reports from the world health organization ( who ) reveal that 17.6 million persons contracted whooping cough in 2003 resulting in 279000 deaths ; however , some authorities estimate the real figures of cases with pertussis to be around 50 million with 90% living in developing countries . according to the report of who regional office for the eastern mediterranean , 19000 deaths have occurred in under 5-year - old children because of pertussis .  
immunization against pertussis is included in the expanded program of immunization developed by the who and has been implemented in iran since 1950 .
whole cell pertussis vaccine is used in combination with diphtheria and tetanus toxoids ( as dwpt ) to vaccinate infants and children against these three deadly diseases . in spite of a coverage of > 95% for dwpt
young children continue to contract whooping cough and are placed at risk for complications and sometimes mortality associated with this vaccine preventable disease .
occurrence of pertussis in vaccinated individuals has raised questions about the protection afforded by the whole cell vaccine ; numerous researchers have suggested different approaches for assessment of vaccine efficacy including estimating the prevalence of pertussis in vaccinated populations . since pertussis may mimic other respiratory diseases such as adenovirus , influenza and mycoplasma infections , resulting in the so - called  pertussis - like 
syndrome , relying solely on the clinical presentation would not be a true measure for documenting the disease , as many cases would be clinically mislabeled as pertussis .
current diagnostic procedures include culture , polymerase chain reaction ( pcr ) , and a rise in antibodies through enzyme - linked immuno - assay ( elisa ) .
isolation of bordetella pertussis which is the gold standard for diagnosis is a difficult and time consuming procedure , making it impractical for epidemiologic studies .
detecting the organism by pcr is rapid and sensitive but sensitivity decreases with time and with antibiotic treatment .
serology , however , appears to be an easily available and reliable technique to document definite infection with bordetella pertussis ; a rise in igg antibodies against pertussis toxin ( igg - pt ) is seen in > 90% of individuals exposed to b. pertussis either through a natural infection or through vaccination .
serum iga , however , does not rise after vaccination and is detectable only in children who acquire natural infection . in vaccinated children
because of the anamnestic response of the immune system after immunization , a rapid increase in anti - pertussis antibodies is seen which prevents a significant difference in antibody concentrations between the acute and recovery sera
. therefore , in vaccinated individuals , detection of anti - pertussis iga , single values of igg antibodies above a certain level , and single high values of igg antibodies 2 to 3 standard deviations exceeding the mean value in vaccinated uninfected individuals have been used to diagnose natural infection .
we aimed to determine the prevalence of pertussis in vaccinated infants and children at different ages ranging from 2 months to 6 years by measuring the anti - pertussis igg and iga antibodies .
we aimed to provide an estimate of the protection afforded by the whole cell pertussis vaccine incorporated in the dwpt vaccine currently used in iran for routine immunization of children .
this cross - sectional study was done in 6 health facility centers affiliated to tehran and shahid beheshti universities of medical sciences , tehran , iran .
the protocol of this study was approved by the ethics committee of shahid beheshti university of medical sciences , tehran , iran .
we included disease - free and afebrile infants and children aged 2 , 4 , 6 , 12 , 18 and 72 months with a valid vaccination record ( card ) , referring to centers for dwpt vaccination .
children with incomplete or poorly documented vaccination records , those with a history of blood transfusion , immune - compromised children or those receiving immunosuppressive drugs were excluded from our study .
the sample size was estimated to be 100 samples from each age group ( power=80% , confidence interval=95% ) .  
the children s vaccination cards showed that their vaccination status was up - to - date .
after documenting the relevant data , 2 ml venous blood was collected from each child and sent to the laboratory where the sample was centrifuged and the serum stored at -70c .
samples were then tested by elisa for the presence of anti - pertussis iga ( anti - pertussis toxin , anti - filamentous hemaglutinin , and anti - lipopolysaccharides antibodies ) and igg ( anti - pertussis toxin , anti - filamentous hemaglutinin , and anti - lipopolysaccharides antibodies ) using the kit supplied by the ibl company , germany ( reference no : re56131 and re 56141 ) .
serum igg and iga levels were measured in 2 , 4 , 6 , 12 , 18 and 72-month - old children before administering the scheduled dwpt vaccine , imported from the serum institute of india and is routinely administered at 2 , 4 , 6 , 18 , and 72 months of age .
the antibody levels were recorded at different ages and compared with baseline levels at 2 months . in further analysis ,
the geometric mean titer ( gmt ) were classified sequentially for both igg and iga at ages 2 , 4 , 6 , 12 , and 18 months as the baseline levels and compared with the gmt of the two antibodies at higher ages .
a natural pertussis infection was determined through any of the following : 1- a positive iga titer , 2- to have an igg level above the mean+2sd level . in each age group , after excluding iga positive individuals , as naturally infected persons in the remaining children , assumed as being uninfected , the mean+2sd level of igg was assigned as the upper limit of vaccine induced antibody and also as a cut - off ( threshold ) . any rise above this level of igg
3- to have an igg level 100 iu / ml .   categorical variables were reported as frequency and percentage and for quantitative variables were presented as meanstandard deviation ( sd ) .
antibody gmts and related standard errors ( se ) were calculated by logarithmic transformation of data .
linear or logistic regression analyses were done according to the type of dependent variable . to evaluate the level of antibodies against burdetella pertussis between the groups , analysis of variance ( anova ) and for pair wise comparisons bonferroni test have been used . to evaluate the association of categorical data with each other ,
p<0.05 was considered as statistically significant . in case of performing multiple comparisons to evaluate a single hypothesis ,
we included 725 children aged 2 , 4 , 6 , 12 , 18 , and 72 months .
the most collected samples ( n=182 ) were from the 6-year - old group .  
mean ( sd ) igg levels ( gmts ) at 2 , 4 , 6 , 12 , 18 , and 72 months were 8.43 ( 1.07 ) , 6.31 ( 1.22 ) , 8.29 ( 1.04 ) , 8.58 ( 1.08 ) , 7.35 ( 1.11 ) , 14.4 ( 1.06 ) u / ml , respectively . the mean ( sd ) iga levels at the same ages were 1.48 ( 1.21 ) , 1.43 ( 1.23 ) , 1.45 ( 1.32 ) , 2.66 ( 1.21 ) , 2.24 ( 1.19 ) , 2.03 ( 1.1 ) u / ml , respectively ( tables 1 and 2 ) .  
comparison of iga geometric meansd titers at different ages with 2 , 4 , 6 , 12 , and 18 months considered sequentially as bases for comparison with higher ages gmt : geometric mean titer ; * statistically significant the estimated frequency of natural pertussis infection using different variables the gmts for igg showed a slight fall after 2 months and a definite rise in children aged 72 months ( figure 1 ) .
the gmts for igg shows a slight fall after 2 months and a definite rise in children aged 72 months .
gmt : geometric mean titer this figure shows the gmt for iga at different ages of 2 , 4 , 6 , 12 , 18 and 72 months .
gmt : geometric mean titer 1% of 2 , 4 , and 6-month - old infants , 6% of the 12 and 18-month - olds and 12% of 6-year - old children had igg levels above the determined cut - off ( derived from mean+2sd ) .
1% of the 2 , 4 , and 6month - old infants , 5% of the 12 and 18month - olds and 10% of the 6-year - old children had igg levels 100 iu / ml .   gmts of serum iga were compared between different age groups , which showed significantly higher gmts at certain ages ( table 2 ) .
gmts for iga reached the highest levels at 12 and 18 months of age ( figure 2 ) .
iga levels above the assay cut - off were detected in 5 , 9 , 6 , 23 , 11 , and 8% of the children at the ages of 2 , 4 , 6 , 12 , 18 and 72 months , respectively .
considering the equivocal results of iga as well as the positive ones ( iga8 u / ml ) , the frequency of natural infection were 5 , 9 , 6 , 23 , 11 , and 8% at the ages of 2 , 4 , 6 , 12 , 18 and 72 months , respectively .
our findings revealed that igg titers declined slightly after 2 months , and then remained constant until 18 months of age .
however , a sharp rise in the igg gmt was seen at 72 months , i.e. before receiving the pre - school booster .
the plateau in the gmt until 18 months can be explained by the repeated vaccinations including the primary series at 2 , 4 and 6 months and the first booster given between 12 and 18 months of age .
however , the unexpected sharp rise in igg before the preschool booster i.e. between 4 - 5 years after the previous immunization implies recent contact with bordetella pertussis , which could only be explained through the acquirement of natural infection .   in france , 360 children were tested for pertussis serology 0.5 to 158 months after complete whole cell pertussis vaccination .
they concluded that unrecognized pertussis is common in france despite massive and sustained immunization in infants and that vaccinated children become susceptible to infection more than 6 years after their last vaccination .
although a rise in serum iga is observed only after a natural infection , all infections are not associated with an iga response .
although , detection of iga against bordetella pertussis has a low sensitivity with a negative predictive value of 61% , it is highly specific for a definite diagnosis of pertussis with a positive predictive value of almost 100% . in our study serum iga level
was elevated in 6 - 12% of infants < 2years of age and in 4% of 6-year - old children .
it has been reported that iga against the pt antigen rises in 20 - 40% and iga against the anti - filamentous hemaglutinin is increased in 30 - 50% of natural infections . in this study we measured iga against three of bordetella pertussis antigens ( anti - pertussis toxin , anti - filamentous hemaglutinin and anti - lipopolysaccharides antibodies ) .
therefore , we presumed that the sensitivity of iga in our study would be higher than the quoted figures for the measurement of separate antigens .
besides , as the half - life of iga antibodies is considerably shorter than igg , the presence of this antibody denotes a recent infection .
based solely on iga levels , we estimated the prevalence of natural infection in our studied population of vaccinated children at ages of 4 , 6 , 12 , 18 , and 72 months to be between 9 - 11% , with the highest percentage was at 18 months . in infants aged 2 months , yet to receive their dwpt vaccination , 5% revealed evidence of recent exposure to bordetella pertussis .
because of the low sensitivity of iga , these records are believed to be only a part of the real figures .
some investigators have used cut - off points for single serum samples derived from the mean+2sds of anti - pertussis igg to document natural infection . in our study
we used a similar strategy to estimate the frequency of the naturally infected vaccinated children through measuring anti - pertussis igg .
then , a cut - off point of mean+2sd of the anti - pertussis igg was assumed in the remaining samples ( uninfected group ) as the maximum level of vaccine induced antibody .
even these figures are an underestimation of naturally infected individuals , because inclusion of iga negative but igg positive individuals in the uninfected group would increase the mean igg levels .
consequently , the cut - off point of mean+2sd would rise , resulting in the underestimation of truly infected children .
the protective effect of the dwpt vaccine is reported to last for a varying period from 4 - 12 years . moreover , only about 52% of children would have a protective level of antibodies 4 years after receiving the vaccine . in a recent study from australia
it was noticed that the peak rate of pertussis had shifted from the age of 8 - 9 years to 12 - 13 years , after the 5 dose of the dwpt vaccine was introduced as a pre - school booster in 4 - 5 year - old children .
the authors concluded that the protection provided by the dwpt vaccine declines 6 - 9 years after the last dose .
immunity against pertussis , either after vaccination or even after natural infection does not bear a direct relationship with the antibody levels , and an individual may be protected against the disease even if the antibodies are undetectable in the blood .
on the other hand , antibody levels may rise after an asymptomatic infection in vaccinated individuals .
the who reports 98 proven cases of pertussis from iran in 2004 , and 125 cases in 2005 .
these figures have grossly underestimated the true numbers and result from under - diagnosis and under - reporting , both of which stem from a lack of definite clinical diagnostic criteria and appropriate laboratory methods .
our figures vary widely from those of ghanaie and colleagues from iran who quote an incidence of 318/100000 in 2008 in tehran among school children between the ages of 6 - 14 years .
this disparity could be explained by the differences in the age of the study population in the 2 studies as well as the use of different criteria used for the diagnosis of pertussis ; theirs being clinical manifestation plus a positive pcr .
a considerable percentage of children had high levels of anti - pertussis igg antibodies , positive anti - pertussis iga , and most importantly a significant rise of anti - pertussis igg gmt at 6 years of age , depicting a natural infection in vaccinated children .
further surveys need to be done to study the medium and long - term protection afforded by the current vaccine , in order to prevent the disease in these age group .
 
 background : it seems that the incidence of pertussis - like illnesses is considerably increasing despite the wide coverage of immunization with the whole cell pertussis vaccine . 
 we aimed to investigate the occurrence of pertussis in vaccinated children by measuring anti - pertussis antibodies . 
 
 methods : in this cross - sectional study , blood samples were taken from vaccinated children aged 2 , 4 , 6 , 12 , 18 , and 72 months . 
 anti - pertussis igg and iga were measured by elisa . 
 p<0.05 was considered significant . 
 
 results : 725 children were enrolled in the study . 
 geometric mean titers for igg that showed a slight decease after 2 months of age and increased distinctly in children aged 72 months . 
 the frequency of the individuals whose igg was above the determined cut - off ( derived from mean+2sd ) was observed in 1% of the 2 , 4 , and 6-month - old infants , 6% of the 12 and 18-month - olds and 12% of the 6-year -old children . 
 positive iga titers were detected in 5 , 9 , 6 , 23 , 11 , and 8% of children aged 2 , 4 , 6 , 12 , 18 , and 72 months , respectively . 
 
 conclusion : since a considerable percentage of children had high levels of anti - pertussis igg antibodies ( 2 sd ) , positive anti - pertussis iga , and most importantly an increased level of anti - pertussis igg geometric mean titer at 6 years of age , further investigations regarding the protection provided by the presently used pertussis vaccine seems necessary . 
Introduction
Subjects and Methods
Results
Discussion
Conclusion
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