Multiplex real-time PCR for diagnosing malaria in a non-endemic setting: a prospective comparison to conventional methods.

Almost a decade ago our diagnostic laboratory implemented an in-house real-time PCR for the detection of Plasmodium DNA to diagnose malaria in parallel with conventional diagnostics, i.e., microscopy (thick and thin smears), quantitative buffy coat microscopy (QBC), and a rapid diagnostic test (RDT). Here we report our experiences and make a comparison between the different diagnostic procedures used in this non-endemic setting. All patients during the period February 2009-December 2017 suspected of malaria were prospectively tested at the moment of sample collection. Both PCR and conventional malaria diagnostics were carried out on a total of 839 specimens from 825 patients. In addition, three Plasmodium falciparum (Pf) patients were closely followed by real-time PCR and microscopy after treatment. Overall, 56 samples (55 patients) tested positive by real-time PCR, of which six were missed by microscopy and seven by QBC. RDT showed fairly good results in detecting Pf, whereas specificity was not optimal. RDT failed to detect 10 of 17 non-Pf PCR positive specimens. One Plasmodium malariae patient would have been missed if only conventional diagnostic tests had been used. The high sensitivity of the PCR was confirmed by the number of PCR positive, microscopy negative post-treatment samples. In conclusion, within our routine diagnostic setting, malaria real-time PCR not only showed a high level of agreement with the conventional methods used, but also showed higher sensitivity and better specificity. Still, for complete replacement of the conventional procedures in a non-endemic setting, the time-to-results of the real-time PCR is currently too long.

Pretransplantation Donor-Recipient Pair Seroreactivity Against BK Polyomavirus Predicts Viremia and Nephropathy After Kidney Transplantation.

Kidney transplant donors are not currently implicated in predicting BK polyomavirus (BKPyV) infection in kidney transplant recipients. It has been postulated, however, that BKPyV infection originates from the kidney allograft. Because BKPyV seroreactivity correlates with BKPyV replication and thus might mirror the infectious load, we investigated whether BKPyV seroreactivity of the donor predicts viremia and BKPyV-associated nephropathy (BKPyVAN) in the recipient. In a retrospective cohort of 407 living kidney donor-recipient pairs, pretransplantation donor and recipient sera were tested for BKPyV IgG levels and correlated with the occurrence of recipient BKPyV viremia and BKPyVAN within 1 year after transplantation. Donor BKPyV IgG level was strongly associated with BKPyV viremia and BKPyVAN (p < 0.001), whereas recipient BKPyV seroreactivity showed a nonsignificant inverse trend. Pairing of high-BKPyV-seroreactive donors with low-seroreactive recipients resulted in a 10-fold increased risk of BKPyV viremia (hazard ratio 10.1, 95% CI 3.5-29.0, p < 0.001). In multivariate analysis, donor BKPyV seroreactivity was the strongest pretransplantation factor associated with viremia (p < 0.001) and BKPyVAN (p = 0.007). The proportional relationship between donor BKPyV seroreactivity and recipient infection suggests that donor BKPyV seroreactivity reflects the infectious load of the kidney allograft and calls for the use of pretransplantation BKPyV serological testing of (potential) donors and recipients.

Comparison of ePlex Respiratory Pathogen Panel with Laboratory-Developed Real-Time PCR Assays for Detection of Respiratory Pathogens.

Infections of the respiratory tract can be caused by a diversity of pathogens, both viral and bacterial. Rapid microbiological diagnosis ensures appropriate antimicrobial therapy as well as effective implementation of isolation precautions. The ePlex respiratory pathogen panel (RP panel) is a novel molecular biology-based assay, developed by GenMark Diagnostics, Inc. (Carlsbad, CA), to be performed within a single cartridge for the diagnosis of 25 respiratory pathogens (viral and bacterial). The objective of this study was to compare the performance of the RP panel with those of laboratory-developed real-time PCR assays, using a variety of previously collected clinical respiratory specimens. A total of 343 clinical specimens as well as 29 external quality assessment (EQA) specimens and 2 different Middle East respiratory syndrome coronavirus isolates have been assessed in this study. The RP panel showed an agreement of 97.4% with the real-time PCR assay regarding 464 pathogens found in the clinical specimens. All pathogens present in clinical samples and EQA samples with a threshold cycle (CT ) value of <30 were detected correctly using the RP panel. The RP panel detected 17 additional pathogens, 7 of which could be confirmed by discrepant testing. In conclusion, this study shows excellent performance of the RP panel in comparison to real-time PCR assays for the detection of respiratory pathogens. The ePlex system provided a large amount of useful diagnostic data within a short time frame, with minimal hands-on time, and can therefore potentially be used for rapid diagnostic sample-to-answer testing, in either a laboratory or a decentralized setting.

[The prevalence of the human rhinoviruses and coronaviruses circulating in the Moscow region during 2007-2012].

The rhinoviruses and coronaviruses are the most common causative agents of the acute upper respiratory tract infection in humans. They include several species that vary in the pathogenicity, some causing severe respiratory tract diseases. In this work, the species prevalence of rhinoviruses and coronaviruses was studied in 92 virus-positive clinical patients that were collected at the area of the Moscow region during the period from 2007 to 2012. Using the real-time PCR the virus circulation has been established for all species common in humans, including three rhinoviruses, HRV A, HRV B, and HRV C, and four coronaviruses, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. For eight patients, the identity of the rhinoviruses, including 4 cases of HRV-C, 3 cases of HRV-A, and a single case of HRV-B, was corroborated using partial sequencing of the 5 non-coding regions and phylogenetic analysis. The viruses of HRV-C, HCoV-NL63, and HCoV-OC43 were prevalent in children with severe respiratory diseases.

Clinical performance of two new, fully integrated molecular platforms used for HIV-1, HBV and HCV viral load analysis, the NeuMoDx 288 and the Alinity m.

BACKGROUND: Viral load (VL) determination in patients with human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) is essential for proper patient management and follow-up. New molecular platforms have been developed to fully automate these diagnostic assays. OBJECTIVE: Evaluation of the clinical performance of HIV-1, HBV and HCV VL assays on the Alinity m (Abbott) and NeuMoDx (Qiagen) molecular platforms. METHOD: Test panels of the three viruses have been compiled of 100 plasma and/or serum samples per target containing non-detectable, non-quantifiable and quantifiable VLs. All samples were retrospectively tested on the Alinity m and NeuMoDx platforms according to manufacturers' instructions. RESULTS: A total of 74, 86 and 66 samples with valid results for both platforms were included in the HIV-1, HBV and HCV analysis respectively. Overall qualitative agreement of the assays on both platforms was 78% for HIV-1, 93% for HBV and 100% for HCV. Quantitative agreement (less than 0.5 log difference) was shown to be 68% for HIV-1, 68% for HBV and 94% for HCV. CONCLUSION: The Alinity m and NeuMoDx HCV assay have a comparable performance. Quantification differences in the HIV-1 assay were mostly apparent in the lower VLs and under-quantification of the NeuMoDx HBV assay was observed.

Evaluation of the sample-to-result, random access NeuMoDx platform for viral load testing of Cytomegalovirus and Epstein Barr virus in clinical specimens.

BACKGROUND: The detection and follow up of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) viral loads (VL) are crucial in the management of immunocompromised patients. Recently, molecular CE-IVD assays for detection and quantification of CMV and EBV have been launched for use on the random-access and sample-to-result NeuMoDx 96 and 288 platforms (Qiagen). OBJECTIVE: Evaluating the qualitative and quantitative performance of the NeuMoDx CMV and EBV assays in clinical specimens compared to a lab developed tests (LDT) and the CE-IVD assays on the Abbott m2000 system. METHOD: Both a prospective and a retrospective panel, compiled of non-detectable (ND), non-quantifiable (NQ) and quantifiable VLs in plasma samples have been evaluated for both CMV and EBV: NeuMoDx versus LDT and NeuMoDx versus Abbott m2000. Quantitative agreement was determined for samples with a quantifiable VL on both systems. RESULTS: Qualitative and quantitative agreement between the NeuMoDx and LUMC's LDT CMV assays was 88%. Qualitative agreement between the NeuMoDx and Abbott m2000 CMV assays was 92% and quantitative agreement was 87%. Qualitative and quantitative agreement between the NeuMoDx and the LDT EBV assays was 87%. Qualitative agreement between the NeuMoDx and Abbott m2000 EBV assays was 91% and quantitative agreement was 0%. CONCLUSION: These data show that the NeuMoDx assays are suitable for both detection and quantification of CMV and EBV in a medium- to high throughput diagnostic setting, but that differences in sensitivity and quantification (for EBV, NeuMoDx versus Abbott m2000) warrant an extensive transition period when using the respective assays for following VL in patient samples.

Lower respiratory tract infection in the community: associations between viral aetiology and illness course.

OBJECTIVES: This study determined associations between respiratory viruses and subsequent illness course in primary care adult patients presenting with acute cough and/or suspected lower respiratory tract infection. METHODS: A prospective European primary care study recruited adults with symptoms of lower respiratory tract infection between November 2007 and April 2010. Real-time in-house polymerase chain reaction (PCR) was performed to test for six common respiratory viruses. In this secondary analysis, symptom severity (scored 1 = no problem, 2 = mild, 3 = moderate, 4 = severe) and symptom duration were compared between groups with different viral aetiologies using regression and Cox proportional hazard models, respectively. Additionally, associations between baseline viral load (cycle threshold (Ct) value) and illness course were assessed. RESULTS: The PCR tested positive for a common respiratory virus in 1354 of the 2957 (45.8%) included patients. The overall mean symptom score at presentation was 2.09 (95% confidence interval (CI) 2.07-2.11) and the median duration until resolution of moderately bad or severe symptoms was 8.70 days (interquartile range 4.50-11.00). Patients with influenza virus, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), coronavirus (CoV) or rhinovirus had a significantly higher symptom score than patients with no virus isolated (0.07-0.25 points or 2.3-8.3% higher symptom score). Time to symptom resolution was longer in RSV infections (adjusted hazard ratio (AHR) 0.80, 95% CI 0.65-0.96) and hMPV infections (AHR 0.77, 95% CI 0.62-0.94) than in infections with no virus isolated. Overall, baseline viral load was associated with symptom severity (difference 0.11, 95% CI 0.06-0.16 per 10 cycles decrease in Ct value), but not with symptom duration. CONCLUSIONS: In healthy, working adults from the general community presenting at the general practitioner with acute cough and/or suspected lower respiratory tract infection other than influenza impose an illness burden comparable to influenza. Hence, the public health focus for viral respiratory tract infections should be broadened.

High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.

Epidemiology and clinical presentations of the four human coronaviruses 229E, HKU1, NL63, and OC43 detected over 3 years using a novel multiplex real-time PCR method.

Four human coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43) are associated with a range of respiratory outcomes, including bronchiolitis and pneumonia. Their epidemiologies and clinical characteristics are poorly described and are often reliant on case reports. To address these problems, we conducted a large-scale comprehensive screening for all four coronaviruses by analysis of 11,661 diagnostic respiratory samples collected in Edinburgh, United Kingdom, over 3 years between July 2006 and June 2009 using a novel four-way multiplex real-time reverse transcription-PCR (RT-PCR) assay. Coronaviruses were detected in 0.3 to 0.85% of samples in all age groups. Generally, coronaviruses displayed marked winter seasonality between the months of December and April and were not detected in summer months, which is comparable to the pattern seen with influenza viruses. HCoV-229E was the exception; detection was confined to the winter of 2008 and was sporadic in the following year. There were additional longer-term differences in detection frequencies between seasons, with HCoV-OC43 predominant in the first and third seasons and HCoV-HKU1 dominating in the second (see Results for definitions of seasons). A total of 11 to 41% of coronaviruses detected were in samples testing positive for other respiratory viruses, although clinical presentations of coronavirus monoinfections were comparable to those of viruses which have an established role in respiratory disease, such as respiratory syncytial virus, influenza virus, and parainfluenza viruses. The novel multiplex assay for real-time pan-coronavirus detection enhances respiratory virus diagnosis, overcomes potential diagnostic problems arising through seasonal variation in coronavirus frequency, and provides novel insights into the epidemiology and clinical implications of coronaviruses.