellular yeast in microscopical preparations, which are irregularly stained through the Giemsa technique, leads to rapid mycological diagnosis, later confirmed by the culturing, and allows the immediate administration of antifungal treatment. All this improves the possibilities of a sucessful treatment.

Diagnóstico micológico en pacientes infectados por el virus de la inmunodeficiencia humana / Mycological diagnosis in patients infected with human immunodeficiency virus

Con el descubrimiento del virus de la inmunodeficiencia humana (VIH) en 1981, la frecuencia de las infecciones oportunistas en pacientes inmunodeprimidos, especialmente los infectados por el VIH, ha experimentado un notable incremento. Dentro de éstas se encuentran las de etiología fúngica, las cuales presentan un alto grado de mortalidad si no son diagnosticadas y tratadas a tiempo. En nuestro país con la instauración de la terapia antirretroviral altamente efectiva a partir del año 2001, se ha notado una reducción significativa en la morbilidad y la mortalidad por infecciones oportunistas, en particular por las micosis, muy comunes en este tipo de pacientes. En el presente estudio se realizó el diagnóstico micológico procedente de pacientes infectados por el VIH/sida, durante el período comprendido entre enero 2005 y diciembre de 2009. De un total de 1173 muestras biológicas procesadas, solamente resultaron positivas 324 para un 27,6% de positividad. La candidiasis orofaríngea y la meningoencefalitis criptococóccica fueron las infecciones diagnosticadas con una mayor incidencia. En las muestras analizadas la concordancia en la positividad entre el examen directo y el examen por cultivo micológico fue de un 56,2%, lo que confirma la importancia del empleo del examen directo en la visualización de estructuras fúngicas a partir de muestras clínicas

With the discovery of the human immunodeficiency virus (HIV) in 1981, the frequency of the opportunistic infections in the immunosuppressed patients, especially the infected ones by the HIV, it has experienced a notable increase. Inside these there are those of micotic aetiology, which present a high grade of mortality, if they are not diagnosed and treated on time. In our country with the instauration of the highly effective antirretroviral therapy since the year 2001, a significant reduction has been seen in the morbidity and the mortality of the opportunistic infections, specialle those caused by fungus, in this type of patients. In the present study the positividad was analyzed in the mycological diagnosis realized by conventional methods of the different clinical samples received in the National Laboratory of Reference of Mycology, from patients infected with HIV/aids during the period between January 2005 and December 2009. Of a whole of 1173 biological samples processed only 324 turned out positive, for 27, 6 % of positivity, being the oropharingeal candidiasis and the cryptococcal meningoencefalitis, those of major incidence observed. In the clinical samples analyzed, a concordance of 56,2 % was obtained in the positividad of the samples studied by means of direct examination and mycologycal cultures, which confirms the importance of direct preparations of clinical samples for the visualization of fungal elements from clinical samples