Summary and Conclusions
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1 Summary and Conclusions
2 SUMMARY In children, the most vulnerable group of populatton, the problem of pneumonla IS Immense In terms of morbtd~ty and mortality. Worldwide, around 5 million children younger than 5 years of age die each year from pneumonia due to S.pneurnoniae, the commonest etiological agent. In India, acute respiratory tract infection is the second most common cause of mortality In children after acute diarrhoea Mortality due to acute lower respiratory lnfectlon In lndla constitute 10-45% of all ch~ldhoo deaths In chlldren w~th pneumonia, establ~sh~ng a bacter~al diagnosis IS dlfficult nasopharyngeal carrlage of th~s pathogen is more common posltive bacterial culture from blood or a needle aspirat~on from the lung conflrms a speclfic dlagnos~s but ~n children both techniques are lnsensttlve Rapld sensitive, speciftc and cost-effective methods are needed for establishing the diagnosis A In llght of the global Importance of Spneumon~ae as a cause of ~llness sequelae and death an attempt was made to develop useful and slmple dlagnostlc test for pneumococcal dlsease Aim of thls study was to evaluate certa~n ~rnrnunolog~cal tests based on a proteln antlgen pneumolysln and to compare ~t wth pneumococcal capsular polysaccharlde based tests for the et~olog~cal diagnosis of pneumonla suspected and radlologtcally diagnosed Cases chosen were cl~n~cally
3 Blood culture and detection of pneumococcal antigens such as pneumolysin and capsular polysaccharide from serum and urine samples by Co-agglutination; detection of pneumolysin and capsular polysaccharide from urlne samples by Latex agglutination; demonstration of IgM and IgG specific ant~bodies to pneumolysin and capsular polysaccharide by enzyme llnked ~mmunosorbent assay; demonstration of immune complexes containing IgM and IgG antibodies to pneumolysln and capsular polysaccharide by ELlSA and detection of antibodies to capsular polysaccharide by Indirect Haemagglutinat~on assay were the battery of tests that were evaluated. Seventy one chlldren adm~tted to the Paediatrlc Ward wth clinical and radlologlcal dlagnosls of pneumonla between the age group 1-10 years were enrolled In the study Controls comprised of 70 age and sex matched ch~ldren cl~nlcally d~agnosed as suffering from lnfect~ons other than pneumonia, like tuberculos~s, enterlc fever matarla hepat~tls and diarrhoea wth septicemla Forty SIX school chlldren who d ~d not have symptoms of any lnfect~on were Included as healthy controls Predom~nant cltnlcal features encountered In this study were cough and fever wth an elevated respiratory rate. Majority of children had consolidation as the radiological finding Routine haematological investigat~ons carried out with the blood samples revealed an elevated total leukocyte count (10,001-20,0001wmm) in 72% and erythrocyte sedimentation rate (51-80 mmlhr) in 50% of children wtth pneumonla
4 C-reactive protein was elevated (> 100 ~glrnl) in 72% of cases and only in 15% of nonpneumonia controls and the results were statistically significant with a 'p' value of <0.01 BACTERIAL ISOLATION Thirty percent of blood cultures from children wth pneumonia yielded S pneumoniae and none from the control group No statistically signlflcant d~fference was observed when the results of blood culture were compared to CRP (p > 0 05) ANTIBIOTIC SUSCEPTIBILITY S.pneumonrae Isolated from blood from cases of pneumonia showed a vaned susceptibility pattern for the different antibiot~cs tested. 14.2% of the lsolates were found to be resistant to oxacillin wth an MIC range of pglml None of the ~solates were sensitive to Co-tr~moxazole. All the stralns were sensitive to cefotaxime and vancomycln. Res~stant strains to erythromycin, tetracycline, ofloxacin, ciprofloxacin and chloramphenicol were lsolated Serotyping was carrled out by Co-agglutination technique using pneumotest kit S pneumoniae belonging to serotype 5 was the predominant Isolate causing bacteremia in children with pneumonia. Other serotypes Isolated were 6, 1, 19, 3, 7 and % of the lsolates belonged to 3nvacclne type penicill~n. Of the 3 nonvacclne serotypes, 2 showed relatlve resistance to
5 ANTIGEN DETECTION Co-agglutination tests for detection of pneumolysin (Plo and Ply) and capsular polysaccharide (CPS) from serum and urine samples were carried out. Tests from serum showed a low sensitivity of 15.4% for Plo and 11.2% each for Ply and CPS A 100% specificity was achieved for the all antigen detectlon systems. There was a statistically significant difference in the results of all the antigen detection between the pneumonia and nonpneumonia group of children. Co-agglutination tests from serum samples had an efficiency of 69% (Plo), 68% (Ply) and 70% (CPS), when compared to blood cultures Antlgen detectlon In urlne samples by Co-agglut~nation had a better sensltivlty than in serum The tests had a sensitlvlty of 43%, 37% and 39% for Plo, Ply and CPS respectively, when comparea to nonpneumonla controls Specificity was 96%, 94% and 97% respectively When compared wth healthy controls the tests had a specificity of 98% for Plo and 100% each for Ply and CPS Efficiency of the assays In comparison to blood culture was found to be 59% 64% and 63% for Plo Ply and CPS respectively Latex agglutrnatlon tests carrled out In urine of cases nonpneumonla controls and healthy controls showed senslt~vlty of 46% 39% and 40% for Plo Ply and CPS respectively compared between the twu groups (p < 0 01) Results were statistically signitlcant when Among the 46 healthy controls none were posltlve for Plo Ply and CPS y~eldlng a specificity of 100% for the antlgen detectlon systems LA tests had an efflc~ency of 64% for Plo 71% for Ply and 73% for CPS when compared wth culture LA tests had better sens~tiv~ty and efficiency than Co-A tests
6 Antlgen detectlon tests overall had a low sensltlvlty from serum when compared to urlne samples However ~t allowed us to plck up around 8%- 38% more poslt~ve cases whlch were not posslble by blood culture Enhancement of the eff~cacy of antlgen detectlon systems can be trled by employing an enrichment step or concentrating the ant~gens from the samples or by uslng a high tltre antlsera to prepare the antlgen delectlon reagents ANTIBODY DETECTION Serological evidence of pneumococcal infection was Indicated by the demonstration of IgM and IgG antibodies to pneumolysin (Plo and Ply) and capsular polysaccharide antlgens IgM detection had a low sensitivity of 8% for Plo, 25% for Ply and 35% for CPS. Specif~city was higher (96%) for the detection of anti-plo antibodies reflecting the poor antigenicity of a highly pur~fied (genetically formulated) protein antigen Detection of anti-cps antibodies showed a statistically significant difference beheen the cases and controls; efficiency of 66% when compared to blood culture. Serum IgG response was poor among the cases Highest sensitlvlty (23%) was achieved for the detectlon of anti-ply IgG antibodies. Specif~city was high (97%) for the detectlon of antl-cps antibodies. Results were not stat~stlcally slgnliicant (P > 0.05) when compared to nonpneumonia controls. Efficiency of anti-ply antibody detection was hlgher (79%) than that of Plo and CPS when compared with blood culture. IgM response to pneurnolysln was demonstrable In 17.25% of chlldren with pneumonia, a value higher than that of CPS (13%) In the younger age group of 1-2 years, Immune response to CPS Improved as age advanced
7 Poor response to polysaccharide ant~gens In the younger age group may be the explanation for this observatron. INDIRECT HAEMAGGLUTINATION ASSAY Anticapsular antibody detection in cases of pneumonia was evaluated The test had a sensitivity of 54% when results were compared between cases and controls. T-test for equality of mean between cases and controls drd not show any statist~cal srgnif~cance. IMMUNE COMPLEXES DETECTION IgM Immune complexes contalnlng ant~bod~es to Plo, Ply and CPS were demonstrable In and 6 ch~ldren respect~vely, who were cl~n~cally and radlolog~cally diagnosed wth pneumonia Spec~f~c~ty of 100% was noted In the detect~on of IgM ICS contarnrng antrbod~es to CPS stat~st~cally s~gn~f~cant (p < 0 01) Results were Detection of Plo IgG ICs had the hrghest sensit~vity (17%) and spec~fic~ty (97%); results were statist~cally signrficant (p < 0.01) in comparison between the cases and nonpneumonia controls. On comparison with blood culture, sensitivity of 24% was achieved for Plo and Ply each. Detection of Ply Ics had an efficiency of 70%. Slmilar finding of better response to pneumolysin than that of CPS was observed in the age group of 1-2 years Totally, 82% of cases could be confirmed by one or more of the laboratory tests wh~ch rncluded blood culture, antigen detection by Co-A and LA in urine and serum, antibody and immune complexes detection by ELSA
8 and IHA. By serology alone, pneumococcal pneumonia could be confirmed in 80% of children, which was very much higher than that confirmed by conventional blood culture (30%) alone. Antlbody detection and IC demonstration assays had limitations owlng to these poor sens~tivtty Llrnitat~ons of indlv~dual tests can be overcome by the use of a panel of tests Including antigen detection, antibody and IC detection assays to confirm the diagnosis of pneumococcal pneumonia. However, this may be necessary only when establishing an etiological diagnosis IS cons~derations of paramount importance for therapeutic and prognostic
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