In-house qPCR Research Articles

P314 TNF gene activation used as criterion to discontinue biologic therapy – A patient series introducing the NovaPrime TNF kit

Abstract Background Severe inflammatory bowel disease can be successfully treated with biologic drugs such as anti-TNF. However, there is no consensus on if and how to stop treatment. Our group has earlier shown that normalization of TNF gene expression may be a beneficial prognostic factor when attempting to discontinue biologic therapy. We here present data from a series of patients in endoscopic remission and with normalized TNF gene expression. Methods Severe inflammatory bowel disease can be successfully treated with biologic drugs such as anti-TNF. However, there is no consensus on if and how to stop treatment. Our group has earlier shown that normalization of TNF gene expression may be a beneficial prognostic factor when attempting to discontinue biologic therapy. We here present data from a series of patients in endoscopic remission and with normalized TNF gene expression. A total of 91 patients were recruited at hospitals in Norway and Italy: 55 patients (32 UC, 23 CD) in remission (picked by normalization of TNF using our in-house TNF qPCR), and for comparison: 14 patients with active disease, and 22 normal controls. Mucosal samples taken prior to drug discontinuation from all patients were re-measured for TNF mRNA by the NovaPrime TNF kit. Results A clear difference could be seen between normal controls and patients with active disease, showing highly elevated TNF mRNA values in the active disease group. The patients in remission had values resembling the normal controls with values generally below a cut-off of 9060 copies/µg total RNA by the NovaPrime TNF kit. The patients in remission were followed for up to 5 years noting two endpoints: relapse triggering any adjustment of medication, and relapse triggering restart of biologics. Median survival time before adjusting medication was 12 months for ulcerative colitis and 17 months for Crohn’s disease. Median time to restart of biologics was 21 months (2 – 40) overall, and after 3 years 43% still were not in need of biologic therapy. Conclusion We conclude that the NovaPrime TNF kit yields consistent readings comparable to our in-house assay, and that this industrial standard kit enables any PCR lab to perform TNF gene expression in samples from intestinal mucosa. A considerable proportion of patients with normalized TNF gene expression can maintain long term remission without biologic therapy, relieving health care costs and potential side effects of long-term therapy.

Increased sensitivity of a new commercial reverse transcriptase-quantitative PCR for the detection of Pneumocystis jirovecii in respiratory specimens

The fungus Pneumocystis needs to be detected early in respiratory samples to prevent pneumonia in immunocompromised hosts. We evaluated a new commercial RTqPCR on 213 respiratory samples to detect Pneumocystis and found it more sensitive and faster than our routine sensitive in-house qPCR assay.

Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant.

IntroductionThe SARS-CoV-2 pandemic has been associated with the occurrence since summer 2020 of several viral variants that overlapped or succeeded each other in time. Those of current concern harbor mutations within the spike receptor binding domain (RBD) that may be associated with viral escape to immune responses. In our geographical area a viral variant we named Marseille-4 harbors a S477 N substitution in this RBD.Materials and methodsWe aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay with a hydrolysis probe that specifically detects the SARS-CoV-2 Marseille-4 variant.ResultsAll 6 cDNA samples from Marseille-4 variant strains identified in our institute by genome next-generation sequencing (NGS) tested positive using our Marseille-4 specific qPCR, whereas all 32 cDNA samples from other variants tested negative. In addition, 39/42 (93 %) respiratory samples identified by NGS as containing a Marseille-4 variant strain and 0/26 samples identified as containing non-Marseille-4 variant strains were positive. Finally, 2018/3960 (51%) patients SARS-CoV-2-diagnosed in our institute, 10/277 (3.6 %) respiratory samples collected in Algeria, and none of 207 respiratory samples collected in Senegal, Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR.DiscussionOur in-house qPCR system was found reliable to detect specifically the Marseille-4 variant and allowed estimating it is involved in about half of our SARS-CoV-2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical characterics based on comprehensive sets of data.

Rapid Testing and Interventions to Control Legionella Proliferation following a Legionnaires' Disease Outbreak Associated with Cooling Towers.

Most literature to date on the use of rapid Legionella tests have compared different sampling and analytical techniques, with few studies on real-world experiences using such methods. Rapid tests offer a significantly shorter feedback loop on the effectiveness of the controls. This study involved a complex of five factories, three of which had a history of Legionella contamination in their cooling water distribution system. Multiple sampling locations were utilised to take monthly water samples over 39 months to analyse for Legionella by both culture and quantitative polymerase chain reaction (qPCR). Routine monitoring gave no positive Legionella results by culture (n = 330); however, samples were frequently (68%) positive by qPCR for Legionella spp. (n = 1564). Legionella spp. qPCR assay was thus found to be a good indicator of cooling tower system health and suitable as a routine monitoring tool. An in-house qPCR limit of 5000 genomic units (GU)/L Legionella spp. was established to trigger investigation and remedial action. This approach facilitated swift remedial action to prevent Legionella proliferation to levels that may represent a public health risk. Cooling tower operators may have to set their own action levels for their own systems; however, in this study, 5000 GU/L was deemed appropriate and pragmatic.

Large fragment demineralization: an alternative pretreatment for forensic DNA typing of bones.

In forensic genetics, the analysis of postmortem bones is one of the most challenging due to the low quantity of degraded endogenous DNA. The most widely used approach for sample preparation, in those cases, is pulverizing the bone. However, processing pulverized bone is extremely delicate, requiring strict laboratory conditions and operating procedures. In fact, several recent publications have focused on non-powder approaches. The objectives of this study were, thus, to validate a non-powder protocol for DNA extraction from forensic bones and an alternative pretreatment, large fragment demineralization (LFD). Thirty human femurs and tibiae received by the Legal Medicine Institute of Brescia, Italy, were included in the study. Bone powder and one transversal section of the diaphysis were sampled from each bone. DNA extraction from the powder was carried out using PrepFiler BTA (BTA), while the transversal section was submitted to the alternative demineralizing pretreatment (LFD) followed by DNA extraction using the QIAamp DNA Investigator. DNA extracts were assessed for human DNA quantity and degradation by means of a validated in-house qPCR assay and amplified with commercial kits. Inhibition assessment was carried out through Quality Sensor analysis using 24plex QS Kit. The differences in quantity, quality of human DNA, and number of alleles detected between both methods were comparable and not statistically significant. We propose the use of the LFD protocol as a complementary approach capable of confirming the genotypes or detect alleles not observed using BTA, without the need for pulverization.

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.

Comparison of the Diagnostic Performance of qPCR, Sanger Sequencing, and Whole-Genome Sequencing in Determining Clarithromycin and Levofloxacin Resistance in Helicobacter pylori.

Helicobacter pylori antibiotic resistance is increasing worldwide, emphasizing the urgent need for more rapid resistance detection prior to the administration of H. pylori eradication regimens. Macrolides and fluoroquinolones are widely used to treat H. pylori. In this study, we aimed to compare the diagnostic performance of A) 23SrDNA qPCR (with melting curve analysis) and an in-house developed gyrA qPCR followed by Sanger sequencing with a commercial IVD-marked hybridization probe assay (for 23SrDNA and gyrA) using 142 gastric biopsies (skipping culturing) and B) the same two qPCR for 23SrDNA and gyrA (including Sanger sequencing) with whole-genome sequencing (WGS) and phenotypic characterization of clarithromycin and levofloxacin resistance using 76 cultured isolates. The sensitivity of both qPCRs was 100% compared to that of the commercial IVD-marked hybridization probe assay for the detection of H. pylori in gastric biopsies (without resistance testing). The specificity of the qPCR gyrA followed by Sanger sequencing was 100%, indicating that the best sequence identity was always H. pylori. The results show good agreement between molecular tests, especially between qPCR (inclusive Sanger sequencing) and WGS. Discrepancies (concerning mutated or wild type of positive H. pylori gastric biopsies) were observed between Sanger sequencing of the gyrA gene and the corresponding commercial hybridization probe assay, mostly because the high sequence diversity of the gyrA gene even at positions adjacent to the relevant codons of 87 and 91 interfered with obtaining correct results from the hybridization probe assay. Interestingly, we found several mixed sequences, indicating mixed populations in the gastric biopsies (direct detection without culturing). There was a high percentage of both levofloxacin and clarithromycin resistance in gastric biopsies (both between 22% and 29%, direct detection in gastric biopsies). Therefore, we recommend analyzing both targets in parallel. We confirmed that phenotypic resistance is highly correlated with the associated mutations. We concluded that the two qPCR followed by Sanger sequencing of the gyrA gene is a fast, cost-effective and comprehensive method for resistance testing of H. pylori directly in gastric biopsies.

Revised Exon Structure of l-DOPA Decarboxylase (DDC) Reveals Novel Splice Variants Associated with Colorectal Cancer Progression.

Colorectal cancer (CRC) is a highly heterogenous malignancy with an increased mortality rate. Aberrant splicing is a typical characteristic of CRC, and several studies support the prognostic value of particular transcripts in this malignancy. l-DOPA decarboxylase (DDC) and its derivative neurotransmitters play a multifaceted role in physiological and pathological states. Our recent data support the existence of 6 DDC novel exons. In this study, we investigated the existence of additional DDC novel exons and transcripts, and their potential value as biomarkers in CRC. Next-generation sequencing (NGS) in 55 human cell lines coupled with Sanger sequencing uncovered 3 additional DDC novel exons and 20 splice variants, 7 of which likely encode new protein isoforms. Eight of these transcripts were detected in CRC. An in-house qPCR assay was developed and performed in TNM II and III CRC samples for the quantification of transcripts bearing novel exons. Extensive biostatistical analysis uncovered the prognostic value of specific DDC novel exons for patients’ disease-free and overall survival. The revised DDC exon structure, the putative protein isoforms with distinct functions, and the prognostic value of novel exons highlight the pivotal role of DDC in CRC progression, indicating its potential utility as a molecular biomarker in CRC.

Time to act–assessing variations in qPCR analyses in biological nitrogen removal with examples from partial nitritation/anammox systems

Quantitative PCR (qPCR) is broadly used as the gold standard to quantify microbial community fractions in environmental microbiology and biotechnology. Benchmarking efforts to ensure the comparability of qPCR data for environmental bioprocesses are still scarce. Also, for partial nitritation/anammox (PN/A) systems systematic investigations are still missing, rendering meta-analysis of reported trends and generic insights potentially precarious. We report a baseline investigation of the variability of qPCR-based analyses for microbial communities applied to PN/A systems. Round-robin testing was performed for three PN/A biomass samples in six laboratories, using the respective in-house DNA extraction and qPCR protocols. The concentration of extracted DNA was significantly different between labs, ranged between 2.7 and 328 ng mg−1 wet biomass. The variability among the qPCR abundance data of different labs was very high (1−7 log fold) but differed for different target microbial guilds. DNA extraction caused maximum variation (3–7 log fold), followed by the primers (1–3 log fold). These insights will guide environmental scientists and engineers as well as treatment plant operators in the interpretation of qPCR data.

The environmental contribution to the dissemination of carbapenem and (fluoro)quinolone resistance genes by discharged and reused wastewater effluents: The role of cellular and extracellular DNA

Wastewater treatment plants (WWTPs) are major reservoirs and sources for the dissemination of antibiotic resistance into the environment. In this study, the population dynamics of two full-scale WWTPs was characterized along different sampling points, including the reused effluents, in both cellular and extracellular DNA samples. The analysis was performed by high throughput sequencing targeting the 16S rRNA V4 gene region and by three in-house TaqMan multiplex qPCR assays that detect and quantify the most clinically relevant and globally distributed carbapenem (bla) and (fluoro)quinolone (qnr) resistance genes. The obtained results identify the biological treatment as the crucial step on tailoring the wastewater bacterial community, which is thereafter maintained in both discharged and reused effluents. The influent bacterial community does not alter the WWTP core community, although it clearly contributes for the introduction and spread of antibiotic resistance to the in-house bacteria. The presence of high concentrations of bla and qnr genes was not only detected in the wastewater influents and discharged effluents, but also in the reused effluents, which therefore represent another gateway for antibiotic resistant bacteria and genes into the environment and directly to the human populations. Moreover, and together with the study of the cellular DNA, it was described for the first time the role of the extracellular DNA in the dissemination of carbapenem and (fluoro)quinolone resistance, as well as the impact of the wastewater treatment process on this DNA fraction. Altogether, the results prove that the current wastewater treatments are inefficient in the removal of antibiotic resistant bacteria and genes and reinforce that targeted treatments must be developed and implemented at full-scale in the WWTPs for wastewater reuse to become a safe and sustainable practice, able to be implemented in areas such as agricultural irrigation.

Mycoplasma genitalium prevalence in Welsh sexual health patients: Low antimicrobial resistance markers and no association of symptoms to bacterial load

ObjectivesMycoplasma genitalium (MG) is a common cause of sexually transmitted infection, however no prevalence data is available for Wales. MG was detected by qPCR (quantitative) as well as two separate SpeeDx commercial assays, and related to clinical symptoms, age, gender and sample type. MethodsCervical swabs, urethral swabs and/or urine were collected from 1000 patients at walk-in sexual health clinics at 3 Welsh health centres from October 2017–October 2018. Extracted DNA was investigated to determine concordance between an in-house quantitative PCR, SpeeDx ResistancePlus® MG and the SpeeDx MG + parC (beta 2) assays; mutations in parC were substantiated by Sanger sequencing. ResultsMG was detected in 17/600 female patients (2.7%) and 13/400 (3.5%) male patients, with a 100% concordance between in-house qPCR and both SpeeDx assays. Macrolide resistance was low (relative to other studies), but more common in males (4/13; 30.8%) than females (2/17; 11.8%) and the only fluoroquinolone resistant sample (3.4% overall) was also macrolide resistant and detected from an MSM. Vaginitis was clinically apparent in 12/17 MG-positive females (2 with additional cervicitis, 1 with additional pelvic inflammatory disease), while 7 MG-positive males were asymptomatic. MG bacterial load did not correlate to clinical symptoms and females (4559 ± 1646/ml) had significantly lower MG load than males (84,714 ± 41,813/ml; p = 0.0429). ConclusionsMG prevalence and antibiotic resistance in Welsh sexual health clinics is low. MG bacterial load did not correlate to clinical presentation, men have higher MG load/ml in urine than women, genders have different age bias for MG prevalence and urine and swabs are equivalent for detecting MG.

The respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease.

IntroductionExacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), and respiratory bacterial and viral infections are an important trigger. However, using conventional diagnostic techniques, a causative agent is not always found. Metagenomic next-generation sequencing (mNGS) allows analysis of the complete virome, but has not yet been applied in COPD exacerbations.ObjectivesTo study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS.Study design88 nasopharyngeal swabs from 63 patients from the Bergen COPD Exacerbation Study (2006–2010) were analysed by mNGS and in-house qPCR for respiratory viruses. Both DNA and RNA were sequenced simultaneously using an Illumina library preparation protocol with in-house adaptations.ResultsBy mNGS, 24/88 samples tested positive. Sensitivity and specificity, as compared with PCR, were 96% and 98% for diagnostic targets (23/24 and 1093/1120, respectively). Additional viral pathogens detected by mNGS were herpes simplex virus type 1 and coronavirus OC43. A positive correlation was found between Cq value and mNGS viral normalized species reads (log value) (p = 0.002). Patients with viral pathogens had lower percentages of bacteriophages (p<0.001). No correlation was found between viral reads and clinical markers.ConclusionsThe mNGS protocol used was highly sensitive and specific for semi-quantitative detection of respiratory viruses. Excellent negative predictive value implicates the power of mNGS to exclude any pathogenic respiratory viral infectious cause in one test, with consequences for clinical decision making. Reduced abundance of bacteriophages in COPD patients with viral pathogens implicates skewing of the virome during infection, with potential consequences for the bacterial populations, during infection.

Automation and standardisation of clinical molecular testing using PCR.Ai – A comparative performance study

BackgroundWe undertook a prospective clinical study to evaluate PCR.Ai’s (www.pcr.ai) accuracy and impact when automating the manual data-analysis and quality control steps associated with routine clinical pathogen testing using real-time PCR (qPCR). ObjectivesWe evaluated the impact of PCR.Ai when used as the final interpretation/verification step for routine in-house qPCR tests for respiratory pathogens and for norovirus for a total of 22,200 interpretations. Study DesignWe compared PCR.Ai to our existing manual interpretation, to determine accuracy and hands-on time savings. PCR.Ai was accurate. Results and ConclusionsThere was 100% concurrence between validated respiratory virus and norovirus detection by our manual routine analysis method and PCR.Ai. Furthermore, there were significant routine savings with PCR.Ai of 45 min/respiratory run and 32 min/norovirus run. Our conclusion is that PCR.Ai is a highly accurate time-saving tool that reduces complexity of qPCR analysis and hence the need for specialists and hands-on time. It demonstrated capabilities to enable us to get results out more quickly with lower costs and less risk of errors.

Use of synthesized double-stranded gene fragments as qPCR standards for the quantification of antibiotic resistance genes

Pollution of various environmental matrices by antibiotic resistance genes (ARGs) has become a growing threat to human health. For the quantitative analysis of the presence of ARGs, there is a need for sensitive and robust qPCR assays which can detect various genes from different types of DNA extracts. Fourteen ARGs were selected as target genes in this study including: blaTEM, blaOXA-1 and blaCTX-M coded for resistance to β-lactams; ermB for macrolides; tetA, tetG, tetM, tetQ, tetW and tetX for tetracyclines; sul I and sul II for sulfonamides; drfA1 and drfA12 d for trimethoprim; and integron gene intI 1 and intI 2. Chemically synthesized double-stranded gene fragments were modified using molecular biology methods and used as real-time PCR standards as well as to establish in-house qPCR assays. The ermB gene from a naturally occurring plasmid was used to compare the performance of qPCR assay with the chemically synthesized ermB. Additionally, environmental water, soil and faeces samples were used to validate the established qPCR assays. Importantly, the study proves the usefulness of rapidly synthesized oligonucleotides serving as qPCR standards for ARG analysis and provides comparable sensitivity and reliability to a traditional amplicon standard.

Human T-cell lymphotropic virus type I and II seroprevalence among volunteer blood donors in Thailand

ABSTRACTHuman T-cell lymphotrophic virus type I and II (HTLV-I/II) are closely related but distinct retroviruses that can infect humans. Both the viruses can be transmitted via transfusion of contaminated blood components. HTLV pre-transfusion screening is not mandatory in Thailand until now. Current epidemiological data for HTLV prevalence is still lacking since the past surveys were done more than a decade ago. The main objective of this study was to determine the seroprevalence of HTLV-I/II among voluntary blood donors in Thailand. 11,057 volunteer blood donors were screened for HTLV-I/II antibodies using the ARCHITECT rHTLV-I/II chemiluminescent immunoassay (CLIA). Initial-reactive (IR) samples were subjected to repeat duplicate testing and were also sent for confirmatory testing at Korean Red Cross Society (KRC), Seoul or National Serology Reference Laboratories (NRL), Australia using alternate HTLV serological assays and immunoblot and/or specific nucleic acid testing respectively.Out of 11,057 plasma samples, 10,080 were low-risk seronegative donors and 977 were first-time/high-risk donors. Twenty of 24 IR samples were repeatedly reactive (RR) in low-risk seronegative donors group. On confirmatory testing of these 24 IR by immunoblot, 13 indeterminate and 11 negative results were observed. One out of 977 samples from first-time/high-risk donors was RR for anti-HTLV-I/II antibodies. This sample was co-reactive for HBsAg, but negative for HTLV by EIA or in-house HTLV-I qPCR. The ARCHITECT rHTLV-I/II assay exhibited a specificity of 99.93% in low-risk donors and 99.90% among high-risk donors.This study concluded that HTLV-I/II prevalence is low among blood donors in Thailand. But periodic surveillance should be continually conducted to ensure high blood safety standards in the country.

Abstract 5902: RNA sequencing analysis of taxane-resistant prostate cancer cells reveals potential candidate genes for therapeutic targeting

Abstract There is no cure for metastatic prostate cancer (PCa), the most commonly diagnosed cancer and third leading cause of cancer mortality among U.S. men. Patients with advanced PCa often develop resistance to conventional therapies resulting in metastatic castration-resistant PCa (mCRPC). Docetaxel (DTX) chemotherapy is the standard-of-care for mCRPC, followed by secondary chemotherapy with cabazitaxel (CBZ), yet chemoresistance and death inevitably occurs. While pharmacological agents targeting single survival pathways have shown promise at the bench side, clinical trials have failed. This is mostly due to the presence of redundant pathways utilized by PCa tumor cells to maintain chemoresistance. Therefore, we are interested in further defining molecular pathways associated with DTX-resistance in order to identify novel therapeutic targets to overcome this resistance. We hypothesized that RNA sequencing (RNAseq) analysis comparing the transcriptomes of chemosensitive and chemoresistant mCRPC cells will reveal potential targets for combinatorial therapies. RNAseq analysis of DTX-sensitive and -resistant PC3 and DU145 cells revealed a number of differentially regulated genes that were either unique to each cell line or common to both. We found that dipeptidyl peptidase 4 (DPP4), fatty acid binding protein 5 (FABP5), nestin (NES), and tetraspanin 8 (TSPAN8) were among the top most robustly and significantly upregulated genes in both DTX-resistant PC3 and DU145 cell lines. In-house qPCR studies validated the RNAseq results for these genes, and Western blotting analysis showed increased expression of these proteins in DTX-resistant cells, which correlated with increased mRNA expression. Interestingly, NES, TSPAN8, DPP4 are genes associated with development of the cancer stem-cell (CSC) phenotype. Other genes associated with CSCs were also upregulated in the DTX-resistant cells, suggesting that in vitro selection of mCRPC cells for DTX resistance may enrich for CSCs. This novel finding is consistent with recent studies implicating CSCs in PCa chemoresistance. Further analysis and validation of our RNAseq data will increase our understanding of DTX-resistance mechanisms in PCa cells, including the yet undefined functional role of CSC-related genes. These studies also reveal potential therapeutic targets to circumvent DTX resistance in PCa. Citation Format: Christina K. Cajigas-Du Ross, Leanne Woods-Burnham, Joshua Ramirez, Xin Chen, Charles Wang, Carlos A. Casiano. RNA sequencing analysis of taxane-resistant prostate cancer cells reveals potential candidate genes for therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5902.

Quantitative detection of BK virus in kidney transplant recipients: a prospective validation study.

Introduction:BK virus (BKV) infection in renal transplant patients may cause kidney allograft dysfunction and graft loss. Accurate determination of BKV viral load is critical to prevent BKV-associated nephropathy (BKVAN) but the cut-off that best predicts BKVAN remains controversial.Objective:To evaluate the performance of a commercial and an in-house qPCR test for quantitative detection of BK virus in kidney transplant recipients.Methods:This was a prospective study with kidney transplant recipients from two large university hospitals in Brazil. Patients were screened for BKV infection every 3 months in the first year post-transplant with a commercial and an in-house real time polymerase chain reaction (qPCR) test. BKVAN was confirmed based on histopathology. The area under the curve for plasma qPCR was determined from receiver operating characteristic analysis.Results:A total of 200 patients were enrolled. Fifty-eight percent were male, 19.5% had diabetes mellitus, and 82% had the kidney transplanted from a deceased donor. BKV viremia was detected in 32.5% and BKVAN was diagnosed in 8 patients (4%). BKVAN was associated with viremia of 4.1 log copies/mL, using a commercial kit. The cut-off for the in-house assay was 6.1 log copies/mL. The linearity between the commercial kit and the in-house assay was R2=0.83.Conclusion: Our study shows that marked variability occurs in BKV viral load when different qPCR methodologies are used. The in-house qPCR assay proved clinically useful, a cheaper option in comparison to commercial qPCR kits. There is an urgent need to make BKV standards available to the international community.

Failure of molecular diagnostics of a keratitis-inducing Acanthamoeba strain

An otherwise healthy 49-year-old female patient presented at the local hospital with severe keratitis in both inflamed eyes. She was a contact lens wearer and had no history of a corneal trauma. In our laboratory for medical parasitology Acanthamoebae were detected microscopically from the cornea scraping and from the fluid of the contact lens storage case after xenical culture and showed the typical cyst morphology of Acanthamoebae group II. The diagnosis of “Acanthamoeba keratitis” was established and successful therapy was provided. While the morphological microscopic method led to the correct diagnosis in this case, an in-house multiplex qPCR and a commercial qPCR showed false negative results regarding Acanthamoeba sp. The subsequent sequencing revealed the Acanthamoeba genotype T4. In the present case report, the inability to detect Acanthamoebae using qPCR only is presented. Therefore, we recommend the utilization of combined different assays for optimal diagnostic purposes.

Evaluation of self-collected rectal swabs for the detection of bacteria responsible for sexually transmitted infections in a cohort of HIV-1-infected patients.

Purpose. The standard approach to screening sexually transmitted infections (STIs) has often been restricted to urogenital specimens. Most current guidelines, however, also recommend testing extra-genital sites, including rectal locations, because asymptomatic rectal carriage of pathogens has often been reported. The aim of our study was to evaluate self-collected rectal swabs to screen bacterial STIs in HIV-infected patients in Marseille, France.Methodology. Between January 2014 and December 2015, 118 HIV-infected patients (93 males and 25 females) agreed to self-sample anal swabs for detection of bacterial STI. Detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Mycoplasma genitalium and Haemophilus ducreyi was performed using in-house qPCR assay.Results/Key findings. Bacterial STIs were found in 8 % (9/118) of the patients. C. trachomatis was the most commonly detected bacterium (4.2 %) followed by N. gonorrhoeae (2.5 %), M. genitalium (1.7 %) and T. pallidum (0.8 %). All the positive patients were males. The rectal carriage of pathogenic bacteria was fortuitously discovered for seven men (78 %) who did not present rectal signs of STIs and was suspected for two men who presented proctitis (22 %).Conclusion. In conclusion, testing extra-genital sites is crucial for the diagnosis of STIs in men and women presenting or not concomitant urogenital infections in order to detect asymptomatic carriage with the aim of controlling and preventing transmission to their sexual partners.

P006 Clinical evaluation of the resistanceplus™ mg kit, for detection of mycoplasma genitalium and screening for macrolide resistance

IntroductionEuropean guidelines onMycoplasma genitalium(MG) infections and on the mangement of non-gonococcal urethritis strongly recommend NAAT testing for MG and screening for macrolide resistance. The ResistancePlus™ MG kit has been developed for the simultaneous detection of MG and five mutations in the 23S rRNA gene associated with azithromycin resistance.MethodsThe ResistancePlus™ MG kit (SpeeDx) was evaluated in a prospective-restrospective study on 182 urogenital samples from patients routinely tested for Chlamydia and gonorrhoea. The ResistancePlus™ MG (550) kit was performed using the 7500 Fast (Applied Biosystems), after sample extraction on the MagNA Pure 96 Instrument (Roche) using the DNA and Viral NA Small Volume Kit following the Universal Pathogen 200 protocol. Results were analysed using the FastFinder ResistancePlus™ MG (7500) analysis software. Results were compared with an in-house qPCR test for MG detection with positives subsequently sequenced to determine 23S rRNA mutation status.ResultsThe ResistancePlus™ MG kit showed high clinical performance compared with the reference methods with sensitivity and specificity for MG detection of 98% and 100%, and 23S rRNA mutation detection of 92.5% and 100%, respectively. The ResistancePlus™ assay has an analytical sensitivity of 10-15 copies for all targets, and no cross-reactivity was seen in a wide range of non-target organisms.DiscussionThe ResistancePlus™ MG kit demonstrated excellent clinical performance for the simultaneous detection of MG and mutations associated with macrolide resistance. Detection of MG with resistance information is capable of guiding personalised treatment at the first health-care visit, reducing clinical-care costs and reducing the spread of antimicrobial resistance.