Rapid Multiplex Polymerase Chain Reaction Research Articles

Diagnostic Accuracy of Blind Bronchial Sample Testing by BioFire Pneumonia plus Panel in Pediatric Intensive Care Unit Patients and Its Impact in Early Adaptation of Antimicrobial Therapy: A Prospective Observational Study.

Community-acquired and nosocomial lower-respiratory-tract infections in critically ill pediatric patients require early appropriate antibiotic therapy to optimize outcomes. Using blind bronchial samples, we assessed the diagnostic performance of the rapid-multiplex polymerase chain reaction (PCR) assay BioFire Pneumonia plus Panel vs. reference standard culturing with antimicrobial susceptibility testing. For this prospective observational study in a single pediatric intensive care unit, we included consecutive patients younger than 18 years admitted for suspected community-, hospital- or ventilator-associated pneumonia in 2021-2022. Sensitivity, specificity, positive predictive value and negative predictive value of the multiplex PCR assay were determined. The kappa coefficient was computed to assess agreement, and univariate analyses were done to identify factors associated with discrepancies between the 2 diagnostic methods. Of the 36 included patients (median age, 1.4 years; interquartile range, 0.2-9.2), 41.7%, 27.8%, and 30.5% had community-, hospital- and ventilator-associated pneumonia, respectively. The overall κ was 0.74, indicating good agreement. Overall, the sensitivity of the multiplex PCR assay was 92% (95% CI: 77%-98%) and specificity 95% (95% CI: 92%-97%), with variations across microorganisms. The median time from sample collection to antimicrobial susceptibility test results was 3.9 (2.5-15) hours with the multiplex PCR assay and 60.5 (47.6-72.2) hours with the reference technique. The BioFire Pneumonia plus Panel used to test blind bronchial samples had satisfactory diagnostic performance in critically ill pediatric patients. The rapid results provided by this test may improve the appropriateness of antimicrobial therapy and help minimize the use of antibiotics.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1.

Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Implementation of a Rapid Multiplex Polymerase Chain Reaction Pneumonia Panel and Subsequent Antibiotic De-escalation.

Net effects of implementation of a multiplex polymerase chain reaction (PCR) pneumonia panel (PNP) on antimicrobial stewardship are thus far unknown. This retrospective study evaluated the real-world impact of the PNP on time to antibiotic de-escalation in critically ill patients treated for pneumonia at an academic medical center. This retrospective, quasi-experimental study included adult intensive care unit (ICU) patients with respiratory culture results from 1 May to 15 August 2019 (pre-PNP group) and adult ICU patients with PNP results from 1 May to 15 August 2020 (PNP group) at Nebraska Medical Center. Patients were excluded for the following reasons: any preceding positive coronavirus disease 2019 PCR test, lack of antibiotic receipt, or non-respiratory tract infection indications for antibiotics. The primary outcome was time to discontinuation of anti-methicillin-resistant Staphylococcus aureus (MRSA) therapy. Secondary outcomes included time to discontinuation of antipseudomonal therapy, frequency of early discontinuation for atypical coverage, and overall duration (in days) of antibiotic therapy for pneumonia. Sixty-six patients in the pre-PNP group and 58 in the PNP group were included. There were significant differences in patient characteristics between groups. The median time to anti-MRSA agent discontinuation was 49.1 hours in the pre-PNP and 41.8 hours in the PNP group (P = .28). The median time to discontinuation of antipseudomonal agents was 134.4 hours in the pre-PNP versus 98.1 hours in the PNP group (P = .47). Other outcomes were numerically but not significantly improved in our sample. This early look at implementation of a multiplex PNP did not demonstrate a statistically significant difference in antibiotic use but lays the groundwork to further evaluate a significant real-world impact on antibiotic de-escalation in ICU patients treated for pneumonia.

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses

Human Respiratory Syncytial Virus (RSV) infections pose a significant risk to human health worldwide, especially for young children. Whole genome sequencing (WGS) provides a useful tool for global surveillance to better understand the evolution and epidemiology of RSV and provide essential information that may impact on antibody treatments, antiviral drug sensitivity and vaccine effectiveness. Here we report the development of a rapid and simplified amplicon-based one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) for WGS of both human RSV-A and RSV-B viruses. Two mRT-PCR reactions for each sample were designed to generate amplicons for RSV WGS. This new method was tested and evaluated by sequencing 206 RSV positive clinical samples collected in Australia in 2020 and 2021 with RSV Ct values between 10 and 32. In silico analysis and laboratory testing revealed that the primers used in the new method covered most of the currently circulating RSV-A and RSV-B. Amplicons generated were suitable for both Illumina and Oxford Nanopore Technologies (ONT) NGS platforms. A success rate of 83.5% with a full coverage for the genome of 98 RSV-A and 74 RSV-B was achieved from all clinical samples tested. This assay is simple to set up, robust, easily scalable in sample preparation and relatively inexpensive, and as such, provides a valuable addition to existing NGS RSV WGS methods.

Rapid multiplex PCR for respiratory viruses reduces time to result and improves clinical care: Results of a systematic review and meta-analysis

The clinical impact of rapid sample-to-answer 'syndromic' multiplex polymerase chain reaction (PCR) testing for respiratory viruses is not clearly established. We performed a systematic literature review and meta-analysis to evaluate this impact for patients with possible acute respiratory tract infection in the hospital setting. We searched EMBASE, MEDLINE, and Cochrane databases from 2012 to present and conference proceedings from 2021 for studies comparing clinical impact outcomes between multiplex PCR testing and standard testing. Twenty-seven studies with 17,321 patient encounters were included in this review. Rapid multiplex PCR testing was associated with a reduction of -24.22hours (95% CI -28.70 to -19.74hours) in the time to results. Hospital length of stay was decreased by -0.82 days (95% CI -1.52 to -0.11 days). Among influenza positive patients, antivirals were more likely to be given (RR 1.25, 95% CI 1.06 to 1.48) and appropriate infection control facility use was more common with rapid multiplex PCR testing (RR 1.55, 95% CI 1.16 to 2.07). Our systematic review and meta-analysis demonstrates a reduction in time to results and length of stay for patients overall along with improvements in appropriate antiviral and infection control management among influenza positive patients. This evidence supports the routine use of rapid sample-to-answer multiplex PCR testing for respiratory viruses in the hospital setting.

539. Impact of a Rapid Diagnostic Assay for the Detection of Bacterial and Viral Agents on Antimicrobial use in Critically Ill Patients with Pneumonia

Abstract Background Conventional diagnostic methods for pneumonia have a long turnaround time, and a pathogen is isolated in only 33% of cases. As a result, empiric therapy with broad-spectrum antimicrobials often becomes definitive therapy. Rapid molecular diagnostics paired with antimicrobial stewardship intervention have the potential to improve outcomes for patients with lower respiratory tract infections (LRTIs). However, data to support this hypothesis are scarce. This study examined the influence of a rapid multiplex polymerase-chain-reaction (PCR) test on antimicrobial use in critically ill patients with LRTIs. Methods This single-center study consisted of pre-implementation (PrIP, December 1, 2018, to February 28, 2019) and post-implementation periods (PoIP, December 1, 2021, to February 28, 2022). Both groups included intensive-care unit (ICU) patients with respiratory cultures from bronchoalveolar lavage (BAL), mini-BAL, or tracheal aspirates. Interventions during the PoIP included concurrent testing of specimens with the BioFire® Pneumonia Panel and clinical decision support tools to streamline treatment recommendations from ICU pharmacists. The primary outcome was the difference in time to optimal therapy (TTOT). Secondary outcomes included time to effective therapy (TTET) and duration of therapy (DOT) for antipseudomonal and anti-MRSA agents. Results A total of 163 patients were included (n = 80, PrIP; n = 83, PoIP). The mean age was 57 + 16 years, and medical ICU patients accounted for 60% (n = 97) of cases. The median APACHE II score was the only significant baseline difference between the two arms (22 vs. 18, interquartile range [IQR] 16, 28 vs. 12, 25, p = 0.01). The median TTOT was 38 hours in the PrIP and 21 hours in the PoIP (p < 0.001). TTET was 5.7 hours in the PrIP and 4.9 hours in the PoIP (p = 0.106). The median DOT for antipseudomonal agents decreased by 52% (4.4 days in the PrIP and 2.1 days in the PoIP, p < 0.001), and anti-MRSA agent DOT was 53% shorter in the PoIP (1.9 days vs. 0.9 days, p < 0.001). Conclusion Implementation of a rapid multiplex PCR panel for lower respiratory-tract pathogens significantly reduced the time to optimal therapy in critically ill patients with pneumonia. Clinically meaningful reductions in anti-MRSA and anti-pseudomonal agent duration were also noted. Disclosures Blake W. Buchan, PhD, Accelerate: Advisor/Consultant|Accelerate: Honoraria|BioFire: Honoraria|ChromaCode: Advisor/Consultant|ChromaCode: Honoraria|Pattern: Advisor/Consultant|Pattern: Honoraria.

Potential of Multiplex Polymerase Chain Reaction Performed on Protected Telescope Catheter Samples for Early Adaptation of Antimicrobial Therapy in ARDS Patients.

Background: Diagnosis of co/superinfection in patients with Acute Respiratory Distress Syndrome (ARDS) is challenging. The FilmArray Pneumonia plus Panel (bioMérieux, France), a new rapid multiplex Polymerase Chain Reaction (mPCR), has never been assessed on a blinded protected telescope catheter (PTC) samples, a very common diagnostic tool in patients under mechanical ventilation. We evaluated the performance of mPCR on PTC samples compared with conventional culture and its impact on antibiotic stewardship. Methods: Observational study in two intensive care units, conducted between March and July 2020, during the first wave of the COVID-19 pandemic in France. Results: We performed 125 mPCR on blinded PTC samples of 95 ARDS patients, including 73 (77%) SARS-CoV-2 cases and 28 (29%) requiring extracorporeal membrane oxygenation. Respiratory samples were drawn from mechanically ventilated patients either just after intubation (n = 48; 38%) or later for suspected ventilator-associated pneumonia (VAP) (n = 77; 62%). The sensitivity, specificity, positive, and negative predictive values of mPCR were 93% (95% CI 84–100), 99% (95% CI 99–100), 68% (95% CI 54–83), and 100% (95% CI 100–100), respectively. The overall coefficient of agreement between mPCR and standard culture was 0.80 (95% CI 0.68–0.89). Intensivists changed empirical antimicrobial therapy in only 14% (18/125) of cases. No new antibiotic was initiated in more than half of the CAP/HAP pneumonia-suspected cases (n = 29; 60%) and in more than one-third of those suspected to have VAP without affecting or delaying their antimicrobial therapy. Conclusions: Rapid mPCR was feasible on blinded PTC with good sensitivity and specificity. New antibiotics were not initiated in more than half of patients and more than one-third of VAP-suspected cases. Further studies are needed to assess mPCR potential in improving antibiotic stewardship.

Population Genetic Structure and Hybridization of Schistosoma haematobium in Nigeria.

Background: Schistosomiasis is a major poverty-related disease caused by dioecious parasitic flatworms of the genus Schistosoma with a health impact on both humans and animals. Hybrids of human urogenital schistosome and bovine intestinal schistosome have been reported in humans in several of Nigeria’s neighboring West African countries. No empirical studies have been carried out on the genomic diversity of Schistosoma haematobium in Nigeria. Here, we present novel data on the presence and prevalence of hybrids and the population genetic structure of S. haematobium. Methods: 165 Schistosoma-positive urine samples were obtained from 12 sampling sites in Nigeria. Schistosoma haematobium eggs from each sample were hatched and each individual miracidium was picked and preserved in Whatman® FTA cards for genomic analysis. Approximately 1364 parasites were molecularly characterized by rapid diagnostic multiplex polymerase chain reaction (RD-PCR) for mitochondrial DNA gene (Cox1 mtDNA) and a subset of 1136 miracidia were genotyped using a panel of 18 microsatellite markers. Results: No significant difference was observed in the population genetic diversity (p > 0.05), though a significant difference was observed in the allelic richness of the sites except sites 7, 8, and 9 (p < 0.05). Moreover, we observed two clusters of populations: west (populations 1–4) and east (populations 7–12). Of the 1364 miracidia genotyped, 1212 (89%) showed an S. bovis Cox1 profile and 152 (11%) showed an S. haematobium cox1 profile. All parasites showed an S. bovis Cox1 profile except for some at sites 3 and 4. Schistosoma miracidia full genotyping showed 59.3% of the S. bovis ITS2 allele. Conclusions: This study provides novel insight into hybridization and population genetic structure of S. haematobium in Nigeria. Our findings suggest that S. haematobium x S. bovis hybrids are common in Nigeria. More genomic studies on both human- and animal-infecting parasites are needed to ascertain the role of animals in schistosome transmission.

Simultaneous Detection of Four Main Foodborne Pathogens in Ready-to-Eat Food by Using a Simple and Rapid Multiplex PCR (mPCR) Assay.

The increasing consumption of organic or ready-to-eat food may cause serious foodborne disease outbreaks. Developing microbiological culture for detection of food-borne pathogens is time-consuming, expensive, and laborious. Thus, alternative methods such as polymerase chain reaction (PCR) are usually employed for outbreaks investigation. In this work, we aimed to develop a rapid and simple protocol for the simultaneous detection of Escherichia coli (E coli), Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus) and Salmonella enterica (S. enterica), by the combination of an enrichment step in a single culture broth and a multiplex PCR (mPCR) assay. The effectiveness of several enrichment media was assessed by culture and PCR. Buffered peptone water (BPW) was selected as the optimum one. Then, mPCR conditions were optimized and applied both to pure co-cultures and artificially inoculated food samples (organic lettuce and minced meat). In the culture medium inoculated at 100 CFU/mL, mPCR was able to detect the four microorganisms. When performed on artificially food samples, the mPCR assy was able to detect E. coli, S. enterica, and L. monocytogenes. In conclusion, BPW broth can effectively support the simultaneous growth of E. coli, S. aureus, L. monocytogenes, and S. enterica and could be, thus, used prior to a mPCR detection assay in ready-to-eat food, thereby considerably reducing the time, efforts and costs of analyzes.

105. Impact of a Multiplex Polymerase Chain Reaction Panel on Duration of Empiric Antibiotic Therapy in Suspected Bacterial Meningitis

BackgroundMultiplex polymerase chain reaction (PCR) panels allow for rapid detection or exclusion of pathogens causing community-acquired meningitis and encephalitis (ME). However, the clinical impact of rapid multiplex PCR ME panel results on the duration of empiric antibiotic therapy is not well characterized.MethodsWe performed a retrospective pre-post study to evaluate the implementation of the FilmArray ME panel (BioFire Diagnostics, LLC) for diagnosis of bacterial meningitis at our institution. We included adults who presented with suspected bacterial meningitis, received empiric antibiotic therapy, and underwent cerebrospinal fluid microbiological testing in the emergency department. The primary outcome was duration of empiric antibiotic therapy. A bivariable analysis that compared baseline demographics, clinical characteristics, and study outcomes between the pre-ME panel and post-ME panel periods was performed using Mann-Whitney tests, chi-squared tests, or Fisher’s exact tests. Time-to-event analysis used the Kaplan-Meier method and log-rank statistics.ResultsIn the pre-ME panel period, the positive detection rate of bacterial pathogens was 2.2% (3/137) by cerebrospinal fluid culture and 4.3% (3/69) in the post-ME panel period. Table 1 shows baseline characteristics of patients. Compared to the pre-ME panel period, there were significant reductions in the post-ME panel period for the duration of empiric antibiotic therapy (median 34.7 h, IQR 8.5–61.7, vs. 12.3 h, IQR 3.3–40.0, P=0.01), time to targeted therapy (59.3 h, IQR 36.5–74.6, vs 7.02 h, IQR 0.9–12.4, P< 0.001), and hospital length of stay (4 d, IQR 2–7, vs. 3 d, IQR 1–5, P=0.03), as shown in Table 2. There was also significant reduction in time to discontinuation or de-escalation of empiric antibiotic therapy (P=0.049) as shown in Figure 1.Table 1. Baseline characteristics for patients with suspected bacterial meningitis Table 2. Antimicrobial use and hospitalization outcomes Compared to the pre-ME panel period, there were significant reductions in the post-ME panel period for the duration of empiric antibiotic therapy (P=0.01), time to targeted therapy (P<0.001), and hospital length of stay (P=0.03).Figure 1. Probability of Empiric Antibiotic Therapy Between Pre- and Post-ME Panel Periods Kaplan-Meier analysis of the time from initiation of empiric antibiotic therapy to discontinuation or de-escalation of empiric antibiotic therapy between the pre- and post-ME panel periods. P value from log-rank test=0.049 (n=206). There was a significant difference in the time to discontinuation or de-escalation of empiric antibiotic therapy between the groups (sex- and immunosuppressant use-adjusted hazard ratio, 1.46 [95% confidence interval, 1.08–1.97]; P=0.01).ConclusionThe implementation of the FilmArray ME panel for suspected bacterial meningitis appears to reduce the duration of empiric antibiotic therapy, time to targeted therapy, and hospital length of stay compared to traditional culture-based microbiological testing methods.Disclosures Justin J. Choi, MD, Allergan (Consultant, Grant/Research Support)Roche (Consultant, Grant/Research Support) Lars Westblade, PhD, Accelerate Diagnostics Inc (Grant/Research Support)BioFire Diagnostics (Grant/Research Support)Hardy Diagnostics (Grant/Research Support)Roche (Consultant, Advisor or Review Panel member)Shionogi Inc (Advisor or Review Panel member)Talis Biomedical (Advisor or Review Panel member) Marshall J. Glesby, MD, Enzychem (Consultant)Gilead (Grant/Research Support)ReAlta Life Sciences (Consultant)Regeneron (Consultant, Grant/Research Support)Sobi (Consultant)Springer (Other Financial or Material Support, Royalties)UpToDate (Other Financial or Material Support, Royalties)

Impact of a Multiplex Polymerase Chain Reaction Panel on Duration of Empiric Antibiotic Therapy in Suspected Bacterial Meningitis.

BackgroundMultiplex polymerase chain reaction (PCR) panels allow for rapid detection or exclusion of pathogens causing meningitis and encephalitis (ME). The clinical impact of rapid multiplex PCR ME panel results on the duration of empiric antibiotic therapy is not well characterized.MethodsWe performed a retrospective prepost study at our institution that evaluated the clinical impact of a multiplex PCR ME panel among adults with suspected bacterial meningitis who received empiric antibiotic therapy and underwent lumbar puncture in the emergency department. The primary outcome was the duration of empiric antibiotic therapy.ResultsThe positive pathogen detection rates were similar between pre- and post-multiplex PCR ME panel periods (17.5%, 24 of 137 vs 20.3%, 14 of 69, respectively). The median duration of empiric antibiotic therapy was significantly reduced in the post-multiplex PCR ME panel period compared with the pre-multiplex PCR ME panel period (34.7 vs 12.3 hours, P = .01). At any point in time, 46% more patients in the post-multiplex PCR ME panel period had empiric antibiotic therapy discontinued or de-escalated compared with the pre-multiplex PCR ME panel period (sex- and immunosuppressant use-adjusted hazard ratio 1.46, P = .01). The median hospital length of stay was shorter in the post-multiplex PCR ME panel period (3 vs 4 days, P = .03).ConclusionsThe implementation of the multiplex PCR ME panel for bacterial meningitis reduced the duration of empiric antibiotic therapy and possibly hospital length of stay compared with traditional microbiological testing methods.

Development of Novel Multiplex PCR for Diagnosis of Co-infected Hemo-parasites in Cattle

Background: A novel, rapid and specific multiplex polymerase chain reaction was developed to diagnose hemo-parasitic infection in bovine blood co-infected with three of the most common hemo-parasites. Methods: The diagnostic process relied on the detection of the three different bovine hemoparasites isolated from red blood cells (RBCs) of cattle (N=30) by conventional Giemsa stained blood smear (GSBS) and confirmed by multiplex PCR. The multiplex PCR system was used to diagnose GSBS positive blood samples (N=12) found infected or co-infected with hemoparasites. The designed multiplex primer sets was attempted to amplify 205, 313 and 422 bp fragments of apocytochrome b, sporozoite and macroschizont 2 (spm2) and 16S rRNA gene for Babesia bigemina, Theileria annulata and Anaplasma marginale, respectively. Result: This multiplex PCR was sensitive with the ability to detect the presence of 150 ng of genomic DNA. The primers used in this multiplex PCR also showed highly specific amplification of specific gene fragments of each respective parasite. Comparing the two detection methods revealed that 58.33% of specimens showed concordant diagnoses with both techniques. The specificity, positive predictive value and kappa coefficient of the agreement was highest for diagnosis of B. bigemina and lowest for A. marginale. The overall Kappa coefficient for diagnosis based on GSBS for multiple pathogens compared to multiplex PCR was 0.56, slightly behind the threshold of 0.6 of agreement. Therefore, confirmation should always be based on PCR to rule out false positives due to differences in subjective observations, stain particles and false negatives due to low parasitemia. The simplicity and rapidity of this specific multiplex PCR method make it suitable for large-scale epidemiological studies and follow-up of drug treatments.

Application of Multiplex Polymerase Chain Reaction for Pathogen Identification and Antibiotic Use in Children With Respiratory Infections in a PICU.

To compare the pathogen identification rate and use of antibiotics before and after the implementation of multiplex polymerase chain reaction testing in children with respiratory infections in a PICU. Single-center, pre-post study. PICU of Osaka Women's and Children's Hospital, Osaka, Japan. Consecutive children with respiratory infections who were admitted to the PICU between December 2017 and November 2018 (premultiplex polymerase chain reaction period) and between March 2019 and February 2020 (postmultiplex polymerase chain reaction period). Conventional rapid antigen tests and bacterial culture tests were performed throughout the study period. Multiplex polymerase chain reaction testing using the FilmArray respiratory panel (BioFire Diagnostics, Salt Lake City, UT) was conducted to detect 17 viruses and three bacterial pathogens. During the postmultiplex polymerase chain reaction period, we did not recommend prescribing antibiotics for stable children, depending on the virus species and laboratory test results. Ninety-six and 85 children were enrolled during the pre- and postmultiplex polymerase chain reaction periods, respectively. Rapid antigen tests identified pathogens in 22% of the children (n = 21) during the premultiplex polymerase chain reaction period, whereas rapid antigen tests and/or multiplex polymerase chain reaction testing identified pathogens in 67% of the children (n = 57) during the postmultiplex polymerase chain reaction period (p < 0.001). The most commonly identified pathogen using multiplex polymerase chain reaction testing was human rhino/enterovirus. Bacterial pathogens were identified in 50% of the children (n = 48) and 60% of the children (n = 51) during the pre- and postmultiplex polymerase chain reaction periods (p = 0.18). There were no differences in antibiotic use (84% vs 75%; p = 0.14), broad-spectrum antibiotic use (33% vs 34%; p = 0.91), or the duration of antibiotic use within 14 days of admission (6.0 vs 7.0 d; p = 0.45) between the pre- and postmultiplex polymerase chain reaction periods. Although the pathogen identification rate, especially for viral pathogens, increased using multiplex polymerase chain reaction testing, antibiotic use did not reduce in children with respiratory infections in the PICU. Definitive identification of bacterial pathogens and implementation of evidence-based antimicrobial stewardship programs employing multiplex polymerase chain reaction testing are warranted.

Evaluation of the accuracy of diagnostic coding for influenza compared to laboratory results: the availability of test results before hospital discharge facilitates improved coding accuracy

BackgroundAssessing the accuracy of diagnostic coding is essential to ensure the validity and reliability of administrative coded data. The aim of the study was to evaluate the accuracy of assigned International Classification of Diseases version 10-Australian Modification (ICD-10-AM) codes for influenza by comparing with patients’ results of their polymerase chain reaction (PCR)-based laboratory tests.MethodA retrospective study was conducted across seven public hospitals in New South Wales, Australia. A total of 16,439 patients who were admitted and tested by either cartridge-based rapid PCR or batched multiplex PCR between January 2016 and December 2017 met the inclusion criteria. We calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of ICD-10-AM coding using laboratory results as a gold standard. Separate analyses were conducted to determine whether the availability of test results at the time of hospital discharge influenced diagnostic coding accuracy.ResultsLaboratory results revealed 2759 positive influenza cases, while ICD-10-AM coding identified 2527 patients. Overall, 13.7% (n = 378) of test positive patients were not assigned an ICD-10-AM code for influenza. A further 5.8% (n = 146) patients with negative test results were incorrectly assigned an ICD-10-AM code for influenza. The sensitivity, specificity, PPV and NPV of ICD-10-AM coding were 93.1%; 98.9%; 94.5% and 98.6% respectively when test results were received before discharge and 32.7%; 99.2%; 87.8% and 89.8% respectively when test results were not available at discharge. The sensitivity of ICD-10-AM coding varied significantly across hospitals. The use of rapid PCR or hospitalisation during the influenza season were associated with greater coding accuracy.ConclusionAlthough ICD-10-AM coding for influenza demonstrated high accuracy when laboratory results were received before discharge, its sensitivity was substantially lower for patients whose test results were not available at discharge. The timely availability of laboratory test results during the episode of care could contribute to improved coding accuracy.

Development of a simple, rapid multiplex PCR tool kit by using the 16S rRNA gene for the identification of faecal and non-faecal coliforms in drinking water

Abstract A multiplex method for the detection of faecal and non-faecal coliforms in drinking water was developed using three primers from the V2, V3 and V9 variable regions of the 16S rRNA gene. 194F, 474F and 1436R are the three primers designed for specific amplification of the V2, V3 and V9 hyper variable regions of the 16S rRNA gene. Multiplex polymerase chain reaction (PCR) allows for differentiation of total coliforms from faecal coliforms by specific amplicons: 1,285 bp of amplicon is specific for six non-faecal coliform genera and 1,009 bp of amplicon is specific for faecal coliform ie. E. coli. If drinking water was contaminated with both faecal and non-faecal coliforms then two amplicons of 1,285 bp and 1,009 bp by combination of the three primers are observed. A multiplex PCR assay based on the 16S rRNA gene should be a beneficial tool kit for the rapid identification of total coliforms in a large number of water samples compared with traditional methods. Results can be acquired within 3 hrs compared with the classic most probable number (MPN) method (3–4 days). This assay will be useful in diversification and detection of seven genera of total coliforms by using variable regions of 16S rRNA.

Nosocomial Pneumonia in the Era of Multidrug-Resistance: Updates in Diagnosis and Management.

Nosocomial pneumonia (NP), including hospital-acquired pneumonia in non-intubated patients and ventilator-associated pneumonia, is one of the most frequent hospital-acquired infections, especially in the intensive care unit. NP has a significant impact on morbidity, mortality and health care costs, especially when the implicated pathogens are multidrug-resistant ones. This narrative review aims to critically review what is new in the field of NP, specifically, diagnosis and antibiotic treatment. Regarding novel imaging modalities, the current role of lung ultrasound and low radiation computed tomography are discussed, while regarding etiological diagnosis, recent developments in rapid microbiological confirmation, such as syndromic rapid multiplex Polymerase Chain Reaction panels are presented and compared with conventional cultures. Additionally, the volatile compounds/electronic nose, a promising diagnostic tool for the future is briefly presented. With respect to NP management, antibiotics approved for the indication of NP during the last decade are discussed, namely, ceftobiprole medocaril, telavancin, ceftolozane/tazobactam, ceftazidime/avibactam, and meropenem/vaborbactam.

Multiplex Solid-Phase Real-Time Polymerase Chain Reaction without DNA Extraction: A Rapid Intraoperative Diagnosis Using Microvolumes

Current polymerase chain reaction (PCR) methods for the diagnosis of infections are time consuming and require large sample volume and skilled technicians. We developed a novel, easy-to-use, and rapid (processing time, 1 minute; total time, 33 minutes) multiplex real-time PCR test (Direct Strip PCR) that did not require DNA extraction to detect 9 pathogens that could cause uveitis in 20-μl samples. Multicenter prospective evaluation of a diagnostic PCR test. A total of 511 participants (patients with infectious uveitis and controls) were examined at 18 institutes worldwide. After validation, intraocular fluid samples were subjected to etiologic or exclusive diagnosis, including intraoperative rapid diagnosis. The concordance and correlations between Direct Strip PCR and quantitative PCR (qPCR) results. Direct Strip PCR exhibited rapid detection, good repeatability and specificity, long storage stability, and detection ability equal to that of qPCR. It also showed low interinstitutional variability compared with qPCR, even when PCR beginners used various real-time PCR machines. The Direct Strip PCR for 9 pathogens exhibited high concordance against the qPCR (positive concordance rate, 98.8%-100%; negative concordance rate, 99.8%-100%; κ coefficient, 0.969-1.000; P < 0.001-0.031). Additionally, results obtained using Direct Strip PCR and qPCR were highly correlated (ρ= 0.748; P < 0.001). This assay was used for rapid intraoperative diagnosis. The Direct Strip PCR test may improve the prognosis of various infectious diseases because it facilitates rapid etiologic evaluation at the first hospital visit and can be used for intraoperative diagnosis.

Effectiveness of rapid multiplex polymerase chain reaction for early diagnosis and treatment of pertussis.

BackgroundPertussis, is an infectious respiratory disease caused by Bordetella pertussis. The incidence of pertussis has been increasing in South Korea to due to waning vaccine-induced immunity. Culture has a low sensitivity and a long turnaround time (TAT). Recently, a rapid multi-polymerase chain reaction (mPCR) test with a TAT of about 1 h was developed for the detection of respiratory pathogens (17 viruses and three bacteria), including B. pertussis. This study aimed to investigate the effectiveness of mPCR for early diagnosis and treatment of pertussis.MethodsWe performed a retrospective study of patients with pertussis diagnosed from May 2017 to June 2019 at a university hospital in South Korea. Nasopharyngeal swab specimens were tested using mPCR. Data were extracted from medical records.ResultsA total of 27 patients with a median age of 48.9 years (range: 3.3–82.2 years) were diagnosed with pertussis, of whom 9 (33.3%) were male. Eleven (40.7%) had fever, 12 (44.4%) had dyspnea, three (11.1%) had paroxysmal cough, and nine (33.3%) had inspiratory whooping. The median interval from symptom onset to diagnosis was 9.0 days (range: 1–31 days). Twenty-four patients (81.5%) were diagnosed within 2 weeks from symptom onset. All but one patient was prescribed macrolide antibiotics. Twenty-two patients (81.5%) required hospitalization, including three (11.1%) who required intensive care unit care for ventilation.ConclusionTesting patients with respiratory symptoms using mPCR can improve early diagnosis of pertussis, ensure proper treatment, and may help with outbreak control.

A rapid and accurate method for the detection of four aminoglycoside modifying enzyme drug resistance gene in clinical strains of Escherichia coli by a multiplex polymerase chain reaction.

BackgroundAntibiotics are highly effective drugs used in the treatment of infectious diseases. Aminoglycoside antibiotics are one of the most common antibiotics in the treatment of bacterial infections. However, the development of drug resistance against those medicines is becoming a serious concern.AimThis study aimed to develop an efficient, rapid, accurate, and sensitive detection method that is applicable for routine clinical use.MethodsEscherichia coli was used as a model organism to develop a rapid, accurate, and reliable multiplex polymerase chain reaction (M-PCR) for the detection of four aminoglycoside modifying enzyme (AME) resistance genes Aac(6′)-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3′)-Ia. M-PCR was used to detect the distribution of AME resistance genes in 237 clinical strains of E. coli. The results were verified by simplex polymerase chain reaction (S-PCR).ResultsResults of M-PCR and S-PCR showed that the detection rates of Aac(6′)-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3′)-Ia were 32.7%, 59.2%, 23.5%, and 16.8%, respectively, in 237 clinical strains of E. coli. Compared with the traditional methods for detection and identification, the rapid and accurate M-PCR detection method was established to detect AME drug resistance genes. This technique can be used for the clinical detection as well as the surveillance and monitoring of the spread of those specific antibiotic resistance genes.

A rapid diagnostic multiplex PCR approach for xenomonitoring of human and animal schistosomiasis in a 'One Health' context.

Studying the epidemiology of schistosomiasis-the most prevalent gastropod-borne human disease and an economic burden for the livestock industry-relies on adequate monitoring tools. Here we describe a molecular assay for detecting human and animal African schistosome species in their planorbid gastropod host (xenomonitoring) using a two-step approach. First, schistosome infections are detected and discriminated from other trematode infections using a multiplex polymerase chain reaction (PCR) that includes a trematode-specific marker (in 18S rDNA), a Schistosoma genus-specific marker (in internal transcribed spacer 2 [ITS2]) and a general gastropod marker (in 18S rDNA) as an internal control. Upon Schistosoma sp. detection, a second multiplex PCR is performed to discriminate among Schistosoma haematobium, Schistosoma mansoni, Schistosoma mattheei and Schistosoma bovis/Schistosoma curassoni/Schistosoma guineensis using markers of differential lengths in the cytochrome c oxidase subunit 1 (COX1) gene. The specificity of these assays was validated with adult worms, naturally infected gastropods and human urine and stool samples. Sensitivity was tested on experimentally infected snail specimens that were sacrificed 10 and 40 days post-infection in order to mimic a natural prepatent and mature infection, respectively. The assay provides a diagnostic tool to support the xenomonitoring of planorbid gastropods for trematode infections in a One Health context, with a focus on the transmission monitoring of schistosomiasis.