Comparison of electronic microarray to enzyme hybridization assay for multiplex reverse-transcriptase PCR detection of common respiratory viruses in children.

A new assay, composed of the NGEN RVA (Nanogen, Inc., San Diego, CA; Prodesse, Inc., Waukesha, WI), which is a pair of analyte-specific reagents that allow the multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and electronic microarray detection of influenza virus A and B, respiratory syncytial virus A and B, and human parainfluenza virus types 1, 2, and 3, was evaluated in comparison with the Hexaplex (Prodesse), a multiplex RT-PCR-enzyme hybridization assay. Comparisons included the detection of respiratory viruses from whole-virus stocks (ATCC) and from frozen pediatric respiratory specimens collected at Children's Hospital of Wisconsin between 1991 and October 1998. After the retesting of six indeterminants and 20 discrepants, overall agreement improved to 96% on the positives and 100% on negatives, with only eight specimens still discrepant. The RVA reagents allow a rapid, sensitive, and specific assay for detecting seven of the most common respiratory viruses in children.

The pneumoplex assays, a multiplex PCR-enzyme hybridization assay that allows simultaneous detection of five organisms, Mycoplasma pneumoniae, Chlamydia (Chlamydophila) pneumoniae, Legionella pneumophila, Legionella micdadei, and Bordetella pertussis, and its real-time counterpart.

Respiratory disease caused by atypical bacteria remains an important cause of morbidity and mortality for adults and children, despite the widespread use of effective antimicrobials agents. Culture remains the "gold standard" for the detection of these agents. However, culture is labor-intensive, takes several days to weeks for growth, and can be very insensitive for the detection of some of these organisms. Newer singleplex PCR diagnostic tests are sensitive and specific, but multiple assays would be needed to detect all of the common pathogens. Therefore, we developed the Pneumoplex assays, a multiplex PCR-enzyme hybridization assay (the standard assay) and a multiplex real-time assay to detect the most common atypical pathogens in a single test. Primer and probe sequences were designed from conserved regions of specific genes for each of these organisms. The limits of detection were as follows: for Bordetella pertussis, 2 CFU/ml; for Legionella pneumophila (serotypes 1 to 15) and Legionella micdadei, 9 and 80 CFU/ml, respectively; for Mycoplasma pneumoniae, 5 CFU/ml; and for Chlamydia (Chlamydophila) pneumoniae, 0.01 50% tissue culture infective doses. Recombinant DNA controls for each of these organisms were constructed, and the number of copies for each DNA control was calculated. The Pneumoplex could detect each DNA control down to 10 copies/ml. The analytical specificity demonstrated no cross-reactivity between 23 common respiratory pathogens. One hundred twenty-five clinical bronchoalveolar lavage fluid samples tested by the standard assay demonstrated that the Pneumoplex yielded a sensitivity and a specificity of 100 and 98.5%, respectively. This test has the potential to assist clinicians in establishing a specific etiologic diagnosis before initiating therapy, to decrease hospital costs, and to prevent inappropriate antimicrobial therapy.

Evaluation of the Hexaplex assay for detection of respiratory viruses in children.

The Hexaplex assay (Prodesse, Inc., Milwaukee, Wis.) is a multiplex reverse transcriptase (RT)-PCR assay for the detection of parainfluenza virus types 1, 2, and 3, respiratory syncytial virus (RSV) types A and B, and influenza virus types A and B. We evaluated the Hexaplex assay in comparison with conventional viral cell cultures and rapid enzyme immunoassays (EIAs) for RSV (Directigen; Becton Dickinson Inc., Cockeysville, Md.) and influenza A virus (Abbott Test Pack; Abbott Laboratories, Abbott Park, Ill.) for the detection of respiratory viruses from pediatric respiratory specimens obtained from children seen at Children's Hospital of Wisconsin from December 1997 through May 1998. A total of 363 respiratory specimens were evaluated. The tissue culture prevalence of parainfluenza virus during this period of time was low (1.1%). The sensitivity, specificity, and positive and negative predictive value of Hexaplex compared to tissue culture for the detection of parainfluenza virus were 100, 95.8, 19.0, and 100%, respectively. Only one specimen was determined to contain influenza B virus by Hexaplex; it was tissue culture negative. A specimen was considered to contain RSV or influenza A virus when it was either culture positive or culture negative but Hexaplex and EIA positive. Prior to the analysis of discrepant results, the sensitivity, specificity, and positive and negative predictive value for the detection of RSV were 91.2, 100, 100, and 98.0%, respectively, for tissue culture; 84.5, 100, 100, and 96.6% for EIA; and 98.5, 91.5, 72.8, and 99.6% for Hexaplex, respectively. The sensitivity, specificity, and positive and negative predictive value for the detection of influenza A virus prior to the analysis of discrepant results were 100, 100, 100, and 100%, respectively, for culture, 78.0, 100, 100, and 89.4% for EIA, respectively, and 95.1, 94.1, 67.2, and 99.3% for Hexaplex, respectively. Culture- and/or EIA-negative, Hexaplex-positive specimens were analyzed by a second RT-PCR assay which used primers specific for a different genomic region than that used in the Hexaplex assay. After analysis of these discrepant results, the sensitivity, specificity, and positive and negative predictive value for the detection of RSV were 74.3, 100, 100, and 93.5%, respectively, for tissue culture; 70.3, 100, 100, and 92.5% for EIA; and 98.6, 97.4, 91.2, and 99.6% for Hexaplex. The sensitivity, specificity, and positive and negative predictive value for the detection of influenza A virus were 83.3, 100, 100, and 97.4%, respectively, for tissue culture; 69.4, 100, 100, and 83.3% for EIA; and 95.8, 98.7, 92.0, and 99.3% for Hexaplex. Hexaplex is a rapid, sensitive, and specific method for the detection of the seven most common respiratory viruses in children.

Prevention of central venous catheter-related infections and thrombotic events in immunocompromised children by the use of vancomycin/ciprofloxacin/heparin flush solution: A randomized, multicenter, double-blind trial.

PURPOSE: To determine whether an antibiotic flush solution containing vancomycin, heparin, and ciprofloxacin (VHC) can prevent the majority of line infections. PATIENTS AND METHODS: A prospective double-blind study was performed comparing VHC to vancomycin and heparin (VH) to heparin alone in 126 pediatric oncology patients. RESULTS: The 153 assessable lines resulted in 36,944 line days studied. There were 58 blood stream infections (43 gram-positive, 14 gram-negative, and one fungal). Forty were defined as line infections (31 heparin, three VH, six VHC). The time to develop a line infection was significantly increased using either antibiotic flush (VH, P =.011; VHC, P =.036). The rate of total line infections (VH, P =.004; VHC, P =.005), gram-positive line infections (VH, P =. 028; VHC, P =.022), and gram-negative line infections (VH, P =.006; VHC, P =.003) was significantly reduced by either VH or VHC. Sixty-two (41%) of the lines developed 119 occlusion episodes (heparin, 3.99 per 1,000 line days; VHC, 1.75 per 1,000 line days; P =.0005). Neither antibiotic could be detected after flushing, and no adverse events were detected, including increased incidence of vancomycin-resistant Enterococcus colonization or disease. CONCLUSION: The use of either VH or VHC flush solution significantly decreased the complications associated with the use of tunneled central venous lines in immunocompromised children and would save significant health care resources.

Rapid simultaneous diagnosis of infections with respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (Hexaplex).

A multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (Hexaplex; Prodesse, Milwaukee) was developed and used to rapidly detect and quantitate RNA of respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 in nasal wash specimens in a single test. Primers and probes originated from highly conserved regions of each viral genome. Six and a half primer pairs were mixed for the simultaneous detection and quantitation of RNA from seven different respiratory viruses. We tested 109 clinical samples with this assay. Twenty-nine virus culture-positive samples were all positive by Hexaplex. Samples from 40 symptomatic patients were negative by virus culture, but eight of these were positive by Hexaplex. Forty samples from asymptomatic children were negative by both virus culture and Hexaplex. No cross-reactions were noted among 17 different respiratory viruses with use of this assay. Hexaplex was 100% sensitive (95% confidence interval [CI], 0.88-1.0) and 98% specific (95% CI, 0.97-0.99). All eight "false-positive" Hexaplex results (in comparison with negative viral culture results) were for symptomatic patients with low numbers of virus RNA copies. This finding suggests that Hexaplex may be more sensitive than virus culture. Our data demonstrate that Hexaplex is a rapid, sensitive, and specific quantitative test for the diagnosis of infections with these seven common respiratory viruses.

Antigenic structure, function, and evolution of the hemagglutinin-neuraminidase protein of human parainfluenza virus type 1.

Twenty-two monoclonal antibodies directed to the hemagglutinin-neuraminidase protein of human parainfluenza virus type 1 (HPIV-1) were used in competition assays to create an antigenic map of neutralization sites. Eighty-seven clinical strains isolated over 35 years from multiple geographic regions were reacted in ELISA, hemagglutinin-inhibition, and microneutralization assays with these monoclonal antibodies. Together these assays revealed 21 epitopes on five nonoverlapping antigenic sites (I, III-VI) with a sixth (II) bridging site connecting sites I, III, and IV. Only 7 (33%) of these epitopes were conserved among all isolates. Previously described HPIV-1 genotypes were associated with the presence or absence of specific antigenic sites and evidence of probable immune selection within genotypes. Two sites were present on all isolates tested (III, V), and one (VI, genotype A) has not been found for 15 years. Forty hemagglutinin-neuraminidase nucleotide sequences were analyzed in terms of homology, structure, and evolution. These data may be useful in future epidemiologic, therapeutic, or vaccine-related work.

Rapid diagnosis of human parainfluenza virus type 1 infection by quantitative reverse transcription-PCR-enzyme hybridization assay.

The detection and quantitation of human parainfluenza virus type 1 (HPIV-1) RNA in nasal wash specimens from 49 children with lower respiratory infections were performed by a reverse transcription-PCR-enzyme hybridization assay (RT-PCR-EHA). The HPIV-1 RT-PCR-EHA was then used to test 40 samples from asymptomatic children. Primers and probes were designed from regions within the HPIV-1 hemagglutinin-neuraminidase gene which are highly conserved among all known genotypes. HPIV-1 was detected in all nine children who were culture positive. Other common respiratory viruses (HPIV-2, -3, and -4, mumps virus, respiratory syncytial virus, and influenza virus) were not detected by the HPIV-1 assay. Forty symptomatic children were negative by culture, and four of these were positive by RT-PCR-EHA. All of the samples from asymptomatic children were negative by culture and RT-PCR-EHA. RT-PCR-EHA was 100% sensitive (95% confidence interval, 0.66 to 1.00) and 95% specific (95% confidence interval, 0.88 to 0.99) compared with culture. The four false-positive results (relative to the results of culture) were in children with lower respiratory infections compatible with HPIV-1 infection and suggest that RT-PCR-EHA may be more sensitive than culture. Our data indicate that HPIV-1 may be underdiagnosed by routine culturing methods. RT-PCR-EHA has been demonstrated to be an easy, rapid, sensitive, and specific test for diagnosing HPIV-1 infection and provides a methodology for the rapid detection of closely related respiratory viruses.

Two distinct human parainfluenza virus type 1 genotypes detected during the 1991 Milwaukee epidemic.

The extent of genetic and antigenic variation found in a population of human parainfluenza virus type 1 (HPIV-1) during a single local epidemic was investigated. Fifteen HPIV-1 strains isolated from children in 1991 were analyzed. Nucleotide sequence variation in the hemagglutinin-neuraminidase protein (HN) gene demonstrated two distinct genotypes (genotypes C and D). Unique patterns were identified involving 62 nucleotide and 10 amino acid positions. These patterns represented 40% of all mutations within the HN gene. The remaining mutations were randomly distributed, and 74% involved only one (55%) or two isolates. Genotypes were statistically different from each other at both the nucleotide (P = 0.001) and amino acid (P = 0.001) levels and demonstrated unique potential N-linked glycosylation patterns. Thirty-eight monoclonal antibodies (MAbs) made to four different viral proteins (22 HN, 2 fusion [F], 1 phosphoprotein, and 13 nucleoprotein) (originating from two different genotypes [genotypes A and D]) were compared for their ability to bind to the clinical isolates in enzyme-linked immunosorbent assays (ELISAs) and hemagglutinin-inhibition (HI) assays. Twenty-one MAbs bound well to all clinical isolates in ELISAs and HI assays. The remaining 17 MAbs showed variation in all four structural proteins. Three HN MAbs demonstrated genotype C- and D-specific antigenic and neutralization differences. Evolutionary analysis using parsimony methods confirmed the differences between the two genotypes. No differences in either clinical presentation or disease severity between the two genotypes were found. Geographically localized HPIV-1 epidemics can be caused by at least two distinct genotypes with minor but specific antigenic changes. The clinical and immunologic roles of HPIV-1 genotypes have not been determined.

Brain abscess caused by Blastomyces dermatitidis in a child with cystic fibrosis.

An 8-year-old girl with moderately severe cystic fibrosis and right upper lobe bronchiectasis developed a cerebellar abscess caused by Blastomyces dermatitidis. To our knowledge, this is the youngest child with cystic fibrosis and a brain abscess, and the first documented case caused by a fungus.

Epidemiology and cost of infection with human parainfluenza virus types 1 and 2 in young children.

To determine the morbidity, costs, and epidemiological features of lower respiratory tract infections (LRIs) due to human parainfluenza virus types 1 and 2 (HPIV-1 and HPIV-2), we evaluated 1,213 children < 6 years of age who were seen for LRIs in the emergency room of the Children's Hospital of Wisconsin and/or were admitted to the hospital for LRIs during the fall quarter of 1991. The age, sex, race, and respiratory syndrome were recorded for each child; 158 patients (13%) had respiratory samples cultured for viruses and were followed clinically for the duration of their illness. Caucasian children had croup diagnosed more often than did African-American children (relative risk [RR] = 3.12; 95% confidence interval [CI], 2.43-4.00; P < .001), while African-American children more often had pneumonia (RR = 1.85; 95% CI, 1.36-2.5; P < .001). Forty-five of 70 viruses recovered were HPIV-1 (17 cases) or HPIV-2 (28 cases). Together these two viruses were recovered from 49% of children presenting with croup, 10% of those presenting with bronchiolitis, and 12% of those presenting with pneumonia. Gender- and race-associated differences were documented in the group of children infected with HPIV-2: specifically, this group included more girls than boys (RR = 1.99; 95% CI, 1.02-3.88; P < .04) and more Caucasian than African-American children (RR = 2.64; 95% CI, 1.05-6.63; P = .027). These data extrapolate nationally to approximately 250,000 emergency-room visits and approximately 70,000 hospitalizations due to HPIV-1 and HPIV-2, with a cost of $50 million for the former and $140 million for the latter.

Recovery of human parainfluenza virus types one and two.

The ability to recover human parainfluenza virus types 1 and 2 (HPIV-1, 2) from infected individuals has been highly variable. During the autumn of 1991, 158 nasal wash specimens collected from children with lower respiratory symptoms were split and cultured independently at two laboratories using different tissue culture techniques. Immunofluorescent antibody (IFA) and hemadsorption (HAd) assays were compared for their speed and efficiency in viral detection. 45 isolates [HPIV-1 (17) and HPIV-2 (28)] were recovered by one laboratory and only one (HPIV-2) by the other. IFA was the most sensitive assay detecting 87% of HPIV-1 and 70% of HPIV-2 by the fourth day of culture. HAd assay detected 87% of HPIV-1 isolates by the time they were positive by IFA, but only 35% of the HPIV-2 isolates. Significant methodologic differences between laboratories were then compared simultaneously for effect on virus recovery from culture positive frozen clinical specimens. Recovery of 100% of the isolates was achieved. Factors that contributed to differences in recovery of HPIV-1 and 2 were: (1) primary African green monkey (AGMK) cells were inferior to cynomolgus monkey kidney or LLC-MK2 cells, (2) addition of trypsin to culture medium for AGMK and LLC-MK2 cells enhanced recovery, (3) use of IFA was essential for rapid detection of HPIV-2, and (4) use of microtiter plate culture without specimen dilution enhanced virus recovery. A survey of clinical virology laboratories demonstrated considerable variability in the use of these techniques for routine respiratory virus culture.

Lower respiratory viral infections in immunocompetent children.

Viral lower respiratory disease causes a heavy burden on our society. Better understanding of the epidemiology of these viruses combined with new rapid diagnostic techniques will provide more rapid and more reliable diagnosis of these agents in the future. Two agents not commonly thought of as causes of LRI in children (rhinoviruses, coronaviruses) should now be added to an already long list. Effective drugs exist for prophylaxis against influenza virus type A and therapy for influenza virus type A, type B, and RSV. While no new antiviral drugs are near clinical use at this time, new antiviral agents are constantly being tested and developed. High-titer, specific antiviral IVIG appears promising for both therapy and prophylaxis. Over the next decade, improved influenza virus vaccines and safe and effective vaccines against HPIV and RSV are expected. Adenoviral vaccines for use in immunocompromised patients are possible, but a generally available vaccine for all children is less likely. Although the basic clinical epidemiology of these viruses has been well investigated over the last 30 years, new molecular techniques are greatly expanding our understanding of these agents. Antigenic and genetic variation is being found in many viruses previously thought homogeneous. The exact role and biologic significance of this variation is just beginning to be explored, but already there is evidence of differences in pathogenicity and immunogenicity in many of these substrains. All of this information will have an impact on future vaccine and antiviral drug development.

Genetic variation and evolution of human parainfluenza virus type 1 hemagglutinin neuraminidase: analysis of 12 clinical isolates.

The extent of genetic variation and evolution in a population of human parainfluenza virus type 1 was investigated. The hemagglutinin neuraminidase genes of 13 isolates collected over a 26-year period were sequenced and compared. All isolates except the 1957 type strain were from a single geographic location and demonstrated significant consistent genetic change from the type strain (47/7 [nucleotide/amino acid] substitutions). Antigenic subgroup A isolates demonstrated minor intragroup differences (9/1 substitutions). However, 18/7 unique substitutions separated subgroup A from B regardless of geographic location or year of isolation. Multiple strains of both subgroups appeared and reappeared over decades with only minor variation. There may be significant genetic differences between clinical isolates based on geographic location, and progressive mutational change may occur. Previously defined antigenic and now genetic subgroups were stable and at least regional in distribution over the period studied. The biologic implications and extent of this variation need further evaluation.

Monoclonal antibodies to human parainfluenza virus type 1 detect major antigenic changes in clinical isolates.

The extent of antigenic diversity within a population of human parainfluenza virus type 1 (HPIV-1) isolates collected over a 26-year period was investigated. Twenty-three monoclonal antibodies (MAbs) made to the hemagglutinin-neuraminidase protein (HN), fusion protein (F), phosphoprotein (P), and nucleoprotein (NP) of a 1957 type strain were compared in their ability to bind to the different clinical isolates in ELISA and hemagglutinin-inhibition (HI) assay. Four HN, one F, and two NP MAbs bound equally to all of the viruses tested, but six of the MAbs demonstrated significant antigenic heterogeneity. Most of these antigenic changes appeared stable over time and noncummulative. Four of the clinical isolates and the type virus had similar reactivity patterns (subtype A) to these MAbs, while the remaining 10 isolates may form a second group (subtype B). Children's sera demonstrated this same subtype specificity in HI assays. One neutralization site was present on the 1957 strain and not on any of the subsequent isolates. The possibility of two or more major subtypes of HPIV-1 should be considered in future epidemiologic, therapeutic, and vaccine-related work.

Neutralizing epitopes of human parainfluenza virus type 3 are conformational and cannot be imitated by synthetic peptides.

The possibility that linear epitopes on the haemagglutinin-neuraminidase (HN) surface glycoprotein of human parainfluenza virus type 3 (PIV-3) might induce neutralizing antibodies after virus infection was investigated. Thirty-seven peptides, representing 64% of the extramembranous portion of the HN molecule of PIV-3, were synthesized. Their ability to bind to 14 neutralizing murine monoclonal antibodies (mAbs) specific for HN or 26 high-titre human serum samples were tested in a direct enzyme-linked immunosorbent assay (ELISA) and in an indirect competition ELISA. None of the synthetic peptides reacted with any of the mAbs or serum samples in the direct test and none of 11 synthetic peptides tested blocked mAbs from binding to HN in the competition ELISA. These findings suggest that synthetic peptides cannot be used to imitate the known neutralizing epitopes on the HN. Analyses of reduced and non-reduced HN in ELISA and immunoblot assays confirmed that protein folding and tertiary structure are essential for epitope formation in these neutralizing sites. However, some children's sera analysed by immunoblotting contained antibodies to an uncharacterized linear epitope(s) not recognized by our panel of mAbs, raising the possibility that a neutralizing linear epitope does exist on the HN of PIV-3.

Prevention of bacteremia attributed to luminal colonization of tunneled central venous catheters with vancomycin-susceptible organisms.

Forty-five children with oncologic or hematologic disorders requiring tunneled central venous catheters (TCVC) for the administration of immunosuppressive therapy were randomized to receive either 10 U/mL heparin (H) (24 patients) or a solution of 10 U/mL H and 25 micrograms/mL vancomycin (H-V) (21 patients) for all catheter flushes. Episodes of fever or suspected sepsis were evaluated to determine whether the addition of vancomycin to the flush solution would alter the incidence of symptomatic bacteremia attributed to luminal colonization of TCVC with vancomycin-susceptible bacteria. Patients were enrolled for 247 +/- 150 days, accounting for a total of 11,095 days of catheter use. Bacteremia attributed to luminal colonization with vancomycin-susceptible organisms occurred in five patients (six infections) receiving H alone compared with zero patients receiving H-V (P = .035). The time to the first episode of bacteremia with vancomycin-susceptible organisms, analyzed by Kaplan-Meier survival curves, was significantly longer in patients receiving H-V (P = .04). There were no differences in the incidence of other infections including bacteremia attributed to luminal colonization with vancomycin-resistant organisms, other bacteremias (including those arising from the catheter exit site), exit-site cellulitis, or fungal infections. No organisms resistant to vancomycin were identified. Vancomycin could not be detected in the peripheral blood of patients receiving vancomycin in the flush solution. No vancomycin-related toxicities were noted. We conclude that the use of an H-V flush solution in immunocompromised patients with TCVC can decrease the frequency of bacteremia attributed to luminal colonization with vancomycin-susceptible bacteria.

Fulminating Staphylococcus epidermidis bacteremia.

We have reported a case of disseminated Staphylococcus epidermidis infection in a patient with leukemia and examined the relation between an acute respiratory arrest and the infection. Plasmid profiles of five isolates of S epidermidis cultured from this patient's blood, bone marrow, and lung before and after the arrest indicate that all isolates were derived from a single strain. This strain was also isolated by culture of the tip of the central venous catheter that was removed from the patient suggesting that the indwelling catheter was the source of infection. Because this patient had rigors during an infusion through the catheter just before the acute respiratory arrest, we suspect that infusion through the colonized catheter precipitated the respiratory arrest.