cough is a common symptom for which medical attention is sought by patients . in the united states ,  cough
 was the third principal reason for office visits after  progress visit  and  general medical examination  ; there were 31 million visits for cough in 2012 ( 1 ) . in uk general practice ,  cough  was the most common symptom in more than half of all new referrals ( 2 ) . in japan ,
 cough  is also a common symptom among patients . among the 35,768 patients who visited the department of general medicine of juntendo university hospital in 2012 ,
4,543 had a  cough  , which was the second most common symptom following a  fever  ( 3 ) .
various guidelines for the diagnosis and management of cough have been published ( 4 - 9 ) .
pertussis is one of the important causes of cough , especially chronic cough ( 10 ) .
pertussis remains one of the top 10 causes of childhood death worldwide , mainly in unvaccinated children , and it causes nearly 300,000 deaths in children every year ( 11 ) .
pertussis is the most dangerous in infants , where the cough can be minimal or absent but can induce apnea , which leads to death . in unvaccinated children ,
clinical suspicion of pertussis is made in patients with a cough - associated illness lasting at least two weeks with one of the following : paroxysms of coughing , inspiratory  whoop  , and post - tussive vomiting . in the initial catarrhal stage ,
the differentiation of pertussis from a common cold is difficult . the paroxysmal whooping cough sets in after one to two weeks and can persist for four weeks or longer . adults and older children
are less likely to have typical symptoms , instead showing only mild , non - specific symptoms like prolonged cough ; therefore , the diagnosis of pertussis is difficult when it is based on symptoms alone .
all studies assessing the relative proportion of pertussis in adults with longer - lasting coughs agree that a significant proportion of them have pertussis ( 12 ) .
the best way to prevent pertussis is to get vaccinated . in japan , the pertussis vaccine has been administered since 1950 , and the number of patients with pertussis decreased from more than 100,000 in 1950 to 206 in 1971 . the first vaccine against pertussis included whole - cell killed bordetella pertussis ( b. pertussis ) bacteria and contained pertussis endotoxin ( surface lipooligosaccharide ) , which had side effects .
severe neurologic disorders led to the suspension of pertussis vaccination in 1975 , and the number of patients increased to about 13,000 in 1979 in japan .
new acellular pertussis vaccines , which include only a few selected pertussis antigens , were introduced in 1981 ( 13 ) .
outbreaks occurred in university students in 2007 , and it was suspected that many young people did not have immunity against b. pertussis , although recent studies have suggested another mechanism : asymptomatic transmission from individuals who were vaccinated with the currently used acellular b. pertussis vaccines ( 14 ) .
identification of the pathogen by culturing nasopharyngeal secretions has been considered the gold standard for diagnosis , but this method is unsuitable in clinical practice for young and adult patients , as b. pertussis loads are very low in these populations , and the isolation and identification of bacteria is very difficult , especially in previously vaccinated persons ( 15,16 ) .
compared with the culture method , nucleic acid amplification tests such as the is481-based real - time polymerase chain reaction ( pcr ) and b. pertussis - specific loop - mediated isothermal amplification ( lamp ) are highly sensitive for the detection of b. pertussis dna ( 17 - 19 ) .
therefore , these nucleic acid amplification tests have been recommended for the accurate diagnosis of pertussis ( 20 ) .
nevertheless , the pcr and lamp are virtually impossible to perform in clinical practice due to their cost and the fact that they are not covered by public health insurance in japan .
the most common pertussis serologic assays use enzyme - linked immunosorbent assay ( elisas ) with highly purified pertussis toxin ( pt ) antigen ( 21,22 ) .
pt antigen is expressed only by b. pertussis , and no cross - reacting antigens have been described ; furthermore , igg responses occur in most patients with b. pertussis infection ( 23,24 ) . a two- to four - fold increase in the igg titers against pertussis toxin antigen ( pt - igg ) in paired acute and convalescent sera
a review reported that a single measurement of pt - igg can be indicative of current pertussis infection , with a sensitivity of 76% and a specificity of 99% for the diagnosis of acute pertussis ( 11 ) .
some reports have proposed cut - off values for pt - igg to indicate recent contact ( 25 ) . in the japan respiratory society 
guidelines for cough  , a cut - off value of 100 eu / ml was used for a definitive diagnosis of pertussis , and pt - igg levels < 10 eu / ml were used to indicate non - immunized status ( 26 ) .
it was reported that concentrations of pt - igg fall below the defined cut - off about 4.5 months after infection on average , and in most patients ( 82% ) within 1 year ( 27 ) .
we herein investigated the effect of previous vaccination for pertussis on the levels of pt - igg .
we also examined the time course of the levels of pt - igg in the japanese population .
we retrospectively identified subjects who had visited ikebukuro otani clinic between december 2012 and october 2014 . in japan ,
about half of the patients were given a final diagnosis of bronchial asthma or cough - variant asthma , and one third were diagnosed with acute bronchitis by viral or bacterial infection .
all patients who had their pt - igg levels measured at multiple time points as of february 2015 were recruited for the present study , as well as those with measurements at a single time point ( fig .
1 ) . information on sex , date of birth , and pt - igg levels with the sampling date were collected from the medical records of each patient .
the patients were followed up for one year , and their data were updated in february 2016 .
we conducted epidemiologic studies of the age distribution for natural and vaccine - induced immunity against b. pertussis to evaluate the correlation between the age and pt - igg levels . to study the natural time course of the pt - igg levels ,
the average change in the logarithm of the pt - igg level versus elapsed time was analyzed for patients who had their pt - igg levels measured at multiple time points .
we excluded the subjects who were deemed inappropriate for the natural time course analysis . to exclude subjects with active b. pertussis infection during the observation period ,
the data from subjects with more than a two - fold increase of pt - igg level were excluded from the analysis .
pt - igg levels exceeding 160 eu / ml were reported as  pt - igg > 160  , and we were unable to obtain the exact values ; therefore , these data were also excluded from the change rate analysis .
the levels of pt - igg antibody were measured using commercially available enzyme immunoassays for pertussis ( denka seiken co. , ltd . ,
briefly , a plate coated with the pertussis toxin antigen was incubated with standard or test sera , washed , incubated with goat anti - human igg conjugated with horseradish peroxidase , and then washed again .
the pt - igg titers were determined using calibration curves obtained from standard serum jnih-10 ( 28 ) from the national institute of infectious diseases ( niid ) in japan .
the measurements were conducted at the laboratories of the showa medical science corporation in accordance with the manual .
pt - igg levels exceeding 160 eu / ml were reported as  pt - igg > 160  ; serum was not diluted , and no reexamination was performed to obtain the exact values .
the robustness of our results was assessed using the graphpad prism 6 software program for mac os x ( graphpad software , inc . , la jolla , usa ) .
the correlation coefficients between the age and pt - igg levels were calculated by spearman 's correlation , a method that makes no assumptions about gaussian - like distributions .
differences in the pt - igg levels between groups were estimated using the kruskal - wallis test .
this study was approved by the ethics committee of the tokyo medical and dental university ( approved number 1957 ) . given the retrospective nature of the study , informed consent from the participating patients was waived by the institutional ethics committee and not obtained , but the public was notified of the study by posters .
a total of 205 ( men , 66 ; mean age , 40.913.8 ; women , 139 ; mean age , 39.811.4 ) patients were recruited who had pt - igg levels measured at multiple time points ( fig .
201 ( men , 88 ; mean age , 42.412.8 ; women , 113 ; mean age , 39.714.1 ) first - visit patients between march 2014 and may 2014 who had pt - igg levels measured once were recruited .
thus , the age distribution of pt - igg levels in a total of 406 patients was determined ( fig .
2 ) . we found no correlation between the age and pt - igg level ( r=0.06 , p=0.20 ) .
3 shows the distribution of the pt - igg levels in the three groups by date of birth .
only a small portion of babies who were born between 1975 and 1985 were vaccinated for pertussis . after more than 30 years
, there was little difference in the levels between those born in or before 1974 ( 51.949.5 eu / ml ) , those born between 1975 and 1984 ( 44.343.6 eu / ml ) , and those born in or after 1985 ( 43.046.7 eu / ml ) ( p=0.15 ) .
the distribution of pt - igg levels in the three groups by date of birth .
pertussis vaccination was suspended in 1975 , and new pertussis vaccines were introduced in 1981 , but only a small number of babies were vaccinated . more than 18% ( 37 out of 205 ) of recruited patients had pt - igg levels exceeding 100 eu / ml . because patients with a high pt - igg level tend to be examined multiple times , we analyzed first - visit patients who had single measurements .
among the first - visit patients with single measurements , 15% ( 29 out of 201 ) had a pt - igg level exceeding 100 eu / ml . the pt - igg levels of very few subjects had diminished over a long period of time , and the average decay rate of pt - igg levels per year was 0.97-fold . in the 37 patients with an initial pt - igg level exceeding 100 eu / ml , only 3 had a pt - igg level that had dropped below 100 eu / ml during follow - up .
most patients continued to show levels exceeding 100 eu / ml for at least several months ( fig .
the temporal changes in the pt - igg levels in patients with an initial pt - igg level exceeding 100 eu / ml . the average annual decay rate of the pt - igg levels was 0.97-fold .
igg - pt levels exceeding 160 eu / ml ( upper shaded area ) were not reported as exact values .
only a small portion of patients showed pt - igg levels that had decreased to below 100 eu / ml ( lower shaded area ) . in patients with pt - igg levels between 10 and 100 eu / ml , more than 6% ( 9 out of 135 ) showed a more than 2-fold increase in the pt - igg level during long - term follow - up and were therefore regarded as having b. pertussis infection . except for these subjects ,
the average change rate for the pt - igg levels per year was 1.11-fold ( fig .
the temporal changes in the pt - igg levels in patients with an initial pt - igg level between 50 and 100 eu / ml . the average annual change rate of the pt - igg levels was 1.18-fold .
the temporal changes in the pt - igg levels in patients with an initial pt - igg level between 20 and 50 eu / ml .
the temporal changes in the pt - igg levels in patients with an initial pt - igg level between 10 and 20 eu / ml .
the average annual change rate of the pt - igg levels was 1.20-fold . in the patients with pt - igg levels below 10 eu / ml , 9%
( 3 out of 32 ) showed a more than 2-fold increase in the pt - igg level during long - term follow - up , resulting in a level exceeding 10 eu / ml during follow - up . except for these subjects ,
the average change rate for the pt - igg levels per year was 1.15-fold ( fig .
the temporal changes in the pt - igg levels in patients with an initial pt - igg level below 10 eu / ml .
we found no correlation between the age and pt - igg levels . because only a small portion of babies who were born between 1975 and 1984 had been vaccinated for pertussis , we stratified subjects according to the date of birth , and the results showed that , after more than 30 years , there was little difference in the levels between the age groups . using a cut - off value of < 10
eu / ml as an indication of non - immunization for pertussis ( 26 ) , the proportion of subjects without immunity for b. pertussis was 14% ( 55 out of 406 ) , and 9% ( 3 out of 32 ) of these subjects were regarded as having b. pertussis infection subsequently .
the patients with pt - igg levels between 10 and 100 eu / ml are usually regarded as having immunity against pertussis . in the present study , more than 5% of subjects
the relation between antibody and protection is not straightforward , with no particular serological correlate of protection  ( 11 ) .
we showed that the values of pt - igg changed very little over a long period of time .
it is unlikely that concentrations of pt - igg had naturally fallen over time and increased again after another infection by b. pertussis . because there is a very low possibility that many subjects would show almost equal concentrations of pt - igg before naturally falling over time and then rising after b. pertussis reinfection .
because patients with a high pt - igg level tend to be examined multiple times , we analyzed the patients who only had single measurements . among them , 15% had a pt - igg level exceeding 100 eu / ml .
in other words , more than 15% of subjects could be definitively diagnosed with pertussis based on a single measurement of pt - igg , according to the japan respiratory society 
however , only a small portion of them were clinically given a diagnosis of active pertussis .
one reason for this is that the pt - igg levels of very few subjects had diminished over a long period of time , and many subjects continued to have a value that exceeded the cut - off after infection or immunization .
our finding that the pt - igg levels of only a few subjects diminished over a long period of time was inconsistent with the results from previous reports ( 27 ) .
this discrepancy in findings may be due to differences in race / ethnicity or epidemic strains of b. pertussis in japan . in the present study
, the levels of pt - igg antibody were measured using a commercially available enzyme immunoassay for pertussis ( denka seiken co. , ltd . ) , which is currently the only way to measure pt - igg level in japan .
this technique is considered to allow the comparison of pt - igg titer data obtained in the iu with values from other countries , as the iu value was approximately 70 - 80% of the eu value in this assay ( 28 ) . since serum antibodies
subtle differences in the measurement methods for pt - igg levels may also explain these discrepancies in findings .
the purities of antigens , purification methods , strains , composition of buffer solutions , etc . used in the test differ among manufacturers . in the present study
, we showed that a pt - igg level exceeding 100 eu / ml might simply indicate past infection or immunization , at least in japan .
a single measurement of pt - igg using cut - off values might therefore lead to the overdiagnosis of pertussis .
commercially available measurement levels of pt - igg range from 0 to 160 eu / ml in japan .
exact values for subjects with a pt - igg exceeding 160 eu / ml might be needed for detailed analyses . the main limitation associated with
this study was that the subjects were retrospectively identified at a single institute that specialized in treating patients with cough .
furthermore , we did not use culture , pcr , or lamp methods to identify the pathogen . while we did perform culture and nucleic acid amplification tests ,
an accurate diagnosis of pertussis was impossible , as b. pertussis loads are very low in adults ( 15,16 ) , creating the possibility of false - negative results .
only a small number of subjects were given a clinical diagnosis of active pertussis , although more than 15% of recruited patients had pt - igg levels exceeding the cut - off value .
about half of the patients were given a final diagnosis of bronchial asthma or cough variant asthma , and one third were diagnosed with acute bronchitis by viral or bacterial infection .
in fact , only one subject was diagnosed with acute pertussis , and he was indistinguishable from the others because he did not present with typical symptoms such as paroxysmal coughing , inspiratory  whoop  , or post - tussive vomiting . based on paired serum titers showing a more than 2-fold increase in igg antibodies , about 7% of subjects were regarded as having b. pertussis infection in the long - term follow - up , which does not necessarily indicate an infection at the time of study entry . in summary ,
a single measurement of pt - igg level for the diagnosis of pertussis is insufficient , and using cut - off values for the diagnosis may run the risk of overdiagnosis .
the guidelines ( 26 ) have three problems associated with diagnosing pertussis : ( a ) patients with a pt - igg level exceeding 100 eu / ml can be definitively diagnosed with pertussis ; ( b ) patients with a pt - igg level exceeding 10 eu / ml who have no history of immunization can be definitively diagnosed with pertussis ; and ( c ) patients with a pt - igg level exceeding 10 eu / ml who previously had a pt - igg level less than 10 eu / ml can be definitively diagnosed with pertussis . with respect to ( a ) , in actual clinical practice in japan , the pt - igg level rarely falls below the defined cut - off value ( i.e. 100 ) ( fig .
4 ) . with respect to ( b ) , pertussis vaccinations were stopped from 1975 to 1981 , and therefore , subjects born between 1975 and 1984 are at high risk of not having been vaccinated for pertussis . in this group ,
86% ( 128 out of 149 ) of subjects showed a pt - igg level exceeding 10 eu / ml ( fig .
3 ) and therefore should have been diagnosed with acute pertussis , according to the guideline ( 26 ) . with respect to ( c ) ,
3 . orange line ) observed in the present study are within normal limits and not caused by pertussis . to make a definitive diagnosis ,
we recommend the use of the b. pertussis - specific lamp or the confirmation of a two- to four - fold increase in the pt - igg values in paired sera .
 
 objective adult patients with pertussis rarely show typical symptoms , such as paroxysmal coughing , inspiratory  whoop  , or post - tussive vomiting . 
 while a culture is regarded as the gold standard for diagnosis , the sensitivity is very low . 
 therefore , the diagnosis of pertussis in adults in clinical practice is mostly based on single - sample serology using an enzyme - linked immunosorbent assay ( elisa ) with the pertussis toxin antigen . 
 various cut - off values for the anti - pertussis toxin igg ( pt - igg ) have been proposed . 
 it has been reported that concentrations of pt - igg fall below the defined cut - off about 4.5 months after infection on average , and within 1 year in most patients . 
 we investigated the distribution and time course of the pt - igg levels . 
 
 methods the data were collected from the medical records . 
 
 patients the study retrospectively identified subjects who had visited ikebukuro otani clinic , which is a specialized clinic for patients with cough . 
 we retrospectively reviewed 406 patients with pt - igg measurements to investigate the age distribution of pt - igg levels . 
 the changes in pt - igg levels over time were assessed in the 205 patients who had more than one pt - igg measurement . 
 
 results pt - igg levels were 100 eu / ml in more than 15% of subjects . 
 the pt - igg levels of a few subjects had diminished over a long period of time . 
 
 conclusion a pt - igg level greater than the defined cut - off value simply indicates past infection or immunization in most subjects . as such 
 , a single measurement of pt - igg using the cut - off values might lead to overdiagnosis of pertussis . 
 further data collection and analysis are required . 
Introduction
Materials and Methods
Results
Discussion
The authors state that they have no Conflict of Interest (COI).