the most common serious pneumococcal disease is pneumonia , an infection of the tissues of the lungs . pneumococci may also cause infections in the ear ( otitis media ) , para nasal sinuses ( sinusitis ) , the tissues covering the brain and spinal cord ( meningitis ) , and , less often , the heart valves , joints , and abdominal cavity . the capsular polysaccharide is the main virulence factor that protects pneumococci against the action of host defense mechanisms . at least one million children die annually from pneumococcal diseases , and most of them are young children in developing countries . several risk factors for pneumococcal infections , such as age , race , immunodeficiency , other illness , socio - economic status , previous antibiotic therapy and day care attendance have been reported . streptococcus pneumoniae is facultative anaerobe , gram positive , capsulated organism that usually grows in pairs or short chain . molecular techniques have significantly improved the diagnostic yield and decreased the time for pathogen identification . these techniques are helpful in the determination of drug sensitivity and the understanding of transmission and outbreaks . the effect of swab composition and compared the sensitivities of detection of s. pneumoniae in simulated and clinical respiratory specimens by culture and pcr - based methods have been evaluated . this work aims to evaluate the sensitivity of pneumolysin - targeted nested - pcr for the detection of s. pneumonae and its comparison with routine clinical methods . pediatric patients with age 1 - 6 months ( group a ) and up to 5 years ( group b ) admitted to mayo hospital , children hospital and services hospital , lahore , pakistan with a clinical and radiological picture suggestive of pneumonia were enrolled in the study . they had fever of 39c and a leukocyte ( wbc ) count of 1510/liter suspected to have invasive bacterial infection . in group a , 98 patients and 145 in group b fulfilled the criteria for suspected invasive pneumococcal infection . blood samples from 60 healthy persons were also collected to test the specificity of the pcr assay . in other study , middle ear fluid ( mef ) samples were taken from the acute otitis media ( aom ) patients from the ent department of mayo hospital . aom was defined by the otoscopic examination suggesting mef and at least one of the symptoms : rubbing of the ear , runny nose , cough , ear pain or temperature of at least 38c . the blood samples were collected in tubes containing edta from each patient during his / her initial visit . middle ear fluid samples were taken from the acute otitis media patients in sterilized cotton swabs . cotton tipped swab was inserted into the ear , aspirated and the secretion was rinsed out with 0.5 ml of phosphate buffered saline . mef bacterial culture was inoculated on selective blood agar media containing 5 g / ml gentamicin and incubated at 37c for 72 h in a 5% co2 atmosphere . blood samples ( 300 l ) were resuspended in a 1.5 ml microfuge tube containing 900 l rbc lysis solution ( 10 mm tris - hcl , 400 mm nacl and 2 mm na2 edta , ph 8.2 ) . the cell suspension was digested with 0.2 ml of 10% sds and 0.5 ml of a proteinase k solution ( 1 mg proteinase k in 1% sds and 2 mm na2 edta ) at 37c for overnight . one ml of 6 m nacl was added and shaken vigorously for 15 sec , followed by centrifugation at 13,000 g for 15 min . the supernatant containing dna was transferred to another tube , added double volume of absolute ethanol and inverted several times until the dna precipitated . dna was washed three times with 70% ethanol and centrifuged at 13,000 g for 1 min . pneumococcal cells from mef samples were suspended in the phosphate buffer saline by rubbing each swab against the wall of tube and centrifuged at 12,000 g for 10 min . the pellet was suspended in a buffer containing 10 mm tris , 0.14 m nacl , 0.1 m sodium citrate and 10 mm na2 edta and incubated at 80c for 10 min . the selection of the primers was based on the published sequence of the pneumolysin gene . pcr amplification of a 50 l reaction mixture containing pneumolysin specific primers was performed in a programmable thermal cycler ( eppendorf ) . the outer primers ( 5-atttctgtaacagctaccaacga-3 ) and ( 5-gaattccctgtcttttcaaagtc-3 ) amplified a 348-bp region of the pneumolysin gene , and the inner primers ( 5-cccacttcttcttgcggttga-3 ) and ( 5-tgagccgttattttttcatactg-3 ) amplified 208-bp region . the reaction mixture contained 10 mm tris - hcl ( ph 8.8 ) , 1.5 mm mgcl2 , 50 mm kcl , 200 m deoxyribonucleotides , 50 pmol of primers , 1u of taq dna polymerase ( fermantas ) and 2 l of dna ( 250 ng ) extracted from whole blood and mef fractions . the amplifications were repeated 35 times as follows : denaturation at 94c for 45 sec , annealing at 50c for 30 sec , and extension at 72c for 30 sec . after 35 cycles , the samples were incubated for a further 6 min at 72c and stored at 4c until analyzed . amplifications were carried out as for the first - round pcr , autoclaved double distilled water and dna of healthy volunteers used as negative control . pcr amplified products mixed with 6 bromophenol blue loading dye were run in a 2% agarose gel , stained with ethidium bromide and visualized under uv light gel documentation system . spss version 17.0 was used to perform binomial test to demonstrate the statistical significance of pcr for diagnosis of pneumococcal infections . pediatric patients with age 1 - 6 months ( group a ) and up to 5 years ( group b ) admitted to mayo hospital , children hospital and services hospital , lahore , pakistan with a clinical and radiological picture suggestive of pneumonia were enrolled in the study . they had fever of 39c and a leukocyte ( wbc ) count of 1510/liter suspected to have invasive bacterial infection . in group a , 98 patients and 145 in group b fulfilled the criteria for suspected invasive pneumococcal infection . blood samples from 60 healthy persons were also collected to test the specificity of the pcr assay . in other study , middle ear fluid ( mef ) samples were taken from the acute otitis media ( aom ) patients from the ent department of mayo hospital . aom was defined by the otoscopic examination suggesting mef and at least one of the symptoms : rubbing of the ear , runny nose , cough , ear pain or temperature of at least 38c . the blood samples were collected in tubes containing edta from each patient during his / her initial visit . middle ear fluid samples were taken from the acute otitis media patients in sterilized cotton swabs . cotton tipped swab was inserted into the ear , aspirated and the secretion was rinsed out with 0.5 ml of phosphate buffered saline . mef bacterial culture was inoculated on selective blood agar media containing 5 g / ml gentamicin and incubated at 37c for 72 h in a 5% co2 atmosphere . blood samples ( 300 l ) were resuspended in a 1.5 ml microfuge tube containing 900 l rbc lysis solution ( 10 mm tris - hcl , 400 mm nacl and 2 mm na2 edta , ph 8.2 ) . the cell suspension was digested with 0.2 ml of 10% sds and 0.5 ml of a proteinase k solution ( 1 mg proteinase k in 1% sds and 2 mm na2 edta ) at 37c for overnight . one ml of 6 m nacl was added and shaken vigorously for 15 sec , followed by centrifugation at 13,000 g for 15 min . the supernatant containing dna was transferred to another tube , added double volume of absolute ethanol and inverted several times until the dna precipitated . dna was washed three times with 70% ethanol and centrifuged at 13,000 g for 1 min . pneumococcal cells from mef samples were suspended in the phosphate buffer saline by rubbing each swab against the wall of tube and centrifuged at 12,000 g for 10 min . the pellet was suspended in a buffer containing 10 mm tris , 0.14 m nacl , 0.1 m sodium citrate and 10 mm na2 edta and incubated at 80c for 10 min . the selection of the primers was based on the published sequence of the pneumolysin gene . pcr amplification of a 50 l reaction mixture containing pneumolysin specific primers was performed in a programmable thermal cycler ( eppendorf ) . the outer primers ( 5-atttctgtaacagctaccaacga-3 ) and ( 5-gaattccctgtcttttcaaagtc-3 ) amplified a 348-bp region of the pneumolysin gene , and the inner primers ( 5-cccacttcttcttgcggttga-3 ) and ( 5-tgagccgttattttttcatactg-3 ) amplified 208-bp region . the reaction mixture contained 10 mm tris - hcl ( ph 8.8 ) , 1.5 mm mgcl2 , 50 mm kcl , 200 m deoxyribonucleotides , 50 pmol of primers , 1u of taq dna polymerase ( fermantas ) and 2 l of dna ( 250 ng ) extracted from whole blood and mef fractions . the amplifications were repeated 35 times as follows : denaturation at 94c for 45 sec , annealing at 50c for 30 sec , and extension at 72c for 30 sec . after 35 cycles , the samples were incubated for a further 6 min at 72c and stored at 4c until analyzed . amplifications were carried out as for the first - round pcr , autoclaved double distilled water and dna of healthy volunteers used as negative control . pcr amplified products mixed with 6 bromophenol blue loading dye were run in a 2% agarose gel , stained with ethidium bromide and visualized under uv light gel documentation system . spss version 17.0 was used to perform binomial test to demonstrate the statistical significance of pcr for diagnosis of pneumococcal infections . the age of the children was 1 - 6 months ( group a ) and above one year ( group b ) . the clinical diagnosis and numbers of pediatric patients in the group a , fulfilling the study inclusion criteria ( n=98 ) were as follows : pneumonia 44 ; acute respiratory tract infection 30 ; pneumococcemia 24 . the study inclusion criteria in group b ( n=145 ) were as follows : pneumonia 80 ; fever without infection focus 8 ; acute respiratory tract infection 28 ; pneumococcemia 22 ; gastroenteristis 3 and acute tonsillitis 4 . a , the highest wbc count values were 23 x 10 to 15 x 10/l and the highest body temperatures 45  1.1 and 38.8  1.2c , respectively . in group b , the highest wbc count values were 20.210 to 15.510/l and the highest body temperatures 451.0 and 381.1c , respectively . the study group of otitis media subjects ( up to 5 years ) were distributed into two groups , group i , mef pneumococcal culture negative ( 90 samples ) and group ii culture positive ( 60 samples ) . among pneumonia patients of group a , pcr gave a clear band of the expected molecular size in the nested - pcr ( fig . the specificity of the pcr assay was demonstrated by its negative results for normal healthy persons . the sensitivity of the assay was evaluated with about 0.06 pg of purified s. pneumoniae genomic dna . amongst 44 pneumonia patient samples , 86.4% showed pcr positive results while 6.6% and 8.3% positive results were obtained in acute respiratory tract infection and pneumococcemia , respectively ( table 1 ) . none of the samples of the controls was found to be positive by pcr and blood culture . amplification of 208-bp fragment by pcr of dna extracted from peripheral blood of pneumonia pediatric patients ( group a ) . lane m , molecular size marker ; lane 1 and 5 pcr positive samples and lane 2 , 3 , 4 , 6 , 7 , 8 , 9 pcr negative s. pneumoniae patients blood samples pcr results for peripheral blood samples from patients with clinical symptoms arti : acute respiratory tract infection in group b , nested - pcr protocol was found to be positive in 62 of the 80 pneumonia cases . four ( 18% ) patients with pneumococcemia and four ( 14.2% ) with acute respiratory tract infection were positive for pneumolysin gene ( fig . pcr amplification results of group b. lane m , dna size marker ; lane 1 , 3 - 6 are positive s. pneumoniae patient 's blood samples . in other study , the results of pcr based amplification of the pneumolysin gene were compared with those obtained from culture method . among 60 mef culture positive samples ( group ii ) , 52 showed the evidence of pneumolysin by nested - pcr . on the other hand , among 90 s. pneumoniae culture - negative mef samples ( group i ) comparison of s. pneumoniae culture and pneumolysin pcr results for mef samples mef : middle ear fluid ; pcr : polymerase chain reaction the age of the children was 1 - 6 months ( group a ) and above one year ( group b ) . the clinical diagnosis and numbers of pediatric patients in the group a , fulfilling the study inclusion criteria ( n=98 ) were as follows : pneumonia 44 ; acute respiratory tract infection 30 ; pneumococcemia 24 . the study inclusion criteria in group b ( n=145 ) were as follows : pneumonia 80 ; fever without infection focus 8 ; acute respiratory tract infection 28 ; pneumococcemia 22 ; gastroenteristis 3 and acute tonsillitis 4 . a , the highest wbc count values were 23 x 10 to 15 x 10/l and the highest body temperatures 45  1.1 and 38.8  1.2c , respectively . in group b , the highest wbc count values were 20.210 to 15.510/l and the highest body temperatures 451.0 and 381.1c , respectively . the study group of otitis media subjects ( up to 5 years ) were distributed into two groups , group i , mef pneumococcal culture negative ( 90 samples ) and group ii culture positive ( 60 samples ) . among pneumonia patients of group a , pcr gave a clear band of the expected molecular size in the nested - pcr ( fig . the specificity of the pcr assay was demonstrated by its negative results for normal healthy persons . the sensitivity of the assay was evaluated with about 0.06 pg of purified s. pneumoniae genomic dna . amongst 44 pneumonia patient samples , 86.4% showed pcr positive results while 6.6% and 8.3% positive results were obtained in acute respiratory tract infection and pneumococcemia , respectively ( table 1 ) . none of the samples of the controls was found to be positive by pcr and blood culture . amplification of 208-bp fragment by pcr of dna extracted from peripheral blood of pneumonia pediatric patients ( group a ) . lane m , molecular size marker ; lane 1 and 5 pcr positive samples and lane 2 , 3 , 4 , 6 , 7 , 8 , 9 pcr negative s. pneumoniae patients blood samples pcr results for peripheral blood samples from patients with clinical symptoms arti : acute respiratory tract infection in group b , nested - pcr protocol was found to be positive in 62 of the 80 pneumonia cases . four ( 18% ) patients with pneumococcemia and four ( 14.2% ) with acute respiratory tract infection were positive for pneumolysin gene ( fig . pcr amplification results of group b. lane m , dna size marker ; lane 1 , 3 - 6 are positive s. pneumoniae patient 's blood samples . in other study , the results of pcr based amplification of the pneumolysin gene were compared with those obtained from culture method . among 60 mef culture positive samples ( group ii ) , 52 showed the evidence of pneumolysin by nested - pcr . on the other hand , among 90 s. pneumoniae culture - negative mef samples ( group i ) comparison of s. pneumoniae culture and pneumolysin pcr results for mef samples mef : middle ear fluid ; pcr : polymerase chain reaction s. pneumoniae is a common human pathogen that may be considered as a part of normal flora of the upper respiratory tract , however , infants , elderly and deliberate patients with other compromising medical conditions of the respiratory tract , or decreased immunological infections are at great risk of acquiring pneumococcal disease . the spectrum of pneumococcal diseases changes in various age groups and different populations[4 , 11 ] . the diagnosis of pneumococcal infection requires s. pneumoniae isolation from infection sites by using insidious procedures such as lung puncture , pleural fluid aspiration , and middle ear fluid ( mef ) aspiration ; however , their presence in peripheral blood is an indirect confirmation . 20 to 30% of adults and 10% of children blood cultures pneumolysin is a species - specific protein toxin produced intracellularly by all clinically relevant pneumococcal strains . the expression of pneumolysin gene is anticipated to specify pneumococcal involvement in the disease , without considering the pneumococcal serotype . therefore , pneumolysin is a good target for diagnosis since it is produced by all pneumococcal strains isolated from clinical samples and its virulence properties are well known . molecular methods have been widely used for the diagnosis of several infectious diseases , including pneumococcal pneumonia , due to their high sensitivity , specificity and speed . the purpose of this study was to develop a simplified molecular strategy for s. pneumoniae detection by dna amplification in clinical specimens of children visiting different hospitals . in group a , among 44 pneumonia patient samples , 86.4% showed pcr positive results and only 2 positive results obtained in 30 acute respiratory tract infection samples . in group b , 62 out of 80 pneumonia patients and 4 out of 28 acute respiratory tract infection patients were found to be pneumolysin positive ( table 1 ) . toikka et al have described 44% pneumolysin pcr positive results out of 25 pneumococcal infected patients . mayoral et al reported that 10/24 serum samples of a group ( children with definitive diagnosis of pneumococcal pneumonia confirmed by blood - culture ) were positive when subjected to first round pcr . however , when the second round of nested - pcr was applied to the same samples , 20/24 ( 83.3% ) were found to be positive . in our study , none of 60 healthy controls when analyzed by pcr , was found to be positive , indicating a good specificity and sensitivity of the pcr procedure . peripheral blood samples from pcr positive pneumonia patients ( group a and b ) were collected prior and subsequent to antibiotic administration . out of 100 patients under antibiotic treatment for less than 48 h , however , after antibiotic treatment for more than three days , pneumococcal dna could not be detected . even after 48 h antibiotic treatment , dagan et al did not detect pneumococcal dna in the serum samples of the patients . lane m , 50bp dna size marker ; lane 2 , 4 and 5 are pneumolysin positive samples the diagnosis of acute otitis media ( aom ) requires a sensitive and consistent method for deterrence of the disease . bacterial culture and pcr - based assay was used to study the presence of s. pneumoniae in middle ear fluid samples from patients with aom . out of total 150 mef culture samples , 60 cases were s. pneumoniae culture - positive and among them 52 samples exhibited the presence of pneumolysin gene by nested - pcr . on the other hand , out of 90 mef culture - negative cases , 8 confirmed pcr positive results demonstrating the sensitivity and specificity of the molecular method to establish the involvement of pneumococcal pathogens in mef samples of children with acute otitis media ( table 2 ) . in other study , 47.1% pcr positive results for pneumolysin gene in mef samples while 27.3% culture positive results for the same samples are reported . virolainen et al have described s. pneumoniae culture positivity in 33 ( 18% ) and pneumolysin gene amplification in 51 out of 180 ( 28% ) mef clinical specimens . only 2 out of 21 pcr - positives , s. pneumoniae culture - negative samples were positive for other otitis pathogens . binomial test of proportionality performed on ( spss 17 ) showed low p values ( < 0.05 ) that suggests pneumolysin gene to be a significant diagnostic genomic marker particular to s. pneumoniae using the pcr technique . the diagnosis of pneumococcal contagion in children has been a challenge for years and largely relying on traditional culture techniques . in recent years to improve the analytic sensitivity and specificity of the diagnostic methods , numerous nucleic acid amplification technologies are developed for the rapid detection of widespread bacterial pathogens . polymerase chain reaction based assessment of pneumococcal pneumonia as compared to conventional methods , has made it a valuable tool in the etiologic diagnosis of pneumonia in the children visiting hospitals of lahore . there are several factors that appreciate using the sophisticated , rapid and accurate diagnostic tools and specifically the methods designed for keeping economic and financial considerations of the developing country like pakistan . the present study describes a comparative analysis of sensitive nested - pcr and bacterial culture in pediatric patients with clinical and radiological indication of s. pneumoniae infection . binomial test of proportionality demonstrate the sensitivity and reliability of pcr for rapid pneumonia evaluation .