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EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR
Published on: January 16, 2016
Determination of enterovirus serotype inferred from sequence analysis of PCR products
Takeshi Takami1, Tetsuo Nakayama, Hisashi Kawashima
1Department of Pediatrics, Tokyo Medical University, 6-7-1, Nishi-Shinjuku, Shinjuku-ku, Tokyo, 160-0023, Japan. t-takami@k8.dion.ne.jp
Background:
Enterovirus infections are common in neonates. Virus isolation is the only diagnostic method to confirm enterovirus serotype infections, however, is not always successful.
Objectives:
A new approach for the diagnosis of enterovirus infections was performed, using the reference strain inferred from sequence analysis of PCR products.
Study Design:
Virus isolation, enterovirus RT-PCR and sequence analysis were performed from clinical samples or stored sera from two neonates with fever and rash. Neutralizing test (NT) antibodies against prototype reference virus were measured in paired sera.
Results:
Virus isolation was negative in both patients but the enterovirus genome was amplified in the acute phase sera obtained from the two patients. From the results of sequence analysis of 109 nucleotides located in the 5'-noncoding of the conserved region of enteroviruses, a high homology to echovirus types 25 and 30 was found. More than a 4-fold increase in NT antibodies against reference viruses was demonstrated in the acute and convalescent phase sera. They were confirmed as echovirus type 25 and 30 infection, respectively.
Conclusions:
These virological examinations are practical and useful for clinical settings for a diagnosis of enterovirus infections because of an insufficient positive rate in virus isolation.
Insights
Diagnosis of enterovirus infections in neonates is challenging due to low virus isolation rates. A new method combining RT-PCR and sequence analysis successfully identified echovirus types 25 and 30 in infants where virus isolation failed.
Area of Science:
- Virology
- Molecular Diagnostics
- Neonatal Medicine
Background:
- Enterovirus infections are frequent in newborns.
- Traditional virus isolation methods for diagnosing enterovirus serotypes have limited success rates.
Observation:
- Two neonates presented with fever and rash.
- Virus isolation attempts were negative for both patients.
- Enterovirus RNA was detected in acute-phase sera via RT-PCR.
Findings:
- Sequence analysis of PCR products revealed high homology to echovirus types 25 and 30.
- Paired sera showed a significant increase in neutralizing antibodies against reference viruses.
- Echovirus type 25 and 30 infections were confirmed in the respective patients.
Implications:
- RT-PCR and sequence analysis offer a practical and effective alternative for diagnosing enterovirus infections in neonates.
- This molecular approach improves diagnostic yield when virus isolation is unsuccessful.
- Accurate and timely diagnosis is crucial for managing neonatal enterovirus infections.
