Clinical applications of spectroscopic techniques in conjunction with multivariate analysis in virus diagnosis

Evaluation of the Cepheid Respiratory Syncytial Virus and Influenza Virus A/B real-time PCR analyte specific reagent

We compared our recently reported reverse transcriptase polymerase chain reaction (PCR)-based assay for detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples with different virus isolation (VI) procedures. Thirty-six blood samples and 1 spleen sample from deer were assessed by the EHDV PCR assay and VI in baby hamster kidney (BHK)-21 cells and embryonated chicken eggs (ECE). The EHDV PCR assay detected EHDV RNA from 6 blood samples obtained from deer during 1988-1989 outbreaks of epizootic hemorrhagic disease and from the spleen and blood samples of a deer with clinical hemorrhagic disease in 1992. The 6 blood samples from the 1988-1989 outbreaks and the spleen sample from the 1992 case were VI positive on BHK-21 cell culture. The blood from the same deer with the PCR- and VI-positive spleen was VI negative in BHK-21 cells and ECE. All EHDV isolates were identified as EHDV serotype 2 by a plaque inhibition test. The results of this study indicate that the sensitivity of the previously described EHDV PCR assay is comparable to or greater than that of the VI method in BHK-21 cell culture or ECE. The EHDV PCR assays could provide a superior diagnostic alternative to the current cumbersome and time-consuming VI procedures.

BackgroundCurrent molecular target-dependent methods are used to detect only known viruses. However, metagenomics based on next-generation sequencing (NGS) technique is a target-independent assay that enables simultaneous detection and genomic characterisation of all microorganisms present in a sample. In this study, we aimed to develop a metagenomics approach using NGS to identify and characterise viruses in stool samples from infants and children with Acute Gastroenteritis (AGE) in Kuwait.MethodsWe have investigated 84 stool samples from infants and children aged one month to ten years old with signs and symptoms of gastroenteritis who attended Mubarak Al-Kabeer and Al-Amiri hospitals in Kuwait from January to December 2017. A metagenomics approach using NGS to characterise viruses in clinical samples was used. Also, the commercial Real-Time PCR assay was used to detect viruses causing gastroenteritis.ResultsMetagenomics analysis revealed an average of 280,768 reads in which 5% of the reads were derived from viruses. The analysis of viral sequences verified that single infection of human adenovirus was the leading cause of gastroenteritis among infants and children, which was detected in 23.2% of the patients, followed by a mixed infection of human adenovirus and other viruses, which was detected in 20.9% of patients. Also, the newly discovered viruses known to cause gastroenteritis were detected, such as astrovirus MLB2, primate bocaparvovirus-1, Aichivirus A, cardiovirus, parechovirus A, astrovirus VA4, cosavirus-F, and bufavirus-3. Our results showed 71% agreement (k = 0.445, P = 0.000) between multiplex Real-Time PCR, which is used as a routine diagnostic test and metagenomics approach in the detection of viruses causing gastroenteritis in clinical samples.ConclusionDespite the difficulties in sample preparation and analysis process, we showed that metagenomics approach is a powerful and promising tool for the detection and characterisation of different viruses in clinical samples.

Advances in molecular testing and microfluidic technologies have opened new avenues for rapid detection of animal viruses. We used a centrifugal microfluidic disk (CMFD) to detect 6 important swine viruses, including foot-and-mouth disease virus, classical swine fever virus, porcine reproductive and respiratory swine virus-North American genotype, porcine circovirus 2, pseudorabies virus, and porcine parvovirus. Through integrating the loop-mediated isothermal amplification (LAMP) method and microfluidic chip technology, the CMFD could be successfully performed at 62℃ in 60 min. The detection limit of the CMFD was 3.2 × 102 copies per reaction, close to the sensitivity of tube-type LAMP turbidity methods (1 × 102 copies per reaction). In addition, the CMFD was highly specific in detecting the targeted viruses with no cross-reaction with other viruses, including porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine rotavirus. The coincidence rate of CMFD and conventional PCR was ~94%; the CMFD was more sensitive than conventional PCR for detecting mixed viral infections. The positive detection rate of 6 viruses in clinical samples by CMFD was 44.0% (102 of 232), whereas PCR was 40.1% (93 of 232). Thirty-six clinical samples were determined to be coinfected with 2 or more viruses. CMFD can be used for rapid, sensitive, and accurate detection of 6 swine viruses, offering a reliable assay for monitoring these pathogens, especially for detecting viruses in widespread mixed-infection clinical samples.

The aim of the present study was to compare traditional methods for the detection of respiratory syncytial virus with a newly developed commercial assay based on real-time nucleic acid sequence based amplification. Respiratory syncytial virus is a major cause of severe respiratory infection in infants and in certain groups of older children and adults. Treatment options are limited, but a rapid diagnosis improves patient management and infection control. The rapid diagnosis of respiratory syncytial virus currently relies on antigen detection assays. These tests are limited to use in certain good-quality types of samples, which are rarely obtained from adult patients. Molecular-based assays for the detection of respiratory syncytial virus are shown to be highly sensitive, specific, and more rapid than cell culture techniques. This retrospective study compared traditional laboratory techniques for the detection of respiratory syncytial virus in 508 respiratory samples collected during the winter months of 2003-2004 against the recently developed, commercially available NucliSens EasyQ Respiratory Syncytial Virus A+B assay (bioMérieux, Marcy l'Etoile, France), which is based on real-time nucleic acid sequence based amplification using molecular beacons and an internal control. Using traditional techniques, the prevalence of respiratory syncytial virus in the samples tested was found to be 21%. Using the real-time nucleic acid sequence-based amplification assay, an additional 41 samples from patients with a clinically diagnosed respiratory illness were found to be positive for respiratory syncytial virus. The NucliSens EasyQ assay was shown to be sensitive and specific for the detection of respiratory syncytial virus A+B in different types of respiratory samples. Moreover, the time required to complete the assay was <4 h, so results could be obtained on the same day as sample receipt in the laboratory.

BackgroundSequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. However, as specific PCR only detects pre-defined targets, novel virus strains or viruses not included in routine test panels will be missed. Recently, advances in high-throughput sequencing allow for virus-sequence-independent identification of entire virus populations in clinical samples, yet standardized protocols are needed to allow broad application in clinical diagnostics. Here, we describe a comprehensive sample preparation protocol for high-throughput metagenomic virus sequencing using random amplification of total nucleic acids from clinical samples.ResultsIn order to optimize metagenomic sequencing for application in virus diagnostics, we tested different enrichment and amplification procedures on plasma samples spiked with RNA and DNA viruses. A protocol including filtration, nuclease digestion, and random amplification of RNA and DNA in separate reactions provided the best results, allowing reliable recovery of viral genomes and a good correlation of the relative number of sequencing reads with the virus input. We further validated our method by sequencing a multiplexed viral pathogen reagent containing a range of human viruses from different virus families. Our method proved successful in detecting the majority of the included viruses with high read numbers and compared well to other protocols in the field validated against the same reference reagent. Our sequencing protocol does work not only with plasma but also with other clinical samples such as urine and throat swabs.ConclusionsThe workflow for virus metagenomic sequencing that we established proved successful in detecting a variety of viruses in different clinical samples. Our protocol supplements existing virus-specific detection strategies providing opportunities to identify atypical and novel viruses commonly not accounted for in routine diagnostic panels.

Isolation of porcine reproductive and respiratory syndrome virus (PRRSV) in cell culture is a primary means of obtaining virus isolates for autogenous vaccine production and other applications. However, it has not been well characterized whether cell culture isolate and the virus in clinical sample are equivalent. This study compared PRRSV ORF5 sequences from 1024 clinical samples (995 PRRSV-2, 26 PRRSV-1, and three PRRSV-1 and PRRSV-2 PCR-positive) and their isolates in MARC-145 and/or ZMAC cells. For three PRRSV-1 and PRRSV-2 PCR-positive clinical samples, both PRRSV-1 and PRRSV-2 were isolated in ZMAC cells, whereas either PRRSV-1 or PRRSV-2, but not both, was isolated in MARC-145 cells, with isolate sequences matching the respective viruses in clinical samples. Twenty-six PRRSV-1 and most of 995 PRRSV-2 PCR-positive clinical samples had matching viral ORF5 sequences with their cell culture isolates. However, 14 out of 995 PRRSV-2 cases (1.4%) had nonmatching viral sequences between clinical samples and MARC-145 isolates, although viral sequences from clinical samples and ZMAC isolates matched. This is concerning because, if the MARC-145 isolate is directly used for autogenous vaccine production without sequencing confirmation against the virus in the clinical sample, it is possible that the produced autogenous vaccine does not include the desired wild-type virus strain found on the farm and instead contains vaccine-like virus. Vaccine-specific PCR and next-generation sequencing performed on six selected cases indicated presence of ≥2 PRRSV-2 strains (mixed infection) in such clinical samples. In summary, PRRSV ORF5 sequences from clinical samples and cell culture isolates matched each other for majority of the cases. However, PRRSV sequences between clinical sample and MARC-145 cell culture isolate could occasionally be different when the clinical sample contains ≥2 PRRSV-2 strains. Characterizing PRRSV sequences from clinical samples and cell culture isolates should be conducted before using isolates for producing autogenous vaccines or other applications.

Rapid detection, culture-amplification and typing of herpes simplex viruses by enzyme immunoassay in clinical samples

Since the first publication of the polymerase chain reaction (PCR) in 1985, there has been a large number of reports on its applications in the study of viral infections. This review outlines the PCR methodology and its variations, as well as the problems that may be encountered during its performance. Its application in the detection of various viruses in clinical samples is also presented.

Parvovirus B19, a pathogenic virus widely distributed in the human population, is responsible for various pathologies and diverse clinical manifestations (1). Diagnosis by detection of virus and quantitative evaluation of viral load is important mainly at the onset of infections before an immune response develops, in cases of atypical immune response, in the course of chronic infections, and in the occurrence of fetal infections (2). In the acute phase of infections, virus can be present in the blood at concentrations >1015 virions/L, posing a risk of transmission through transfusions and therapeutic use of blood products. Quantitative evaluation of B19 virus contamination of plasma pools for the production of blood derivatives is required as a measure to reduce the risk of transmission of infections (3). Fluorescence-based real-time PCR assays can combine specific detection and quantitative evaluation of the viral load. For quantitative evaluation of the viral load, real-time PCR assays must be carefully designed to give reliable results. In particular, the mode of quantification, whether absolute or relative, and an appropriate calibration method need to be firmly established (4)(5). Absolute quantification can be obtained by referring to a calibration curve. To take into account the variability attributable to cumulating effects both in sample processing and in the analytical phases, the calculated amount of target nucleic acids can be normalized to the calculated amount of a reference target that is likely to be present in constant amounts in all samples analyzed. Alternatively, relative quantification can be obtained from the ratio of target present in experimental samples to target in a calibrator sample. To compensate for sample variability, ratios can be further normalized with respect to a parallel, relative quantification of a reference target (6). Different real-time PCR assays for the detection of B19 virus in clinical samples …

Influenza viruses cause respiratory infection, spread through respiratory secretions, and are shed into the nasal secretion and saliva specimens. Therefore, nasal fluid and saliva are effective clinical samples for the diagnosis of influenza virus-infected patients. Although several methods have been developed to detect various types of influenza viruses, approaches for detecting mutant influenza viruses in clinical samples are rarely reported. Herein, we report for the first time a surface-enhanced Raman scattering (SERS)-based sensing platform for oseltamivir-resistant pandemic H1N1 (pH1N1) virus detection in human nasal fluid and saliva. By combining SERS-active urchin Au nanoparticles and oseltamivir hexylthiol, an excellent receptor for the pH1N1/H275Y mutant virus, we detected the pH1N1/H275Y virus specifically and sensitively in human saliva and nasal fluid samples. Considering that the current influenza virus infection testing methods do not provide information on the antiviral drug resistance of the virus, the proposed SERS-based diagnostic test for the oseltamivir-resistant virus will inform clinical decisions about the treatment of influenza virus infections, avoiding the unnecessary prescription of ineffective drugs and greatly improving therapy.

A metagenomic approach based on target independent next‐generation sequencing has become a known method for the detection of both known and novel viruses in clinical samples. This study aimed to use the metagenomic sequencing approach to characterize the viral diversity in respiratory samples from patients with respiratory tract infections. We have investigated 86 respiratory samples received from various hospitals in Kuwait between 2015 and 2016 for the diagnosis of respiratory tract infections. A metagenomic approach using the next‐generation sequencer to characterize viruses was used. According to the metagenomic analysis, an average of 145, 019 reads were identified, and 2% of these reads were of viral origin. Also, metagenomic analysis of the viral sequences revealed many known respiratory viruses, which were detected in 30.2% of the clinical samples. Also, sequences of non‐respiratory viruses were detected in 14% of the clinical samples, while sequences of non‐human viruses were detected in 55.8% of the clinical samples. The average genome coverage of the viruses was 12% with the highest genome coverage of 99.2% for respiratory syncytial virus, and the lowest was 1% for torque teno midi virus 2. Our results showed 47.7% agreement between multiplex Real‐Time PCR and metagenomics sequencing in the detection of respiratory viruses in the clinical samples. Though there are some difficulties in using this method to clinical samples such as specimen quality, these observations are indicative of the promising utility of the metagenomic sequencing approach for the identification of respiratory viruses in patients with respiratory tract infections.

Purpose. To evaluate the Luminex NxTAG respiratory pathogen panel (NxTAG RPP) for the detection of respiratory viruses in clinical samples from patients with the symptoms of respiratory infection.Methodology. The NxTAG RPP was compared to an in-house multiplex real-time PCR panel (LDT) for the detection of respiratory viruses in 314 clinical samples from patients with the symptoms of respiratory infection.Results. Thirty-one samples were negative in both tests and 193 samples contained a single virus that was detected in both tests. Polymicrobial infections were detected in 74 samples, with 268 samples overall having concordant results in both assays, and there were a total of 51 discordant results in 44 samples. Two samples were invalid in the NxTAG RPP assay and were excluded from the final analysis. The overall agreement between the NxTAG RPP and LDT was very high, as indicated by the Kappa coefficients, which ranged from 0.85 for metapneumovirus up to 0.96 for RSV A, and the overall percentage agreement values of 96.2 % for enterovirus/rhinovirus and 100 % for influenza A, influenza B, PIV 4 and RSV B.Conclusion. The NxTAG RPP is a sensitive and specific test for the detection of respiratory viruses and the high sample throughput and low hands-on time make the NxTAG RPP assay suitable for screening clinical samples for respiratory pathogens.

BackgroundPrompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV.ResultsThe LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture. A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive. Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis. The typing results obtained with the LC-PCR and by culture amplified test were completely concordant.ConclusionsThis study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube. In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days).

BackgroundDengue is the most frequent arthropod-borne viral disease worldwide. Because dengue manifestations are similar to those of many other febrile syndromes, the availability of dengue-specific laboratory tests is useful for the differential diagnosis. Timely and accurate diagnosis of dengue virus (DENV) infection is important for appropriate management of complications, pathophysiological studies, epidemiological investigations and optimization of vector-control measures. Several “in-house” reverse transcriptase-polymerase chain reaction (RT-PCR) methods have been developed to detect, type and/or quantify DENV. Standardized dengue RT-PCR kits with internal controls have been recently introduced, but need clinical evaluation. We assessed the performances of 4 commercial DENV real-time RT-PCR kits.FindingsThe 4 kits were evaluated using a panel of 162 samples positive with an existing in-place hemi-nested RT-PCR used for routine DENV-infection diagnosis in patients with acute-febrile disease. The panel included 46 DENV-1, 37 DENV-2, 33 DENV-3, and 46 DENV-4. Also, 70 negative serum specimens were used to determine specificity. Geno-Sen’s Dengue 1–4 Real-Time RT-PCR kit was the only assay to provide quantification using standards, but lacked sensitivity for DENV-4 detection. The SimplexaTM Dengue RT-PCR assay, with 151 (93.2% [95% confidence interval, 89.3–97.1]) positive samples, had significantly higher sensitivity than the other 3 kits; in a complementary evaluation of 111 consecutive patients’ samples, its performance and genotyping agreed with the hemi-nested gold-standard assay.ConclusionsThe SimplexaTM Dengue RT-PCR’s good performance to detect and genotype DENV1–4 requires further evaluation in multicenter and prospective studies, particularly in settings of clinical diagnosis during dengue outbreaks.