Minutes of PCR Users Working Group
Tuesday 20th March 2012
Parklands Hotel, Perth
Present:
Dave Yirrell (Chair – Dundee) DY
Lesley Dargie (Inverness) LD
Naomi Gadsby (Edinburgh) NG
Rory Gunson (Gartnavel) RG
Kathleen Harvey-Wood (Yorkhill) KHW
Alison Hunt (Aberdeen) AH
Geraldine Kaminski (Dundee) GK
Juliet Kenicer (Edinburgh) JK
Susan McDonagh (Inverness) SM
Ben Parcell (Dundee) BP
Kate Templeton (Edinburgh) KT
Oxford Biosystems Cadama representative: Fiona Alcock
Fiona presented the meeting with information on the Focus/3M Integrated
cycler and the PCR assays available.
Apologies: Fiona Hamilton, Sally Taylor
Minutes of last meeting: Approved
Subjects discussed:
1.
Current Respiratory Season
The current season seems to have been quieter than last year. DY noted that
contingency measures in Tayside had been agreed with management and
were in place but were not required in the end; also that pandemic flu
planning meetings had been re-started. KT noted Edinburgh frequently doing
respiratory screening on children who present to A&E but rapidly discharged,
therefore more community-type screening; clinicians like to have result to
phone out. Therefore, numbers tested are still high.
The predominant flu has been H3. KT estimated about 150 cases so far from
Edinburgh mainly from February onwards; will be sending 100 positives to
Glasgow, including the few pregnant and ITU cases. Gartnavel had similar
numbers or slightly less. Yorkhill sending IF negatives to Gartnavel for
respiratory PCR. RG noted about a third of H3 positives had a 1 to 3 log drop
in Ct with the typing assay, not related to inhibition, unclear why. Edinburgh
and Gartnavel have influenza H1N1v H275Y resistance typing assays in
place.
GK noted Tayside haven’t seen the increase in Mycoplasma pneumoniae
noted in Edinburgh and Glasgow in winter 2011/12, however, testing is by
serology not PCR (see Euro Surveill. 2012;17(10). pii: 20110). Edinburgh
project looking at macrolide resistance in M. pneumoniae by genotyping,
noted 1 resistant so far but clear majority are sensitive.
DY raised the issue of how to pick up drift in (particularly) RNA viral detection
now we have a lack of viral culture, using example of RSV-A variants coming
to light with QCMD panel. However, should caution against over-reaction,
requires assessment of evidence first. Maintaining diversity of PCR assays
between laboratories and ensuring communication of any changes is helpful.
2.
ABI software issue
GK described her experience of ABI software repeatedly not reading FluB
positives correctly. These give a clearly positive trace but the software sees
them as undetected, unless the detector is changed to that of another assay
with the same fluorophore. Therefore, there is a danger of missing positives if
staff just print off the report and do not check individual traces. Problem has
been noted on their other ABI machines and ABI have been contacted. NG
noted that Edinburgh recently had a different glitch (one-off) with a positive
trace that disappeared and re-appeared on the graph, with no line apparent at
the crossing point although a Ct value was generated. Illustrates again the
importance of checking the traces.
3.
Commercial assay evaluations
JK reported her experience evaluating the Beckton Dickenson BDMax for an
in-house Norovirus real-time PCR assay at Edinburgh. The BDMax enables
automated extraction and real-time PCR on the same instrument. JK noted
that it was easy to use, cartridge-based, load-and-walk-away. However less
control in the analysis and less sensitivity than the routine method (easyMAG
extraction and ABI7500 fast PCR). Several specimens from same patient
were inhibitory therefore contributing to the decreased sensitivity overall.
Machine uses whole extracted volume for PCR, therefore nothing left at the
end. Not random access; can do 48 specimens in a day; can feed in 2
batches of 12 at a time. Discussion about usefulness for out of hours testing,
cost, comparison to antigen testing.
RG has evaluated the Focus Diagnostics/ 3M integrated cycler with
FA/FB/RSV assay which offers automated extraction and real-time PCR on
the same instrument. Can do 8 tests at a time, results in one hour. RG found it
was easy to use, very small, fast, however 1 to 2 log less sensitive than
Gartnavel routine assay and problems with extraction from purulent sputum
and pre-processed sputum. Suitable for low through-put use only, also looked
at the 96-well version but on many assays only does PCR not extraction
currently.
DY noted that Tayside have tried a commercial HSV PCR however this
missed several strongly positive specimens due to design based on
sequences in Genbank not clinical isolates.
Edinburgh are evaluating commercial gastroenteritis panel (viruses bacteria
and parasites) assays from different suppliers. Also looking at Alere rapid flu
detection method.
4.
Internal controls
Laboratories are now actively working towards putting in internal controls,
generally in a piece-meal way as assays come up for development /
modification. Discussion as to whether internal control should go into each
well of a multiplex or only one well if all assays are routinely carried out on a
specimen e.g. respiratory screen. RG queried the level of variation in IC Ct
deemed acceptable, working on basis of +/- 3 Ct (1 log) – not 5 as in previous
minutes.
SM reported that Inverness are using a commercial DNA IC and for RNA they
received EAV from the Netherlands and have grown up a stock.
GK noted a partial block failure leading to a 2-month look back exercise would
probably have been spotted earlier if internal controls were present –
particular wells in one area were failing due to problem with block, run
controls not always put in this area.
NG noted PhHV IC not amplified where some bacterial targets positive in inhouse bacterial meningitis PCR; RG suggested use of specialist multiplex
PCR mastermix e.g. Qiagen Quantitect, DY noted that commercial assays will
accept positive sample if IC not amplified.
5.
In vitro diagnostics European announcement
Some discussion of interpretation of the announcement and what it means for
laboratory testing; will also be further discussed e.g. Scottish consultants
group. Issues e.g. CE marking for all tests or primarily those for BBV /
transplant testing; unable to use CE marked test where no equivalent e.g.
dried blood spot testing, emerging viruses; requirement for CE marking of
instruments e.g. current ABI 7500 is for research use only.
6.
Staffing
General picture is one of senior staff retiring and not being replaced, or being
replaced by lower-skilled posts. Some laboratories have a top-heavy age
structure with some senior staff not doing senior roles. Inverness are
undergoing some combining of laboratories and are cross-training staff. Darrel
Ho-Yen has retired and has been replaced by Emma Wilson, other consultant
posts not yet filled. Dundee have been able to replace a couple of posts
recently, through combining of laboratories with bacteriology. Edinburgh
moving to 7 day working, probably from summer 2012. Yorkhill moving to new
space at Southern General from 25th April 2012.
7.
Chlamydia spp. PCR
DY raised the issue of the psittacosis family cluster in Tayside; positive
reports by serology then Edinburgh Chlamydia spp. research assay positive.
Dundee and Inverness doing CFTs for Chlamydia and M. pneumoniae but
may move to molecular in future. Gartnavel have research-based Chlamydia
spp PCR assay. Yorkhill have PCR for C. pneumoniae / B. pertussis /
Aspergillus which they do if other tests negative and clinically indicated.
8.
New developments
Edinburgh: Updating RhV and RSV primers and probes for improved RhV-C
and RSV-A detection. PhHV IC now in EBV and CMV viral load assays and
bacterial meningitis multiplex. Running new H. influenzae target in bacterial
meningitis multiplex to bring into line with reference laboratory SHLMPRL.
Running routine PCRs for T. pallidum (where indicated by GUM) and B.
pertussis (hospitalised children). Plan to introduce Chlamydia spp PCR in few
specific cases e.g. ITU where everything negative; previously screened the
respiratory archive and only found 2 positives, therefore not doing routinely.
Doing typing of AdV in severe cases, seeing AdV3 particularly, AdV14 last
seen in December. 16S pan-bacterial PCR will be sent to GOSH now, not
HPA. Noted trouble with Legionella spp. in easyMAG buffer has resolved as
supplier has found new source. Plan to introduce IL-28B genotyping from
June/July. Looking at ion torrent sequencing for HIV resistance typing.
Dundee: looking to add M. pneumoniae to AdV respiratory PCR assay. MSc
project validated PCR for Pseudomonas aeruginosa in CF patients direct from
culture isolates. Now in place and may add Burkholderia cepacia PCR in
future. Running own in-house GC/CT PCR following easyMAG extraction
where indeterminate on the Cobas assay (less inhibition). Evaluating BKV and
CMV quantification kits. Faecal bacterial targets possibility for the future.
Inverness: Current state of flux due to management change and laboratory
reconfiguration. Likely to be introducing more molecular testing in the future,
either in-house or commercial.
Gartnavel: Looking at IL-28B genotyping, evaluating different assays, may be
done initially by biochemistry. Reporting HCV genotyping as 1a and 1b
subtypes by PCR now. Evaluating automated sequence reading /
interpretation. Looking at ion torrent / 454 sequencing for HIV resistance
typing also. Moving to Abbott for detection of lower level HCV viral loads on PI
treatment, but will increase costs. Developed universal FluA, plus H3 and
resistance typing in single multiplex. RhV respiratory assay is a picornavirus
assay therefore will pick up EV but also RhV-Cs; do EV-specific assay if
clinically relevant.
Yorkhill: Evaluating gastroenteritis bacterial multiplex. Doing norovirus PCR
now also. An MSc project is looking at molecular testing for ESBLs inc. CTXM from blood culture isolates. Assessing new requirements following the
move to the Southern General at the end of April, not solely paediatric
anymore.
Aberdeen: Running own HCV viral load assays (QiaSymphony + in-house
assay) and testing HIV viral loads in parallel with sending them to Gartnavel.
Looking at B. pertussis in-house PCR, also BK virus and Flu A H3.
9.
Miscellaneous
DY noted that with the Olympics in the summer, HPA have been developing
in-house PCR assays for respiratory, gastroenteritis etc.
Discussion around gastroenteritis PCR. Drivers are now infection control,
decreased number and skill of staff, rather than turn-around times and
sensitivity. Is there an evidence base for use in infection control? KT noted
infection control act on basis of symptoms not results. Automated systems
required for extraction e.g. Roche Magnapure, QiaSymphony. However, no
fully walk-away solution, all modular except BDMax, but requirement for high
through-put.
Discussion as to whether the meeting should retain a predominantly viral
focus or should include bacterial molecular testing. Acknowledgement that
most development currently is around bacterial PCR. Two parallel groups
were suggested as an alternative. There would be overlap as most
laboratories are increasingly working in a cross-disciplinary manner for
“molecular”. This issue will be raised at the Scottish Consultants meeting.
Date of Next Meeting: Wednesday 19th September 2012.