Multiplex Reverse Transcriptase-polymerase Chain Reaction Research Articles

The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids

With current methodology, DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) that can identify blood, saliva, semen and menstrual blood in individual stains or in mixtures of body fluids. Messenger RNA transcripts specific to each type of body fluid have been identified and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) system developed to identify these body fluids along with three housekeeping genes. This multiplex can detect semen and seminal fluid (semen without spermatozoa present). Furthermore, we have targeted the co-isolation of RNA and DNA from the same sample and, with the RT-PCR multiplex, we can determine the type of body fluid present as well as generate a DNA profile(s) from the same stain.

The presence of mature granulocytes/monocytes derived from leukemic cells in MLL-associated leukemia

We observed the mature granulocytes/monocytes derived from leukemic cells in patients with acute myeloid leukemia who present mixed lineage leukemia gene (MLL). Morphologic observation and fluorescence in situ hybridization analysis (FISH) for chromosome 11q23 abnormality were studied, and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was done to identify the fusion partners with MLL. The bone marrow cells with FISH signals of MLL showed the cell differentiation of the myeloid and/or monocytic lineages in 4 of 6 AML patients. MLL partner genes were AF6, AF9, ELL, and ENL, respectively. There was no correlation between the fusion partner and the appearance of mature cells derived from MLL clones. RT-PCR showed the fusion between MLL exon 9 or 10 and the partner genes in mature granulocytes/monocytes. These findings suggest that subgroup of leukemia cells with MLL rearrangement has the differentiation potential of leukemic cells and mature granulocytes/monocytes derived from MLL clones may be biologically different from normal mature cells.

An assessment of molecular variability and recombination patterns in South African isolates of Potato virus Y

The coat protein (CP) gene of 75 South African Potato virus Y (PVY) isolates was amplified using reverse-transcriptase polymerase chain reaction (RT-PCR). The resulting cDNA products were cloned and sequenced. These sequences were used to identify the strains to which the isolates belonged. Some, when compared to reference sequences, belonged to the PVY(N) and PVY(O) strains. A number of isolates were found to demonstrate significant homology to PVY(N) strains from China. A large number of South African isolates possessed CP sequences showing evidence of recombination between PVY(N) and PVY(O) strains, similar to those of PVY(NTN) isolates. Multiplex RT-PCR analysis allowed further differentiation of PVY(O) isolates and revealed that the majority were of the PVY(N)-Wilga strain. It was deduced that the most likely way in which these isolates reached South Africa was via the importation of infected material.

Are Meteorological Parameters Associated with Acute Respiratory Tract Infections?

Information on the onset of epidemics of acute respiratory tract infections (ARIs) is useful in timing preventive strategies (eg, the passive immunization of high-risk infants against respiratory syncytial virus [RSV]). Aiming at better predictions of the seasonal activity of ARI pathogens, we investigated the influence of climate on hospitalizations for ARIs. Samples obtained from 3044 children hospitalized with ARIs in Mainz, Germany, were tested for pathogens with a multiplex reverse-transcriptase polymerase chain reaction enzyme-linked immunosorbent assay from 2001 through 2006. Hospitalizations for ARIs were correlated with meteorological parameters recorded at the University of Mainz. The frequency of hospitalization for RSV infection was predicted on the basis of multiple time series analysis. Influenza A, RSV, and adenovirus were correlated with temperature and rhinovirus to relative humidity. In a time series model that included seasonal and climatic conditions, RSV-associated hospitalizations were predictable. Seasonality of certain ARI pathogens can be explained by meteorological influences. The model presented herein is a first step toward predicting annual RSV epidemics using weather forecast data.

Development and Validation of a Short‐Time Cell Culture and Multiplex Reverse Transcriptase Polymerase Chain Reaction Assay for Infectious Pancreatic Necrosis Virus in Mexican Farm‐Sampled Rainbow Trout

The infectious pancreatic necrosis virus (IPNV) affects several species of freshwater and marine fish. In Mexico, IPNV has an important impact on farming of rainbow trout Oncorhynchus mykiss; however, IPNV distribution in Mexico is unclear. The diagnosis of IPNV is laborious; usually it is based on isolation tests in cell culture followed by immunological identification using techniques of serum neutralization, immunofluorescence, or enzyme-linked immunosorbent assay. It has recently been demonstrated that reverse transcriptase polymerase chain reaction (RT-PCR) is an adequate method for the detection of aquatic birnaviruses. However, its diagnostic use is still limited because very low titers of viable virus cannot be easily detected. In this study, a combination of short-time cell culture and multiplex RT-PCR was established for the diagnosis of IPNV in rainbow trout obtained from farms in the state of Mexico. Three primer sets were used in a single reaction in the multiplex RT-PCR to increase the probability of identifying all serotypes of IPNV serogroup A as well as to help prevent a false-negative result. This approach was able to identify samples with an IPNV concentration of just 0.01 tissue culture infective dose with 50% endpoint (TCID50)/mL, and it identified more infected fish than RT-PCR alone or first-passage cell culture alone. Moreover, this technique made the same identifications as second-passage cell culture but in approximately 30% of the time needed for second-passage cell culture. Consequently, the time and cost efficiency of IPNV diagnosis were greatly reduced.

Dermatofibrosarcoma protuberans: clinical, pathological, and genetic (COL1A1‐PDGFB ) study with therapeutic implications

To analyse the presence of collagen type I alpha 1-platelet-derived growth factor beta (COL1A1-PDGFB) transcripts in 20 cases of dermatofibrosarcoma protuberans (DFSP) and to assess the relationship between COL1A1 breakpoints and clinical and histopathological variables. Multiplex reverse transcriptase-polymerase chain reaction was carried out using frozen tissue. Our series contained 14 men and six women. Histologically, most cases were of conventional type (n = 9), followed by fibrosarcoma (n = 4), Bednar tumour (n = 2), sclerosing (n = 2), myoid (n = 1) and atrophic (n = 1) DFSP, and giant cell fibroblastoma (n = 1). Immunohistochemistry revealed CD34 expression in 90% of cases. COL1A1-PDGFB fusion transcripts were present in 89% of cases (exons 18, 19, 20, 25, 26, 31, 33/34, 39, 40, 46, 47 and 48 of COL1A1 with exon 2 of PDGFB). There was no recurrence of DFSP in any of the 19 patients treated by Mohs surgery. A partial response was obtained in the two patients treated with imatinib. The COL1A1-PDGFB fusion was present in all histological subtypes of DFSP, but not all cases expressed the fusion transcript. No association was observed between different COL1A1 breakpoints and clinicopathological parameters. Imatinib mesylate can be useful in locally advanced tumours and metastases.

Detection of a novel CBFB/MYH11 variant fusion transcript (K-type) showing partial insertion of exon 6 of CBFB gene using two commercially available multiplex RT-PCR kits

We report on a 20-year-old man with acute myeloid leukemia (AML) showing a distinct novel CBFB/MYH11 variant fusion transcript. Initial results of bone marrow, chromosome, and flow cytometric analyses were not in accordance with the diagnosis of acute myelomonocytic leukemia with eosinophilia (AML-M4Eo) or AML with a CBFB/MYH11 rearrangement. However, results from 2 commercially available multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) tests repeatedly showed an unusual PCR product from his bone marrow specimen. Not only does this case show a partial insertion of exon 6 of the CBFB (ENSG00000067955) gene, but it also involves novel breakpoints within both exon 6 of the CBFB gene and exon 28 (previously exon 7) of the MYH11 (ENSG00000133392) gene, which is regarded as a previously non-reported, new type (K-type) of CBFB/MYH11 fusion transcript. In addition, our study result was in agreement with the recent report of Schnittger et al. that rare fusion transcripts of CBFB/MYH11 are correlated with an atypical cytomorphology and other aberrant characteristics. Therefore, multiplex RT-PCR and sequence analysis of these atypical products should be performed to diagnose atypical AML with CBFB/MYH11 rearrangement, to predict prognosis of these patients as well as to elucidate the molecular mechanism.

Detection of nasopharyngeal carriages in children by multiplex reverse transcriptase-polymerase chain reaction

Purpose : The aim of this study was to determine the prevalence of asymptomatic nasopharyngeal carriages in children using a multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) assay kit. Methods : We obtained nasopharyngeal swabs from 33 children without any underlying disease from July 25 to July 28, 2008. The children were free from the signs of respiratory tract infections at the time of sampling. DNA was extracted from the swabs and subjected to multiplex RT-PCR using a primer set for the detection of pneumococci (Seeplex ® PneumoBacter ACE Detection Seegene, Seoul, Korea). The amplified PCR products were separated on 2% agarose gels and stained with either ethidium bromide or screen tape system (Lab901 Scotland, UK). Results : A total of 33 children (male, 15 female, 18) aged between 3.2 and 16.3 (median, 8.2) years were included in this study. The mRT-PCR detected colonized bacteria (Streptococcus pneumoniae, Hemophilus influenzae, Chlamydia pneumoniae, and Bordetella pertussis) in 30 children (90.9%). Of these, 13 children (39.4%) showed more than 2 bacteria: 12 children were positive for 2 bacteria (S. pneumoniae and H. influenzae) and 1 child was positive for 3 bacteria (S. pneumoniae, H. influenzae, and C. pneumoniae). Conclusion : mRT-PCR was found to be a sensitive tool for the detection of asymptomatic nasopharyngeal carriages. Clinical significances of the bacteria detected by mRT-PCR will have to be evaluated in the future. (Korean J Pediatr 2009; 52:1358-1363)

Epidemiologic and clinical features in children with acute lower respiratory tract infection caused by human metapneumovirus in 2006-2007

Purpose : The causes of acute lower respiratory tract infection (ALRTI) are mostly attributable to viral infection, including respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus A/B (IFV A/B), or adenovirus (ADV). Several Korean studies reported human metapneumovirus (hMPV) as a common pathogen of ALRTI. However, studies on seasonal distribution and clinical differences relative to other viruses are insufficient, prompting us to perform this study. Methods : From November 2006 to October 2007, we tested nasopharyngeal aspiration specimens in children hospitalized with ALRTI with the multiplex reverse transcriptase-polymerase chain reaction to identify 6 kinds of common pathogen (hMPV, RSV, PIV, IFV A/B, and ADV). We analyzed positive rates and clinical features by respiratory chart review. Results : We detected 38 (8.4%) hMPV-positive cases out of 193 (41.8%) virus-positive specimens among 462 patients. HMPV infection prevailed from March to June with incidence peaking in April. HMPV-positive patients were aged 15 years (76.3%), and the ratio of boys to girls was 1.2:1. The median age was 27 months. HMPV primarily caused pneumonia (76.3 %) (P=0.018). Average hospitalization of HMPV-associated ALRTI patients was 5.8 days. In addition, they showed parahilar peribronchial infiltration (100%) on chest X-ray, normal white blood cell count (73.7%), and negative C-reactive protein (86.8 %) (P>0.05). All hMPV-positive patients recovered without complication. Conclusion : HMPV is a common pathogen of ALRTI in Korean children, especially in 1-5 year olds, from March to May. Immunocompetent children diagnosed with hMPV-associated ALRTI may have a good prognosis.

Association between Kawasaki disease and acute respiratory viral infections

Purpose:The etiology of Kawasaki disease (KD) is still unknown. Recently, an association between human coronavirus NL63 (HCoV-NL63) and KD was implicated. Hence, we attempted to determine the association between KD and acute respiratory viral infections. Methods:Nasopharyngeal aspirate samples were obtained from 54 patients diagnosed with KD at the Seoul National University (SNU) Children’s Hospital and SNU-Bundang Hospital between October 2003 and September 2006. Viral diagnoses of 11 respiratory viruses were made using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR): respiratory syncytial virus (RSV), adenovirus, rhinovirus (RV), parainfluenza viruses (PIVs) 1 and 3, influenza viruses (IFVs) A and B, human metapneumovirus (HMPV), human bocavirus (HBoV), HCoV OC43/229E, and HCoV-NL63. Clinical data were reviewed retrospectively. Results:The median age was 32 months (6 months-10.4 years). Respiratory symptoms were observed in 37 patients (69%). The following respiratory viruses were identified in 12 patients (22%): RV (n=4), PIV-3 (n=2), HBoV (n=2), and adenovirus, RSV, PIV-1, IFV-A, and HCoV-NL63 (n=1). Co-infection with PIV-3 and RV was observed in one patient. Respiratory symptoms were observed in 7 (58.3%) and 30 (71.4%) patients of the virus-positive and virus-negative groups (P> 0.05). Response rate to intravenous immunoglobulin administration was 67% (n=8) and 86% (n=36) in the virus- positive and virus-negative groups (P>0.05). Conclusion:Respiratory symptoms were commonly observed in KD patients but the association between respiratory viruses and KD were not found. Large multicenter-based investigations are required to confirm the association between acute respiratory viral infections and KD.

The Clinical Significance of Nasopharyngeal Carriages in Immunocompromised Children as Assessed

Background: The aim of this study was to determine the prevalence of asymptomatic nasopharyngeal carriages in immunocompromized children by using a multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) assay kit. Methods: We obtained clinical samples by nasopharyngeal swabs from 42 patients with underlying immune deficiency from May 20, 2008 to May 22, 2008. The children were free from signs of respiratory tract infections at the time of sampling. Isolated cDNA was extracted and after this the DNA was examined using a multiplex primer set for pneumonial bacteria detection (SeeplexR PneumoBacter ACE Detection, Seegene, Seoul, Korea). The amplified PCR products were separated on 2% agarose gels and stained with ethidium bromide and a screentape system (Lab901, Scottland, UK) and then they were compared. The nasopharyngeal swab culture was done simultaneously and this was compared with the results of mRT-PCR. Results: A total of 42 patients (males: 24, females: 18) aged between 1.2 and 16.3 years (median: 9.2 years) were included in this study. The mRT-PCR detected bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae, and Bordetella pertussis) in 28 patients (66.6%). Of these 28 patients, 4 patients (14.3%) showed more than 2 bacteria: 2 patients were positive for two bacteria (S. pneumoniae and H. influenzae, H.influenzae and B. pertussis) and 2 patients were positive for three bacteria (S. pneumoniae, B. pertussis and C. pneumoniae, C. Pneumoniae, H. influenzae and B. pertussis). S. pneumoniae was cultured in one patient (2.4%). Conclusions: The mRT-PCR is a sensitive tool for the detection of the asymptomatic nasopharyngeal carriages. The clinical significance of the bacteria detected in immunocompromized patients by mRT-PCR will need further evaluation. (Korean J Hematol 2009;44:220-226) 󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏

Efficient assessment of the utility of immortalized Fa2N-4 cells for cytochrome P450 (CYP) induction studies using multiplex quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and substrate cassette methodologies

Induction of cytochrome P450 (CYP) 1A2, CYP2B6, and CYP3A4 by 22 prototypical inducers was evaluated in the Fa2N-4 immortalized human hepatic cell line. To facilitate this a duplex one-step quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for CYP1A2 and CYP3A4 and a substrate cassette allowing simultaneous monitoring of CYP1A2, CYP2B6, CYP2C9, and CYP3A4 activity were developed. CYP1A2 messenger RNA (mRNA) and activity were induced by the prototypical aryl hydrocarbon receptor (AhR) ligand β-naphthoflavone (Emax = 217- and 11-fold, respectively, and EC50 = 8 µM). CYP3A4 mRNA and activity were induced by the prototypical pregnane X receptor (PXR) ligands, rifampicin (Emax = 36- and 6-fold, respectively, and EC50 = 4 µM) and phenobarbital (Emax = 12- and 4-fold, respectively, and EC50 = 205 µM). No induction of CYP2B6 was detected with several prototypical constitutive androstane receptor (CAR) ligands. A large mRNA–activity Emax ratio was observed for some time-dependent inhibitors of CYP3A4, whereas EC50 determinations appeared to be independent of the endpoint. In conclusion, Fa2N-4 cells are a good surrogate for primary human hepatocytes for assessing AhR and PXR-mediated CYP1A2 and CYP3A4 induction, respectively, but not for CAR-mediated CYP2B6 induction. The sensitive and selective methodologies presented in this paper afford maximal data generation and enhanced throughput capability and are readily transferable to primary human hepatocytes or alternate cellular systems.

Establishment and characterization of a new human acute myelocytic leukemia cell line SH-2 with a loss of Y chromosome, a derivative chromosome 16 resulting from an unbalanced translocation between chromosomes 16 and 17, monosomy 17, trisomy 19, and p53 alteration

To report here a new acute myelocytic leukemia (AML) cell line SH-2 and describe its biological characteristics. Mononuclear cells isolated from a patient with AML-M2 subtype were passaged by liquid culture medium. Interleukin-3 and bone marrow stromal cells were used to support cell proliferation at the first 3 months. Various methods, including cytogenetic analysis, fluorescence in situ hybridization (FISH), multiplex FISH (M-FISH), reverse transcriptase polymerase chain reaction (RT-PCR), multiplex RT-PCR, short tandem repeat (STR)-PCR, direct sequencing of DNA, clonogenic assay, and tumorigenicity in nude and severe combined immunodeficient (SCID) mice were employed to identify and characterize SH-2 cell line. SH-2 cells were maintained without cytokine and stromal cells for 3 years. It had no Epstein-Barr virus or mycoplasma contamination. The SH-2 cell line showed typical myelocytic features in morphology and simultaneous strongly expressed myeloid antigens (CD13, 99.6% and CD33, 99.26%) and natural killer (NK)-related antigens (CD56, 99.5% and CD16/56, 99.62%) suggesting that SH-2 is an AML cell line with NK-antigen expression. SH-2 cell line initially showed a karyotype of 45, X, -Y, der(16)t(16;17)(q24;q12), -17, +19. During the passage period, the cells with a hypodiploid karyotype gradually decreased and were replaced by the near-tetraploid cells with a karyotype of 71-105(86), XX, -Y, -Y, der(16)t(16;17)x2, -17, -17, +19, +19. FISH and M-FISH delineated all abnormalities. SH-2 cells had the approximately same morphological, immunophenotypical, and cytogenetic features as the patient's leukemia cells had. STR-PCR provided powerful evidence for the derivation of SH-2 cell line from the patient's leukemia cells. SH-2 cells showed multiple drug resistance (MDR), which may be related to the p53 gene alteration, including the loss of one p53 allele due to the monosomy 17 and a point mutation of CAG to CAT at codon 576 of exon 5 in another p53 allele resulting in the loss of p53 gene function. In addition, SH-2 cell line did not express MDR-related genes, such as MDR1, multidrug resistance-related protein, and lung resistance protein, but expressed apoptosis-related genes, such as Bcl-2, Fas, glutathione S-transferase-pi, and p21, which were also related to the MDR. SH-2 cell line had tumorigenic capacities in nude and SCID mice. Because SH-2 cell line had a clear biology background, it will provide a useful tool for the study of the pathogenesis and treatment strategy of AML with MDR.

Wheezing Rhinovirus Illnesses in Early Life Predict Asthma Development in High-Risk Children

Virus-induced wheezing episodes in infancy often precede the development of asthma. Whether infections with specific viral pathogens confer differential future asthma risk is incompletely understood. To define the relationship between specific viral illnesses and early childhood asthma development. A total of 259 children were followed prospectively from birth to 6 years of age. The etiology and timing of specific viral wheezing respiratory illnesses during early childhood were assessed using nasal lavage, culture, and multiplex reverse transcriptase-polymerase chain reaction. The relationships of these virus-specific wheezing illnesses and other risk factors to the development of asthma were analyzed. Viral etiologies were identified in 90% of wheezing illnesses. From birth to age 3 years, wheezing with respiratory syncytial virus (RSV) (odds ratio [OR], 2.6), rhinovirus (RV) (OR, 9.8), or both RV and RSV (OR , 10) was associated with increased asthma risk at age 6 years. In Year 1, both RV wheezing (OR, 2.8) and aeroallergen sensitization (OR, 3.6) independently increased asthma risk at age 6 years. By age 3 years, wheezing with RV (OR, 25.6) was more strongly associated with asthma at age 6 years than aeroallergen sensitization (OR, 3.4). Nearly 90% (26 of 30) of children who wheezed with RV in Year 3 had asthma at 6 years of age. Among outpatient viral wheezing illnesses in infancy and early childhood, those caused by RV infections are the most significant predictors of the subsequent development of asthma at age 6 years in a high-risk birth cohort.

Enrichment of cerebrospinal fluid samples on cell culture for enhancement of sensitivity of mumps and enterovirus detection by multiplex RT-PCR

It has been demonstrated that the detection of enteroviruses and mumps virus nucleic acid in cerebrospinal fluid (CSF) specimens improves the management of the patients with aseptic meningitis. To determine the effect of overnight enrichment of mumps and enteroviruses in CSF samples on cell culture for increasing the sensitivity of viral detection, we developed a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR). CSFs were collected from 100 patients younger than 7 years. The samples were tested for the presence of enteroviruses and mumps virus by multiplex RT-PCR method. Negative samples in Multiplex RT-PCR were enriched for viral template RNAs by overnight culture of CSF samples on cells and followed by the optimized Multiplex RT-PCR. Overall, 35% of the CSF samples were positive for enteroviruses, whereas only 1% of CSF samples were positive for both viruses. After enriching in cell culture, 34% of the negative samples showed a positive polymerase chain reaction band for enteroviruses, and a 3% increase was observed for both viruses. The results showed that enrichment of viral template RNAs in cell culture can increase the sensitivity of the multiplex RT-PCR assay and provide a rapid and sensitive method for detection of viral infections.

A Novel Real-Time Multiplex Reverse Transcriptase-Polymerase Chain Reaction for the Detection of HIV-1 RNA by Using Dual-Specific Armored RNA as Internal Control

Objective: Transmission of HIV-1 and diagnosis of infection in hospitals and public health settings remains a worldwide concern. HIV-1 detection is sometimes not possible using current commercial assays, probably due to mismatches between the selected primers and probes. Methods: By screening primers and probes, we developed a dual-specificity probe real-time reverse transcriptase-polymerase chain reaction (DSPrtRT-PCR) assay using dual-specific armored RNA as the internal control. The specificity and sensitivity were compared between the monospecificity probe real-time and DSPrtRT-PCR techniques. Results: The sensitivity of DSPrtRT-PCR improved significantly, with no effect on its specificity. The detection limit was 173 IU/ml. All the HIV-1 group M and group O could be detected. In clinical assays, 1,000 copies/ml of armored RNA was required as internal control. When applied to negative samples, 100% specificity was achieved. Among 60 samples from the tested patients, DSPrtRT-PCR demonstrated high sensitivity, accurately detecting 50 positives and 10 negatives that were confirmed by the COBAS AmpliScreen assay. Conclusion: DSPrtRT-PCR is a more efficient and effective viral assay with high sensitivity and specificity as compared to monospecificity probe PCR. It can be widely applied in blood donor screening and qualitative individual detection of HIV-1 RNA.

Risk Factors Associated with Human Infection during the 2006 West Nile Virus Outbreak in Davis, a Residential Community in Northern California

We collected a total of 15,329 mosquitoes during weekly sampling in Davis, CA, from April through mid-October 2006 at 21 trap sites uniformly spaced 1.5 km apart over an area of approximately 26 km(2). Of these mosquitoes, 1,355 pools of Culex spp. were tested by multiplex reverse transcriptase-polymerase chain reaction, of which 16 pools (1.2%) were positive for West Nile virus (WNV). A degree-day model with a developmental threshold of 14.3 degrees C accurately predicted episodic WNV transmission after three extrinsic incubation periods after initial detection. Kriging interpolation delineated that Culex tarsalis were most abundant at traps near surrounding agriculture, whereas Cx. pipiens clustered within residential areas and greenbelt systems in the old portion of Davis. Spatial-temporal analyses were performed to test for clustering of locations of WNV-infected dead birds and traps with WNV-positive Cx. tarsalis and Cx. pipiens; human case incidence was mapped by census blocks. Significant multivariate spatial-temporal clustering was detected among WNV-infected dead birds and WNV-positive Cx. tarsalis, and a WNV-positive Cx. pipiens cluster overlapped areas with high incidences of confirmed human cases. Spatial analyses of WNV surveillance data may be an effective method to identify areas with an increased risk for human infection and to target control efforts to reduce the incidence of human disease.

Diagnostic performance of a rapid antigen test for RSV in comparison with a 19-valent multiplex RT-PCR ELISA in children with acute respiratory tract infections

A rapid and reliable diagnosis of respiratory syncytial virus (RSV) infection in childhood is very important for clinical management. In this study we compared a rapid antigen test (enzyme immunoassay, EIA) for the detection of RSV with a reverse transcriptase polymerase chain reaction (RT-PCR) [19-valent multiplex RT-PCR enzyme-linked immunosorbent assay (ELISA)] to assess the diagnostic performance. Furthermore the diagnostic value of the EIA in terms of age and season relation was analyzed. A total of 400 nasopharyngeal or tracheal secretions from pediatric patients with clinical signs of lower respiratory tract infection were included. The specimen had to be taken in a standardized manner within 72 h after admission. Specimens were tested in parallel with the EIA and the multiplex RT-PCR ELISA. The RT-PCR technique was used as the target assay. The EIA reached a sensitivity of 58% and a specificity of 90% for all samples tested. For patients < [corrected] 1 year the post-test probability for a positive EIA was 91% during the RSV season; a negative test result decreased disease probability from 53 to 25%. For older patients a positive test raised disease probability from 23 to 45% during the RSV season. Negative test results did not markedly change disease probability.

A Multiplex RT-PCR Test for the Differential Identification of Turkey Astrovirus Type 1, Turkey Astrovirus Type 2, Chicken Astrovirus, Avian Nephritis Virus, and Avian Rotavirus

Recent studies have revealed the presence ofastroviruses and rotavirus in numerous poorly performing and healthy chicken and turkey flocks in the United States. The phylogenetic analysis of the sequence data produced during these studies has identified four groups of avian astroviruses circulating in the United States: turkey astrovirus types 1 and 2 (TAstV-1 and TAstV-2), avian nephritis virus (ANV), and a chicken-origin astrovirus (CAstV). As the molecular epidemiology of poultry enteric disease is poorly understood, the development of updated diagnostic assays is crucial to the continued surveillance and management of enteric disease in affected as well as healthy flocks. This report details the development of a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay specific for astroviruses and avian rotavirus in turkey-origin and chicken-origin samples. The assay consists of two multiplex tests, one for turkey-origin samples and one for chicken-origin samples. The turkey sample test differentially identifies TAstV-1, TAstV-2, ANV, and avian rotavirus. The test for chicken-origin samples differentially identifies CAstV, ANV, and avian rotavirus. Assay sensitivity varied by target sequence between approximately 10 copies for avian rotavirus alone and approximately 2 x 10(6) copies for TAstV-2 in the presence of a heterologous competitor RNA sequence. Each test was shown to be specific for the intended target by testing for cross-reaction with other common avian enteric viruses. The specificity was further shown by testing 109 chicken specimens and 32 turkey specimens from commercial flocks with the appropriate test and sequencing the RT-PCR amplicons to confirm amplification of the correct target.

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.