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

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.

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Rapid Shell Vial Culture Technique for Detection of Enteroviruses and Adenoviruses in Fecal Specimens: Comparison with Conventional Virus Isolation Method

Journal of Clinical Microbiology ◽

10.1128/jcm.36.10.2865-2868.1998 ◽

1998 ◽

Vol 36 (10) ◽

pp. 2865-2868 ◽

Cited By ~ 33

Author(s):

G. J. J. Van Doornum ◽

J. C. De Jong

Keyword(s):

Cell Culture ◽

Cerebrospinal Fluid ◽

Virus Isolation ◽

Adenovirus Type ◽

Isolation Procedure ◽

Enzyme Linked Immunosorbent Assay ◽

Shell Vial ◽

Clinical Specimens ◽

Conventional Culture ◽

Stool Specimens

Detection of enteroviruses and adenoviruses mainly in fecal specimens by rapid culture with inoculation onto cell monolayers in flat-bottom tubes by centrifugation and immunofluorescence staining with genus-specific monoclonal antibodies was compared with that by the conventional virus isolation procedure. For both conventional culture and shell vial culture human lung fibroblast cells and tertiary monkey kidney cells were used. For enterovirus detection, 979 clinical specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection rate for enteroviruses by rapid cell culture after 2 to 3 days of incubation was 42 of 74 (57%). The genus-specific enterovirus monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of adenoviruses was performed with 567 clinical specimens (536 stool specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by conventional culture. Nine of the 42 (21%) adenovirus isolates were detected by conventional culture within 3 days after inoculation, whereas 21 (50%) were found by rapid cell culture within 2 to 3 days. Only two of the nine specimens found to be positive for the enteric adenovirus type 41 by conventional culture as well by a type-specific enzyme-linked immunosorbent assay (ELISA) tested positive by rapid cell culture. In conclusion, the rapid shell vial assay allows the early detection and identification of enteroviruses and adenoviruses in clinical specimens but is markedly less sensitive than the conventional isolation procedure according to the eventual results of the conventional isolation procedure. Conventional cell culture remains a prerequisite for serotyping of enteroviral isolates. On the basis of the results for adenovirus type 41, the rapid detection of adenoviruses was not considered to be useful for the detection of clinically relevant adenoviruses in fecal samples.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.36.9.2408-2412.1998 ◽

1998 ◽

Vol 36 (9) ◽

pp. 2408-2412 ◽

Cited By ~ 36

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.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.36.6.1741-1745.1998 ◽

1998 ◽

Vol 36 (6) ◽

pp. 1741-1745 ◽

Cited By ~ 27

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.

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Enrichment of cerebrospinal fluid samples on cell culture for enhancement of sensitivity of mumps and enterovirus detection by multiplex RT-PCR

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2007.10.023 ◽

2008 ◽

Vol 60 (4) ◽

pp. 375-379

Author(s):

Masoumeh Kermanian ◽

Hoorieh Soleimanjahi ◽

Ehsan Arefian ◽

Taravat Bamdad

Keyword(s):

Cell Culture ◽

Cerebrospinal Fluid ◽

Rt Pcr

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Propagation and isolation of group A coxsackieviruses in RD cells

Journal of Clinical Microbiology ◽

10.1128/jcm.2.3.183-185.1975 ◽

1975 ◽

Vol 2 (3) ◽

pp. 183-185

Author(s):

N J Schmidt ◽

H H Ho ◽

E H Lennette

Keyword(s):

Cell Culture ◽

Cell Line ◽

Clinical Specimens ◽

Culture Systems ◽

Suckling Mice ◽

Cell Culture Systems ◽

Rd Cells ◽

Group A ◽

Group B

The RD cell line, derived from a human rhabdomyosarcoma, supported replication of a number of group A coxsackieviruses, including types A5 and A6 which heretofore have been propagable only in suckling mice. A number of the group A coxsackievirus types which replicated in RD cells had higher titers in this cell line than in other cell culture systems. In tests on a limited number of clinical specimens, RD cells were slightly less sensitive than suckling mice for isolation of group A coxsackieviruses, but they did permit the recovery of certain virus types which previously could be isolated only in suckling mice. Group B coxsackieviruses replicated poorly or not at all in RD cells.

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Amplicor Enterovirus Polymerase Chain Reaction in Patients With Aseptic Meningitis

Archives of Pathology & Laboratory Medicine ◽

10.5858/1999-123-0882-aepcri ◽

1999 ◽

Vol 123 (10) ◽

pp. 882-884

Author(s):

Emilia Hadziyannis ◽

Nancy Cornish ◽

Colleen Starkey ◽

Gary W. Procop ◽

Belinda Yen-Lieberman

Keyword(s):

Cell Culture ◽

Polymerase Chain Reaction ◽

Cerebrospinal Fluid ◽

Chart Review ◽

Culture Method ◽

Final Diagnosis ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Enteroviral Meningitis

Abstract Objective.—To evaluate the sensitivity and specificity of reverse-transcription polymerase chain reaction (RT-PCR) and routine cell culture for the detection of enterovirus in cerebrospinal fluid. Methods.—Thirty-eight cerebrospinal fluid specimens were included. Cell culture was inoculated immediately and incubated for 14 days. An aliquot was kept frozen for Amplicor RT-PCR. Chart review was performed to determine the validity of the results. Results.—Nine of 38 specimens were positive for enterovirus by culture, and 14 were positive by RT-PCR. There were 7 discrepancies between the 2 methods. Six specimens were positive by RT-PCR and negative by the culture method. The 1 culture-positive but RT-PCR–-negative specimen was determined to contain PCR inhibitors. All discrepant results were confirmed as true positives by chart review. Patients whose cerebrospinal fluid was negative by both methods had a final diagnosis other than enterovirus infection. Conclusion.—Amplicor PCR is more sensitive than cell culture (93.3% vs 60%) and is very specific. With the incorporation of appropriate controls for the detection of amplification inhibitors, RT-PCR could be a valuable tool in the diagnosis of enteroviral meningitis.

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Rapid Detection of Human Rhinoviruses in Nasopharyngeal Aspirates by a Microwell Reverse Transcription-PCR–Hybridization Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.37.9.2813-2816.1999 ◽

1999 ◽

Vol 37 (9) ◽

pp. 2813-2816 ◽

Cited By ~ 39

Author(s):

S. Blomqvist ◽

A. Skyttä ◽

M. Roivainen ◽

T. Hovi

Keyword(s):

Cell Culture ◽

Reverse Transcription ◽

Solid Phase ◽

Culture Method ◽

Hybridization Assay ◽

Confirmatory Test ◽

Rt Pcr ◽

Clinical Specimens ◽

Rolling Tube ◽

Tube Cell

A rapid and sensitive microwell reverse transcription (RT)-PCR–hybridization assay was developed to detect human rhinoviruses in clinical specimens and cell culture suspensions. Two hundred three nasopharyngeal aspirates collected from children with symptoms of respiratory disease were analyzed by a classical rolling-tube cell culture method, microwell culture of HeLa Ohio cell monolayers, and RT-PCR with detection of the amplicons in a microwell hybridization assay. The RT-PCR was also done with harvests of the microwell cultures. RNA was extracted with a commercial kit, and the RT-PCR procedure was carried out with microtiter-format equipment. A confirmatory test that exploited a blocking oligonucleotide at the hybridization step was developed to reliably identify marginally positive specimens. Of the 203 nasopharyngeal aspirate specimens, rhinovirus or rhinoviral RNA was detected in 111 specimens (55%). Ninety-eight specimens (48%) were found to be positive by RT-PCR of the original nasopharyngeal aspirates, while the conventional rolling-tube cell culture method yielded 52 (26%) positive specimens. This RT-PCR method with solid-phase hybridization is easy to perform, sensitive, and specific and will be especially useful for analysis of large numbers of clinical specimens.

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Detection of enteroviruses in marine waters by direct RT-PCR and cell culture

Water Science & Technology ◽

10.2166/wst.1995.0635 ◽

1995 ◽

Vol 31 (5-6) ◽

pp. 323-328 ◽

Cited By ~ 8

Author(s):

K. A. Reynolds ◽

C. P. Gerba ◽

I. L. Pepper

Keyword(s):

Cell Culture ◽

Polymerase Chain Reaction Amplification ◽

Cost Effective ◽

The United States ◽

Water Runoff ◽

Rt Pcr ◽

Marine Waters ◽

Storm Water Runoff ◽

Routine Monitoring ◽

Reaction Amplification

Sewage outfalls and storm water runoff introduces pathogenic human enteric viruses into marine coastal waters, which may pose a potential public health risk. Although members of the enterovirus group have been suggested as possible indicators of sewage pollution in marine waters, the lack of rapid, sensitive and cost effective methods have prevented routine monitoring in the United States. This study compared traditional cell culture and direct RT-PCR (reverse transcriptase-polymerase chain reaction) amplification for detection of an enterovirus. Poliovirus could be recovered from 100 L of artificial seawater with an average efficiency of 77%, using adsorption and elution from electronegative filters. Viruses were eluted from the filters with 1.5% beef extract for viruses (BEV) adjusted to pH 9.5 and reconcentrated by organic flocculation to a volume of 30 mL. Substances which interfered with detection by RT-PCR were removed by treatment of the concentrates with sephadex and chelex resins. Direct RT-PCR could detect 2.5 and 0.025 PFU (plaque forming units) for single (25 cycles) and double PCR (2 × 25 cycles) in 10 μL of pure culture poliovirus samples, respectively. These methods are currently being applied to assess the occurrence of enteroviruses at marine bathing beaches influenced by sewage discharges.

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SARS-CoV-2 Is Not Detected in the Cerebrospinal Fluid of Encephalopathic COVID-19 Patients

Frontiers in Neurology ◽

10.3389/fneur.2020.587384 ◽

2020 ◽

Vol 11 ◽

Author(s):

Dimitris G. Placantonakis ◽

Maria Aguero-Rosenfeld ◽

Abdallah Flaifel ◽

John Colavito ◽

Kenneth Inglima ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Viral Entry ◽

Cell Types ◽

Host Cells ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Neurologic Sequelae ◽

Show Evidence ◽

Novel Coronavirus ◽

Transmembrane Serine Protease

Neurologic manifestations of the novel coronavirus SARS-CoV-2 infection have received wide attention, but the mechanisms remain uncertain. Here, we describe computational data from public domain RNA-seq datasets and cerebrospinal fluid data from adult patients with severe COVID-19 pneumonia that suggest that SARS-CoV-2 infection of the central nervous system is unlikely. We found that the mRNAs encoding the ACE2 receptor and the TMPRSS2 transmembrane serine protease, both of which are required for viral entry into host cells, are minimally expressed in the major cell types of the brain. In addition, CSF samples from 13 adult encephalopathic COVID-19 patients diagnosed with the viral infection via nasopharyngeal swab RT-PCR did not show evidence for the virus. This particular finding is robust for two reasons. First, the RT-PCR diagnostic was validated for CSF studies using stringent criteria; and second, 61% of these patients had CSF testing within 1 week of a positive nasopharyngeal diagnostic test. We propose that neurologic sequelae of COVID-19 are not due to SARS-CoV-2 meningoencephalitis and that other etiologies are more likely mechanisms.

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