TABLE 16.

PCR for viruses

VirusReferenceCultureReal-time PCRComments
No. of specimensNo. positive (%)Cells or antigenNo. of specimensNo. positive (%)Test platform/ gene target
Influenza virusaWard et al. 2004 (527)233 throat swabs50 (21.5)MDCK, tertiary cynomolgus233140 (60.0)ABI Prism/M1 matrix geneReal-time assays were designed for both A and B viruses. Large-scale screening and identification of influenza virus using real-time PCR was carried out as part of the development of zanamivir.
van Elden et al. 2001 (507)Tertiary rhesus monkey kidney cells27 reference strains (A), 9 reference strains (B), and other isolatesA, 36 (37), B, 4 (4)ABI Prism/matrix protein (A), hemagglutinin gene (B)Influenza virus could be detected in nasal wash specimens up to 7 days after initial presentation of influenza-like symptoms.
98 clinical specimens22 (22.4)Shell vial98
Spackman et al. 2002 (458)1,550 cloacal, tracheal, or environmental swabs from various avian species266 (17.2)Embryonated eggs1,550303 (19.6)ABI Prism/matrix gene (conserved for all A matrix genes; in addition, primer sets were developed to H5 and H7 strains of avian influenza virus; broad-range detection including both North American and Eurasian lineage avian viruses and isolates of human, equine, and swine origin influenza A virusRecovery of influenza virus requires 1-2 weeks in embryonated eggs.
Smith et al. 2003 (453)58 throat and nasal swabs35 (60.3) (25 A; 10 B)Rhesus monkey kidney cells/tube culture5851 (88.0) (41 A; 10 B)LightCycler/A matrix (300 bp) protein (5′ end of the matrix gene M1), B nucleoprotein (184 bp); LC run parameters were identical for A and B4 LightCycler (positive only) results sequenced. Analysis of fragment matched the M1 gene of influenza virus A. B strains, frozen supernatants previously known to contain influenza virus B assay tested with H3N2 and H1N1 strains and two influenza B viruses (Beijing-like).
Boivin et al. 2003 (37)Nasal and pharyngeal swabsLightCycler/hemagglutinin geneStudy assessed kinetics of the influenza virus load in respiratory tract samples of infected individuals receiving early treatment with neuraminadase inhibitors compared to those receiving deferred treatment. The mean pretreatment virus load was significantly lower in 24 patients who initiated treatment within 24 h of the onset of symptoms than it was in 26 patients who initiated treatment between 24 and 48.
Boivin et al. 2004 (36)172 (subset of nasopharyngeal aspirates from hospitalized children)A: 19 (11.0), B: 0Compared to rapid antigen assays for A and B (Becton Dickinson)17242 (24.4)LightCycler/matrix genes of both A and BMelting curve feature of LightCycler instrument was used to differentiate influenza strains from RSV. Pediatric study ≤3 yr of age.
Frisbie et al. 2004 (132)75 children ≤4 yr; archived nasal aspirates22 (29.3) archived nasal aspiratesRhesus monkey kidney cells7518 (24)ABI Prism/A matrix gene; B hemagglutinin geneRepeat testing by culture of 21 of 22 positive archival specimens revealed only 11 positive results; all 21 were PCR positive by repeat testing.
Espy et al.b 2004557 respiratory tract specimens (throat swabs, nasal washes, bronchoalveolar lavage, sputum, nasal swabs)51 (9.2%) R-mix 24 (4.3%) BINAX antigen testR-mix55792 (16.5%)LightCycler/matrix geneLightCycler PCR was rapid (3 h extraction and analytic time) and more sensitive than R-mix cell cultures and BINAX for the detection of influenza virus type A (H3N2) strains from clinical specimens.
Respiratory syncytial virusBoivin et al. 2003 (36)204 nasopharyngeal aspirates from children <3 yr of age94 (46)RSV TestPack antigen test204104 (51)ABI Prism/F geneAuthors developed a multiplex test to differentiate RSV and influenza A and B virus amplicons by melting curve analysis.
Borg et al. 2003 (38)62 acute respiratory tract infection (children); nasopharyngea1 secretions29 (46.8)ABI Prism/F1 gene (subunit of fusion protein)Real-time (quantitative) assay specific for subgroup A was developed. The median viral load of the specimens from patients with chronic obstructive pulmonary disease was 6.1 × 10 copies/ml compared with median of 1.2 × 107 copies/ml in children with respiratory tract infection.
125 adults with chronic obstructive pulmonary disease, nasal lavage fluid and induced sputum35 (28)
Falsey et al. (122)169, 13 adult volunteers, nasal washes58 (34)HEp-216973 (43)ABI Prism/F geneReal-time assay detects both RSV A and B subgroups.
Gueudin et al. 2003 (154)75 nasal aspirates34 (45.3)MRC-5 and A-5497542 (56%)LightCycler/N geneDirect immunofluorescence was positive in 31/75 (41.3%) of samples.
Hu et al. 2003 (189)175 nasopharyngeal aspirates from children21 (12), immunofluorescence17536 (20.3) (10 RSV A, 26 RSV B)ABI Prism/N geneAssay detects RSV A and B subgroups. Immunofluorescence technique identified 32/75 (42.7%) RSV-positive samples.
Mentel et al. 2003 (313)71 71 7125 (35.2%) (Real-Time) 19 (26.7%) Nested, conventional PCR 10 (14.1) Antigen ELISA (VirionSerion)iCycler/F gene71 consecutive specimens were processed and tested by PCR from hospitalized children with clinical symptoms of acute respiratory distress to obtain a rapid laboratory diagnosis of RSV infection.
VirusReferenceCultureReal-time PCRComments
No. of specimensNo. positive (%)Cells or antigenNo. of specimensNo. positive (%)Test platform/gene target
van Elden et al. 2003 (508)168 (during period of symptoms), combined nose and throat swabs from immunocompromised adults (autologous or allogenic stem cell transplant patients (n = 73), hematologic malignancies (n = 17)4 (2.4%)Shell vial rhesus monkey kidney cells16813 (4.9%) (Real-time PCR) 13 (4.9%) Laboratory developed nested PCRABI Prism/N geneReal-time assay detects both RSV A and B subgroups. Detection of RSV cDNA by nested PCR and real-time PCR is equivalent.
AdenovirusHoung et al. 2002 (187)96 throat swabs from military personnel with acute respiratory tract disease72 (previously positive Ad4 strains isolated in cell cultures)A-549 cell cultures (human lung carcinoma)96 specimens selected for positive results by previous inoculation in cell cultures from 1953 through 1998 and then tested by PCR in 200171ABI Prism/Ad4 hexon geneQuantitative real-time PCR assay used to detect Ad4 (subgroup E) DNA in specimens from military recruits during an acute respiratory disease outbreak. The assay did not crossreact with representative members of adenovirus subgroups A, B, C, D, and F.
Gu et al. 2003 (151)4520 (44.4)ABI Prism/hexon geneReal-time quantitative PCR was designed to detect adenovirus DNA from all major subgroups of the virus.
45 (conventional PCR)20 (44.4)
Heim et al. 2003 (170)234 (real-time PCR)53 (22.6)LightCycler/hexon geneAdenovirus DNA was detected in blood by real-time (quantitative) PCR in 4/27 (14.8%) pediatric and 8/93 (8.6%) of adult stem cell transplant patients but only in 5/306 healthy blood donor controls (1.6%). Detection of high virus loads in blood holds promise for simplified and earlier diagnosis of disseminated adenovirus disease in immunosuppressed patients.
234 (conventional PCR)39 (16.6)
Lion et al. 2003 (277)132 (patients)36 (27.2) (patients); positive results from at least one specimenABI Prism/hexon geneIn this series, 8/11 (73%) children with adenovirus DNA in blood specimens, but none of the patients with adenovirus DNA detectable at sites other than blood, developed fatal disseminated adenoviral disease.
Faix et al. 2004 (121)140 throat swabs from 86 subjects99 (70.7)A-549 cell cultures14098 (70%)SmartCycler/Ad4 hexon geneAssay developed by Houng et al. (187) was adapted to the SmartCycler instrument for the detection of adenovirus DNA in specimens from military recruits during an acute respiratory disease outbreak. Overall, rapid PCR results had a sensitivity of 100% and a specificity of 100% compared with viral culture.
Leruez-Ville et al. 2004 (261)44 plasma8 (18.1)ABI Prism/hexon geneAll 8 patients for whom PCR detected adenovirus DNA in blood samples had disseminated adenovirus infection.
Lankester et al. 2004 (246)iCycler IQ/hexon geneFour allogeneic stem cell transplant patients were treated with ribavirin and subsequently with cidofovir. Quantitative real-time PCR determinations indicated that these antiviral drugs did not reduce levels of adenovirus DNA.
Metapneumo-virusCôté et al. 2003 (84)LLC-MK2 (continuous monkey kidney cell line)20 specimens selected as probable positive cultures for metapneumovirus20 (100%) with nucleoprotein, N gene targetLightCycler/nucleoprotein, matrix, fusion, phosphoprotein, polymerase gene targetsPCR is method of choice for metapneumovirus laboratory diagnosis because of poor replication of the virus in cell culture.
10 nasopharyngeal aspirates10 (100%) with nucleoprotein, N gene target
Mackay et al., 2003 (295)62 nasopharyngeal aspirates 6 known PCR-positive and 56 known PCR-negative specimens (conventional methods)12 (19.4) real-time PCR 6 (9.7) Conventional PCRLightCycler/nucleoprotein gene targetReal-time PCR was the preferred choice for detecting metapneumovirus and was considerably more reliable than cell culture in the routine clinical laboratory.
Maertzdorf et al., 2004 (296)38 clinical samples found positive by conventional PCR38 (100)ABI Prism/nucleoprotein gene targetHuman metapneumoviruses can be divided into two main genetic lineages (A and B) representing two serotypes and each comprising two sublineages (A1, A2, B1, B2). This assay detects all strains of the virus.
54 clinical samples known as negative by conventional PCR0
Parainfluenza virusTempleton et al. 2004 (483)35811 (3.1%)HEp-2, HEL, and LLCMK23584 (1.1)iCycler IQ/accession nos. PI-1-70948 , PI-2-AF213352 , PI-3-M18760 , PI-4-M55976Rapid real-time multiplex PCR assay was developed for the detection of influenza A and influenza B viruses, RSV, and parainfluenza viruses 1, 2, 3, and 4.
Templeton et. al. 2004 (482)Case report: parainfluenza virus type 3 detected by direct immunofluorescence from nasal wash specimens.Parainfluenza virus 1 (accession no. 70948 ); parainfluenza virus 2 (AF213352); parainfluenza virus 3 (M18760); parainfluenza virus 4 (M55976); iCycler IQ (molecular beacon technology)PCR was found to be far more sensitive than culture or immunofluorescence in an immunocompromised host (post-stem cell transplantation) and results of this assay (real-time PCR) could improve management of patients.
    Parainfluenza virus type 3 detected by culture (child with acute lymphoblastic leukemia who received a hematopoietic stem cell transplant)
SARS-CoVPoon et al. 2004 (382)98 nasopharyngeal aspirates43 (44)ABI/TaqMan/ORF16
36 stool samples21 (57)
Drosten et al. 2004 (99)66 samples from 29 confirmed SARS patients; 31 respiratory specimens47 (70.8)LightCycler/replicase gene (Real/Art LC kit)
35 stool and other specimens44 (67.1)LightCycler/nucleocapsid gene
Chan et al. 2004 (64)531 respiratory tract45 (8.4)Vero E6471 respiratory tract122 (25.9)No specimen was positive by culture and negative by PCR.
526 nonrespiratory4 (0.76)365 nonrespiratory83 (22.7)
Ng et al. 2003 (337)36 plasma15 (41.6)ABI/TaqMan/ polymerase geneSamples collected over 2 weeks.
23 serum18 (78.2)ABI/TaqMan/ nucleocapsid geneSamples collected on day of hospital admission.
  • a In this section, A and B refer to influenza virus types A and B, respectively.

  • b M. J. Espy, S. K. Schneider, P. A. Wright, S. Kidiyala, M. F. Jones, and T. F. Smith, Program Abstr. 20th Clin. Virol. Symp., abstr. M51, 2004.