Report of RV Laboratory technical working group meeting, 11-12 Nov 2013
Meeting Report of the laboratory Technical Working Group of the WHO
Rotavirus Surveillance Network
Bangkok, 11-12 November 2013
EXECUTIVE SUMMARY
Background
Following recommendations made at the Global Surveillance Meeting, Geneva 2011, WHO
established a laboratory technical working group (TWG) to assist with technical guidance needed
to strengthen laboratory capacities within the Global Rotavirus Network, improve the quality of
diagnostic and genotyping data, standardize lab methods/training modules and improve the
Quality Assurance (QA)/Quality Control (QC) systems.
Members of the TWG include WHO Global and Regional Laboratory coordinators and selected
technical experts in the field of rotavirus surveillance from the WHO Rotavirus Surveillance
Network. The previous in-person meeting was held in Bangkok in September 2012, which
reviewed progress, discussed issues with data quality, approaches to genotyping and to
decreasing the proportion of untypable samples and to improving the quality assurance program.
In 2013, the group met via conference call in June and was informed of the ongoing strategic
review being conducted for both rotavirus and the invasive bacterial diseases networks in all
WHO regions with a planned presentation to SAGE in November 2013 and to plan
standardization of activities across all regions. Minutes from the meeting were circulated and a
face-to-face meeting was planned. This meeting was held November 11-12, 2013 in Bangkok.
Meeting objectives
 Update on Strategic Review
 Global and regional updates
 Standardization of strain selection for genotyping
 Finalisation of guidelines on external quality control for confirmatory testing
 Participation in a Bill and Melinda Gates Foundation program for additional pathogens
detection.
Background documents in participant file
Draft documents for
 Guidelines for sending RV samples from Regional Reference Labs (RRLs) to the
Global Reference Lab (GRL) for confirmatory testing
 Guidelines for Rotavirus strain characterization for the WHO Rotavirus
Surveillance Network
Report of RV Laboratory technical working group meeting, 11-12 Nov 2013

Laboratory variables for standard reporting of genotype data
Meeting structure
1. Update on the Strategic Review
The rotavirus and invasive bacterial disease sentinel hospital surveillance networks transitioned
to WHO in 2008 and completed 5 years of activities in 2013. In the context of competing health
priorities and scarce human and financial resources, the need to evaluate performance and
identify strengths and weaknesses to develop a strategic approach for future work was
recognized. The strategic review was launched in February 2013, in partnership with the
informal Technical Advisory Group (iTAG) for New and Under-utilized Vaccine Initiative with
monthly calls between the WHO HQ, regional offices and the iTAG, was discussed in detail at a
meeting in Geneva in September 2013 and presented to SAGE in November 2013. The primary
assessment was whether the original 2008 objectives were met. These were as described below:
Pre-vaccine introduction period
 Document presence of disease, describe disease epidemiology, provide data for
estimating disease burden
 Establish system to measure impact after vaccine introduction
 Identify circulating serotypes & measure serotype distribution
Post-vaccine introduction period
 Assess disease trends over time
 Monitor vaccination program impact
 Monitor changes in circulating strains/serotypes
 Platform for effectiveness and safety evaluation
Overall, the rotavirus network met the 2008 objectives for presence of disease, contributed to
estimation of disease burden and monitoring disease trends over time and assessment of
genotype distribution. In some regions, the surveillance networks were used as platforms to
monitor impact and safety. Countries valued and were using the surveillance platform and data.
Countries in regions that have introduced vaccine have been able to use the surveillance network
for special studies to demonstrate impact.
However, there were some lack of cohesiveness in the separate regional networks, limited ability
to use data for real time monitoring, lack of case level data in standard format and difficulty
linking laboratory results with the clinical and epidemiologic data on the same patient.
Key recommendations from the strategic review included:
• Report surveillance data, including genotype data, twice yearly (where possible)
• Report case-based data to WHO HQ, and link genotype data to cases
• As feasible, build additional technical capacity at the national laboratory (NL) level
• Review standard definition of a sentinel site to ensure that sites meet those criteria and
thus can meet surveillance objectives in the post-vaccine introduction era
• Initiate zero reporting if it does not already exist
• 12 months reporting and >100 specimens at a single site (no satellite sites)
• Develop strategies to improve performances at all sites (GAVI and non-GAVI)
• Standardize sample selection for genotyping
• Examine country-level genotype distribution, in addition to distribution at regional and
global levels
Report of RV Laboratory technical working group meeting, 11-12 Nov 2013
•
•
•
•
Examine current performance indicators for usefulness
Improve data management and quality
Modify future Global Bulletins to show all reporting countries on map, but limit analysis
to a subset of reporting sites
Network across all regions for lessons learned.
2. Plans for testing of additional enteric pathogens
Duncan Steele from BMGF followed up on the previous year’s discussion on additional
testing for enteric pathogens to describe the options for multiple testing including multiplex
PCRs, Luminex systems and the Taqman low density array cards (TAC). He stated that Dr.
Eric Houpt’s laboratory at the University of Virginia had evaluated the TAC cards with
specimens from several of the Global Enteric Multicenter Study (GEMS) and other sites and
believed that these were the best option in terms of ease of use and numbers of targets tested,
but were expensive and required several pieces of equipment which might not be readily
available in all regional reference laboratories. BMGF has awarded funds to the CDC
Foundation for the collaborative study between Dr. Houpt's lab, WHO and participating
Global and Regional Reference (GRL and RRL) Labs using specimens collected by sentinel
sites of the surveillance network. The Grant has funds to purchase the needed equipment for
2 Reference labs and there is interest in supporting one laboratory each in Asia and Africa to
validate the findings from the GEMS study. The GEMS study which was published in May
2013, demonstrated that while rotavirus was the primary pathogen in moderate to severe
diarrhea in infancy, the contributions of other pathogens not previously considered as
significant causes of diarrheal disease burden, needed to be evaluated. This will be taken
forward in discussion with WHO.
3. Overview and update of the Global Rotavirus Surveillance Network
Nine regional reference laboratories and one global reference laboratory support the WHO
RV laboratory network. Earlier data from the network was published during 2013 in the
Weekly Epidemiologic Record (http://www.who.int/wer/2013/wer8821/en/index.html and
MMWR (24 May 2013, Volume 62, N°20). The 2012 Rotavirus data collected via the
network was presented by each of the regions. The findings of the strategic review and the
implications for the regional networks were discussed.
4.
Comparison of Bioline and Qiagen One Step RT-PCR kits
Based on 2012 TWG meeting recommendations, WHO had procured kits for comparison of
the less expensive Bioline kits with the Qiagen one step RT-PCR. Data presented by the
network laboratories, while limited, indicated that the kits gave comparable results. Results
are still preliminary and additional analysis is needed to make a decision about kit use
recommendations.
In addition some EIA kit comparisons were presented and contrasting results were shown
between the labs on rates of false-positives associated with the RIDASCREEN and VIKIA
while Prospect and Rotaclone seem to have similar performance. The group has agreed that
all results from the different analysis done should be shared with WHO for comparison to
guide decisions on the kits use. WHO will collect the different studies and present them in
order to have a better interpretation and conclusions.
Report of RV Laboratory technical working group meeting, 11-12 Nov 2013
5. Selection of samples for genotyping
It was recognized that genotyping data being generated at the RRL/National labs is
considered by MOH and partners to be useful, there is a need to rationalize the number of
samples selected for genotyping. The draft document on selection of samples for genotyping
was discussed. The number 50 samples per country was initially proposed in the draft
document which would be able to detect a 30% change in a genotype, but there was no
clarity on the precision with which this number of genotyped samples would be able to track
strain distribution over time. It was suggested that an estimate be developed based on strain
prevalence at a minimum defined percentage and evaluated on real data. Additional analysis
will be done to come up with estimate for number of samples to be genotyped by
country/site. Selection criteria proposed in the draft guidelines were agreeable to all members
of the group and include:
 Random selection of rotavirus antigen positive stool specimens; random selection will
ensure that factors such as age, disease severity, seasonality, among others, do not bias
the strain characterization results
 Only specimens with adequate volume for several tests (more than 1 ml) should be
chosen in order to avoid running out of material before testing is complete
 Consult with the Reference Laboratories to determine the maximum number of
specimens that the Reference Laboratory is willing to accept on an annual basis.
6. External quality assessment
Thermostable stool matrix panels were shipped for the first time by the GRL in 2013 for the
EQA program. The shipments were made from May 2013 onwards and laboratories were
expected to report within 20 business days of receipt. All regional laboratories met the 80%
criteria, but there was a discussion on whether the GRL’s assigned genotype should be used
as the expected result and it was decided that this should be done in consultation with the
referee laboratories. Improving the timing of feedback with the expected results in addition to
the currently provided percentage was also discussed as this would allow labs to investigate
reason for potential typing issues identified and take remedial action in a timely way.
There was a brief description of the plans to create a non-infectious panel to further increase
the ease of use of the EQA panels.
Some WHO Regional Offices and RRLs in AFRO proposed GRL explore possibility of
transferring technology of PT preparation to RRL and support RRL to take over and manage
this program at the RRL in the future.
7. Confirmatory testing of rotavirus as part of implementing QC programme
The draft document on confirmatory testing was discussed. As was decided during the
Technical Working Group discussion in June 2013, for the first year alone (2014) the GRL
would receive selected samples from the regional laboratories for confirmatory testing. By
end of 2014, the QC results will be reviewed and shared with the TWG to make
recommendations for the way forward. The aim is that in 2015, this function will be served
by the RRLs for the region or willing RRLs would serve as the confirmatory testing
laboratory for the network in rotation with support from GRL. It was recognized that this
exercise might face some challenges due to the differences in strain prevalence and the use of
Report of RV Laboratory technical working group meeting, 11-12 Nov 2013
different primers sets in the various laboratories. However, this will be a learning and helpful
exercise for all labs.
Meeting Recommendations
I- Data Quality
 All RRL to use standardized core laboratory lab variables agreed upon during the
meeting to report genotype data to WHO and countries
 Increase genotype reporting to twice a year which may involve organizing shipping of
samples from countries to RRL twice a year in some regions; it was discussed that this
might be challenging in the AFR context as genotyping is done during annual
workshops at the RRL. In this Region, RO colleagues would study alternatives to
implement a system of samples referral to RRL while keeping the workshops as
opportunities of capacity building
 Previous calendar year data, testing, analysis and reporting at all levels to WHO HQ
to be completed by end of July
 Link the clinical and lab data by using a unique ID in all levels from sentinel site
hospital to the regional reference laboratory; RRL genotyping should be only done on
samples that have a unique ID number.
 Standardise sample selection for genotyping at RRL
o Goal is not to burden RRL
o Number to be defined as quickly as possible; timeline proposed March 2014
o Selection method agreed on - random selection as proposed in the draft guidelines
document
o Number of samples limited by available resources and the differences in number
of countries/RRL.
II_ Use of kits for RT-PCR and EIA
 Complete the comparative analysis between Bioline and Qiagen one step RT-PCR
and share results with the group for discussions and recommendations
 All RRLs who have data comparing RIDASCREEN, ProspecT, VIKIA and
Rotaclone EIA kits to share their results with WHO and the group to discuss
discrepancies and make recommendations on EIA kit use.
III- QA/QC system strengthening
 EQA:
o Global programme to continue as the programme has been valuable for all
participating labs
o PT panels to be validated at the referee labs before distribution at big scale
o GRL to continue testing the development of non-infectious panels to decrease
logistical burden related to shipments
o Regional level shipment to be discussed with regional offices for 2014 survey to
allow all labs to participate at the same time to the survey and avoid delays in
global results
 QC for confirmatory testing between RRL and GRL to be tested in 2014 according to
guidelines agreed upon during the meeting:
Report of RV Laboratory technical working group meeting, 11-12 Nov 2013
o This exercise to be transferred to regional labs in the future
o In year 1, the RRL/RO will generate a random set of 50 samples to be tested
o Minimal data set (S. No. and date of collection) ) to be provided; WHO to
review genotyping results from both GRL and RRLs and highlight discrepancies
for further discussion to agree on true result
o Volume of sample should be sufficient to retain an aliquot for future testing if
needed
o Random selection within set of adequate samples
o MTA to be obtained (GRL/RRL/NL) and organized through WHO HQ and
Regional offices to link with
TORs and TSAs with GRL/RRLs and
agreement/MTAs with the countries
o Samples will be sent to the GRL, tested and reported within 3 months
o It was recognized that referral of samples from RRL to GRL is also needed for
difficult or untyped samples; additional samples can be added to the confirmatory
testing up to a number decided in discussion with WHO and GRL.
Additional issues will need future discussions to address strategic review recommendations such
as funding timelines, working towards national capacity strengthening and future dissemination
of technical capacity.
Report of RV Laboratory technical working group meeting, 11-12 Nov 2013
Annex 1. Meeting Agenda
Technical Working Group for WHO Rotavirus Surveillance Network
November 11-12, 2013
Pathumwan Princess Hotel
Bangkok, Thailand
9:00-10:00
10:0010:30
10:3011:00
11:0013:00
13:0014:00
14:0015:30
15:3016:00
16:0017:30

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
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Monday 11 November 2013
Chair: Gagandeep Kang
Rapporteur: Miren Iturizza
Welcome and Introductions
Global network overview
Strategic review outcomes
Meeting objectives and expected outcomes
Discussion
BMGF discussion Grant
F. Serhan
M. Agocs
D. Steele
COFFEE BREAK
Global and Regional updates GRL and RRLs
 Global
 AFR
 AMR
 EMR
 EUR
 SEAR
 WPR
J. Gentsch
G. Armah
M. Seheri
G. Rey
El Mohammady
G. Semeiko
G. Kang
C. Kirkwood
J. Hyesook
LUNCH
Improve quality of RV laboratory data
1. Implementing the strategic review recommendations:
 Regional feedbacks and adaptation to regional contexts: role of
RRLs in implementation
 Discussions and agreements
Group discussions
COFFEE BREAK
2. Improving strategy for referral of samples for GT at reference lab:
 Standardization of RV samples selection for GT: refer to guidelines M. Agocs
M. Iturizza
on RV strain characterization draft document
Report of RV Laboratory technical working group meeting, 11-12 Nov 2013
 Continued: Discussions and recommendations
17:30
9:00-10:30
Group discussions
END DAY 1
Tuesday 12 November 2013
Chair: Carl Kirkwood
Rapporteur: Gagandeep Kang
3. Improving data collection system
 Core lab variables (Please refer to core variables list in participant F. Serhan
folder)
Group discussions
 Data flow and timeliness of reporting
Quality Assurance / Quality Control
10:3011:00
11:0013:00
13:0014:00
14:0015:00
15:0015:30
15:30
COFFEE BREAK
1. EQA (30 min) (and preliminary results of one step RT-PCR kits
comparative analysis study at GRL)
M. Bowen
 2013 results
 Feedbacks from group
 Discussion and recommendations
F. Serhan
2. External Quality Control for confirmatory testing:
 Guidelines for referral of samples to RL for confirmatory testing J. Gentsch
Group Discussions
(please refer to guidelines in participant folder)
 Discussion and recommendations
LUNCH
Network expectations, funding, way forward
Summary of recommendations and meeting conclusions
Meeting Closing
Report of RV Laboratory technical working group meeting, 11-12 Nov 2013
Annex 2: WHO Rotavirus Regional and Global Reference Laboratories, 2013
Region
Name of Laboratory
Location
Countries Served
Ghana
Benin, Ghana, Liberia,
Niger, Nigeria, Sierra
Leone, Togo, Gambia,
Senegal, Cote d’Ivoire,
Guinea Bissau
University of Limpopo Medunsa
Campus, WHO Regional Reference
Laboratory for Rotavirus
South Africa
Burkina Faso, Cameroon,
Côte d'Ivoire, Democratic
Republic of the Congo
(the), Ethiopia, Gambia
(the), Kenya, Mauritius,
South Africa, United
Republic of Tanzania
(the), Togo, Uganda,
Zambia, Zimbabwe
Centers for Disease Control and
Prevention (CDC), WHO Regional
Reference Laboratory for Rotavirus
Atlanta, USA
Guatemala, Honduras
Nicaragua, Suriname
National Public Health Institute of Rio
de Janeiro, Acting WHO Regional
Reference Laboratory for Rotavirus*
Rio de Janeiro,
Brazil
Brazil
Cairo, Egypt
Sudan (the), Syrian Arab
Republic (the)
Armenia, Azerbaijan,
Belarus, Georgia,
Kyrgyzstan, Republic of
Moldova, Tajikistan,
Ukraine
Noguchi Institute for Medical Research,
WHO Regional Reference Laboratory
for Rotavirus
African Region
(AFR)
Region of the
Americas
(AMR)
Eastern
Mediterranean
Region
(EMR)
Naval Medical Research Unit No. 3
(NAMRU), WHO Regional Reference
Laboratory for Rotavirus
European
Region
(EUR)
Republican Research and Practical
Center for Epidemiology and
Microbiology, WHO Regional
Reference Laboratory for Rotavirus
Minsk, Belarus
South-East
Asia Region
(SEAR)
Christian Medical College, WHO
Regional Reference Laboratory for
Rotavirus
Vellore, India
Myanmar, Nepal, Sri
Lanka, Indonesia
Report of RV Laboratory technical working group meeting, 11-12 Nov 2013
Western
Pacific
Region
(WPR)
Global
Reference
Laboratory
Murdoch Children's Research Institute
(MCRI),
Royal Children's Hospital, WHO
Regional Reference Laboratory for
Rotavirus
Melbourne,
Australia
Korea Centers for Disease Control and
Prevention (KCDC), WHO Regional
Reference Laboratory for Rotavirus
Chungcheongbukdo, Republic of
Korea
Cambodia, Lao Peolple's
Democratic Republic
(the), Mongolia
Institute for Viral Disease Control and
Prevention, China Centers for Disease
Control and Prevention, Nominated as
WHO Regional Reference Laboratory
for Rotavirus
Beijing, China
China
Centers for Disease Control and
Prevention (CDC)
Atlanta, USA
Worldwide
Fiji, Mongolia, Papua
New Guinea, Philippines