Evaluation of the ceftazidime/avibactam and meropenem susceptibility by MALDI-TOF MS directly from positive blood cultures.

The purpose of this study was to evaluate the MBT-ASTRA to determine susceptibility to ceftazidime/avibactam (CZA) and meropenem (MEM) of Enterobacterales directly from positive blood cultures (BC). Bacterial suspension was incubated with antibiotic and analyzed by MALDI-TOF MS. The relative growth was calculated and cutoff values were determined to categorize isolates as "S," "I," and "R." Klebsiella spp. with CZA 20/8 mg/L and 1.5-h incubation presented 1 (5.9%) major discrepancy and 96.3% category agreement; other species required 2.5 h for 100% category agreement. For MEM, 4 mg/L and 1.5h were necessary, demonstrating 2 (6.67%) minor discrepancies and 93.3% categorical agreement.

Performance of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Targeting the RNA Polymerase Gene for the Direct Detection of SARS-CoV2 in Nasopharyngeal Swabs.

In 2020, a global pandemic caused by SARS-CoV-2 was declared. Different institutes proposed diagnostic molecular methods to detect the virus in clinical samples. This study aims to validate and standardize the use of a loop-mediated isothermal amplification (LAMP)-based methodology targeting the viral RP gene, as a faster and low-cost diagnostic method for SARS-CoV-2 infections. The results obtained with RT-LAMP (Reverse Transcriptase) were compared to the results of real-time polymerase chain reaction (RT-PCR) to assess its sensitivity and specificity. In total, 115 samples (nasopharyngeal samples) were used for detecting SARS-CoV-2 by RT-LAMP, with 43 positives and 72 negatives. The study showed a positive predictive value (PPV) of 90.7% and a negative predictive value (VPN) of 100%. The LAMP assay also demonstrated a high sensitivity of 90.7% and a specificity of 100% (confidence interval 77.9-97.4%) when using the lower detection limit of 40 copies/µL. The RT-LAMP described here has the potential to detect even the new variants of SARS-CoV-2, suggesting that it may not be significantly affected by gene mutations. The RT-LAMP targeting the RP viral region is faster and less expensive than other molecular approaches, making it an alternative for developing countries.

Evaluation of Aztreonam and Ceftazidime/Avibactam Synergism against Klebsiella pneumoniae by MALDI-TOF MS.

INTRODUCTION: Resistance to carbapenems due to the co-production of NDM and ESBL or NDM and KPC is increasing. Therefore, combined therapy with aztreonam (ATM) plus ceftazidime/avibactam (CZA) has been recommended. Then, it is necessary to develop and evaluate fast and simple methods to determine synergism in vitro in microbiology laboratories. OBJECTIVE: To develop a method to determine the synergism of ATM and CZA by MALDI-TOF MS (SynMALDI). METHOD: Klebsiella pneumoniae (n = 22) isolates with blaNDM and/or blaKPC genes were tested. The time-kill curve assay was performed for four isolates (three positives for blaNDM and blaKPC and one positive for blaNDM only). For SynMALDI, each isolate was incubated for 3 h in 4 tubes containing brain-heart infusion broth with the following: (1) no antibiotic; (2) ATM at 64 mg/L; (3) CZA at 10/4 mg/L; and (4) ATM at 64 mg/L plus CZA at 10/4 mg/L. After incubation, the bacterial protein extract was analyzed by MALDI-TOF MS, and the relative growth (RG) was determined for each isolate, considering intensities of the peaks of the bacterium incubated with antibiotic (tubes 2, 3, and 4) to the same bacterium incubated without antibiotic (tube 1), as follows: RG = IntensityWith antibiotic/IntensityWithout antibiotic. The combination was determined as synergistic when there was an RG decrease of 0.3 in the antibiotic combination in relation to the RG of the most active antibiotic alone. RESULTS: The combination of ATM plus CZA proved to be synergic by time-kill curve assay. All isolates tested with the SynMALDI method also presented synergism. CONCLUSIONS: Detection of synergism for ATM plus CZA combination can be determined by MALDI-TOF MS, providing fast results in order to improve patient treatment.

A Rapid and Easy Method of MALDI Biotyper Antibiotic Susceptibility Test Rapid Assay To Provide Early Meropenem Susceptibility Profile in Enterobacterales.

Antimicrobial susceptibility testing (AST) that can provide faster results is necessary. The MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) can provide early AST results but still needs to be simplified in order to facilitate its execution by microbiology laboratories. The aim of this study was to evaluate an adaptation of MBT-ASTRA. Isolates of Enterobacterales were tested for meropenem susceptibility by MBT-ASTRA using a solution prepared from meropenem disks and performing a manual spectrum analysis. The relative growth (RG) was calculated for each isolate, and a cutoff value was established to determine the susceptibility profile of the isolates. Results of the adapted method were compared with the standard susceptibility method (broth microdilution). An adapted method of MBT-ASTRA was developed. The RG cutoff values for meropenem were ≤0.1510 for susceptibility and >0.6272 for resistance, presenting 95.65% categorical agreement, with 2.9% (2/69) minor discrepancy and 3.23% (1/31) very major discrepancy. MBT-ASTRA can be used to provide rapid AST results with a simpler and more accessible protocol, especially regarding spectrum analysis. IMPORTANCE The simplification of the MBT-ASTRA technique, especially in spectrum analysis, can considerably allow more laboratories to rapidly determine antimicrobial susceptibility profiles.

Rapid detection of the main carbapenemases in Brazil directly from spiked blood culture using the RESIST-3 O.K.N. immunoassay.

The emergence of carbapenem-resistant Enterobacterales (CRE) is a matter of public health concern. Carbapenemases are the main mechanism of resistance among CRE, and its rapid detection is essential. The detection of carbapenemases usually requires culture-based methods and molecular assays, which may be costly and need long turnaround times. Recently, an easy and rapid immunochromatographic assay for carbapenemases (OXA-48, KPC, and NDM) detection based in lateral flow immunoassay with specific monoclonal antibodies on a nitrocellulose membrane has been developed. We aimed to evaluate the RESIST-3 O.K.N. in colonies from pure culture as well as in spiked blood cultures with Enterobacterales. All carbapenemase producers (CP) presenting the OXA-48-like, KPC, and NDM enzymes presented positive results in both pure colonies and spiked blood cultures. None of the carbapenemase non-producers (CNP) presented positive results in the tests. A total of 97% CP isolates presented positive results in pure colonies in less than 5 min. For CP directly from blood culture, the mean time to positivity for OXA-48-like and KPC was 1 min, whereas it was 25 min for NDM. Our results indicate that this immunoassay can be used to detect carbapenemases directly from blood culture bottles in a routine diagnostic laboratory, which would reduce the turnaround time of CP detection.

Cutaneous infection caused by Corynebacterium pseudodiphtheriticum: a microbiological report.

We report here a rare case of cutaneous infection due to Corynebacterium pseudodiphtheriticum. The patient presented to the clinical laboratory with a skin ulcer on his left leg. Gram-stained preparation of the purulent secretion revealed the presence of numerous rod-shaped Gram-positive organisms in the absence of any other species. The organism was grown in pure culture on sheep blood agar and was further identified as C. pseudodiphtheriticum using a commercial identification system (API-Coryne, BioMérieux, France). The infection was successfully treated with ciprofloxacin. This case emphasizes the importance of the clinical microbiology laboratory in correctly identifying Gram-positive organisms obtained in pure culture from skin ulcers.

Cutaneous infection caused by Corynebacterium pseudodiphtheriticum: a microbiological report / Infecção cutânea causada por Corynebacterium pseudodiphtheriticum: relato microbiológico

We report here a rare case of cutaneous infection due to Corynebacterium pseudodiphtheriticum. The patient presented to the clinical laboratory with a skin ulcer on his left leg. Gram-stained preparation of the purulent secretion revealed the presence of numerous rod-shaped Gram-positive organisms in the absence of any other species. The organism was grown in pure culture on sheep blood agar and was further identified as C. pseudodiphtheriticum using a commercial identification system (API-Coryne, BioMérieux, France). The infection was successfully treated with ciprofloxacin. This case emphasizes the importance of the clinical microbiology laboratory in correctly identifying Gram-positive organisms obtained in pure culture from skin ulcers.

Reportamos o isolamento de Corynebacterium pseudodiphtheriticum de um caso de infecção cutânea. O paciente apresentou-se ao laboratório clínico com uma úlcera na perna esquerda. A coloração de Gram do material revelou a presença de bacilos Gram-positivos e ausência de outras espécies bacterianas. O organismo foi isolado em cultura pura no ágar sangue de carneiro e foi identificado como C. pseudodiphtheriticum através de um sistema de identificação comercial (API-Coryne, BioMérieux, França). A infecção foi tratada com sucesso através do uso de ciprofloxacina. Este caso reforça a importância do laboratório de microbiologia clínica na identificação de organismos Gram-positivos isolados de cultura pura de amostras de úlceras cutâneas.

Utility of the ceftazidime-imipenem antagonism test (CIAT) to detect and confirm the presence of inducible AmpC beta-lactamases among enterobacteriaceae.

Detection of AmpC beta-lactamase production by enterobacteria has been problematic. Contrary to ESBLs, no specific guidelines are available for detection and confirmation of AmpC production by clinical relevant microorganisms. Moreover, some bacterial species may produce inducible AmpC beta-lactamases that can be easily overlooked by routine susceptibility tests. We reported here a new test based on the strong inducible effect of imipenem on AmpC genes and the consequent antagonism with ceftazidime. This test is very simple and proved to be helpful in detecting AmpC-inducible enzymes among several species of clinical isolates.

Utility of the ceftazidime-imipenem antagonism test (CIAT) to detect and confirm the presence of inducible AmpC beta-lactamases among enterobacteriaceae

Detection of AmpC beta-lactamase production by enterobacteria has been problematic. Contrary to ESBLs, no specific guidelines are available for detection and confirmation of AmpC production by clinical relevant microorganisms. Moreover, some bacterial species may produce inducible AmpC beta-lactamases that can be easily overlooked by routine susceptibility tests. We reported here a new test based on the strong inducible effect of imipenem on AmpC genes and the consequent antagonism with ceftazidime. This test is very simple and proved to be helpful in detecting AmpC-inducible enzymes among several species of clinical isolates.

Fatal case of bacteremia caused by an atypical strain of Corynebacterium mucifaciens

Corynebacterium species have often been considered normal skin flora or contaminants; however, in recent years they have been increasingly implicated in serious infections. Moreover, many new species have been discovered and old species renamed, especially after molecular biology techniques were introduced. Corynebacterium mucifaciens is mainly isolated from blood and from other normally-sterile body fluids; it forms slightly yellow, mucoid colonies on blood agar. We report a fatal case of bacteremia due to an atypical strain of C. mucifaciens. This strain had atypical colony morphology; analysis of the 16S rRNA gene was used to define the species.

[Diphyllobothrium latum: case report in Brazil]. / Diphyllobothrium latum: relato de caso no Brasil.

Diphyllobothriasis is caused in humans by infection with adult tapeworms of the genus Diphyllobothrium acquired by consuming raw or undercooked freshwater fish. Diphyllobothrium latum was confirmed by examination of the gravid proglottids and typical operculated eggs in the stool. The patient had a history of eating crustaceans and fish. This is the case report of the first Brazilian infected.

Diphyllobothrium latum: relato de caso no Brasil / Diphyllobothrium latum: case report in Brazil

Difilobotriose é causada em humanos pela infeccão com vermes adultos do gênero Diphyllobothrium adquiridos pelo consumo de peixe cru ou mal cozido. Diphyllobothrium latum foi confirmado pelo exame dos proglotes grávidos e típicos ovos operculados nas fezes. O paciente havia comido crustáceos e peixes. É o relato do primeiro brasileiro infectado.

Fatal case of bacteremia caused by an atypical strain of Corynebacterium mucifaciens.

Corynebacterium species have often been considered normal skin flora or contaminants; however, in recent years they have been increasingly implicated in serious infections. Moreover, many new species have been discovered and old species renamed, especially after molecular biology techniques were introduced. Corynebacterium mucifaciens is mainly isolated from blood and from other normally-sterile body fluids; it forms slightly yellow, mucoid colonies on blood agar. We report a fatal case of bacteremia due to an atypical strain of C. mucifaciens. This strain had atypical colony morphology; analysis of the 16S rRNA gene was used to define the species.

Fatal bacteremia due to Anaerobiospirillum succiniciproducens: first description in Brazil.

Anaerobiospirillum succiniciproducens is an anaerobic, Gram-negative, spiral shaped bacteria, which is motile by means of bipolar tuffs of flagella. This organism appears to be a rare cause of bacteremia in humans, and it usually affects patients submitted to immunosuppressive therapy. Anaerobiospirillum succiniciproducens resembles Campylobacter spp. in Gram-stained preparations, however, it is considered resistant to most antimicrobial drugs that are used to treat Campylobacter infections. We observed Gram-negative, spiral shaped bacteria in Gram-stained preparations from blood culture flasks. Growth occurred only under anaerobic incubation, and identification to the species level was achieved by PCR amplification of the 16S rRNA gene, followed by direct sequencing and a GenBank homology search. To the best of our knowledge, this is the first reported Brazilian case of Anaerobiospirillum succiniciproducens bacteremia.

Fatal bacteremia due to Anaerobiospirillum succiniciproducens: first description in Brazil

Anaerobiospirillum succiniciproducens is an anaerobic, Gram-negative, spiral shaped bacteria, which is motile by means of bipolar tuffs of flagella. This organism appears to be a rare cause of bacteremia in humans, and it usually affects patients submitted to immunosuppressive therapy. Anaerobiospirillum succiniciproducens resembles Campylobacter spp. in Gram-stained preparations, however, it is considered resistant to most antimicrobial drugs that are used to treat Campylobacter infections. We observed Gram-negative, spiral shaped bacteria in Gram-stained preparations from blood culture flasks. Growth occurred only under anaerobic incubation, and identification to the species level was achieved by PCR amplification of the 16S rRNA gene, followed by direct sequencing and a GenBank homology search. To the best of our knowledge, this is the first reported Brazilian case of Anaerobiospirillum succiniciproducens bacteremia.

Avaliação de um sistema automatizado na identificação de espécies de Enterococcus / Evaluation of an automated system for the identification of Enterococci

O uso de métodos automatizados tem freqüentemente levado a falhas na identificação do gênero Enterococcus em laboratórios de microbiologia clínica. Neste estudo foi avaliada a utilização de um sistema automatizado Vitek (bioMérieux) em dois laboratórios de microbiologia clínica para identificação de diferentes espécies de enterococos. Os resultados foram comparados com os testes fisiológicos convencionais. As amostras (80) foram inoculadas em testes bioquímicos convencionais e no cartão Vitek GPI. No geral, a concordância entre os dois métodos foi de 83,7% (67/80). Entre as amostras de E. faecalis, o sistema Vitek identificou corretamente 35/40 (87,5%) e entre os E. faecium, a concordância foi 12/14 (85,7%). Em 20/26 amostras (76,9%) pertencentes a espécies não-E. faecalis e não-E. faecium, o sistema chegou à identificação correta. Os resultados do presente estudo mostram que o sistema Vitek necessita de melhorias para a identificação de enterococos, especialmente diante de espécies menos freqüentes.

Automated systems may present problems in the identification of members of the genus Enterococcus in clinical laboratories. Having conventional physiological tests as the reference method, we evaluated the use of an automated system (VITEK – bioMérieux) in the identification of 80 isolates belonging to different species of Enterococcus. The general agreement between results obtained by the conventional method and by the Vitek GPI card was 83.7% (67/80). Among isolates of E. faecalis and E faecium we observed that the automated system correctly identified 35/40 (87.5%) and 12/14 (85.7%) of the strains, respectively. Among isolates belonging to species which are neither E. faecalis, nor E. faecium, it was observed an agreement of 20/26 (76.9%). Results point to the need of improvement in the automated systems to identify enterococci. Special consideration must be taken regarding less frequently isolated species.

Quality control for microbiological culture media: Is it enough to follow the NCCLS M22-A2 procedures?

O uso de protocolos de controle de qualidade com padrões rígidos devem ser utilizados para garantir o isolamento dos microrganismos. Nosso objetivo foi o de avaliar um procedimento alternativo que nos permitisse detectar falhas de crescimento bacteriano em termos quantitativos, e comparação com o protocolo estabelecido pelo documento M22-A2 do NCCLS. Haemophilus influenzae apresentou diferença significativa de crescimento entre meio de cultivo de fontes distintas. Nos concluímos que, para alguns microrganismos fastidiosos, seja feita uma verificação quantitativa da capacidade de crescimento de cada meio de cultivo.

Quality control for microbiological culture media: Is it enough to follow the NCCLS M22-A2 procedures?

Stringent quality control protocols must be used in order to guaranty that a particular medium is able to recover all sort of organism that may be present in clinical samples. Our aim was to evaluate an alternative protocol that would allow us to detect medium failure to yield quantitative growth of selected pathogens, and compare with the document M22-A2 from NCCLS. The detection limit of Haemophilus influenzae was significantly different depending on media source. We conclude that for some fastidious microorganisms, quantitative verification of the growth capacity of the culture medium is advised.

O uso de protocolos de controle de qualidade com padrões rígidos devem ser utilizados para garantir o isolamento dos microrganismos. Nosso objetivo foi o de avaliar um procedimento alternativo que nos permitisse detectar falhas de crescimento bacteriano em termos quantitativos, e comparação com o protocolo estabelecido pelo documento M22-A2 do NCCLS. Haemophilus influenzae apresentou diferença significativa de crescimento entre meio de cultivo de fontes distintas. Nos concluímos que, para alguns microrganismos fastidiosos, seja feita uma verificação quantitativa da capacidade de crescimento de cada meio de cultivo.

Quality control for microbiological culture media: Is it enough to follow the NCCLS M22-A2 procedures?

Stringent quality control protocols must be used in order to guaranty that a particular medium is able to recover all sort of organism that may be present in clinical samples. Our aim was to evaluate an alternative protocol that would allow us to detect medium failure to yield quantitative growth of selected pathogens, and compare with the document M22-A2 from NCCLS. The detection limit of Haemophilus influenzae was significantly different depending on media source. We conclude that for some fastidious microorganisms, quantitative verification of the growth capacity of the culture medium is advised.

O uso de protocolos de controle de qualidade com padrões rígidos devem ser utilizados para garantir o isolamento dos microrganismos. Nosso objetivo foi o de avaliar um procedimento alternativo que nos permitisse detectar falhas de crescimento bacteriano em termos quantitativos, e comparação com o protocolo estabelecido pelo documento M22-A2 do NCCLS. Haemophilus influenzae apresentou diferença significativa de crescimento entre meio de cultivo de fontes distintas. Nos concluímos que, para alguns microrganismos fastidiosos, seja feita uma verificação quantitativa da capacidade de crescimento de cada meio de cultivo.