1
Cha ac e iza ion o S ep ococcus pneumoniae
se o ypes in pos accine e a in Pakis an
Depa men o Biology
Syed Baba Ali School o Science and Enginee ing
Laho e Uni e si y o Managemen Sciences (LUMS)
Submi ed in Pa ial Ful ilmen o he Requi emen s o he deg ee o
Bachelo o Sciences
Is a Razzak
24100306
Ad iso :
D . Shape Mi za
2
DECLARATION
This is o ce i y ha his disse a ion en i led as “Cha ac e iza ion o S ep ococcus pneumoniae
se o ypes in pos accine e a in Pakis an” submi ed by Is a Razzak, unde he supe ision o D .
Shape , is accep ed in i s p esen o m by he Depa men o Biology, Laho e uni e si y o
managemen sciences, Laho e, Pakis an as sa is ying he disse a ion o he deg ee o BS in
Biology. I he eby decla e ha his submission is my own wo k and ha , o he bes o my
knowledge and belie , con ains no ma e ial p e iously published o w i en by any o he pe son
nei he has i been accep ed o ewa d o any o he deg ee a a uni e si y o any o he ins i u e
o highe lea ning, excep whe e due acknowledgemen has been made in he ex .
- Is a Razzak
Supe iso :
D . Shape Mi za
Associa e P o esso , Depa men o Biology
Laho e Uni e si y o Managemen Sciences (LUMS)
3
Acknowledgemen s:
I am p o oundly g a e ul o a numbe o indi iduals whose guidance and suppo ha e been
in aluable du ing he cou se o my hesis.
Fi s and o emos , I ex end my deepes g a i ude o my pa en s, whose unwa e ing suppo
ha e been my cons an sou ce o s eng h h oughou . Thei sac i ices ha e no gone unno iced,
and his achie emen is as much hei s as i is mine.
I am especially hank ul o my supe iso , D . Shape Mi za, o p o iding me wi h he
oppo uni y o wo k unde he guidance. He expe ise and insigh s ha e been c ucial in my
lea ning and g ow h.
My hea el hanks go o my iends, pa icula ly Yus a and Fa ima, whose p esence has been
my s eng h in he mos challenging imes.
I would also like o exp ess my g a i ude o D . Asia, who has been an inc edible men o
h oughou his p ocess. He de ailed eedback has been in aluable in imp o ing my wo k.
I would also like o hank Zuha Bilal, who ini ia ed his p ojec . He ea ly con ibu ions we e
i al in shaping he di ec ion o my esea ch.
Finally, I ex end my app ecia ion o all he membe s o he MEM lab o p o iding a
suppo i e wo k en i onmen .
Thank you all o you con ibu ions, which ha e been i al o he comple ion o his hesis.
Bes ,
Is a Razzak - 24100306
4
Table o Con en s
Abs ac : .......................................................................................................................................... 6
In oduc ion: .................................................................................................................................... 7
Me hodology: ................................................................................................................................ 11
Ma e ials and Me hods:................................................................................................................. 13
Me hods: ....................................................................................................................................... 13
1.1: G ow h o Pneumococcal S ain on Blood Aga : ............................................................... 14
1.2: DNA Isola ion: ................................................................................................................... 14
1.3: ly A and pspA PCR: ........................................................................................................... 15
1.4: Housekeeping genes PCR: ................................................................................................. 15
1.5: Mul iplex PCR: .................................................................................................................. 17
1.6: Gel Elec opho esis: ........................................................................................................... 21
Resul s: .......................................................................................................................................... 22
2.1: G aphs: ............................................................................................................................... 22
2.2: DNA Isola ion: ................................................................................................................... 26
2.3: ly A PCR: ............................................................................................................................ 27
2.4: pspA PCR: .......................................................................................................................... 28
2.5: Housekeeping genes PCR: ................................................................................................. 29
2.6: Mul iplex PCR: .................................................................................................................. 31
Discussion: .................................................................................................................................... 34
Re e ences: .................................................................................................................................... 35
Appendix A .................................................................................................................................. 38
Table A1: Blood Aga ................................................................................................................ 38
Table A2: Todd Hewi B o h (THY) ........................................................................................ 38
Table A3: 1X TE Bu e ............................................................................................................ 39
Table A4: 10% SDS ................................................................................................................... 39
Table A5: 5M Po assium ace a e ............................................................................................... 40
Table A6: 95% E hanol ............................................................................................................. 40
Table A7: 70% E hanol ............................................................................................................. 40
5
Table A11: 1% Aga ose gel ....................................................................................................... 42
APPENDIX B ............................................................................................................................... 43
Table B1: Demog aphic da a o pneumococcal isola es ........................................................... 43
6
Abs ac :
S ep ococcus pneumoniae is he leading cause o dea h and kills an es ima ed 1.1 million
child en unde he i e yea s e e y yea which is mo e han AIDS, mala ia and ube culosis
combined. The o ganism is esponsible o causing a ious in ec ions including mild espi a o y
in ec ions like sinusi is and o i is media o mo e se e e diseases such as pneumonia, meningi is,
and bac e emia. Popula ion a isk o in ec ion include child en unde he age o 5, adul s abo e
65 yea s o age and immunocomp omised indi iduals. Pneumococcal in ec ions a e accine
p e en able and cu en ly he e a e ou accines (PCV 7,10,13 and 24) a ailable in ma ke o
immunizing child en and adul s a isk o in ec ions. Pakis an in oduced PCV 10 in yea 2012 o
lowe he bu den o in ec ions. While educing he bu den o in ec ion by accine s ains,
immuniza ion also esul ed in phenomenon including, se o ype eplacemen and accine escape.
The e is a weal h o in o ma ion a ailable on how hese phenomenon a e e ol ing globally,
howe e , he e is signi ican dea h o in o ma ion on bo h o hese o Pakis an.
O e all objec i e o his s udy is o in es iga es pos - accine se o ype eplacemen and quan i y
bu den o in ec ion caused by non- accine s ains. We hypo hesize ha immuniza ion agains S.
pneumoniae is changing he popula ion biology o S. pneumoniae esul ing in inc eased bu den
o in ec ion by non- accine s ains. To es his hypo hesis, we ha e designed he ollowing aims.
The aim o he s udy includes:
1. To de e mine he p opo ion o in ec ions caused by accine se o ypes o S.pneumoniae.
2. To measu e he bu den o accine escape and se o ype eplaced s ains.
3. Cha ac e ize se o ypes on he basis o geog aphic dis ibu ion, demog aphic and clinical
cha ac e is ics.
Samples ob ained om a e ia y and a clinical labo a o y we e subjec ed o cul u e, op ochin
esis ance o di e en ia ion be ween pneumococci and species o i idians g oup and inally o
PCR using p ime s o ly A and pspA wo genes ha a e p esen in all pneumococci iden i ied o
da e. Fo con i ma ion o se o ypes, we used mul iplex PCR. O e all, ou esul s demons a ed
a dec ease in he bu den o in ec ion by accine se o ypes, as obse ed by he low abundance o
PCV se o ypes in bo h ca iage and in asi e s ains. Ne e heless, clinical samples exhibi ed a
signi ican p e alence o PCV13 se o ypes, indica ing he eme gence o accina ion escape
7
s ains. Non- accine se o ypes we e mo e common in pus, spu um, and blood samples, and non-
ypeable s ains we e p edominan ly de ec ed in acheal sec e ions, sugges ing a possible
de iciency in he p esen accine's e ec i eness.
Geog aphical dis ibu ion in Pakis an showed di e ences in he dis ibu ion o se o ypes,
emphasizing he impo ance o de eloping accina ion egimens ha a e speci ically a ge ed o
di e en egions.
In oduc ion:
S.pneumoniae causes pneumonia, o i is media and o he in asi e diseases such as bac e emia and
meningi is, collec i ely called pneumococcal diseases (PD). In 2017, he WHO included S.
pneumoniae as one o 12 p io i y pa hogens (Weise e al.). I is an oppo unis ic pa hogen ha
colonizes he mucosal su aces o he human uppe espi a o y ac . Pneumococcus plays a
majo ole in he mo bidi y and mo ali y o child en as well as he elde ly wo ldwide. I is
es ima ed ha ~ 14.5 million cases o PD occu each yea , causing he dea hs o ~ 1 million
child en < 5 yea s o age (Khan e al.). Pneumonia is he leading cause o dea h o child en
unde i e, especially in Pakis an whe e one- i h o he popula ion alls in o his age ange.
Acco ding o es ima es ound in nume ous Pakis ani esea ch, he [annual] incidence o acu e
espi a o y in ec ion (ARI) among child en unde i e in Pakis an is 4% in he communi y,
which makes up a ound 22% o he 160 million people li ing in he na ion. Using his 4%
es ima e, we may de e mine ha he e a e 15 million ARI episodes among child en unde i e
annually (“Pneumonia – Fede al Di ec o a e o Immuniza ion, Pakis an”).
S ep ococcus genus is di ided in o 49 species and eigh subspecies, om which as many as 35
ha e been iden i ied as sou ces o in asi e in ec ions in humans. The s udy o S ep ococcus
spans ac oss unde s anding i s pa hogenic mechanisms, an ibio ic esis ance pa e ns, and
de eloping e ec i e accines, highligh ing i s signi ican impac on medical mic obiology and
public heal h.
S ep ococci a e G am-posi i e, nonmo ile, non-spo e o ming, ca alase-nega i e cocci ha occu
in pai s o chains. While some s ep ococci a e obliga e anae obes, he majo i y a e acul a i e
anae obes. S ep ococci a e classi ied on he basis o colony mo phology, hemolysis,
biochemical eac ions, and se ologic speci ici y. Thei hemolysis on blood aga is classi ied in o
8
h ee g oups: β-hemoly ic - comple e, clea lysis o ed cells, α hemoly ic - incomple e, g een
hemolysis, and γ hemoly ic - no hemolysis (Pa e son).
Pneumococcal su i es in nasopha yngeal ca i y howe e , i can in ade in o deepe issues, an
abili y ha is impa ed by a ious cell su ace and in acellula p o eins e med as i ulence
ac o s. Among he mos impo an i ulence ac o s a e capsula polysaccha ides, pneumolysin,
pneumococcal su ace p o ein A (PspA), pneumococcal su ace p o ein C (PspC), and
pneumococcal su ace adhesin A (PsaA). Capsula polysaccha ides a e conside ed he mos
essen ial i ulence ac o because hey su ound and p o ec pneumococci om phagocy es
(Song e al.).
S. pneumoniae is no able o i s wide a ie y o se o ypes— o e 100 —each de ined by a unique
chemical and gene ic composi ion o i s capsule. This di e si y is signi ican o bo h i s
i ulence and he challenges i poses o accine de elopmen . S. pneumoniae belongs o he α
hemoly ic g oup as i has g eenish colonies on blood aga (Yesilkaya e al.). Op ochin sensi i i y
is a hallma k ai o S.pneumoniae, inhibi ing i s g ow h nea op ochin discs on blood aga due
o i s unique cell wall p ope ies, leading o cell lysis.
The capsula polysaccha ides a e composed o epea ing uni s o oligosaccha ides. The s uc u e
o he majo i y o his capsula polysaccha ide is al eady known. Capsula ype is known o be
immunogenic and elici an immune esponse agains bo h ca iage and in asi e disease. While
an ibodies elici ed o mos capsula ypes do no c oss eac , capsules wi h iden ical chemical
composi ions a e known o ha e c oss eac i e an ibodies. Fo ins ance, se o ypes 6A and 6B
ha e almos iden ical chemical composi ions, excep o a single bond be ween wo suga s, and
hey exhibi ex ensi e c oss- eac i i y (Robbins e al.). On he o he hand, se o ypes 19F and
19A show less c oss- eac i i y (Ta aso a e al.).
9
Figu e 1: Illus a ion o he a angemen o he pneumococcal cell wall and capsule. (KALIN)
Molecula analysis o he genes esponsible o he syn hesis o ce ain capsula componen s has
demons a ed ha hey a e o de ed in casse es and con ain all he gene ic ma e ial necessa y o
capsule syn hesis (Dilla d e al.). The pneumococcus, as well as se e al o he s ep ococci, is
na u ally ans o mable and exchange gene ic ma e ial wi h and ac oss species. By such a
mechanism he capsule speci ici y, in a casse e ype ecombina ion e en , can be eplaced in
i o as well as in i o. Occasionally pneumococcal bac e ia may possess bo h o wo dis inc
polysaccha ide coding gene casse es a he same ime. I has been pos ula ed ha subsequen
ecombina ion e en s in such s ains may c ea e no el capsule ypes (Aus ian).
Figu e 2: P og ession o Pneumococcal in ec ions (Weise e al.)
De elopmen o pneumococcal accines was a medical ad ancemen ha signi ican ly impac ed
he bu den o bo h mo bidi y and mo ali y due o in asi e pneumococcal in ec ions. (Wiese e
al.). The i s accine o pneumococcal in ec ions con ained 23 pneumococcal se o ypes (PPV-
23)and was de eloped in he yea 1977. While he accine p o ided minimal p o ec ion agains
in asi e in ec ion, owing o i T-cell independen na u e he accine p o ided empo a y
immuni y wi hou inducing memo y B cells o iso ype swi ching. Al hough he accine was
16
Loci
Func ion o gene
P ime Sequence
P oduc
Size (bp)
a oE
shikima e dehyd ogenase
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-phospha e
dehyd ogenase
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
ecP
anske olase
F: 5'-GCC AAC TCA GGT CAT CCA GG
R: 5'- TGC AAC CGT AGC ATT GTA AC
450
spi
signal pep idase I
F: 5'-TTA TTC CTC CTG ATT CTG TC
R: 5'-GTG ATT GGC CAG AAG CGG AA
474
xp
xan hine
phospho ibosyl ans e ase
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: P ime sequence and P oduc size o he Housekeeping genes
Fo his, 25 μl o eac ion mix u e was p epa ed. In which 2.5 μl o 25 mM MgCl₂, 2.5 μl o Taq
bu e , 0.5 μl o Taq polyme ase, 0.5 μl o 2.5mM dNTPs along wi h 2 μl o empla e DNA and
15 μl o PCR wa e while 1 μl o o wa d and e e se p ime s we e added in each PCR ial.
PCR ubes we e placed in he PCR machine o 35 cycles wi h he ollowing condi ions:
Ini ial Dena u a ion 95⁰C o 5 minu es
17
Dena u a ion 95⁰C 30 seconds
Annealing 50⁰C o 30 seconds
Ex ension 72⁰C o 45 seconds
Final ex ension 72⁰C o 10 minu es
1.5: Mul iplex PCR:
Mul iplex PCR o se o yping was pe o med using 39 di e en se s o p ime pai s in 8 di e en
PCR eac ions. The eac ion condi ions we e used acco ding o Cen e o Disease (CDC) which
we e gi en on hei websi e.
De ails o he p ime s used o mul iplex is gi en below:
Se o ypes
P ime sequences
P oduc
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: Lis o p ime s used in mul iplex PCR, hei sequence and P oduc size.
Fo his, 25 μl o eac ion mix u e was p epa ed. In which 25 μl o 2.5 mM MgCl, 2.5 μl o KCI
bu e , 0.2 μl o Taq polyme ase, 2.5 μl o 2.5 mM dNTPs along wi h 2 μl o
empla e DNA while in each eac ion p ime s whe e added in PCR ial acco ding o
ecommended ecipe o CDC. In each eac ion 0.1 μl o cpsA p ime was used as a posi i e
con ol.
PCR ubes we e placed in he PCR machine o 35 cycles wi h he ollowing condi ions:
Ini ial Dena u a ion 95⁰C o 15 minu es
Dena u a ion 94⁰C 30 seconds
Annealing 54⁰C o 1:30 minu e
Ex ension 72⁰C o 1:00 minu e
Final ex ension 72⁰C o 10 minu es
This able shows he p ime se s used in each Mul iplex PCR. In each eac ion 0.1μl o cpsA
p ime was used as a posi i e con ol.
Pool
P ime
P ime conc.
(μM)
Reac ions
P ime s
P ime 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: P ime se used in each mul iplex PCR.
1.6: Gel Elec opho esis:
A 1% aga ose gel was p epa ed using 1X TAE bu e and solidi ied by hea ing in he p o ided
o en. A e cooling, he gel was pou ed in o a cas ing ay, and a comb was inse ed o c ea e
wells.
Fo sample p epa a ion, 5 µl o PCR ampli ied DNA was mixed wi h 3 µl o 6X DNA loading
dye. The p epa ed 8 µl o each sample was hen ca e ully loaded in o he wells o he aga ose
gel, which was subme ged in 1X TAE bu e wi hin he elec opho esis ank. Alongside he
samples, 3 µl o a 100 bp DNA ladde was also loaded o acili a e he de e mina ion o DNA
sizes. The gel ank was hen closed, he powe sou ce swi ched on, and he gel was un a 120V
and 300 mA o 40 minu es, allowing o he sepa a ion o DNA agmen s by size.
22
Resul s:
2.1: G aphs:
G aph 1: Pe cen age p e alence o Pneumococcal s ain
The g aph depic s he pe cen age p e alence o a ious pneumococcal se o ypes iden i ied in he
sample popula ion. I is di ided in o h ee ca ego ies: se o ypes included in he PCV-13 accine,
non- accine se o ypes, and non- ypeable s ains.
Se o ypes con ained in PCV-13 a e known as PCV-13 se o ypes. The lowe p e alence o PCV-
13 accine se o ypes suppo s he ongoing e icacy o he PCV-13 in con olling hese a ge ed
s ains.
Non-Vaccine Se o ypes a e cu en ly in no accine o mula ions. Se o ypes 8 was ound a la ge
pe cen ages han o he s, indica ing a high p e alence. These se o ypes may ep esen na u al
di e si y and e olu ion o pneumococcal se o ypes o sugges possibili ies o inclusion in u u e
accines.
Non-Typeable S ains (NT) a e he s ains which could no be se o yped using mul iplex PCR
echnique. The la ges p e alence is shown in he NT g oup, which may poin o a wide a ie y
o pneumococcal s ains ha a e no de ec ed by he mul iplex PCR se o yping panels ha a e
being employed.
23
G aph 2: Age wise dis ibu ion o se o ypes in Pakis ani popula ion
The g aph ep esen s he dis ibu ion o di e en se o ypes o S ep ococcus pneumoniae ac oss
a ious age g oups in he Pakis ani popula ion. The age anges a e di ided in o h ee ca ego ies:
less han 15 yea s, 16 o 50 yea s, and o e 50 yea s.
The age-wise se o ype dis ibu ion demons a es a ma ked dec ease in he se o ypes among
child en unde 15 yea s in Pakis an. This pa e n sugges s ha he ecen pedia ic immuniza ion
campaigns ha e been success ul. The dec ease in he incidence o accine-included se o ypes
among he accina ed coho s can be a ibu ed o he di ec e ec o he pneumococcal
conjuga e accina ion (PCV-13). This decline highligh s he signi icance o ongoing and
inc eased accina ion campaigns and he accine's con ibu ion o changing he pneumococcal
disease pic u e in his age g oup.
The ise o non- accine se o ypes and a sizable ac ion o non- ypeable s ains, especially in he
16-50 yea s age g oup poses a di icul p oblem. Acco ding o hese esul s, accina ion has been
e ec i e in educing he p e alence o some se o ypes, bu i is also impo an o keep an eye on
and comp ehend he dynamics o s ains ha a e no accina ed o ypeable. The inc eased
equency o hese s ains in he elde ly may be due o p e ious accina ion co e age 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 Se o ypes Non Typeable
Age wise dis ibu ion
<15 yea s 16-50 yea s >50 yea s
24
G aph 3: Gende wise dis ibu ion o se o ypes in Pakis ani popula ion
The g aph ep esen s he Gende wise dis ibu ion o di e en se o ypes o S ep ococcus
pneumoniae in he Pakis ani popula ion.
The dis ibu ion o pneumococcal se o ypes in Pakis an acco ding o gende shows ha he
dis ibu ion o accine-included se o ypes is compa a i ely equal be ween males and emales.
On he o he hand, males ha e a signi ican ly g ea e equency o non- accine and non- ypeable
s ains. This aises he possibili y o a ia ions in exposu e o suscep ibili y o ce ain s ains
based on gende . The esul s emphasize he necessi y o ongoing moni o ing o comp ehend he
ac o s behind hese a ia ions and gua an ee ha public heal h ini ia i es, such as immuniza ion
campaigns, a e app op ia ely ca e ed o bo h gende s.
G aph 4: Geog aphical dis ibu ion o se o ypes in Pakis ani popula ion
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 Se o ypes Non Typeable
Gende wise dis ibu ion
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 Se o ypes Non Typeable
Geog aphical dis ibu ion
Bahawalnaga Chinio Faisalabad Gujja Khan Haza a
Jhelum KPK Laho e Lala Musa Mul an
Pindi Bha ian Sahiwal Sadiqabad Sialko Sukku
25
The g aph ep esen s he Geog aphical dis ibu ion o di e en se o ypes o S ep ococcus
pneumoniae in Pakis ani popula ion.
I shows a a iable incidence o non- ypeable s ains, accine-included se o ypes (PCV-13), and
non- accine se o ypes. No able indings include he la ge occu ence o non- ypeable s ains in
se e al loca ions, wi h Sahiwal ha ing he g ea es equency o hese s ains, and he
concen a ed p e alence o some non- accine se o ypes in pa icula places. This egional
a ia ion could e lec di e ences in accina ion co e age, public heal h in as uc u e, o local
en i onmen al ac o s ha in luence pneumococcal ansmission.
G aph 5: Dis ibu ion o se o ypes by Si e o In ec ion
The g aph ep esen s he Dis ibu ion o se o ypes by Si e o In ec ion in Pakis ani popula ion.
Se o ypes included in he PCV-13 accine a e ound in pleu al luid, spu um, blood and CSF
samples only. Non- accine se o ypes ound in pus, spu um, and blood sugges he ongoing need
o assess he spec um o se o ype co e age p o ided by cu en accines. The p edominance o
non- ypeable s ains in acheal sec e ions highligh s a po en ial gap in ou unde s anding o
pneumococcal epidemiology and may indica e he ci cula ion o no el o uncha ac e ized s ains.
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 Se o ypes Non Typeable
Si e o In ec ion
Blood CSF Ea Swab Pleu al Fluid Pus Spu um T acheal Sec e ions
32
Mul iplex PCR 3: (F om le o igh )
Leade , (+), 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
Mul iplex PCR 4: (F om le o igh )
Leade , (+), 16/24, 17/24, 18/24, 70/23, 71/23, 73/23, 74/23 , 75/23, 76/23, 77/23, 24/24, 25/24
Figu e 11: Mul iplex PCR 5 and 6 o Se o yping
Mul iplex PCR 5: (F om le o igh )
Leade , (+), 16/24, 17/24, 18/24, 70/23, 71/23, 74/23, 75/23, 76/23, 77/23, 24/24, 25/24
Mul iplex PCR 6: (F om le o igh )
Leade , (+), 16/24, 17/24, 18/24, 70/23, 71/23, 74/23, 75/23, 76/23, 25/24
33
Figu e 12: Mul iplex PCR 7 and 8 o Se o yping
Mul iplex PCR 7: (F om le o igh )
Leade , (+), 16/24, 17/24, 18/24, 70/23, 71/23, 74/23, 76/23, 25/24
Mul iplex PCR 8: (F om le o igh )
Leade , (+),16/24, 17/24, 18/24, 70/23, 74/23, 76/23, 25/24
Each mul iplex PCR a ge s a g oup o se o ypes, and i a sample es s posi i e o a se o ype, i
is no p ocessed u he in subsequen PCRs. He e is an explana ion o each gel:
Mul iplex PCR 1: The se o yping indings o se o ypes 6A/B, 3, 19A, 22F, and 16F a e
displayed on his gel. The p esence o se o ype 3 in sample 53/23 is shown by a dis inc band a
371bp. The cpsA gene (160 bp) was posi i e in all samples, indica ing ha S. pneumoniae is
p esen .
Mul iplex PCR 2: Se o ypes 8, 33F, 15A, 23A, and 7F/A a e he a ge ed in his PCR. Sample
19/24 ma ked shows he p esence o se o ype 8, wi h a co esponding band nea 294bp.
Mul iplex PCR 3: Se o ypes 19F, 12F, 11A, 38, and 35B a e a ge ed in his PCR. Sample 72/23
ma ked shows he p esence o se o ype 35B, wi h a co esponding band nea 677 bp.
Mul iplex PCR 4: Se o ypes 24, 7C, 4, 18, and 9V a e a ge ed in his PCR. Sample 71/23
ma ked shows he p esence o se o ype 7, wi h a co esponding band nea 260 bp.
Mul iplex PCR 5: Se o ypes 14, 1, 23F, 15B, and 10A a e a ge ed in his PCR. Two samples
(77/23 and 24/24) ma ked show he p esence o se o ype 1, wi h a co esponding band nea
280bp.
Mul iplex PCR 6: Se o ypes 39, 10F, 5, 35, and 17F a e a ge ed in his PCR. Sample 75/23
ma ked shows he p esence o se o ype 17F wi h a co esponding band nea 693 bp.
Mul iplex PCR 7: Se o ypes 23B, 35A, 34, 9N, and 31 a e a ge ed in his PCR. Sample 74/23
ma ked shows he p esence o se o ype 35A wi h a co esponding band nea 574 bp.
34
Mul iplex PCR 8: This inal gel a ge s se o ypes 21, 2, 20, and 13. Th ee samples (18/24, 70/23
and 71/23) we e posi i e o se o ypes 2,13 and 20 as indica ed by bands a 290 bp, 655 bp and
514 bp espec i ely.
Discussion:
This hesis has desc ibed he dis ibu ion o pos -PCV13 accine se o ypes o S ep ococcus
pneumoniae in Pakis an. The esul s om he s udy on he dis ibu ion o S ep ococcus
pneumoniae se o ypes in Pakis an e eal signi ican insigh s in o he epidemiology o
pneumococcal disease. No ably, he e has been a subs an ial dec ease in he p e alence o
accine-included se o ypes (PCV-13) among child en unde 15 yea s o age, indica ing he
success o pedia ic accina ion campaigns. Howe e , he s udy iden i ied a ise in non- accine
se o ypes and non- ypeable s ains, pa icula ly among adul s aged 16 o 50 and he elde ly. This
pa e n sugges s ha while accina ion has e ec i ely con olled ce ain s ains, o he s no
included in he accine a e becoming mo e p e alen , likely due o accine-d i en selec ion
p essu es. The e olu ion o pneumococcal disease in Pakis an is indica i e o a dynamic
in e ac ion be ween accina ion s a egies and he na u al adap a ion o he pneumococcus
popula ion. This is simila o ends likely obse ed in neighbo ing coun ies wi h compa able
accina ion p og ams, hough speci ic di e ences in public heal h in as uc u e and
en i onmen al ac o s could lead o dis inc e olu iona y pa hs. I has also ound a signi ican
dec ease in child en's accine-included se o ype in ec ions, bu i has also no ed pe sis en
b eak h ough in ec ions, highligh ing he necessi y o ongoing epidemiological su eillance.
Va iable egional se o ype dis ibu ion sugges s he necessi y o speci ic public heal h
in e en ions, and he ad en o non- accine se o ypes and he non- ypeable s ains indica es
possible se o ype eplacemen and e olu ion beyond p esen accine co e age.
35
Re e ences:
1. Pa e son, Ma ia Je i z. “S ep ococcus.” Nih.go , Uni e si y o Texas Medical B anch a
Gal es on, 2010, www.ncbi.nlm.nih.go /books/NBK7611/.
2. Song, Joon Young, e al. “Clinical Implica ions o Pneumococcal Se o ypes: In asi e
Disease Po en ial, Clinical P esen a ions, and An ibio ic Resis ance.” Jou nal o Ko ean
Medical Science, ol. 28, no. 1, 2013, p. 4, h ps://doi.o g/10.3346/jkms.2013.28.1.4.
3. Yesilkaya, Hasan, e al. “S ep ococcus Pneumoniae: “Cap ain o he Men o Dea h” and
Financial Bu den.” Mic obiology, ol. 168, no. 12, 2 Dec. 2022,
h ps://doi.o g/10.1099/mic.0.001275. Accessed 24 Dec. 2022.
4. Robbins, J. B., e al. “Conside a ions o Fo mula ing he Second-Gene a ion
Pneumococcal Capsula Polysaccha ide Vaccine wi h Emphasis on he C oss-Reac i e
Types wi hin G oups.” Jou nal o In ec ious Diseases, ol. 148, no. 6, 1 Dec. 1983, pp.
1136–1159, h ps://doi.o g/10.1093/in dis/148.6.1136. Accessed 20 Ma . 2021.
5. Ta aso a, Alla A., e al. “App oaches o Vaccine P ophilaxis o Respi a o y In ec ions in
Pa ien s wi h Diabe es Melli us in he Mode n Epidemiological Si ua ion.” Diabe es
Melli us, ol. 22, no. 5, 17 Jan. 2020, pp. 473–480, h ps://doi.o g/10.14341/dm9820.
Accessed 11 Feb. 2020.
6. Dilla d, J P, e al. “Cha ac e iza ion o he Casse e Con aining Genes o Type 3
Capsula Polysaccha ide Biosyn hesis in S ep ococcus Pneumoniae.” The Jou nal o
Expe imen al Medicine, ol. 181, no. 3, 1 Ma . 1995, pp. 973–983,
h ps://doi.o g/10.1084/jem.181.3.973. Accessed 26 Ap . 2020.
7. Aus ian, Robe . “Some Obse a ions on he Pneumococcus and on he Cu en S a us o
Pneumococcal Disease and I s P e en ion.” Clinical In ec ious Diseases, ol. 3, no.
Supplemen _1, 1 Ma . 1981, pp. S1–S17,
h ps://doi.o g/10.1093/clinids/3.supplemen _1.s1. Accessed 16 Sep . 2022.
8. Weise , Je ey N., e al. “S ep ococcus Pneumoniae: T ansmission, Coloniza ion and
In asion.” Na u e Re iews Mic obiology, ol. 16, no. 6, 2018, pp. 355–367,
www.ncbi.nlm.nih.go /pmc/a icles/PMC5949087/, h ps://doi.o g/10.1038/s41579-018-
0001-8.
9. Khan, Faidad, e al. “Molecula Cha ac e iza ion o Pneumococcal Su ace P o ein a
(PspA), Se o ype Dis ibu ion and An ibio ic Suscep ibili y o S ep ococcus Pneumoniae
36
S ains Isola ed om Pakis an.” In ec ious Diseases and The apy, ol. 7, no. 2, 9 Ma .
2018, pp. 277–289, h ps://doi.o g/10.1007/s40121-018-0195-0.
10. De ance, Thie y, e al. “T Cell-Independen B Cell Memo y.” Cu en Opinion in
Immunology, ol. 23, no. 3, June 2011, pp. 330–336,
h ps://doi.o g/10.1016/j.coi.2011.03.004.
11. Rennels, Ma ga e B, e al. Sa e y and Immunogenici y o Hep a alen Pneumococcal
Vaccine Conjuga ed o CRM197 in Uni ed S a es In an s. Vol. 101, no. 4, 1 Ap . 1998,
pp. 604–611, h ps://doi.o g/10.1542/peds.101.4.604. Accessed 31 May 2023.
12. Di ec and Indi ec E ec s o Rou ine Vaccina ion o Child en wi h 7-Valen
Pneumococcal Conjuga e Vaccine on Incidence o In asi e Pneumococcal Disease--
Uni ed S a es, 1998-2003.” MMWR. Mo bidi y and Mo ali y Weekly Repo , 16 Sep .
2005, pubmed.ncbi.nlm.nih.go /16163262/.
13. Hausdo , W. P., e al. “Which Pneumococcal Se og oups Cause he Mos In asi e
Disease: Implica ions o Conjuga e Vaccine Fo mula ion and Use, Pa I.” Clinical
In ec ious Diseases, ol. 30, no. 1, 1 Jan. 2000, pp. 100–121,
h ps://doi.o g/10.1086/313608.
14. Wiese, And ew D., e al. “Impac o Pneumococcal Conjuga e Vaccines on
Hospi aliza ions o Pneumonia in he Uni ed S a es.” Expe Re iew o Vaccines, ol. 18,
no. 4, 20 Ma . 2019, pp. 327–341, h ps://doi.o g/10.1080/14760584.2019.1582337.
Accessed 15 May 2020.
15. Te eziu, S ela, and Da id A. Min e . “Pneumococcal Vaccine.” PubMed, S a Pea ls
Publishing, 2020, www.ncbi.nlm.nih.go /books/NBK507794/.
16. Spanelo a, Pe a, e al. “Whole Genome Sequencing o Mac olide Resis an
S ep ococcus Pneumoniae Se o ype 19A Sequence Type 416.” BMC Mic obiology, ol.
20, no. 1, 25 July 2020, h ps://doi.o g/10.1186/s12866-020-01909-1. Accessed 20 Ap .
2022.
17. Pneumonia – Fede al Di ec o a e o Immuniza ion, Pakis an.” Epi.go .pk, 2018,
epi.go .pk/ accine-p e en able-diseases/pneumonia/.
18. McAllis e , Da id A, e al. “Global, Regional, and Na ional Es ima es o Pneumonia
Mo bidi y and Mo ali y in Child en Younge han 5 Yea s be ween 2000 and 2015: A
37
Sys ema ic Analysis.” The Lance Global Heal h, ol. 7, no. 1, Jan. 2019, pp. e47–e57,
h ps://doi.o g/10.1016/s2214-109x(18)30408-x.
19. Shakoo , Sadia, e al. “Pneumococcal Se o ypes and Se og oups Causing In asi e
Disease in Pakis an, 2005–2013.” PLoS ONE, ol. 9, no. 6, 3 June 2014, p. e98796,
h ps://doi.o g/10.1371/jou nal.pone.0098796. Accessed 6 Ma . 2023.
20. Shahid, Shahi a, e al. “Pneumococcal Ca iage in In an s Pos -PCV10 In oduc ion in
Pakis an: Resul s om Se ial C oss-Sec ional Su eys.” Vaccines, ol. 10, no. 6, 18 June
2022, p. 971, h ps://doi.o g/10.3390/ accines10060971. Accessed 19 Aug. 2022.
38
Appendix A
Ma e ials
Media o cul u ing o S.pnemoniae
Table A1: Blood Aga
S .no
Ing edien s
Quan i y (g/l)
1
Pep one
10
2
Bee Ex ac
10
3
NaCl
5
4
Aga
15
5
Dis illed Wa e
Up o 1000ml
Mix all he con en s well, hen s e ilize by au ocla ing. Fo blood aga , cool o oom empe a u e
and add sheep blood o i .
Table A2: Todd Hewi B o h (THY)
S .no
Ing edien s
Quan i y (g/l)
1
Pep one
20
2
Yeas Ex ac
5
39
3
NaCl
2
4
Dex ose
2
5
Disodium Phospha e
0.4
6
Hea in usion
3.1
7
Sodium ca bona e
2.5
8
Dis illed Wa e
Up o 1000ml
Reagen s o DNA isola ion:
Table A3: 1X TE Bu e
S .no
Ing edien s
Quan i y (g/l)
1
1M T is (pH 8)
10 ml
2
0.5M EDTA (pH 8)
2 ml
3
Dis illed Wa e
Up o 1000ml
Table A4: 10% SDS
S .no.
Ing edien s
Quan i y (g/Li e )
1
Sodium dodecyl sulpha e
10
2
Dis illed wa e
Up o 100 ml
40
Table A5: 5M Po assium ace a e
S .no.
Ing edien s
Quan i y (g/Li e )
1
Po assium ace a e
294.42 g
2
Dis illed wa e
Up o 100 ml
Adjus he pH o solu ion a 4.6 hen make up he olume up o 1000s ml wi h dis illed wa e .
Table A6: 95% E hanol
S .no.
Ing edien s
Quan i y (pe Li e )
1
Absolu e E hanol
95 ml
2
Dis illed wa e
5 ml
Table A7: 70% E hanol
S .no.
Ing edien s
Quan i y (pe
Li e )
1
Absolu e E hanol
70 ml
2
Dis illed wa e
30 ml
Reagen s o Mul iplex PCR
Table A8: 25μl Reac ion mix u e
S .no.
Ing edien s
Quan i y
1
25mM MgCl₂
2.5μl
41
2
Taq bu e
2.5μl
3
2.5mM dNTPS
2.5μl
4
Taq polyme ase
0.2μl
5
P ime F
Acco ding o
eac ion
6
P ime R
Acco ding o
eac ion
7
DNA Templa e
2μl
8
PCR wa e
Up o 25μl
Reagen s o PCR o Housekeeping genes
Table A9: 25μl Reac ion mix u e
S .no
.
Ing edien s
Quan i y
1
25nM MgCl₂
2.5μl
2
Taq bu e
2.5μl
3
2.5mM dNTPS
0.5μl
4
Taq polyme ase
0.5μl
5
P ime F
1μl
6
P ime R
1μl
7
DNA Templa e
2μl
8
PCR wa e
15μl
