??????? ?????? ??????????????????????????? ???? ?? (H1N1)

1
??????? ?????? ??????????????????????????? ????
?? (H1N1) ? ??????????? ??????? ???.
How to Deal with Oseltamivir Drug Resistance
???????????? ?? ??????? ???? ???? ????
???? ?. ?.??.??? ????????? ??????????????????
?????????????????????????? ????????????????
2
??????? ?????? ??????????????????????????? ????
?? (H1N1) ? ??????????? ??????? ???.
How to Deal with Oseltamivir Drug Resistance

• Background current status of oseltamivir
  resistance
• How to select antivirals for treatment of current
  influenza/ILI
• How to minimize problem of drug resistance
• Do we need to stockpile alternative antiviral?
• What do we have in future to combat antiviral
  resistance?

3
Current Antiviral Drugs

• Oseltamivir tablet
• Zanamivir inhaler

For treatment, chemoprophylaxis and stockpile
4
A. Neuraminidase Activity
B. Neuraminidase Inhibitor
Anne Moscona. Neuraminidase Inhibitors for
Influenza 20053531363-73
5
Anne Moscona. Global transmission of
oseltamivir-resistant influenza. NEJM
2009360953-6.
6
His274Tyr resistance mutation H1N1 viruses with
the 20072008 influenza season
H1N1 viruses containing the His274Tyr resistance
mutation became widespread beginning with the
20072008 influenza season
Approximate prevalence of
Northern Hemisphere
His274Tyr resistance - United States
10 - Europe
25 - Norway (exceptional) 70
Southern Hemisphere's predominated 2008
influenza season United States 2009, H1N1
predominates virtually 100
Anne Moscona. Global transmission of
oseltamivir-resistant influenza. NEJM
2009360953-6.
7
(No Transcript)
8
Therapeutic options to cope with this season's
influenza viruses 2009
H1N1, the major circulating influenza strain in
USA, is still sensitive to zanamivir, the
inhaled neuraminidase inhibitor adamantanes, an
older class of compounds. but
resistant to oseltamivir H3N2, less frequently
isolated in USA 2009 than in the past, is
sensitive to zanamivir oseltamivir
but resistant to the adamantanes
Adamantane derivatives amantadine, rimantadine
with inhibition of the M2 channel, a viroporin of
influenza A. All H5N1 human isolates tested were
resistant to amantadine rimantadine
9
Interim recommendations for the selection of
antiviral treatment using laboratory test results
and viral surveillance data, United
States, 2008-09 season
Rapid antigen/ Predominant
virus(es) Preferred
Alternative other laboratory test in
community medication(s)
(combination antiviral Rx) Not done or
negative, H1N1 or unknown
Zanamivir Oseltamivir
Rimantadine but clinical suspicion for
influenza Not done or negative,
H3N2 or B Oseltamivir or Zanamivir
None but clinical suspicion for
influenza Positive A H1N1 or
unknown Zanamivir Oseltamivir
Rimantadine Positive A H3N2 or B
Oseltamivir or Zanamivir None Positive
B Any Oseltamivir or
Zanamivir None
These interim recommendations are available at
http//www2a.cdc.gov/HAN/ArchiveSys/ViewMsgV.asp?A
lertNum00279 Pneumonia and Influenza (PI)
Mortality Surveillance
10
Resistance Rate to Oseltamivir
One study identified resistant isolates in 4
percent of treated children In a recent study of
children treated with oseltamivir in Japan, 9 of
50 (18) treated children harbored viruses with
mutations in the neuraminidase gene that encoded
drug-resistant neuraminidase proteins If this
frequent emergence of resistant mutants is found
to be a general occurrence in children, it is a
serious concern, especially since children are an
important source of the spread of influenza in
the community
11
Resistance to Oseltamivir
Animal study to show stability and
transmissibility
In the ferret model, resistant variants with the
same mutation that is found in some children grew
well in - the index ferret - the
contact animals - the OR-flu viruses were
readily transmitted raising concern that some
oseltamivir-resistant mutant viruses might be
transmissible during an epidemic.
It is not clear whether the spread of
antiviral-resistant viruses was due to a direct
selective advantage of the mutations responsible
for antiviral resistance or a hitchhiker effect
in which drug resistance mutations were carried
along with others that offered an advantageous
immunological niche for a particular influenza
strain.
12
??????????????????????? ??????? ?.?.-??.?.?.?.
2552 ???????????????????????? ???????????????????
???????????? ??????????????????????
WWW.thainihnic.org
???????????????????????????????????????
Oseltamivir Neuraminidase Inhibitor
Susceptibility Analysis ???????????????
Oseltamivir ??????? ??????-?????? 2552
??????????????????????? ?????????????????????
?????????????????? ???????????? n n
A/H1N1 33 33 100
A/H3N2 11 0 0 B 16
0 0
13
Antiviral Resistance During the Influenza Season
2008/2009 in USA
Centre for Disease Prevention and Control
(CDC) 2008-2009 Influenza Season (1st Oct 2008 -
Week 19 ending 16th May 2009)
H274Y mutation a histidine to tyrosine
substitution at amino acid 274
2008-2009 Influenza Season Week 19 ending May 16,
2009. Available at http//www.cdc.gov/flu/weekly/
Accessed May 2009
14
Published at www.nejm.org May 7, 2009
(10.1056/NEJMoa0903810) Emergence of a Novel
Swine-Origin Influenza A (H1N1) Virus in
HumansNovel Swine-Origin Influenza A (H1N1)
Virus Investigation Team
15
??????????????????? oseltamivir
???????????????????????????????? ???? A H1N1 2009
?????????????????????? ???????????????????????????
???????? ???????? gene sequencing (genetic
analysis)
????????????????? ?????????????????????????????
???? A H1N1 2009 ???????????????? ?????????
oseltamivir
16
Oseltamivir-resistant H1N1 2009 Virus A Reality
Denmark June 29, 2009 a swine flu patient treated
with oseltamivir in Denmark showed resistance to
the drug for the first time. Japan July 1, 2009
Japans health ministry reported a case of
resistance in a woman from Osaka who had taken a
10-day course. Studies have shown that
oseltamivir-resistant flu viruses develop in 0.4
to 4 of adults and children treated for
seasonal influenza. The reported cases of
oseltamivir-resistant swine flu emerged
independently.
17
Oseltamivir-resistant H1N1 2009 Virus A Reality
Hong Kong A 16-year-old girl flew from San
Francisco and was intercepted by officials at
Hong Kong International Airport on June 11,
2009. (News released on July 3, 2009) She was
admitted to Queen Mary Hospital for isolation,
tested positive for the new H1N1 flu strain and
opted not to take oseltamivir. She had mild
symptoms and was discharged upon recovery on
June 18, 2009. Oseltamivir-resistant swine flu
was found and marked the first known case of
oseltamivir resistance in a swine flu patient not
treated with the drug.
18
Oseltamivir-resistant H1N1 2009 Virus A Reality
TORONTO - Canada has recorded a case of
oseltamivir-resistant swine flu virus, in a
Quebec man who had been given the drug to prevent
infection. JAPAN - meanwhile, revealed Tuesday
it had found a second such case of oseltamivir
resistance, in a person who has no ties to the
country's earlier reported case. The reported
cases are the fourth and fifth globally since the
new H1N1 virus was discovered in April. Quebec
first case of oseltamivir-resistant swine
flu Japan second case of oseltamivir-resistant
swine flu
19
Oseltamivir-resistant H1N1 2009 Virus A Reality
Key question Will the oseltamivir-resistant
strains become dominant around the world ?. At
this moment, these findings certainly heighten
surveillance for similar resistance and
independent swine Flu The new H1N1 pandemic
virus and a seasonal H1N1 variant are more
likely to develop resistance to oseltamivir than
other common flu strains. About 95 of the
seasonal flu viruses circulating around the
world are oseltamivir-resistant flu strains (WHO
report on March 21, 2009)
Saturday 8, 2009 Ramathibodi Hospital reports
the first oseltamivir-resistant isolate of the
novel Flu A 2009
20
Antiviral Resistance During the Influenza Season
2008/2009 in USA
Centre for Disease Prevention and Control
(CDC) 2008-2009 Influenza Season (1st Oct 2008 -
Week 19 ending 12th Sep 2009)
The adamantanes (amantadine and rimantadine) are
not effective against influenza B viruses. Two
screening tools were used to determine
oseltamivir resistance sequence analysis of
viral genes or a neuraminidase inhibition
assay. Three state public health laboratories
perform antiviral resistance testing and report
their results to CDC. An additional oseltamivir
resistant 2009 influenza A (H1N1) viruses has
been identified by these laboratories, bringing
the total number to 9.
2008-2009 Influenza Season Week 36 ending Sep 12,
2009. Available at http//www.cdc.gov/flu/weekly/
Accessed Sep 2009
21
Flu Seen Heard at 49th ICAAC
Secondary bacterial infection with H1N1 was
infrequent and the cause of death was related to
viral damage with subsequent multi organ system
failure. A viral factor (PB1-F2) that drives an
exuberant inflammatory response with subsequent
respiratory tract damage, and is associated with
a predisposition to bacterial superinfection is
NOT present in pandemic H1N1 perhaps somewhat
reducing the odds of bacterial pneumonia
complicating H1N1. However, other factors are
also in play and bacterial superinfections,
especially S. aureus in children, have been
reported with H1N1. 20 reported cases of H1N1
2009 confirmed oseltamivir resistance all with
the 275Y mutation.
49th ICAAC, San Francisco USA, 11-15 September
2009
22
Oseltamivir Resistance of Novel Influenza A H1N1
2009 in Thailand
?????????????????? ????????????????????????????
influenza ????????????? ?? ?????????? mutation H
275 Y 1 ??? ???????????????????? 50 ???
?????????????????????? ?????????????????????????
????????????????????? 2552 ??????? ?????????
?????????????????? mutation ????????
??????????????????????????????????????????
????????????? ?????????? ??????????? ??? ?.??
????????? ????????????????? influenza A 2009
(H1N1) ??????? H 275 Y ????? 218 ???
??????????????? mutation ????????
????????????????????????????????????????????????
??? IC50 ??????? phenotypic expression
???????????????????
23
Oseltamivir-resistant H1N1 2009 Virus in Thailand
Saturday 8th August 2009 Ramathibodi Hospital
reports the first oseltamivir-resistant isolate
of the novel Flu A H1N1 2009

• Send another sample for confirmation of the
  finding
• Test the susceptibility of other novel Flu A H1N1
  2009 isolates that were obtained from the same
  place where this case acquired the novel Flu A
  H1N1 2009 as well as other isolates
• Strict implement of infection control to curb the
  spreading of this oseltamivir-resistant isolate
• Check if the resistance is a consequence of
  mutation or another re-assortment with a seasonal
  flu As NA gene
• Susceptibility surveillance of other isolates of
  novel Flu A H1N1 2009 with the IC50 phenotypic
  technique in order to detect other mechanisms of
  antiviral resistance

24
Easy Detection of Oseltamivir Resistance by RT-PCR
Detect only H275Y mutant that confers resistance
to oseltamivir
FAM signal 275H
From Yong.P_at_chula.ac.th Subject Fwd H1N1 2009
Drug resistance To peak_y111_at_hotmail.com Date
Sat, 8 Aug 2009 215025 0700
JOE signal 275Y
control
Samples tested
25
Office test for oseltamivir-resistant pandemic
influenza A(H1N1) available
August 28, 2009 By Michele G. Sullivan A
Montreal biosciences company The test uses
genetic sequencing to detect the H275Y mutation
of the neuraminidase gene Oseltamivir- resistant
pandemic influenza A(H1N1). The test results,
available in 2 working days But physicians
shouldnt wait on test results to initiate
treatment, the short turnaround time would give
quick notice on any need to switch drugs due to
resistance.
26
Office test for oseltamivir-resistant pandemic
influenza A(H1N1) available
The prevalence of oseltamivir-resistant mutations
is unclear but we do believe there is a
potential for increased resistance as
oseltamivir is used more frequently. Some
isolated incidents of resistance have been seen
globally, including in Denmark, Japan, Hong
Kong, Canada, USA in California Seattle. All
tested viruses retain their sensitivity to
zanamivir, and is universally resistant to the
adamantane antiviral medications, amantadine and
rimantadine.
27
Office test for oseltamivir-resistant pandemic
influenza A(H1N1) available
Oseltamivir or zanamivir is recommended for all
patients with confirmed, probable, or suspected
cases of 2009 influenza A (H1N1) virus infection
who are hospitalized or who are at higher risk
for seasonal Flu complications The resistance
testing costs 272 performed in conjunction
with an H1N1 Flu virus subtyping, which costs an
additional 180
28
Oseltamivir Rx For H5N1 Flu Control
Oseltamivir needs higher doses longer
duration? The standard recommended dose
incompletely suppresses viral replication in at
least some patients with H5N1 avian influenza,
increasing the risk of viral resistance and
rendering therapy less effective. Accordingly, it
has been suggested that higher doses and longer
durations of therapy should be used for treatment
of patients with the H5N1 virus. Clinical trials
for an increased dosage were set to begin by May
2007. All avian influenza cases in Indonesia,
Thailand, and Vietnam will be inducted into the
trial. The trial will also include 100 cases of
severe seasonal influenza from each of those
countries, plus the United States. Half of cases
will receive the current standard dosage, and
half will receive a double dosage, but for the
standard length of time. Some strains have been
resistant to oseltamivir
29
How Does Oseltamivir Resistance Develop
Especially after Chemoprophylaxis ??
Initial Infection After Os- exposure Flu A
virus population Flu A virus population
1
100 Os-susceptible Os-resistant mutant
97-99.9 Os-susceptible Os-resistant
virus becomes dominant
2
30
How to Deal with Oseltamivir Resistance?

• Doctors IC nurses
• Viral laboratory gov. private sectors
• MOPH
• Division of Epidemiology
• 4. Government or MOPH drug stockpile
• 5. Public response limit Flu transmission

31
How to Minimize Problem of Drug Resistance for
Influenza/ILI
At this moment, any type of chemoprophylaxis of
influenza with any antiviral (oseltamivir in this
case) is strongly NOT recommended Use the
antivirals whenever indicated, for example,
according to the CPG.
32
Who Should Get the Antivirals for Flu(s)
Severe H1N1 Disease Should Get Antivirals WHO
document on clinical management of novel
H1N1 (Free PDF) The World Health Organization
is reiterating its position that antivirals are
appropriate for adults and children over age 1
who have severe novel H1N1 disease or who are at
risk for H1N1 complications. The WHO took this
step in response to a BMJ analysis, published
earlier this week (2nd week of August 2009),
calling into question the wisdom of giving
antivirals to children with mild seasonal
influenza.
33
How to Select Antivirals for Treatment of Current
Influenza/ILI
Cause of Current Influenza
34
Low Overall Sensitivity for Novel Influenza A
Virus
Rapid influenza diagnostic tests have a low
overall sensitivity for novel influenza A (H1N1),
MMWR reports (August 2009). RT-PCR ve for
influenza A (H1N1) No. of sample / or
seasonal influenza A (H1N1 or H3N2) 65
(100) For novel influenza A (Flu A 2009) Three
Flu rapid diagnostic tests - Overall sensitivity
40 to 69 For 9 specimens with high viral
titers - Sensitivity ranged from 89 to 100
For seasonal Flu A (H1N1 or H3N2) - Overall
sensitivity ranged from 60 to 83
35
Influenza Virus with LRTI
Influenza virus A H2N2, (H3N1, H1N1, H3N2, H5N1
etc.?)
incubation period 1-3 days
Tracheobronchitis No radiographic pneumonia
Viral infection followed by bacterial pneumonia
Rapidly progressive diffuse influenza pneumonia
Concomitant viral bacterial pneumonia
Louria et al. J Clin Invest 195938213-65.
36
How to Select Antivirals for Treatment of Current
Influenza/ILI/Pneumonia
Flu virus
Asymp
ILI
Pneumonia
ARDS
Zana

Osel
No
Yes
Osel
Zana
Relapse/ no improvement
37
Reason for Using Zanamivir/ Oseltamivir
Zanamivir in Severe Pneumonia or ARDS
It could be seasonal Flu A (H1N1) (1-5 of severe
Flu??) Rarely, it could be dual infection with
Flu A 2009 and seasonal Flu A (H1N1) Very
unlikely, but it could be infected with
oseltamivirresistant Flu A 2009 Once pneumonia
/ ARDS develops, patient needs effective
antiviral the soonest. Uncertainty of adequate
intestinal absorption of oseltamivir intake in
severe cases (nausea, vomiting, gastric
distention, hypotension, diarrhea) Zanamivir
delivered by nebulizer in intubated patient is
not a proven method of effective drug delivery We
do not expect synergy between oseltamivir
zanamivir So what do you think???
38
How to Select Antivirals for Treatment of Current
Influenza/ILI

• Most ILI-suspected and proven cases have
  responded satisfactory to GPO-Flu A intake within
  24-48 hours recently.
• Only one report of oseltamivir resistance in
  novel Flu virus 2009.
• Nearly all seasonal Flu A H1N1 viruses are
  resistant to oseltamivir
• My recommendation
• Oseltamivir therapy for general cases with
  ILI/severe ILI
• Zanamivir given empirically in the cases with
• Pneumonia or ARDS
• No response within 48 hrs of oseltamivir therapy
  for proven ILI
• Relapsing Flu who are highly having suspected or
  proven Flu
• Since we do not know we are dealing with
  seasonal Flu A or oseltamivir-resistant Flu A
  2009 or in very rare case, dual infections of
  both strains.
• Zanamivir may be added to oseltamivir Rx in cases
  with ARDS and uncertain pKs of both drugs

39
Who are at Risk Populations in Influenza?

• Populations with/are
• Chronic lung disease such as asthma, emphysema,
  chronic
• bronchitis, bronchiectasis, or cystic fibrosis
• Heart disease or congestive heart failure
• Chronic kidney disease or renal failure
• Diabetes or another chronic metabolic disorder
• Any condition that can compromise the respiratory
  system
• Severe anemia
• Diseases or are having treatments that depress
  immunity,
• including HIV infection
• Residents in a nursing home or other chronic or
  long-term care facility

40
Who are at Risk Populations in Influenza?

• Populations with/are
• Women who will be in the second or third
  trimester during the influenza season
• All children 6 months through 18 years of age
  (especially those 6 months to 5 years)
• Persons 6 months to 18 years of age receiving
  chronic aspirin therapy
• 50 years of age or older
• Extreme obesity
• Pregnant woman

??????????????????????????????????????????(??????)
?????????
41
Severe or Mild Flu 2009 The Indicator

• ????????????? ?????????????????????????????? 2009
  ????????? ??????? ????????????????????????
• ???????????????? ?????????????????????????????????
  ???????????????
• ??????????????????? ????????????????????
  ???????????????????????????????? 3 ?????
• ????????????????????? 3 ?????????? 3
  ??????????????
• ?????????? 2-3 ???????????????????????????????????
  ?????????? ??????????? (???? ????????????? 3 ???
  6 ????????????????)
• ????????????????????????????????????????????
  (???????????????????????????????????????)
• ????????????? (??????) ???????????????????
• ??????????????? ???????????????????????
  ????????????????

42
When to Collect Send Specimen to Viral
Laboratory

• ??????????????????
• ????????????????????????????????????????????
  ??????????????????????????????????????????????????
  ?
• ????????????????????????????????????????
  ????????????????
• ????????????????????????????????
• ????????????????????????????????? ???? ??????? ?
  ??????????-???? ??????????????????????????????????
  ?????????????????????????????????
• ??????????????????????? ??????????????????
• ??????????????????????????????????????????????????
  ????????????
• ?????????????????????????????????????

43
Viral Laboratory Government Private Sectors

• ??????????????????????????????????????????????????
  ???????????????????????? ???? RT-PCR ?? H275Y
• ????????????????????????????? ???????????????????
  ????????????????????????????????
• ????????????????????????????????????
  ??????????????????????????????????????????????????
  ??????????
• ????????????????????????????????
• ????????????????????????????????? ???? ??????? ?
  ??????????-???? ??????????????????????????????????
  ?????????????????????????????????
• ??????????????????????????????????
  ??????????????????????????
• ??????????????????????????? IC50 ??????????????
• ?????????????????? ?????????? ????????????????

44
MOPH Division of Epidemiology

• ???????????????????????????? (oseltamivir)
  ?????????????????
• ??????????????????????????????????????
  (oseltamivir)
• ??????????????????????????????????????????????????
  ????????????????????????????????
• ??????????????????????????????????????????????????
  ?????????? ???????????????
• ??????????????????????????????????????????????????
  ????? ???????????????????????????????
  ?????????????????????????????????????
• ??????????????????????? ???????? ???? zanamivir
• ????????????????? ????????????????????????

45
The Next Pandemic When or Never ???

• World Health Organizations belief and plan
• The world was closer to another influenza
  pandemic than it has been any time since 1968,
  when the last of the 20th century's three
  pandemics swept the globe
• Has developed a global influenza preparedness
  plan
• Outline the role of the WHO in the crisis
• Set up recommendations for national measures
  before and during pandemics

From Wikipedia, the free encyclopedia quoted 22
July 2009
46
The Next Pandemic When or Never ???

• World Health Organizations belief and plan
• The world was closer to another influenza
  pandemic than it has been any time since 1968,
  when the last of the 20th century's three
  pandemics swept the globe
• Influenza field has evolved into two sub fields
• 1. Re-emerging disease (seasonal flu)
• 2. Newly emerging disease (potential
  pandemic flu)

47
The Next Pandemic When or Never ???
Notable influenza pandemics 1. Spanish Flu
(19181920) 2. Asian Flu (19571958) 3. Hong
Kong Flu (19681969)
4.1 Bird Flu Pandemic in Avians,
Global Spread in Human (2004-2007) 4.2 Swine Flu
Pandemic (Since 2009)
48
History of Reassortment Events in the Evolution
of the 2009 Influenza A (H1N1) Virus. The eight
segments shown within each virus code for the
following proteins of the influenza A virus (top
to bottom) 1. polymerase PB2 2. polymerase
PB1 3. polymerase PA 4. hemagglutinin 5.
nuclear protein 6. neuraminidase 7. matrix
proteins 8. nonstructural proteins The
segments of the human 2009 influenza A (H1N1)
virus have coexisted in swine influenza A virus
strains for more than 10 years. The ancestors
of neuraminidase have not been observed for
almost 20 years. The mixing vessel for the
current reassortment is likely to be a swine host
but remains unknown.
49
Letter to Nature nfv, (2009) doi10.1038/nature0
8260. Published online 13 July 2009
50
Virus Titres in Organs of Infected Cynomolgus
Macaques
Letter to Nature nfv, (2009) doi10.1038/nature0
8260. Published online 13 July 2009
51
Isolated in year 2009
Letter to Nature nfv, (2009) doi10.1038/nature0
8260. Published online 13 July 2009
52
WHO director-general Margaret Chan said the
pandemic now looks beyond anyones ability to
control.
53
WHO Recommends National Stockpiles of Antivirals
for Treatment and Prophylaxis

• Purpose Response to outbreaks within the country
  or the treatment and prophylaxis of citizens
  during a pandemic
• Timing Pandemic phase III and later
• Application Stockpile under the control of a
  specific nation and positioned within its
  borders.Treatment of individuals with
  confirmedor suspected avian or pandemic
  influenzavirus infection and high risk
  exposuregroups

Oseltamivir or zanamivir or both
54
Age-Specific Death Rates Should Determine Swine
Flu Treatment
July 27, 2009 -- Governments without sufficient
stockpiles of antiviral drugs should prioritize
treatment for swine flu based on age-specific
fatality rates, a study said. Giving antivirals
to the elderly should be a priority if the
current outbreak follows the patterns of the
1969-1970 flu pandemic in Italy, where deaths
occurred primarily among those older than 65,
research published in the BioMed Central
Infectious Diseases medical journal found.
Treatment of younger adults should be the focus
if the flu is more like the 1918-1919 pandemic in
Copenhagen, the study said.
55
Tackling Antiviral Resistance of Influenza Viruses

• Alternative more effective antiviral strategies
  are needed
• Parenteral/newer neuraminidase inhibitors
  Peramivir, LANI,
• Sialidases DAS181 (Fludase)
• Polymerase inhibitors Favipiravir
• Anti-flu antibodies or broadly cross-neutralizing
  human monoclonal antibody, human anti-M2 mAbs,
• Activation of innate immunity
• Polyinosinic-polycytidylic acid (a ds DNA
  or)Poly-ICLC)
• Combination therapies (synergistic/additive
  antiviral activity) for creating genetic barriers
  to resistance development and for using
  combinations of existing drugs.
• Each of the existing neuraminidase inhibitors
  targets different aspects of the neuraminidase
• Unpublished data (a triple combination of
  oseltamivir amantadine ribavirin showed
  greater synergy than double combinations against
  H1N1, H3N2 and H5N1 viruses)

56
Thank you
Guinness World Record of the Largest Aqua
Aerobics Display held at Pattaya Beach, Thailand
by the RCPT ( n2,047 persons ) on April 25, 2009