Rapid Polymerase Chain Reaction Assay Research Articles

SG-APSIC1123: Diagnostic and infection prevention and control (IPC) performance of rapid polymerase chain reaction (PCR) compared to conventional culture PCR methods for detecting carbapenemase-producing organisms (CPOs)

Objectives: In this study, we compared the performance of a rapid polymerase chain reaction (PCR) method in detecting carbapenemase-producing organisms (CPOs) and its impact on infection prevention and control (IPC) measures compared with a culture PCR method. Methods: All patients requiring CPO screening were included. Rectal swabs were collected with double rayon swabs (Copan 139C). They were simultaneously analyzed for the presence of CPOs using rapid PCR assay (Xpert Carba-R assay, Cepheid, Sunnyvale, CA) and a culture–PCR method (ChromID CARBA-SMART, bioMerieux, Marcy-l’Etoile, France). For CARBA-SMART, only colored colonies (ie, Enterobacterales) were evaluated for CPOs according to the prevailing institutional protocol. We tracked time to CPO detection. Using CPO positivity from either the rapid PCR or the culture PCR method as the gold standard, we calculated the sensitivity and specificity of both tests. We calculated the number of epidemiologically linked contacts generated when the first test results were known. We prospectively followed the ward census to identify the putative additional number of contacts generated by the later known result. Contacts were patients who shared the same ward (with overlapping time) as the CPO patients. Results: Between April 2019 and June 2020, culture PCR method detected CPOs in 316 (1.3%) of 24,514 samples (blaOXA48, N = 211; blaNDM, N = 51; blaIMI, N = 21; blaIMP, N = 10; blaKPC, N = 9; mixed genotypes, N = 14). The rapid PCR test detected CPOs in 605(2.5%) of 24,514 samples (blaOXA48, N = 266; blaNDM, N = 161; blaIMP, N = 99; blaVIM, N = 29; blaKPC, N = 15; mixed genotypes, N = 35). The sensitivity of direct PCR and culture PCR methods were 94.2% (95% CI, 92.1%–95.8%) and 43.5% (95% CI, 39.6%–47.4%), respectively. Both tests had 100% specificity. The median times to detection for the rapid PCR and culture PCR methods were 3–4 hours and 4 days, respectively. Compared with rapid PCR, the culture PCR method generated additional 7,415 contacts when it also tested positive for CPOs and an additional 23,135 contacts when it tested negative for CPOs. Conclusions: In our study, the rapid PCR test was more sensitive, identified CPO faster, and generated fewer epidemiologically linked contacts than the culture PCR method.

A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species

Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., Rhizopus, Lichtheimia, Mucor, and Rhizomucor) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.

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.

Impact of Rapid PCR Influenza Testing on the Rate of Inpatient Admissions During Influenza Season at a Tertiary-Care Center

Background: Influenza causes a high burden of disease in the United States, with an estimate of 960,000 hospitalizations in the 2017–2018 flu season. Traditional flu diagnostic polymerase chain reaction (PCR) tests have a longer (24 hours or more) turnaround time that may lead to an increase in unnecessary inpatient admissions during peak influenza season. A new point-of-care rapid PCR assays, Xpert Flu, is an FDA-approved PCR test that has a significant decrease in turnaround time (2 hours). The present study sought to understand the impact of implementing a new Xpert Flu test on the rate of inpatient admissions. Methods: A retrospective study was conducted to compare rates of inpatient admissions in patients tested with traditional flu PCR during the 2017–2018 flu season and the rapid flu PCR during the 2018–2019 flu season in a tertiary-care center in greater Detroit area. The center has 1 pediatric hospital (hospital A) and 3 adult hospitals (hospital B, C, D). Patients with influenza-like illness who presented to all 4 hospitals during 2 consecutive influenza seasons were analyzed. Results: In total, 20,923 patients were tested with either the rapid flu PCR or the traditional flu PCR. Among these, 14,124 patients (67.2%) were discharged from the emergency department and 6,844 (32.7%) were admitted. There was a significant decrease in inpatient admissions in the traditional flu PCR group compared to the rapid flu PCR group across all hospitals (49.56% vs 26.6% respectively; P < .001). As expected, a significant proportion of influenza testing was performed in the pediatric hospital, 10,513 (50.2%). A greater reduction (30% decrease in the rapid flu PCR group compared to the traditional flu PCR group) was observed in inpatient admissions in the pediatric hospital (Table 1) Conclusions: Rapid molecular influenza testing can significantly decrease inpatient admissions in a busy tertiary-care hospital, which can indirectly lead to improved patient quality with easy bed availability and less time spent in a private room with droplet precautions. Last but not the least, this testing method can certainly lead to lower healthcare costs.Funding: NoneDisclosures: None

Rapid, highly accurate and cost‐effective open‐source simultaneous complete HLA typing and phasing of class I and II alleles using nanopore sequencing

Accurate rapid genotyping of the genes within the HLA region presents many difficulties because of the complexity of this region. Here we present the results of our proof of concept nanopore-based long read polymerase chain reaction (PCR) solution for HLA genotyping. For 15 HLA anthropology-based samples and 13 NHS Blood and Transplant derived samples 40 ng of genomic DNA underwent long-range PCR for class I and II HLA alleles. Pooled PCR products were sequenced on the Oxford Nanopore MinIoON R9.4.1 flow cell. Sequenced reads had HLA genotype assigned with HLA-LA. Called genotypes were compared with reference derived from a combination of short-read next-generation sequencing, Sanger sequence and/or single-site polymorphism (SSP) typing. For concordance, accuracy was 100%, 98.4%, 97.5% and 95.1% for the first, second, third and fourth fields, respectively, to four field accuracy where it was available, otherwise three field in 28 samples for class I calls and 17 samples for class II calls. Phasing of maternal and paternal alleles, as well as phasing based identification of runs of homozygosity, was shown successfully. Time for assay run was 8 hours and the reconstruction of HLA typing data was 15 minutes. Assay cost was £55 ($80USD)/sample. We have developed a rapid and cost-effective long-range PCR and nanopore sequencing-based assay that can genotype the genes within HLA region to up to four field accuracy, identify runs of homozygosity in HLA, reconstruct maternal and paternal haplotypes and can be scaled from multi-sample runs to a single sample.

Targeted Assessment of the EGFR Status as Reflex Testing in Treatment-Naive Non-Squamous Cell Lung Carcinoma Patients: A Single Laboratory Experience (LPCE, Nice, France).

Background: Assessment of actionable EGFR mutations is mandatory for treatment-naïve advanced or metastatic non-squamous lung carcinoma (NSLC), but the results need to be obtained in less than 10 working days. For rapid EGFR testing, an EGFR-specific polymerase chain reaction (PCR) assay is an alternative and simple approach compared to next generation sequencing (NGS). Here, we describe how a rapid EGFR-specific PCR assay can be implemented in a single laboratory center (LPCE, Nice, France) as reflex testing in treatment-naïve NSLC. Methods: A total of 901 biopsies from NSLC with more than 10% of tumor cells were prospectively and consecutively evaluated for EGFR mutation status between November 2017 and December 2019 using the Idylla system (Biocartis NV, Mechelen, Belgium). NGS was performed for nonsmokers with NSLC wild type for EGFR, ALK, ROS1, and BRAF and with less than 50% PD-L1 positive cells using the Hotspot panel (Thermo Fisher Scientific, Waltham, MA, USA). Results: Results were obtained from 889/901 (97%) biopsies with detection of EGFR mutations in 114/889 (13%) cases using the Idylla system. Among the 562 EGFR wild type tumors identified with Idylla, NGS detected one actionable and one nonactionable EGFR mutation. Conclusions: Rapid and targeted assessment of EGFR mutations in treatment-naïve NSLC can be implemented in routine clinical practice. However, it is mandatory to integrate this approach into a molecular algorithm that allows evaluation of potentially actionable genomic alterations other than EGFR mutations.

Utility of Rapid Influenza Molecular Testing in an Outpatient Hemodialysis Unit: A Prospective Cohort Study.

Background:Early initiation of antiviral therapy for individuals at risk for severe influenza infection is important for improving patient outcomes. Current guidelines recommend empiric antiviral therapy for patients with end-stage kidney disease presenting with suspected influenza infection. Rapid molecular influenza assays may reduce diagnostic uncertainty and improve patient outcomes by providing faster diagnostics compared to traditional batched polymerase chain reaction (PCR) testing.Objective:To determine the utility of implementing a rapid influenza PCR assay compared to the standard of care in a hemodialysis unit.Design:This is a prospective cohort study.Setting:A hospital-based dialysis unit in a tertiary care hospital.Patients:Adult patients with end-stage kidney disease on intermittent hemodialysis.Measurements:Patient characteristics, influenza PCR swab results, antibiotic prescriptions, antiviral prescriptions, emergency room visits and hospitalizations.Methods:From November 1, 2017 to March 31, 2018, we assigned samples collected from a single center, hemodialysis unit to be processed using a rapid influenza PCR (cobas® Influenza A/B & respiratory syncytial virus assay) or the standard of care (in-house developed multiplex PCR). Samples were assigned to the rapid PCR if the patient received dialysis treatment in the morning dialysis shift, while the remainder were processed as per standard of care. Study outcomes included the time from collection to result of nasopharyngeal swab, prescription of influenza antiviral therapy, time to receiving prescription, and the need for emergency department visit or hospitalization within 2 weeks of presentation.Results:During the study period, 44 patients were assessed (14 with the rapid PCR and 30 with the standard of care assay). Compared to conventional testing, the time to result was shorter using rapid PCR compared to conventional testing (2.3 vs 22.6 hours, P < .0001). Individuals who were tested using the rapid PCR had a tendency to shorter time to receiving antiviral prescriptions (0.7 days vs 2.1 days, P = .11), and fewer emergency department visits (7.1% vs 30%, P = .13) but no difference in hospitalizations (14.3% vs 30%, P = .46) within 2 weeks of testing.Limitations:This is a single center non-randomized study with a relatively small sample size. Patients who were tested using the standard of care assay experienced a delay in the prescription of antiviral therapy which deviates from recommended clinical practice.Conclusions:Rapid influenza molecular testing in the hemodialysis unit was associated with a shorter time to a reportable result and with a tendency to reduced time to prescription of antiviral therapy. Rapid molecular testing should be compared with standard of care (empiric therapy) in terms of economic costs, adverse events, and influenza-related outcomes.

Clinical Utilization of the FilmArray Meningitis/Encephalitis (ME) Multiplex Polymerase Chain Reaction (PCR) Assay.

Objective: To assess the clinical utilization and performance of the FilmArray® Meningitis/Encephalitis (ME) multiplex polymerase chain reaction (PCR) panel in a hospital setting.Background: Rapid diagnosis and treatment of central nervous system (CNS) infections are critical to reduce morbidity and mortality. The ME panel is a Food and Drug Administration (FDA) approved rapid multiplex PCR assay that targets 14 bacteria, viruses, and fungi. Previous studies show an overall agreement of 93–99% between the ME panel and conventional diagnostic testing. However, few studies have evaluated the clinical implementation of the ME assay, which is available for routine use at our institution.Methods: We performed a single center retrospective chart review of inpatients who underwent ME panel testing from August 2016 to May 2017. Clinical, radiologic, and laboratory data were reviewed to determine the clinical significance of results. Indication for lumbar puncture (LP), time to results of the ME panel, and duration of antimicrobial therapy were evaluated.Results: Seven hundred and five inpatients underwent ME testing, of whom 480 (68.1%) had clinical suspicion for CNS infection with 416 (59.0%) receiving empiric antimicrobial treatment for CNS infection. The median time-to-result of the ME panel was 1.5 h (IQR, 1.4–1.7). Overall agreement between the ME panel results and clinico-laboratory assessment was 98.2%. Forty-five patients tested positive by ME, of which 12 (26.6%) were determined likely to be clinically insignificant.Conclusions: Routine availability of the ME panel led to overutilization of diagnostic test ordering, as demonstrated by the fact that over one-third of ME panel tests performed were ordered for patients with little or no suspicion for CNS infection. The median time from LP to ME panel result was 1.5 h (IQR, 1.4–1.7). The ME panel's rapid turn-around time contributed to the overuse of the test. Approximately one-quarter of positive ME results were deemed clinically insignificant, though the impact of these positive results requires additional evaluation. Twenty-four and forty-eight hours after the ME panel resulted, 68 and 25% of patients started on empiric therapy remained on antibiotics, respectively. The median time from diagnosis to discontinuation and/or narrowing of antibiotic coverage was 25.6 h (IQR, 3.6–42.5). Further consideration of the appropriate indications for use of the ME panel in clinical settings is required.

Detection of Talaromyces macrosporus and Talaromyces trachyspermus by a PCR assay targeting the hydrophobin gene.

Talaromyces species are typical fungi capable of producing the heat-resistant ascospores responsible for the spoilage of processed food products. Hydrophobins, which are unique to fungi, are small secreted proteins that form amphipathic layers on the outer surface of fungal cell walls. In this study, species-specific primer sets for detecting and identifying Talaromyces macrosporus and Talaromyces trachyspermus were designed based on hydrophobin gene sequences. A conventional polymerase chain reaction (PCR) assay using these primer sets produced species-specific amplicons for T. macrosporus and T. trachyspermus. The detection limit for each primer set was 100pg template DNA. This assay also detected fungal DNA extracted from blueberries inoculated with T. macrosporus. Other heat-resistant fungi, including Byssochlamys, Neosartorya and Talaromyces species, which cause food spoilage, were not detected in PCR amplifications with these primer sets. Furthermore, a conventional PCR assay using a crude DNA extract as the template also yielded amplicons specific to T. macrosporus and T. trachyspermus. The simple and rapid PCR assay described herein is highly species-specific and can reliably detect T. macrosporus and T. trachyspermus, suggesting it may be relevant for the food and beverage industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The heat-resistant ascospores of Talaromyces macrosporus and Talaromyces trachyspermus are responsible for food spoilage after pasteurization. Traditional methods for detecting fungal contamination based on morphological characteristics are time-consuming and exhibit low sensitivity and specificity. In this study, a conventional polymerase chain reaction (PCR) assay based on hydrophobin gene sequences was developed for the specific detection of T. macrosporus and T. trachyspermus. This detection method was simple, rapid and highly specific. These results suggest that the conventional PCR assay developed in this study may be useful for detecting T. macrosporus and T. trachyspermus in raw materials and processed food products.

Development and validation of a multiplex polymerase chain reaction assay for detection of the five families of plasmid-encoded colistin resistance

Plasmid-mediated colistin resistance is increasingly described worldwide in Enterobacteriaceae from animal and human isolates. Diffusion of these resistance traits among carbapenem-resistant enterobacterial isolates is of particular concern as colistin has become the last resort antibiotic for treating human infections with these organisms. Therefore, being able to monitor the presence of these transferable colistin resistance genes (mcr-1 to mcr-5-variants) is crucial. This paper describes the development of a multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes in Enterobacteriaceae. Five primer pairs were designed to amplify mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 gene products in a multiplex PCR. This assay was validated retrospectively on colonies of 50 Escherichia coli, 44 Klebsiella pneumoniae and 12 Salmonella enterica isolates of animal and human origin, all well characterized, and validated prospectively on 450 carbapenem-resistant enterobacterial isolates received by the French National Reference Centre. In addition, 82 Aeromonas spp. and 10 Shewanella spp. known to be the progenitors of mcr-3 and mcr-4 alleles, respectively, were screened. Mcr-multiplex PCR assay displayed 100% specificity, sensitivity, negative predictive value and positive predictive value. The assay was able to detect all variants of the different mcr alleles, and was able to detect chromosomally encoded mcr-4-like variants present in two Shewanella bicestrii JAB-1 and Shewanella woodyi S539. In conclusion, a rapid and robust multiplex PCR assay able to detect all known mcr gene families described in Enterobacteriaceae was developed and validated. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance, especially in carbapenem-resistant bacteria.

Accuracy of a rapid intrapartum group B Streptococcus test: A new immunochromatographic assay

ObjectivesTo decrease the incidence of early-onset group B streptococcal (GBS) disease, a culture-based screening of all pregnant women at 35–37 weeks is recommended. This gold standard test requires 24–72hours culture. This delay precludes its use for intrapartum screening. This study assesses a new immunoassay, the DIMA test, for identifying GBS-positive patients in the labor ward. Materials and methodsThis was a prospective observational study of 195 pregnant women presenting with full-term labor at a single site in France between June and August 2012. We assessed the diagnostic accuracy of intrapartum DIMA testing as compared to intrapartum GBS culture and prenatal screening at 35–38 weeks. ResultsThe DIMA test sensitivity and specificity were 57.1% and 83.2%, respectively, as compared to 42.9% and 97% for prenatal culture screening. ConclusionThe DIMA test assay is a rapid and inexpensive test for the detection of maternal GBS colonization in the labor ward. Its sensitivity is higher than antepartum culture but its specificity is lower. Its performance was inferior to that reported for rapid polymerase chain reaction assays.

Ruptured aortic aneurysm due to Mycobacterium bovis BCG with a delayed bacteriological diagnosis due to false negative result of the MPB 64 immunochromatographic assay

BackgroundAdjuvant therapy with bacillus Calmette–Guerin (BCG), a live attenuated strain of Mycobacterium bovis, has become the treatment of choice for low-risk superficial bladder carcinoma following transurethral resection of the bladder. Complications following vesical BCG instillations are uncommon but, in some cases, severe side-effects can occur such as sepsis or mycotic aneurysm. Besides usual laboratory techniques used for the diagnosis of Mycobacterium tuberculosis complex (MTBC) infections (smear microscopy and cultures), commercial immunochromatographic assays detecting MBP64, a 24 kDa M. tuberculosis complex-specific secretory protein, can rapidly distinguish MTBC and non-tuberculosis mycobacteria (NTM). MPB64 is found in M. tuberculosis, M. bovis and some but not all substrains of M.bovis BCG. Therefore, these immunochromatographic tests can lead to false negative results and delayed bacteriological diagnosis depending on the presence or absence of MPB64 protein in BCG substrains used for intravesical therapy.Case presentationWe report the case of a 78-year-old male patient who was admitted to the hospital because of a 1-month history of unexplained fever, thrill, weight-loss and general malaise. His past medical history was marked by a non-muscle-invasive bladder carcinoma treated by transurethral resection followed by BCG instillations (Oncotice, Merck, USA). The patient was initially treated for a urinary tract infection but as fever persists after 72 h of antibiotherapy, urinary tract ultrasound was performed and revealed a large abdominal aortic aneurysm confirmed by computed tomography. Surgery was performed after multidisciplinary discussion. Direct smear of perioperative samples revealed acid-fast bacilli and both solid and liquid cultures were massively positive. Rapid identification of the positive mycobacterial culture was performed using an immunochromatographic assay based on the detection of the Mycobacterium tuberculosis MPB 64 antigen. The result was negative for Mycobacterium tuberculosis complex. After review of the medical record, a polymerase chain reaction (PCR) was performed and gave a positive result for M. tuberculosis complex. Anti-tuberculosis therapy was started immediately and the patient evolved favorably.ConclusionsThrough this case, we showed how the utilisation of MPB64 immunochromatographic assays can provide misleading information due to the variable presence of this protein among the different BCG strains. This case further illustrates the utility of rapid TB complex-specific PCR assays which provide a more reliable identification of all MTBC species.

Rapid MRSA PCR on respiratory specimens from ventilated patients with suspected pneumonia: a tool to facilitate antimicrobial stewardship.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of pneumonia in ventilated patients. Our objective was to evaluate the GeneXpert MRSA/SA SSTI Assay (Xpert MRSA/SA) (Cepheid, Sunnyvale, CA) for use in lower respiratory tract (LRT) specimens for rapid MRSA detection and to determine the potentially saved antibiotic-days if a culture-based identification method was replaced by this assay. Remnant LRT samples from ventilated patients submitted to the microbiology laboratory for routine culture were tested using conventional culture and Xpert MRSA/SA. One hundred of 310 LRT specimens met the inclusion criteria. Ten samples were positive for MRSA by Xpert MRSA/SA, while six were positive by routine culture methods. Xpert MRSA/SA correctly identified 5/6 positive and 89/94 negative MRSA specimens, for a sensitivity of 83.3%, specificity of 94.7%, positive predictive value of 45.6%, and negative predictive value of 98.9%. The assay also correctly detected 3/3 positive and 90/97 negative methicillin-susceptible S. aureus (MSSA) specimens, for a sensitivity of 100%, specificity of 92.8%, positive predictive value of 30%, and negative predictive value of 100%. A total of 748 vancomycin and 305 linezolid antibiotic-days were associated with the enrolled specimens. Vancomycin and linezolid utilization could decrease by 68.4% and 83%, respectively, if discontinued 1day after negative polymerase chain reaction (PCR) results. The Xpert MRSA/SA SSTI rapid MRSA PCR assay performed well in respiratory samples from ventilated patients with suspected pneumonia and has the potential to facilitate stewardship efforts such as reducing empiric vancomycin and linezolid therapy.

Test de dépistage rapide intra partum du portage vaginal de streptocoque du groupe B (SGB) pour le repérage des nouveau-nés à risque d’infection néonatale précoce à SGB. Étude observationnelle analytique dans une maternité de type III

Group B streptococcus (GBS) is the most common infectious agent responsible for early-onset bacterial sepsis (EOS) in term newborns. French prevention of perinatal GBS disease guidelines recommend screening for maternal vaginal GBS colonization at the 9th month of pregnancy, and use of intrapartum antibiotic prophylaxis (IAP) in case of detected GBS vaginal colonization. Peripheral bacterial sampling (gastric aspiration, ear, or meconium) and measurement of C-reactive protein (CRP) are performed in asymptomatic newborns in case of infectious risk factors and/or incomplete IAP. The aim of this study was to investigate the relation between a rapid intrapartum screening test for GBS during labor in term parturients and infants developing GBS EOS and in comparison to current recommendations. We conducted an observational analytic single-center study, with use of a rapid intrapartum GBS screening test, at Toulouse University Hospital. A total of 1416mother-newborn dyads were prospectively included between 31/01/2012and 17/08/2012. Vaginal GBS colonization was found at the 9th month of pregnancy in 148mothers (10.6%), and 176mothers (12.5%) were screened positively at delivery using intrapartum GBS rapid polymerase chain reaction assay (GBS PCR) (P=0.025). No confirmed neonatal GBS EOS was found. Nine infants had suspected GBS EOS because of a positive peripheral bacterial finding and elevated CRP. In these infants, seven pregnant mothers were GBS-positive with GBS PCR assay during labor, and four women were positive on prenatal culture at the 9th month of pregnancy. The diagnostic values of the two tests highlighted a nonsignificant superiority of intrapartum GBS PCR assay (AUC=0.83 [0.68-0.97] vs. 0.67 [0.50-0.84]), (P=0.057). The negative predictive value was improved with intrapartum PCR assay (negative likelihood ratio [LR]: 0.3 [0.1-0.9] vs. 0.6 [0.4-1.1]). Intrapartum GBS PCR assay provided its best positive predictive value in the absence of complete AIP and without other infectious factors (positive LR: 21.3 [15.4-29.5]). These results suggest that the intrapartum GBS PCR assay offers a better predictive value of GBS EOS than the usual vaginal culture swab at the 9th month but requires confirmation by large studies.

Rapid multiplex polymerase chain reaction for simultaneous detection of Vibrio harveyi, V. parahaemolyticus, and V. vulnificus in pacific white shrimp (Litopenaeus vannamei)

Context: A comparatively small number of species, e.g., Vibrio parahaemolyticus and V. vulnificus , cause disease in both aquatic animals and humans. V. harveyi is marine animal pathogen and rarely causes infections in humans; however, it might become a reservoir of antibiotic-resistant bacteria forms and virulence genes. Aims: 1) to develop rapid multiplex polymerase chain reaction (PCR) assay for the simultaneous detection of V. harveyi, V. parahaemolyticus , and V. vulnificus by using vhhP2, tl , and rpoS genes as the respective target genes and 2) to evaluate specificity and determined detection of multiplex PCR technique. Materials and Methods: The multiplex PCR assay was developed and evaluated for specificity on 36 isolates of V. harveyi , 30 isolates of V. parahaemolyticus , and 14 isolates of V. vulnificus , along with other species of Vibrio and non- Vibrio bacterial isolates. Sensitivity of test was described as detection limit of pathogens in lowest amount of sample (CFU/mL or CFU/g) was determined by diluted DNA extracts of the pure cultures and spiked pacific white shrimp (Litopenaeus vannamei) samples Results: This developed multiplex PCR was proved as an accurate method, which was specific for three Vibrio species. The detection limits of V. harveyi , V. parahaemolyticus , and V. vulnificus in pure cultures and spiked shrimp samples ranged 1.05-4.8 × 10 3 CFU/mL and 1.9-7 × 10 4 CFU/g, respectively. Conclusions: This rapid multiplex PCR assay can decrease amount and process of sample preparation, which was time-consuming, and had preferable accuracy. This developed technique will be suitable and useful for food-borne pathogen detection in shrimp and horizontal gene transfer study among different Vibrio species in aquatic animals.

Evaluation of saliva as diagnostic materials for influenza virus infection by PCR-based assays

BackgroundImmunochromatographic antigen tests have been widely used for detection of influenza virus; however its low sensitivity restricts the use of clinical materials other than nasopharyngeal swabs. Saliva is obtained non-invasively and has utility for diagnosis of influenza. Polymerase chain reaction (PCR) is not typically used for rapid testing because it is time consuming. We evaluated the utility of saliva as diagnostic materials for influenza virus infection by PCR-based assays. MethodsNasopharyngeal swabs and saliva were simultaneously collected from 144 patients and investigated by reverse transcription-quantitative PCR (RT-qPCR) and droplet-RT-PCR. ResultsOverall concordance of results from nasopharyngeal swabs and saliva were 95.8%. Influenza gene was detectable in less than 12min in saliva by the droplet-RT-PCR. Saliva as well as nasopharyngeal swabs contained more than 1×102 copies/μl of the influenza gene. About half of the patients provided positive results in nasopharyngeal swabs and saliva within 24h from the onset of the symptoms. ConclusionThe study demonstrates that saliva can be used as an alternative specimen source to nasopharyngeal swabs. When rapid PCR assay including RNA extraction to be full-automation in a miniaturized machine, point-of-care test based on PCR may be realized using saliva without restriction of materials.

Update on management of skin and soft tissue infections in the emergency department.

Skin and soft tissue infections (SSTIs) are frequently treated in the emergency department (ED) setting. Recent studies provide critical new information that can guide new approaches to the diagnosis and treatment of SSTIs in the ED. Rapid polymerase chain reaction assays capable of detecting MRSA in approximately 1h hold significant potential to improving antibiotic stewardship in SSTI care. Emergency ultrasound continues to demonstrate value in guiding appropriate management of SSTIs, including the early diagnosis of necrotizing infections. Since emerging in the 1990s, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) continues to increase in prevalence, and it represents a significant challenge to optimizing ED antibiotic use for SSTI management. Growing literature reinforces the current recommendation of incision and drainage without antibiotics for uncomplicated abscesses. Selecting antibiotics with CA-MRSA coverage is recommended when treating purulent SSTIs; however, it is generally not necessary in cases of nonpurulent cellulitis. Future advances in ED SSTI care may involve expansion of outpatient parenteral antimicrobial therapy protocols and the recent development of a novel, once weekly antibiotic with activity against MRSA.

Rapid detection of methicillin-resistant Staphylococcus aureus by a newly developed dry reagent-based polymerase chain reaction assay

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for significant numbers of nosocomial and community-acquired infections worldwide. Molecular diagnosis for MRSA nasal carriers is increasingly important for rapid detection and screening of MRSA colonization because the conventional methods are time consuming and labor intensive. However, conventional polymerase chain reaction (PCR) tests still require cold-chain storage as well as trained personnel, which makes them unsuitable for rapid high-throughput analysis. The aim of this study was to develop a thermostabilized PCR assay for MRSA in a ready-to-use form that requires no cold chain. The thermostabilized PCR assay detects the following targets simultaneously: (1) 16S rRNA of the Staphylococcus genus; (2) femA gene specific for S. aureus; (3) mecA gene conferring methicillin resistance; and (4) lukS gene, which encodes the virulent toxin. The thermostabilized PCR incorporates an internal amplification control that helps to verify the presence of PCR inhibitors in samples. PCR reagents and specific primers were lyophilized into a pellet form with an enzyme stabilizer. The PCR was validated with 235 nasal swabs specimens and was found to be 100% sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 6 months at 24 °C. The limit of detection of thermostabilized PCR assay was determined by probit regression (95% confidence interval) was 10(6) colony forming units at the bacterial cell level and 10 ng of DNA at the genomic DNA level, which is comparable with conventional PCR methods. A rapid thermostabilized PCR assay that requires minimal pipetting steps and is cold chain-free was developed for detecting MRSA nasal carriers.

Evaluation of an ethidium monoazide–enhanced 16S rDNA real‐time polymerase chain reaction assay for bacterial screening of platelet concentrates and comparison with automated culture

BACKGROUNDCulture-based systems are currently the preferred means for bacterial screening of platelet (PLT) concentrates. Alternative bacterial detection techniques based on nucleic acid amplification have also been developed but these have yet to be fully evaluated. In this study we evaluate a novel 16S rDNA polymerase chain reaction (PCR) assay and compare its performance with automated culture.STUDY DESIGN AND METHODSA total of 2050 time-expired, 176 fresh, and 400 initial-reactive PLT packs were tested by real-time PCR using broadly reactive 16S primers and a “universal” probe (TaqMan, Invitrogen). PLTs were also tested using a microbial detection system (BacT/ALERT, bioMérieux) under aerobic and anaerobic conditions.RESULTSSeven of 2050 (0.34%) time-expired PLTs were found repeat reactive by PCR on the initial nucleic acid extract but none of these was confirmed positive on testing frozen second aliquots. BacT/ALERT testing also failed to confirm any time-expired PLTs positive on repeat testing, although 0.24% were reactive on the first test. Three of the 400 “initial-reactive” PLT packs were found by both PCR and BacT/ALERT to be contaminated (Escherichia coli, Listeria monocytogenes, and Streptococcus vestibularis identified) and 14 additional packs were confirmed positive by BacT/ALERT only. In 13 of these cases the contaminating organisms were identified as anaerobic skin or oral commensals and the remaining pack was contaminated with Streptococcus pneumoniae.CONCLUSIONThese results demonstrate that the 16S PCR assay is less sensitive than BacT/ALERT and inappropriate for early testing of concentrates. However, rapid PCR assays such as this may be suitable for a strategy of late or prerelease testing.

Intrapartum Group B streptococcus detection by rapid polymerase chain reaction assay for the prevention of neonatal sepsis

Group B streptococcus (GBS) is a leading cause of infectious neonatal morbidity and mortality. Timely and accurate identification of colonized mothers is imperative so that antibioprophylaxis can be implemented during labour to reduce the risk of neonatal sepsis. We planned our study to analyse the diagnostic accuracy of an intrapartum PCR assay to identify GBS-colonized women and to allow the implementation of correct (i.e. at least 4 h) intrapartum antibiotic prophylaxis based on the PCR results. We included 695 women in labour who were tested for rectovaginal GBS carriage by culture and PCR. Women were also screened at 35–37 weeks of gestation. Intrapartum GBS colonization was 19.3%. Assay sensitivity was 81.0% for antenatal culture and 85.0% for intrapartum PCR; p 0.72. GBS colonization (n = 107) was known at least 4 h before delivery in 68 (64%) and 73 (68%) women based on antenatal culture and intrapartum PCR, respectively. Among 43 women delivering preterm, correct status was known at least 4 h before delivery in 10 (23%) and 32 (74%) women according to antenatal culture and intrapartum PCR, respectively. These results support the concept that GBS screening can be performed routinely during labour in a clinical setting. The intrapartum approach is at least as accurate as the antenatal screening, with the additional advantage of identifying women delivering preterm or not followed during pregnancy.