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

Study Objectives Understand the serotype distribution of IPD in children <5 using PCR methodology (Nov. 2006 – 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.

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.

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. 44:124-131, 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.

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.

Invasive Pneumococcal Disease Oklahoma, 2001-2006 3510 IPD cases were reported to the Oklahoma State Department of Health Annual Average IPD Children <5: 23.97/100,000 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.

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 2007. 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: 2006 N=73 2007 N=74 No changes in reporting between 2005 and 2006, starting reporting via PHIDDO. Electronic lab reporting 2005

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 2003. However, In 2006 Oklahoma was noticeably higher than the US. 2001 2002 2003 2004 2005 OK16.5 20.7 33.0 21.1 20.31 US 38.9 23.2 22.3 22 21.2 Year *US Data from ABC Surveillance Data

Pneumococcal Disease and Vaccination in Oklahoma, Children Under Five IPD 56.6% of the vaccine-preventable diseases Oklahoma PCV7 3+ vaccination rates and rank: 2004 44.1% 50th (US: 73.2%) 2005 54.4% 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.

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.

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

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

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

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

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

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.

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.

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.

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.

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.

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.

Pneumococcal Conjugate Vaccine (PCV7) Conjugate vaccine, covers 7 serotypes that cause >80% of disease 4, 6B, 9V, 14, 18C, 19F, 23F Licensed in Feb. 2000 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 12-15 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 12-15 months of age.

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.

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

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.