Finding significant pathogens in blood cultures in children: Should we set the timer to 36 hours?

Background: Knowledge of time to positivity (TTP) for blood cultures is useful to assess timing of discontinuation of empiric antimicrobials for suspected bacteremia with no focus. Methods: An audit of positive blood cultures from the Children's Hospital of Eastern Ontario (CHEO) from November 1, 2019, to October 31, 2020, was performed to determine TTP, defined as the start of incubation to a positive signal from automated incubators. Results: Three hundred seventy-six positive blood cultures were identified from 248 patients (average age: 6.27 [SD 6.24] years). Of these, 247 isolates were speciated; 90 (36.4%) were definitive/probable (DP) pathogens (median TTP 12.75 hours) and 157 (63.6%) possible/probable (PP) contaminants (median TTP 24.08 hours). At each time point, the adjusted rate of positive blood culture was significantly higher for DP pathogens compared to PP contaminants (hazard ratio [HR] 1.80 [95% CI 1.37, 2.36]) and for children ≤27 days old compared to the oldest age group (HR 1.94 [95% CI 1.19, 3.17]). By 36 hours, the proportion of positive cultures was significantly higher in the youngest age group (≤27 days) compared with the 3-11 years old age group (91.7% [95% CI 68.6%, 97.8%] versus 58.2% [95% CI 46.91%, 68.06%]). Conclusion: Across all ages, the TTP was significantly shorter for blood cultures with DP pathogens compared to those with PP contaminants (HR 1.80 [95% CI 1.37, 2.36]). In newborns, 90% of blood cultures were positive by 36 hours supporting this re-assessment time for empiric antimicrobials. TTP was longer in children ≥12 months, possibly related to other factors such as blood culture volume.

Historique: Il est utile de connaître le délai de positivité (DdP) des hémocultures pour évaluer le moment de mettre un terme aux antimicrobiens empiriques en cas de présomption de bactériémie sans source apparente. Méthodologie: Les chercheurs ont procédé à un audit des hémocultures positives du Centre hospitalier pour enfants de l'est de l'Ontario (CHEO) entre le 1er novembre 2019 et le 31 octobre 2020 pour déterminer le DdP, défini comme la période entre le début de l'incubation et le signal positif d'incubateurs automatisés. Résultats: Les chercheurs ont extrait 376 hémocultures positives provenant de 248 patients (d'un âge moyen de 6,27 ± 6,24 ans). De ce nombre, ils ont différencié 247 isolats, dont 90 (36,4 %) étaient des agents pathogènes confirmés ou probables (CP) (DdP médian de 12,75 heures) et 157 (63,6 %), des contaminants possibles ou probables (PP) (DdP médian de 24,08 heures). À chaque point temporel, le taux corrigé d'hémocultures positives était sensiblement plus élevé à l'égard des agents pathogènes CP que des contaminants PP (rapport de risque instantanés [RRI] : 1,80 [IC à 95 % 1,37,2,36]) et des nouveau-nés de 27 jours de vie ou moins que des enfants plus âgés (RRI 1,94 [IC à 95 % 1,19,3,17]). Au bout de 36 heures, la proportion de cultures positives était sensiblement plus élevée dans le groupe le plus jeune (27 jours de vie ou moins) que dans celui des enfants de trois à 11 ans, soit de 91,7 % (IC à 95 % 68,6 %, 97,8 %) par rapport à 58,2 % (IC à 95 % 46,91 %, 68,06 %). Conclusion: À tout âge, le DdP était sensiblement plus court, à l'égard des hémocultures contenant des agents pathogènes CP que des contaminants PP (RRI 1,80 [IC à 95 % 1,37,2,36]). Chez les nouveau-nés, 90 % des hémocultures sont positives au bout de 36 heures, ce qui appuie ce moment pour réévaluer la prise d'antimicrobiens empiriques. Le DdP était plus long chez les enfants âgés de plus de 12 mois, peut-être à cause d'autres facteurs comme le volume de l'hémoculture.

Effect of delayed entry of blood culture bottles in BACTEC automated blood culture system in the context of laboratory consolidation.

Delayed entry of blood culture bottles is frequent in consolidated laboratories. A retrospective study evaluated time from insertion to detection and total detection time as a function of preincubation time, and we prospectively looked for false negative results. 69,604 blood culture bottles were reviewed for preincubation time, incubation time and total detection time. Positive cultures for specific bacterial subtypes were reviewed to assess the effect of preincubation time on likelihood of detection. 492 negative blood cultures were prospectively tested by 16S RNA PCR and Staphylococcus-specific PCR for the presence of bacterial DNA. Mean preincubation time for samples collected within the city-limits was 3.94 h versus 9.49-18.89 h for other client sites. Higher preincubation times were partially mitigated by a lower incubation time, with an overall increase in total detection time. A lower odds ratio of recovery of Staphylococcus spp was identified, but not confirmed by terminal subcultures and molecular assays. Prolonged preincubation of blood cultures affects total detection time despite a reduction in incubation time. Successful centralization of microbiological services may depend upon optimization of courier routes for inoculated blood culture bottles. Our data supports consideration for an increase in suggested maximum preincubation times.

Therapeutic Drug Monitoring of Vancomycin in Adult Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia or Pneumonia.

BACKGROUND: Vancomycin remains widely used for methicillin-resistant Staphylococcus aureus (MRSA) infections; however, treatment failure rates up to 50% have been reported. At the authors' institution, monitoring of trough concentration is the standard of care for therapeutic drug monitoring of vancomycin. New guidelines support use of the ratio of 24-hour area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) as the pharmacodynamic index most likely to predict outcomes in patients with MRSA-associated infections. OBJECTIVES: To determine the discordance rate between trough levels and AUC24/MIC values and how treatment failure and nephrotoxicity outcomes compare between those achieving and not achieving their pharmacodynamic targets. METHODS: This retrospective cohort study involved patients with MRSA bacteremia or pneumonia admitted to the study hospital between March 1, 2014, and December 31, 2018, and treated with vancomycin. Data for trough concentrations were collected, and minimum concentrations (C min) were extrapolated. The AUC24/MIC values were determined using validated population pharmacokinetic models. The C min and AUC24/MIC values were characterized as below, within, or above pharmacodynamic targets (15-20 mg/L and 400-600, respectively). Discordance was defined as any instance where a patient's paired C min and AUC24/MIC values fell in different ranges (i.e., below, within, or above) relative to the target ranges. Predictors of treatment failure and nephrotoxicity were determined using logistic regression. RESULTS: A total of 128 patients were included in the analyses. Of these, 73 (57%) received an initial vancomycin dose less than 15 mg/kg. The discordance rate between C min and AUC24/MIC values was 21% (27/128). Rates of treatment failure and nephrotoxicity were 34% (43/128) and 18% (23/128), respectively. No clinical variables were found to predict discordance. Logistic regression identified initiation of vancomycin after a positive culture result (odds ratio [OR] 4.41, 95% confidence interval [CI] 1.36-14.3) and achievement of target AUC24/MIC after 4 days (OR 3.48, 95% CI 1.39-8.70) as modifiable predictors of treatment failure. CONCLUSIONS: The relationship between vancomycin monitoring and outcome is likely confounded by inadequate empiric or initial dosing. Before any modification of practice with respect to vancomycin monitoring, empiric vancomycin dosing should be optimized.

CONTEXTE: La vancomycine reste largement utilisée contre les infections dues au Staphylococcus aureus méthicillinorésistant (SAMR); cependant, on rapporte des taux d'échec de traitement allant jusqu'à 50 %. Dans l'institution où travaillent les auteurs, la surveillance de la concentration minimale constitue la norme de soins du suivi thérapeutique pharmacologique de la vancomycine. De nouvelles lignes directrices soutiennent l'utilisation du ratio de 24 h de l'aire sous la courbe de concentration-temps à concentration minimale inhibitrice (AUC24/MIC) en tant qu'indice pharmacodynamique, vraisemblablement pour prédire certains résultats concernant les patients présentant des infections associées au SAMR. OBJECTIFS: Déterminer le taux de discordance entre la concentration minimale et les valeurs de l'AUC24/MIC et la manière dont les échecs de traitement et les résultats de néphrotoxicité se comparent entre les personnes atteignant leurs cibles pharmacodynamiques et celles qui ne l'atteignent pas. MÉTHODES: Cette étude de cohorte rétrospective impliquait des patients atteints d'une bactériémie au SAMR ou d'une pneumonie au SAMR, admis à l'hôpital où se déroulait l'étude entre le 1er mars 2014 et le 31 décembre 2018 et traités à l'aide de vancomycine. Les données relatives aux concentrations minimales ont été recueillies, et les concentrations minimales (C min) extrapolées. Les valeurs de l'AUC24/MIC ont été déterminées à l'aide de modèles de population pharmacocinétiques validés. La caractérisation des valeurs de la C min et des valeurs de l'AUC24/MIC se décrit comme suit: « en dessous ¼, « à l'intérieur ¼ ou « au-dessus ¼ des cibles pharmacodynamiques (respectivement 15­20 mg/L et 400­600). La discordance était définie comme une situation où les valeurs associées de la C min et de l'AUC24/MIC tombaient dans des plages différentes (c.-à-d., en dessous, à l'intérieur ou au-dessus) par rapport aux plages cibles. Une régression logistique a permis de déterminer les prédicteurs d'échecs de traitement et de néphrotoxicité. RÉSULTATS: Au total, 128 patients ont été inclus dans les analyses. De ceux-ci, 73 (57 %) ont reçu une dose initiale de vancomycine de moins de 15 mg/kg. Le taux de discordance entre les valeurs de la C min et de l'AUC24/MIC était de 21 % (27/128). Les taux d'échec de traitement et de néphrotoxicité se montaient respectivement à 34 % (43/128) et 18 % (23/128). Aucune variable clinique n'a pu prédire la discordance. La régression logistique a permis de déterminer le début de l'administration de la vancomycine après un résultat de culture positif (rapport de cotes [RC] 4,41, 95 % intervalle de confiance [IC] 1,36­14,3) et l'atteinte de la cible de l'AUC24/MIC après quatre jours (RC 3,48, 95 % IC 1,39­8,70) en tant que prédicteurs modifiables de l'échec du traitement. CONCLUSIONS: Il existe probablement une confusion relative à la relation entre la surveillance de la vancomycine et le résultat à cause d'un dosage empirique ou initial inadéquat. Avant de modifier la pratique relative à la surveillance de la vancomycine, le pharmacien doit optimiser son dosage empirique.

A comparison of the Quidel Solana GAS assay, the Luminex Aries Group A Strep assay and the Focus Diagnostics Simplexa Group A Strep Direct assay for detection of Group A Streptococcus in throat swab specimens.

We compared three commercially available group A Streptococcus (GAS) nucleic acid amplification tests, the Quidel Solana GAS assay, the Luminex Aries Group A Strep assay and the Focus Diagnostics Simplexa Group A Strep Direct assay, with GAS bacterial culture. A true positive result was defined as one positive by culture or positive by ≥2/3 molecular methods. Two hundred eighty-seven throat swabs (207 children, 80 adults) were collected. The sensitivity of culture was 84.8% (95% CI 77.7-90.3%) with a specificity of 100% (95% CI 97.5-100%). The Solana assay sensitivity was 94.2% (95% CI 88.9-97.5%) with a specificity of 98.7% (95% CI 95.2-99.8%). Simplexa assay sensitivity was 99.3% (95% CI 96.0-99.9%) with a specificity of 95.3% (95% CI 90.6-98.1%). Aries assay sensitivity was 96.4% (95% CI 91.8-98.8%) with a specificity of 98.0% (95% CI 94.2-99.6%). In summary, all the molecular methods evaluated showed high sensitivity and specificity and were more sensitive than culture.

A comparison of the Quidel Solana HSV 1 + 2/VZV Assay, the Focus Diagnostics Simplexa HSV 1 & 2 Direct Assay and the Luminex Aries HSV 1&2 Assay for detection of herpes simplex virus 1 and 2 from swab specimens.

BACKGROUND: Molecular methods enable more rapid and sensitive detection of herpes simplex virus (HSV) than viral culture. OBJECTIVE: Three commercial molecular methods, all of which detect both HSV-1 and HSV-2, were compared to viral culture for the detection of HSV from swab specimens. STUDY DESIGN: Pediatric and adult patient viral swab specimens were cultured for HSV. Residual swab fluid was frozen at -80 °C until tested with the 3 molecular methods: the Quidel Solana HSV 1 + 2/VZV Assay, the Focus Diagnostics Simplexa HSV 1 & 2 Direct Assay and the Luminex Aries HSV 1&2 Assay. A true positive was defined as positive by culture or positive by ≥ 2/3 molecular methods. RESULTS: 177 specimens were studied. The sensitivity of culture was 81.3% (61/75, 95% CI 70.7-89.4%) and specificity was 100% (102/102, 95% CI 96.4-100%). The sensitivities of both the Solana and Simplexa were 100% (75/75, 95% CI 95.2-100%) and specificities were also both 100% (102/102, 95% CI 96.4-100%). The Aries had a sensitivity of 98.7% (74/75, 95% CI 92.8-99.97%) and specificity 99.0% (101/102, 95% CI 94.7-99.98%). All three molecular methods were significantly more sensitive than culture (p ≤ 0.0005 for Solana and Simplexa and p ≤ 0.0012 for Aries). CONCLUSION: All the molecular methods studied provided a significantly higher sensitivity than culture. In addition, the molecular methods took 1-2 hours to perform compared to a mean of 2.1 days for culture results. Use of any of the three molecular methods could lead to improved patient care.

A comparison of the Allplex™ bacterial and viral assays to conventional methods for detection of gastroenteritis agents.

OBJECTIVE: Molecular methods to detect diarrheal pathogens are increasingly being used in place of conventional methods. We compared a new multiplex real-time PCR assay for detection of both bacterial and viral gastroenteritis agents, the Allplex™ Gastrointestinal Panel Assays (AGPA), to conventional methods (stool culture for bacterial pathogens and electron microscopy (EM) for viral pathogens). RESULTS: Gastrointestinal viruses, in particular norovirus genogroup II viruses, were detected by the AGPA in a high number of specimens that were negative by EM. For bacterial pathogens, the AGPA was able to detect the organisms grown in culture with high sensitivity and additionally detected several types of E. coli, such as enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and non-O157 Shiga toxin-producing E. coli (STEC), that could not be detected with conventional culture methods. Overall, the AGPA had a > 2-fold higher detection rate than the conventional methods, with 24/135 (17.8%) samples positive by conventional methods and 60/135 (44.4%) by AGPA. Thus, diarrhea pathogen detection rates increased substantially with the use of the AGPA as compared to conventional methods.

Inverse association of repressor growth factor independent-1 with CD8 T cell interleukin (IL)-7 receptor [alpha] expression and limited signal transducers and activators of transcription signaling in response to IL-7 among [gamma]-chain cytokines in HIV patients.

BACKGROUND: CD8 T lymphocytes from chronically infected HIV-positive patients degenerate into a preapoptotic state and exhibit impaired functionality. Particularly in viremic patients, this was associated with an increased proportion of interleukin-7 receptor-alpha low-expressing (IL-7Ralpha(low)) effector-like CD8 T cells. As cytokine signaling through signal transducers and activators of transcription (STAT) is essential for cellular function, we hypothesized that activation of this pathway may be impaired in these cells. OBJECTIVES: To evaluate cytokine-induced STAT activation in IL-7Ralpha(low) and IL-7Ralpha(high) CD8 T cells from chronically infected HIV-positive patients and investigate the potential molecular mechanism involved in the reduced IL-7Ralpha expression. METHODS: CD8 T cells from HIV-positive patients on and off antiretroviral therapy were assayed respectively for STAT activation, cytokine receptor, and transcription factor expression by flow cytometry and real-time PCR. RESULTS: IL-7 stimulation failed to activate STAT5 in a substantial proportion of patient CD8 T cells. This correlated with reduced IL-7Ralpha mRNA and surface protein expression. Interestingly, IL-7Ralpha(low) cells appeared to be fully capable of recruiting the STAT pathway in response to IL-2, IL-4, IL-10, and IL-15. mRNA expression suggested a potential role for growth factor independent (Gfi)-1 as an IL-7Ralpha transcriptional repressor, but not that of other transcriptional regulators studied, including Gfi-1B and GA-binding protein alpha. Programmed death-1 inhibitory receptor, though upregulated in CD8 T cells from HIV-positive patients, appeared unrelated to IL-7Ralpha expression and STAT signaling capacity.

Amplification of the signal transducer and activator of transcription I signaling pathway and its association with apoptosis in monocytes from HIV-infected patients.

BACKGROUND: Monocytes/macrophages play a major role in inflammation and pathogen clearance. However, chronic immune activation observed during HIV infection may also cause cellular dysfunction and tissue pathology. Indeed, several defects have been reported in these cells during HIV infections. As cytokine responsiveness via the signal transducer and activator of transcription (STAT1) signaling pathway is critical for these functions, we hypothesized that its activation in monocytes from HIV-positive patients may be disrupted. OBJECTIVES: To evaluate cytokine-dependent STAT signaling in monocytes from HIV-positive patients and study the biological impact and molecular mechanisms responsible for the alterations in the interferon (IFN)-gamma-induced STAT1 pathway observed. METHODS: Monocytes from chronically infected HIV-positive patients on and off antiretroviral therapy were assayed respectively for STAT activation, apoptosis, and other downstream effects by flow cytometry, real-time PCR and enzyme-linked immunosorbent assay. RESULTS: Unlike IFN-alpha, interleukin-10, granulocyte macrophage colony-stimulating factor, and interleukin-4, only IFN-gamma-induced STAT1 activation was upregulated in monocytes from off-therapy patients compared with those on antiretroviral therapy and HIV-negative controls, correlating with increased total STAT1 expression. Among the IFN-gamma responsive genes (IRF-1, CXCL9, CXCL10) studied, differential effects were observed, likely reflecting the more complex regulatory control over their expression. Interestingly, spontaneous monocyte apoptosis was elevated in HIV-positive patients off-therapy compared with HIV-negative controls and correlated with STAT1 expression. IFN-gamma-induced apoptosis was also increased and persisted despite seemingly effective antiretroviral therapy. CONCLUSION: Amplification of STAT1 signaling and apoptosis may reflect the chronic nature of immune activation in HIV-positive patients and contribute to the functional impairment observed in monocytes through the course of the disease.