Increased Detection of Respiratory Syncytial Virus, Influenza Viruses, Parainfluenza Viruses, and Adenoviruses with Real-Time PCR in Samples from Patients with Respiratory Symptoms

Abstract

Respiratory samples (n = 267) from hospitalized patients with respiratory symptoms were tested by real-time PCR, viral culture, and direct immunofluorescence for respiratory syncytial virus, influenza virus, parainfluenza viruses, and adenoviruses. Compared with conventional diagnostic tests, real-time PCR increased the diagnostic yields for these viruses from 24% to 43% and from 3.5% to 36% for children and adults, respectively.

Determining etiological diagnoses for patients admitted to the hospital with respiratory symptoms remains a clinical and laboratory challenge. Infections with respiratory syncytial virus (RSV), influenza viruses (IVs), parainfluenza viruses (PIVs), and adenoviruses (AdVs) are traditionally diagnosed using viral culture and direct immunofluorescence (DIF) tests. However, over the last 2 decades, nucleic acid amplification techniques, particularly real-time PCR, have become available as diagnostic tools. The aim of the present study was to determine the diagnostic yields of real-time PCR for RSV, IV, PIVs and AdVs in clinical practice, compared with those of traditional tests.

For this purpose, respiratory specimens from hospitalized pediatric and adult patients with respiratory symptoms were collected during one respiratory season (December 2004 through May 2005). Follow-up samples (taken within 4 weeks after a first sample) were excluded. Samples were divided into three aliquots. One aliquot of the samples from children was used for DIF assays to detect RSVs A and B, IVs A and B, PIVs 1 to 3, and AdV using Imagen kits (DaKo, Glostrup, Denmark) according to the manufacturer's protocol. DIF was not performed for samples from adults, because it has been shown to have a very low sensitivity in an adult population (2). One aliquot of the samples was used for immediate viral culture of RSV, IVs, PIVs 1 to 3, and AdV on LLC-MK2, RD, R-HELA, and HEp-2C cells. Cultures were examined every other day for the development of a cytopathologic effect for 14 days, and positive cultures were confirmed by DIF with monoclonal antibodies specific for RSV A or B; IV A or B; PIV 1, 2, or 3; or AdV (DaKo, Glostrup, Denmark). Finally, an aliquot of the samples was used to extract viral nucleic acids using the MagNA Pure LC total nucleic acid isolation kit (Roche Diagnostics, Basel, Switzerland) as described previously (6). Subsequently, cDNA was synthesized using MultiScribe reverse transcriptase and random hexamers (both from Applied Biosystems, Foster City, CA). Detection of viruses was performed using parallel real-time PCR assays for RSVs A and B (8) and IVs A and B as previously described (7, 10). In addition, in-house real-time PCR methods were developed for the detection of PIVs 1 to 4 and AdVs using Primer Express (Applied Biosystems). Conserved target regions were identified using BLAST (www.ncbi.nlm.nih.gov/blast). Sequences of the primers and probes used are summarized in Table 1. Real-time PCR procedures were performed as described previously (6).

TABLE 1.

Primers and probes used for real-time PCR detection

Virus(es)	Target gene	Forward primer(s) (5′-3′)	Reverse primer(s) (5′-3′)	Probe(s)a	Reference
RSV A	Nucleocapsid	AGA TCA ACT TCT GTC ATC CAG CAA	TTC TGC ACA TCA TAA TTA GGA GTA TCA AT	FAM-CAC CAT CCA ACG GAG CAC AGG AGA T-TAMRA	8
RSV B	Nucleocapsid	AAG ATG CAA ATC ATA AAT TCA CAG GA	TGA TAT CCA GCA TCT TTA AGT ATC TTT ATA GTG	FAM-TTC CCT TCC TAA CCT GGA CAT AGC ATA TAA CAT ACC T-TAMRA	8
IV A	Matrix	AAG ACC AAT CCT GTC ACC TCT GA	CAA AGC GTC TAC GCT GCA GTC C	FAM-TTT GTG TTC ACG CTC ACC GT-TAMRA	10
IV B	Hemagglutinin	AAA TAC GGT GGA TTA AAC AAA AGC AA	CCA GCA ATA GCT CCG AAG AAA	FAM-CAC CCA TAT TGG GCA ATT TCC TAT GGC-TAMRA	7
PIV 1	Hemagglutinin-neuraminidase	TGA TTT AAA CCC GGT AAT TTC TCA T	CCT TGT TCC TGC AGC TAT TAC AGA	FAM-ACG ACA ACA GGA AAT C-MGB
PIV 2	Hemagglutinin-neuraminidase	AGG ACT ATG AAA ACC ATT TAC CTA AGT GA	AAG CAA GTC TCA GTT CAG CTA GAT CA	FAM-ATC AAT CGC AAA AGC TGT TCA GTC ACT GCT ATA C-TAMRA
PIV 3	Hemagglutinin-neuraminidase	TGA TGA AAG ATC AGA TTA TGC ATA TC	CCG GGA CAC CCA GTT GTG	FAM-TGG ACC AGG GAT ATA CTA CAA AGG CAA AAT AAT ATT TCT C-TAMRA
PIV 4	Nucleocapsid	CAA AYG ATC CAC AGC AAA GAT TC	ATG TGG CCT GTA AGG AAA GCA	FAM-GTA TCA TCA TCT GCC AAA TCG GCA ATT AAA CA-TAMRA
AVs	Hexon	TTT GAG GTG GAY CCM ATG GA	AGA ASG GSG TRC GCA GGT A	FAM-ACC ACG TCG AAA ACT TCG AA-MGB
		TTT GAG GTY GAY CCC ATG GA	AGA ASG GTG TRC GCA GAT A	FAM-ACC ACG TCG AAA ACT TCA AA-MGB
				FAM-ACA CCG CGG CGT CA-MGB

^aFAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; MGB, minor groove binding.

A total of 267 samples from patients with respiratory symptoms were analyzed. Of these samples, 181 were from children (152 nasopharyngeal aspirate, 25 sputum, and 4 bronchoalveolar lavage specimens) and 86 from adults (77 nasal and/or throat swab, 6 bronchoalveolar lavage, and 3 sputum specimens). Twelve children and four adults contributed two samples.

For children, viral culture identified viruses in 25 (14%) samples, whereas DIF detected viruses in 35 (19%) samples. Thirty-eight (21%) samples were inadequate for analysis by DIF because of the absence of intact cells. The combination of viral culture and DIF detected viruses in 43 (24%) samples. A total of 78 (43%) samples were positive by real-time PCR; in these samples, 89 viruses were identified. Mixed viral infections were detected by real-time PCR only (seven [3.9%] double infections and two [1.1%] triple infections).

In adults, viral culture identified respiratory viruses in only three (3.5%) samples. Respiratory viruses were detected in 31 (36%) samples by real-time PCR; no mixed viral infections were detected.

The diagnostic yields of conventional methods compared to that of real-time PCR as the reference method are presented in Table 2. The sensitivities of conventional methods (culture and DIF) in diagnosing the pediatric population were 0.46 (confidence interval [CI], 0.38 to 0.53) for RSVs, 0.44 (CI, 0.37 to 0.52) for IVs, 0.63 (CI, 0.55 to 0.70) for PIVs, and 0.24 (CI, 0.17 to 0.30) for AdVs. The sensitivities of conventional methods (viral culture) for detection in adults appeared to be much lower (Table 2) and were 0.00 (CI, 0.00 to 0.00) for RSVs, 0.11 (CI, 0.04 to 0.18) for IVs, 0.00 (CI, 0.00 to 0.00) for PIVs, and 1.00 (CI, 1.00 to 1.00) for AdVs. The specificities of conventional methods for the different respiratory viruses were 0.98 to 1.00 for children and 1.00 for adults.

TABLE 2.

Comparison of the results of conventional diagnostic tests and real-time PCR

Patient population	Conventional test(s)	Result of conventional test	No. of specimens with indicated PCR result for:
			RSVs		IVs		PIVs		AdVs
			+	−	+	−	+	−	+	−
Children	Culture + DIF	+	21	1	8	1	5	0	4	3
−		25	134	10	162	3	173	13	161
Adults	Culture	+	0	0	2	0	0	0	1	0
		−	10	76	16	68	2	84	0	85

Since the viral load in a sample can be relatively assessed by determining the cycle threshold (C_T) value, we compared the values for the samples that were culture/DIF positive with values for the samples that were culture/DIF negative. High C_T values are representative of a low viral load, while a low C_T value reflects a high viral load. For the pediatric population, the mean C_T value of samples positive for RSV by real-time PCR but in which RSV was not detected by conventional tests was 3.9 cycles higher (CI, 0.36 to 7.4) than that of positive samples subjected to detection by conventional tests (P = 0.03). For AdV, the C_T value was 13 cycles higher (CI, 5.4 to 22; P = 0.003). For IV, the trend was the same (5.7 cycles higher [CI, −0.25 to 12]; P = 0.06). For PIVs, no difference was observed. For adults, this analysis could not be performed due to the small number of positive results by viral culture.

In the present study, we showed that real-time PCR for RSV, IV, PIV, and AdV increased the diagnostic yields for these viruses substantially in clinical practice, in comparison with those of conventional diagnostic tests. However, some limitations of our study deserve further discussion. First, there is no gold standard for the detection of respiratory viruses to which both conventional tests and real-time PCR can be compared. This is a problem encountered in all studies evaluating real-time PCR (3, 4). Second, we did not use blind antibody fluorescence staining for viral culture and therefore may have missed positive cultures without cytopathogenic effects.

The findings of our study are in accordance with those of previous reports. van Kraaij et al. identified RSV, IV, and PIV as present in 2% of 52 episodes of respiratory tract infection in adults after stem cell transplantation, whereas real-time PCR was positive for 12%, 4%, and 6% of the samples, respectively (9). Templeton et al. (5) identified RSV, IV, and/or PIV in 19% of 358 respiratory samples by viral culture, compared with 24% of samples by real-time PCR (5). In the present study, we found an association between C_T values and conventional tests being positive. This was in agreement with the work of Bredius et al., who found C_T values of 27 to 42 for samples positive only by real-time PCR versus C_T values of 18 to 22 for culture-positive samples (1).

Apart from being more sensitive than conventional methods, real-time PCR is also able to detect microorganisms that are difficult to culture, microorganisms for which no (commercial) DIF assay is available, or microorganisms that cannot be cultured at all. At our center, real-time PCR is routinely performed for rhinoviruses, coronaviruses, human metapneumovirus, and Mycoplasma pneumoniae. These agents were found, respectively, in 47, 23, 13, and 5 pediatric samples and in 16, 6, 1, and 1 adult samples (unpublished data), increasing the detection rate further to 69% and 57% for children and adults, respectively.

In conclusion, real-time PCR considerably increases the diagnostic yields for respiratory viruses from patients admitted with respiratory symptoms within a clinically relevant time frame. This allows clinicians to initiate optimal patient management and to initiate adequate (future) use of antiviral therapy and optimal infection control.