Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT04831788 |
| Other study ID # |
01-1093/2 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
June 2, 2021 |
| Est. completion date |
January 7, 2022 |
Study information
| Verified date |
January 2023 |
| Source |
University of Novi Sad |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial 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
Description:
Research center(s):
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 5.0 ml Todd Hewitt broth containing 0,5%
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 0.85% 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 37°C for 30 min (to
overnight), heated for 10 min at 100°C and spinned for 4 min. No more than 2,5 μl of
supernatant will be used as DNA template. Extracts will be stored at -20°C 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 targets/serotypes 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, www.eucast.org).
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 120,000, and therefore we expect to collect the sample up to 0.4% 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 21.0 (IBM Corp., Armonk, NY, USA).
