scholarly journals Detection by PCR of Enteroviruses in Cerebrospinal Fluid during a Summer Outbreak of Aseptic Meningitis in Switzerland

1998 ◽  
Vol 36 (9) ◽  
pp. 2408-2412 ◽  
Author(s):  
Meri Gorgievski-Hrisoho ◽  
Jean-Daniel Schumacher ◽  
Nevenka Vilimonovic ◽  
Daniel Germann ◽  
Lukas Matter
Keyword(s):  
Cell Culture ◽  
Cerebrospinal Fluid ◽  
Aseptic Meningitis ◽  
Diagnostic Techniques ◽  
Rt Pcr ◽  
Echovirus 30 ◽  
Reverse Transcription Pcr ◽  
Viral Culture ◽  
Test Kit ◽  
Virus Culture

Enteroviruses (EV) are among the most common causes of aseptic meningitis. Standard diagnostic techniques are often too slow and lack sensitivity to be of clinical relevance. EV RNA can be detected within 5 h by a commercially available reverse transcription-PCR (RT-PCR) test kit. Cerebrospinal fluid (CSF) samples from 68 patients presenting with aseptic meningitis during a summer outbreak in Switzerland were examined in parallel with cell culture and commercial RT-PCR. RT-PCR was positive in all 16 CSF specimens positive by cell culture (100%). In addition, 42 of 52 (80%) CSF samples negative by cell culture were PCR positive. In 26 of these 42 (62%) patients, viral culture from other sites (throat swab or stool) was also positive. The CSF virus culture took 3 to 7 days to become positive. Echovirus 30 was the type most often isolated in this outbreak. The sensitivity of CSF RT-PCR based on clinical diagnosis during this aseptic meningitis outbreak in patients with negative bacterial culture results was 85%, i.e., considerably higher than the sensitivity of CSF virus culture (24%). We conclude that this commercial RT-PCR assay allows a positive diagnosis with minimal delay and may thus influence clinical decisions.

scholarly journals Evaluation of a Commercially Available Reverse Transcription-PCR Assay for Diagnosis of Enteroviral Infection in Archival and Prospectively Collected Cerebrospinal Fluid Specimens

1998 ◽  
Vol 36 (6) ◽  
pp. 1741-1745 ◽  
Author(s):  
Francisco Pozo ◽  
Inmaculada Casas ◽  
Antonio Tenorio ◽  
Gloria Trallero ◽  
Jose M. Echevarria
Keyword(s):  
Cell Culture ◽  
Cerebrospinal Fluid ◽  
Aseptic Meningitis ◽  
Reverse Transcription ◽  
Diagnostic Systems ◽  
Rt Pcr ◽  
Enteroviral Infection ◽  
Reverse Transcription Pcr ◽  
Sporadic Cases ◽  
Pcr Method

A commercially available reverse transcription (RT)-PCR method (AMPLICOR EV; Roche Diagnostic Systems, Inc., Branchburg, N.J.) was evaluated for detection of enteroviruses in cerebrospinal fluid from patients with neurological disease. This assay was compared with virus isolation in cell culture and an in-house RT-PCR method designed with a nonoverlapping region of the enteroviral genome. A panel of 200 cerebrospinal fluid specimens prospectively collected from patients with a wide variety of neurological symptoms, including 50 patients involved in three different outbreaks of acute aseptic meningitis, was assayed. A second panel of 97 archived cerebrospinal fluid specimens, stored for 2 to 5 years, from patients with aseptic meningitis associated with several enterovirus outbreaks was also studied. From the first panel, enteroviruses were detected in 13 of 50 specimens by cell culture (26%), in 43 of 50 specimens by AMPLICOR EV (86%), and in 46 of 50 specimens by the in-house assay (92%) from patients with aseptic meningitis associated with outbreak and 1 of 29, 3 of 29, and 4 of 29 specimens, respectively, from sporadic cases of aseptic meningitis. The remaining 121 cerebrospinal fluid specimens from patients with other neurological syndromes were negative by all tests. From the second panel, enteroviral RNA was detected by the AMPLICOR test (31 of 97 specimens, 32%) and the in-house assay (39 of 97 specimens, 40%). According to our results, patients with aseptic meningitis should be analyzed for enteroviral infection in cerebrospinal fluid by RT-PCR methods, and the AMPLICOR EV test is a suitable tool for performing such studies. Archival cerebrospinal fluid specimens are less suitable for evaluation of the performance of RT-PCR methods designed for enterovirus detection.

scholarly journals The Utility of Caco-2 cells in Isolation of Enteroviruses from Environmental and Clinical Material

2014 ◽  
Vol 63 (1) ◽  
pp. 69-73
Author(s):  
MAGDALENA WIECZOREK ◽  
AGNIESZKA CIĄĆKA ◽  
AGNIESZKA WITEK ◽  
BOGUMIŁA LITWIŃSKA
Keyword(s):  
Cell Culture ◽  
Cerebrospinal Fluid ◽  
Rate Increase ◽  
Clinical Material ◽  
Rt Pcr ◽  
Viral Titre ◽  
Clinical Specimens ◽  
Strain Isolation ◽  
Two Samples ◽  
Rd Cells

The work presented here demonstrates the utility of Caco-2 cells in the isolation of enteroviruses (EVs) from environmental and clinical materials. Thirty-two samples of cerebrospinal fluid positive in Pan-entero RT-PCR were taken for EV strain isolation in cell culture. Out of the 32 samples analysed, 22 (68.75%) were positive for enteroviruses by isolation in Caco-2 cells, and 10 (31.25%) were positive by isolation in RD cells. High viral titre in clinical specimens resulted in rate increase for isolation in Caco-2 cells and RD cells (87.5% and 50%, respectively). Also, the probability of isolation of enteroviruses from sewage in Caco-2 cells was 20 times higher that in RD cells. We proved that Caco-2 cells were more effective than RD cells in enterovirus isolation, irrespective of the material used in the inoculation process.

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 DIRECT DETECTION OF ENTEROVIRUS GENOME IN CELL-CULTURE NEGATIVE CEREBROSPINAL FLUID FROM ASEPTIC MENINGITIS CASES IN BRAZIL

2012 ◽  
Vol 17 (1-2) ◽  
Author(s):  
Gina Peres Lima dos Santos ◽  
Fernanda Marcicano Burlandy ◽  
Eliane Veiga da Costa ◽  
Edson Elias Da Silva
Keyword(s):  
Cell Culture ◽  
Cerebrospinal Fluid ◽  
Aseptic Meningitis ◽  
Direct Detection ◽  
Culture Negative

scholarly journals Culture-Based Virus Isolation To Evaluate Potential Infectivity of Clinical Specimens Tested for COVID-19

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Chung-Guei Huang ◽  
Kuo-Ming Lee ◽  
Mei-Jen Hsiao ◽  
Shu-Li Yang ◽  
Peng-Nien Huang ◽  
...  
Keyword(s):  
Real Time ◽  
Viral Genome ◽  
Copy Number ◽  
Virus Isolation ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Genome Copy Number ◽  
Virus Culture

ABSTRACT Real-time reverse transcription-PCR (RT-PCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19). However, the correlation between detectable viral RNA and culturable virus in clinical specimens remains unclear. Here, we performed virus culture for 60 specimens that were confirmed to be positive for SARS-CoV-2 RNA by real-time RT-PCR. The virus could be successfully isolated from 12 throat and nine nasopharyngeal swabs and two sputum specimens. The lowest copy number required for virus isolation was determined to be 5.4, 6.0, and 5.7 log10 genome copies/ml sample for detecting the nsp12, E, and N genes, respectively. We further examined the correlation of genome copy number and virus isolation in different regions of the viral genome, demonstrating that culturable specimens are characterized by high copy numbers with a linear correlation observed between copy numbers of amplicons targeting structural and nonstructural regions. Overall, these results indicate that in addition to the copy number, the integrity of the viral genome should be considered when evaluating the infectivity of clinical SARS-CoV-2 specimens.

scholarly journals Comparison of a New Gold Immunochromatographic Assay for the Rapid Diagnosis of the Novel Influenza A (H7N9) Virus with Cell Culture and a Real-Time Reverse-Transcription PCR Assay

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Changzhong Jin ◽  
Nanping Wu ◽  
Xiaorong Peng ◽  
Hangping Yao ◽  
Xiangyun Lu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Influenza A ◽  
High Specificity ◽  
High Viral Load ◽  
Immunochromatographic Assay ◽  
Clinical Samples ◽  
H7n9 Virus ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Viral Culture

We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings.

scholarly journals Detection and Identification of Mammalian Reoviruses in Surface Water by Combined Cell Culture and Reverse Transcription-PCR

2001 ◽  
Vol 67 (7) ◽  
pp. 3016-3020 ◽  
Author(s):  
Michael L. Spinner ◽  
George D. Di Giovanni
Keyword(s):  
Cell Culture ◽  
Surface Water ◽  
Sequence Analysis ◽  
Reverse Transcription ◽  
Mammalian Species ◽  
Sampling Site ◽  
Water Sources ◽  
Sequence Diversity ◽  
Rt Pcr ◽  
Reverse Transcription Pcr

ABSTRACT Reoviruses are a common class of enteric viruses capable of infecting a broad range of mammalian species, typically with low pathogenicity. Previous studies have shown that reoviruses are common in raw water sources and are often found along with other animal viruses. This suggests that in addition to the commonly monitored enteroviruses, reoviruses might serve as an informative target for monitoring fecal contamination of drinking water sources. Mammalian reoviruses were detected and identified by a combined cell culture–reverse transcription-PCR (RT-PCR) assay with novel primers targeting the L3 gene that encodes the λ3 major core protein. Five of 26 (19.2%) cytopathic effect-positive cell culture lysates inoculated with surface water were positive for reoviruses by RT-PCR. DNA sequence analysis of RT-PCR products revealed significant sequence diversity among isolates, which is consistent with the sequence diversity among previously characterized mammalian reoviruses. Sequence analysis revealed persistence of a reovirus genotype at a single sampling site, while a sample from another site contained two different reovirus genotypes.

Rapid Detection Method for Hepatitis A Virus from Lettuce by a Combination of Filtration and Integrated Cell Culture–Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (10) ◽  
pp. 1756-1761 ◽  
Author(s):  
JI-YEON HYEON ◽  
JUNG-WHAN CHON ◽  
CHANKYU PARK ◽  
JUNG-BOK LEE ◽  
IN-SOO CHOI ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rt Pcr ◽  
Cytopathic Effects ◽  
Reverse Transcription Pcr ◽  
Charged Membrane ◽  
Polyethylene Glycol Precipitation ◽  
Positively Charged

We have developed a rapid and simple method for filtration using a positively charged membrane to concentrate hepatitis A virus (HAV) from lettuce and an integrated cell culture–real-time reverse transcription PCR (ICC–real-time RT-PCR) to detect infectious HAV. The most suitable buffer for HAV concentration by filtration was 100 mM Tris-HCl, 50 mM glycine (pH 9.5). Filtration using the NanoCeram matrix was compared with polyethylene glycol precipitation for viral concentration from lettuce inoculated with 6 log RNA copies of HAV. The recovery rate of filtration was statistically higher than that of polyethylene glycol precipitation (47.3 versus 24.9%, respectively). The sensitivity of ICC–real-time RT-PCR for detection of infectious HAV was determined by inoculation of FRhK-4 cells with HAV (4 log to 0 log RNA copies). ICC–real-time RT-PCR detected infectious HAV on average 5 days earlier than cytopathic effects at all inoculation levels. HAV recovered from lettuce (approximately 3 log RNA copies) was also analyzed with ICC–real-time RT-PCR. Infectious HAV was detected within 2 days postinfection by ICC–real-time RT-PCR, whereas cytopathic effects were not observed until 7 days postinfection. Coupled with a virus concentration and purification system using a positively charged membrane, ICC–real-time RT-PCR has the potential to become a novel and rapid method for the detection of infectious HAV in vegetables.

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