Ignite Creation Date:
2024-05-06 @ 4:00 PM
Last Modification Date:
2024-10-26 @ 2:01 PM
Study NCT ID:
NCT04831788
Status:
COMPLETED
Last Update Posted:
2023-11-01
First Post:
2021-04-02
Brief Title:
Pneumococcal Nasopharyngeal and Oropharyngeal Carriage in Adults
Sponsor:
University of Novi Sad
Organization:
University of Novi Sad
Study Overview
Official Title:
Pneumococcal Nasopharyngeal and Oropharyngeal Carriage in Adults Older Than 50 Years of Age in Outpatient Health Care Facility in Novi Sad
Status:
COMPLETED
Status Verified Date:
2023-01
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
This is the first baseline pilot-study that will evaluate the NP and OP colonization with the underline of pneumococcal serotype distribution among adults older than 50 years of age in Serbia and Southeastern Europe Results of this project will serve as additional evidence in order to increase coverage among adults and elderly
Detailed Description:
Research centers
The research will be carried out by the
1 Institute of Public Health of Vojvodina
Centre for Disease Control and Prevention and the Centre for Microbiology
2 Health Care Centre of Novi Sad
Department of General Medicine
Sample processing To confirm the NP and OP carriage sampling will include NP and OP swabs Swabs will be collected by the general practitioners in the Health Care Centre of Novi Sad and will transport within 24h of collection to the Centre for Microbiology of Institute of Public Health of Vojvodina Novi Sad The sterile cotton-tipped wire swabs will be inserted into the anterior nares gently rubbed on the NP swab wall and immediately placed in transport medium Copan Venturi Transystem Brescia Italy
Laboratory procedure Within the laboratory NP swabs will be analyzed by 200 μl of swab-inoculated STGG media will be transferred to 50 ml Todd Hewitt broth containing 05 yeast extract THY and 1 ml of rabbit serum and incubated at 35-37 C for six hours Cultured broth will be plated on sheep blood agar and incubated in 5 CO2 at 35-37 C After 18-24 hours of incubation plates will be examined for the appearance of alpha-haemolytic colonies resembling streptococci Positive samples will be cultured and optochin disc and bile solubility test will be performed
Molecular serotyping
DNA Extraction To obtain DNA extracts for PCR reactions an overnight growth of blood agar plate will be suspended in in 300 μl of 085 NaCl heated to 70 C for 15 min spinned for 2 min and supernatant will be removed Pellet will be suspended in 50 μl TE buffer with an addition of 10 μl mutanolysin and 8 μl of hyaluronidase kept at 37C for 30 min to overnight heated for 10 min at 100C and spinned for 4 min No more than 25 μl of supernatant will be used as DNA template Extracts will be stored at -20C until PCR testing 9
Identification of S pneumoniae Identification of S pneumoniae will be achieved by amplifying the lytA gene using primers and probes recommended by CDC and Quanta Biosciences PerfeCTa1 qPCR ToughMix1 Low RoxTM Quanta Biosciences Beverly USA 10 LytA positive samples will be further analyzed for serotype identification
Serotype identification Conventional multiplex PCR assays will be performed as a series of multiplex reactions using CDC recommended schemes and primers for pneumococcal serotype deduction and 2X PCR Buffer - QIAGEN Multiplex PCR Kit Qiagen Hilden Germany The PCR products will be analyzed on 2 NuSieve agarose gels Cambrex Bio Science Inc Rockland ME stained with ethidium bromide Gel images will be recorded BioDocAnalyze system Analytik Jena Jena Germany
Real Time PCR will be performed optionally in case of any difficulties with conventional PCR IT will be performed as a series of 21 monoplex assays encompassing 21 serotypes using CDC recommended schemes based on geographic prevalence of serotypes The assays are multiplexed in a sequential triplex format three targetsserotypes in one reaction each detecting targets on FAM Rox or Cy5 and Hex channels Reactions will be performed on ABI 7500 Instruments Thermo Fisher Scientific Waltham USA using specific primers and probes and Invitrogen-Platinum Quantitive PCR SuperMix Thermo Fisher Scientific Waltham USA
Susceptibility testing Susceptibility of Spn isolates to Optochin Oxacilin Norfloxacin Erytromycin Clindamycin Tetracycline and Trimetroprim-sulfometoxazole will be determined by the Diffusion method according to the European Committee on Antimicrobial Susceptibility Testing EUCAST wwweucastorg
This baseline assessment is taking into consideration to present descriptive only current results but they are not separately taken into account for comparative analysis
The results of laboratory testing conducted for each patient will forwarded to elected physicians who indicated sampling of patient material
Study procedures Adults will be recruited during their health examination at Health Care Centre of Novi Sad by their elected physician
After providing a verbal and written explanation of the research aim Appendix 1 informed consent Appendix 2 will be obtained from subjects before enrolment Personal and confidential information obtained from participants will be removed except for demographic information including date of sampling age gender data on previous upper respiratory tract infection number of children siblings aged 0-10 years residing in the household of participants with pneumococcal and influenza immunization history because influenza immunization may prevent pneumococcal superinfections smoking habits of participants and their household members and the information about home residence Survey Questionnaire- Appendix 3
Samples will be provided as one NP swab per study subject Every participant will be assigned only once
Confirmed case is every laboratory tested participant in which after testing PCR or ELISA a positive result is obtained
Statistical Analysis and Sample Size Justification Collection of the data Characteristics of all respondents acquired by questionnaire from Novi Sad the test results of laboratory testing of samples of participants and the final characterization of Spn serotypes will be entered in specially designed database Personal information of participants will be removed
Investigator of the research along with statistician will analyze the collected data
Sample size justification In accordance with the sample size calculation for a study estimating a population prevalence 11 between 350 and 500 samples are planned to be collected If more than 500 will be eligible they will all be included We presume to detect pneumococci in up to 10 PCR-positive to Spn Total number of adults older than 50 years of age is around 120000 and therefore we expect to collect the sample up to 04 of the total targeted population
Expected results After carefully implementation of the research we expected that Spn carriage among adults older than 50 years of age will be present in less than 10 of the total population In addition we presume that Spn serotypes covered by PPV or PCV will represent more than 60 and 50 of the serotypes in overall distribution among tested subjects
Statistical Methods We will examine associations between risk factors and NP and OP carriage of pneumococci in participants aged older than 50 years The following factors will be examined as possible risk factors in covered population age gender data on previous upper respiratory tract infection number of children siblings aged 0-10 years residing in the household of participants with pneumococcal and influenza immunization history smoking habits of participants and their household members and the information about home residence These factors will be analyzed by univariate and multivariate analyses if appropriate Also the prevalence of NP and OP carriage as the proportion of participants whose nasopharyngeal cultures were positive for Spn and corresponding 95 confidence intervals will be estimated All descriptive analyses will be performed using the SPSS Statistics software Version 210 IBM Corp Armonk NY USA
The research will be carried out by the
1 Institute of Public Health of Vojvodina
Centre for Disease Control and Prevention and the Centre for Microbiology
2 Health Care Centre of Novi Sad
Department of General Medicine
Sample processing To confirm the NP and OP carriage sampling will include NP and OP swabs Swabs will be collected by the general practitioners in the Health Care Centre of Novi Sad and will transport within 24h of collection to the Centre for Microbiology of Institute of Public Health of Vojvodina Novi Sad The sterile cotton-tipped wire swabs will be inserted into the anterior nares gently rubbed on the NP swab wall and immediately placed in transport medium Copan Venturi Transystem Brescia Italy
Laboratory procedure Within the laboratory NP swabs will be analyzed by 200 μl of swab-inoculated STGG media will be transferred to 50 ml Todd Hewitt broth containing 05 yeast extract THY and 1 ml of rabbit serum and incubated at 35-37 C for six hours Cultured broth will be plated on sheep blood agar and incubated in 5 CO2 at 35-37 C After 18-24 hours of incubation plates will be examined for the appearance of alpha-haemolytic colonies resembling streptococci Positive samples will be cultured and optochin disc and bile solubility test will be performed
Molecular serotyping
DNA Extraction To obtain DNA extracts for PCR reactions an overnight growth of blood agar plate will be suspended in in 300 μl of 085 NaCl heated to 70 C for 15 min spinned for 2 min and supernatant will be removed Pellet will be suspended in 50 μl TE buffer with an addition of 10 μl mutanolysin and 8 μl of hyaluronidase kept at 37C for 30 min to overnight heated for 10 min at 100C and spinned for 4 min No more than 25 μl of supernatant will be used as DNA template Extracts will be stored at -20C until PCR testing 9
Identification of S pneumoniae Identification of S pneumoniae will be achieved by amplifying the lytA gene using primers and probes recommended by CDC and Quanta Biosciences PerfeCTa1 qPCR ToughMix1 Low RoxTM Quanta Biosciences Beverly USA 10 LytA positive samples will be further analyzed for serotype identification
Serotype identification Conventional multiplex PCR assays will be performed as a series of multiplex reactions using CDC recommended schemes and primers for pneumococcal serotype deduction and 2X PCR Buffer - QIAGEN Multiplex PCR Kit Qiagen Hilden Germany The PCR products will be analyzed on 2 NuSieve agarose gels Cambrex Bio Science Inc Rockland ME stained with ethidium bromide Gel images will be recorded BioDocAnalyze system Analytik Jena Jena Germany
Real Time PCR will be performed optionally in case of any difficulties with conventional PCR IT will be performed as a series of 21 monoplex assays encompassing 21 serotypes using CDC recommended schemes based on geographic prevalence of serotypes The assays are multiplexed in a sequential triplex format three targetsserotypes in one reaction each detecting targets on FAM Rox or Cy5 and Hex channels Reactions will be performed on ABI 7500 Instruments Thermo Fisher Scientific Waltham USA using specific primers and probes and Invitrogen-Platinum Quantitive PCR SuperMix Thermo Fisher Scientific Waltham USA
Susceptibility testing Susceptibility of Spn isolates to Optochin Oxacilin Norfloxacin Erytromycin Clindamycin Tetracycline and Trimetroprim-sulfometoxazole will be determined by the Diffusion method according to the European Committee on Antimicrobial Susceptibility Testing EUCAST wwweucastorg
This baseline assessment is taking into consideration to present descriptive only current results but they are not separately taken into account for comparative analysis
The results of laboratory testing conducted for each patient will forwarded to elected physicians who indicated sampling of patient material
Study procedures Adults will be recruited during their health examination at Health Care Centre of Novi Sad by their elected physician
After providing a verbal and written explanation of the research aim Appendix 1 informed consent Appendix 2 will be obtained from subjects before enrolment Personal and confidential information obtained from participants will be removed except for demographic information including date of sampling age gender data on previous upper respiratory tract infection number of children siblings aged 0-10 years residing in the household of participants with pneumococcal and influenza immunization history because influenza immunization may prevent pneumococcal superinfections smoking habits of participants and their household members and the information about home residence Survey Questionnaire- Appendix 3
Samples will be provided as one NP swab per study subject Every participant will be assigned only once
Confirmed case is every laboratory tested participant in which after testing PCR or ELISA a positive result is obtained
Statistical Analysis and Sample Size Justification Collection of the data Characteristics of all respondents acquired by questionnaire from Novi Sad the test results of laboratory testing of samples of participants and the final characterization of Spn serotypes will be entered in specially designed database Personal information of participants will be removed
Investigator of the research along with statistician will analyze the collected data
Sample size justification In accordance with the sample size calculation for a study estimating a population prevalence 11 between 350 and 500 samples are planned to be collected If more than 500 will be eligible they will all be included We presume to detect pneumococci in up to 10 PCR-positive to Spn Total number of adults older than 50 years of age is around 120000 and therefore we expect to collect the sample up to 04 of the total targeted population
Expected results After carefully implementation of the research we expected that Spn carriage among adults older than 50 years of age will be present in less than 10 of the total population In addition we presume that Spn serotypes covered by PPV or PCV will represent more than 60 and 50 of the serotypes in overall distribution among tested subjects
Statistical Methods We will examine associations between risk factors and NP and OP carriage of pneumococci in participants aged older than 50 years The following factors will be examined as possible risk factors in covered population age gender data on previous upper respiratory tract infection number of children siblings aged 0-10 years residing in the household of participants with pneumococcal and influenza immunization history smoking habits of participants and their household members and the information about home residence These factors will be analyzed by univariate and multivariate analyses if appropriate Also the prevalence of NP and OP carriage as the proportion of participants whose nasopharyngeal cultures were positive for Spn and corresponding 95 confidence intervals will be estimated All descriptive analyses will be performed using the SPSS Statistics software Version 210 IBM Corp Armonk NY USA
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None