Assessment of VITEK® MS IVD database V3.0 for identification of Nocardia spp. using two culture media and comparing direct smear and protein extraction procedures.

We assessed the performance of the VITEK® MS IVD V3.0 matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-ToF MS) V3.0 database for the identification of Nocardia spp. as compared with targeted DNA sequencing. A collection of 222 DNA sequence-defined Nocardia spp. strains encompassing 18 different species present or not in the database was tested. Bromocresol purple agar (BCP) and Columbia agar +5% sheep's blood (COS) culture media were used together with two different preparation steps: direct smear and a "3 attempts" procedure that covered (1) spotting of an extract, (2) new spotting of the same extract, and (3) spotting of a new extract. The direct smear protocol yielded low correct identification rates (≤ 15% for both media) whereas protein extraction yielded correct identification results (> 67% regardless of the media used.). The use of 2 additional attempts using repeat or new extracts increased correct identification rates to 87% and 91% for BCP and COS, respectively. When using the 3 attempts procedure, the best identification results, independent of media types, were obtained for N. farcinica and N. cyriacigeorgica (100%). Identification attempts 2 and 3 allowed to increase the number of correct identifications (BCP, +20%; COS, +13%). The enhancement in performance during attempts 2 and 3 was remarkable for N. abscessus (81% for both media) and low prevalence species (BCP, 70%; COS, 85%). Up to 3.4% and 2.4% of the strains belonging to species present in the database were misidentified with BCP and COS media, respectively. In 1.9% of the cases for BCP and 1.4% for COS, these misidentifications concerned a species belonging to the same phylogenetic complex. Concerning strains that are not claimed in the V3.0 database, N. puris and N. goodfellowi generated "No identification" results and 100% of the strains belonging to N. arthritidis, N.cerradoensis, and N. altamirensis yielded a misidentification within the same phylogenetic complex. Vitek® MS IVD V3.0 is an accurate and useful tool for identification of Nocardia spp.

First case of cerebral abscess due to a novel Nocardia species in an immunocompromised patient.

We report the first case of cerebral abscess due to a novel species of Nocardia in a heart transplant patient and describe the antimicrobial susceptibility of this isolate. As our patient was intolerant to trimethoprim-sulfamethoxazole, we also discuss alternative therapeutic options in brain abscess due to Nocardia sp.

[Actinomycetoma of the knee due to Nocardia otitidiscaviarum described in the Comoro Islands]. / Actinomycétome du genou àNocardia otitidiscaviarum aux Comores.

BACKGROUND: Mycetoma is a chronic skin and soft tissue infection encountered in the dry tropical regions and are caused by fungi (eumycetoma) or bacteria (actinomycetoma). PATIENTS AND METHODS: A 25-year-old man consulted at the hospital on Mayotte Island for a left knee injury sustained 10 years earlier in a motorcycle accident with broken skin occurring in Anjouan in the Comoro Islands. Clinical and histological diagnosis of mycetoma was made, and in the absence of microbiological diagnosis, empirical antifungal therapy was initiated. Given the poor outcome, new biopsies were performed and resulted in the identification of Nocardia otitidiscaviarum. More than 1 year later, the patient had fully recovered and after administration of several and extended antibiotic courses including cotrimoxazole and linezolid. DISCUSSION: Bacterial mycetomas are usually described in semi-arid regions and the occurrence of this disease is unexpected in humid tropical areas such as the Comoro Islands. N. otitidiscaviarum is rarely involved in this infection, particularly in Africa.

Evaluation of the Andromas matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of aerobically growing Gram-positive bacilli.

Matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

Cavitary pneumonia caused by Nocardia otitidiscaviarum.

We report a case of cavitary pneumonia caused by N. otitidiscaviarum in a man with diabetes mellitus and thrombocytopenia treated with systemic corticosteroid. Taxonomic identification involved phenotypic testing and molecular identification that was carried out by DNA sequencing of the 16SrRNA gene.

Actinomycetoma caused by Gordonia westfalica: first reported case of human infection.

Bacteria of the genus Gordonia are rarely involved in human infections. We report here the case of a 30-year-old man from Guinea Buissau with mycetoma of the foot. 16S DNA sequencing after surgical biopsy identified Gordonia westfalica. To our knowledge, this is the first report of human infection caused by G. westfalica.

First Spanish case of nocardiosis caused by Nocardia takedensis.

Nocardia takedensis is a recently described species isolated from soil. The first clinical isolate in Japan has recently been reported. This report describes the first clinical isolate of N. takedensis in Spain from a respiratory specimen.

[Nocardia aroensis and lung infection: first case report in Africa ]. / Infection pulmonaire à Nocardia aroensis: première observation en Afrique.

Nocardiosis is a rare disease that is difficult to diagnose. Pulmonary forms are most common in association with a variety of nonspecific symptoms. Up to now isolation of the offending species, i.e., Nocardia aroensis, has been reported only once during the first description in Japan. The purpose of this article is to report the second world case of isolation of the Nocardia aroensis in a 50-year-old immunocompetent African woman.

Antibiotic susceptibility testing and species identification of Nocardia isolates: a retrospective analysis of data from a French expert laboratory, 2010-2015.

OBJECTIVES: Nocardia, a Gram-positive bacterium, is responsible for rare and severe infections. Accurate microbiological data are essential to guide antibiotic treatment. Our primary objective was to describe species identification and results of antimicrobial susceptibility testing (AST) for Nocardia isolates analysed over a 6-year period. Secondary objectives were to study temporal trends in species distribution and AST results. METHODS: We retrospectively analysed results from Nocardia isolates sent between January 2010 and December 2015 to a French laboratory dedicated to Nocardia (Observatoire Français des Nocardioses). Species identification was obtained by amplification and sequencing of a 600-bp fragment of the 16S rRNA gene (for all isolates) and of hsp65 (when required). AST was performed using disk diffusion. RESULTS: We included 793 Nocardia isolates, mostly from the lungs (53.8%). The most frequent species were Nocardia farcinica (20.2%), Nocardia abscessus complex (19.9%) and Nocardia nova complex (19.5%). The proportion of N. farcinica increased significantly over time from 13% in 2010 to 27.6% in 2014. Linezolid, amikacin, trimethoprim-sulfamethoxazole, minocycline and imipenem were the most frequently identified active antibiotics with, respectively, 0% (0/734), 2.9% (21/730), 5.4% (40/734), 9.4% (69/734) and 19.5% (143/732) of isolates not susceptible. Nocardia farcinica was frequently not susceptible to cefotaxime (118/148, 79.7% of the isolates), but only about 5% of Nocardia cyriacigeorgica and N. abscessus complex isolates were not susceptible to cefotaxime. CONCLUSIONS: In this first epidemiological study of Nocardia isolated from human samples in France, N. farcinica was the species most frequently identified and its prevalence increased over time.

[Hemophagocytic lymphohistiocytosis with granulomatosis and diffuse T-cell infiltration associated with disseminated Nocardiosis and pulmonary infection due to Streptomyces spp]. / Syndrome d'activation macrophagique associé à une granulomatose et une infiltration lymphocytaire T CD4 diffuse révélant une nocardiose disséminée et une infection pulmonaire à Streptomyces spp.

INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome frequently secondary to infectious disease, especially in immuno-compromised patients. We report a HLH secondary to disseminated nocardiosis and Streptomyces spp pulmonary infection. CASE REPORT: A 69-years-old women had recent subcutaneous nodules of the forearms and loins associated with peripheral neuropathy and pulmonary nodule of the right upper lobe. Cutaneous biopsy revealed granuloma. Cutaneous lesions worsened and the patient developed a HLH with probable cardiac and neurological involvement, associated with cutaneous granulomatosis and diffuse polyclonal lymphocyte proliferation. Nocardia PCR was positive in cutaneous biopsy. Pulmonary samples revealed Streptomyces in culture and Nocardia in PCR. The evolution under antibiotic treatment was favorable. CONCLUSION: Recent diagnosis of HLH without obvious etiology should lead to etiological investigation, including the search for infections with slow-growing bacteria such as Nocardia or Streptomyces spp.

[Disseminated nocardiosis in a child with acute lymphoblastic leukemia]. / Nocardiose viscérale disséminée chez un enfant atteint de leucémie aiguë lymphoblastique.

Nocardiosis is a rare infectious disease in children. We report here a disseminated nocardiosis in a child with acute lymphoblastic leukemia. The patient presented prolonged febrile neutropenia and nodular pneumopathy. Based on the amplification of a 16S rDNA, a PCR assay detected Nocardia sp. in the patient's bronchoalveolar lavage (BAL) fluid. Culture of BAL samples yielded Nocardia nova colonies after 2 weeks of incubation. Hepatic, splenic, renal and cerebral localisations were detected on extension checkup. trimethoprime-sulfamethoxazole and amikacine were started given the results of PCR assay, with a good response. Improvement of the patient's general condition led to complete chemotherapy under ciprofloxacine and ceftriaxone treatment, without nocardiosis reactivation. Nocardiosis is a rare complication in children with acute lymphoblastic leukemia. trimethoprime-sulfamethoxazole prophylaxis is widely used to prevent Pneumocystis jiroveci infection in children with haematologic malignancies. As Nocardia species are usually sensible, trimethoprime-sulfamethoxazole could play a role in Nocardia prophylaxis in such population. In our patient, compliance with trimethoprime-sulfamethoxazole had been low. Nocardia species are relatively fastidious growth bacteria and are difficult to isolate with classical bacteriological techniques. Molecular methods are now available, with a good sensitivity and fast results allowing to start an appropriate antibiotherapy before culture results, as early treatment is a major prognosis factor in nocardiosis. Nocardia infection should be suspected in case of nodular pneumopathy in immunocompromised children. An extension checkup should be performed to detect secondary localisations.

High Intraspecific Genetic Diversity of Nocardia brasiliensis, a Pathogen Responsible for Cutaneous Nocardiosis Found in France: Phylogenetic Relationships by Using sod and hsp65 Genes.

This study aims at genetic characterization and phylogenetic relationships of Nocardia brasiliensis focusing by using housekeeping rrs, hsp65, and sodA genes. N. brasiliensis is the species responsible for 80% of cases of actinomycetoma, one form of cutaneous nocardiosis which occurs mainly in tropical regions reaching immunocompetent patients in which the disease can lead to amputation. We analyze 36 indigenous cases of N. brasiliensis that happened in France. Phylogenetic analysis targeting rrs gene showed no robustness at phylogenetic nodes level. However, the use of a concatenation of hsp65 and sodA genes showed that the tested strains surprisingly ranked in 3 well-defined genotypes. Genotypes 2 and 3 were phylogenetically closer to each other and both diverged from genotype 1 sustained by a high bootstrap of 81%. This last genotype hosts all the cases of pulmonary forms (3), the sole cerebral form, and almost all the cases of immunocompromised patients (3 out of 4). Moreover, excepting one of them, all the strains belonging to this group present a susceptibility to imipenem which is not the case in the other genotypes that rarely count among them strains being susceptible to this drug. The haplotype diversity (Hd) of hsp65 (0.927) and sodA (0.885) genes was higher than that of rrs (0.824). For this gene, we obtained 16 polymorphic sites whereas, for hsp65 and sodA genes, up to 27 and 29 were identified, respectively. This study reveals that these two genes have an important genetic discriminatory power for the evaluation of the intraspecies genetic variability of N. brasiliensis and they may be useful for identification purposes at species level. This study also reveals the possible existence of a new species harbored by genotype 1.

[Cerebral and pulmonary nocardiosis to Nocardia abscessus in an immunocompetent Algerian patient]. / Nocardiose cérébrale et pulmonaire à Nocardia abscessus chez un patient algérien immunocompétent.

Nocardial brain abscess is often occurring in immunocompromised patients. It is uncommon in immunocompetent individuals. Here, the authors describe a case of cerebral and pulmonary nocardiosis mimicking a metastatic tumor in an apparently health 40-year-old Algerian male. The patient presented multiple brain abscess revealed by inaugural epileptic seizure. He was afebrile and presented with left hemiparesis. Staging imaging showed a nodular lung lesion in the apical segment of the right lower lobe. The patient underwent double craniotomy for resection of the lesion. Culture of the resected specimen isolated Nocardia abscessus. The patient was initially started on intravenous trimethoprim-sulfamethoxazole and intravenous amikacine. He was switched to oral trimethoprim-sulfamethoxazole. He finished seven months of antibiotic therapy with a good clinical response. Imaging revealed reduction in the brain abscess and a complete resolution of the lung lesion. Cotrimoxazole was stopped after twelve months of therapy. After two years, the health status of our patient improves day after day. He is however regularly under medical supervision for control exams.

Molecular identification of Nocardia species using the sodA gene: Identificación molecular de especies de Nocardia utilizando el gen sodA.

Currently for bacterial identification and classification the rrs gene encoding 16S rRNA is used as a reference method for the analysis of strains of the genus Nocardia. However, it does not have enough polymorphism to differentiate them at the species level. This fact makes it necessary to search for molecular targets that can provide better identification. The sodA gene (encoding the enzyme superoxide dismutase) has had good results in identifying species of other Actinomycetes. In this study the sodA gene is proposed for the identification and differentiation at the species level of the genus Nocardia. We used 41 type species of various collections; a 386 bp fragment of the sodA gene was amplified and sequenced, and a phylogenetic analysis was performed comparing the genes rrs (1171 bp), hsp65 (401 bp), secA1 (494 bp), gyrB (1195 bp) and rpoB (401 bp). The sequences were aligned using the Clustal X program. Evolutionary trees according to the neighbour-joining method were created with the programs Phylo_win and MEGA 6. The specific variability of the sodA genus of the genus Nocardia was analysed. A high phylogenetic resolution, significant genetic variability, and specificity and reliability were observed for the differentiation of the isolates at the species level. The polymorphism observed in the sodA gene sequence contains variable regions that allow the discrimination of closely related Nocardia species. The clear specificity, despite its small size, proves to be of great advantage for use in taxonomic studies and clinical diagnosis of the genus Nocardia.

Actualmente, para la identificación y clasificación bacteriana se utiliza como método de referencia la secuenciación el gen rrs que codifica al rRNA16S, en el caso del análisis de cepas del género Nocardia, sin embargo, no tiene el suficiente polimorfismo para diferenciarlas a nivel de especie lo que hace necesaria la búsqueda de blancos moleculares que puedan proporcionar una mejor identificación. El gen sodA (que codifica la enzima superóxido dismutasa) ha tenido buenos resultados en la identificación de especies de otros Actinomicetos. En este estudio se propone para la identificación y diferenciación a nivel de especie del género Nocardia. Se utilizaron 41 especies Tipo de diversas colecciones, se amplificó y secuenció un fragmento de 386 pb del gen sodA y se realizó un análisis filogenético comparando los genes rrs (1171 pb) hsp65(401pb) secA1 (494pb), gyrB (1195pb) y rpoB (401pb), las secuencias fueron alineadas utilizando el programa Clustal X, los árboles evolutivos de acuerdo con el método de "Neighbor-Joining"se hicieron con el programa Phylo_win y Mega 6. Se analizó la variabilidad específica del gen sodA del género Nocardia presentando una alta resolución filogenética, una variabilidad genética importante, especificidad y confiabilidad para la diferenciación de los aislados a nivel de especie. El polimorfismo observado en la secuencia del gen sodA contiene regiones variables que posibilitan la discriminación de especies de Nocardia estrechamente relacionadas, y una clara especificidad, a pesar de su pequeño tamaño, demostrando ser de gran ventaja para utilizarse en estudios taxonómicos y en el diagnóstico clínico del género Nocardia.

Phenotypic and genotypic evaluation of 18 Nocardia isolates from human clinical samples in Mexico.

BACKGROUND: Mexico has the largest number of clinical cases of actinomycetoma in North and South America. Species originally identified by less specific methods have been recently reclassified as other known species or as new species. OBJECTIVE: To assess, by 16S rRNA gene sequencing and phenotypic methods, the species distribution of 18 human clinical isolates originally identified as N. brasiliensis, some of them isolated between 1947 and 1959 in Mexico City. METHODS: Clinical isolates came from the Hospital General, "Dr. Manuel Gea Gonzalez", and Instituto Nacional de Diagnóstico y Referencia Epidemiológica (INDRE) in Mexico, D.F. The strains used in this study included 15 clinical strains isolated between 1947 and 1959 that were originally identified as N. brasiliensis and three more strains obtained in 2007 identified as Nocardia spp. The isolates were identified genotypically by sequencing the 16S rRNA gene, and their phenotypic profiles were obtained with the API Coryne(®) system. Antibiotic susceptibility patterns were tested according to the protocol of the Comité de l'antibiogramme de la Société française de microbiologie[4]. RESULTS: According to 16S rRNA gene, sequencing were identified among 18 human clinical isolates as Nocardia farcinica (n=11) and Nocardia brasiliensis (n=7). A high number of the strains were susceptible to the majority of the antibiotics tested. The phenotypic profiles of the strains were quite uniform for N. farcinica and some variability was observed for N. brasiliensis strains. CONCLUSION: N. farcinica was the most prevalent species identified. Modern methodologies should be applied in clinical laboratories to accurately identify etiological agents.

Cavitary pneumonia caused by nocardia otitidiscaviarum

We report a case of cavitary pneumonia caused by N. otitidiscaviarum in a man with diabetes mellitus and thrombocytopenia treated with systemic corticosteroid. Taxonomic identification involved phenotypic testing and molecular identification that was carried out by DNA sequencing of the 16SrRNA gene.