Laboratory Diagnostic Options and Challenges for Chikungunya Viruses

Pulse ◽  
2018 ◽  
Vol 10 (1) ◽  
pp. 18-24
Author(s):  
MM Rahman
Keyword(s):  
Viral Disease ◽  
Medical Attention ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Specific Pcr ◽  
Highly Sensitive

Background: Chikungunya infection is a Aedes mosquito transmitted viral disease caused by Chikungunya virus (CHIKV), a member of the Alphavirus genus. It is an important human pathogen that causes a syndrome characterized by fever, chills, headache and severe joint pain usually of the smaller joints, with or without swelling. Though the disease is almost self-limiting, during the recent outbreak CHIKV was also found to cause long-term arthralgia, neurological disease and even few fatalities. Despite the fact that CHIKV is associated with epidemics of unprecedented magnitude, only a few specific serological and molecular diagnostic tools are available.Objective: CHIKV diagnosis is essentially based on virus isolation, reverse transcription (RT)-PCR and ELISA assays. The gold standard of CHIKV diagnosis is culture, however, required facilities and skills are not available in any routine laboratory in the country. Highly sensitive and specific PCR assays for CHIKV have been developed and commercially available.Conclusion: Although the reagents and equipment are costly for widespread use RT-PCR is the method of choice for the early detection and confirmation of virus in clinical samples as most acutely infected patients seek medical attention within the first few days of illness when role of serology based diagnosis is minimum.Pulse Vol.10 January-December 2017 p.18-24

scholarly journals Characterization of Genomic Variants Associated with Resistance to Bedaquiline and Delamanid in Naive Mycobacterium tuberculosis Clinical Strains

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
S. Battaglia ◽  
A. Spitaleri ◽  
A. M. Cabibbe ◽  
C. J. Meehan ◽  
C. Utpatel ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genetic Variants ◽  
Protein Structures ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Clear Understanding ◽  
Data Set ◽  
Gene Target ◽  
Genomic Regions

ABSTRACT The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants’ role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

A Norovirus Outbreak at a Long-Term-Care Facility: The Role of Environmental Surface Contamination

2005 ◽  
Vol 26 (10) ◽  
pp. 802-810 ◽  
Author(s):  
Henry M. Wu ◽  
Mary Fornek ◽  
Kellogg J. Schwab ◽  
Amy R. Chapin ◽  
Kristen Gibson ◽  
...  
Keyword(s):  
Risk Factors ◽  
Long Term Care ◽  
Care Facility ◽  
Rt Pcr ◽  
Term Care ◽  
Term Care Facility ◽  
Long Term Care Facility ◽  
Environmental Surface

AbstractBackground:The role of environmental surface contamination in the propagation of norovirus outbreaks is unclear. An outbreak of acute gastroenteritis was reported among residents of a 240-bed veterans long-term-care facility.Objectives:To identify the likely mode of transmission, to characterize risk factors for illness, and to evaluate for environmental contamination in this norovirus outbreak.Methods:An outbreak investigation was conducted to identify risk factors for illness among residents and employees. Stool and vomitus samples were tested for norovirus by reverse transcription polymerase chain reaction (RT-PCR). Fourteen days after outbreak detection, ongoing cases among the residents prompted environmental surface testing for norovirus by RT-PCR.Results:One hundred twenty-seven (52%) of 246 residents and 84 (46%) of 181 surveyed employees had gastroenteritis. Case-residents did not differ from non-case-residents by comorbidities, diet, room type, or level of mobility. Index cases were among the nursing staff. Eight of 11 resident stool or vomitus samples tested positive for genogroup II norovirus. The all-cause mortality rate during the month of the outbreak peak was significantly higher than the expected rate. Environmental surface swabs from case-resident rooms, a dining room table, and an elevator button used only by employees were positive for norovirus. Environmental and clinical norovirus sequences were identical.Conclusion:Extensive contamination of environmental surfaces may play a role in prolonged norovirus outbreaks and should be addressed in control interventions.

scholarly journals Comparison of commercial RT-PCR diagnostic kits for COVID-19

2020 ◽  
Author(s):  
Puck B. van Kasteren ◽  
Bas van der Veer ◽  
Sharon van den Brink ◽  
Lisa Wijsman ◽  
Jørgen de Jonge ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Cross Reactivity ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Specific Method ◽  
Preliminary Evaluation ◽  
Rt Pcr ◽  
Accurate Identification ◽  
Clinical Sensitivity ◽  
Pcr Efficiency

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

scholarly journals Improved Detection of Rhinoviruses in Clinical Samples by Using a Newly Developed Nested Reverse Transcription-PCR Assay

1999 ◽  
Vol 37 (3) ◽  
pp. 524-530 ◽  
Author(s):  
Arno C. Andeweg ◽  
Theo M. Bestebroer ◽  
Martijn Huybreghs ◽  
Tjeerd G. Kimman ◽  
Jan C. de Jong
Keyword(s):  
Reverse Transcription ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Noncoding Regions ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Virus Culture ◽  
Culture Positive

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

scholarly journals Development, characterization and clinical validation of new sensitive immunofluorometric assay for the measurement of serum thyroglobulin

2012 ◽  
Vol 56 (9) ◽  
pp. 658-665 ◽  
Author(s):  
Cláudia C. D. Nakabashi ◽  
Rosa Paula M. Biscolla ◽  
Teresa S. Kasamatsu ◽  
Teresinha T. Tachibana ◽  
Rafaela N. Barcelos ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
High Sensitivity ◽  
Differentiated Thyroid Carcinoma ◽  
Serum Thyroglobulin ◽  
Additional Advantage ◽  
Highly Sensitive ◽  
Long Term Follow Up

OBJECTIVE: In the last decade, data published stressed the role of highly-sensitive thyroglobulin (Tg) assays in the follow-up of differentiated thyroid carcinoma (DTC) patients. The present study describes a new, highly-sensitive Tg assay, compares it with an available commercial assay, and validates it in the follow-up of DTC patients. SUBJECTS AND METHODS: The immunofluorometric high-sensitivity Tg assay is based on monoclonal and polyclonal antibodies produced at our laboratories. It was validated in 100 samples of 87 patients with DTC submitted to total thyroidectomy, 87% of whom also received radioiodine. For correlation, all samples were also tested using a commercial Tg assay (Beckman Access) with functional sensitivity (FS) of 0.1 ng/mL. RESULTS: The new method showed FS of 0.3 ng/mL. The correlation between the two methods was good (r = 0.74; p < 0.0001). The diagnostic sensitivity was 88.9%, and it was increased to 100% when combined with neck US. CONCLUSION: This new, high-sensitivity Tg assay presented a good correlation with Beckman Access assay and with the clinical outcome of the patients. The continuous availability of a validated assay is an additional advantage for long term follow-up of DTC patients. Arq Bras Endocrinol Metab. 2012;56(9):658-65

scholarly journals Rose Rosette Disease: Recent Advances on Molecular Diagnostic Tools

HortScience ◽  
2018 ◽  
Vol 53 (5) ◽  
pp. 596-600 ◽  
Author(s):  
Binoy Babu ◽  
Gary Knox ◽  
Mathews L. Paret ◽  
Francisco M. Ochoa-Corona
Keyword(s):  
The United States ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Department Of Agriculture ◽  
Specialty Crop ◽  
Detection And Identification ◽  
Research Initiative ◽  
Highly Sensitive ◽  
Rose Rosette ◽  
Rose Rosette Disease

Rose rosette emaravirus (RRV, genus Emaravirus), the causal agent of rose rosette disease, is the topmost pathogen of concern for the rose industry in the United States. The only strategy available for disease management is early identification and eradication of the infected plants. Highly reliable, specific, and sensitive detection assays are thus required to test and confirm the presence of RRV in suspected plant samples. RRV is only a recently characterized virus and hence limits the diagnostic tools available for its early detection. With a U.S. Department of Agriculture (USDA) Specialty Crop Research Initiative (SCRI) project sponsorship, several diagnostic tools including end-point reverse transcription-polymerase chain reaction (RT-PCR) and RT-qPCR assays targeting single and multiple genes targets were developed for routine diagnostics. This review introduces an overall view of the different diagnostic tools developed, which are reliable, highly sensitive, and can be easily implemented for detection and identification in laboratories providing diagnostic services and confirmation of RRV-infected samples.

Elucidating the Pivotal Role of Immune Players in the Management of COVID-19: Focus on Mesenchymal Stem Cells and Inflammation

2020 ◽  
Vol 15 ◽  
Author(s):  
Seidu A. Richard ◽  
Sylvanus Kampo ◽  
Marian Sackey ◽  
Maite Esquijarosa Hechavarria ◽  
Alexis D. B. Buunaaim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Disease Burden ◽  
Viral Disease ◽  
Inflammatory Factors ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Paracrine Secretion ◽  
Polymerase Chain

: The world is currently engulfed with a viral disease with no cure. So, far, millions of people are infected with the virus across the length and breadth of world with thousand losing their lives each passing day. The WHO is February 2020 classified the virus as a coronavirus and the name Coronavirus-19 (CoV-19) was offered to the virus. The disease caused by the virus was termed coronavirus disease-19 (COVID-19). The pathogenesis of COVID-19 is associated with elevation of several immune plays as well as inflammatory factors which contributes to cytokine storms. Currently, the detection of CoV-19 RNA is through reverse transcriptase polymerase chain reaction (RT-PCR). Mesenchymal stem cells (MSCs) are capable of suppressing several kinds of cytokines via the paracrine secretion system. Therefore, MSCs therapy could be game charges in the treatment of the current COVID-19 pandemic. Also, intravenous IG may be capable of suppressing the high expression of IL-6 by the CoV-19 resulting in lessen disease burden. Anti-inflammatory medications like, corticosteroids, tocilizumab, glycyrrhetinic acid, as well as etoposide may be very advantageous in decreasing the COVID-19 burden because, their mode of action targets the cytokine storms initiated by the CoV-19. It is important to indicate that, these medication does not target the virus itself. Therefore, potent CoV-19 anti-viral medications are needed to completely cure patients with COVID-19. Also, a vaccine is urgently needed to stop the spread of the virus. This review therefore elucidates the immune players in the management of COVID-19; focusing principally on MSCs and inflammatory mediators.

scholarly journals Comparison of SARS-CoV-2 RT-PCR on a high-throughput molecular diagnostic platform and the cobas SARS-CoV-2 test for the diagnostic of COVID-19 on various clinical samples

2020 ◽  
Vol 78 (8) ◽  
Author(s):  
Onya Opota ◽  
René Brouillet ◽  
Gilbert Greub ◽  
Katia Jaton
Keyword(s):  
High Throughput ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Test Results ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Rapid Spread ◽  
Bronchial Aspirate ◽  
Very High

ABSTRACT Objectives:In order to cope with the rapid spread of the COVID-19 pandemic, we introduced on our in-house high-throughput molecular diagnostic platform (MDx Platform) a real-time reverse transcriptase PCR (RT-PCR) to detect the SARS-CoV-2 from any clinical specimens. The aim of this study was to compare the RT-PCR results obtain with the MDx Platform and the commercial assay cobas SARS-CoV-2 (Roche) on nasopharyngeal swab and other clinical specimens including sputum, bronchial aspirate, bronchoalveolar lavage and anal swabs. Methods: Samples received in our laboratory from patients suspected of COVID-19 (n = 262) were tested in parallel with our MDx platform SARS-CoV-2 PCR and with the cobas SARS-CoV-2 test. Results: The overall agreement between the two tests for all samples tested was 99.24% (260/262), which corresponded to agreements of 100% (178/178) on nasopharyngeal swabs, 95.45% (42/44) on lower respiratory tract specimen with discordant resultS obtained for very high cycle threshold (Ct) value and 100% (40/40) on anorectal swabs. The Ct values for nasopharyngeal swabs displayed an excellent correlation (R2 &gt; 96%) between both tests. Conclusions: The high agreements between the cobas SARS-CoV-2 test and the MDx platform supports the use of both methods for the diagnostic of COVID-19 on various clinical samples. Very few discrepant results may occur at very low viral load.

scholarly journals Role of Nucleocapsid Protein Antigen Detection for Safe End of Isolation of SARS-CoV-2 Infected Patients with Long Persistence of Viral RNA in Respiratory Samples

2021 ◽  
Vol 10 (18) ◽  
pp. 4037
Author(s):  
Antonella Mencacci ◽  
Alessio Gili ◽  
Anna Gidari ◽  
Elisabetta Schiaroli ◽  
Carla Russo ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Infectious Virus ◽  
Viral Rna ◽  
Concomitant Disease ◽  
Rt Pcr ◽  
Antigen Assay ◽  
Diagnosis Of Infection ◽  
Better Than

Background. In SARS-CoV-2 infection, viral RNA may persist in respiratory samples for several weeks after the resolution of symptoms. Criteria to assess the end of infectivity are not unequivocally defined. In some countries, time from diagnosis is the unique criterion used, in addition to symptom cessation. This study evaluates the role of the Lumipulse® Antigen Assay (LAA) for the safe end of isolation of patients ≥21 days after the diagnosis of infection. Methods. A total of 671 nasopharyngeal swabs from patients diagnosed with infection at least 21 days before were assessed by RT-PCR and LAA, and the role of LAA in predicting the absence of infectivity was evaluated by virus cell culture. Results. Viable virus was present in 10/138 cultured samples. Eight out of ten infective patients suffered from a concomitant disease, predisposing them to long-term shedding of infective virus. In particular, infectious virus was isolated from 10/20 RT-PCR+/LAA+ cultured samples, whereas no viable virus was found in all 118 RT-PCR+/LAA– cultured swabs. LLA and RT-PCR agreed in 484/671 (72.1%) samples, with 100% and 26.7% concordance in RT-PCR negative and positive samples, respectively. Conclusions. Viable virus can be found ≥21 days after diagnosis in immunocompromised or severely ill patients. LAA better than RT-PCR predicts non-infectivity of patients and can be safely used to end isolation in cases with long persistence of viral RNA in the respiratory tract.

scholarly journals Accuracy of Roche SARS-CoV-2 Rapid Antigen Test in Nasopharyngeal Swab: Clinical Impression Matters

2021 ◽  
Vol 2 (10) ◽  
pp. 929-938
Author(s):  
Khin Phyu Pyar ◽  
Khine Khine Su ◽  
Kyaw Wunna ◽  
Myo Thant ◽  
Kaung Myat ◽  
...  
Keyword(s):  
Predictive Value ◽  
False Negative ◽  
Hospital Setting ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Clinical Impression ◽  
Antigen Test ◽  
Rt Pcr ◽  
Ct Value

Background: In COVID-19 pandemic, the diagnosis and treatment must be as early as possible to save the life of each patient. Moreover, screening of asymptomatic carriers, close contacts or healthy subjects must not be delay to prevent transmission to publics. For confirmation of diagnosis of SARS-CoV-2 infection, nasopharyngeal swab must be tested either by real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) tests or Rapid Antigen Test (RAT). RAT is faster, easier and cheaper; thus, it is suitable for health service in developing country. Objectives: The aim of this study was to assess the diagnostic accuracy of Roche SARS-CoV-2 Rapid Antigen Test (RAT) in diagnosing SARS-CoV-2 infection. Methods: Hospital based exploratory study was done in out-patient department and fever clinic, and molecular laboratory of No. (1) Defence Services General Hospital. Nasopharyngeal swabs were taken, and the Roche SARS- CoV-2 RAT was conducted in parallel with RT-PCR test (reference standard). Results: Among the 932 patients/subjects recruited, RT-PCR was positive in 468 individuals, corresponding to a prevalence of 50.2%. The RAT was positive in 363 patients (60.4%), false positive in 120 patients; it was negative in 569 individuals (39.6%), false negative in 225 patients. The overall sensitivity of the RAT was 51.9% (95% Confidence Interval [CI] 47.29-56.53) and, the specificity was 74.1% (95% CI 69.9-78.07); positive predictive value was 66.9% and negative predictive value was 60.5%. The sensitivity varied with Ct value; 78% in clinical samples with Ct values < 20, 57.5% in those with Ct values between 21 and 25, 41.8% in samples with Ct values between 26 and 30, and, 36.4% in samples with Ct value > 30. Conclusion: The accuracy of the SARS-CoV-2 Roche RAT in diagnosing SARS-CoV-2 infections was inferior to RT-PCR and manufacturer’s data. The sensitivity was with low Cycle threshold values < 20 which were inversely related to the viral load. RAT test should be used in association with clinical impression of physicians. In hospital setting especially in emergency department, the role of RAT should be reconsidered in those patients presenting with anosmia and some cases of dyspnoea, late symptoms in the course of disease, as the RAT results would be false negative. Other errors may arise if the operator for RAT has to handle more than recommended tests per hour especially in the peak of epidemics.

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