Genetic variability of the 16S rRNA gene of Nocardia brasiliensis, the most common causative agent of actinomycetoma in Latin America and the Caribbean

ABSTRACT Mycetoma is a neglected tropical disease (NTD) declared by the World Health Organization (WHO) in 2016. It is characterized by the progressive growth of nodules and granulomatous lesions on the legs, arms, and trunk. It is potentially disfiguring and causes disability or amputations in working-age people from marginalized areas. The causative agents can be fungi (eumycetoma) or actinobacteria (actinomycetoma), the latter being the most common in America and Asia. Nocardia brasiliensis is the most important causal agent of actinomycetoma in the Americas. Taxonomic problems have been reported when identifying this species, so this study aimed to detect the 16S rRNA gene variations in N. brasiliensis strains using an in silico enzymatic restriction technique. The study included strains from clinical cases of actinomycetoma in Mexico, isolated from humans and previously identified as N. brasiliensis by traditional methods. The strains were characterized microscopically and macroscopically, then subjected to DNA extraction and amplification of the 16S rRNA gene by PCR. The amplification products were sequenced, and consensus sequences were constructed and used for genetic identification and in silico restriction enzyme analysis with the New England BioLabs® NEBcutter program. All study strains were molecularly identified as N. brasiliensis; however, in silico restriction analysis detected a diversity in the restriction patterns that were finally grouped and subclassified into 7 ribotypes. This finding confirms the existence of subgroups within N. brasiliensis. The results support the need to consider N. brasiliensis as a complex species.

Acid-fast bacteria as causative agents of skin and soft tissue infections: case presentations and literature review

ABSTRACT Acid-fast bacteria can be implicated in skin and soft tissue infections. Diagnostic identification can be challenging or not feasible by routine laboratory techniques, especially if there is no access to the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technology. Here, we present two cases of skin and soft tissue infections caused by two different acid-fast bacteria, Nocardia brasiliensis and Mycobacterium marinum. They both grew on Löwenstein-Jensen medium, Sabouraud agar medium and blood agar medium. Both bacteria appeared acid-fast by Ziehl-Neelsen stain and Gram-positive by Gram stain. The identification was performed by MALDI-TOF MS and gene analysis. N. brasiliensis and nontuberculous mycobacterium M. marinum represent rare pathogens that cause severe skin and soft tissue infections. Failure to identify the causative agent and subsequent inappropriate or inadequate treatment may lead to severe complications or even disseminated disease, especially in immunocompromised individuals.

Nocardiosis linfocutánea en paciente inmunocomprometido

RESUMEN Se presenta el caso de un paciente, agricultor, adulto mayor, portador de hipertensión arterial, cardiopatía isquémica y cáncer de próstata con metástasis en hueso en tratamiento conservador. Acude por cuadro de 15 días de evolución con flictenas en miembro superior y fiebre. Durante su internación se aísla en la secreción de piel Nocardia brasiliensis y es tratado con trimetoprin - sulfametoxazol. En el 15° de internación se agrega una neumonía asociada a los cuidados de la salud. Posteriormente se detecta una lesión ósea destructiva en columna a nivel de L3. Sale de alta con el diagnóstico de nocardiosis linfocutánea en paciente inmunocomprometido a los 31 días de internación con buena respuesta clínica y con planes de continuar antibióticoterapia.

ABSTRACT The case of a patient, an elderly farmer who has hypertension, ischemic heart disease and prostate cancer with bone metastases in conservative treatment, is presented. He consults for a 15-day evolution case with flictenas in the upper limb and fever. During hospitalization, Nocardia basiliensis is isolated in the secretion of skin and is treated with trimethoprine - sulfamethoxazole. In the 15th day of hospitalization, health care-associated pneumonia is added. Subsequently, a destructive bone lesion in the spine is detected at the L3 level. He is discharged with the diagnosis of lymphocutaneous nocardiosis in an immunocompromised patient after 31 days of hospitalization with a good clinical response and with plans to continue antibiotic therapy.

Actinomicetoma en el embarazo. Reporte de un caso: ¿Qué hacer y cómo tratarlo? / Actinomycetoma in pregnancy. Case report: what to do, how to handle?

Resumen ANTECEDENTES: El micetoma es una infección causada por hongos y actinomicetos aeróbicos. Es un padecimiento frecuente en México, con mayor afectación en hombres que en mujeres (3:1). Se caracteriza por aumento de volumen, deformidad del sistio de lesión y formación de fístulas. OBJETIVO: Reportar un caso de actinomicetoma durante el embarazo y analizar el protocolo de tratamiento. CASO CLINICO: Paciente de 29 años de edad, en su cuarto embarazo, con 29 semanas de gestación. El padecimiento se inició 13 años atrás, con dermatosis localizada en la extremidad inferior izquierda, constituida por un nódulo indoloro, que permaneció sin cambios hasta el inicio de la gestación actual, cuando le aparecieron múltiples lesiones nodulares y fístulas. En el examen directo de la secreción se observaron granos y en el cultivo se identificó Nocardia brasiliensis. Después del embarazo a término y con recién nacido sano, si indicó lactancia durante 4 meses y se interrumpió para prescribir bromocriptina. La dermatosis se extendió al doble, sin afectación ósea; se indicó tratamiento con sulfametoxazol-trimetoprima y dapsona. El tiempo total de tratamiento fue de 15 meses y seguimiento sin medicación durante un año. Se obtuvo curación clínica y microbiológica. CONCLUSIONES: El micetoma en mujeres embarazadas es excepcional. La mayor parte de los portocolos de tratamiento deben contraindicarse durante el embarazo. Cuando el micetoma se localiza en una zona que no afecta otros órganos ni se extiende, se sugiere continuar el embarazo y la lactancia sin prescripción de medicamentos pero iniciarla posterior al nacimiento.

Abstract BACKGROUND: Mycetoma is an infection caused by fungi and aerobic actinomycetes. It is a frequent condition in Mexico; it presents less in women than men (1:3). It is characterized by increased volume deformity of the region and sinuses. OBJECTIVE: We present a case of actinomycetoma in a pregnant patient and to analyze the behavior in its therapeutic management CLINICAL CASE: We present female, 29 years old, attending her fourth pregnancy at 29 weeks of gestation. It began 13 years ago with a localized dermatosis of the lower left limb, constituted by a painless nodule, remained unchanged until the beginning of the current gestation, developed multiple nodules and sinuses. A direct examination of the secretion was performed, observing grains, Nocardia brasiliensis was identified. After product birth, lactation was allowed for 4 months and discontinued with bromocriptine. The dermatosis extended to double without bone affection, treatment with sulfamethoxazole/trimethoprim + dapsone was given. Total time was 15 months and follow-up without medication for one year. Clinical and microbiological cure was achieved. CONCLUSIONS: The development of mycetoma in pregnant women is rare, it is important to know the etiology, in eumycetoma all the antimycotics are teratogenic and in actinomycetoma most antibiotics cannot be used in pregnancy with some exceptions. If mycetoma is located in an area that does not compromise other organs or does not spread it is best to leave the course of pregnancy and lactation and then start treatment.

Minimicetoma por Nocordia brasiliensis: un caso infantil en Veracruz / Minimicetoma by Nocordia brasiliensis: a children's event in Veracruz

El término minimicetoma designa a los micetomas de pequeño tamaño, con morfología atípica y superficial, causados por Nocardia brasiliensis. Se comunica un paciente de sexo masculino, de 10 años de edad, originario y residente de Xalapa (Veracruz), que presentó un minimicetoma por N. brasiliensis, confirmado por estudio micológico e histopatológico. El tratamiento con penicilina procaínica durante 10 días, en conjunto con trimetoprima/sulfametoxazol y diaminodifenisulfona por 6 meses, tuvo resultados satisfactorios.

Minimycetoma is an atypical small mycetoma usually caused by Nocardia brasiliensis. We report a 10-year-old male from Xalapa (Veracruz) with a minimycetoma due to N. brasiliensis confirmed by mycological and histopathological studies. Simultaneous treatment with procainic penicillin for ten days and trimethoprim/sulfamethoxazol and dapsone showed satisfactory results after 6 months.

Nocardia brasiliensis primary pulmonary nocardiosis with subcutaneous involvement in an immunocompetent patient.

This is a report of an unusual case of Nocardia brasiliensis causing primary pulmonary nocardiosis with disseminated subcutaneous lesions in an immunocompetent patient. This case highlights the importance of considering nocardiosis as a differential diagnosis in patients with pulmonary and cutaneous lesions and the need for vigorous management for complete cure.

Primary Cutaneous Nocardiosis Caused by Nocardia brasiliensis / 대한피부과학회지

Nocardiosis is an uncommon, potentially life-threatening infectious disease caused by several species of the genus Nocardia, which are Gram-positive branched bacilli. Most infections enter through the respiratory tract and then disseminate systemically but rarely has a primary infection occurred resulting from direct inoculation. Isolation of Nocardia from clinical specimens and identification of species is difficult and require a specialized microbiologist. We report a case of primary cutaneous nocardiosis caused by N. brasiliensis in a 68-year-old man who was diagnosed by bacterial culture and 16S ribosomal RNA sequencing. The skin lesions improved with trimethoprim- sulfamethoxazole antibiotic therapy for 6 months.

Pleural nocardiosis.

Pleural involvement in nocardiosis is rarely documented in India. We report two cases of pleural nocardiosis. In both the cases, the aetiologic agent was isolated from pleural fluid and was later identified as Nocardia asteroides in one case and as Nocardia brasiliensis in the second case. The former case of pyopneumothorax died before the diagnosis was made and the latter with pleural effusion showed excellent response to six months of therapy with trimethoprim-sulfamethoxazole.

Nocardiose disseminada em paciente leucêmico / Disseminated nocardiosis in leucemic patient

Atualmente, a análise citológica deste líquido é feita de forma manual, utilizando a câmara Fuchs-Rosenthal, o que torna o exame demorado e com grande possibilidade de erros. Entretanto, outras análises citológicas podem ser realizadas através de contadores automáticos, garantindo uma análise mais rápida e confiável. O objetivo do trabalho é a comparação entre a contagem manual e automatizada de células no LCR, para verificação da possibilidade de ser realizada na automação. Para tanto, foram analisadas 100 amostras de LCR, sendo que 50 amostras foram analisadas no contador hematológico Pentra® 60 (ABX) e 50 amostras analisadas no contador hematológico CellDyn® 3200, em comparação com a contagem manual de referência. Os resultados demonstraram que 62 amostras não reproduziram adequadamente os dados na contagem automatizada, o que nos leva a imaginar que o LCR não deve ser analisado em automação. Assim, é importante a realização de novos trabalhos, com números maiores de amostras e aparelhos para definirmos a possibilidade do uso da automação na contagem citológica de LCR.

Inmunoglobulinas en pacientes con actinomicetoma por Nocardia brasiliensis / Immunoglobulins in patients with Nocardia brasiliensis actinomycetoma

Considerando que algunos autores han reportado un aumento en la cantidad de algunas inmunoglobulinas en los pacientes con actinomicetoma, en este trabajo nos propusimos determinar diferencias en la producción de IgG1, IgG2, IgG3, IgG4 e IgM en 25 pacientes con actinomicetoma por Nocardia brasiliensis y 25 personas sanas provenientes de una zona endémica de micetoma. La determinación de inmunoglobulinas se realizó por medio de la técnica de ELISA. Para sensibilizar las placas se emplearon 6 antígenos de N. brasiliensis: un antígeno crudo denominado NB y cinco derivados del mismo (NB2, NB4, NB6, NB8 y NB10) separados por punto isoeléctrico. Los niveles de las cuatro subclases de IgG fueron mayores en los sueros de los pacientes que en el suero de los controles, con una diferencia máxima en IgG3 e IgG4; para esta última subclase, los seis antígenos fueron altamente reactivos. La concentración de IgM fue igual en ambos grupos. Es probable que como ocurre en otras infecciones, en la fisiopatogenia del actinomicetoma influya no sólo el aumento o deficiencia de una clase de inmunoglobulina, sino la relación que existe entre las diferentes subclases.

Considering that some authors have reported an increasing of some immunoglobulins in actinomycetoma patients, in this study we propose to determine differential production of IgG1, IgG2, IgG3, IgG4 and IgGM in 25 patients with actinomycetoma and 25 healthy individuals from a mycetoma endemic area. Immunoglobulins were determined by ELISA technique. To sensibilize the plates, six Nocardia brasiliensis antigens were used: a crude antigen denominated NB and five derivatives (NB2, NB4, NB6, NB8 and NB10) obtained by their isoelectric point. Results showed that all IgG subclasses were higher in the patients’ sera than in control sera, with a maximal difference to IgG3 and IgG4. To the latter subclass, six antigens were highly reactives. IgM levels were similar in both groups. As it occurs in other infections, in the actinomycetoma pathogenesis probably participate the increase or deficiency of a determined immunoglobulin class, as well as the relationship between different subclasses.