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Human rhabdomyosarcoma cells for rapid detection of enteroviruses by shell-vial assay

Servicio de Microbiología, Hospital Universitario Virgen de las Nieves, Avda Fuerzas Armadas s/n, 18014 Granada, Spain

Correspondence M. Pérez-Ruiz mercedes.perez.ruiz.sspa{at}juntadeandalucia.es

Received February 25, 2003
Accepted June 11, 2003

The ability of the RD (rhabdomyosarcoma) and MRC-5 cell-lines to detect enteroviruses in 33 clinical samples (cerebrospinal fluid, stools and throat swabs) was evaluated. The samples had previously tested enterovirus-positive by traditional tube-culture and had been frozen after their initial processing. By traditional tube-culture, 100 and 85 % of samples were positive for enterovirus in RD and MRC-5 cells, respectively. By rapid shell-vial assay, 94 and 45.5 % were positive after 48 h incubation in RD and MRC-5 cells, respectively. RD cells supported growth of all enterovirus serotypes, whereas MRC-5 cells were not able to detect any of the three coxsackieviruses that were found (one coxsackievirus A9 and two coxsackievirus B5). The shell-vial assay with RD cell-lines may be a useful tool for rapid diagnosis of enteroviral infection.

	Introduction

TOP Introduction Methods Results and Discussion References

 
Enteroviruses, which belong to the Picornaviridae family, comprise five human pathogenic species [poliovirus, human enterovirus (HEV)-A, HEV-B, HEV-C and HEV-D] with 65 antigenically distinct serotypes: poliovirus (three serotypes), coxsackievirus A (23 serotypes), coxsackievirus B (six serotypes), echovirus (28 serotypes) and enterovirus serotypes 68–71 and 73 (King et al., 2000; Romero & Rotbart, 2003). Non-polio HEVs are responsible for a wide variety of clinical syndromes in humans, ranging from asymptomatic or mild upper respiratory illness to more severe diseases, such as aseptic meningitis (Melnick, 1996). Aseptic meningitis is associated with many infectious and non-infectious causes, although 80–92 % of cases for which the aetiological agent is identified are due to HEVs (Berlin et al., 1993; Rotbart, 1995). Meningitis caused by HEVs appears mainly in summer and autumn and leads to an increase in hospitalization of both children and adults (Sawyer, 1999).

HEV infections can be clinically indistinguishable from other, more severe viral and bacterial infections. Therefore, HEV-infected patients undergo inappropriate treatment and prolonged hospital stays (Sawyer, 1999). To avoid this, it is important that a rapid aetiological diagnosis is available (Marshall et al., 1997).

Viral isolation in cell-culture remains the standard method to diagnose infection caused by HEVs (Melnick, 1996). The traditional method is tube-culture (TC) in appropriate cell-lines, although growth of the virus is slow and results are usually only available when the patient has been discharged (Sawyer, 1999). Rapid techniques such as shell-vial assay (SV) can reach a definite diagnosis in 48 h (Van Doornum & De Jong, 1998), but there is no consensus cell-line that supports growth of most HEV serotypes.

We have analysed the ability of human rhabdomyosarcoma (RD) cells to detect HEVs by SV, compared to MRC-5-SV and TC in both cell-lines.

	Methods

TOP Introduction Methods Results and Discussion References

 
The study was conducted on 33 samples: seven cerebrospinal fluid (CSF) samples, 11 stools and 15 throat swabs, received between June and September 2000, which corresponded to 22 patients with clinical suspicion of meningitis. The samples had previously tested HEV-positive and had been frozen at -80 °C after their initial processing.

Initial isolation of HEVs was carried out by TC. For this purpose, 200 µl sample was inoculated into RD, MRC-5 and Vero cell-lines by standard protocols (Clarke et al., 1992). Tubes were examined daily for 14 days to observe the appearance of cytopathic effect (CPE). Identification of isolates from cell-lines with CPE was performed by indirect immunofluorescence assay (IFA) with a mAb against the enteroviral VP1 protein (clone 5-D8/1; Dako). All viral cultures without CPE were subjected to IFA for HEV detection at the end of the incubation period (as described above) before discarding them as negative. This was considered to be the reference method. All positive samples (n = 33) and 15 HEV isolates from these samples were frozen at -80 °C for comparative analysis of cell-lines and serotyping, respectively.

The 33 positive samples were inoculated in duplicate into RD and MRC-5 cells for TC [as described by Clarke et al. (1992)] and SV. Briefly, 200 µl was inoculated into flat-bottomed tubes and centrifuged at 800 g for 45 min at 35 °C, followed by addition of 1 ml serum-free maintenance medium (minimal essential medium, MEM) and incubation at 35–37 °C with continuous shaking. After 48 h incubation, IFA with specific antibodies was carried out on the monolayers.

HEV serotypes were determined for 15 clinical isolates (five from CSF samples, four from stools and six from throat swabs). RD cells were inoculated with isolated strains and when CPE appeared, IFA was carried out with mAbs against echovirus 4, 6, 9, 11 and 30 and coxsackievirus A9, A24 and B1–B6 (Chemicon).

	Results and Discussion

TOP Introduction Methods Results and Discussion References

 
Of the 33 samples studied, 33 (100 %) and 28 (85 %) tested positive in RD and MRC-5 cells by TC, respectively. By SV, 31 (94 %) and 15 (45.5 %) samples tested positive in RD and MRC-5 cells, respectively. No samples were simultaneously positive by SV and negative by TC, nor positive in MRC-5 and negative in RD cells by SV (Table 1). Detection times of HEVs by TC ranged from 3 to 9 days for RD cells (mean time, 4.9 days) and from 3 to 11 days for MRC-5 cells (mean time, 5.7 days), compared to 2 days for SV. No HEVs were detected by ‘blind’ IFA after 14 days incubation. CPE was present in all positive samples before the end of the incubation period.

HEV serotypes detected in the 15 strains analysed were as follows: two echovirus-6 (13.3 %), five echovirus-30 (33.3 %), one coxsackievirus A9 (6.7 %), two coxsackievirus B5 (13.3 %) and five non-typable strains (33.3 %) (Table 2). All strains grew from the original sample in RD cells by TC and SV. Originally, none of the three coxsackieviruses was recovered from the sample cultured in MRC-5 cells; however, both coxsackievirus B5 strains grew in MRC-5 subcultures, but coxsackievirus A9 did not.

The most suitable samples for diagnosis of aseptic meningitis by HEV are CSF samples, stools and throat exudates (Melnick, 1996; Terletskaia-Ladwig et al., 2000), although the isolation of HEVs from the latter two offers only a probable diagnosis; this may sometimes be due to asymptomatic excretion of the virus (Melnick, 1996). However, lower viral loads in CSF samples may yield false-negative results by cell-culture, or positive results may not be available in less than 7 days. For all these reasons, the use of rapid techniques such as SV, which allows a prompt diagnosis of infection, has been recommended (Klespies et al., 1996; Van Doornum & De Jong, 1998; Lipson et al., 2001). Several cell-lines support growth of HEVs in TC, e.g. MRC-5, RD, RMK, BGMK and A-549 (Chonmaitree et al., 1988; Van Doornum & De Jong, 1998; Huang et al., 2002).

In this study, RD cells were more sensitive than MRC-5 cells for HEV isolation in all cases, by both TC and SV. HEVs in all CSF samples were detected by RD-SV, whilst none grew in MRC-5 cells by SV; this demonstrated a higher sensitivity of RD cells, mainly in samples with suspected lower titres of virus. This is supported by the fact that the number of fluorescent foci was higher with RD than with MRC-5 cells, as observed by immunofluorescence from samples that grew in both cell-types (5–20-fold greater by 40x field examination; data not shown). This comparative study was carried out with frozen samples; freezing may have contributed to a decrease in viral load compared to fresh samples. This may explain the discrepancy of our results with those of other authors who achieved better results with MRC-5-SV (Chonmaitree et al., 1988; Reina et al., 2000).

The sensitivity of RD-SV was 94 % with respect to RD-TC, but failed in two samples: one stool sample and one throat swab. However, we do not know whether this was due to lower sensitivity of SV compared to TC, or to other factors that influenced the isolation of HEVs from these samples, e.g. enhanced toxicity of these types of sample in SV. Furthermore, CSF samples (with lower expected toxicity and viral loads) showed a correlation between RD-SV and RD-TC of 100 %. Indeed, the advantage of RD-SV with respect to TC is that the problems caused by rapid overgrowth of RD cells in TC are solved.

With respect to the ability of RD and MRC-5 cells to detect different HEV serotypes, 100 and 71 % of echoviruses grew in both cell lines, respectively, which demonstrates that these cells are appropriate for the detection of these HEV serotypes, as reported previously (Dagan & Menegus, 1986; Chonmaitree et al., 1988). However, no coxsackieviruses were isolated by MRC-5-TC or -SV from the original sample, but they were all isolated by RD-SV and -TC. These results were expected for coxsackievirus A9 but not for the coxsackievirus B5 strains, as MRC-5 cells are similar or even better than RD cells for this serotype (Hsiung, 1994; Romero & Rotbart, 2003).

RT-PCR and real-time RT-PCR from clinical samples, especially CSF, have been demonstrated to be better methods for diagnosis of aseptic meningitis due to HEVs than TC (Buck et al., 2002; Verstrepen et al., 2002). However, cell-culture is still the method used by many laboratories, as molecular techniques may not be available in routine practice. In these situations, SV with RD cells may be a good alternative as it reduces the detection time of HEV compared to TC without a significant deleterious effect on sensitivity.

In summary, we consider that RD-SV may be a useful tool for rapid diagnosis of aseptic meningitis due to HEVs in our area, although the real use of this method in other geographical areas needs to be evaluated as there are differences in distribution of HEV serotypes.

	References

TOP Introduction Methods Results and Discussion References