Annexures

ANNEXURE - I PNEUMOCOCCAL PNEUMONIA IN CHILDREN: A STUDY OF IMMUNOLOGICAL

METHODS OF DIAGNOSIS

PROFORMA

INDEX CASE I CONTROL HISTORY

Name.

Hospital No.:

Fathets Name:

Date of Adm~ss~on: Durat~on of Hospital Stay(Days)

Present~ng complaints

Treatment I days.

1. SYMPTOM:

Cough, Fever Rapid Breathing (Rate) Runny Nose Decreased Appetite Vomibng D~arrhoea Red Eyes

Y I N Y 1 N Y 1 N Y I N Y / N Y I N Y 1 N Y I N

2. FEEDING:

Breast feed~ng alone:

Breast feeding plus non-breast feed~ng

Non-breast fed:

3. CHEST X-RAY FINDINGS:

4. OTHERS:

Study No

Age:

Past H~story

Pnor Antibiotics. (within last week)

Duration(Days) Diagnosis

Haematology' CRP.

WBC. Hb:

ESR:

LABORATORY RESULTS

I. BACTERIOLOGY:

A. Bacteria isolated from Blood:

Streptococcus pneumon~ae suscepbb~l~ty Serotype

Others.

II. SEROLOGY:

CPSlC

Serum

A Antigen detect~on:

Serum

Unne

Co-agglut~nation at ex-agglutinat~or- Pneumolysin PI0 1 Ply

I

Pneumolys~n CPS

ANNEXURE - I1

Chemicals used for the experiments were of reagent grade and were

purchased from HI-media (Mumbai), S~gma Chem~cals Co , GlBCO BRL

Quallgens, SD Fine Chem Ltd , E Merck (Indla) Ltd etc Water used to

make up the buffers were reagent grade (mllli-Q M~llrpore Chromic USA)

All the glasswares used were cleaned rn soap and acld before rlnslng well In

runnlng water Prlor to the experiments glasswares were rinsed in dlst~lled

water and drled Other requirements were eppendorfs prpette t~ps and

plast~c ware Molecular B~ology Manual (Sambrook et a1 1989) was followed

for the standard protocols and procedures of SDS-PAGE

1.Protein Estimation by Lowry's method:

This method is based on the combination of Biuret reaction and folin-

ciocalteau reaction The protein fractions obtained after gel filtration and

ultrafiltration were estimated for their orotein content

In the flrst step of the Lowry reaction prote~n binds to copper in

alkal~ne medlum and produces Cu Ions In the 2" step Cu ion catalyses

oxidation of aromatic aminoacids by reducing phosphomolbdotungstate

heterop~lymolybdenum blue. This reaction produces strong blue colour

which predominantly depends upon tyrosine and tryptophan content of

protein and to a lesser extent cystelne and other residues in protein.

Reagents required:

1 Protein Standard BSA

2. Solution I : Copper solution

3. Solution II : Alkaline tartarate

4. Solution Ill . Folin reagent

Other requirements:

Micropipette

Spectrophotometer

Standard Protein Stock Preparation:

5 rng of BSA IS dissolved in 1 rnl distilled water to get 5 mglrnl This

stock was kept at 4'C. Dllute 0 1 rnl of 5 mglrnl BSA solution with 0 1 ml

distilled water to get 0.5 mglrnl just before use.

Complex Forming Reagents:

To one volume of solution I add 100 volume of solution II just before

use Fohn reagent (Solution Ill) to be used d~rectly.

Protocol:

1 Plpette standard BSA 0 5 rnglrnl samples and dlst~lled water referrrng to

the protocol glven below (Table I) and adjust the volume to 0 2 ml

2 Add 2 rnl complex formlng reagent mlx and keep for 10 mlnutes

3 Add 0 2 rnl of solutlon Ill wlth vortexlng and ~ncubate for 30 mlnutes

4 Read the optical denslty on spectrometer at 660 nrn

Table I. Assay Protocol

Serial Standard sample, Amount in pl Water in pl No. figltube

1 Blank 0 200

2 10 20 180

3 20 40 160

4 40 80 120

5 60 120 80

6 80 160 40

7 Sample "V' 200-V

CALCULATION:

1 A callbratlon curve was constructed by plotting optlcal dens~ty read~ng

on Y' axis against standard proteln In pgltube

2 Record the value 'X from the graph corresponding to the optical dens~ty

readlng for indlv~dual sample

3 Calculation of the sample concentratton was done using the followng

formula

Sample concentration = XN mglrnl

Water X - Value from the graph in pg

V - Volume of sample in pl

SDS-PAGE:

Composition:

Followng volumes (ml) of reagents were used for castlng a 10% gel In

a Bangalore Gene1 vert~cal electrophores~s apparatus (Bangalore Genel)

Gel components : For I00 ml

-. -. -- Separating Gel - Stacking Gel

30% acrylamide and 0.8% bisacrylamide

0.614 M Tris (pH 8.8) 0.147 M Tris (pH 6.8)

SDS

APS

TEMED

SDS-PAGE RUNNING BUFFER (0.025 M Tris, 0.192 M glyc~ne, pH 8 3)

Trls 3.03 g

Glycine 14.4 g

SDS 1 o g

Salts were dissolved in 800 ml water and made up to 1 litre.

Sample Loading Buffer:

2.5 ml of 0.5 M Tris (pH 6.8), 2 ml of glycerol, 0.4 g of SDS, 0.2 ml of

@-ME and 0.1 mg of Bromophenol blue in water made up to 10 mi.

Protocol:

Preparation and Electrophoresis of Acrylamide gel:

For cast~ng an SDS-polyacrylam~de gel the glass plates spacers and

comb were thoroughly washed wth soap solutlon and water The plates

were rlnsed wth chloroform and assembled wth the s~de spacers In the gel

castlng set up 10% separat~ng and stack~ng gel was prepared as described

above The separatlng solutlon was poused before the gel hardens few

drops of water was layered on top of the gel solutlon After 10-20 mln an

Interface could be seen ~nd~catrng that the gel has solldlf~ed Just before use

the water layer was removed and the stackrng solut~on poured lmmedlately

the comb was Inserted and the gel was allowed to polymerize Afler 30 mrn

the comb was removed and the wells were r~nsed wth 2X runnlng buffer

Samples were mrxed wth equal volume of sample load~ng buffer and heated

In a bolllng water bath for 5 mln before load~ng on to the gel Electrophores~s

was carr~ed out at 25 mA untll the proteln entered the separatlng gel after

h ~ c h the current was ~ncreased to 30 mA

Rapid Coomassie-Blue Staining:

Afler the electrophoresis, the gel was soaked in fixing solution 150%

merhanol and 50 p1 of formaldehyde (0 05%)] for 1 hour wth constant

sllaking. The gel was later soaked in rap~d blue stain [lo% acetic acid and

0.006% Coomassie brilliant blue stain R-250 for 30 mln. The bands were

visualized after destaining with 10% acetic acid unt~l a clear background was

obtained.

Buffer Composition:

Buffer used for Co-agglutination:

0.03 M Phosphate Buffered Saline (pH 7 2)

A) Na2HP04 + 2H20 (MW - 178.16) - 5 34 glL

8) KH2P04 (MW - 136.09) - 4.08 glL

715 ml of solution(A) + 285 rnl of solution(B) = 1000 ml

C) NaCI (MW - 58.44) - 7.0128 g1L

To 1000 rnl of phosphate buffer, add 7 0128 gms of NaCl and 1 0 gm

of sod~um azide as preservative. This will form 1000 rnl of PBS, pH 7 2

Buffer used for Latex agglutination:

GBS-BSA.

Glycine 0 75 g1100 rnl

NaCl 0 876 gl l 00 mi

BSA 2.5 gllOO ml

PH 8.0

Th~omersal 1 :10,000

Reaction Buffer:

GBS-BSA (pH 8.0) 100 ml

Urea (0.2 M) , 1.221 gms

PEG 4 gms

Buffers used for ELISA:

0.01 M PBS pH(7.2).

NaCl 8 0 gm

KC I 0 2 gm

KHz Po4 0.2 gm

Na2 HP04.2Hz0 1.5 gm

Distilled water 1000 ml

Wash Buffer (PBST)

0.01M PBS (pH 8 2). 1 litre

Tween 20 . . 500 ~1

Serum d~luent

0 01M PBS (PH 7 2) 100 ml

Tween 20 50 pi

BSA 1 gm

Conjugate diluent.

PBS (pH 7.2) 100 ml

BSA 0.5 gm

ANNEXURE - Ill

CULTURE MEDIA, CHEMICALS AND KITS

Braln Heart lnfuslon Broth - HI-Media, Mumbai

Blood Aqar - Blood agar base + 5% citrated sheep blood Sterile blood IS

added to autoclaved blood agar base at 50% and poured 20 mllplate.

Chocolate Aaar - Blood agar base + 5% citrated sheep blood. Sterile blood

is added to autoclaved blood agar base at 75% and kept for 10 minutes.

Cooled to 50°C and poured 20 mllplate.

Mac Conkev's aqar - Hi-Media. Mumba~

Trvptic Soy aqar - 'Difco' Laboratories; Detroit MI 48232-7058, USA

Mueller-Hinton s h e e ~ blood aoar - Mueller Hinton agar (HiMed~a) autoclaved - at 12I0C for 15 mln. 5% sterile defibrinated sheep blood added following

cooling to 50°C. 20 ml/plate is poured into 100 mm petridshes to a depth of

4-5 mm

Todd Hewitt Broth (for Co-A reagent):

Beef heart Infusion Neopeptone Dextrose NaCl Sodium carbonate Disodlum phosphate Distilled water pH

Antibiotic powder for MIC:

Penicillin - RM132

Hi-media, Mumbai

Pneumotest:

Pneumococcal antisera for typing of Streptococcus pneumoniae

Statens Serum lnstitut

Artillerivej 5

2300 Copenhagen S

Denmark.

PNEUMO 23:

Vaccin Pneurnococcique polyvalent pneurnococcal vaccine

Pasteur Merieux S.V.

58 a\., Ledere

69007 Lyon-France

Distributed by: Pasteur Merieux India Ltd., New Delhi, India.

C-reactive protein kit:

Elitech Diagnostic Kit (96 tests)

Elltech Diagnostics Zone lndustrielle

G1500, Sees. France

Latex Beads:

Part~cle diameter - 3 798

Sigma Chemical Co

P 0 Box 14508,

St. Louis, MO 63178

USA. 314-771 -5750.

Molecular Markers.

Slgma Chem~cal Company

P 0 Box 14508

St Louls MO 63178 USA

B~otlnylated SDS Molecular wt Markers - Phosphorylase b - 97 400 Bov~ne

albumln - 66 000 Ovalbumln - 43 000 Carbonlc anhydrase - 29 000

Nunc plates:

F8 Maxlsorp 469949

Nunc - 1mmunoTM Modules

Nunc Brand products

Denmark

IgM Conjugate:

Peroxldase conjugated ant~human IgM

Dako NS

DK - 2600

GI OSTRUP

Denmark

IgG Conjugate:

Peroxidase conjugated anti-human IgG

Dako - A/S GLOSTRUP

Denmark

3,3',5,5'-Tetramethyl benzidine dihydrochloride (TMB):

Dako, Glostrup, Denmark

Master Chart

KEY TO MASTER CHART

A.No DOHS Clin diagnosis :

CXR 1 2 3 4 TLC ESR CRP ND BC Spn Ster GS OPT BS S R I sty ox Va E T Cx Co C C f Of MIC ST NV AA CPloSe C-Plo-Ur C-PlySs C-Ply-Ur

CASES SERIAL NUMBER DURATION OF HOSPITAL STAY CLINICAL DIAGNOSIS

CHEST X-RAY FINDINGS OPACITY R -RIGHT HAZINESS L - LEFT CONSOLIDATION B - BILATERAL EFFUSION TOTAL LEUKOCYTE COUNT ERYTHROCYTE SEDIMENTATION RATE

C-REACTIVE PROTEIN NOT DONE BLOOD CULTURE Streptococcus pneumoniee STERILE GRAM STAINING OPTOCHIN TEST

BILE SOLUBILITY SENSITIVE RESISTANT INTERMEDIATE ANTIBIOTIC SUSCEPTIBILITY TEST

OXAClLLlN

VANCOMYCIN

ERYTHROMYCIN

TETRACYCLINE CEFOTAXIME COTRIMOWOLE CHLORAMPHENICOL CIPROFLOXACIN

O F L O W I N MINIMUM INHIBITORY CONCENTRATION SEROTYPE NONVACCINE TYPE

AUTOAGGLUTINATION

PNEUMOLYSOID IN SERUM BY COAGGLUTINATION PNEUMOLYSOID IN URINE BY COAGGLUTINATION PNEUMOLYSIN IN SERUM BY COAGGLUTINATION

PNEUMOLYSIN IN URINE BY COAGGLUTINATION

CCPS-se CCPS-ur L-Plo-Ur LPly-Ur Plo-lgM Plo-lgG Ply-lgM Ply-lgG CPS-lgM CPS-lgG ICPlo-lgM IC-Plo-lgG ICPly-lgM IC-Ply-lgG ICCPS-IgM IC-CPS-lgG IHA Tt 1

CAPSULAR POLYSACCHARIDE IN SERUM BY COAGGLUTINATION CAPSULAR POLYSACCHARIDE IN URINE BY COAGGLUTINATION PNEUMOLYSOID IN URINE BY LATEXAOGLUTINATION PNEUMOLYSIN IN URINE BY LATEX AGGLUTINATION ANTIPNEUMOLYSOID lgM

ANTIPNEUMOLYSOID lgG ANTIPNEUMOLYSIN lgG

ANTIPNEUMOLYSIN lgG ANTICAPSULAR POLYSACCHARIDE IgM ANTICAPSULAR POLYSACCHARIDE IgG IgM PNEUMOLYSOID IMMUNE COMPI-EXES IgG PNEUMOLYSOID IMMUNE COMPLEXES IgM PNEUMOLYSIN IMMUNE COMPLEXES IgG PNEUMOLYSIN IMMUNE COMPLEXES IgM CAPSULAR POLYSACCHARIDE IMMUNE COMPLEXES IgG CAPSULAR POLYSACCHARIDE IMMUNE COMPLEXES INDIRECT HAEMAGGLUTINATION ASSAY TREATMENT CEFOTAxlME AMOXYClLLlN ERYTHROMYCIN

CRYSTALLINE PENICILLIN OTHERS (GENTAMYCIN I CLOMCILLIN I AMPICILLIN)

OUTCOME DISCHARGED EXPIRED

US1 Of PAPERS PRESENTED IN NAWNAL CONFERENCE I IN PRESS

1 Rejslakshm~ B Kmmgo R Snntvasan S. Badnnath S Pneurnolysin

in unne A rapd anttgen detect~on method to diagnose ~umococca l

pneurnonla In children (Presented In the XXlV Nahonal Confererne of

lndan Aswat lon of Medical M ~ ~ o l o g ~ s t s , MOO) (Abstract

endOsed)

2 Kanungo R Dorothy D Llma Rajalakshm~ B Kumar A. Badrinath S

Emerging ant~b~otlc resistant poeurnococcl in lnvaslve lnfedlons tn

South indla Need fw monitoring ind~an J Pharmecol2001 (In press)

PIIEUYOLVSRI IN URH4E. A R I P M ANTIGEN DETECIW MElHOD TO MAGNOSE PIIEUYOCOCCAL PIIEUMWIIA WI CHILLREN I

1 Aultms RAJAUKStlMI B REBA KAIIUIICO S SRIIIVASAII S BADRlNAM rrru, I I n s l l M h DEPT OF MICROBI(KM;V, JIPMER. PW4MCHERRV

- I Pt~eunlolystn and capsulat ydysaccl~attde atlltgen o l s l ~~p lococcus poeunantee *ere &kid by

co agg lu l t na l~o~~ [CU A) ltottl IIIP utllie 01 c i ~ t~~ca l l y d iaq~~osed cases 01 p t i e u t t i ~ ~ o ~ c u i l l P~I~UIIIOIII~ {11:331

Cot~tl iattr ig l l te tesun w t l l ~ 45 Ihealll~y cot~ltols, a senstltvtly o l 60 60%. 15 is'; and 66 66'0. a spec~ltctty ol

IOU% 9 i 82% and 100'. were rtolod when anl~seta ayalnsl puu l~ed pneutnolys~n (Ply) ]et~elically dettved

p t~eu l l ~o l ys~n (pla) at14 caysulat polysacchartde ICPSI were used In Co A reayenls respecllvel) Wtieti

corl~pated a1111 42 conlrols. ( w ~ l l ~ r1o11 pt~eutnmoccal lower tespltaloty Iracl ~ t ~ l r c l ~ o t i s a t ~ d olliet IIII~CIIOIISI

~IPI~L~IOII 111 lily ~IIIN/-II l n ' l a CIYISIII,II~ 01 Gu 613', PIO 75 i5', and CPS 5fi 66'0 C ~ ~ t t ~ l i a i ~ r l w ~ l h blouil

ru l lu le l l le setisl!lvllf atid s ~ t e c ~ l ~ ~ ~ l i e s wete GU'. atrd 39 13:. lor ply 70'a and 21 10'0 lot 1310 1Ub 811d

3 4 ill', lor CPS tesl,eclivel) P t \ e u ~ t ~ o l ~ s i n wl~tcl! 18 1101 slraltl s l ~ r c t l ~ c , lhas an a l v a i ~ l a g ~ over CI'S 111 81,

ati i~yert deiecltot~ sysler!, A s~ f t~p l e , rapid and eca t i o~ t~~ca l tnell~od In d ~ l c c l p r ~ e t t ~ v l y ~ ~ r l 1r1 llle ut le lu l l l ie

diayttos~s ol ~ineutriococcal ptleutuotila 11oi.J~ pfotrtlse