Detection and Identification of Human Parainfluenza Viruses 1, 2, 3, and 4 in Clinical Samples of Pediatric Patients by Multiplex Reverse Transcription-PCR

Virus.

Prototype strains of HPIV-1 (strain C35), HPIV-2 (strain Greer), HPIV-3 (strain C-243), HPIV-4A (strain M-25), and HPIV-4B (strain 19.153) were obtained from the Centers for Disease Control and Prevention collections. Wild-type HPIV isolates (two isolates each of HPIV-1, -2, and -3) from cell cultures inoculated with samples from multiple respiratory disease outbreak seasons were obtained from the Spanish National Center for Microbiology archives, as were three individual isolates of influenza A virus (two of subtype H3 and one of subtype H1), three individual isolates of influenza B virus, two individual isolates of adenovirus, two individual isolates of mumps virus, two individual isolates of measles virus, and three individual isolates of respiratory syncytial virus.

Clinical samples.

Two hundred thirty nasopharyngeal aspirate specimens were collected from pediatric patients who had a lower tract respiratory illness and who were recruited for a long-term prospective study of severe respiratory infections. These patients were referred to the emergency room or required hospitalization at the Severo Ochoa Hospital in Leganés (Madrid, Spain). These samples were collected during the periods of maximum HPIV activity detected by viral isolation and antigen detection. One hundred eighty-four specimens obtained from September 1997 to January 1998 were tested retrospectively and 46 specimens obtained from June 1998 to July 1998 were tested prospectively (see below for study design). Specimens were obtained with an aspirator device, placed in viral transport medium, and processed within 24 h of collection. When the specimens arrived in the laboratory, they were diluted to 5 ml with phosphate-buffered saline solution and homogenized before testing. Three 0.5-ml aliquots were stored at −70°C.

IF assay.

The indirect immunofluorescence (IF) assay was performed directly with cells from respiratory secretions that were concentrated by centrifugation and stained by standard methods. Commercial reagents (Chemicon, Temecula, Calif.) were used; however, monoclonal antibody (MAb) specific for HPIV-4 (MAb 531-3F) was obtained from the Centers for Disease Control and Prevention (17). For each MAb the results for two 7-mm-diameter wells were read before a sample was considered negative.

Virus isolation.

Human laryngeal epidermoid carcinoma (HEp-2) cells, human lung mucoepidermoid carcinoma (NCI-H292) cells, Madin-Darby canine kidney (MDCK) cells, and human embryonic lung fibroblast (Fp) cell cultures were used for primary viral isolation. Tubes with 80% confluent monolayers were inoculated with 0.3 ml of homogenized samples. The adsorption of HEp-2, Fp, and MDCK cell cultures was enhanced by centrifugation at 3,000 rpm (Labofuge GL, Heraeus Sepatech) for 45 min. HEp-2 and Fp cells were fed 2 ml of 2% fetal calf serum in Eagle basal medium containing antibiotics. For MDCK cells, Eagle minimal essential medium with antibiotics was supplemented with 3 μg of trypsin per ml. NCI-H292 cells were adsorbed for an hour without centrifugation and were fed Eagle minimal essential medium supplemented with 1.5 μg of trypsin per ml (4). Cell monolayers were observed for cytopathic effect every 48 h. When a cytopathic effect was observed or after 10 days, the monolayer was scraped and tested for respiratory viruses by IF assay, as described above. The monolayers with IF assay-negative culture results were subcultured and again submitted to blind IF assay after 10 days.

Primers.

Primers specific for HPIV-1, -2, and -3 (8) and internal control primers (2) were published previously. For design of the primer specific for HPIV-4, the sequences of the phosphoprotein P gene were obtained from GenBank and were aligned by using the Wisconsin Analysis Package, version 8 (Genetics Computer Group, Madison, Wis.). External generic primers PI4P+ (5′-CTGAACGGTTGCATTCAGGT-3′ [genome sense; bases 11 to 39]) and PI4P− (5′-AGGACTCATTCTTGATGCAA-3′ [genome antisense; bases 433 to 452]) were chosen from regions conserved between the HPIV-4A and HPIV-4B subtypes. A second pair of internal generic primers, PI4S+ (5′-AAAGAATTAGGTGCAACCAGTC-3′ [genome sense; bases 158 to 179]) and PI4S− (5′-GCTGCTTATGGGATCAGACAC-3′ [genome antisense; bases 382-402]) were selected for the nested reaction by using the same criteria described above. Subtype A- and B-specific primers PI4SA+ (5′-ATGATGGTGGAACCAAGATT-3′ [genome sense; bases 226 to 245]) and PIASB+ (5-AACCAGGGAAACAGAGCTC-3′ [genome sense; bases 320 to 339]), whose sequences were within the first amplification fragment, were also selected; PI4P+ annealed to the 5′ noncoding region, and the rest of the primers annealed to the P/V common region of phosphoprotein P (19).

RNA extraction, RT, and primary amplification.

RNA was extracted from clinical samples and virus isolates as described previously (3). Briefly, 50 μl of each sample was treated with 200 μl of guanidinium thiocyanate extraction buffer including 100 molecules of a plasmid with an insert of the polymerase gene of the pseudorabies herpesvirus DNA as an internal control template (2), followed by alcohol precipitations. The pellet was resuspended in 10 μl of RNase-free water. A single-step RT-amplification reaction was performed by using the Promega Access RT-PCR system kit (Promega, Madison, Wis.), which consisted of a PCR mixture containing 3 mM MgSO₄, 500 μM each dATP, dGTP, dCTP, and dTTP, 0.5 μM HPIV type 1- to 4-specific primers, 0.2 μM internal control-specific primary reaction primers, 10 μl of avian myeloblastosis virus-Tfl 5× reaction buffer, 5 U of avian myeloblastosis virus reverse transcriptase, and 5 U of Tfl DNA polymerase. The PCR mixtures were overlaid with mineral oil, and 5 μl of extracted RNA was added to a final volume of 50 μl. The tubes were centrifuged for a few seconds and were placed in an Autocycler Plus Termocycler (Linus, Cultek S. L., Spain). Cycling conditions were as published previously (8).

Nested amplification and product detection.

Analytical conditions were as published previously (8), but generic HPIV-4-specific and internal control-specific primers were added.

PCR products were sized by gel electrophoresis on 2% agarose. Expected band sizes were 317 bp for HPIV-1, 203 bp for HPIV-2, 102 bp for HPIV-3, 246 bp for HPIV-4, and 140 bp for the internal control (Fig. 1). For subtyping of HPIV-4, a nested reaction that included only primers PIS4A+, PIS4B+, and PIS4− was performed under the same conditions used for the nested PCR mentioned above with the first reaction products. Expected band sizes were 178 bp for HPIV-4A and 84 bp for HPIV-4B (Fig. 1). Positive samples showed the specific HPIV band and the internal control band. When only the internal control band appeared, samples were considered negative. Samples that showed no band were retested, and those that lacked any band after repeat testing were assumed to contain enzyme inhibitors.

Standard precautions were taken throughout the procedure to avoid cross-contamination. Negative and low-titer positive controls were included in every assay. All positive results were confirmed by retesting a different aliquot of the specimen. In case of disagreement of the results, a third m-RT-PCR was performed to resolve the results.

PCR product cloning.

Primary amplification products from prototype strains of HPIVs were purified by using the GeneClean II kit and were ligated into pGEM-T plasmid vectors with the pGEM-T plasmid vector system (Promega) by following the manufacturer's directions. Plasmids were transformed into high-efficiency competent cells (Epicurian coli XL1-Blue; Stratagene Cloning Systems, La Jolla, Calif.) by electroporation. Transformants were selected in Luria broth–ampicillin–isopropyl-β-d-thiogalactopyranoside–5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside plates, and the presence of the expected insert was confirmed by PCR. Plasmids were purified with the Wizard Plus SV Minipreps kit (Promega). The number of plasmid copies in the final suspensions was estimated by UV spectroscopy at an optical density of 260 nm.

Study design. (i) Prospective panel.

Viral isolation and antigen detection by IF assay with all HPIV-specific MAbs and m-RT-PCR were performed with all fresh specimens (n = 46) that arrived at the laboratory during the study period; the results for m-RT-PCR-positive specimens were confirmed with frozen aliquots.

(ii) Retrospective panel.

Because an HPIV-4-specific MAb was not used for cell culture screening prior to the study, frozen aliquots from all archived specimens (n = 184) were recultured in NCI-H292 cells, and the cultures were rescreened for all HPIVs by IF assay. The results were the same as those obtained with the fresh samples except for the results for one specimen from which HPIV-1 was previously isolated but for which the result was negative on rescreening. Only rescreening results are considered for further analysis. The IF assay was performed only with fresh samples. Consequently, no IF assay results are available for HPIV-4. The m-RT-PCR was performed with frozen aliquots, and the viruses in samples positive for HPIV-4 were subtyped by using the primary amplification products as templates.

Evaluation of m-RT-PCR sensitivity and specificity.

DNA bands of the expected size were obtained by m-RT-PCR with all reference HPIV strains and all wild-type HPIV isolates. No amplification was observed with the other respiratory viruses tested. A comparison of the results of m-RT-PCR with those of virus culture in NCI-H292 cells by using serial 10-fold dilutions of the reference HPIV strains obtained sensitivities of 0.01 50% tissue culture infective dose (TCID₅₀), calculated by the Reed-Muench method, for HPIV-1, 0.02 TCID₅₀ for HPIV-2, 32 TCID₅₀s for HPIV-3, 0.001 TCID₅₀ for HPIV-4A, and 0.0004 TCID₅₀ for HPIV-4B. In dilution experiments with plasmids containing cloned HPIV cDNA, m-RT-PCR was able to detect as few as 10 molecules for all HPIVs.

Evaluation of m-RT-PCR with clinical specimens.

Of the 230 clinical specimens, 22 from the retrospective panel and 7 from the prospective panel were not suitable for use in comparisons of the techniques. Sixteen (6.9%) exhibited microbial contamination in the cell culture, 8 (3.4%) had scarce respiratory tract epithelial cells by IF assay, 1 (0.4%) had both problems, and 4 (1.7%) were suspected of having enzymatic inhibition when tested by m-RT-PCR. Among the samples with contamination in the cell cultures, m-RT-PCR and IF assay detected two HPIVs (one HPIV-1 and one HPIV-3), and m-RT-PCR alone detected five HPIVs (two HPIV-1, one HPIV-2, and two HPIV-3). Both culture and m-RT-PCR detected one HPIV-1 and one HPIV-3 in samples for which results were not available by IF assay. One HPIV-3 in the specimen with both problems was amplified by m-RT-PCR. No HPIVs were detected by any other technique in samples for which the m-RT-PCR was inhibited.

The remaining 201 samples for which results were available by all techniques were analyzed further. For all samples, the same virus was detected by m-RT-PCR and culture. Other respiratory viruses were identified in 50 specimens (Table 1). Similar monthly distributions of positive results for the different HPIVs were observed by cell culture isolation and m-RT-PCR (Fig. 2).

HPIVs were recovered from 32 of the 162 suitable samples belonging to the retrospective panel; HPIV-1 from 21 (12.9%), HPIV-2 from 6 (3.7%), HPIV-3 from 2 (1.2%), and HPIV-4 from 1 (0.6%). The IF assay detected 16 (9.8%) HPIV-1, 2 (1.2%) HPIV-2, and 1 (0.6%) HPIV-3. m-RT-PCR yielded 23 (14.2%) HPIV-1, 6 (3.7%) HPIV-2, 3 (1.8%) HPIV-3, and 10 (6.1%) HPIV-4 (Table 1). All 10 HPIV-4 were subtyped as HPIV-4A with the subtype-specific primers. One sample previously positive for respiratory syncytial virus was found to be HPIV-4 positive by m-RT-PCR. Dual infections were not observed by either culture or IF assay.

Only HPIV-3 was detected within the prospective panel. Twelve (30.7%) of the 39 suitable clinical samples yielded viral isolates, with 7 (17.9%) of these being virus positive by IF assays. Twenty-one (53.8%) of the total had a positive result by m-RT-PCR (Table 1). One sample had a positive result by IF assay but negative results by both cell culture isolation and m-RT-PCR. HPIV-3 could be amplified from three samples known to contain adenoviruses. As for the retrospective panel, no coinfection was observed by IF assay or cell culture.