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Clinical characteristics of Pneumocystis pneumonia in non-HIV patients and prognostic factors including microbiological genotypes
Clinical characteristics of Pneumocystis pneumonia in non-HIV patients and prognostic factors including microbiological genotypes
Matsumura, Yasufumi
Shindo, Yuichiro
Iinuma, Yoshitsugu
Yamamoto, Masaki
Shirano, Michinori
Matsushima, Aki
Nagao, Miki
Ito, Yutaka
Takakura, Shunji
Hasegawa, Yoshjnori
Ichiyama, Satoshi
info:doi/10.1186/1471-2334-11-76
BMC Infectious Diseases 2011, 11:76
2011-03-25
BMC Infectious Diseases
2011-03-25
11
1
Research article
76

–>Research article

Yasufumi Matsumura , Yuichiro Shindo , Yoshitsugu Iinuma , Masaki Yamamoto , Michinori Shirano , Aki Matsushima , Miki Nagao , Yutaka Ito , Shunji Takakura , Yoshjnori Hasegawa and Satoshi Ichiyama

BMC Infectious Diseases 2011,
11:76doi:10.1186/1471-2334-11-76

Abstract (provisional)

EID Journal Home Volume 17, Number 4–April 2011

Volume 17, Number 4–April 2011

Dispatch

Polly W.Y. Mak, Chloe K.S. Wong, Olive T.W. Li, Kwok Hung
Chan, Chung Lam Cheung, Edward S. Ma, Richard J. Webby, Yi Guan, Joseph S.
Malik Peiris, and Leo L.M. Poon
Author affiliations: The University of Hong Kong, Hong Kong
Special Administrative Region, People’s Republic of China (P.W.Y. Mak, C.K.S.
Wong, O.T.W. Li, K.H. Chan, C.L. Cheung, E.S. Ma, Y. Guan, J.S.M. Peiris,
L.L.M. Poon); St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
(R.J. Webby); and Hong Kong University–Pasteur Research Centre, Hong Kong (J.S.M. Peiris)

Suggested citation for this article

Abstract
The emergence of pandemic (H1N1) 2009 virus highlighted the
need for enhanced surveillance of swine influenza viruses. We used real-time
reverse–transcription PCR–based genotyping and found that this rapid and simple
genotyping method may identify reassortants derived from viruses of Eurasian
avian-like, triple reassortant-like, and pandemic (H1N1) 2009 virus lineages.

Co-infection of influenza A viruses enables viral gene reassortments,
thereby generating progeny viruses with novel genotypes. Such reassortants may
pose a serious public health threat, as exemplified by the emergence of
pandemic influenza (H1N1) in 2009 (1). Transmission of pandemic (H1N1)
2009 virus from humans to pigs has been reported (2–5). We recently
identified a reassortment between pandemic (H1N1) 2009 virus and swine
influenza viruses in pigs (6). These results emphasize the potential
role of pigs as a mixing vessel for influenza viruses and the need for screening
tests that can identify major reassortment events in pigs.

We previously developed 8 monoplex SYBR green–based
quantitative reverse transcription–PCRs to detect all 8 gene segments derived
from the pandemic (H1N1) 2009 virus or virus segments that are closely related
to this lineage (i.e., neuraminidase [NA] and matrix protein from the Eurasian
avian-like swine linage and polymerase basic protein [PB] 2, PB1, polymerase
acidic protein [PA], hemagglutinin [HA], nucleocapsid protein [NP], and nonstructural
protein [NS]) from triple reassortant swine linage (5). Using these
PCRs, we identified swine viruses of atypical genotypes. However, with the
exception of the HA-specific assay, the melting-curve signals of pandemic
(H1N1) 2009 virus may be indistinguishable from the positive signals generated
from its sister clade as indicated above. To differentiate between these
closely related groups of viruses, we further optimized these assays by adding
sequence-specific hydrolysis probes in the SYBR green assays.

The Study

For this study, all SYBR green assays were modified from the
previously described assays (5), with the exception of the reverse
primers for the newly designed PB1 and NS segments (Table 1). The subtype H1N1
swine influenza viruses isolated in Hong Kong during the past few years were
mainly derived from the Eurasian avian-like swine lineage (6,7). To
generate more precise genotyping data for our ongoing surveillance, the NA
segment–specific assay was specifically designed to react with the pandemic
(H1N1) 2009 virus and a portion of Eurasian avian-like swine viruses that are
circulating in southeastern China (Technical Appendix Figure 1 [ 563 KB, 11 pages]).
To avoid overlapping the emission spectrum of SYBR green, we labeled all
pandemic (H1N1) 2009 virus–specific hydrolysis probes (Integrated DNA
Technologies, Inc., Coralville, IA, USA) with cyanine 5 (Cy5) and Black Hole
Quencher-2 dyes at their 5′ and 3′ ends, respectively (Table 1). To
enable use of short oligonucleotide sequences without compromising the
annealing temperature of these probes, we modified the probes with locked
nucleic acids (7). RNA extraction and complimentary DNA synthesis were
identical to the protocols described (5,8). One microliter of 10-fold
diluted complimentary DNA sample was amplified in a 20-μL reaction containing
10 μL of Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA)
and the corresponding primer probe set (0.5 μmol/L each). All reactions were
optimized and performed simultaneously in a 7500 Sequence Detection System
(Applied Biosystems) with the following conditions: 20 s at 95°C, followed by
30 cycles of 95°C for 3 s and 62°C for 30 s. SYBR green and Cy5 signals from
the same reaction were captured simultaneously at the end of each amplification
cycle. The expected PCR results of virus segment derived from different swine
viral lineages are shown in Table 2.

The dissociation kinetics of PCR amplicons were studied by a
melting curve analysis at the end of the PCR (60°C–95°C; temperature increment
0.1°C/s). We also tested various probe and SYBR green concentrations under
different PCR conditions. The condition described above gave the most robust and
consistent DNA amplification (data not shown). We tested 31 human pandemic
(H1N1) 2009 and 63 human seasonal influenza viruses (33 subtype H1N1, 30
subtype H3N2) as controls. As expected, all human pandemic influenza viruses
were double positive (i.e., positive with SYBR green and Cy5) and all seasonal
influenza samples were double negative in all 8 assays.

To evaluate the sensitivity of the assays, we tested serial
diluted plasmid DNA of the corresponding segments of influenza A/California/4/2009
virus as a standard. The fluorescent signals generated from the SYBR green
reporter dye in all assays were highly similar to those previously reported (5),
and the modified assays had a linear dynamic detection range from 10² to 10⁸ copies/reaction (Technical Appendix Figure 2 [ 563 KB, 11 pages]). As
expected, the threshold cycle values deduced from the Cy5 reporter signal were
generally higher than those from the SYBR green reporter (Technical Appendix Figure 2 [ 563 KB, 11 pages]) (9). This finding can be partly explained by the
nature of these 2 kinds of real-time PCR chemistries: a single Cy5 fluorophore
of the hydrolysis probe was released from quenching for each amplicon
synthesized while multiple SYBR green dyes bound to a single amplicon (10).
After 35 PCR amplification cycles, the linear dynamic detection range of Cy5
signals generated from these reactions was 10² to 10⁸ copies/reaction (data not shown). However, to avoid nonspecific SYBR green
signals, we purposely limited the number of amplification cycles to 30.

Using these assays, we tested 41 swine virus isolates
collected during January 2009–January 2010. In all 8 reactions, 10 pandemic
(H1N1) 2009 virus samples transmitted from humans to pigs (6) were
double positive (Technical Appendix Figure 1, pink [ 563 KB, 11 pages]). In these assays,
gene segments of another 31 swine isolates were either SYBR green positive/Cy5
negative (Technical Appendix Figure 1, yellow [ 563 KB, 11 pages]) or double negative (Technical Appendix, green [ 563 KB, 11 pages]), indicating that these virus segments were
derived from the sister clade of pandemic (H1N1) 2009 virus or other swine
lineages (except NA), respectively. For example, the reassortant of pandemic
(H1N1) 2009 virus (A/swine/Hong Kong/201/2010 [H1N1]) was double positive for
NA, double negative for HA and matrix protein, and SYBR green positive/Cy5
negative for PB2, PB1, PA, NP, and NS (Figure; other data not shown). All
genotyping results of the studied viruses were consistent with results of previous
phylogenetic analyses (5,6), indicating that our modified probes and
SYBR green assays can provide more accurate genotyping results. With these
genotyping data, viruses with atypical positive signal patterns might suggest a
novel viral reassortment event and can be highlighted for investigation with
sequencing-based methods.

To demonstrate the potential use of these assays in studying
swine viruses circulating in other geographic locations, we tested 7 recent
swine isolates (1 pandemic influenza subtype H1N1, 4 subtype H1N2, and 2
subtype H3N2) collected in the United States. Genotyping results agreed 100%
with data deduced from sequence analyses (Technical Appendix Table 1 [ 563 KB, 11 pages]).
We also analyzed all 436 contemporary (2008–2010) US swine virus segments
available from the National Center for Biotechnology Information influenza
virus sequence database. On the basis of the in silico analysis of sequences
targeted by our primers and probes, 95% of the sequences (n = 413) are
predicted to yield the expected results (Technical Appendix Table 2 [ 563 KB, 11 pages]).

Conclusions

The emergence of pandemic (H1N1) 2009 has highlighted the
need for global systematic influenza surveillance in swine. Our results
demonstrated that the addition of locked nucleic acid hydrolysis probes specific
for pandemic (H1N1) 2009 virus into previously established SYBR green assays
can help differentiate segments of pandemic (H1N1) 2009, Eurasian avian-like,
and triple reassortant virus lineages. These assays might provide a rapid and
simple genotyping method for identifying viruses that need to be fully genetically
sequenced and characterized. They may also help provide better understanding of
the viral reassortment events and viral dynamics in pigs. Although at present,
genes derived from human seasonal viruses cannot be characterized with our
modified assays, the performance of our assays warrants similar investigations
for genotyping human influenza viruses.

Acknowledgment

This study was supported by the Area of Excellence Scheme of
the University Grants Committee Hong Kong (grant AoE/M-12/06), the Research
Grant Council of Hong Kong (HKU 773408M to L.L.M.P.), the Seed Funding for
Basic Research (Hong Kong University), Research Fund for the Control of
Infectious Disease Commissioned Project Food and Health Bureau (Hong Kong), and
the National Institutes of Health (National Institute of Allergy and Infectious
Diseases contract HHSN266200700005C).

Ms Mak is a postgraduate student in the Department of
Microbiology, The University of Hong Kong. Her research focuses on molecular
diagnosis of influenza virus.

References

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Figure

Figure. Genotyping of A) polymerase acidic protein, B)
hemagglutinin, and C) neuraminidase segments of A/swine/Hong Kong/201/2010 influenza
(H1N1) virus…

Tables

Table 1. Primer–probe sets selective for pandemic (H1N1) 2009 virus gene segments
Table 2. Summary of expected genotyping results of swine and human influenza viruses

Suggested Citation for this Article

Mak PWY, Wong CKS, Li OTW, Chan
KH, Cheung CL, Ma ES, et al. Rapid genotyping of swine influenza viruses. Emerg
Infect Dis [serial on the Internet]. 2011 Apr [date cited].

http://www.cdc.gov/EID/content/17/4/691.htm

DOI: 10.3201/eid1704.101726

Please use the form below to submit correspondence to the authors or contact them at the following address:

Leo L.M. Poon, Department of
Microbiology, University Pathology Building, Queen Mary Hospital, Pokfulam,
Hong Kong Special Administrative Region, People’s Republic of China; email: llmpoon@hkucc.hku.hk

Contact us at eideditor@cdc.gov

The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.

This page posted March 25, 2011

How to Cite

Huang, L.-W., Lin, Y.-H., Pan, H.-S., Seow, K.-M. and Lin, C.-Y. , Human papillomavirus genotyping as a predictor of high‐grade cervical dysplasia in women with mildly cytologic abnormalities: A two‐year follow‐up report. Diagnostic Cytopathology, n/a. doi: 10.1002/dc.21591

By a GenomeWeb Staff Reporter

NEW YORK (GenomeWeb News) – Lumigenix has launched what it said is the first personal genomics testing service provided directly to consumers in Australia.

The private California firm, which is owned by an Australian parent, has launched a saliva sample testing kit that provides information about predisposition to common diseases, traits such as caffeine metabolism and taste perception, and ancestry information based on haplogroups.

According to the company’s website, the introductory test kit is currently being marketed at a US$99 sale price that is marked down from a regular price of $279.

The Lumigenix genotyping services are conducted through partnerships with Illumina and the Australian Genome Research Facility, and its service includes a risk report based on peer-reviewed content from Mayo Clinic.

The company said that its tests are for 76 risks for common diseases that can be caused by gene-environment interaction, and that it is intended to provide consumers with information that can motivate them to make changes to minimize their risks or increase health.

Lumigenix also said that it will not provide results for carrier status for diseases, such as BRCA mutation status for breast cancer, but it plans to launch in the future a separate product for such tests that will incorporate physician and geneticist review.

A company official acknowledged in an e-mail to GenomeWeb Daily News that the firm is launching in an unclear regulatory environment, as the direct-to-consumer genomics field has come under scrutiny from the US Food and Drug Administration and may become subject to regulation by that agency. The official added that Lumigenix believes it is currently operating in accordance with Australia’s laws.

The company said during its launch that it believes that developing standards for the industry, instead of banning DTC tests, would be the best regulatory approach for governments to pursue.

“We believe people are both capable of understanding, and should have access to, their own genetic information,” Lumigenix CEO Romain Bonjean said in a statement announcing the launch. “We consider our responsible approach to be one that the Australian health and medical community will start to embrace, as we are not trying to replace their expertise, but to arm interested people with additional information.”

“In the near future we also hope to develop relationships with research bodies to allow our customers to anonymously provide their data to advance Australian genetic research,” he added. “Therefore, we consider a one-size-fits-all regulatory approach ineffective for such a complex industry.”