First epidemic of echovirus 16 meningitis in Cuba.

From April to September 2000, an epidemic of aseptic meningitis spread throughout Cuba, with 16,943 reported cases. Virologic studies identified echovirus 16 as the cause of this epidemic. This is the first reported isolate of echovirus 16 from patients with viral meningitis in Cuba.

cell culture fluids infected with polioviruses sabin 1-3, coxsackievirus A9 and A16, and the most common epidemic types of echoviruses (echovirus 4, 6,9,11,30), as well as by the absence of amplified cDNA with RNA extracted from herpesvirus family-infected and noninfected monkey kidney cells (Vero). The recognized sensitivity (0.01 50% tissue culture infective dose) and specificity of our enterovirus-RNA detection assay allowed us to detect specific enterovirus RNA sequences in 46.3% (25 of 54) of CSF specimens. We do not believe that positive results were due to PCR contamination because universal precautions were adopted (4). An equal number of test samples and reagent controls were processed in each test batch to prevent contamination of RNA extrac-Address for correspondence: Luis Sarmiento Perez, Department of Virology, "Pedro Kouri" Tropical Medicine Institute, Autopista Novia del Mediodía, km 6, PO Box 601, Marianao 13, Havana, Cuba; fax: 53-7-220633 and 53-7-246051; e-mail: sarmiento@ipk.sld.cu  tion by extraneous nucleic acid sequences; PCR reagents were amplified by PCR. The reagent controls' reaction verified the absence of contamination at all stages of the PCR process.
The application of this in-house RT-N-PCR assay guaranteed the rapid etiologic diagnosis of this epidemic. However, our RT-N-PCR is unable to determine the enterovirus serotype, which is necessary to understand the epidemiology of enterovirus infections.
For enterovirus isolation, 200 µL of CSF and fecal specimens were inoculated in duplicate into tubes covered with monolayers of fibroblastic diploid embryonic human cells (PhuE-1) and Vero cells. From the 76 fecal specimens inoculated, 45 (59.2%) induced cytopathic effect (CPE). This CPE was only evident in the PhuE-1 cell monolayers; Vero cells remained unchanged. Viral isolation was possible from all the tubes showing CPE. However, no enterovirus strains were recovered by cell culture isolation from any CSF specimen. These discrepancies could be explained by the massive excretion of viral particles in feces. Moreover, previous studies have demonstrated that enteroviruses are isolated from CSF in only a few cases with acute aseptic meningitis because viral particles are present in low titers in CSF (5-7). Nevertheless, a higher positivity was obtained from 20 CSF samples when RT-N-PCR was used than for 20 samples of feces from the same patients when evaluated by viral isolation. Only 10 of the 16 CSF specimens positive by PCR could be correlated with a positive fecal culture, whereas the 4 PCR-negative CSF samples correlated well with the absence of viral isolation from the related fecal specimens.
CPEs produced by isolated strains were typical of those characterizing enteroviruses (e.g., cell rounding followed by shrinkage and degeneration of the cell sheet). However, at the beginning of the epidemic, isolated strains produced a CPE that progressed slower (slow-CPE) than typical enterovirus isolates do. The inoculated cultures had to be subpassaged weekly at least 5 times to obtain the typical degenerative CPE of enterovirus. By the late stages of the epidemic, CPE produced by isolated strains became evident at the first passage (fast-CPE). No slow-CPE isolates were detected late in the epidemic, and there was no evidence of "fast CPE-to-slow CPE" reversion of isolates late in the epidemic. Nevertheless, no changes in the clinical outcome of the infection were observed during the epidemic. It is tempting to speculate that the genetic constitution of the selected viral population could be substantially different from that of the original strain. This phenomenon could explain the variation in CPE during the epidemic.
All strains from the epidemic were identified as echovirus 16 by a neutralization test using Lim-Benyesh-Melnick antisera pools. To corroborate the infecting serotype, presence of a fourfold or greater increase of type-specific virusneutralizing antibody titers between acute-and convalescent-phase serum specimens was determined; 54.8% (17 of 31) of the patients' sera exhibited a significant rise of neutralizing antibody titer against the isolated strains. The geometric mean titers of the first and second sera were 1:3.4; and 1:22.4, respectively.
Nevertheless, the age distribution in this outbreak suggests previous exposure and immunity to echovirus 16 in older persons (Figure 2). The age group distribution was also similar to that in previously described outbreaks of aseptic meningitis in Cuba (6). A possible explanation for this age distribution is that an antigenically related virus without potential to cause acute aseptic meningitis circulated in the population before year 2000 and induced an immune response. Alternatively, maternal antibodies against this virus may protect neonates from infection.
Factors influencing the prevalence of enteroviruses are poorly understood. The ease with which a type of nonpolio enterovirus can be isolated, however, is likely to be a major determinant of the frequency with which it is reported (5,8). Echovirus 16 isolates associated with sporadic cases of aseptic meningitis have probably been infrequently reported in previous years because of the difficulties in tissue culture propagation.
According to data collected through the U.S. National Enterovirus Surveillance System, echovirus 16 is routinely isolated, but the frequency of its isolation is low in most years (i.e., 3 to 5 cases per year) (Centers for Disease Control and Prevention, unpub. data).
To our knowledge, the last Cuban outbreak of aseptic meningitis associated with echovirus 16 infections occurred in Tajimi City, Gifu prefecture, in 1984. Diverse etiologic agents (coxsackievirus B1, B4, and B5) were also reported during the outbreak (9).
The emergence of echovirus 16 associated with a very large-scale meningitis epidemic in Cuba should alert public health officials to the potential for epidemics associated with this serotype in other areas of the world.
The overall genetic diversity and molecular evolution in echovirus 16 strains and the correlation with the epidemiologic features of echovirus 16-associated disease have not yet been studied. The availability of the viral isolates, together with the massive clinical and epidemiologic data from this epidemic, represents an unprecedented opportunity to study the emergence of echovirus 16 strains and their subsequent molecular evolution.