Luke T. Daum

Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes

ABSTRACT A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 103 to 104 gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.

Real-Time PCR Detection of Salmonella in Suspect Foods from a Gastroenteritis Outbreak in Kerr County, Texas

ABSTRACT In June 2001, an outbreak of acute gastroenteritis among 109 attendees of a church picnic in Kerr County, Texas, was reported. A 5′-nuclease PCR assay was used to screen for Salmonella in nine food items from the buffet line. Barbeque chicken B tested positive for Salmonella, and no amplification was detected in the remaining food items. These PCR findings were consistent with culture results and were confirmed by direct nucleotide sequencing. Salmonella enterica serotype Panama was cultured from both food and patient stool samples.

Next-Generation Ion Torrent Sequencing of Drug Resistance Mutations in Mycobacterium tuberculosis Strains

ABSTRACT A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes—rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)—were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.

Real-time RT-PCR assays for type and subtype detection of influenza A and B viruses

Influenza viruses type A (H3N2 and H1N1 subtypes) and B are the most prevalently circulating human influenza viruses. However, an increase in several confirmed cases of high pathogenic H5N1 in humans has raised concerns of a potential pandemic underscoring the need for rapid, point of contact detection. In this report, we describe development and evaluation of ‘type,’ i.e., influenza virus A and B, and ‘subtype,’ i.e., H1, H3, and H5, specific, single‐step/reaction vessel format, real‐time RT‐PCR assays using total RNA from archived reference strains, shell‐vial cultured and uncultured primary (throat swab/nasal wash) clinical samples. The type A and B specific assays detected all 16 influenza type A viruses and both currently circulating influenza B lineages (Yamagata and Victoria), respectively. ‘Type’ and ‘subtype’ specific assays utilize one common set of thermocycling conditions, are specific and highly sensitive (detection threshold of approximately 100 target template molecules). All clinical specimens and samples were evaluated using both the unconventional portable Ruggedized Advanced Pathogen Identification Device (RAPID) and standard laboratory bench LightCycler instruments. These potentially field‐deployable assays could offer significant utility for rapid, point of care screening needs arising from a pandemic influenza outbreak.

A rapid, single-step multiplex reverse transcription-PCR assay for the detection of human H1N1, H3N2, and B influenza viruses.

BACKGROUND Influenza is a viral respiratory pathogen responsible for frequent seasonal epidemics. There are currently three major human influenza viruses in global circulation, H1N1, H3N2 and B. OBJECTIVES A one-step multiplex reverse transcription (RT)-polymerase chain reaction (PCR) assay targeting the HA1 segment of the human hemagglutinin gene was developed as a rapid surveillance method. STUDY DESIGN A researcher-blind study was performed using 112 randomly selected, culture-positive clinical samples collected through the Department of Defense (Global Emerging Infectious Surveillance (DOD-GEIS) influenza network during the 2000-2001 influenza season. Three subtype specific primer sets capable of producing PCR products with base-pair lengths of 585, 402 and 290 corresponding to influenza H1, H3, and B subtypes, respectively, were utilized together in a one step, one tube, reaction. RESULTS Multiplex primers were able to simultaneously type, and subtype 100% (112/112) of positive cultures. CONCLUSIONS The results confirm that this assay is a highly sensitive and timely surveillance tool for rapid detection and simultaneous subtyping of clinical influenza specimens isolated worldwide.

Surveillance of transcriptomes in basic military trainees with normal, febrile respiratory illness, and convalescent phenotypes

Gene expression profiles permit analysis of host immune response at the transcriptome level. We used the Pax gene Blood RNA (PAX) System and Affymetrix microarrays (HG-U133A&B) to survey profiles in basic military trainees and to classify them as healthy, febrile respiratory illness (FRI) without adenovirus, FRI with adenovirus, and convalescent from FRI with adenovirus. We assessed quality metrics of RNA processing for microarrays. Class prediction analysis discovered nested sets of transcripts that could categorize the phenotypes with optimized accuracy of 99% (nonfebrile vs febrile, P<0.0005), 87% (healthy vs convalescent, P=0.001), and 91% (febrile without vs with adenovirus, P<0.0005). The discovered set for classification of nonfebrile vs febrile patients consisted of 40 transcripts with functions related to interferon induced genes, complement cascades, and TNF and IL1 signaling. The set of seven transcripts for distinguishing healthy vs convalescent individuals included those associated with ribosomal structure, humoral immunity, and cell adhesion. The set of 10 transcripts for distinguishing FRI without vs with adenovirus had functions related to interferon induced genes, IL1 receptor accessory protein, and cell interactions. These results are the first in vivo demonstration of classification of infectious diseases via host signature transcripts and move us towards using the transcriptome in biosurveillance.

Genetic and Antigenic Analysis of the First A/New Caledonia/20/99-like H1N1 Influenza Isolates Reported in the Americas

From February through May of 1999, 13 cases of Influenza A virus (FLUAV), type H1N1 were reported at a Department of Defense influenza surveillance sentinel site in Lima, Peru. Genetic and antigenic analysis by hemagglutination inhibition and direct nucleotide sequencing of the HA1 region of the hemagglutinin gene were performed on two isolates, A/Peru/1641/99 and A/Peru/1798/99. Both isolates were distinct from the Bayern/7/95-like viruses circulating in the Americas and closely related to a Beijing/262/95-like variant, A/New Caledonia/20/99. With the exception of travel-related cases, the detection of these isolates represents the first appearance of New Caledonia/20/99-like viruses in the Americas. Since the characterization of these Peru isolates, a number of New Caledonia/20/99-like viruses have been reported worldwide. For the 2000/01 and 2001/02 influenza seasons, the World Health Organization (WHO) has recommended the inclusion of A/New Caledonia/20/99 as the H1N1 vaccine component for both the southern and northern hemispheres.

Influenza A (H3N2) Outbreak, Nepal

Worldwide emergence of variant viruses has prompted a change in the 2005–2006 H3N2 influenza A vaccine strain.

Performance of five FDA-approved rapid antigen tests in the detection of 2009 H1N1 influenza A virus.

Rapid antigen tests are commonly used by clinicians for rapid, simple, point‐of‐care testing. Five rapid antigen tests were shown to have low sensitivity (40.3–58.8%) when compared to real‐time RT‐PCR using nasal wash specimens from patients with influenza‐like‐illness (N = 167) that were collected previously and confirmed as 2009 pandemic influenza A (H1N1)‐positive by PCR. Rapid antigen test sensitivity correlated with virus levels in nasal secretions when comparisons were made to cycle threshold (CT) values obtained from real‐time RT‐PCR. When CT values are <25 (equating to viral concentrations of >104 TCID50/ml) sensitivity for all five rapid antigen kits was high (range: 83–94% positive); however, when CT values are >30 (102 TCID50/ml), sensitivities of only 16–18% were observed for four of five rapid antigen kits. The Directigen EZ Flu A + B test detected more positive samples (35%) at lower viral concentrations with CT values >30 when compared with other commercial kits (P = 0.05). Rapid antigen test results must be interpreted with caution, and negative specimens may need confirmation by sensitive molecular assays. J. Med. Virol. 84:1699–1702, 2012.

Molecular analysis of isolates from influenza B outbreaks in the U.S. and Nepal, 2005

Summary.Currently circulating influenza B viruses can be divided into two antigenically and genetically distinct lineages referred to by their respective prototype strains, B/Yamagata/16/88 and B/Victoria/2/87, based on amino acid differences in the hemagglutinin surface glycoprotein. During May and July 2005, clinical specimens from two early season influenza B outbreaks in Arizona and southeastern Nepal were subjected to antigenic (hemagglutinin inhibition) and nucleotide sequence analysis of hemagglutinin (HA1), neuraminidase (NA), and NB genes. All isolates exhibited little reactivity with the B/Shanghai/361/2002 (B/Yamagata-like) vaccine strain and significantly reduced reactivity with the previous 2003/04 B/Hong Kong/330/2001 (B/Victoria-like) vaccine strain. The majority of isolates were antigenically similar to B/Hawaii/33/2004, a B/Victoria-like reference strain. Sequence analysis indicated that 33 of 34 isolates contained B/Victoria-like HA and B/Yamagata-like NA and NB proteins. Thus, these outbreak isolates are both antigenically and genetically distinct from the current Northern Hemisphere vaccine virus strain as well as the previous 2003–04 B/Hong Kong/330/2001 (B/Victoria lineage) vaccine virus strain but are genetically similar to B/Malaysia/2506/2004, the vaccine strain proposed for the coming seasons in the Northern and Southern Hemispheres. Since these influenza B outbreaks occurred in two very distant geographical locations, these viruses may continue to circulate during the 2006 season, underscoring the importance of rapid molecular monitoring of HA, NA and NB for drift and reassortment.