Characterization of Streptococcus pneumoniae serotypes in post vaccine era in Pakistan

1 Characterization of Streptococcus pneumoniae serotypes in post vaccine era in Pakistan Department of Biology Syed Babar Ali School of Science and Engineering Lahore University of Management Sciences (LUMS) Submitted in Partial Fulfilment of the Requirements for the degree of Bachelor of Sciences Isra Razzak 24100306 Advisor: Dr. Shaper Mirza 2 DECLARATION This is to certify that this dissertation entitled as “Characterization of Streptococcus pneumoniae serotypes in post vaccine era in Pakistan” submitted by Isra Razzak, under the supervision of Dr. Shaper, is accepted in its present form by the Department of Biology, Lahore university of management sciences, Lahore, Pakistan as satisfying the dissertation for the degree of BS in Biology. I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, contains no material previously published or written by any other person neither has it been accepted for reward of any other degree at a university or any other institute of higher learning, except where due acknowledgement has been made in the text. - Isra Razzak Supervisor: Dr. Shaper Mirza Associate Professor, Department of Biology Lahore University of Management Sciences (LUMS) 3 Acknowledgements: I am profoundly grateful to a number of individuals whose guidance and support have been invaluable during the course of my thesis. First and foremost, I extend my deepest gratitude to my parents, whose unwavering support have been my constant source of strength throughout. Their sacrifices have not gone unnoticed, and this achievement is as much theirs as it is mine. I am especially thankful to my supervisor, Dr. Shaper Mirza, for providing me with the opportunity to work under her guidance. Her expertise and insights have been crucial in my learning and growth. My heartfelt thanks go to my friends, particularly Yusra and Fatima, whose presence has been my strength in the most challenging times. I would also like to express my gratitude to Dr. Asia, who has been an incredible mentor throughout this process. Her detailed feedback has been invaluable in improving my work. I would also like to thank Zuha Bilal, who initiated this project. Her early contributions were vital in shaping the direction of my research. Finally, I extend my appreciation to all the members of the MEM lab for providing a supportive work environment. Thank you all for your contributions, which have been vital to the completion of this thesis. Best, Isra Razzak - 24100306 4 Table of Contents Abstract: .......................................................................................................................................... 6 Introduction: .................................................................................................................................... 7 Methodology: ................................................................................................................................ 11 Materials and Methods:................................................................................................................. 13 Methods: ....................................................................................................................................... 13 1.1: Growth of Pneumococcal Strain on Blood Agar: ............................................................... 14 1.2: DNA Isolation: ................................................................................................................... 14 1.3: lytA and pspA PCR: ........................................................................................................... 15 1.4: Housekeeping genes PCR: ................................................................................................. 15 1.5: Multiplex PCR: .................................................................................................................. 17 1.6: Gel Electrophoresis: ........................................................................................................... 21 Results: .......................................................................................................................................... 22 2.1: Graphs: ............................................................................................................................... 22 2.2: DNA Isolation: ................................................................................................................... 26 2.3: lytA PCR: ............................................................................................................................ 27 2.4: pspA PCR: .......................................................................................................................... 28 2.5: Housekeeping genes PCR: ................................................................................................. 29 2.6: Multiplex PCR: .................................................................................................................. 31 Discussion: .................................................................................................................................... 34 References: .................................................................................................................................... 35 Appendix A .................................................................................................................................. 38 Table A1: Blood Agar ................................................................................................................ 38 Table A2: Todd Hewitt Broth (THY) ........................................................................................ 38 Table A3: 1X TE Buffer ............................................................................................................ 39 Table A4: 10% SDS ................................................................................................................... 39 Table A5: 5M Potassium acetate ............................................................................................... 40 Table A6: 95% Ethanol ............................................................................................................. 40 Table A7: 70% Ethanol ............................................................................................................. 40 5 Table A11: 1% Agarose gel ....................................................................................................... 42 APPENDIX B ............................................................................................................................... 43 Table B1: Demographic data of pneumococcal isolates ........................................................... 43 6 Abstract: Streptococcus pneumoniae is the leading cause of death and kills an estimated 1.1 million children under the five years every year which is more than AIDS, malaria and tuberculosis combined. The organism is responsible for causing various infections including mild respiratory infections like sinusitis and otitis media to more severe diseases such as pneumonia, meningitis, and bacteremia. Population at risk of infection include children under the age of 5, adults above 65 years of age and immunocompromised individuals. Pneumococcal infections are vaccine preventable and currently there are four vaccines (PCV 7,10,13 and 24) available in market for immunizing children and adults at risk of infections. Pakistan introduced PCV 10 in year 2012 to lower the burden of infections. While reducing the burden of infection by vaccine strains, immunization also resulted in phenomenon including, serotype replacement and vaccine escape. There is a wealth of information available on how these phenomenon are evolving globally, however, there is significant dearth of information on both of these for Pakistan. Overall objective of this study is to investigates post-vaccine serotype replacement and quantify burden of infection caused by non-vaccine strains. We hypothesize that immunization against S. pneumoniae is changing the population biology of S. pneumoniae resulting in increased burden of infection by non-vaccine strains. To test this hypothesis, we have designed the following aims. The aim of the study includes: 1. To determine the proportion of infections caused by vaccine serotypes of S.pneumoniae. 2. To measure the burden of vaccine escape and serotype replaced strains. 3. Characterize serotypes on the basis of geographic distribution, demographic and clinical characteristics. Samples obtained from a tertiary and a clinical laboratory were subjected to culture, optochin resistance for differentiation between pneumococci and species of viridians group and finally to PCR using primers for lytA and pspA two genes that are present in all pneumococci identified to date. For confirmation of serotypes, we used multiplex PCR. Overall, our results demonstrated a decrease in the burden of infection by vaccine serotypes, as observed by the low abundance of PCV serotypes in both carriage and invasive strains. Nevertheless, clinical samples exhibited a significant prevalence of PCV13 serotypes, indicating the emergence of vaccination escape 7 strains. Non-vaccine serotypes were more common in pus, sputum, and blood samples, and nontypeable strains were predominantly detected in tracheal secretions, suggesting a possible deficiency in the present vaccine's effectiveness. Geographical distribution in Pakistan showed differences in the distribution of serotypes, emphasizing the importance of developing vaccination regimens that are specifically targeted to different regions. Introduction: S.pneumoniae causes pneumonia, otitis media and other invasive diseases such as bacteremia and meningitis, collectively called pneumococcal diseases (PD). In 2017, the WHO included S. pneumoniae as one of 12 priority pathogens (Weiser et al.). It is an opportunistic pathogen that colonizes the mucosal surfaces of the human upper respiratory tract. Pneumococcus plays a major role in the morbidity and mortality of children as well as the elderly worldwide. It is estimated that ~ 14.5 million cases of PD occur each year, causing the deaths of ~ 1 million children < 5 years of age (Khan et al.). Pneumonia is the leading cause of death for children under five, especially in Pakistan where one-fifth of the population falls into this age range. According to estimates found in numerous Pakistani research, the [annual] incidence of acute respiratory infection (ARI) among children under five in Pakistan is 4% in the community, which makes up around 22% of the 160 million people living in the nation. Using this 4% estimate, we may determine that there are 15 million ARI episodes among children under five annually (“Pneumonia – Federal Directorate of Immunization, Pakistan”). Streptococcus genus is divided into 49 species and eight subspecies, from which as many as 35 have been identified as sources of invasive infections in humans. The study of Streptococcus spans across understanding its pathogenic mechanisms, antibiotic resistance patterns, and developing effective vaccines, highlighting its significant impact on medical microbiology and public health. Streptococci are Gram-positive, nonmotile, non-spore forming, catalase-negative cocci that occur in pairs or chains. While some streptococci are obligate anaerobes, the majority are facultative anaerobes. Streptococci are classified on the basis of colony morphology, hemolysis, biochemical reactions, and serologic specificity. Their hemolysis on blood agar is classified into 8 three groups: β-hemolytic - complete, clear lysis of red cells, α hemolytic - incomplete, green hemolysis, and γ hemolytic - no hemolysis (Patterson). Pneumococcal survives in nasopharyngeal cavity however, it can invade into deeper tissues, an ability that is imparted by various cell surface and intracellular proteins termed as virulence factors. Among the most important virulence factors are capsular polysaccharides, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), and pneumococcal surface adhesin A (PsaA). Capsular polysaccharides are considered the most essential virulence factor because they surround and protect pneumococci from phagocytes (Song et al.). S. pneumoniae is notable for its wide variety of serotypes— over 100 —each defined by a unique chemical and genetic composition of its capsule. This diversity is significant for both its virulence and the challenges it poses for vaccine development. S. pneumoniae belongs to the α hemolytic group as it has greenish colonies on blood agar (Yesilkaya et al.). Optochin sensitivity is a hallmark trait of S.pneumoniae, inhibiting its growth near optochin discs on blood agar due to its unique cell wall properties, leading to cell lysis. The capsular polysaccharides are composed of repeating units of oligosaccharides. The structure of the majority of this capsular polysaccharide is already known. Capsular type is known to be immunogenic and elicit an immune response against both carriage and invasive disease. While antibodies elicited to most capsular types do not cross react, capsules with identical chemical compositions are known to have cross reactive antibodies. For instance, serotypes 6A and 6B have almost identical chemical compositions, except for a single bond between two sugars, and they exhibit extensive cross-reactivity (Robbins et al.). On the other hand, serotypes 19F and 19A show less cross-reactivity (Tarasova et al.). 9 Figure 1: Illustration of the arrangement of the pneumococcal cell wall and capsule. (KALIN) Molecular analysis of the genes responsible for the synthesis of certain capsular components has demonstrated that they are ordered in cassettes and contain all the genetic material necessary for capsule synthesis (Dillard et al.). The pneumococcus, as well as several other streptococci, is naturally transformable and exchange genetic material with and across species. By such a mechanism the capsule specificity, in a cassette type recombination event, can be replaced in vitro as well as in vivo. Occasionally pneumococcal bacteria may possess both of two distinct polysaccharide coding gene cassettes at the same time. It has been postulated that subsequent recombination events in such strains may create novel capsule types (Austrian). Figure 2: Progression of Pneumococcal infections (Weiser et al.) Development of pneumococcal vaccines was a medical advancement that significantly impacted the burden of both morbidity and mortality due to invasive pneumococcal infections. (Wiese et al.). The first vaccine for pneumococcal infections contained 23 pneumococcal serotypes (PPV23)and was developed in the year 1977. While the vaccine provided minimal protection against invasive infection, owing to it T-cell independent nature the vaccine provided temporary immunity without inducing memory B cells or isotype switching. Although the vaccine was 16 Loci Function of gene Primer Sequence Product Size (bp) aroE shikimate dehydrogenase F: 5'-GCC TTT GAG GCG ACA GC R: 5'-TGC AGT TCA (G/A)AA ACA T(A/T)T TCT AA 405 gdh glucose-6-phosphate dehydrogenase F: 5'-ATG GAC AAA CCA GC(G/A/T/C) AG(C/T) TT R: 5'-GCT TGA GGT CCC AT(G/A) CT(G/A/T/C) CC 460 gki glucose kinase F: 5'-GGC ATT GGA ATG GGA TCA CC R: 5'-TCT CCC GCA GCT GAC AC 483 recP transketolase F: 5'-GCC AAC TCA GGT CAT CCA GG R: 5'- TGC AAC CGT AGC ATT GTA AC 450 spi signal peptidase I F: 5'-TTA TTC CTC CTG ATT CTG TC R: 5'-GTG ATT GGC CAG AAG CGG AA 474 xpt xanthine phosphoribosyltransferase F: 5'-TTA TTA GAA GAG CGC ATC CT R: 5'-AGA TCT GCC TCC TTA AAT AC. 486 ddl D-alanine-D-alanine ligase F: 5'-TGC (C/T)CA AGT TCC TTA TGT GG R: 5'-CAC TGG GT(G/A) AAA CC(A/T) GGC AT 441 Table 1: Primer sequence and Product size of the Housekeeping genes For this, 25 μl of reaction mixture was prepared. In which 2.5 μl of 25 mM MgCl₂, 2.5 μl of Taq buffer, 0.5 μl of Taq polymerase, 0.5 μl of 2.5mM dNTPs along with 2 μl of template DNA and 15 μl of PCR water while 1 μl of forward and reverse primers were added in each PCR vial. PCR tubes were placed in the PCR machine for 35 cycles with the following conditions: Initial Denaturation 95⁰C for 5 minutes 17 Denaturation 95⁰C 30 seconds Annealing 50⁰C for 30 seconds Extension 72⁰C for 45 seconds Final extension 72⁰C for 10 minutes 1.5: Multiplex PCR: Multiplex PCR for serotyping was performed using 39 different sets of primer pairs in 8 different PCR reactions. The reaction conditions were used according to Centre of Disease (CDC) which were given on their website. Details of the primers used for multiplex is given below: Serotypes Primer sequences Product Size (bp) 1 F: CTC TAT AGA ATG GAG TAT ATA AAC TAT GGT TA R: CCA AAG AAA ATA CTA ACA TTA TCA CAA TAT TGG C 280 2 F: TAT CCC AGT TCA ATA TTT CTC CAC TAC ACC R: ACA CAA AAT ATA GGC AGA GAG AGA CTA CT 290 3 F: ATG GTG TGA TTT CTC CTA GAT TGG AAA GTA G R: CTT CTC CAA TTG CTT ACC AAG TGC AAT AAC G 371 4 F: CTG TTA CTT GTT CTG GAC TCT CGA TAA TTG G R: GCC CAC TCC TGT TAA AAT CCT ACC CGC ATT G 430 5 F: ATA CCT ACA CAA CTT CT ATT ATG CCT TTG TG R: GCT CGA TAA ACA TAA TCA ATA TTT GAA AAA GTA TG 362 6A/B F: AAT TTG TAT TTT ATT CAT GCC TAT ATC TGG R: TTA GCG GAG ATA ATT TAA AAT GAT GAC TA 250 7F/A F: CCT ACG GGA GGA TAT AAA ATT ATT TTT GAG R: CAA ATA CAC CAC TAT AGG CTG TTG AGA CTA AC 826 7C/B/40 F: CTA TCT CAG TCA TCT ATT GTT AAA GTT TAC GAC GGG A R: GAA CAT AGA TGT TGA GAC ATC TTT TGT AAT TTC 260 18 8 F: GAT GCC ATG AAT CAA GCA GTG GCT ATA AAT C R: ATC CTC GTG TAT AAT TTC AGG TAT GCC ACC 294 9V F: GGG TTC AAA G TC AGA CAG TG A AT TTA A R: CCA TGA ATG A AA TCA ACA TT G TCA GTA GC 816 10A F: GGT GTA GAT TTA CCA TTA GTG TCG GCA GAC R: GAA TTT CTT CTT TAA GAT TCG GAT ATT TCT C 628 10F/C/33C F: GGA GTT TAT CGG TAG TGC TCA TTT TAG CA R: CTA ACA AAT TCG CAA CAC GAG GCA ACA 248 11A F: GGA CAT GTT CAG GTG ATT TCC CAA TAT AGT G R: GAT TAT GAG TGT AAT TTA TTC CAA CTT CTC CC 463 12F F: GCA ACA AAC GGC GTG AAA GTA GTT G R: CAA GAT GAA TAT CAC TAC CAA TAA CAA AAC 376 13 F: TAC TAA GGT AAT CTC TGG AAA TCG AAA GG R: CTC ATG CAT TTT ATT AAC CG C TTT TTG TTC 655 14 F: CTT GGC GCA GGT GTC AGA ATT CCC TCT AC R: GCC AAA ATA CTG ACA AAG CTA GAA TAT AGC C 208 15A/F F: ATT AGT ACA GCT GCT GGA ATA TCT CTT C R: GAT CTA GTG AAC GTA CTA TTC CAA AC 436 15B/C F: TTG GAA TTT TTT AAT TAG TGG CTT ACC TA R: CAT CCG CTT ATT AAT TGA AGT AAT CTG AAC C 496 16F F: GAA TTT TTC AGG CGT GGG TGT TAA AAG R: CAG CAT ATA GCA CCG CTA AGC AAA TA 717 17F F: TTC GTG ATG ATA ATT CCA ATG ATC AAA CAA GAG R: GAT GTA ACA AAT TTG TAG CGA CTA AGG TCT GC 693 18C/F/B/A F: CTT AAT AGC TCT CAT TAT TCT TTT TTT AAG CC R: TTA TCT GTA AAC CAT ATC AGC ATC TGA AAC 573 19A F: GAG AGA TTC ATA ATC TTG CAC TTA GCC A R: CAT AAT AGC TAC AAA TGA CTC ATC GCC 566 19F F: GTT AAG ATT GCT GAT CGA TTA ATT GAT ATC C 304 19 R: GTA ATA TGT CTT TAG GGC GTT TAT GGC GAT AG 20 F: GAG CAA GAG TTT TTC ACC TGA CAG CGA GAA G R: CTA AAT TCC TGT AAT TTA GCT AAA ACT CTT ATC 514 21 F: CTA TGG TTA TTT CAA CTC AAT CGT CAC C R: GGC AAA CTC AGA CAT AGT ATA GCA TAG 192 22F F: GAG TAT AGC CAG ATT ATG GCA GTT TTA TTG TC R: CTC CAG CAC TTG CGC TGG AAA CAA CAG ACA AC 643 23A F: TAT TCT AGC AAG TGA CGA AGA TGC G R: CCA ACA TGC TTA AAA ACG CTG CTT TAC 722 23B F: CCA CAA TTA G CG CTA TAT TCA TTC AAT CG R: GTC CAC GCT GAA TAA AAT GAA GCT CCG 199 23F F: GTA ACA GTT GCT GTA GAG GGA ATT GGC TTT TC R: CAC AAC ACC TAA CAC ACG ATG GCT ATA TGA TTC 384 24A/F/B F: GCT CCC TGC TAT TGT AAT CTT TAA AGA G R: GTG TCT TTT ATT GAC TTT ATC ATA GGT CGG 99 31 F: GGA AGT TTT CAA GGA TAT GAT AGT GGT GGT GC R: CCG AAT AAT ATA TTC AAT ATA TTC ТА СТС 701 33F/A/37 F: GAA GGC AAT CAA TGT GAT TGT GTC GCG R: CTT CAA AAT GAA GAT TAT AGT ACC CTT CTA C 338 34 F: GCT TTT GTA AGA GGA GAT TAT TTT CAC CCA AC R: CAA TCC GAC TAA GTC TTC AGT AAA AAA CTT TAC 408 35A/35C/42 F: ATT ACG ACT CCT TAT GTG ACG CGC ATA R: CCA ATC CCA AGA TAT ATG CAA CTA GGT T 574 35B F: GAT AAG TCT GTT GTG GAG ACT TAA AAA GAA TG R: CTT TCC AGA TAA TTA CAG GTA TTC CTG AAG CAA G 677 35F F: GAA CAT AGT CGC TAT TGT ATT TTA TTT AAA GCA A R: GAC TAG GAG CAT TAT TCC TAG AGC GAG TAA ACC 517 38 F: CGT TCT TTT ATC TCA CTG TAT AGT ATC TTT ATG R: ATG TTT GAA TTA AAG CTA ACG TAA CAA TCC 574 20 39 F: TCA TTG TAT TAA CCC TAT GCT TTA TTG GTG R: GAG TAT CTC CAT TGT ATT GAA ATC TAC CAA 98 cpsA F: GCA GTA CAG CAG TTT GTT GGA CTG ACC R: GAA TAT TTT CAT TAT CAG TCC CAG TC 160 Table 2: List of primers used in multiplex PCR, their sequence and Product size. For this, 25 μl of reaction mixture was prepared. In which 25 μl of 2.5 mM MgCl, 2.5 μl of KCI buffer, 0.2 μl of Taq polymerase, 2.5 μl of 2.5 mM dNTPs along with 2 μl of template DNA while in each reaction primers where added in PCR vial according to recommended recipe of CDC. In each reaction 0.1 μl of cpsA primer was used as a positive control. PCR tubes were placed in the PCR machine for 35 cycles with the following conditions: Initial Denaturation 95⁰C for 15 minutes Denaturation 94⁰C 30 seconds Annealing 54⁰C for 1:30 minute Extension 72⁰C for 1:00 minute Final extension 72⁰C for 10 minutes This table shows the primer sets used in each Multiplex PCR. In each reaction 0.1μl of cpsA primer was used as a positive control. Pool Primer Primer conc. (μM) Reactions Primers Primer conc. (μM) 1 6A/B/C 0.3 5 14 0.3 3 0.3 1 0.3 19A 0.3 23F 0.5 22F 0.5 15B 0.3 16F 0.4 10A 0.5 21 2 8 0.2 6 39 0.2 33F 0.3 10F 0.3 15A 0.3 5 0.3 23A 0.5 35 0.3 7F/A 0.4 17F 0.5 3 19F 0.5 7 23B 0.2 12F 0.5 35A 0.3 11A 0.3 34 0.3 38 0.3 9N 0.5 35B 0.5 31 0.5 4 24 0.2 8 21 0.2 7C 0.3 2 0.3 4 0.3 20 0.3 18 0.3 13 0.4 9V 0.5 Table 3: Primer set used in each multiplex PCR. 1.6: Gel Electrophoresis: A 1% agarose gel was prepared using 1X TAE buffer and solidified by heating in the provided oven. After cooling, the gel was poured into a casting tray, and a comb was inserted to create wells. For sample preparation, 5 µl of PCR amplified DNA was mixed with 3 µl of 6X DNA loading dye. The prepared 8 µl of each sample was then carefully loaded into the wells of the agarose gel, which was submerged in 1X TAE buffer within the electrophoresis tank. Alongside the samples, 3 µl of a 100 bp DNA ladder was also loaded to facilitate the determination of DNA sizes. The gel tank was then closed, the power source switched on, and the gel was run at 120V and 300 mA for 40 minutes, allowing for the separation of DNA fragments by size. 22 Results: 2.1: Graphs: Graph 1: Percentage prevalence of Pneumococcal strain The graph depicts the percentage prevalence of various pneumococcal serotypes identified in the sample population. It is divided into three categories: serotypes included in the PCV-13 vaccine, non-vaccine serotypes, and non-typeable strains. Serotypes contained in PCV-13 are known as PCV-13 serotypes. The lower prevalence of PCV13 vaccine serotypes supports the ongoing efficacy of the PCV-13 in controlling these targeted strains. Non-Vaccine Serotypes are currently in no vaccine formulations. Serotypes 8 was found at larger percentages than others, indicating a high prevalence. These serotypes may represent natural diversity and evolution of pneumococcal serotypes or suggest possibilities for inclusion in future vaccines. Non-Typeable Strains (NT) are the strains which could not be serotyped using multiplex PCR technique. The largest prevalence is shown in the NT group, which may point to a wide variety of pneumococcal strains that are not detected by the multiplex PCR serotyping panels that are being employed. 23 Graph 2: Age wise distribution of serotypes in Pakistani population The graph represents the distribution of different serotypes of Streptococcus pneumoniae across various age groups in the Pakistani population. The age ranges are divided intro three categories: less than 15 years, 16 to 50 years, and over 50 years. The age-wise serotype distribution demonstrates a marked decrease in the serotypes among children under 15 years in Pakistan. This pattern suggests that the recent pediatric immunization campaigns have been successful. The decrease in the incidence of vaccine-included serotypes among the vaccinated cohorts can be attributed to the direct effect of the pneumococcal conjugate vaccination (PCV-13). This decline highlights the significance of ongoing and increased vaccination campaigns and the vaccine's contribution to changing the pneumococcal disease picture in this age group. The rise of non-vaccine serotypes and a sizable fraction of non-typeable strains, especially in the 16-50 years age group poses a difficult problem. According to these results, vaccination has been effective in reducing the prevalence of some serotypes, but it is also important to keep an eye on and comprehend the dynamics of strains that are not vaccinated or typeable. The increased frequency of these strains in the elderly may be due to previous vaccination coverage gaps. 0 1 2 3 4 5 6 1 3 14 15 B 8 15B 2 13 34 6C 7B 11A 16F 17F 19B 35A 7C NT PCV-13 Non Vaccine Serotypes Non Typeable Age wise distribution <15 years 16-50 years >50 years 24 Graph 3: Gender wise distribution of serotypes in Pakistani population The graph represents the Gender wise distribution of different serotypes of Streptococcus pneumoniae in the Pakistani population. The distribution of pneumococcal serotypes in Pakistan according to gender shows that the distribution of vaccine-included serotypes is comparatively equal between males and females. On the other hand, males have a significantly greater frequency of non-vaccine and non-typeable strains. This raises the possibility of variations in exposure or susceptibility to certain strains based on gender. The results emphasize the necessity of ongoing monitoring to comprehend the factors behind these variations and guarantee that public health initiatives, such as immunization campaigns, are appropriately catered to both genders. Graph 4: Geographical distribution of serotypes in Pakistani population 0 1 2 3 4 5 6 1 3 14 15 B 8 15B 2 13 34 6C 7B 11A 16F 17F 19B 35A 7C NT PCV-13 Non Vaccine Serotypes Non Typeable Gender wise distribution Female Male 0 1 2 3 4 5 1 3 14 15 B 8 15B 2 13 34 6C 7B 11A 16F 17F 19B 35A 7C NT PCV-13 Non Vaccine Serotypes Non Typeable Geographical distribution Bahawalnagar Chiniot Faisalabad Gujjar Khan Hazara Jhelum KPK Lahore Lala Musa Multan Pindi Bhatian Sahiwal Sadiqabad Sialkot Sukkur 25 The graph represents the Geographical distribution of different serotypes of Streptococcus pneumoniae in Pakistani population. It shows a variable incidence of non-typeable strains, vaccine-included serotypes (PCV-13), and non-vaccine serotypes. Notable findings include the large occurrence of non-typeable strains in several locations, with Sahiwal having the greatest frequency of these strains, and the concentrated prevalence of some non-vaccine serotypes in particular places. This regional variation could reflect differences in vaccination coverage, public health infrastructure, or local environmental factors that influence pneumococcal transmission. Graph 5: Distribution of serotypes by Site of Infection The graph represents the Distribution of serotypes by Site of Infection in Pakistani population. Serotypes included in the PCV-13 vaccine are found in pleural fluid, sputum, blood and CSF samples only. Non-vaccine serotypes found in pus, sputum, and blood suggest the ongoing need to assess the spectrum of serotype coverage provided by current vaccines. The predominance of non-typeable strains in tracheal secretions highlights a potential gap in our understanding of pneumococcal epidemiology and may indicate the circulation of novel or uncharacterized strains. 0 1 2 3 4 5 6 1 3 14 15 B 8 15B 2 13 34 6C 7B 11A 16F 17F 19B 35A 7C NT PCV-13 Non Vaccine Serotypes Non Typeable Site of Infection Blood CSF Ear Swab Pleural Fluid Pus Sputum Tracheal Secretions 32 Multiplex PCR 3: (From left to right) Leader, (+), 16/24, 17/24, 18/24, 70/23, 71/23, 72/23, 73/23, 74/23, 75/23, 76/23, 77/23, 24/24, 25/24 Multiplex PCR 4: (From left to right) Leader, (+), 16/24, 17/24, 18/24, 70/23, 71/23, 73/23, 74/23 , 75/23, 76/23, 77/23, 24/24, 25/24 Figure 11: Multiplex PCR 5 and 6 for Serotyping Multiplex PCR 5: (From left to right) Leader, (+), 16/24, 17/24, 18/24, 70/23, 71/23, 74/23, 75/23, 76/23, 77/23, 24/24, 25/24 Multiplex PCR 6: (From left to right) Leader, (+), 16/24, 17/24, 18/24, 70/23, 71/23, 74/23, 75/23, 76/23, 25/24 33 Figure 12: Multiplex PCR 7 and 8 for Serotyping Multiplex PCR 7: (From left to right) Leader, (+), 16/24, 17/24, 18/24, 70/23, 71/23, 74/23, 76/23, 25/24 Multiplex PCR 8: (From left to right) Leader, (+),16/24, 17/24, 18/24, 70/23, 74/23, 76/23, 25/24 Each multiplex PCR targets a group of serotypes, and if a sample tests positive for a serotype, it is not processed further in subsequent PCRs. Here is an explanation of each gel: Multiplex PCR 1: The serotyping findings for serotypes 6A/B, 3, 19A, 22F, and 16F are displayed on this gel. The presence of serotype 3 in sample 53/23 is shown by a distinct band at 371bp. The cpsA gene (160 bp) was positive in all samples, indicating that S. pneumoniae is present. Multiplex PCR 2: Serotypes 8, 33F, 15A, 23A, and 7F/A are the targeted in this PCR. Sample 19/24 marked shows the presence of serotype 8, with a corresponding band near 294bp. Multiplex PCR 3: Serotypes 19F, 12F, 11A, 38, and 35B are targeted in this PCR. Sample 72/23 marked shows the presence of serotype 35B, with a corresponding band near 677 bp. Multiplex PCR 4: Serotypes 24, 7C, 4, 18, and 9V are targeted in this PCR. Sample 71/23 marked shows the presence of serotype 7, with a corresponding band near 260 bp. Multiplex PCR 5: Serotypes 14, 1, 23F, 15B, and 10A are targeted in this PCR. Two samples (77/23 and 24/24) marked show the presence of serotype 1, with a corresponding band near 280bp. Multiplex PCR 6: Serotypes 39, 10F, 5, 35, and 17F are targeted in this PCR. Sample 75/23 marked shows the presence of serotype 17F with a corresponding band near 693 bp. Multiplex PCR 7: Serotypes 23B, 35A, 34, 9N, and 31 are targeted in this PCR. Sample 74/23 marked shows the presence of serotype 35A with a corresponding band near 574 bp. 34 Multiplex PCR 8: This final gel targets serotypes 21, 2, 20, and 13. Three samples (18/24, 70/23 and 71/23) were positive for serotypes 2,13 and 20 as indicated by bands at 290 bp, 655 bp and 514 bp respectively. Discussion: This thesis has described the distribution of post-PCV13 vaccine serotypes of Streptococcus pneumoniae in Pakistan. The results from the study on the distribution of Streptococcus pneumoniae serotypes in Pakistan reveal significant insights into the epidemiology of pneumococcal disease. Notably, there has been a substantial decrease in the prevalence of vaccine-included serotypes (PCV-13) among children under 15 years of age, indicating the success of pediatric vaccination campaigns. However, the study identified a rise in non-vaccine serotypes and non-typeable strains, particularly among adults aged 16 to 50 and the elderly. This pattern suggests that while vaccination has effectively controlled certain strains, others not included in the vaccine are becoming more prevalent, likely due to vaccine-driven selection pressures. The evolution of pneumococcal disease in Pakistan is indicative of a dynamic interaction between vaccination strategies and the natural adaptation of the pneumococcus population. This is similar to trends likely observed in neighboring countries with comparable vaccination programs, though specific differences in public health infrastructure and environmental factors could lead to distinct evolutionary paths. It has also found a significant decrease in children's vaccine-included serotype infections, but it has also noted persistent breakthrough infections, highlighting the necessity of ongoing epidemiological surveillance. Variable regional serotype distribution suggests the necessity for specific public health interventions, and the advent of non-vaccine serotypes and the non-typeable strains indicates possible serotype replacement and evolution beyond present vaccine coverage. 35 References: 1. Patterson, Maria Jevitz. “Streptococcus.” Nih.gov, University of Texas Medical Branch at Galveston, 2010, www.ncbi.nlm.nih.gov/books/NBK7611/. 2. Song, Joon Young, et al. “Clinical Implications of Pneumococcal Serotypes: Invasive Disease Potential, Clinical Presentations, and Antibiotic Resistance.” Journal of Korean Medical Science, vol. 28, no. 1, 2013, p. 4, https://doi.org/10.3346/jkms.2013.28.1.4. 3. 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Accessed 19 Aug. 2022. 38 Appendix A Materials Media for culturing of S.pnemoniae Table A1: Blood Agar Sr.no Ingredients Quantity (g/l) 1 Peptone 10 2 Beef Extract 10 3 NaCl 5 4 Agar 15 5 Distilled Water Upto 1000ml Mix all the contents well, then sterilize by autoclaving. For blood agar, cool to room temperature and add sheep blood to it. Table A2: Todd Hewitt Broth (THY) Sr.no Ingredients Quantity (g/l) 1 Peptone 20 2 Yeast Extract 5 39 3 NaCl 2 4 Dextrose 2 5 Disodium Phosphate 0.4 6 Heart infusion 3.1 7 Sodium carbonate 2.5 8 Distilled Water Upto 1000ml Reagents for DNA isolation: Table A3: 1X TE Buffer Sr.no Ingredients Quantity (g/l) 1 1M Tris (pH 8) 10 ml 2 0.5M EDTA (pH 8) 2 ml 3 Distilled Water Upto 1000ml Table A4: 10% SDS Sr.no. Ingredients Quantity (g/Liter) 1 Sodium dodecyl sulphate 10 2 Distilled water Upto 100 ml 40 Table A5: 5M Potassium acetate Sr.no. Ingredients Quantity (g/Liter) 1 Potassium acetate 294.42 g 2 Distilled water Upto 100 ml Adjust the pH of solution at 4.6 then make up the volume upto 1000s ml with distilled water. Table A6: 95% Ethanol Sr.no. Ingredients Quantity (per Liter) 1 Absolute Ethanol 95 ml 2 Distilled water 5 ml Table A7: 70% Ethanol Sr.no. Ingredients Quantity (per Liter) 1 Absolute Ethanol 70 ml 2 Distilled water 30 ml Reagents for Multiplex PCR Table A8: 25μl Reaction mixture Sr.no. Ingredients Quantity 1 25mM MgCl₂ 2.5μl 41 2 Taq buffer 2.5μl 3 2.5mM dNTPS 2.5μl 4 Taq polymerase 0.2μl 5 Primer F According to reaction 6 Primer R According to reaction 7 DNA Template 2μl 8 PCR water Upto 25μl Reagents for PCR of Housekeeping genes Table A9: 25μl Reaction mixture Sr.no . Ingredients Quantity 1 25nM MgCl₂ 2.5μl 2 Taq buffer 2.5μl 3 2.5mM dNTPS 0.5μl 4 Taq polymerase 0.5μl 5 Primer F 1μl 6 Primer R 1μl 7 DNA Template 2μl 8 PCR water 15μl