Related Experiment Videos
RT-PCR based analysis of cell culture negative stools samples from poliomyelitis suspected cases
Ana P Santos1, Eliane V Costa, Silas S Oliveira
1Laboratório de Enterovirus, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil.
Background:
Routine diagnosis of acute flaccid paralysis (AFP) is still based on classical virological procedures. Several serotypes of enterovirus which possess the potential to cause neurological disorders are not easily isolated in the cell culture systems used for the AFP diagnosis.
Objectives:
Our goal was to look into the presence of enterovirus genomes in fecal suspensions previously considered negative by cell culture procedures, using RT-PCR.
Study Design:
One hundred and seventy-three fecal samples collected from AFP cases and contacts occurring in Brazil, Peru and Bolivia and tested negative regarding viral isolation, after inoculation in the cell lines RD and Hep2C, were analyzed by RT-PCR using a pair of primers which specifically detects enterovirus genome RNA's.
Results:
Twenty-six samples (15%) showed amplicons compatible with those observed for enterovirus RNA amplification. The identity of these amplicons were confirmed by nucleotide sequencing. By using RT-PCR directly in the fecal suspensions we were able to detect enterovirus RNA's in twenty-six additional samples. These samples would be considered as negative if only the standard cell-culture-based methodology had been utilized. No polioviruses were detected among the positive samples.
Insights
Routine diagnosis of acute flaccid paralysis (AFP) often misses enteroviruses. Reverse transcription-polymerase chain reaction (RT-PCR) detected enterovirus RNA in 15% of samples previously negative by cell culture, improving diagnostic sensitivity.
Area of Science:
- Virology
- Molecular Biology
- Infectious Diseases
Background:
- Classical virological procedures are standard for diagnosing acute flaccid paralysis (AFP).
- Certain enterovirus serotypes causing neurological disorders are difficult to isolate using conventional cell culture methods.
Purpose of the Study:
- To investigate the presence of enterovirus genomes in fecal samples that tested negative via cell culture.
- To evaluate the utility of reverse transcription-polymerase chain reaction (RT-PCR) for detecting enteroviruses in AFP cases.
Main Methods:
- 173 fecal samples from AFP cases and contacts in Brazil, Peru, and Bolivia were analyzed.
- Samples initially tested negative for viral isolation in RD and Hep2C cell lines.
- RT-PCR was performed using primers specific for enterovirus genome RNA.
Main Results:
- Enterovirus RNA was detected in 26 samples (15%) using RT-PCR.
- Nucleotide sequencing confirmed the identity of the amplified enterovirus sequences.
- RT-PCR identified enterovirus RNA in 26 additional samples missed by standard cell culture, demonstrating increased sensitivity.
Conclusions:
- RT-PCR significantly enhances the detection of enteroviruses in fecal samples from AFP cases.
- This molecular method improves diagnostic yield for enterovirus infections compared to traditional cell culture.
- No polioviruses were identified in the enterovirus-positive samples detected by RT-PCR.