Journal of Clinical Virology 23 (2002) 149 – 152 RT-PCR based analysis of cell culture negative stools samples from poliomyelitis suspected cases Ana P. Santos, Eliane V. Costa, Silas S. Oliveira, Michele C. Souza, Edson E. Da Silva * Laborato ´rio de Enteroirus, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil Received 20 March 2001; received in revised form 31 May 2001; accepted 15 June 2001 Abstract 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. Objecties: 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. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Enterovirus; Acute flaccid paralysis; RT-PCR www.elsevier.com/locate/jcv Viral isolation in cell culture followed by iden- tification of the isolate is the routine procedure for acute flaccid paralysis (AFP) diagnosis within the World Health Organization (WHO) network of laboratories inside the initiative to eradicate poliomyelitis. The identification of the isolate is heavily dependent on serological procedures while intratypic differentiation using molecular methods is generally necessary in case of poliovirus isola- tion. At least two passages showing negative cyto- pathic effect (CPE) are necessary for a given sample to have the result reported as negative for poliovirus or non-polio enteroviruses isolation (WHO, 1998). Polioviruses, members of the enterovirus genus, family Picornairidae, are the main causal agent of poliomyelitis, an acute viral disease, whose clinical expression may varies from a sub-clinical * Corresponding author. Tel./Fax: +055-21-564-7638. E-mail address: eesilva@gene.dbbm.fiocruz.br (E.E. Da Silva). 1386-6532/01/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S1386-6532(01)00211-6