Pandemic H1N1 (2009) laboratory response

Singapore

• Containment phase– All patients with travel history to affected

countries go to one hospital (TTSH)– Later more hospitals opened up– Severe symptoms or high-risk groups: admit

to hospital– Others wait at Emergency Department for

laboratory test results: 6-8 hours

• Transition and mitigation phase– Test only patients in high-risk groups, or those

sick enough to be admitted to hospital– Influenza-like illness (ILI): stay home, MC x 7

days– ILI in vulnerable groups: empiric treatment

with antiviral

Laboratory testing methods

• PCR for influenza A– H1N1 (2009) + H3 + H1 (seasonal)– US CDC protocol (modified for primers,

machines, reagents)– Self-designed real-time and end-point PCR– Roche kit

• Virus isolation (MDCK) for positive PCR cases

Quality assurance of H1N1 PCR

• Test with virus samples– Seasonal and H1N1(2009)– Other respiratory viruses– Samples known to be positive for seasonal flu

• Control strain from Melbourne WHO CC• External quality assurance (9 labs)

– Known positive samples / RNA sent to participating labs

• First cases – M gene sequencing• New testing labs – first 20 cases require re-

testing by NPHL

Sample collection

• Nasopharyngeal swabs– Preferred flocked swabs in universal transport

medium (UTM)

• Alternatives– Combined throat + nose swab– Throat swab only

• Diagnostic testing: 2 swabs taken within 10 minutes– Concordance almost 100%, so reduced to one swab

Testing laboratories

• NPHL (National Public Health Laboratory)– Obtain test protocol– Recommend and distribute primers/ probes– Obtain control strains; make RNA– Perform sequencing and virus isolation– Re-test problem cases

• 6 hospital labs with PCR capability– TTSH, SGH, KKH, NUH, AH, CGH

Increasing lab capacity

• Bought more– Robotic extractors (Qiagen, EasyMag,

Qiacube, liquid handler)– Thermocyclers

• Hired temporary and permanent staff

• Use other labs: National Environment Agency, Defence Science Organization

• Improve workflow

Increasing lab capacity

• Working hours– Extended 8 am to 12 midnight + weekends– One hospital team: 24 hour service

• Before pandemic– <50 influenza PCR samples/ day

• At the peak– >1 000 samples/day

Testing objectives

• Diagnostic testing: 6-8 h turnaround time

• Test before discharge

• Surveillance testing

• Coverage for major international meetings

• Coverage for mass sports events– Asian Youth Games

• + other non-recommended situations!

Surveillance testing

• Before pandemic: weekly data, 20-50 samples/week

• Pandemic– *daily* monitoring– Achieve statistical confidence to detect 1%

change = 160 samples/ day

Surveillance testing

• Based on ILI symptoms

• Sentinel sites– Polyclinics (government clinics)– Private GPs– Emergency departments

Surveillance testing

• Support decision-making• Examples

– 0 to 1% : ? Signal of community spread – transit into mitigation *

– >15% : justifies empiric use of antivirals in susceptible groups

– Future: return to 10-15% - tailing of pandemic?

• Monitor other parameters as well e.g. total ARI (acute respiratory illness) attendance

*Estimated 20 000 ARIs/ day, est. 4 000 ILIs/ day, therefore 1% = 40 cases 5% = 200 cases; x6 for private GPs

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Weekly Polyclinic Attendances of ARI, Year 2006 to 2009

7000

9000

11000

13000

15000

17000

19000

21000

23000

25000

27000

1 2 3 4 5 6 7 8 9 10 1112 13 1415 1617 18 1920 2122 23 2425 26 2728 2930 31 3233 3435 36 3738 3940 41 4243 4445 46 4748 4950 51 5253

Week No.

Wee

kly

atte

nd

ance

s

Year 2007

Year 2008

Year 2009

Daily proportion (%) of influenza A/H1N1/2009 among all samples based on influenza surveillance of

polyclinics, GP clinics, and hospitals

3643

32332626

3134

20241819

11141451010910

5 4 2 2 6 6 7 5 2 3 3 3 7 4 4 4 06 3 1 2 1 1 4 0 0 4 0 2 1 2 2 1 1 1 2 0 2 0 2 1 0 0 0 0 0 0 1 2

12 2

55 4 4 2 8 1315

99 9

141415

21

243038

45

433348

46

69

4444

5250524146

5246

3935

5247

53

6366

31

54

625655

63

43

2927

575356

2228

0

10

20

30

40

50

60

70

80

1 Ju

n3

Jun5

Jun9

Jun

11 Ju

n

15 Ju

n

17 Ju

n

19 Ju

n

21 Ju

n

23 Ju

n

25 Ju

n

27 Ju

n

29 Ju

n1

Jul3

Jul5

Jul7

Jul9

Jul

11 Ju

l

13 Ju

l

15 Ju

l

17 Ju

l

19 Ju

l

21 Ju

l

23 Ju

l

25 Ju

l

27 Ju

l

29 Ju

l

31 Ju

l

2 Aug

4 Aug

6 Aug

8 Aug

10 A

ug

12 A

ug

Flu

A p

ositi

vity

(%

)

% Flu A seasonal strains % H1N1/2009

Flu A typing results from Polyclinics

0%

20%

40%

60%

80%

100%

4 M

ay (

n=

3)

6 M

ay (

n=

9)

8 M

ay (

n=

17)

12 M

ay (

n=

19)

14 M

ay (

n=

15)

18 M

ay (

n=

17)

20 M

ay (

n=

8)

22 M

ay (

n=

8)

26 M

ay (

n=

7)

28 M

ay (

n=

1)

1 J

un (

n=

8)

3 J

un (

n=

5)

5 J

un (

n=

4)

9 J

un (

n=

7)

11 J

un (

n=

16)

15 J

un (

n=

6)

17 J

un (

n=

5)

19 J

un (

n=

1)

21 J

un (

n=

0)

23 J

un (

n=

6)

25 J

un (

n=

11)

29 J

un (

n=

9)

1 J

ul (n

=16)

3 J

ul (n

=14)

7 J

ul (n

=12)

9 J

ul (n

=44)

13 J

ul (n

=62)

15 J

ul (n

=50)

17 J

ul (n

=44)

21 J

ul (n

=37)

23 J

ul (n

=73)

25 J

ul (n

=5)

28 J

ul (n

=45)

30 J

ul (n

=51)

3 A

ug (

n=

51)

5 A

ug (

n=

51)

7 A

ug (

n=

47)

12 A

ug (

n=

28)

H1N1 H3N2 Undetermined (Low viral titre) H1N1(2009)

Flu A positivity (%) from Polyclinics

33

45

6052

6252

65

76

59

7175

4740

50

64

404045

3342

50

312821

9

2114

70 0

12813 15

2111

25 28

39

5056

6759

746764

5262

70726359

54

687276

66

78

58

86

44

13 22

71

2016

1515

88

70

0.0

20.0

40.0

60.0

80.0

100.04 M

ay (

N=

9)

6 M

ay (

N=

9)

8 M

ay (

N=

33)

12 M

ay (

N=

22)

14 M

ay (

N=

23)

18 M

ay (

N=

29)

20 M

ay (

N=

18)

22 M

ay (

N=

17)

26 M

ay (

N=

14)

28 M

ay (

N=

8)

1 J

un (

N=

20)

3 J

un (

N=

15)

5 J

un (

N=

18)

9 J

un (

N=

14)

11 J

un (

N=

57)

15 J

un (

N=

65)

17 J

un (

N=

35)

19 J

un (

N=

15)

21 J

un (

N=

9)

23 J

un (

N=

51)

25 J

un (

N=

87)

29 J

un (

N=

61)

1 J

ul (N

=77)

3 J

ul (N

=56)

7 J

ul (N

=43)

9 J

ul (N

=88)

13 J

ul (N

=93)

15 J

ul (N

=68)

17 J

ul (N

=69)

21 J

ul (N

=71)

23 J

ul (N

=104)

25 J

ul (N

=8)

28 J

ul (N

=84)

30 J

ul (N

=71)

3 A

ug (

N=

67)

5 A

ug (

N=

73)

7 A

ug (

N=

60)

12 A

ug (

N=

48)

Daily Influenza Surveillance and Flu A Typing Results

Note: N refers to daily no. of samples tested and n refers to daily no. tested positive for Flu A.

Continued importance of H3N2

• In May 2009, we were in the middle of an H3N2 epidemic

• Vaccine failures in longstay residents caused influenza clusters

Position 78 86 160

174 205 276 277

Vaccine strain and 2008 isolates

E L N K N I R

2009 isolates K I K N/R K M Q

% (Jan to Feb)(n=9) 22 Kindergarten sample

only

22 56 22 0 0

%(Mar to May)(n=16) 69 69 81 75 31 64

2008 Isolates 0 0 0 R was seen in Nov and Dec

0 0 0

Emergence of new H3N2 in 2009 associated with vaccine failure

• View 3D structure with a lot of features

Oseltamivir-resistant H1N1 (2009) with H275Y

Mutations in seasonal H3N2 isolates

Blue : residues within 3A of antibody in complex structure 2VITCyan: residues within 3A of antibody in complex structure 1KENGreen: residues within 3A of antibody in complex structure 1EO8Yellow residues: mutations in your patient/sample