1. Epidemiology of Invasive Streptococcus pneumoniae (IPD) Serotypes in Oklahoma, Children Under Five
Jolianne Stone, MPH
L Smithee, K Bradley, M McDermott
Acute Disease Service
Oklahoma State Department of Health

2. Study Objectives
Understand the serotype distribution of IPD in children <5 using PCR methodology (Nov – Dec. 2007)
Determine number of vaccine-preventable IPD cases
Current vaccine PCV7
Candidate vaccine PCV13
The objectives of this study are to understand the serotype distribution of IPD in children less than 5 using PCR methodology in Oklahoma between November 1, 2006 and December 31, 2007; and determine the number of vaccine-preventable IPD cases with PCV7 and the candidate vaccine PCV13.

3. Oklahoma Reporting Requirements
Passive surveillance system
June 2000, required reporting of all invasive S. pneumoniae cases
November 2006, required facilities to forward sterile site isolates of S. pneumoniae in children <5
Oklahoma uses a passive surveillance system to report cases of IPD. In June of 2000, Oklahoma added invasive Streptococcus pneumoniae to the list of reportable diseases and conditions; requiring all sterile site cases to be reported. This reporting did not include submission of the isolate. In November 2006, Oklahoma enhanced surveillance requiring all sterile site isolates of IPD in children under five to be forwarded to the Oklahoma Public Health Laboratory for serotyping.

4. Laboratory Methods
OSDH Public Health Laboratory followed CDC’s PCR deduction of pneumococcal serotypes with modifications
Sequential Multiplex PCR for S. pneumoniae Serotyping (Pai et. al. J. Clin. Microbiol. 44: , 2006)
Isolates unable to be typed by PCR were forwarded to CDC
Isolates frozen for batch testing
Using reagents provided by the CDC Streptococcal Laboratory and following the protocol described in the article “Sequential mulitplex PCR for Streptococcus pneumoniae serotyping”, the Oklahoma Public Health Laboratory serotyped the majority of the received pneumococcal isolates. Isolates unable to be typed by our laboratory were forwarded to CDC for further testing. To effectively utilize person-time and reagents, the isolates were frozen and batched until a minimum of seven were available.

5. Epidemiologic Methods
Investigated all reported cases from November 1, 2006 – December 31, 2007
Contacted laboratories to confirm submission of sterile site isolates
Determined vaccine status using state immunization registry or by contacting family or physician
Epidemiologists investigated all reports of IPD cases in children under five for symptoms, hospitalization, death status, and infection types. The epidemiologists contacted the laboratory where the isolate was identified and confirmed the submission to the public health laboratory. Immunization status was gathered on each case using the state immunization registry. If still unable to determine complete vaccination history, immunization services contacted providers and parents of the case to confirm vaccination status.

6. Invasive Pneumococcal Disease Oklahoma, 2001-2006
3510 IPD cases were reported to the Oklahoma State Department of Health
Annual Average IPD
Children <5: /100,000
Adults 65: 49.67/100,000
Hospitalized: 3052 (87%)
Deaths: 398 (11%)
Since 2001, 3, 510 cases of IPD were reported to the Oklahoma State Department of Health. The annual average incidence of IPD were highest in adults greater than or equal to 65 years of age and children under five. 87 percent of cases were hospitalized, and 11% expired.

7. Rate of IPD by Age Group Oklahoma, 2001-2007
In this graph the rate of IPD is compared between children less than 5 (in blue) and persons greater than or equal to 5 (in red) in Oklahoma between 2001 and As you can see, the rates of IPD in children under five are higher than those greater than or equal to 5, and has increased since 2005.
Case numbers: N=73
2007 N=74
No changes in reporting between 2005 and 2006, starting reporting via PHIDDO.
Electronic lab reporting 2005

8. Rate of IPD Cases Children <5, Oklahoma and US, 2001-2007
This graph compares the rate of IPD in children less than 5 years of age between Oklahoma (in red) and the US (in blue). As you can see, the rates of IPD in Oklahoma have varied, with Oklahoma rates lower than the US except for However, In 2006 Oklahoma was noticeably higher than the US. OK US
Year
*US Data from ABC Surveillance Data

9. Pneumococcal Disease and Vaccination in Oklahoma, Children Under Five
IPD 56.6% of the vaccine-preventable diseases
Oklahoma PCV7 3+ vaccination rates and rank:
% 50th (US: 73.2%)
% 50th (US: 82.8%)
Question for evaluation:
Low vaccine coverage rates in Oklahoma; is IPD disease incidence due to PCV7 serotypes?
Between 2001 and 2006, among diseases that are vaccine preventable in children under five, over half were IPD. In 2004 and 2005, Oklahoma ranked 50th in the US for 3+ pneumococcal vaccination coverage. Because of Oklahoma’s low vaccine coverage rates, Oklahoma initiated the serotype project to determine if disease incidence was due to PCV7 serotypes.

10. IPD Reported Cases in Children <5 Oklahoma, Nov 2006 – Dec 2007
88 laboratory confirmed cases of IPD
Median Age: 12 months (Range: 1 Day – 4 Years)
Sex:
Female: 37 (42%)
Male: 51 (58%)
Hospitalizations: 66 (82%)
Deaths: 4
Received 70 (79%) isolates for serotyping
During the study period November 1, 2006 to December 31, 2007 a total of 88 confirmed cases of IPD were reported. The median age of the cases was 12 months, with fifty-eight percent male. Eighty-two percent were hospitalized with four deaths. Of these 88 cases, our Public Health laboratory received 70 of the isolates for serotyping.

11. IPD Serotypes Submitted Isolates (N=70), Children Under 5 Oklahoma, Nov 2006 – Dec 2007
COUNT
PERCENT
CUMULATIVE PERCENT 4
5.7%
23F 3
4.3%
10% 1
15.7% 7F 8
11.4%
27.1%
19A 22
31.4%
58.5%
6A/6B
1.4%
59.9% 7C 2
2.9%
62.8%
64.2%
12F
65.6%
15B
67%
17F
69.9%
23B
71.3% 38
72.7%
Not Typeable by PCR 19
100%
PCV7
PCV13**
Nonvaccine
Not typeable by PCR
This table shows individual serotypes isolated using the PCR methods. The red shows serotypes included in PCV7, and the ones coded in blue will be included in the PCV13 candidate vaccine. The 6A/6B is highlighted in yellow because 6B is covered in PCV7, and 6A will be covered in PCV13. Substantial cross-reactivity from the 6B component to 6A exists, but given current laboratory reagents we cannot separate the two serotypes for categorization. The serotypes coded in green are non-vaccine serotypes. As you can see, 22 isolates were 19A, constituting 31.4% of the isolates.
POINT AT DIFFERENT GROUPS
**Candidate vaccine by Wyeth, projected to cover PCV7 serotypes plus 1,3,5,6A, 7F,19A

12. IPD Serotypes by Vaccine Type (N=70), Children Under Five, Oklahoma, Nov 2006 – Dec 2007
This chart shows the percentage of cases by serotype and vaccine coverage. 10 percent of isolates were PCV7 serotypes. Those NOT covered under PCV7 but will be included in the candidate vaccine PCV13 make up 49 percent of isolates. In summary, a total of 50% of cases could potentially be prevented under the candidate vaccine PCV13.
POINT AT PCV7, PCV13, AND 6A/6B
*Candidate vaccine: includes 1,3,5,6A, 7F,and 19A

13. Too young for vaccination: 8 (11.4%)
PCV7 Vaccination Status at Time of Disease (N=70)*, Oklahoma, Nov 2006 – Dec 2007
Too young for vaccination: 8 (11.4%)
Children eligible for vaccination: 62 (88.6%)
No vaccination: 3 (4.8%)
Any PCV7 vaccination: 59 (95.2%)
39 (62.9%) age appropriately vaccinated
19 (30.6%) not age appropriately vaccinated
1 (1.6%) vaccination history incomplete
Among the 70 cases with isolates submitted to the public health laboratory, 8 were too young for vaccination at time of disease. Of the 62 cases eligible to receive PCV7, three cases were unvaccinated. Approximately 63% were age appropriately vaccinated, 19 were not age appropriately vaccinated, and complete vaccination history was unable to be obtained for 1 case.
*Among those with submitted isolates

14. Frequency of PCV7 and PCV13 Serotypes by Vaccination History and Age*
PCV7 Serotypes (N=7)
PCV13 Serotypes (N=31)
Vaccination Status
Age Appropriate
Not Age
Appropriate
Age (months)
2-3
(1dose)
1 (20%)
Serotype 4
1 (50%)
Serotype 23F
1 (5.9%)
2 (14.3%)
4-5
(2 dose)
2 (11.8%)
6-11
(3 dose)
3 (17.6%)
4 (28.6%)
12+
(4 dose)
3 (60%)
Serotypes 4
11 (64.7%)
8 (57.1%)
5 (71.4%)
2 (28.9%)
17 (54.8%)
14 (45.2%)
This table breaks down the isolates into PCV7 and PCV13 serotypes by vaccination status and age categories according to the recommended ACIP schedule. Seven isolates were PCV7 serotypes, either serotype 4 or 23F. Five cases of PCV7 serotype disease were age-appropriately vaccinated, with 4 cases having appropriate time to mount antibody response to vaccination indicating vaccine failure. 31 cases had PCV13 serotypes, with 17 age appropriately vaccinated and potentially preventable with the new vaccine.
(58% of children in chart up to date on vaccination)
*Includes only vaccine-eligible cases and those with known vaccination history

15. Days Since PCV7 Vaccination for IPD Cases with Vaccine Serotypes*
Age at time of Disease
Number PCV7 Doses
Days Since last Vaccination
Clinical Presentation** 1
3 mo 38
otitis media 2
5 mo 8
meningitis, pneumonia 3
16 mo 4
143
meningitis
23 mo
153 5
3 yrs
445
pneumonia
This table shows all five age appropriately vaccinated cases with PCV7 serotypes. The age at time of disease ranged from 3 months to 3 years, with the number of days since last vaccination ranging from 8 to 445 days. All cases presented with bacteremia/sepsis, and were subsequently diagnosed with other presentations including meningitis, otitis media and pneumonia. Case 2 would be considered breakthrough disease, since it takes approximately 2 weeks to mount an immune response after vaccination.
*Those with age-appropriate vaccinations only
**All cases had isolates from blood, clinical presentation of bacteremia/sepsis

16. PCR Protocol Time and Effort
Time Analysis:
5-6 hours of person-time for 7 or less samples
Includes all aspects of the protocol from extraction to gel and analysis
Time increases after 7 samples
Cost Analysis:
CDC provided the set of 60 primers
Cost for other equipment per isolate is $32.29
Tech time per isolate is approximately $25-30
Total laboratory cost per isolate $57-62
To complete the PCR protocol, for every seven samples a total of 5-6 hours of person-time was needed. This time included all aspects of the protocol from culturing and DNA extraction to gel and analysis. CDC provided the set of 60 primers. Included in the equipment cost were all incidentals such as reagents, agar, and media. Add the tech time per isolate, and the total cost per isolate is between 57 and 62 dollars.

17. Conclusion
50% of serotyped cases could potentially be prevented by PCV13
19A is responsible for the largest percentage of serotypes at 31.4%, similar to what is seen across the US
PCR testing complemented IPD surveillance
In conclusion, 50 percent of serotyped isolates could potentially be prevented by the candidate vaccine PCV13. Similar to the US, Oklahoma is seeing a high proportion of non-vaccine serotypes with 19A representing the largest percentage of cases at 31.4%. Oklahoma is the first public health laboratory utilizing PCR methodology for routine IPD surveillance, and it has complimented surveillance activities to help identify serotypes of high incidence.

18. Conclusion
Of the children eligible for vaccination in the study, 63% were age appropriately vaccinated with PCV7
Determined complete immunization history on all but 1 case using other methods along with state immunization registry
Four vaccine failures
Of the 62 children eligible for vaccination in the study, 63% were age appropriately vaccinated with PCV7. Despite no comprehensive vaccine registry, vaccination status was confirmed on all but one case. Seven isolates were vaccine serotypes, with 4 age-appropriately vaccinated and considered vaccine failures.

19. Limitations
Unable to receive all isolates to the OSDH PHL for serotyping
Unable to serotype all isolates received to the OSDH PHL
Short study time frame, unable to determine serotype trends
Despite follow-up efforts made by the epidemiologists, not all isolates were received for serotyping. Over one quarter of the isolates were nontypeable, either because they were nonviable or were non-reactive with the reagents. The study incorporated14 months of data, so we were unable to determine serotype trends.

20. Recommendations Comprehensive vaccine registry
Isolate serotyping useful in determining trends of state-specific disease burden by serotype
Methodology feasible for other states to adopt
Obtaining complete vaccination history was time-intensive, and a comprehensive vaccine registry would be beneficial to track vaccination status of cases. Isolate serotyping of invasive Streptococcus pneumoniae is useful to determine trends of disease burden by serotype in Oklahoma, and is likely to be very useful after the PCV13 is implemented. Like all molecular assays PCR is an expensive test, but overall the methodology used in this project is feasible for other states thinking about adopting a PCR protocol for IPD serotyping.

21. Thank You OSDH CDC Lauri Smithee, MES, MS Kristy Bradley, DVM, MPH
Michael McDermott
Sue Mallonee, RN, MPH
CDC
Melissa VanDyke, PhD, MPH
Matthew Moore, MD, MPH
Bernard Beall, MD, PhD
Gloria Carvalho, PhD
I would like to acknowledge my coauthors and others for their involvement.

22. Pneumococcal Conjugate Vaccine (PCV7)
Conjugate vaccine, covers 7 serotypes that cause >80% of disease
4, 6B, 9V, 14, 18C, 19F, 23F
Licensed in Feb as PREVNAR® by Wyeth Pharmaceuticals
A child is considered age appropriately vaccinated with PCV7 if they have the recommended doses by the ACIP schedules
2,4,6 months of age with a booster dose months of age
PCV7, licensed as PREVNAR by Wyeth, is a pneumococcal conjugate vaccine covering 7 serotypes that cause >80% of disease. Unlike a polysaccharide vaccine, conjugate vaccines have a longer duration of immunity and can reduce carriage of the organism. A child is considered currently vaccinated with PCV7 if they receive the recommended doses at the appropriate intervals according to the ACIP. Despite previous shortages of PCV7 in the past, ACIP currently recommends the vaccine to all children aged 2 months to 2 years of age given in 4 doses; at 2,4, and 6 months with a booster dose given at months of age.

23. Laboratory Methods
Modifications to CDC’s PCR deduction of pneumococcal serotypes
Used Qiagen's EZ1 and DNA tissue kit to extract the DNA of the isolates
Used Promega PCR Master mix Cat# M7502 instead of the separate Taq polymerase and dNTPs
Increased the reaction size to 30ul to accommodate all of the primers
Our laboratory followed CDC’s PCR protocols with the following modifications. To extract the DNA from the bacterial isolates, we used Qiagen’s EZ1 and DNA tissue kit. To setup the master reaction mix, instead of using the separate Taq polymerase and dNTPs, the Promega PCR Master Mix was used. Since the PCR graded water volume varies due to primer concentrations, we increased the reaction size volume from 25 to 30 microliters to accommodate all the primers.

24. Sequential Multiplex PCR Protocol
Isolate* (N=62)
Cumulative %
37%
45%
62.8%
67.6%
74%
80.4%
Reaction 1
19A, 3, 22F, 6A/B
23 Isolates
Reaction 2
4, 12F, 14, 9V
5 Isolates
Reaction 3
23F, 7F, 11A, 33F
11 Isolates
Reaction 4
19F, 16F, SG18, 35B
0 Isolates
Reaction 5
8, 15B/C, 38, 31
3 Isolates
Seven reactions are included in the PCR protocol for pneumococcal serotyping. Each reaction identifies certain serotypes, with the the sequence order based upon the ABCs serotype distribution. In our study, over 60% of isolates were serotyped by reactions 1,2, and 3.
Reaction 6
1, 10A, 35F, 34
4 Isolates
Reaction 7
20, 7C, 17F, 15A
4 Isolates
Nonreactive
11 Isolates
Other Serotype
*8 were non-viable

25. Vaccination History Time and Effort
56% (N=35) eligible for vaccination had complete vaccination history in OSIIS
After obtaining primary care physician information, average time per vaccination follow-up was 30 minutes per case
Total time of vaccination history follow-up was approximately 14 hours person time
Of the 62 cases eligible for vaccination in our study, approximately 56% had complete vaccination history in OSIIS. Primary care physician information was obtained from the chart record at the hospital (if listed), and time contacting the family and the physician to obtain accurate vaccination history was approximately 30 minutes per case. The total person time to follow up on the incomplete immunization cases was approximately 14 hours.