Culture-Based Virus Isolation and Potential Infectivity of Clinical Specimens for COVID-19
Culture-Based Virus Isolation and Potential Infectivity of Clinical Specimens for COVID-19. Jefferson T, Heneghan C.
Published on July 30, 2020
Transmission Dynamics of COVID-19
||Huang C-G, Lee K-M, Hsiao M-J et al. Culture-Based Virus Isolation To Evaluate Potential Infectivity of Clinical Specimens Tested for COVID-19. J Clin Microbiol. 2020;58(8):e01068-20.
||Research Center for Emerging Viral Infections from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan, the Ministry of Science and Technology (MOST), and Taiwan CDC and grants from Chang Gung Memorial Hospital
Cycle threshold may be a predictor of culturability but the small sample size of this study needs developing to propose an acceptable threshold or ways to calibrate PCR to achieve a high PPV on culturability and hence infectiousness. Viral genome integrity isolation is also an important pointer to infectivity
In total 28 isolates were cultured. The culture rate was low (3/19, 16%) for samples from patients with a longer duration between the date of symptom onset to sampling collection.
Specimens collected closer to the start of the illness date tended to be more culturable.
The results suggested that culturable specimens are characterized by a lower Ct value in RT-PCR analysis. This indicated the presence of more viral RNAs allowing harvesting more virus isolates for culture, which is a finding consistent with those of Bullard et al.
Because of gene variability, a threshold copy number required for virus isolation could not be defined, but the overall copy numbers were clearly higher in culturable specimens.
However, the authors remark that Ct alone is not predictive of culturability. The integrity of the viral genome may be important too as non-culturable specimens containing higher or lower concentrations of genes might reflect the detection of degraded genomes or replication intermediates. To address this problem correlation of copy number among SARS-CoV-2 genes could be a useful parameter to determine whether the virus from a given specimen can be cultured.
What did they do?
The study tested the correlation between the culturability of the virus from 60 clinical specimens and the RNA copy number from 60 specimens from 50 laboratory-confirmed COVID-19 collected from January 25 to the end of March 2020 in Taiwan.
The cases were from clusters identified either at a port of entry or in hospital. The specimens were from oropharyngeal (OP) or nasopharyngeal (NP) swabs, or sputum (SP). The authors assessed the association between the cycle threshold (Ct) value and RNA levels for genes encoding RNA-dependent RNA polymerase (nsp12), envelope (E), and nucleocapsid (N) proteins according to World Health Organization guidelines (15) from respiratory specimens of the throat, including oropharyngeal (OP) and
nasopharyngeal (NP) swabs, or sputum (SP). SARS-CoV-2 cDNA was prepared using RNA extracted from the specimens of the first patient with confirmed COVID-19. RT was performed using the MMLV Reverse transcription kit.
All procedures for viral culture were conducted in a biosafety level-3 facility. Vero-E6 and MK-2 (ATCC) cells were maintained in a virus culture medium and the cells were maintained in a 37°C incubator with daily observations of the cytopathic effect.
The study is small and needs repeating. The choice of sampled cases is not clear.
|Clearly defined setting
||Demographic characteristics described
||Follow-up length was sufficient
||Transmission outcomes assessed
||Main biases are taken into consideration
What else should I consider?
This is a small study that needs replication
About the authors
Carl is Professor of EBM & Director of CEBM at the University of Oxford. He is also a GP and tweets @carlheneghan. He has an active interest in discovering the truth behind health research findings
Tom Jefferson, epidemiologist.