In-house PCR Research Articles

Use of a Multiplex PCR Assay To Assess the Presence of Treponema pallidum in Mucocutaneous Ulcerations in Patients with Suspected Syphilis.

We evaluated the utility of the commercial Allplex genital ulcer real-time PCR multiplex assay for detecting Treponema pallidum, herpes simplex virus 1 (HSV-1) and 2 (HSV-2), and Chlamydia trachomatis serovar L (lymphogranuloma venereum [LGV]) DNA in mucosal and genital ulcers in the context of suspected syphilis. In total, 374 documented genital and mucosal ulcers from patients with and without syphilis presenting at several sexually transmitted infection (STI) centers in France from October 2010 to December 2016 were analyzed at the National Reference Center (CNR) for Bacterial STIs at Cochin Hospital in Paris. T. pallidum subsp. pallidum detection results were compared with the final diagnosis based on a combination of clinical examination, serological results, and in-house nested PCR (nPCR). Detections of HSV and LGV were validated against reference methods. We found that 44.6% of the 374 samples tested were positive for T. pallidum subsp. pallidum, 21% for HSV, and 0.8% for LGV. No positive results were obtained for 30.7% of samples, and 4.8% presented coinfections. For T. pallidum subsp. pallidum detection, the overall sensitivity was 80% (95% confidence interval [CI], 76.1 to 84.1%), specificity was 98.8% (95% CI, 97.7 to 99.9%), positive predictive value was 98.8% (95% CI, 97.7 to 99.9%) and negative predictive value was 80.2% (95% CI, 76.2 to 84.2%), with a rate of concordance with the reference method of 92.5% (k = 0.85). This PCR multiplex assay is suitable for T. pallidum subsp. pallidum detection in routine use and facilitates the simultaneous rapid detection of a broad panel of pathogens relevant in a context of suspected syphilis lesions.

Arboviruses in the Astrakhan region of Russia for 2018 season: The development of multiplex PCR assays and analysis of mosquitoes, ticks, and human blood sera.

The Astrakhan region of Russia is endemic for the number of arboviruses. In this paper, we describe the results of the detection of the list of neglected arboviruses in the Astrakhan region for the 2018 season. For the purpose of the study in-house PCR assays for detection of 18 arboviruses have been developed and validated using arboviruses obtained from Russian State Collection of Viruses. Pools of ticks (n=463) and mosquitoes (n=312) as well as 420 samples of human patients sera have been collected and analyzed. Using developed multiplex real-time PCR assays we were able to detect RNA of eight arboviruses (Crimean-Congo hemorrhagic fever virus, Dhori (Batken strain) virus, Batai virus, Tahyna virus, Uukuniemi virus, Inkoo virus, Sindbis virus and West Nile fever virus). All discovered viruses are capable of infecting humans causing fever and in some cases severe forms with hemorrhagic or neurologic symptoms. From PCR-positive samples, we were able to recover one isolate each of Dhori (Batken strain) virus and Crimean-Congo hemorrhagic fever virus which were further characterized by next-generation sequencing. The genomic sequences of identified Dhori (Batken strain) virus strain represent the most complete genome of Batken virus strain among previously reported.

Clinical Care of COVID-19 Patients in a Front-line ICU

No abstract available. Article truncated after 150 words. These are some clinical observations made after over the past 10 months, working in a busy COVID-19 ICU unit in Scottsdale, AZ. The opinions expressed here are those of the private practice authors. Overview of triage and rounding on large numbers of COVID-19 patients in the ICU service. Our approach to bedside care of our ICU service has required abbreviation for the sake of efficiency in the face of more than a doubling of our census. Our approach to rounding is opinion-based. We’ve been forced to cut corners where we can in order to survive. Our hospital uses the Abbott ID-Now® rapid point-of -are test for screening all COVID-19-asymptomatic patients admitted to our hospital, but due to its low sensitivity in hospitalized patients (1). We do not trust it to rule-out COVID-19 in patients with pneumonia being admitted to the ICU and also order an in-house PCR for such patients …

Clinical validation of an in-house quantitative real time PCR assay for cytomegalovirus infection using the 1st WHO International Standard in kidney transplant patients.

Introduction:Cytomegalovirus (CMV) is one of the most common agents of infection in solid organ transplant patients, with significant morbidity and mortality.Objective:This study aimed to establish a threshold for initiation of preemptive treatment. In addition, the study compared the performance of antigenemia with qPCR results.Study design:This was a prospective cohort study conducted in 2017 in a single kidney transplant center in Brazil. Clinical validation was performed by comparing in-house qPCR results, against standard of care at that time (Pp65 CMV Antigenemia). ROC curve analysis was performed to determine the ideal threshold for initiation of preemptive therapy based on the qPCR test results.Results:Two hundred and thirty two samples from 30 patients were tested with both antigenemia and qPCR, from which 163 (70.26%) were concordant (Kappa coefficient: 0.435, p<0.001; Spearman correlation: 0.663). PCR allowed for early diagnoses. The median number of days for the first positive result was 50 (range, 24-105) for antigenemia and 42 (range, 24-74) for qPCR (p<0.001). ROC curve analysis revealed that at a threshold of 3,430 IU/mL (Log 3.54), qPCR had a sensitivity of 97.06% and a specificity of 74.24% (AUC 0.92617 ± 0.0185, p<0.001), in the prediction of 10 cells/105 leukocytes by antigenemia and physician's decision to treat.Conclusions:CMV Pp65 antigenemia and CMV qPCR showed fair agreement and a moderate correlation in this study. The in-house qPCR was revealed to be an accurate method to determine CMV DNAemia in kidney transplant patients, resulting in positive results weeks before antigenemia.

Programmed cell death-1 rs11568821 and interleukin-28B rs12979860 polymorphisms in autoimmune hepatitis

BackgroundAutoimmune hepatitis (AIH) is a relatively rare chronic liver disease of unknown etiology. The genetic background affects susceptibility, clinical phenotype, and prognosis. The programmed cell death-1 rs11568821 polymorphism (PD1.3) has been associated with susceptibility to autoimmune diseases. The interleukin-28B (IL28B) rs12979860 polymorphism has been associated with steatosis, inflammation, and fibrosis in liver diseases. AimOur aim was to investigate for the first time the incidence and clinical significance of PD1.3 and IL28B rs12979860 in AIH. MethodsTwo hundred patients with AIH were evaluated, while 100 healthy subjects were used as controls. Genotyping was performed with in-house allelic discrimination End-Point PCR. ResultsThe SNP PD1.3/A was present in 36/200 (18%) AIH patients compared to 28/100 (28%) healthy controls (p = 0.065). The AA/GA genotypes were not associated with the mode of presentation of AIH, the histological grade or stage, the presence of cirrhosis, risk of disease progression, response to treatment and survival. The IL28B rs12979860 genotype distribution was CC 79/200 (39.5%), TT 36/200 (18%) and CT 85/200 (42.5%), in similar rates with healthy controls (p = 0.878). Inflammatory activity and fibrosis stage did not differ between CC homozygotes and CT/TT carriers. LDL cholesterol was significantly higher in CC than CT/TT patients (P = 0.027), though no differences was found regarding the presence of steatosis or steatohepatitis. On-treatment response to immunosuppressive treatment was not affected by the IL28B rs12979860 polymorphism. However, CC homozygotes AIH patients achieved treatment withdrawal in significantly higher rates (OR 2.3, 95%CI: 1.1–4.7, P = 0.02) irrespective of the presence of steatosis or steatohepatitis. ConclusionsThe PD1.3 and IL28B rs12979860 variants are unlikely to contribute to AIH susceptibility, disease presentation and prognosis. The IL28B rs12979860 is not associated with the presence of concurrent steatosis or steatohepatitis. However, although on-treatment response rates to immunosuppression were not affected by the IL28B rs12979860 polymorphism, AIH patients with CC homozygosity were more likely to achieve complete treatment withdrawal. This novel finding needs validation and further clarification from larger multicenter studies.

Çeşitli Klinik Örneklerden İzole Edilen Vankomisine Direçli Enterokok (VRE) Suşlarının Fenotipik ve Genotipik Değerlendirilmesi

Objective: Enterococci are among the important microorganism group worldwide due to their natural resistance to many antibiotics, especially their acquired resistance mechanisms against glycopeptides (vancomycin, teicoplanin). In this study, vancomycin and teicoplanin resistance were phenotypically determined in Vancomycin-resistant Enterococci strains obtained from various clinical specimens, and the presence of VanA, VanB and VanC was investigated by molecular methods. Method: A total of 30 VRE strains isolated from various clinical specimens at Microbiology Laboratory of Van Yuzuncu Yil University Faculty of Medicine Hospital between 2015 and 2018 were included in the study. MicroScan WalkAway 96 Plus (Beckman Coulter, USA) automated system was used for the identification of strains and determination of antibiotic resistance. Antimicrobial resistance to vancomycin and teicoplanin were also determined by gradient test method. Resistance genes were investigated by in-house PCR method performed in Inonu University Faculty of Medicine, Molecular Diagnostics Laboratory of The Department of Medical Microbiology using appropriate primers. Results: Identification tests revealed 29 strains as Enterococcus faecium and 1 as Enterococcus faecalis. While all strains were resistant to vancomycin and teicoplanin in the automated identification system, three isolates tested by gradient test method were found to be susceptible to these antibiotics. Molecular method of these three strains showed that there were no VanA, VanB and VanC genes. While VanA gene was detected in 27 strains, VanB gene was detected in one strain, and VanC gene was not detected. Resistance detection by gradient test and molecular method were found to be 100% compatible in the identification of vancomycinresistance in enterococci isolates. Conclusion: Gradient test method is reliable in determining glycopeptide resistance of enterococcal isolates in clinical microbiology laboratory.

1221. Evaluation of the FilmArray® Global Fever Panel

BackgroundAcute Febrile Illness (AFI) is caused by a diverse set of pathogens. The FilmArray Global Fever (GF) Panel, developed by BioFire Defense in collaboration with the U.S. Department of Defense and NIAID, uses an automated, multiplex nested PCR system to evaluate whole blood samples for multiple pathogens simultaneously in under an hour.MethodsBioFire Defense conducted analytical performance studies to show sensitivity (LoD), inclusivity, and specificity (exclusivity), and a prospective clinical study to evaluate the positive percent agreement (PPA) and negative percent agreement (NPA) of the GF Panel. The results of these studies will be reported in two submissions to the US FDA.ResultsThe analytical performance demonstrated the ability to accurately detect multiple pathogens, including Category A biothreat pathogens. Eleven locations around the world tested 1,865 specimens on the GF Panel. The rate of positive detections was 35% (652/1865), with Plasmodium spp. accounting for the majority of positives (53.4%, 348/652) and dengue virus the second most (40.5%, 264/652). Other detected pathogens include Leptospira spp., West Nile virus, Zika virus, Leishmania spp., Crimean-Congo hemorrhagic fever virus, and chikungunya virus. Twenty-eight (28) specimens had more than one detected pathogen (4.3% of positive specimens). Comparator testing consisted of in-house developed PCR assays followed by bidirectional sequencing. PPA between GF Panel and comparator testing ranged between 92.7-100%, and the NPA ranged between 99.3-100%. In all cases, discrepancies coincided with analytes that were near the limit of detection of the GF Panel and comparator assays. When the GF Panel result was compared to site-specific malaria testing, the PPA ranged between 94.7-100% and the NPA ranged between 43.3-100%. Analysis of the NPA suggests that the GF Panel is more sensitive than microscopy, producing “discrepancies” for this comparison. The wide range in NPA between sites could be due to variation in microscopy technique; the GF Panel eliminates such variation because it is fully automated.ConclusionThe results show that the FilmArray GF Panel could aid in rapid and actionable AFI diagnosis caused by multiple, sometimes co-occurring, pathogens.DisclosuresJared R. Helm, PhD, BioFire Defense (Employee) Brian Jones, PhD, BioFire Defense, LLC (Employee, own stock) Corike Toxopeus, PhD, BioFire Defense, LLC. (Employee, stock owner) David S. Rabiger, PhD, BioFire Defense (Employee) Mark Gurling, PhD, BioFire Defense, LLC (Employee) Olivia Jackson, n/a, BioFire Defense (Employee) Marissa Burton, BS Biology, Biomerieux, Inc. (Shareholder) Cynthia Andjelic, PhD, BioFire Defense (Employee, Other Financial or Material Support, Own stocks) Cynthia L. Phillips, PhD, BioFire Defense (Employee, Scientific Research Study Investigator, Shareholder)BioFire Defense (Employee, Scientific Research Study Investigator, Shareholder)

56. Could Maturation Effect Contribute to In-hospital Mortality Improvement During COVID-19 Pandemic?

BackgroundThe surge of COVID-19 cases overwhelms hospital systems necessitating rapid learning of the disease process and management. During the course of a novel pandemic, multiple interventions are rapidly implemented to improve patient outcomes. When evaluating efficacy of individual interventions, one should account for the simultaneous improvements in knowledge and experience of healthcare providers (HCP), known as the maturation effect. We hypothesized that multiple processes rapidly implemented, along with the maturation effect would result in improved survival of COVID-19 patients hospitalized over the course of the pandemic.MethodsThis retrospective study was done at Henry Ford Hospital (HFH), a 900-bed tertiary care facility in Detroit, Michigan. The first COVID-19 patient was hospitalized on March 10, 2020 followed by a rapid surge of cases. We evaluated the trends of in-hospital case fatality rate of COVID-19 PCR positive patients through April 28, 2020. Time-points of sequential implementation of key measures for the management of COVID-19 patients were recorded.ResultsA total of 1023 COVID-19 patients were hospitalized during the study period with 165 deaths (16 %). Case fatality rate during week one was 42% and down trended over time (Figure 1). Key measures were sequentially implemented over the course of the study period as shown in Figure 1. These included development and implementation of in-house PCR testing, dedicated infectious diseases COVID-19 rounding teams, treatment guidelines and algorithms, and early steroid use in hypoxic patients. Figure 2 demonstrates that despite the surge of COVID-19 admissions, mortality continued to improve over time.Figure 1. Trend line of all-cause in-house morality over time Figure 2. Mortality by admission date over time ConclusionMaturation effect takes into consideration that regardless of individual interventions, HCP improve their knowledge of the disease process and treatment over time leading to better outcomes. Our study shows the possibility of the maturation effect leading to improved survival in hospitalized COVID-19 patients. The maturation effect should be accounted for when evaluating the effect of specific interventions for COVID-19.DisclosuresMarcus Zervos, MD, Melinta Therapeutics (Grant/Research Support)

Comparison of two commercial and one in-house real-time PCR assays for the diagnosis of bacterial gastroenteritis.

IntroductionThe aim of the study was a comparative evaluation of in-house real-time PCR and commercial real-time PCR (Fast Track Diagnostics (FTD), ampliCube/Mikrogen) targeting enteropathogenic bacteria from stool in preparation of Regulation (EU) 2017/746 on in vitro diagnostic medical devices.MethodsBoth 241 stool samples from patients and 100 samples from German laboratory control schemes (“Ringversuche”) were used to comparatively assess in-house real-time PCR, the FTD bacterial gastroenteritis kit, and the ampliCube gastrointestinal bacterial panels 1&2 either with the in-house PCRs as gold standard and as a test comparison without gold standard applying latent class analysis. Sensitivity, specificity, intra- and inter-assay variation and Cohen’s kappa were assessed.ResultsIn comparison with the gold standard, sensitivity was 75–100% for strongly positive samples, 20–100% for weakly positive samples, and specificity ranged from 96 to 100%. Latent class analysis suggested that sensitivity ranges from 81.2 to 100% and specificity from 58.5 to 100%. Cohen’s kappa varied between moderate and nearly perfect agreement, intra- and inter-assay variation was 1–3 to 1–4 Ct values.ConclusionAcceptable agreement and performance characteristics suggested replaceability of the in-house PCR assays by the commercial approaches.

Evaluation of modified carbapenem inactivation method with CARBA NP, MHT and real-time PCR for detection of carbapenemase-producing Enterobacteriaceae

Background: Carbapenemase-producing Enterobacteriaceae (CPE) are a global emerging threat with high transmissibility, multidrug-resistance and limited treatment options. Reliable detection is important for prevention, containment of these pathogens as well as for antimicrobial stewardship. The modified carbapenem inactivation method (mCIM) is an inexpensive and straightforward phenotypic tool for detecting carbapenemase activity. The aim of this study was to evaluate the performance of mCIM for detecting carbapenemase activity by comparing with CARBA NP, Modified Hodge Test (MHT) and Real-time PCR. Methods and materials: A total of 94 carbapenem-nonsusceptible Enterobacteriaceae comprising of E. coli (n = 43), Klebsiella species (n = 33) and Enterobacter species (n = 18) were analyzed by mCIM, MHT and CARBA NP according to the Clinical and Standard Institute (CLSI) guidelines (M100-S27). In-house Real-time PCR was performed to confirm carbapenemase genes (KPC, NDM, VIM, IMP, OXA-48). The sensitivity and specificity of mCIM, MHT and CARBA NP was determined by comparing the results with Real-time PCR. Klebsiella pneumoniae BAA-1706 strain and Klebsiella pneumoniae ATCC 700603 both carbapenemase non-producers were used as negative controls. Results: Of the ninety-four isolates analyzed by real-time PCR, 47 isolates expressed NDM and 5 isolates expressed OXA. 38 isolates showed co-expression of NDM and OXA. Two showed co-expression of KPC and NDM. One Enterobacter aerogenes showed co-expression of NDM and VIM, while another showed co-expression of NDM and IMP. The sensitivity of mCIM, CARBA NP and MHT were 100%, 84% and 74% respectively. mCIM showed higher sensitivity than CARBA NP & MHT. The mCIM assay perfectly correlated with the genotypic findings of the 94 isolates with 100% sensitivity and specificity for CPE detection. Conclusion: The results of our study show that mCIM is suitable and reliable for detecting carbapenemase-producing Enterobacteriaceae than CARBA NP and MHT. Compared to molecular method, mCIM is less expensive and can be readily adopted in clinical microbiology laboratory.

Introduction of an in-house real-time PCR assay to detect severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) in Sri Lanka: challenges and issues

Official publication of the College of Community Physicians of Sri Lanka and was established in 1995. Full text articles available.Journal of the College of Community Physicians of Sri Lanka (JCCPSL) has been recognised by the International Committee of Medical Journal Editors (ICMJE) as a publication following the ICMJE Recommendations. The journal is indexed in DOAJ.Manuscripts are only accepted online through the portal of the JCCPSL https://jccpsl.sljol.info/ . The prospective authors must read carefully the section on Author Guidelines and prepare their manuscript accordingly. To submit a manuscript or to check the status of a submission the authors must first be registered in the JCCPSL Editorial Management System using an existing or newly created username and password. Next, choose the ‘Start Submission’ link and follow the five-step article submission process which is a simple and self-guided process.

Evaluation of the molecular detection of ciprofloxacin resistance in Neisseria gonorrhoeae by the ResistancePlus GC assay (SpeeDx)

There is growing concern due to the emergence of multidrug resistance in Neisseria gonorrhoeae. A rapid molecular test which guides and provides antimicrobial susceptibility knowledge prior to start of treatment is needed. This study evaluated the clinical performance of the ResistancePlus GC assay compared to in-house PCR and antimicrobial susceptibility results for ciprofloxacin resistance. Samples were selected from a range of sites with corresponding cultures isolated from the same patient episode. The ResistancePlus GC assay displayed high sensitivity for N. gonorrhoeae detection (98.5%) and gyrA detection (97.1%). There was high agreement (98.9%) between the ResistancePlus GC assay and culture phenotype. Mixed population testing showed that the assay was able to detect resistance in a sample containing a minority variant of 27% resistant. The ResistancePlus GC assay performed well and could be used to provide a clinically relevant indication of ciprofloxacin susceptibility for the treatment of gonorrhoea.

Performance Evaluation of a Commercial Real-Time PCR Assay and of an In-House Real-Time PCR for Trypanosoma cruzi DNA Detection in a Tropical Medicine Reference Center, Northern Italy.

Chagas disease, a neglected protozoal disease endemic in Latin America, is also currently considered an emerging threat in nonendemic areas because of population movements. The detection of Trypanosoma cruzi DNA is increasingly being considered as important evidence to support Chagas disease diagnoses. However, further performance evaluation of molecular assays is useful for a standardization of strategy considering the whole process in routine diagnosis, especially for the different settings such as endemic and nonendemic countries. Seventy-five samples were collected from subjects screened for Chagas disease in Italy. The DNA was isolated from blood using automated extraction. We evaluated the performance of the commercial RealCycler® CHAG kit (pmPCR) based on satellite DNA (SatDNA) and of an in-house real-time PCR (ihPCR) targeting Sat and kinetoplast (k) DNAs, using the concordance of two serology assays as a reference standard. The sensitivity of kDNA and SatDNA tests by ihPCR and SatDNA by pmPCR were 14.29% (95% confidence interval (CI) 6.38 to 26.22), 7.14% (95% CI 1.98 to 17.29), and 7.14% (95% CI 1.98 to 17.29), respectively. Specificity was 100% for all PCR assays and targets. Overall, our results suggest that the preferred approach for clinical laboratories is to combine the kDNA and SatDNA as targets in order to minimize false-negative results increasing sensitivity.

Evaluation of the performance of an in-house duplex PCR assay targeting the IS6110 and rpoB genes for tuberculosis diagnosis in Cameroon

BackgroundTuberculosis (TB) remains a major public health concern in many low-income countries accounting for approximately two-thirds of deaths in people living with human immunodeficiency virus (HIV) infection. With prompt, accurate and appropriate treatment, almost all TB disease can be cured. The present study was to evaluate the diagnostic performance of an in-house duplex PCR (D-PCR) using IS1610 and rpoB specific primers in sputum samples from TB suspected patients.MethodsA hospital-based cross-sectional study was conducted at the Limbe and Buea Regional Hospitals of the South West Region of Cameroon from June 2016 to April 2017. Sputum samples, decontaminated with hypertonic saline/sodium hydroxide solution were centrifuged and pellets processed for smear microscopy, culture and DNA extraction. Suspected inhibition was resolved by serial dilution of genomic DNA. Results were compared to culture as gold standard as well as a Composite Reference Standard (CRS).ResultsA total of 129 participants aged between 5 to 82 years were enrolled in to the study. The median age of the participants was 37 years (interquartile range, IQR: 27–50 years), with 54.3% being male. Forty-seven samples (36.4%) were positive by direct sputum microscopy, 49 (38%) by microscopy after concentration, 51 (39.5%) by culture and 62 (40.1%) by D-PCR. PCR inhibition was resolved in 85.7% (18/21) of the samples that had inhibition. The overall sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios and area under the curve AUC) of the D-PCR was 93.5, 94, 94, 94%, 15.6, 0.005 and 89.0% respectively using CRS as reference. The sensitivities of D-PCR observed among different sample categories were 95.7, 87.5 and 87.5% for smear-and culture-positives, smear-negative/culture-positive, and clinically diagnosed cases respectively.ConclusionIS1610 and rpoB duplex PCR using relatively cheap decontamination and DNA extraction methods in addition to simple serial dilutions to resolve PCR inhibition shows high sensitivity in the diagnosis of paucibacillary tuberculosis.

Increasing Mupirocin Resistance Among MRSA Nasal Surveillance Isolates in the Chicago Area

Background: In 2005, our healthcare system began universal admission screening for nasal colonization with MRSA and decolonization of MRSA positive patients with mupirocin. In 2010–2012, we studied the impact of nasal MRSA decolonization and concluded that it does not add benefit when contact precautions are used; plus, it resulted in increased rates of mupirocin resistance up to 9.4% in 2012. In September 2012 routine decolonization of hospitalized patients was discontinued. In the 2 years following discontinuation of mupirocin use for decolonization of MRSA carriers, the rate of mupirocin resistance gradually declined. We undertook a contemporary review of mupirocin resistance rates to ensure that the rates were stable. Methods: NorthShore University HealthSystem, Illinois, consists of 4 hospitals in the northern suburbs of Chicago, with 750 beds and 60,000 annual admissions. Admission nasal swab samples were collected from at-risk hospitalized patients based on a risk-adjusted algorithm. Nasal swabs were tested using the BD MAX MRSA assay. Positive samples were cultured onto BD BBL CHROMagar MRSA to recover the organism and were tested for the mupA gene, which confers high-level mupirocin resistance using an in-house PCR test. Data for mupirocin orders were provided by the pharmacy. Results: Mupirocin resistance rates and prescription orders are shown in Figs. 1 and 2. Conclusions: Mupirocin resistance rates plateaued between 2012 and 2014 and then increased from 9.1% in 2015 to 18.1% in 2019, despite discontinuation of routine decolonization of hospitalized patients. The reason for the increase is unclear; inpatient mupirocin orders were stable from 2015 to 2017.Funding: NoneDisclosures: None

Development, Optimization, and Validation of a Multiplex Real-Time PCR Assay on the BD MAX Platform for Routine Diagnosis of Acanthamoeba Keratitis

The reported number of cases of Acanthamoeba amebic keratitis (AK) is continually increasing. Molecular diagnosis has become the first choice of ophthalmologists for identifying and confirming this clinically problematic diagnosis. However, in-house molecular diagnostic procedures are time-consuming and may not be compatible with the urgency of the situation. In this study, a previous in-house AK-PCR technique was adapted for use on BD MAX (Becton Dickinson, Heidelberg, Germany), a fully integrated, automated platform for molecular biology, for the rapid routine diagnosis of AK. Different protocols were compared to optimize DNA extraction from Acanthamoeba cysts. The analytical parameters of the AK-BD MAX PCR were evaluated. Thirty-two samples were simultaneously tested with AK-BD MAX PCR and the original AK-PCR from which it was developed. A thermal-shock pretreatment protocol was validated. The analytical parameters obtained with BD MAX were similar to those obtained with the previous in-house AK-PCR method. The performance of AK-BD MAX PCR was then assessed for routine testing on 40 clinical samples, mostly corneal scrapings. Frozen, ready-to-use, in-house PCR premixes were stable over 8 months. Overall, 34 of the 40 clinical samples (85%) were considered to be true negatives; 4 (10%), probable AK; and 2 (5%), possible AK. This newly developed AK-BD MAX PCR is reliable, rapid, and efficient, and should facilitate Acanthamoeba keratitis diagnosis.

Early Detection and Control of Methicillin resistant Staphylococcus aureus Outbreak in an Intensive Care Unit.

Although Methicillin resistant Staphylococcus aureus (MRSA) is one of the major pathogens of healthcare associated infections, we had only sporadic cases in our intensive care unit (ICU) for years. To investigate the sudden increase in the number of MRSA cases in ICU. Descriptive study. From the 5th December 2016 to 26th January 2017, we detected 11 new MRSA cases in ICU. Screening of 73 ICU healthcare workers (HCWs) and screening of 13 patients was performed for outbreak investigation. Nine clinical isolates available in stocks and eight screening MRSA isolates were included in molecular studies. PFGE, spa-mecA-mecC-PVL in-house multiplex PCR assay and spa typing, SCCmec typing were performed for all isolates. Sequence type of the representative strain was determined by Multi-Locus Sequence typing (MLST). All strains were mecA positive, PVL negative, and have the same antimicrobial susceptibility pattern except for two strains. All clinical, two patient screening and three nasal isolates of HCWs showed the same pulsotype, named clone A. The spa type of outbreak isolates is t030 and the SCCmec type is SCCmecIII; the MLST type of representative strain is ST239 (PFGE pulsotype A, ST239-SCCmecIII-t030). Unrelated three isolates had PFGE pulsotype B-SCCmecI-t030, PFGE pulsotype C-SCCmecIII-t459, PFGE pulsotype D-SCCmecIII. Molecular typing techniques are the cornerstones for the investigation of outbreaks. Infection control measures, such as enhancing cleaning procedures, promoting hand hygiene, should be enforced in the ICU unit. All patients, including those who have already been discharged to other departments, must be put on contact isolation. HCWs carrying the MRSA strains could be offered decolonization.

Occult Hepatitis C Virus Infection among Hemodialysis Patients: An Iranian Experience.

Patients with chronic kidney failure and those undergoing chronic hemodialysis (CHD) treatment are at high risk of infection with hepatitis C virus (HCV). The incidence of occult HCV infection (OCI) in CHD remains controversial and the real burden of HCV in this population may be affected by the rate of OCI. This study evaluates the molecular assessment of OCI in CHD in an Iranian population. All subjects on CHD in the South Khorasan province of Iran were invited for participation in the study. Whole blood samples were taken and serological, clinical, and demographic information was recorded. HCV-RNAs were checked in serum and peripheral blood mononuclear cells (PBMCs) using an in-house semi-nested PCR assay. Viral load was determined using a real-time PCR-based quantification kit. Sequencing was performed to determine genotypes. Overall, 120 cases participated in the study; 57.5% were male and the rest were female. In serum samples, no positive case was found for HCV-RNA. In PBMC samples, 2/120 (1.6%) were positive for HCV-RNA (95% CI, 0.002 to 0.059); the mean age of OCI positive cases was 37.5 ± 19.2 years which was significantly lower than OCI negative cases (P = 0.026). Only one case had detectable viral load which was 49 IU/mL. The only HCV genotype identified was 1a. This study showed that there is a risk of OCI among CHD patients; the very low and undetectable viral loads of OCI warrant further follow-up molecular testing for earlier diagnosis and treatment in the era of DAA.

Redondovirus DNA in human respiratory samples

Redondovirus (ReDoV) is a recently discovered circular, Rep-encoding single-stranded DNA (CRESS-DNA) virus in humans. Its pathogenesis and clinical associations are still completely unknown. The presence of ReDoV DNA was investigated in biological specimens of 543 Italian subjects by in-house developed PCR assays. The overall ReDoV prevalence was about 4% (23 of 543 samples). The virus was detected in 22 of 209 (11 %) respiratory samples. One stool sample was also ReDoV positive. Viral DNA was not found in blood samples from immunocompetent and immunosuppressed subjects and cerebrospinal fluids from patients with neurological diseases. Genomic nucleotide differences were detected among the ReDoV isolates by sequencing a 582-nucleotide fragment of the capsid gene of the viral genome. The results demonstrate that ReDoV is mainly present in the respiratory tract of infected people. Further investigations are needed to reveal possible clinical implications of this new CRESS-DNA virus in humans.

Evaluation of four commercial real-time PCR assays for the detection of lymphogranuloma venereum in Chlamydia trachomatis-positive anorectal samples

ObjectivesLymphogranuloma venereum (LGV) is a sexually transmitted infection (STI) caused by Chlamydia trachomatis (CT) genovars L. The identification of LGV is of therapeutic interest because treatment requires 3 weeks of doxycycline compared with 1 week for infection with a non-L strain. The aim of this study was to evaluate the performance of four commercial real-time PCR kits in comparison with the reference methods used for LGV diagnosis by the French National Reference Centre (NRC) for bacterial STIs. MethodsA total of 215 French CT-positive anorectal specimens collected consecutively in 2017 were used (66 LGV and 149 non-LGV). Among these, 92 were collected from symptomatic men who have sex with men (MSM) and 123 from asymptomatic MSM using pre-exposure prophylaxis. Four commercial assays were evaluated; a single-plex assay RealCycler CHSL kit (Progenie Molecular), tested on all the specimens, and three multiplex kits, the RealCycler Universal ULCGEN (Progenie Molecular), the Allplex Genital Ulcer Assay (Seegene) and the VIASURE Haemophilus ducreyi + CT LGV Real Time PCR Detection kit (CerTest Biotec), tested on the 92 samples from symptomatic MSM. Clinical performance was determined in comparison to the in-house real time PCR targeting the pmpH and the ompA gene sequencing. ResultsOverall agreement ranged between 91.3% and 100% (95% CI 83.7–100%) with very good Kappa index values (>0.8). The clinical sensitivities and specificities varied between 91% and 100% (95% CI 80.8–100%), and 97% and 100% (95% CI 87.1–100%), respectively, with some kits performing better than others. DiscussionThe four assays showed very good performance for the detection of LGV on anorectal specimens.