462 Archives of Disease in Childhood 1996; 74: 462-463
Enteroviral pharyngitis diagnosed by reverse
transcriptase-polymerase chain reaction
M Sharland, J Hodgson, E G Davies, J Booth, S Jeffery
Abstract MOLECULAR METHODS
The role of enteroviruses in childhood Briefly, 100 RI aliquots from the rhesus mon-
pharyngitis was investigated using key kidney tissue culture specimens were
enteroviral specific reverse transcriptase- thawed and pretreated with RNasin and
polymerase chain reaction (RT-PCR). dithiothreitol. Nucleic acid was extracted using
Viral/bacterial throat swabs were taken phenol:chloroform, and the RNA precipitated
from 50 children with acute pharyngitis using ethanol. All RT and PCR steps were per-
and 26 controls. A positive culture was formed blind. A 20 R1 reverse transcriptase
identified in only 26% of children with (RT) mix was made from 14 pI of sample, 2 p1
pharyngitis (adenovirus 10%, group A of nucleoside triphosphates, 2 pl of RT buffer,
streptococci 20/%), and none ofthe controls. 0*5 U of RNAsin, 0-2 pI of random hexamers,
Enteroviral RT-PCR was positive in 8% of 50 U murine Moloney leukaemia virus
the pharyngitis group and none of the (MMLV) RT (BRL), and 1-5 pLI of 50 mM
controls. Enteroviruses are an important MgCl2. This was incubated at 42°C for 15
cause of pharyngitis in childhood. min, 99°C for 5 min, and 4°C for 5 min. First
(Arch Dis Child 1996; 74: 462-463) round PCR reaction was in 100 pI, using 0-2
units of SuperTaq (HT Biotechnology), with
Keywords: enterovirus, pharyngitis, polymerase chain 0-5 p,m of primer E14 and CX35 with a final
magnesium concentration of 1-5 mM. Two
minutes at 95°C were followed by 40 cycles of
1 min 950C/l min 60°C. Nested PCR used 2
Pharyngitis is one of the commonest illnesses plI of first round product in a 50 pl mix con-
in childhood. While nasopharyngitis usually taining O15 U of SuperTaq, 1 5 mM MgCl2,
has a viral aetiology, pharyngitis may be caused 0-15 pRM E2,4 and CX105 for 30 cycles.
by a range of agents including group A strepto- The PCR product (15 pI) was electro-
cocci, adenoviruses, influenza, parainfluenza, phoresed in 1-5% agarose gels. DNA was
and Epstein-Barr viruses, and Mycoplasma visualised with ethidium bromide and ultravio-
pneumoniae. Enteroviruses have been recog- let. First round products were 287 base pairs,
nised as an important cause of pharyngitis, but and nested products were 154 base pairs. DNA
acute infections are difficult to diagnose using was transferred to Zetaprobe by alkali transfer
either culture or serology. 1 A highly conserved with 0 4 M NaOH. The oligo E3 (100 ng) was
5' region of the enteroviral genome has endlabelled with r32P CTP, and used to probe
recently been identified, allowing amplification the filters.4 Prehybridisation was at 37°C for
of enteroviral specific sequences using reverse 1 h, and hybridisation for 2 h, in 5 ml
transcriptase-polymerase chain reaction (RT- 20XSSPE, 1 ml l00XDenhardt's solution,
PCR).23 The purpose of this study was to and 1 ml 10% SDS made up to 20 ml in
identify the viral and bacterial causes of distilled water. Membranes were washed in
pharyngitis in the local population, and to 2XSSC with 0O1% SDS up to 48°C then
investigate the role of enteroviruses in this con- exposed overnight to x ray film.
dition. The detection limit of the enteroviral nested
PCR was analysed using the positive control of
coxsackie B3/030893 (107 TCID50/ml), and
Methods was shown to be approximately 0 5 TCID50.
St George's Hospital,
Over a one year period all children seen in the As published primer pairs were used, not all
London SW17: paediatric casualty department of St George's enteroviral serotypes were tested. Several other
Hospital and at a local general practice were positive tissue cultures infected with non-
Paediatric Infectious eligible for the study. Children were excluded enteroviral DNA and RNA viruses were tested,
M Sharland if they had received antibiotics, or had but no positive results were obtained. Standard
E G Davies nasopharyngitis. Simultaneous bacterial and negative controls and precautions to avoid
S Jeffery viral throat swabs were taken from children aged contamination were used.
Department of 1-10 years with pharyngitis. Standard culture of
Virology both swabs was performed in the microbiology
J Hodgson and virology laboratories. Viral swabs were cul- Results
J Booth tured in rhesus monkey kidney (RMK) and Over the one year period 50 children with
Dr M Sharland, Paediatric
human epithelial (HEp) cell lines for 14 days. A pharyngitis were swabbed (26 M, 24 F; mean
Infectious Diseases Unit, 5th 2 ml aliquot of the RMK tissue culture only age 4d1 years, range 1 to 10 years). Control
Floor Lanesborough Wing, was stored at -70°C. Control bacterial and throat swabs were taken from 26 children (22
St George's Hospital,
Blackshaw Road, London viral swabs were taken during anaesthesia from M, 4 F; mean age 4 7 years, range 1 to 10 years).
SWl7 OQT. children admitted for routine minor surgery. All swabs from control children were negative
Accepted 23 January 1996 Ethics consent was obtained for the study. for bacterial culture, viral cell culture, and
Enteroviral pharyngitis diagnosed by RT-PCR 463
Frequency of organisms identifledfrom pharyngits group infections would not have been detected. The
enteroviral nested PCR gave clear bands on
Organism isolated Frequency (Y.)
agarose gel and Southern blots, detecting
samples that were negative by cell culture.
Haemophilus influenzae 2 (4) Although it is possible that these positives were
due to contamination, all repeat PCR reactions
Adenovirus 5 (10) gave identical results, despite their lack of diag-
Parainfluenza 3 2 (4) nosis in cell culture. Only the RMK cell culture
was analysed by PCR, as it is unusual for
enteroviruses to grow just in HEp cell lines.
enteroviral PCR. A positive viral or bacterial There have been few prospective studies of
culture was identified in only 13 children (26%) the relative importance of different viral infec-
(table). There were no dual positive cultures. tions in childhood pharyngitis. Adenovirus was
No positive case of enteroviral infection was the commonest pathogen identified in this
detected by viral cell culture. study, confirning a previous report.' Entero-
Four samples (8%) were positive on viral infections were the second commonest
enteroviral nested PCR. All four specimens documented infection, confirming their import-
were repeated on the RMK tube frozen speci- ance as a cause of pharyngitis in childhood.
men, and confirmed to be positive on a second
nested PCR. All four samples that were posi- 1 Cherry JD. Pharyngitis (pharyngitis, tonsillitis, tonsil-
lopharyngitis, and nasopharyngitis). In: Feigin RD, Cherry
tive on nested PCR were also positive on blot- JD, eds. Textbook of paediatnic infectious diseases, 3rd ed.
ting. No sample was positive by blotting, and Philadelphia: W B Saunders, 1992: 159-66.
2 Rotbart HA. Enzymatic RNA amplification of the
not by nested PCR. enteroviruses. J Clin Microbiol 1990; 28: 438-42.
3 Nicholson F, Neetoo G, Aiyar S, Banatvala J, Muir P.
Detection of enterovirus RNA in clinical samples by nested
polymerase chain reaction for rapid diagnosis of enteroviral
Discussion infections. J Virol Methods 1994; 48: 155-66.
4 Chapman NM, Tracy S, Gauntt CJ, Fortmueller U.
In this prospective study, a positive viral or Molecular detection and identification of enteroviruses
bacterial diagnosis was obtained in only one using enzymatic amplification and nucleic acid hybridisa-
tion. J Clin Microbiol 1994; 28: 843-50.
third of children studied. Group A streptococ- 5 Severini GM, Mestroni L, Falaschi A, Camerini F, Giacca
cal infection was very rare (2%). Mycoplasma, M. Nested polymerase chain reaction for high sensitivity
detection of enteroviral RNA in biological samples. J Clin
chlamydia, Epstein-Barr virus, and anaerobic Mic7biol 1993; 31: 1345-9.