Association of Xpert MTB/RIF Cycle Threshold Values with Tuberculosis Treatment Outcomes.

PURPOSE: Considering the current recommendation of the World Health Organization to replace sputum smear microscopy with Xpert MTB/RIF as an initial diagnostic test for tuberculosis (TB), and that culture takes time to provide results, the cycle threshold (CT) of the Xpert test may be the only way to assess bacillary load. The objective of this study is to evaluate the association of bacillary load, measured by the Xpert CT, with the TB treatment outcomes. METHODS: In cohort study, Xpert CT values were evaluated in cured and non-cured (failure and death) patients. Multivariate analysis was performed to evaluate if CT is independently associated with TB treatment outcomes. RESULTS: During this study period, 155 patients (84 cured and 71 non-cured) met the inclusion and were included in the analysis. The mean CT value for Xpert MTB/RIF test was 20.7 ± 5.6 in cured patients and 17.1 ± 5.6 in non-cured patients (p < 0.0001). Previous TB was more frequent in non-cured (28.2%) than in cured patients (7.1%) (p < 0.0001). Non-cured patients were younger than cured ones (37.1 ± 13.3 vs 43.6 ± 16.2; p = 0.006). HIV was more frequent in non-cured (28.2%) than in cured patients (15.5%), although this difference was not statistically significant (p = 0.054). In multivariate analysis, CT values, age, previous TB, and HIV were independently associated with non-cure. CONCLUSIONS: Lower Xpert MTB/RIF CT values were independently associated with worse treatment outcomes. The information from even a single test performed before starting treatment proved to be a relatively good predictor of TB treatment outcome.

Staphylococcus hominis subsp. novobiosepticus strains causing nosocomial bloodstream infection in Brazil.

OBJECTIVES: To report the isolation of six Staphylococcus hominis subsp. novobiosepticus (SHN) strains from hospitalized patients with bloodstream infections in two Brazilian hospitals and to characterize their susceptibility profile to several antimicrobials. METHODS: Species identification was performed by biochemical methods and sodA gene sequencing. The MICs of antimicrobials were determined by broth and agar dilution methods and by Etest. Isolates were typed by PFGE and PCR amplification was used to detect the ccr gene complex and the mec class. Morphometric evaluation of cell wall was performed by transmission electron microscopy (TEM). RESULTS: Susceptibility profiles indicated that the majority of isolates (five) were multidrug-resistant. Overlapping and multiplex PCR showed that five out of the six strains harboured SCCmec type III with class A mec and type 3 ccr. The initial vancomycin MIC value of 4 mg/L for these strains increased to 16-32 mg/L after growth for 10 days in BHI broth supplemented with this antimicrobial. TEM indicated that vancomycin resistance was associated with cell wall thickening and to another mechanism not fully elucidated. Only one SHN strain was oxacillin- and vancomycin-susceptible. The nosocomial infections in at least five of the patients from both hospitals were caused by a single clone of SHN. CONCLUSIONS: It is very important to consider SHN strains as the cause of nosocomial infections. The clinical implications resulting from the pattern of multidrug resistance in these strains may be complicated by the emergence of vancomycin resistance.

Feasible identification of Staphylococcus epidermidis using desferrioxamine and fosfomycin disks.

Coagulase-negative Staphylococcus spp. (CoNS) have emerged as predominant pathogens in hospital-acquired infections, as well as reservoirs of antimicrobial resistance, increasing the necessity of developing reliable methods for identification of the most frequent species. The aim of this study was to propose a simplified method for identification of Staphylococcus epidermidis. A total of 490 isolates of CoNS were identified by Bannerman's method. Taking into account distinct approaches for identification of S. epidermidis, among CoNS, we proposed the use of only two disks: desferrioxamine for the initial trial, and fosfomycin to match the final identification. Of the 320 isolates susceptible to desferrioxamine, Bannerman's method identified 238 S. epidermidis and 73 S. hominis, while we achieved identification of 239 S. epidermidis and 76 S. hominis. Compared to Bannerman's method, the method proposed here obtained a sensitivity of 99.5%, and had a positive predictor value of 99.2%. We also used a genotypic method for identification of S. epidermidis by polymerase chain reaction (PCR) targeting the tuf gene. In conclusion, the method proposed here has proved to be useful for the identification of S. epidermidis, the most frequent species of CoNS isolated from blood cultures in clinical microbiology laboratories.

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.

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.

[Bacteremia due to Rhodococcus equi in a patient with acquired immunodeficiency syndrome: case report]. / Bacteremia por Rhodococcus equi em paciente com síndrome da imunodeficiência adquirida: relato de caso.

Rhodococcus equi is an important agent for zoonotic infections, and may cause serious infections in humans, especially immunocompromised patients. In this study, a case of fatal bacteremia due to Rhodococcus equi in a patient with acquired immunodeficiency syndrome (HIV positive) is reported.

[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.

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.

Rapid detection of Van genes in rectal swabs by real time PCR in Southern Brazil.

INTRODUCTION: Laboratory-based surveillance is an important component in the control of vancomycin resistant enterococci (VRE). METHODS: The study aimed to evaluate real-time polymerase chain reaction (RT-PCR) (genes vanA-vanB) for VRE detection on 115 swabs from patients included in a surveillance program. RESULTS: Sensitivity of RT-PCR was similar to primary culture (75% and 79.5%, respectively) when compared to broth enriched culture, whereas specificity was 83.1%. CONCLUSIONS: RT-PCR provides same day results, however it showed low sensitivity for VRE detection.

Evaluation of the automated system Vitek2 for identification and antimicrobial susceptibility testing of Brazilian Gram-positive cocci strains.

Automated instruments offer many advantages for clinical laboratories. Nevertheless, they can have problems identifying and determining susceptibilities of some pathogens. Vitek2 (bioMérieux) is an automated system that was recently introduced to Brazil. We evaluated the performance of this equipment for Brazilian isolates that had been characterized using reference identification and antimicrobial susceptibility testing methods. Ninety-nine strains of Gram-positive cocci from a local reference center collection were analyzed, consisting of 50 coagulase-negative Staphylococcus (CoNS) and 49 Enterococcus and related species. Vitek2 correctly identified 79.8% (79/99) of the isolates. Oxacillin resistance was detected in 76% (19/25) of resistant S. epidermidis strains and in 88% (22/25) of other resistant CoNS species strains. Vancomycin resistance was detected in 100% (20/20) of resistant Enterococcus and related species strains. Vitek2 performed very well for the identification of S. epidermidis and non-epidermidis staphylococci, and for the detection of vancomycin resistance in Enterococcus and related species. However, the system needs improvement in order to provide reliable results for the characterization of some CoNS species, identification of Enterococcus and related species and for detecting oxacillin resistance in CoNS.

Identification and detection of methicillin resistance in non-epidermidis coagulase-negative staphylococci.

The NCCLS (2004) presented a new methodology to detect, by disk-diffusion agar, oxacillin-resistance using a cefoxitin disk. We identified coagulase-negative staphylococci (SCoN) to the species level and compared the use of cefoxitin disks (30 microg) with oxacillin disks (1 microg), agar dilution (minimum inhibitory concentration of oxacillin) and mecA gene detection in isolates of coagulase-negative bacteria other than Staphylococcus epidermidis (SCoNne). A total of 238 SCoNne was evaluated; oxacillin-resistance (the mecA gene) was detected in 71% of the isolates. All methods gave 100% sensitivity, based on presence of the mecA gene. The specificity of the cefoxitin disk was 100%, while the oxacillin disk gave a specificity of 91% and agar dilution oxacillin gave a specificity of 88%. We conclude that the cefoxitin disk is an efficient test, and it is an easy method for use in clinical laboratories to detect oxacillin-resistance in staphylococci.

Evaluation of oxacillin and cefoxitin disks for detection of resistance in coagulase negative staphylococci.

Coagulase-negative Staphylococcus spp. was considered nonpathogenic until the emergence of multiresistance and the demonstration of their participation as infectious agents. In Brazil, oxacillin resistance may be present in over 80% of isolates, and the Clinical and Laboratory Standards Institute standardized a disk-diffusion method to predict this resistance in Staphylococcus. The aim of this study was to evaluate the variability among commercial disks of oxacillin (1 microg) and cefoxitin (30 microg) widely used in clinical laboratories of microbiology, compared with mecA gene and minimum inhibitory concentration (MIC) of oxacillin. The use of oxacillin and cefoxitin disks simultaneously allowed the detection of important differences, particularly, in less frequent species such as S. cohnii, S. haemolyticus, S. saprophyticus, and S. sciuri. Disks of cefoxitin of the brand 2 displayed good correlation with the mecA gene (98.7%) and oxacillin MIC (97.8%), while major discrepancies were observed using disks of brand 1. One of the critical points in the diffusion disk test is the quality of the disks: the use of better quality disks associated with molecular methods lead to better results to define the best antibiotic therapy.

Rapid detection of Van genes in rectal swabs by real time PCR in Southern Brazil / Rápida detecção de genes Van em swabs retais por PCR real-time na região sul do Brasil

INTRODUCTION: Laboratory-based surveillance is an important component in the control of vancomycin resistant enterococci (VRE). METHODS: The study aimed to evaluate real-time polymerase chain reaction (RT-PCR) (genes vanA-vanB) for VRE detection on 115 swabs from patients included in a surveillance program. RESULTS: Sensitivity of RT-PCR was similar to primary culture (75 percent and 79.5 percent, respectively) when compared to broth enriched culture, whereas specificity was 83.1 percent. CONCLUSIONS: RT-PCR provides same day results, however it showed low sensitivity for VRE detection.

INTRODUÇÃO: Vigilância com base em detecção laboratorial é um componente importante no controle de enterococos resistentes a vancomicina (ERV). MÉTODOS: Avaliamos procedimento da reação em cadeia da polimerase real time (PCR-RT) (genes vanA-vanB) para detecção de ERV em 115 swabs de pacientes incluídos em um programa de vigilância. RESULTADOS: A sensibilidade do RT-PCR foi semelhante a da cultura primária (75 por cento e 79,5 por cento, respectivamente) quando comparada com a cultura em caldo enriquecido, enquanto a especificidade foi de 83,1 por cento. CONCLUSÕES: O RT-PCR fornece resultados no mesmo dia, contudo mostra baixa sensibilidade para a detecção de VRE.

Evaluation of the automated system Vitek2 for identification and antimicrobial susceptibility testing of Brazilian Gram-positive cocci strains

Automated instruments offer many advantages for clinical laboratories. Nevertheless, they can have problems identifying and determining susceptibilities of some pathogens. Vitek® 2 (bioMérieux) is an automated system that was recently introduced to Brazil. We evaluated the performance of this equipment for Brazilian isolates that had been characterized using reference identification and antimicrobial susceptibility testing methods. Ninety-nine strains of Gram-positive cocci from a local reference center collection were analyzed, consisting of 50 coagulasenegative Staphylococcus (CoNS) and 49 Enterococcus and related species. Vitek® 2 correctly identified 79.8 percent (79/99) of the isolates. Oxacillin resistance was detected in 76 percent (19/25) of resistant S. epidermidis strains and in 88 percent (22/25) of other resistant CoNS species strains. Vancomycin resistance was detected in 100 percent (20/20) of resistant Enterococcus and related species strains. Vitek® 2 performed very well for the identification of S. epidermidis and non-epidermidis staphylococci, and for the detection of vancomycin resistance in Enterococcus and related species. However, the system needs improvement in order to provide reliable results for the characterization of some CoNS species, identification of Enterococcus and related species and for detecting oxacillin resistance in CoNS.

Identification and detection of methicillin resistance in Non-Epidermidis coagulase-negative staphylococci

The NCCLS (2004) presented a new methodology to detect, by disk-diffusion agar, oxacillin-resistance using a cefoxitin disk. We identified coagulase-negative staphylococci (SCoN) to the species level and compared the use of cefoxitin disks (30 µg) with oxacillin disks (1 µg), agar dilution (minimum inhibitory concentration of oxacillin) and mecA gene detection in isolates of coagulase-negative bacteria other than Staphylococcus epidermidis (SCoNne). A total of 238 SCoNne was evaluated; oxacillin-resistance (the mecA gene) was detected in 71 percent of the isolates. All methods gave 100 percent sensitivity, based on presence of the mecA gene. The specificity of the cefoxitin disk was 100 percent, while the oxacillin disk gave a specificity of 91 percent and agar dilution oxacillin gave a specificity of 88 percent. We conclude that the cefoxitin disk is an efficient test, and it is an easy method for use in clinical laboratories to detect oxacillin-resistance in staphylococci.

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.

Evaluation of oxacillin and cefoxitin disks for detection of resistance in coagulase negative staphylococci

Coagulase-negative Staphylococcus spp. was considered nonpathogenic until the emergence of multiresistance and the demonstration of their participation as infectious agents. In Brazil, oxacillin resistance may be present in over 80 percent of isolates, and the Clinical and Laboratory Standards Institute standardized a disk-diffusion method to predict this resistance in Staphylococcus. The aim of this study was to evaluate the variability among commercial disks of oxacillin (1 mug) and cefoxitin (30 mug) widely used in clinical laboratories of microbiology, compared with mecA gene and minimum inhibitory concentration (MIC) of oxacillin. The use of oxacillin and cefoxitin disks simultaneously allowed the detection of important differences, particularly, in less frequent species such as S. cohnii, S. haemolyticus, S. saprophyticus, and S. sciuri. Disks of cefoxitin of the brand 2 displayed good correlation with the mecA gene (98.7 percent) and oxacillin MIC (97.8 percent), while major discrepancies were observed using disks of brand 1. One of the critical points in the diffusion disk test is the quality of the disks: the use of better quality disks associated with molecular methods lead to better results to define the best antibiotic therapy.

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.