Cost Analysis of Multiplex PCR Testing for Diagnosing Respiratory Virus Infections

We performed a cost analysis study using decision tree modeling to determine whether the use of multiplex PCR testing for respiratory viruses (xTAG RVP test) is a more or less costly strategy than the status quo testing methods used for the diagnosis of respiratory virus infections in pediatric patients. The decision tree model was constructed by using four testing strategies for respiratory virus detection, viz., direct fluorescent-antibody staining (DFA) alone, DFA plus shell vial culture (SVC), the xTAG RVP test alone, or DFA plus the xTAG RVP test. A review of the charts of 661 pediatric patients was used to determine the length of hospital stay, the number of days in isolation, antibiotic usage, and all other medical procedures performed. The cost of hospitalization by diagnostic status was determined on the basis of the average cost per patient and the number of patients in each arm of the decision tree. The cost per case was the highest for DFA plus SVC at $3,914 (in Canadian dollars), and the lowest was for the xTAG RVP test alone at $3,623, while the costs of DFA alone ($3,911) and DFA plus RVP ($3,849) were intermediate. When all four diagnostic strategies were compared, the least costly strategy was the xTAG RVP test alone when the prevalence of infection was 11% or higher and DFA alone when the prevalence was under 11%. These data indicate a savings of $291 per case investigated if the strategy of using the xTAG RVP test alone was used to replace the status quo test of DFA plus SVC, resulting in a savings of $529,620 per year in direct costs for the four Hamilton, Ontario, Canada, hospitals on the basis of the testing of specimens from 1,820 pediatric inpatients. We conclude that the use of the xTAG RVP test is the least costly strategy for the diagnosis of respiratory virus infections in children and would generate a significant savings for hospitals.Clinical virology laboratories have historically used traditional methods, such as culture, direct fluorescent-antibody staining (DFA), and enzyme immunoassay, for the diagnosis of respiratory tract infections (3). DFA offers a rapid turnaround time for results but is labor-intensive and subjective and requires specific monoclonal antibodies and trained technologists. Both DFA and shell vial culture (SVC) are limited by the availability of monoclonal antibodies, precluding their use for the detection of newly discovered viruses. DFA has a low sensitivity for the detection of some viruses, especially adenovirus, and many laboratories reflex DFA-negative specimens into SVCs to improve the detection rates. For traditional methods such as DFA and SVC, turnaround times for results can be slow for laboratories handling large volumes of specimens. Rapid enzyme immunoassays have been used for the detection of influenza virus and respiratory syncytial virus (RSV), but these tests are only 50 to 70% sensitive (5, 15), which limits their use to specific point-of-care settings at times when the prevalence of infection is high.Over the past 10 years, nucleic acid amplification tests have been developed for an increasing number of respiratory viruses. Nucleic acid amplification tests, including PCR and nucleic acid-sequence based amplification, have shown enhanced sensitivity compared with the sensitivities of DFA and culture for the detection of a number of respiratory viruses (8). The emergence of five new respiratory viruses since 2000, including human metapneumovirus, the sudden acute respiratory syndrome-associated coronavirus, avian influenza virus H5N1, coronaviruses NL63 and HKU1, and human bocavirus, has presented new challenges for clinical laboratories. The absence of commercially available tests for the detection of these emerging viruses often leaves laboratories without the ability to diagnose these important virus infections. Multiplex PCR assays for the detection of multiple respiratory viruses have recently been introduced (for a review, see reference 8). These multiplex assays have heralded a new era in the molecular diagnostics of respiratory virus infections. Some of these tests are now commercially available and can detect up to 18 different respiratory viruses (6). A multiplex PCR test for respiratory viruses (the xTAG RVP test) is the first multiplex PCR to be cleared by the U.S. Food and Drug Administration and has been approved for use for the detection of 12 different respiratory viruses (16). The xTAG RVP test detects 30 to 40% more virus infections than DFA and culture, in part because it is more sensitive for the detection of traditional respiratory viruses, but it also detects nine additional viruses not detected by DFA and SVC (6, 7, 9, 10, 14). These newer multiplex tests are often costly, and the clinical and economic impacts of their implementation in routine hospital laboratories have not been evaluated. We therefore conducted a cost analysis study to compare the costs of the xTAG RVP test to those of conventional tests for the diagnosis of respiratory virus infections in hospitalized patients.     

多重PCR检测肺炎链球菌血清型方法及临床应用

目的:建立一种可检测常见肺炎链球菌(SP)重要血清型的多重PCR(mPCR)方法,并初步应用于临床。方法:根据SP荚膜多糖基因序列(cps)的保守序列cpsA,设计8种SP血清型特异性引物,优化PCR条件后,对SPDNA进行扩增,通过电泳分离出型特异性条带,用于检测8种已知血清型和126株未知血清型SP菌株,并与产物纯化克隆后的测序结果比对。结果:8种已知血清型SP的mPCR分型结果与传统方法分型结果及测序结果一致;mPCR可一次检测出87.50%的血液来源SP血清型和31.81%呼吸道来源SP血清型。结论:mPCR方法可快速检测8种重要SP血清型,对初筛血液来源SP血清型有较高应用价值。     

多重PCR技术检测小儿咽拭子呼吸道病毒的临床分析

目的 研究多重荧光聚合酶链式反应（PCR）技术对小儿咽拭子呼吸道病毒、病原菌检测的应用价值。方法 筛选2018年1月~12月到乌鲁木齐市社区服务门诊就诊的社区获得性肺炎（CAP）患儿524例,采用多重PCR技术检测患儿咽拭子病原菌,分析CAP病原菌的分布情况及其与年龄、季节等的关系。结果 CAP病原检测结果大部分属于病毒性,以乙型流感病毒、肺炎衣原体、肺炎支原体等为主,细菌类以肺炎克雷伯菌等为主;与患儿年龄、季节有关,3岁以下患儿仍是CAP的主要发病年龄段,尤其是婴幼儿最多,发病多集中在秋季和冬季;2种以上病原混合感染形式严峻。结论 多重PCR技术对小儿咽拭子病原菌检测的应用效果良好,快捷方便。     

Improved Multiplex PCR Strategy for Rapid Assignment of the Four Major Escherichia coli Phylogenetic Groups

Using data from whole-genome projects, an updated multiplex PCR strategy was developed to assign Escherichia coli isolates rapidly to major phylogenetic groups. This assay accommodates sequence variations detected within target sequences, thereby increasing sensitivity and reliability. It was validated using 185 isolates of known sequence types and showed improved congruence with multilocus sequence typing data.     

Clonogrouping,a Rapid Multiplex PCR Method for Identification of Major Clones of Listeria monocytogenes

Three multiplex PCR assays were developed to identify the 11 most common Listeria monocytogenes clones in clinical and food samples; 270 (95.7%) of 282 strains of serogroups IVb, IIb, IIa, and IIc were identified accurately. This novel tool is a rapid and efficient alternative to multilocus sequence typing for identification of L. monocytogenes clones.     

Utility of Major Leukocyte Subpopulations for Monitoring Secondary Cytomegalovirus Infections in Renal-Allograft Recipients by PCR

The feasibility of the major peripheral blood leukocyte (PBL) subsets for use in qualitative and quantitative PCR to monitor secondary cytomegalovirus (CMV) infection and ganciclovir therapy was assessed with 188 blood samples derived from 40 CMV immunoglobulin G-positive renal-allograft recipients. In pp65 antigen-positive patients all leukocyte fractions, but only 79.5% of plasma preparations, were PCR positive. In pp65 antigen-negative samples from patients after antiviral treatment only 7.3% of polymorphonuclear cell (PMNL) samples, but 81.8% of peripheral blood mononuclear cells (PBMC), and 10.9% of plasma samples remained PCR positive. Similarly, in patients with latent infections only 5.0% of PMNL, but 51.7% of PBMC preparations, and 8.0% of plasma samples were PCR positive. Regarding patients with active CMV infection, CMV DNA copy numbers in PMNL correlated significantly with pp65 antigen-positive cell counts before and after onset of ganciclovir therapy. Significant differences in CMV DNA copy numbers in PMNL and plasma were observed (i) between patients with symptomatic infection and those with asymptomatic infection and (ii) between patients with active infection and those with latent infection. In contrast, PBMC harbored equally low CMV DNA levels both in patients with active infection and those with latent infections, and no decline of CMV DNA load in PBMC was observed during antiviral treatment. We conclude that detection of CMV DNA in PMNL, not in PBMC, is associated with active infections and is more sensitive than detection of CMV DNA in plasma. Negative PCR results for PMNL after antiviral therapy indicate recovery, and fewer unwanted positive results occur compared to PBMC and plasma. Therefore, purified PMNL should be preferred for analysis by qualitative CMV PCR to avoid unwanted positive results. The CMV DNA load in PBMC compared with that in PMNL is negligible during active infection, so mixed PBL are sufficient for use in quantitative PCR.     

Comparison of a Commercial Multiplex Real-Time PCR to the Cell Cytotoxicity Neutralization Assay for Diagnosis of Clostridium difficile Infections

A commercial multiplex real-time PCR assay (Cepheid Xpert C. difficile assay) for the diagnosis of Clostridium difficile infection was evaluated. The sensitivity and specificity of the Cepheid assay were 97.1% and 93.0% for fresh stools, using the cell cytotoxicity neutralization assay as the reference. Using PCR ribotyping as the reference for ribotype 027 strains, the corresponding figures were 100% and 98.1%, respectively.Clostridium difficile infection (CDI) has increased in frequency and severity in North America and Europe over the last 5 years, largely due to the emergence of the epidemic PCR ribotype 027 strain (10, 11). The diagnosis of CDI is usually based on a clinical history of recent antimicrobial usage and diarrhea in combination with laboratory tests (9). Therefore, rapid and accurate microbiological diagnosis is urgently needed. The Cepheid Xpert C. difficile assay (Sunnyvale, CA) is a real-time multiplex PCR assay performed on the Cepheid GeneXpert Dx system. Proprietary primers specific for the toxin B gene (tcdB), binary toxin genes (cdtA and cdtB), and tcdC gene single-base deletion at nucleotide 117 were designed to detect toxigenic C. difficile and the presumptive PCR ribotype 027 strain. The purpose of this study was to evaluate the Cepheid Xpert C. difficile multiplex real-time PCR assay for the detection of toxigenic C. difficile strains and the presumptive ribotype 027.There were four serial investigations in the present study. In investigation 1, 205 frozen C. difficile strains collected during 2007 and 2008 were analyzed. In investigation 2, 195 frozen stool specimens belonging to different categories were selected based on direct cell cytotoxicity neutralization assay (CCNA) and toxigenic anaerobic culture results. Because PCR ribotype 027 is uncommon in Sweden, 40 frozen stool specimens collected in the United States were also analyzed. In investigation 3, 30 pairs of fresh-frozen stool specimens were analyzed. The fresh stool was analyzed within 24 h of collection, and then the leftover was stored at −20°C for 3 days and retested. In investigation 4, 220 consecutive fresh, unformed stool specimens (Bristol Stool Chart grade 5 to 7) from patients older than 2 years were analyzed within 24 h of collection. Eligible participants were those symptomatic patients who had a stool sample submitted to the Karolinska University Hospital for routine C. difficile testing.Unrepeated strains and stools were determined for C. difficile test by CCNA with a commercial C. difficile toxin/antitoxin kit (TechLab, Blacksburg, VA). For the stool specimens, anaerobic cultures on selective taurocholate cycloserine-cefoxitin-fructose agar plates were also performed (13). All isolates were typed by PCR ribotyping (19).Concurrently, the Cepheid Xpert C. difficile assay was performed according to protocols provided by the manufacturer. Each kit contained single-use disposable cartridges with integrated reaction chambers and reagents. A sterile Copan swab was dipped into the stool specimen or used to pick one fresh C. difficile colony from the blood agar plate, and the stool sample or colony was resuspended in sample buffer and then transferred to the cartridge. The cartridge was placed in the GeneXpert Dx module and run. Every PCR run included a sample-processing control and a probe check control. Each day, a positive control (C. difficile ATCC 9689 or the ribotype 027 strain) and a negative control (diluted C. difficile-negative stool sample) provided by Cepheid were tested. Results were automatically interpreted by the software as follows: “C. difficile positive,” “C. difficile 027 NAP1 presumptive positive,” “C. difficile negative,” “invalid,” “error,” or “no result.” If any of the test results was “invalid,” “error,” or “no result,” the sample was retested.CCNA and strain typing of the isolates were used as the reference standards for all of the investigations. If discrepant results for stool specimens were obtained with the Cepheid assay and CCNA, the results of CCNA for recovered isolates were taken into consideration. Sensitivity, specificity, positive and negative predictive values, and 95% confidence interval (CI) were calculated using SAS.The agreement between the Cepheid assay and CCNA for 205 isolates, 235 frozen stool specimens, and 220 fresh stools was 99.5%, 89.4%, and 93.6%, respectively (Table ​(Table1).1). Mixed populations of isolates with different toxin profiles (toxin B negative and toxin B positive) and different PCR ribotypes were found in four fecal samples. The real-time PCR cycle threshold values for frozen-thawed samples matched those for fresh samples (data not shown). In investigation 4, the initial evaluation of the stool specimens yielded 95.7% sensitivity and 87.3% specificity, respectively. The discrepancies between the assays are listed in Table ​Table2.2. Upon reevaluation by toxigenic anaerobic culture, the sensitivity and specificity increased to 97.1% and 93.0%, respectively.

TABLE 1.

Comparison between the Cepheid Xpert C. difficile assay and CCNA

Multiplex PCR for detection and typing of porcine circoviruses

Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified. A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain only. By both mPCR methods, a PCV-2 infection was demonstrated in tissues of 94.2% (33 of 35) of the sick pigs tested, in agreement with previous findings showing the close association of this new genotype of PCV with outbreaks of PMWS in Europe and North America. On the other hand, a PCV-1 infection was confirmed in only 5.7% (2 of 35) of the pigs, and confirmation of a mixed infection with PCV-2 was obtained by a single PCR with PCV-2-specific primers.     

Multiplex PCR for sexing Schistosoma japonicum cercariae and its utility

Accurate discrimination of the Schistosoma japonicum cercariae gender is very important for establishing monosexual infection animal models and for standardizing the real intensity of infection. In this study, a multiplex PCR technique consisting of two pairs of primers, of which one amplifies a 185-bp band specific for the W chromosome and the other amplifies a 420-bp band for the Z chromosome, was established to sex the S. japonicum cercariae. For male cercariae (ZZ), a single 420-bp band is expected, and for female cercariea (ZW), two distinct 185-bp and 420-bp bands can be observed. There was no cross-reaction with S. mansoni, S. haematobium, Clonorchis sinensis, Paragonimus westermani, and Trichinella spiralis. After sexing the cercariae escaped from a single snail, mice in group A were infected with 60 male cercariae and mice of group B were infected with 40 female cercariae. Meanwhile, mice in group C were infected with 10 male and 10 female cercariae that were sexed by multiplex PCR. At 45 days postinfection, male and female adult worms were recovered to verify the accuracy of multiplex PCR for sexing S. japonicum cercariae and to calculate the male and female survival rate and paired worm ratio. Our results showed that the multiplex PCR technique could distinguish male cercariae with 100% accuracy. However, sometimes the discrimination results of multiplex PCR mis-scored mixed sexual cercariae as female cercariae. The mean male adult worm burden in mice of group C was 10.7 ± 2.4, and the mean female adult worm burden was 7.7 ± 2.5. There was a significant difference between the male worm burden and female worm burden in group C. The P value was 0.013. The real paired worm ratio of group C was 74.2% (95%CI 56.6~91.8%). These results demonstrated a male-biased sex ratio in the mice model with equilibrated sex ratio cercariae infection, as predicted by our multiplex PCR technique. In conclusion, our multiplex PCR technique is an effective tool for sexing S. japonicum cercariae, especially for distinguishing male cercariae, which is of great value for establishing monosexual cercariae infection mice models to harvest male adult worms for anti-schistosomal drug screening.

     

Viral Respiratory Infections Diagnosed by Multiplex PCR after Allogeneic Hematopoietic Stem Cell Transplantation: Long-Term Incidence and Outcome

Viral respiratory infections (VRIs) are frequent after hematopoietic stem cell transplantation and constitute a potential cause of mortality. We analyzed the incidence, risk factors, and prognosis of VRIs in a cohort of transplanted patients. More frequent viruses were human coronavirus and human rhinovirus followed by flu-like viruses and adenovirus. Risk factors for death were lymphocytopenia and high steroid dosage.     

Lower Respiratory Multiplex Panels for the Detection of Bacterial and Viral Infections

Development of commercial multiplex panels for the detection and diagnosis of lower respiratory tract infections is rapidly progressing, and FDA-cleared assays are currently available. This review provides a comprehensive overview of the current or soon-to-be available commercial assays, focusing on their analytical performance, advantages, and challenges and the potential impact on patient outcomes when laboratories deploy the assays.     

Multiplex real-time PCR for detection of anaplasma phagocytophilum and Borrelia burgdorferi

A multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilum and Borrelia burgdorferi. The assay was tested on various Anaplasma, Borrelia, Erhlichia, and Rickettsia species, as well as on Bartonella henselae and Escherichia coli, and the assay was found to be highly specific for A. phagocytophilum and the Borrelia species tested (B. burgdorferi, B. parkeri, B. andersonii, and B. bissettii). The analytical sensitivity of the assay is comparable to that of previously described nested PCR assays (A. phagocytophilum, 16S rRNA; B. burgdorferi, fla gene), amplifying the equivalent of one-eighth of an A. phagocytophilum-infected cell and 50 borrelia spirochetes. The dynamic range of the assay for both A. phagocytophilum and B. burgdorferi was >/=4 logs of magnitude. Purified DNA from A. phagocytophilum and B. burgdorferi was spiked into DNA extracted from uninfected ticks and from negative control mouse and human bloods, and these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. The assay was tested on field-collected Ixodes scapularis ticks and shown to have 100% concordance compared to previously described non-probe-based PCR assays. To our knowledge, this is the first report of a real-time multiplex PCR assay that can be used for the simultaneous and rapid screening of samples for A. phagocytophilum and Borrelia species, two of the most common tick-borne infectious agents in the United States.     

Rapid Detection of Hepatitis B Virus Variants Associated with Lamivudine and Adefovir Resistance by Multiplex Ligation-Dependent Probe Amplification Combined with Real-Time PCR

Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice.     

Multiplex,quantitative, real-time PCR assay for cytomegalovirus and human DNA

We created a multiplex, quantitative, real-time PCR assay that amplifies cytomegalovirus (CMV) and human DNA in the same reaction tube, allowing for a viral load determination that is normalized to measured human DNA. The assay targets a conserved region of the CMV DNA polymerase gene that is not affected by known drug resistance mutations. All 36 strains of CMV detected by culture or qualitative PCR in a population of lung transplant recipients were detected. The assay detected 1 to 10 copies of CMV plasmid DNA. The analytic sensitivity was not affected by the presence of DNA from 10(6) human cells but was reduced approximately 10-fold by alkaline lysates of leukocyte preparations. CMV quantitation was linear over a range of 10(1) to 10(6) copies. The intraassay and interassay coefficients of variation were 29 and 40%. Human DNA was regularly detected in patient plasma samples, and the amount was increased by storage of blood at room temperature before plasma separation and by plasma separation techniques that allowed leukocyte contamination. Applied to whole blood, the assay provides a measurement of CMV DNA in relation to cellular content without a need for cell counting procedures. Applied to plasma, the assay can reveal artifactual increases in plasma CMV levels resulting from leukocyte contamination. Further study of the utility of this assay to monitor patient populations at risk for CMV disease is warranted.     

Multiplex PCR for diagnosis of AIDS-related central nervous system lymphoma and toxoplasmosis.

A nested multiplex PCR assay was designed for the simultaneous detection of Epstein-Barr virus and Toxoplasma gondii DNA from the cerebrospinal fluid of AIDS patients. T. gondii DNA was detected in 8 of 8 patients with Toxoplasma encephalitis and in 0 of 6 patients without toxoplasmosis, and Epstein-Barr virus DNA was found in 9 of 14 patients with central nervous system lymphoma and in 2 of 38 patients without disease.     

Multiplex PCR assay for rapid and accurate capsular typing of group B streptococci

We developed a simple, specific, and sensitive two-multiplex-PCR assay that enabled the detection of all known group B streptococcal (GBS) capsular polysaccharides. This test is well adapted for GBS capsular polysaccharide typing in large-scale epidemiological studies.     

Multiplex real-time PCR assay for detection of influenza and human respiratory syncytial viruses

A multiplex real-time PCR assay was developed with a LightCycler instrument for detection of influenza viruses A and B and the human respiratory syncytial virus (HRSV). Detection of each viral product and of an internal control was based on determination of specific melting temperatures by the LightCycler software. The lower limit of detection in the multiplex PCR assay was found to be 50 copies for each viral target. In an evaluation of nasopharyngeal samples collected from hospitalized children (ages, 0 to 3 years) with acute respiratory tract infections during the winter of 2001 to 2002, a viral pathogen was detected by the multiplex PCR test in 139 (66.8%) of 208 cases, including 45 (21.6%) influenza A virus infections, no (0%) influenza B virus infections, 106 (51%) HRSV infections, and 12 (5.8%) coinfections. The multiplex PCR test was compared to rapid antigen detection assays for influenza viruses A and B (Directigen; Becton Dickinson, Sparks, Md.) and HRSV (RSV TestPack; Abbott Laboratories, Abbott Park, Ill.) in 172 and 204 samples, respectively. After resolution of discrepant test results by use of additional PCR assays targeting other viral genes, the sensitivity (Se) and specificity (Sp) of the multiplex PCR assay for influenza A virus were 100 and 97.7% compared to 43.6 and 98.5% for the antigenic test. Similarly, the Se and Sp of the multiplex PCR assay for HRSV were 94.5 and 98.9% compared to 81.6 and 94.7% for the antigenic test. In conclusion, our multiplex real-time PCR assay combines both rapidity and sensitivity for detecting the most important respiratory viral pathogens in children.     

Real-Time Multiplex PCR Assay and Melting Curve Analysis for Identifying Diarrheagenic Escherichia coli

A real-time multiplex PCR assay was designed to amplify the virulence genes eae, pEAF, aatA, daaC, elt, est, ipaH, stx₁, and stx₂ for the detection of all diarrheagenic Escherichia coli pathotypes. This assay proved to be more sensitive and rapid than a conventional multiplex PCR for diarrheagenic E. coli isolates from children with diarrhea.