Surveillance of Childhood Influenza Virus Infection: What Is the Best Diagnostic Method To Use for Archival Samples?

Despite the clinical importance of influenza virus in pediatric respiratory infections, the optimal set of diagnostic tests to use when conducting studies using archival samples is not clear. In this study, we compared diagnostic tests for influenza virus in 75 children younger than 5 years of age who presented with symptomatic respiratory infection during one of four influenza seasons, had negative viral cultures for other respiratory pathogens, and had both an archival nasal aspirate obtained at the time of illness and serology spanning that influenza season. For all eligible children, we compared the results of viral culture performed at the time of collection with serology and PCR of archival nasal aspirates. Using real-time viral culture as the "gold standard," the test characteristics of PCR of archival nasal aspirates (sensitivity, 82%; specificity, 100%) and serology (sensitivity, 82%; specificity, 87%) were similar. The relatively low sensitivity of PCR of archival nasal samples in this study compared to that of PCR of fresh samples in a previous study suggests that RNA degradation occurred despite storage of the specimens at -70 degrees C. RNA degradation would also explain why only 11 (52%) of 21 archival nasal samples that had positive influenza virus cultures at the time of collection had positive repeat cultures in the summer of 2000. Thus, in archival specimens stored at -70 degrees C, PCR was more sensitive than viral culture. However, testing of fresh specimens had the highest yield in this study. Studies of optimal methods for specimen storage are needed.     

The diagnosis of cat-scratch-disease-associated adenitis: diagnostic value of serology and polymerase chain reaction

The diagnosis of cat scratch disease (CSD) associated adenitis relies classically on the association of clinical, epidemiological and bacteriological criteria. The polymerase chain reaction (PCR) looks like a more competitive diagnostic trial than serology. We evaluated the sensitivity, specificity and predictive positive and negative values of serology in routine diagnosis of CSD. A retrospective study over five years was led among patients presenting a suspicion of CSD and having a serology and/or a PCR. The Gold standard for diagnosis was PCR. The serological tests of Bartonella henselae was performed once in 482 patients, of which 2% (11 out of 482) were positive, and twice in only 39 patients (8%). The PCR diagnosis method for B. henselae was performed in biopsy of specimen lymph nodes in 28 patients and 14 out of 28 were positive. In nine patients, the diagnosis was exclusively made by PCR. Among the 14 patients whose PCR was negative, two had a positive serology and in three others patients, the serology was not performed. The sensitivity of serology was 35%, this confirms the low sensitivity of the serology in the CSD diagnosis. The diagnosis was confirmed in 56% of cases where PCR was performed. This led us to propose to perform systematically the PCR test for B. henselae in case of adenitis possibly associated with CSD.     

Amplification of P1 and 16S rRNA genes by nested PCR for detection of Mycoplasma pneumoniae in paediatric patients

Mycoplasma (M.) pneumoniae is the most frequent atypical pathogen responsible for community-acquired respiratory infection in children and adults. The etiologic diagnosis of these infections still remains difficult. This is mainly due to the absence of characteristic clinical findings, and to the available detection methods (serology and culture) which are time consuming, insensitive and non-specific. To improve the detection of this infectious agent, nested polymerase chain reaction (PCR) analysis was developed. A total of 46 nasal aspirates, from children hospitalised with severe lower respiratory tract infection and in whom M. pneumoniae was suspected, were analysed for the presence of M. pneumoniae DNA by PCR. Routine microbiological investigations revealed no virus in these 46 samples. Using nested PCR, two targets were amplified: the sequences of 16S ribosomal (r) RNA gene (rDNA) and P1 adhesin gene. Evidence of M. pneumoniae infection was identified in four paediatric patients. The amplification of 16S rDNA was found to be more sensitive for the detection of M. pneumoniae. Our results suggest that amplification of the 16S rDNA by nested PCR and detection of the amplification products by visual inspection of the polyacrylamide gel should allow the rapid diagnosis of M. pneumoniae in respiratory tract infection in paediatric patients.     

Comparison of serological and real-time PCR assays to diagnose Bordetella pertussis infection in 2007

Bacterial culture for diagnosing pertussis infection has high specificity but poor sensitivity and is slow. Highly sensitive real-time PCR assays and single-serum pertussis serology have been developed to overcome these limitations, but there are few data available on the relative sensitivities and specificities of such assays for pertussis diagnosis. Using data on 195 participants (>or=7 years old) from an epidemiological study, we assessed the sensitivity, specificity, and performance (Youden index) for pertussis diagnosis of the pertussis toxin enzyme-linked immunosorbent assay (using single and paired serology) and of real-time PCR assays (using the IS481 and ptxA-Pr targets). All available diagnostic information (clinical and laboratory) was pooled to serve as the gold standard. Single serology was the most efficient diagnostic test (Youden index, 0.57 to 0.58), with relatively high sensitivity (>64%) and high specificity (>90%), independent of the cutoff level. IS481 PCR performance was superior to that of ptxA-Pr PCR, and it was the second-most-efficient tool (Youden index, 0.30). Performing both ptxA-Pr and IS481 PCRs did not improve diagnostic performance. The greatest test efficiency (Youden index, 0.69 to 0.74) was achieved when single-serum serology was used in combination with IS481 or ptxA-Pr PCR or paired serology. Combining single serology with one PCR or paired serology increased the sensitivity with an associated limited decrease in specificity. The most specific tests for diagnosis of pertussis were single serology and ptxA-Pr PCR, and the most sensitive diagnostic tool was the combination of IS481 PCR with single serology.     

Congenital HCMV infection: a collaborative and comparative study of virus detection in amniotic fluid by culture and by PCR.

Cytomegalovirus (HCMV) infection is the leading cause of congenital virus infection in developed countries, affecting an estimated 1% of births. This antenatal infection can cause serious sequelae. Strategies for prevention and treatment must, therefore, be agreed upon, entailing a preliminary performance assessment of antenatal virus diagnosis techniques. Between 1992 and 1999, HCMV serology status was established for 19456 pregnant women in four French hospitals. Seronegative patients (55.4%) were given serology screening, and antenatal diagnosis was given to 152 women who had shown seroconversion during their pregnancies (1.4%). The detection of HCMV transmission from mother to fetus was finally established in 95 cases, using polymerase chain reaction (PCR) and viral culture methods for detecting HCMV in the amniotic fluid. These results were compared with viral culture of children's urine after birth, enabling us to distinguish between children really infected in utero (30%) and non-infected children (70%). The results of the virus culture and those of PCR were identical in 94 of the 95 cases, with one discrepancy (culture-/PCR+). The two diagnosis techniques had identical sensitivity (72%), with culture proving slightly more specific than PCR (98.4% as opposed to 96.9%). Positive prediction values for culture and for PCR were, respectively, 95.6 and 91.3%. Antenatal virus diagnosis on amniotic fluid was negative with both techniques in 8 out of 29 cases of children born with HCMV infection (VPN=89%). Over half of these wrongly negative results can be explained by amniocentesis carried out too early in the pregnancy or too early with respect to the mother's primary infection.     

16S rRNA based polymerase chain reaction compared with culture and serological methods for diagnosis ofMycoplasma pneumoniae infection

The use of a 16S rRNA based polymerase chain reaction (PCR) for the detection ofMycoplasma pneumoniae infection was investigated. Sputum samples from 34 patients with respiratory illness and evidence of pneumonia as judged by chest X-ray were analyzed by PCR and microbiological culture. Throat swabs from 14 healthy individuals were used as controls. For serology, an enzyme immunoassay for the detection of immunoglobulin M antibodies and a complement fixation assay were performed. Evidence ofMycoplasma pneumoniae infection was obtained in ten patients (29 %), eight of whom were found positive by both PCR and serology. Two of the sputum samples from these eight patients were negative by culture. Of the remaining two patients positive forMycoplasma pneumoniae, one was positive by PCR and culture but negative by serology, and one was found positive by serology but negative by PCR and culture. Thirteen of the 14 controls were negative by both PCR and serology. One control, however, was negative by serology but positive by PCR, which was probably due to asymptomatic carriage ofMycoplasma pneumoniae. The results of this study indicate the suitability of the PCR for the detection ofMycoplasma pneumoniae in clinical samples as well as its potential value as an additional tool for the diagnosis of infection.     

Comparison of real-time PCR assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children

Conventional fluorescent-antibody (FA) methods were compared to real-time PCR assays for detection of respiratory syncytial virus (RSV), influenza virus type A (FluA), parainfluenza virus types 1, 2, and 3 (PIV1, PIV2, and PIV3), human metapneumovirus (MPV), and adenovirus (AdV) in 1,138 specimens from children with respiratory illnesses collected over a 1-year period. At least one virus was detected in 436 (38.3%) specimens by FA and in 608 (53.4%) specimens by PCR (P<0.001). Specimen quality was inadequate for FA in 52 (4.6%) specimens; 13 of these (25%) were positive by PCR. In contrast, 18 (1.6%) specimens could not be analyzed by PCR; 1 of these was positive by FA. The number of specimens positive only by PCR among specimens positive by PCR and/or FA was 18 (7.0%) of 257 for RSV, 18 (13.4%) of 134 for FluA, 25 (64.1%) of 39 for PIV1, 8 (88.9%) of 9 for PIV2, 17 (30.1%) of 55 for PIV3, and 101 (76.5%) of 132 for AdV. MPV was detected in 6.6% of all specimens and in 9.5% of the 702 specimens negative by FA. The mean number of virus copies per milliliter in specimens positive by both PCR and FA was significantly higher, at 6.7x10(7), than that in specimens positive only by PCR, at 4.1x10(4) (P<0.001). The PCR assays were significantly more sensitive than FA assays for detecting respiratory viruses, especially parainfluenza virus and adenovirus. Use of real-time PCR to identify viral respiratory pathogens in children will lead to improved diagnosis of respiratory illness.     

Evaluation of a commercial real-time PCR kit for detection of dengue virus in samples collected during an outbreak in Goiania, Central Brazil, in 2005

In the past 2 decades, dengue has reemerged in Brazil as a significant public health problem. Clinicians demand a diagnostic test with high sensitivity that is applicable during the early symptomatic phase. We aimed to test two distinct molecular methods on samples from suspected dengue cases during an outbreak in Central Brazil. Acute-phase serum specimens from 254 patients suspected of having dengue were collected during 2005 in the city of Goiania, Central Brazil. Samples were blindly evaluated by real-time and multiplex PCR in addition to routine immunoglobulin M serology and virus culture. Overall, acute dengue was confirmed by serology, multiplex PCR, or virus isolation for 80% of patients (203/254). Another four patients presented real-time PCR-positive results as the unique marker of dengue. Higher real-time PCR positivity levels and viral loads were observed in the early symptomatic phase of disease (< or =5 days) than after this period. Multiplex and real-time PCR assays presented a high kappa agreement (0.85). According to multiplex PCR, 60 samples harbored dengue virus type 3 (DEN-3), 4 samples harbored DEN-2, and 1 sample displayed a pattern compatible with a double infection with DEN-2 and -3. The dengue virus real-time kit was found to be practical and adjustable for high throughput, to display the best performance in the early symptomatic phase of dengue cases, and to be valuable for confirming dengue diagnosis in a timely manner.     

多重PCR检测方法和液态芯片技术在呼吸道病毒检测中的应用研究

目的 评估澳大利亚维多利亚感染性疾病参比实验室（The Victorian Infectious Diseases Reference Laboratory,VIDRL）的呼吸道病毒多重PCR检测方法和Luminex公司多指标同步分析液态芯片技术-呼吸道病毒检测试剂盒（xTAG RVP）,应用于多种常见呼吸道病毒检测的优势和缺点,为更好地、有针对性地选择合适技术进行临床实验诊断或公共卫生应急检测提供依据.方法 使用VIDRL的呼吸道病毒多重PCR检测方法和Luminex公司xTAG RVP试剂盒对198份临床流感样症状患者的咽拭子标本进行常见呼吸道病毒的核酸检测.结果 198份临床标本经VIDRL多重PCR方法和xTAG RVP方法检测,阳性率分别为38.89％和45.45％,符合率为72.22％.单一病毒的符合率为87.88％～100％.结论 xTAG RVP方法与VIDRL多重PCR方法相比,两者在常见呼吸道病毒检测中的特异性和敏感性相近,xTAG RVP方法具有明显的快速和高通量的优势.     

The value of polymerase chain reaction for the diagnosis of viral respiratory tract infections in lung transplant recipients.

BACKGROUND: Respiratory viruses cause severe infections in lung transplant recipients, which require rapid and accurate diagnosis for appropriate management. OBJECTIVES: To evaluate the added benefit of a multiplex PCR for respiratory viruses (influenza [FLU] A and B, respiratory syncytial virus [RSV] A and B and parainfluenza virus [PIV] 1, 2, and 3) complementing rapid respiratory viral culture (RRV) and FLU-A antigen detection (EIA) in this transplant population. RESULTS: Over 6 months, 116 nasal washes and bronchoalveolar lavages, obtained from 72 lung transplant recipients with symptoms of upper or lower respiratory tract infections, were tested in real time by RRV and FLU-A EIA, and batched frozen by PCR. One or more methods recognized a respiratory virus in 31 (27%) specimens, including 15 FLU-A, nine RSV and seven PIV. PCR identified 26 of 31 positive samples demonstrating a sensitivity of 84%, higher than RRV (67%) or EIA (54%). PCR, RRV and EIA detected 60, 80 and 54%, respectively, FLU-A samples. PCR and RRV were equivalent for RSV-A, PIV-2 and 3, but PCR found a significantly higher number of RSV-B and PIV-1. CONCLUSIONS: These data indicate that routine use of PCR will enhance the number and speed with which viral respiratory tract infections are diagnosed in lung transplant recipients.     

Mycoplasma pneumoniae infections: retrospective study in Basse-Normandie, 1997-2005. Epidemiology--diagnostic utility of serology and PCR for a rapid diagnostic

OBJECTIVES: The objective of this study was to evaluate the epidemiology of Mycoplasma pneumoniae (Mpn) infections in Basse-Normandie by a retrospective analysis of serological and PCR data, and to confirm the diagnostic utility of PCR and serology. METHODS: From 1997 to August 2005, 6156 serum samples and 6123 respiratory tract samples were collected from hospitalised patients and evaluated for the diagnosis of Mpn infection by PCR, serological assays, or by the two tests. During the epidemic period (2004-2005), the results of 1489 patients were analysed. RESULTS: Over the 9-y period, the seroprevalence was 40,4% and we reported on 525 cases with serologically or/and PCR proven Mpn infection, according a cyclic pattern spaced out 7 years. During the epidemic period, the seroprevalence increased to 50,2% and the rate of infections was 8.3%. The analysis of the 124 cases of Mpn infection showed typical epidemiological characteristics: a peak of incidence among the children and young adults, a summer-winter pattern and some coinfections with viral strains. For diagnosis of Mpn infection, the comparison of PCR and serological assays among 36 patients showed a concordance of only 41.7%. CONCLUSION: Mpn infections were endemic and outbreaks were observed according cyclic pattern with a high incidence specially in the children. Sensitive and specific tests were now available for early and reliable diagnosis. In children, the combination of the PCR on nasopharyngeal samples and the IgM EIA serology test were recommended. In adults, the PCR was privilegiated.     

Comparison of polymerase chain reaction, culture, and western immunoblot serology for diagnosis of Bordetella pertussis infection.

Polymerase chain reaction (PCR) amplification of the pertussis toxin promoter region was used to detect Bordetella pertussis infection in nasopharyngeal aspirates collected from 24 infants and children infected with pertussis and 13 adult contacts during an epidemiological study. The sensitivity of this PCR assay was approximately one bacterium, and the assay was specific for B. pertussis in tests with other Bordetella species and other respiratory pathogens. The pertussis case definition required a cough with a duration of more than 21 days for infants and children and laboratory confirmation by serology as the primary detection method for infants, children, and adults. The sensitivity of PCR and culture on Bordet-Gengou agar medium was assessed with regard to the case definitions. In the group of infants and children (index cases), the sensitivities of the culture and the PCR were 54.1% (13 of 24) and 95.8% (23 of 24), respectively. In the adult group (household contacts), the sensitivities of the two methods were 15.4% (2 of 13) and 61.5% (8 of 13), respectively. PCR combined with pertussis-specific serology appears to be a useful tool for diagnosis of pertussis especially in epidemiological studies.     

Detection of bovine leukosis provirus in blood and semen samples of bulls

Enzootic bovine leukosis is a chronic lymphoproliferative disorder in cattle caused by the exogenous retrovirus, bovine leukemia virus (BLV). There are numerous molecular protocols for the detection of bovine leukosis provirus in blood and semen. The aim of this study was to investigate the serology status of BLV infection in bulls in Tabriz, Iran and polymerase chain reaction (PCR) detection of viral genome of BLV in seminal cellular fraction and blood samples in sero-positive and sero-negative bulls. In this study, 45 serum samples from bulls from 18 different herds referred to the Artificial Insemination Centre were used to evaluate the serology infection of BLV by enzyme-linked immunosorbent assay. In addition, blood and semen samples of related bulls were used for the detection of BLV DNA. Antibodies against BLV were detected in five of 45 blood samples (4.4%), and PCR demonstrated that semen and blood samples of five sero-positive and one sero-negative bull were infected by BLV. Our findings support the power of a PCR test for BLV detection in bovine semen and blood samples and could be easily used for routine diagnosis. This data strongly suggest that semen from sero-positive bulls could result positive by PCR.     

Improved detection of rhinoviruses in nasal and throat swabs by seminested RT-PCR

A seminested RT-PCR (nRT-PCR) was used to detect picornavirus (PV) RNA in cell cultures inoculated with rhinoviruses (HRVs) and enteroviruses (EVs). PCR tests in which a primary "touchdown" PCR was followed by secondary reactions using PV or HRV specific primers were able to differentiate HRVs of 48 serotypes from EVs. PVnRT-PCR and HRVnRT-PCR were then used to test nasal and throat swabs from adult subjects with naturally acquired respiratory virus infections. The swabs were also analysed for respiratory viruses by cell culture techniques and the rates of PV identification by the two methods were compared. PVnRT-PCR was found to be at least five times more sensitive than cell culture for the detection of PVs in these clinical specimens. Paired acute and convalescent serum samples were tested for complement fixing antibodies to adenovirus, influenza A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, and 3, Myco plasma pneumoniae, and Chlamydia psittaci. An enzyme-linked immunosorbent assay (ELISA) was used to detect rises in antibody level to coronavirus types 229E and OC43. The overall rate of pathogen identification in 159 swabs from adult asthmatics increased from 28% when only cell culture and serology were used to 57% when these methods were supplemented by PVnRT-PCR. © 1993 Wiley-Liss, Inc.     

Poor performance characteristics of conventional PCR in detection of respiratory syncytial virus-experience of a tertiary care centre in Southern India

Respiratory syncytial virus (RSV) is a significant cause of contagious acute respiratory infections in children and older adults. Since there are contradictory reports regarding the efficacy of different methods to detect RSV, we evaluated the performance of the conventional PCR versus real-time PCR in 222 patients with acute respiratory infections (ARI) recruited between January 2012 and March 2013. Conventional PCR had a very poor sensitivity of 40% (95% CI: 19.2-63.9%) and failed to detect RSV in respiratory samples with low viral load. Thus, it may be prudent to replace it with real-time PCR to achieve precise diagnosis.