Analytical performances of the AMPLIQUICK® Respiratory Triplex assay for simultaneous detection and differentiation of SARS-CoV-2, influenza A/B and respiratory syncytial viruses in respiratory specimens

Although patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B and respiratory syncytial virus (RSV) show comparable or very similar manifestations, the therapeutic approaches of these respiratory viral infections are different, which requires an accurate diagnosis. Recently, the novel multiplex real-time reverse transcription-polymerase chain reaction assay AMPLIQUICK® Respiratory Triplex (BioSynex SA, Illkirch-Graffenstaden, France) allows simultaneous detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory tract samples. We herein evaluated the performance of the AMPLIQUICK® Respiratory Triplex for the detection of the four viruses in respiratory specimens, using Allplex™ Respiratory Panel 1 and 2019-nCoV assays (Seegene, Seoul, Korea) as reference comparator assays. A total of 359 archived predetermined respiratory samples, including 83, 145, 19 and 95 positive specimens for SARS-CoV-2, influenza A, influenza B and RSV respectively, were included. The AMPLIQUICK® Respiratory Triplex showed high concordance with the reference assays, with an overall agreement for SARS-CoV-2, influenza A, influenza B, and RSV at 97.6%, 98.8%, 98.3% and 100.0%, respectively, and high κ values ranging from 0.93 to 1.00, indicating an almost perfect agreement between assays. Furthermore, high correlations of cycle threshold (Ct) values were observed for positive samples of the four viruses between the AMPLIQUICK® Respiratory Triplex and comparator assays, with an overall high agreement between Ct values assessed by Bland-Altman analyses. In conclusion, these observations demonstrate that the multiplex AMPLIQUICK® Respiratory Triplex is a reliable assay for the qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory specimens, which may prove useful for streamlining diagnostics during the winter influenza-seasons.

Development of the one-step qualitative RT-PCR assay to detect SARS-CoV-2 Omicron (B.1.1.529) variant in respiratory specimens

A new SARS-CoV-2 Omicron (B.1.1.529) Variant of Concern has been emerging worldwide. We are seeing an unprecedented surge in patients due to Omicron in this COVID-19 pandemic. A rapid and accurate molecular test that effectively differentiates Omicron from other SARS-CoV-2 variants would be important for both epidemiologic value and for directing variant-specific therapies such as monoclonal antibody infusions. In this study, we developed a real-time RT-PCR assay for the qualitative detection of Omicron from routine clinical specimens sampling the upper respiratory tract. The limit of detection of the SARS-CoV-2 Omicron variant RT-PCR assay was 2 copies/μl. Notably, the assay did not show any cross-reactivity with other SARS-CoV-2 variants including Delta (B.1.617.2). This SARS-CoV-2 Omicron variant RT-PCR laboratory-developed assay is sensitive and specific to detect Omicron in nasopharyngeal and nasal swab specimens.

Evaluation of Three Automated Extraction Systems for the Detection of SARS-CoV-2 from Clinical Respiratory Specimens

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly contagious and causes coronavirus disease 2019 (COVID-19). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is the most accurate and reliable molecular assay to detect active SARS-CoV-2 infection. However, a rapid increase in test subjects has created a global bottleneck in testing capacity. Given that efficient nucleic acid extraction greatly affects reliable and accurate testing results, we compared three extraction platforms: MagNA Pure 96 DNA and Viral NA Small Volume kit on MagNA Pure 96 (Roche, Basel, Switzerland), careGENETM Viral/Pathogen HiFi Nucleic Acid Isolation kit (WELLS BIO Inc., Seoul, Korea) on KingFisher Flex (Thermo Fisher Scientific, Rocklin, CA, USA), and SGRespiTM Pure kit (Seegene Inc., Seoul, Korea) on Maelstrom 9600 (Taiwan Advanced Nanotech Inc., Taoyuan, Taiwan). RNA was extracted from 245 residual respiratory specimens from the different types of samples (i.e., NPS, sputum, and saliva) using three different kits. The 95% limits of detection of median tissue culture infectious dose per milliliter (TCID50/mL) for the MagNA Pure 96, KingFisher Flex, and Maelstrom 9600 were 0.37–3.15 × 101, 0.41–3.62 × 101, and 0.33–1.98 × 101, respectively. The KingFisher Flex platform exhibited 99.2% sensitivity and 100% specificity, whereas Maelstrom 9600 exhibited 98.3–100% sensitivity and 100% specificity. Bland–Altman analysis revealed a 95.2% concordance between MagNA Pure 96 and KingFisher Flex and 95.4% concordance between MagNA Pure 96 and Maelstrom 9600, indicating that all three platforms provided statistically reliable results. This suggests that two modifying platforms, KingFisher Flex and Maelstrom 9600, are accurate and scalable extraction platforms for large-scale SARS-CoV-2 clinical detection and could help the management of COVID-19 patients.

Multipathogen Detection in Patients with Respiratory Tract Infection: Identification of Non-respiratory Viruses Using Multiplex Real-time Polymerase Reaction

Background: Due to the overlapping clinical characteristics of respiratory tract infections (RTIs) and the unavailability of appropriate diagnostic techniques, the diagnosis of RTIs is controversial. Objectives: The study aimed to prompt the diagnosis of RTIs using commercial multiplex real-time PCR. Methods: The survey undertook for two years (2019 - 2020) on 144 flu-negative immunocompetent outpatients. Respiratory samples were examined by multiplex PCR assays. Results: Study population consisted of females (n = 77, 53.5%) and males (n = 67, 46.5%). The mean age was 42.8 ± 23.7 years. Thirty-one (21.5%) patients were infected with only one viral or bacterial infection. Eighty-two (57%) were infected with more than one pathogen. Ninety-five (37%) and 161 (62%) tests were positive for bacterial and viral pathogens, respectively. Community-acquired Pneumonia (CAP) and atypical CAP pathogens included 17% and 10% of respiratory specimens, respectively. The predominant pathogens consisted of Human Herpes Virus 7 (HHV-7) (n = 38, 15.5%), Epstein-Barr Virus (EBV) (n = 34, 13.8%), Mycoplasma pneumoniae (n = 24, 9.8%), and Human Herpes Virus 6 (HHV-6) (n = 21, 8.5%). There were associations between pathogen findings and special age categories. Fever, cough, dyspnea, and hemoptysis were associated with certain pathogens. There was no substantial difference between viral and bacterial Ct concerning gender, age group, and comorbidities. Conclusions: Multiplex diagnostic assays significantly increased the rate of appropriate diagnosis of respiratory pathogens. However, further investigation is needed to find non-respiratory viruses' significance in respiratory specimens of immunocompetent symptomatic patients.

High genetic heterogeneity of Mycobacterium intracellulare isolated from respiratory specimens

Background M. intracellulare is a frequent causative pathogen of nontuberculous mycobacteria infection that causes infections in the respiratory tract, whose incidence is increasing in many countries. This study aimed at determining the VNTR-based genetic diversity of a collection of 39 M. intracellulare human strains isolated from respiratory specimens over the last 5 years. Results The VNTR analysis showed that M. intracellulare strains displayed a high genetic diversity, indicating that the M. intracellulare genotypes are quite heterogeneous in our geographical area. Moreover, a comparison with VNTR profiles of strains from other countries confirmed that genotypes of clinical strains of M. intracellulare are not related to geographical origin. Conclusions VNTR typing has proved to be a highly discriminatory method for better understanding the molecular epidemiology of M. intracellulare.

Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 test for bronchoalveolar lavage

Accurate and rapid laboratory tests are essential for the prompt diagnosis of COVID-19, which is important to patients and infection control. The Xpert Xpress SARS-CoV-2 test is a real-time RT-PCR intended for the qualitative detection of nucleic acid from SARS-CoV-2 in upper respiratory specimens. In this study, we assessed the analytical and clinical performance characteristics of this rapid test for SARS-CoV-2 in 60 bronchoalveolar lavage (BAL) specimens. BAL is a specimen type that is not authorized under EUA for the Xpert Xpress SARS-CoV-2 test. The limit of detection of the Xpert Xpress SARS-CoV-2 test was 500 copies/ml. The overall agreement of the Xpert Xpress SARS-CoV-2 test was 100%. The Xpert Xpress SARS-CoV-2 test is sensitive and specific to aid in diagnosis of COVID-19 using bronchoalveolar lavage.

Detection of SARS-CoV-2 RNA in Urine by RT-LAMP: A Very Rare Finding

Detection of SARS-CoV-2 is routinely performed in naso/oropharyngeal swabs samples from patients via RT-qPCR. The RT-LAMP technology has also been used for viral RNA detection in respiratory specimens with both high sensitivity and specificity. Recently, we developed a novel RT-LAMP test for SARS-CoV-2 RNA detection in nasopharyngeal swab specimens (named, N15-RT-LAMP) that can be performed as a single-tube colorimetric method, in a real-time platform, and as dry-LAMP. To date, there has been very little success in detecting SARS-CoV-2 RNA in urine by RT-qPCR, and the information regarding urine viral excretion is still scarce and not comprehensive. Here, we tested our N15-RT-LAMP on the urine of 300 patients admitted to the Hospital of Salamanca, Spain with clinical suspicion of COVID-19, who had a nasopharyngeal swab RT-qPCR-positive (n = 100), negative (n = 100), and positive with disease recovery (n = 100) result. The positive group was also tested by RT-qPCR for comparison to N15-RT-LAMP. Only a 4% positivity rate was found in the positive group via colorimetric N15-RT-LAMP and 2% via RT-qPCR. Our results are consistent with those obtained in other studies that the presence of SARS-CoV-2 RNA in urine is a very rare finding. The absence of SARS-CoV-2 RNA in urine in the recovered patients might suggest that the urinary route is very rarely used for viral particle clearance.

Duration of respiratory sample stability at -80ºC for SARS-CoV-2 PCR

Objective: To determine the stability of respiratory samples for SARS-CoV-2 PCR at standard laboratory ultra-freezer temperatures (-80°C). Methods: Five hundred and sixty-five archived, SARS-CoV-2 PCR positive patient specimens received at the Pathology Department of the Indus Hospital & Health Network between January 2021 and June 2021 were retested in June 2021. Samples had been stored at -70°C or below throughout this duration. Sample integrity following storage was assessed as the percentage of samples with reproducible results, and as consistency of cycle threshold (Ct) values between the original testing and the repeat testing. Results: Of the 565 samples evaluated in this study, 86% gave reproducible results upon retesting. However, there was no correlation between the duration of storage and result reproducibility, though the majority (69% for PCR Target-I and 78% for PCR Target-II respectively) of non-reproducible results had Ct values above 30. Similarly, there was a consistent increase of Ct values upon storage at ultra-freezer temperatures, though the effect again was more contingent upon freezing the sample in the ultra-freezer rather than the duration of storage. Conclusion: SARS-CoV-2 positive respiratory specimens for PCR can be stored for up to six months at -70°C or below without loss of sample integrity, though there is some loss of PCR-detected viral targets as evidenced by an immediate increased in the PCR-generated Ct values. In addition, samples with initial Ct values above 30 are more likely to give non-reproducible results. doi: https://doi.org/10.12669/pjms.38.ICON-2022.5777 How to cite this:Aijaz J, Naseer F, Dojki M, Jamal S. Duration of respiratory sample stability at -80ºC for SARS-CoV-2 PCR. Pak J Med Sci. 2022;38(2):393-398. doi: https://doi.org/10.12669/pjms.38.ICON-2022.5777 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Long-term specific IgG response to SARS-CoV-2 nucleocapsid protein in recovered COVID-19 patients

This study monitored the long-term immune response to severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection in patients who had recovered from coronavirus disease (COVID)-19. Anti-nucleocapsid immunoglobulin G (anti-N IgG) titer in serum samples collected at a single (N = 302) or multiple time points (N = 229) 3–12 months after COVID-19 symptom onset or SARS-CoV-2 detection in respiratory specimens was measured by semiquantitative chemiluminescent microparticle immunoassay. The 531 patients (966 specimens) were classified according to the presence or absence of pneumonia symptoms. Anti N IgG was detected in 87.5% of patients (328/375) at 3 months, 38.6% (93/241) at 6 months, 23.7% (49/207) at 9 months, and 26.6% (38/143) at 12 months. The anti-N IgG seropositivity rate was significantly lower at 6, 9, and 12 months than at 3 months (P < 0.01) and was higher in the pneumonia group than in the non-pneumonia/asymptomatic group at 6 months (P < 0.01), 9 months (P = 0.04), and 12 months (P = 0.04). The rate started to decline 6–12 months after symptom onset. Anti-N IgG sample/cutoff index was positively correlated with age (r = 0.192, P < 0.01) but negatively correlated with interval between symptom onset and blood sampling (r = − 0.567, P < 0.01). These findings can guide vaccine strategies in recovered COVID-19 patients.