Detection Of Pathogens In Blood Research Articles

Clinical evaluation of cell-free and cellular metagenomic next-generation sequencing of infected body fluids

IntroductionPrevious studies have evaluated metagenomic next-generation sequencing (mNGS) of cell-free DNA (cfDNA) for pathogen detection in blood and body fluid samples. However, no study has assessed the diagnostic efficacy of mNGS using cellular DNA. ObjectivesThis is the first study to systematically evaluate the efficacy of cfDNA and cellular DNA mNGS for pathogen detection. MethodsA panel of seven microorganisms was used to compare cfDNA and cellular DNA mNGS assays concerning limits of detection (LoD), linearity, robustness to interference, and precision. In total, 248 specimens were collected between December 2020 and December 2021. The medical records of all the patients were reviewed. These specimens were analysed using cfDNA and cellular DNA mNGS assays, and the mNGS results were confirmed using viral qPCR, 16S rRNA, and internal transcribed spacer (ITS) amplicon next-generation sequencing. ResultsThe LoD of cfDNA and cellular DNA mNGS was 9.3 to 149 genome equivalents (GE)/mL and 27 to 466 colony-forming units (CFU)/mL, respectively. The intra- and inter-assay reproducibility of cfDNA and cellular DNA mNGS was 100%. Clinical evaluation revealed that cfDNA mNGS was good at detecting the virus in blood samples (receiver operating characteristic (ROC) area under the curve (AUC), 0.9814). In contrast, the performance of cellular DNA mNGS was better than that of cfDNA mNGS in high host background samples. Overall, the diagnostic efficacy of cfDNA combined with cellular DNA mNGS (ROC AUC, 0.8583) was higher than that of cfDNA (ROC AUC, 0.8041) or cellular DNA alone (ROC AUC, 0.7545). ConclusionOverall, cfDNA mNGS is good for detecting viruses, and cellular DNA mNGS is suitable for high host background samples. The diagnostic efficacy was higher when cfDNA and cellular DNA mNGS were combined.

Comparative analysis of conventional and direct antimicrobial susceptibility testing of blood cultures in a tertiary care cancer hospital: the way forward

ABSTRACT Bloodstream infections (BSI) are associated with high mortality rates, especially in immunocompromised patients. Identifying pathogens early and selecting appropriate antimicrobials to treat BSI is integral in reducing the mortality rate. There is a need to reduce the turnaround time (TAT) of pathogen identification as well as to accelerate the antimicrobial susceptibility testing (AST) of blood cultures, which can be achieved by following relevant identification methods and performing the direct AST (DAST) by the disk diffusion method. In this study, blood samples were collected from patients with suspected bacteremia/septicemia, and aseptic precautions were taken to prevent contamination. Samples containing gram-negative bacilli (GNB) were then analyzed by DAST and conventional AST (CAST). We tested 118 GNB-positive isolates in total to compare the results of DAST and CAST. DAST and CAST showed good categorical agreement (CA) for various groups of microorganisms: 98.9% and 99.6% for Enterobacterales and Pseudomonas spp., respectively. Early detection of pathogens in blood along with the determination of their antibiotic susceptibility patterns is a need of the hour. By performing DAST on positive blood culture broth, clinical teams can obtain the information necessary for switching from empirical therapy to definitive treatment one day faster. This rapid identification of the pathogen, along with corresponding AST results, will help clinicians to accelerate targeted antimicrobial therapy for critical patients and, thus, reduce mortality and morbidity rates in patients with bloodstream infections.

Comparative study of manual conventional blood cultures versus automated blood culture system in cases of septicemia

: Blood cultures are a proven gold standard method for the identification of causative agents of bloodstream infections. Identification of causative organism along with antibiotic susceptibility plays a pivotal role in proposing suitable antibiotic therapy. Automated blood culture systems show improved monitoring of blood cultures by reducing the time and by ensuring more accurate results when compared to the conventional blood culture system.To isolate the organism from given blood samples of a suspected case of septicemia and to compare the results of conventional and automated blood culture systems and to study the antimicrobial susceptibility pattern of the pathogens isolated.A prospective study of 6 months period was conducted among 100 subjects attending the Department of Microbiology in a tertiary care hospital. Subjects with symptoms and signs of septicemia were included. 25ml of venous blood was drawn aseptically from the venipuncture site, of which 5ml of blood was inoculated into 50ml of Brain Heart Infusion bottle in conventional blood culture system and 10ml each into aerobic and anaerobic BACTEC PLUS bottle in Automated blood culture system BACTEC FX40.Overall, 48% and 60% of the samples revealed positive growth by the conventional and automated blood culture system BACTEC FX40, respectively. Gram Positive Cocci were 52.08% and Gram Negative Bacilli were 47.91% isolated by conventional blood culture system, whereas automated blood culture system BACTEC FX40 isolated 45% and 55%, respectively. Isolates were detected within 24-48hrs and 12-24 hrs by conventional and automated blood culture systems, respectively. The anti-microbial susceptibility pattern of the pathogens isolated was also recorded by Kirby Bauer disc diffusion method of antimicrobial susceptiblity testing.Automated blood culture systems are a trustworthy substitute to conventional blood culture systems. The automated blood culture systems being more sensitive and rapid in detecting septicemia in subjects acts as an appropriate means for the initial identification and detection of blood pathogens and improved provision of antimicrobial therapeutic options for septic Patients especially in Critical Care and Intensive Care Units where positive culture reporting is crucial.

Detection of pathogens in blood or feces of adult horses with enteric disease and association with outcome of colitis.

BackgroundRates of detecting ≥1 potential enteric pathogens (PEP) or toxins (PEP‐T) in feces, blood, or both of horses ≥6 months of age with enteric disease and impact of multiple detections on outcome of horses with colitis has not been reported.ObjectiveTo determine detection rates of PEP/PEP‐T in feces, blood, or both of horses with enteric disease and effect of detecting multiple agents on outcome of horses with colitis.AnimalsThirty‐seven hundred fifty‐three fecal samples submitted to IDEXX Laboratories and 239 fecal and blood samples submitted to Michigan State University's Veterinary Diagnostic Laboratory (MSUVDL).MethodsRetrospective evaluation of PEP/PEP‐T testing results was performed to determine rates of detection of 1 or more PEP/PEP‐T. Impact of detecting multiple agents on outcome was assessed in 239 horses hospitalized for colitis.ResultsOne or more PEP/PEP‐T was detected in 1175/3753 (31.3%) and 145/239 (60.7%) of samples submitted to IDEXX Laboratories and MSUVDL, respectively. In a hospitalized cohort, survival to discharge was lower (76%) in horses with 1 agent, compared to horses with either no (88%) or multiple (89%) agents. There was no difference (P = .78) in days of hospitalization between horses with 0 (1–17), 1 (1–33), and > 1 positive (1–20) result. There was no difference in cost of hospitalization (P = .25) between horses with 0 ($2357, $1110‐15 553), 1 ($2742, $788‐11 005), and >1 positive ($2560, $1091‐10 895) result.Conclusions and Clinical ImportanceDetection rates of PEP/PEP‐T in horses with colitis vary with cohorts and tests performed. Detection of more than 1 PEP or PEP‐T did not affect outcome.

Are Severe Mastitis Cases in Dairy Cows Associated with Bacteremia?

Simple SummaryThe occurrence of bacteremia associated with cases of severe mastitis in dairy cows is an under-researched topic and of great practical importance for the development of evidence-based strategies in mastitis therapy. The aim of this study was to determine the occurrence of bacteremia in lactating dairy cows with severe mastitis cases. For this purpose, we worked on the detection of culturable pathogens in the blood to obtain information on whether systemic antibiotic therapy is necessary in cases of severe mastitis. Detection of culturable pathogens in the blood of cows with severe clinical mastitis was apparently rare (1.4%). Further studies are necessary to estimate the occurrence of bacteremia in severe bovine mastitis.The objective of this study was to investigate the occurrence of bacteremia in dairy cows with severe mastitis. Milk samples were collected from affected udder quarters, and corresponding blood samples were collected from dairy cows with severe mastitis at the time of diagnosis before any therapeutic measures were undertaken. The cultural detection of pathogens in blood classified a bacteremia. Further diagnostic tests were performed to provide evidence of bacteremia. This was realized by PCR with regard to S. aureus, E. coli and S. uberis and the Limulus test. Detection of culturable pathogens in the blood of cows with severe clinical mastitis was rare and occurred in only one of 70 (1.4%) cases. Overall, bacterial growth was detected in 53 of 70 (75.7%) milk samples. S. uberis (22/70), E. coli (12/70) and S. aureus (4/70) were the most frequently isolated pathogens from milk of cows with severe mastitis. PCR was performed in 38 of 70 (54.3%) blood samples. PCR was positive in eight of 38 cases. S. uberis was found most frequently in six blood samples (8.6%). E. coli was found on PCR in one blood sample (1.4%). S. aureus was identified in one blood sample (1.4%). When Coliforms were detected in the quarter milk sample, a Limulus test was performed in the corresponding blood sample. In three of 15 cases, the Limulus test was positive (4.3% of samples). Further studies are needed to investigate the occurrence of bacteremia in cows with severe mastitis in a higher population size.

Metagenomic next-generation sequencing technology for detection of pathogens in blood of critically ill patients

ObjectiveTo explore the applicability of metagenomic next-generation sequencing (mNGS) technology for the detection of blood pathogens in intensive care unit patients. MethodsThe clinical data of 63 critically ill patients who could not be diagnosed with blood culture (BC) and who underwent mNGS blood sample testing were retrospectively analyzed. The diagnostic efficacy of mNGS was compared with that of traditional detection methods; the distribution of the pathogens identified by mNGS was analyzed; and the differences in laboratory tests, comorbidities, treatment, and prognosis between the mNGS-positive and mNGS-negative groups were compared. ResultsThe positive rate of mNGS was 41.3% (26/63), and 16 patients were found to have mixed infections. However, the positive rate of BCs performed simultaneously with mNGS was only 7.9% (5/63). The results of univariate analysis showed that the average length of intensive care unit stay (β, −8.689 [95% CI, −16.176, −1.202]; P = 0.026) and the time from onset to sequencing (β, −5.816 [95% CI,−9.936, −1.696]; P = 0.007) of the mNGS-positive group were significantly shorter than those of the mNGS-negative group. More patients in the positive group were adjusted for anti-infective treatment after mNGS (OR, 3.789 [95% CI,1.176, 12.211]; P < 0.001). ConclusionsDetection of blood pathogens by mNGS has good applicability for critically ill patients who cannot be diagnosed by BC in the early stages of infection, and mNGS should be performed as early as possible to obtain higher pathogen detection rates.

Detection of blood pathogens in camels and their associated ectoparasitic camel biting keds, Hippobosca camelina: the potential application of keds in xenodiagnosis of camel haemopathogens.

Background: Major constraints to camel production include pests and diseases. In northern Kenya, little information is available about blood-borne pathogens circulating in one-humped camels ( Camelus dromedarius) or their possible transmission by the camel haematophagous ectoparasite, Hippobosca camelina, commonly known as camel ked or camel fly. This study aimed to: (i) identify the presence of potentially insect-vectored pathogens in camels and camel keds, and (ii) assess the potential utility of keds for xenodiagnosis of camel pathogens that they may not vector. Methods: In Laisamis, northern Kenya, camel blood samples (n = 249) and camel keds (n = 117) were randomly collected from camels. All samples were screened for trypanosomal and camelpox DNA by PCR, and for Anaplasma, Ehrlichia, Brucella, Coxiella, Theileria, and Babesia by PCR coupled with high-resolution melting (PCR-HRM) analysis. Results: In camels, we detected Trypanosoma vivax (41%), Trypanosoma evansi (1.2%), and " Candidatus Anaplasma camelii" (68.67%). In camel keds, we also detected T. vivax (45.3%), T. evansi (2.56%), Trypanosoma melophagium (1/117) (0.4%), and " Candidatus Anaplasma camelii" (16.24 %). Piroplasms ( Theileria spp. and Babesia spp.), Coxiella burnetii, Brucella spp., Ehrlichia spp., and camel pox were not detected in any samples. Conclusions: This study reveals the presence of epizootic pathogens in camels from northern Kenya. Furthermore, the presence of the same pathogens in camels and in keds collected from sampled camels suggests the potential use of these flies in xenodiagnosis of haemopathogens circulating in camels.

Detection of blood pathogens in camels and their associated ectoparasitic camel biting keds, Hippobosca camelina: the potential application of keds in xenodiagnosis of camel haemopathogens

Background: Major constraints to camel production include pests and diseases. In northern Kenya, little information is available about blood-borne pathogens circulating in one-humped camels ( Camelus dromedarius) or their possible transmission by the camel haematophagous ectoparasite, Hippobosca camelina, commonly known as camel ked or camel fly. This study aimed to: (i) identify the presence of potentially insect-vectored pathogens in camels and camel keds, and (ii) assess the potential utility of keds for xenodiagnosis of camel pathogens that they may not vector. Methods: In Laisamis, northern Kenya, camel blood samples (n = 249) and camel keds (n = 117) were randomly collected from camels. All samples were screened for trypanosomal and camelpox DNA by PCR, and for Anaplasma, Ehrlichia, Brucella, Coxiella, Theileria, and Babesia by PCR coupled with high-resolution melting (PCR-HRM) analysis. Results: In camels, we detected Trypanosoma vivax (41%), Trypanosoma evansi (1.2%), and “ Candidatus Anaplasma camelii” (68.67%). In camel keds, we also detected T. vivax (45.3%), T. evansi (2.56%), Trypanosoma melophagium (1/117) (0.4%), and “ Candidatus Anaplasma camelii” (16.24 %). Piroplasms ( Theileria spp. and Babesia spp.), Coxiella burnetii, Brucella spp., Ehrlichia spp., and camel pox were not detected in any samples. Conclusions: This study reveals the presence of epizootic pathogens in camels from northern Kenya. Furthermore, the presence of the same pathogens in camels and in keds collected from sampled camels suggests the potential use of these flies in xenodiagnosis of haemopathogens circulating in camels.

Detection of blood pathogens in camels and their associated ectoparasitic camel biting keds, Hippobosca camelina: the potential application of keds in xenodiagnosis of camel haemopathogens

Background: Major constraints to camel production include pests and diseases. In northern Kenya, little information is available about blood-borne pathogens circulating in one-humped camels ( Camelus dromedarius) or their possible transmission by the camel haematophagous ectoparasite, Hippobosca camelina, commonly known as camel ked or camel fly. This study aimed to: (i) identify the presence of potentially insect-vectored pathogens in camels and camel keds, and (ii) assess the potential utility of keds for xenodiagnosis of camel pathogens that they may not vector. Methods: In Laisamis, northern Kenya, camel blood samples (n = 249) and camel keds (n = 117) were randomly collected from camels. All samples were screened for trypanosomal and camelpox DNA by PCR, and for Anaplasma, Ehrlichia, Brucella, Coxiella, Theileria, and Babesia by PCR coupled with high-resolution melting (PCR-HRM) analysis. Results: In camels, we detected Trypanosoma vivax (41%), Trypanosoma evansi (1.2%), and " Candidatus Anaplasma camelii" (68.67%). In camel keds, we also detected T. vivax (45.3%), T. evansi (2.56%), Trypanosoma melophagium (1/117) (0.4%), and " Candidatus Anaplasma camelii" (16.24 %). Piroplasms ( Theileria spp. and Babesia spp.), Coxiella burnetii, Brucella spp., Ehrlichia spp., and camel pox were not detected in any samples. Conclusions: This study reveals the presence of epizootic pathogens in camels from northern Kenya. Furthermore, the presence of the same pathogens in camels and in keds collected from sampled camels suggests the potential use of these flies in xenodiagnosis of haemopathogens circulating in camels.

A 3D-Printed Millifluidic Platform Enabling Bacterial Preconcentration and DNA Purification for Molecular Detection of Pathogens in Blood.

Molecular detection of pathogens in clinical samples often requires pretreatment techniques, including immunomagnetic separation and magnetic silica-bead-based DNA purification to obtain the purified DNA of pathogens. These two techniques usually rely on handling small tubes containing a few millilitres of the sample and manual operation, implying that an automated system encompassing both techniques is needed for larger quantities of the samples. Here, we report a three-dimensional (3D)-printed millifluidic platform that enables bacterial preconcentration and genomic DNA (gDNA) purification for improving the molecular detection of target pathogens in blood samples. The device consists of two millichannels and one chamber, which can be used to preconcentrate pathogens bound to antibody-conjugated magnetic nanoparticles (Ab-MNPs) and subsequently extract gDNA using magnetic silica beads (MSBs) in a sequential manner. The platform was able to preconcentrate very low concentrations (1–1000 colony forming units (CFU)) of Escherichia coli O157:H7 and extract their genomic DNA in 10 mL of buffer and 10% blood within 30 min. The performance of the platform was verified by detecting as low as 1 CFU of E. coli O157:H7 in 10% blood using either polymerase chain reaction (PCR) with post gel electrophoresis or quantitative PCR. The results suggest that the 3D-printed millifluidic platform is highly useful for lowering the limitations on molecular detection in blood by preconcentrating the target pathogen and isolating its DNA in a large volume of the sample.

PII: S1201-9712(18)33639-7

Sepsis is one of the most common severe infections and is associated with high morbidity and mortality. Several studies have underlined the importance of rapid initiation of adequate antimicrobial therapy. This is hampered by growing antimicrobial resistance and the long turnaround time of blood culture. Rapid and accurate diagnosis of the causing pathogen directly in blood together with its associated resistance genes would be highly valuable to improve survival. In this presentation an overview of important aspects of the direct detection of pathogens in blood including blood volume, method of isolation and detection, turnaround time and workflow is provided. Currently available commercial assays (SeptiFast, IRIDICA, T2 Sepsis Solution) are discussed as well as assays still in development. In addition, attention is given to specific patient populations as neonates, ICU patients and neutropenic patients. An important additional benefit of molecular detection could be the quantification of bacterial DNA. This bacterial DNA load can be used as a marker of severity of infection and for monitoring of treatment and will be illustrated with several examples. Despite the large number of studies investigating the molecular diagnosis of sepsis directly in blood, it is still not part of routine practice in many laboratories. Factors that are hampering its introduction in clinical practice (costs, suboptimal sensitivity, contamination and implementation) are presented in the last part of the presentation which includes future prospects.

Time to Pathogen Detection for Non-ill Versus Ill-Appearing Infants ≤60 Days Old With Bacteremia and Meningitis.

We sought to determine the time to pathogen detection in blood and cerebrospinal fluid (CSF) for infants ≤60 days old with bacteremia and/or bacterial meningitis and to explore whether time to pathogen detection differed for non-ill-appearing and ill-appearing infants. We included infants ≤60 days old with bacteremia and/or bacterial meningitis evaluated in the emergency departments of 10 children's hospitals between July 1, 2011, and June 30, 2016. The microbiology laboratories at each site were queried to identify infants in whom a bacterial pathogen was isolated from blood and/or CSF. Medical records were then reviewed to confirm the presence of a pathogen and to extract demographic characteristics, clinical appearance, and the time to pathogen detection. Among 360 infants with bacteremia, 316 (87.8%) pathogens were detected within 24 hours and 343 (95.3%) within 36 hours. A lower proportion of non-ill-appearing infants with bacteremia had a pathogen detected on blood culture within 24 hours compared with ill-appearing infants (85.0% vs 92.9%, respectively; P = .03). Among 62 infants with bacterial meningitis, 55 (88.7%) pathogens were detected within 24 hours and 59 (95.2%) were detected within 36 hours, with no difference based on ill appearance. Among infants ≤60 days old with bacteremia and/or bacterial meningitis, pathogens were commonly identified from blood or CSF within 24 and 36 hours. However, clinicians must weigh the potential for missed bacteremia in non-ill-appearing infants discharged within 24 hours against the overall low prevalence of infection.

Potential of High-Affinity, Slow Off-Rate Modified Aptamer Reagents for Mycobacterium tuberculosis Proteins as Tools for Infection Models and Diagnostic Applications.

ABSTRACTDirect pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for Mycobacterium tuberculosis proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native M. tuberculosis proteins was confirmed by using M. tuberculosis culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant (P < 0.0001) differences in the median M. tuberculosis signals and in specific pathogen markers, such as antigen 85B. Samples where many M. tuberculosis aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals (P < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct M. tuberculosis antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of M. tuberculosis SOMAmers using other platforms and sample types is warranted.

Performance and economic evaluation of the molecular detection of pathogens for patients with severe infections: the EVAMICA open-label, cluster-randomised, interventional crossover trial

PurposeMicrobiological diagnosis (MD) of infections remains insufficient. The resulting empirical antimicrobial therapy leads to multidrug resistance and inappropriate treatments. We therefore evaluated the cost-effectiveness of direct molecular detection of pathogens in blood for patients with severe sepsis (SES), febrile neutropenia (FN) and suspected infective endocarditis (SIE).MethodsPatients were enrolled in a multicentre, open-label, cluster-randomised crossover trial conducted during two consecutive periods, randomly assigned as control period (CP; standard diagnostic workup) or intervention period (IP; additional testing with LightCycler®SeptiFast). Multilevel models used to account for clustering were stratified by clinical setting (SES, FN, SIE).ResultsA total of 1416 patients (907 SES, 440 FN, 69 SIE) were evaluated for the primary endpoint (rate of blood MD). For SES patients, the MD rate was higher during IP than during CP [42.6% (198/465) vs. 28.1% (125/442), odds ratio (OR) 1.89, 95% confidence interval (CI) 1.43–2.50; P < 0.001], with an absolute increase of 14.5% (95% CI 8.4–20.7). A trend towards an association was observed for SIE [35.4% (17/48) vs. 9.5% (2/21); OR 6.22 (0.98–39.6)], but not for FN [32.1% (70/218) vs. 30.2% (67/222), P = 0.66]. Overall, turn-around time was shorter during IP than during CP (22.9 vs. 49.5 h, P < 0.001) and hospital costs were similar (median, mean ± SD: IP €14,826, €18,118 ± 17,775; CP €17,828, €18,653 ± 15,966). Bootstrap analysis of the incremental cost-effectiveness ratio showed weak dominance of intervention in SES patients.ConclusionAddition of molecular detection to standard care improves MD and thus efficiency of healthcare resource usage in patients with SES.ClinicalTrials.gov registration number: NCT00709358.

Detection of Pathogens in Blood for Diagnosis of Sepsis and Beyond

Detection of Pathogens in Blood for Diagnosis of Sepsis and Beyond

Real-time PCR TaqMan assay for rapid screening of bloodstream infection

BackgroundSepsis is one of the main causes of mortality and morbidity. The rapid detection of pathogens in blood of septic patients is essential for adequate antimicrobial therapy and better prognosis. This study aimed to accelerate the detection and discrimination of Gram-positive (GP) and Gram-negative (GN) bacteria and Candida species in blood culture samples by molecular methods.MethodsThe Real-GP®, -GN®, and -CAN® real-time PCR kit (M&D, Wonju, Republic of Korea) assays use the TaqMan probes for detecting pan-GP, pan-GN, and pan-Candida species, respectively. The diagnostic performances of the real-time PCR kits were evaluated with 115 clinical isolates, 256 positive and 200 negative blood culture bottle samples, and the data were compared to results obtained from conventional blood culture.ResultsEighty-seven reference strains and 115 clinical isolates were correctly identified with specific probes corresponding to GP-bacteria, GN-bacteria and Candida, respectively. The overall sensitivity and specificity of the real-time PCR kit with blood culture samples were 99.6% and 89.5%, respectively.ConclusionsThe Real-GP®, -GN®, and -CAN® real-time PCR kits could be useful tools for the rapid and accurate screening of bloodstream infections (BSIs).

Rapid Identification of Pathogens with the hemoFISH Test Applying a Novel Beacon-Based Fluorescence in Situ Hybridization (bbFISH) Technology in Positive Blood Culture Bottles

Rapid and accurate identification of pathogens responsible for sepsis is essential for early and targeted antimicrobial therapy. Blood cultures are the current reference standard for detection of pathogens in blood, but culture-based identification methods are time consuming. We evaluated the hemoFISH assay by using the novel bbFISH technology for rapid and accurate identification of a broad range of microorganisms in positive blood cultures. A total of 103 positive blood culture bottles were investigated. In total, 106 bacterial species were detected in the blood cultures and subsequently identified with conventional methods. The Gram-staining indicated monomicrobial growth in 95.1% (98/103) and polymicrobial growth in 4.9% (5/103) blood cultures. In 65.0% (67/103) cultures Gram-positive, 32.0% (33/103) Gram-negative, and 3.0% (3/103) both Gram-positive and Gram-negative bacteria were identified. Depending on the Gram-staining results, either the hemoFISH Gram-positive or the hemoFISH Gram-negative panel was used. In case of a polymicrobial infection, both panels were applied. The hemoFISH assay showed a sensitivity and specificity of 100% (95% CIs of 96.34% to 100% and 30.48% to 100%, respectively). Of the 106 bacterial species, the hemoFISH assay correctly identified 55.7% (n= 59) to species level, 34.0% (n = 36) to genus level, and 7.5% (n = 8) to family level. The novel hemoFISH using bbFISH technology appears to be a valuable rapid tool for the identification of a broad range of microorganisms in positive blood cultures.

Non-culture-based methods to diagnose bloodstream infection: Does it work?

Bloodstream infections are a major cause of morbidity and mortality worldwide. Molecular methods for the detection of pathogens in blood have been developed. The clinical utility of these methods and their integration into the clinical workflow is discussed.

Potential Clinical Usefulness of the Polymerase Chain Reaction Test to Detect Pathogens Causing Sepsis

Objective: A real-time polymerase chain reaction (PCR) test is expected for early and precise detection of pathogens in blood. In this study, we compared the ability of the PCR test and blood culture to detect pathogens in the blood of patients with sepsis. Methods: Patients who were diagnosed as or suspected of having sepsis were included in this prospective observational study. A whole blood sample for PCR test was obtained serially simultaneously with the blood culture sample, and the results were compared. Results: We obtained 93 samples from 26 patients; 69 samples were obtained during the septic condition, and 24 samples were from the non-septic condition. Origins of sepsis were pneumonia in 9 patients, necrotizing fasciitis in 5 patients, and other causes in 12 patients. In the septic condition, rates of positive results were 29.0% for the PCR test and 23.2% for blood culture. Sample contamination occurred in 1 PCR test sample and 5 blood culture samples. In positive PCR samples, 16 of 20 samples were obtained during sepsis after beginning administration of broad- spectrum antibiotics, whereas 5 of 12 samples, except for contaminated samples, were positive in blood culture. Conclusion: In sepsis, the PCR test detected more bacteria than did blood culture even after administration of empirical antibiotics, which might contribute to precise diagnosis of the bacteremic cause of sepsis

MOLECULAR IDENTIFICATION OF BLOODSTREAM PATHOGENS IN PATIENTS PRESENTING TO THE EMERGENCY DEPARTMENT WITH SUSPECTED SEPSIS

The rapid detection of pathogens in blood is critical for a favorable outcome of patients with suspected sepsis. Although blood culture (BC) is considered the criterion standard for diagnosis of bloodstream infection, it often takes several days to detect the causative organism. In this study, we compared BC with a commercially available multiplex real-time polymerase chain reaction (PCR) assay to detect bacteria and fungi in blood samples from 144 patients admitted to the emergency department with suspected sepsis. Of 144 blood samples examined, 91 (63%) were negative by both methods and 53 (37%) were positive by at least one of the two methods. In 30 among all positive cases (56.6%),both methods identified the same organisms, in 13 cases (24.5%), BC identified organisms not detected by real-time PCR,and in 10 cases (18.9%), SeptiFast PCR assay gave positive results, whereas the BC was negative. In this study, we wished to compare SeptiFast results obtained by standard procedures, but future clinical studies are necessary to define SeptiFast PCR as support for BC in the early diagnosis of severe bloodstream infections.