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pertussis ( whooping cough ) is a major acute respiratory infection caused by bordetella pertussis bacteria and is associated with severe respiratory illness in children and persistent cough in adolescents and adults .
the most effective method to prevent and control pertussis is immunization ; however , the incidence of pertussis has increased in several developed countries despite high vaccination coverage .
bordetella parapertussis and bordetella holmesii , which are closely related to b. pertussis , also cause pertussis - like coughs , .
acellular pertussis vaccines ( acvs ) have little or no protection against b.  parapertussis and b.  holmesii infections , both of which have been detected recently with significant percentages in pertussis - like illness , , .
in addition , mycoplasma pneumoniae is a bacterial agent of atypical pneumonia in children and adults that also causes a persistent pertussis - like cough .
the clinical diagnosis of b.  pertussis infection is complicated by other respiratory pathogens . in japan ,
the incidence of pertussis cases in adolescents and adults has significantly increased since the early 2000s , and a large pertussis epidemic occurred between 2008 and 2010 despite high vaccination coverage with acvs .
a total of 17  349 cases were reported from approximately 3000 sentinel clinics and hospitals in the epidemic .
the national pertussis surveillance data regarding individuals diagnosed mainly on the basis of clinical symptoms , bacterial culture and/or serologic testing are collected . in comparison to culture and serology , which are the classic tests for pertussis diagnosis
, nucleic acid amplification tests have improved sensitivity and specificity ; however , these tests have not yet been widely introduced into japan .
therefore , our laboratory , a national reference laboratory for pertussis , developed a multitarget real - time pcr assay ( 4plex rt - pcr ) to discriminate among b.  pertussis , b. parapertussis , b. holmesii and m.  pneumoniae and then introduced the assay into our diagnostic service starting in january 2013 .
the present study reports 2 years of experience of the service for 355 patients with clinically suspected pertussis .
between january 2013 and december 2014 , we conducted a laboratory - based surveillance study of 355 patients clinically diagnosed with pertussis . clinical specimens ( nasopharyngeal swab or aspirate )
all the nasopharyngeal aspirates ( 2% of the total number of specimens ) were collected from young infants hospitalized with severe respiratory distress .
nasopharyngeal swabs were immersed in 0.5  ml of saline , vortexed and subjected to centrifugation ( 20  000    g for 10 minutes ) .
nasopharyngeal aspirates ( 50100  l ) were suspended in 1  ml of saline , vortexed , and centrifuged at the same conditions .
total dna was extracted from the pellet using the qiaamp dna micro kit ( qiagen ) and eluted with 25  l of the ae elution buffer .
the overall result was considered positive if any of these specimens tested positive . in japan , pertussis cases are reported based on clinical diagnosis .
the clinical criteria are cough lasting for  2 weeks with one or more of the following symptoms : whoop and staccato cough , apneic paroxysm or posttussive vomiting . however , in the present study , patients were clinically diagnosed based on their physician 's judgement , so not all patients met the reporting criteria .
this study was considered exempt from institutional review board approval because clinical specimens were obtained for diagnostic and surveillance purposes .
target sequences of the 4plex rt - pcr were insertion sequence is481 ( detection for b.  pertussis and b.  holmesii ) , reca ( b.  holmesii ) , is1001 ( b.  parapertussis ) and atpd ( m.  pneumoniae ) . published primers and probes were used for reca and atpd 
 , , and those for is481 and is1001 were used with minor modifications   ( table  1 ) .
4plex rt - pcr was performed using an abi 7500 fast real - time pcr system ( applied biosystems ) .
the pcr amplifications were carried out in 20  l reaction volumes containing 10  l of 2 premix ex taq ( perfect real time , takara bio ) , 0.2  l of 50 rox reference dye ii , 2  l of dna samples and optimized concentrations of primers and probes ( table  1 ) .
the pcr conditions were 10  s at 95c , followed by 40 cycles of 95c for 3 seconds and 60c for 30 seconds .
the cutoff threshold rn ( relative fluorescent intensity ) value was set to 0.3 for all fluorescence signals , and the sample was considered positive with a change in rn of  0.3 , corresponding to a threshold cycle ( ct ) cutoff value of approximately 36 with automatic calculation . in each assay , purified dnas ( 10  pg of b.  pertussis tohama , 100  pg of b.  holmesii atcc51542 , 10  pg of b.  parapertussis baa-587 , 1  pg of m.  pneumoniae nbrc14401 ) were used as positive controls , whereas the negative control was sterile distilled water .
the analytical sensitivities of the assay were not affected by multiplexing ( supplemental fig . 
the measured pcr amplification efficiencies of 98% to 103% for the targets b.  holmesii is481 and reca and m.  pneumoniae atpd were in good agreement with the theoretical value of 100% .
the efficiencies for b.  pertussis is481 and b.  parapertussis is1001 showed somewhat higher values ( 116117% ) , the reason for which is currently unclear . in the 4plex rt - pcr assay , b.  pertussis infection was defined as positive for is481 and negative for reca ( is481/reca ) , whereas b.  holmesii was defined as positive for both is481 and reca ( is481/reca ) .
b.  parapertussis and m.  pneumoniae were defined as positive for is1001 and atpd , respectively .
among 355 patient samples , 94 ( 26% ) , four ( 1.1% ) and two ( 0.6% ) were positive for b.  pertussis , b. parapertussis and m. pneumoniae , respectively .
no positive samples for b.  holmesii were detected , but 4plex rt - pcr targeting reca is less sensitive than is481 .
b.  pertussis  positive samples were observed in relatively large numbers from april to june 2014 ( 34% of the total ) and also showed a tendency to decrease in the winter season from december to february .
all of them were collected in the same hospital , with no apparent seasonal variations .
two m.  pneumoniae  positive samples were detected in october 2013 and 2014 , and confirmed using a separate genetic test ( loopamp mycoplasma p detection kit ; eiken chemical , japan ) .
our pertussis surveillance data did not fully correspond with national pertussis surveillance data , especially in the period from april to august 2014 . as shown in table  2 , of 94 b.  pertussis - positive samples ,
21 were from infants aged  3 months , and 20 were from children aged 5 to 9 years .
of four b.  parapertussis - positive samples , three and one were from children aged 1 to 4 years and 5 to 9 years , respectively .
similarly , two m.  pneumoniae  positive samples belonged to the same age groups .
infants aged  3 months had the highest rate ( 49% ) , whereas children aged 1 to 4 years had the lowest rate ( 16% ) . of note ,
the positive rates of the age groups 4 to 11 months , 1 to 4 years , 5 to 9 years , and  20 years were statistically lower than that of infants aged  3 months ( p  <  0.05 , fisher 's exact test ) .
in contrast , the positive rates of the age groups 10 to 14 and 15 to 19 years were not statistically significant with that of infants aged  3 months ( p 0.06 and 0.18 , respectively ) .
this study confirmed that b. pertussis is the most common aetiologic agent in cases of suspected pertussis and that b.  parapertussis and m.  pneumoniae are also responsible for causing a small proportion of pertussis - like illnesses . in this study , no positive samples for b.  holmesii were detected , although respiratory infection with b.  holmesii has recently increased worldwide , , .
4plex rt - pcr has high analytical sensitivities for b.  pertussis , b. parapertussis and m.  pneumoniae ( detection limits of 10100 fg genomic dna ) , but it has a relatively low sensitivity for b.  holmesii reca ( detection limit of 1  pg dna , corresponding to a ct value of 35.6 ) .
we therefore can not exclude the possibility that some patients could have low b.  holmesii dna loads ( ct value of > 35.6 ) in their clinical specimens .
however , we previously reported that patients not treated with antibiotics had high b.  holmesii dna loads ( ct values of 21.6 to 28.7 ) in their nasopharyngeal swab samples .
thus , 4plex rt - pcr has sufficient sensitivity to detect b.  holmesii in clinical specimens .
a recent study has found that an increasing number of b.  holmesii bacteraemia cases were associated with outbreaks of b.  pertussis 
 .
this suggests that b.  holmesii epidemics occur periodically , similar to b. pertussis . in japan ,
a b.  pertussis epidemic has not occurred from 2011 to the present ; hence , continuous laboratory - based surveillance is required for b. holmesii . in the present study ,
b. pertussis  positive samples were detected from individuals over a wide age range , consistent with previous reports , , , .
infants aged  3 months had the highest positive rate ( 49% ) ; interestingly , persons aged 10 to 14 and 15 to 19 years also showed high positive rates ( each 29% ) . in japan , acvs are used to control pertussis with a schedule of three primary doses and a single booster dose at ages 3 , 4 , 6 and 18 to 23 months , respectively .
the vaccination is done under the japanese preventive vaccination law , and consequently immunization rates among children are high (  90% ) . the duration of immunity after vaccination is estimated to be in the range of 4 to 12 years , ; accordingly , preteens and teens are considered to be at high risk of b.  pertussis infection .
moreover , this study also demonstrates that children aged 1 to 4 years had the lowest positive rate ( 16% ) .
this age group covers children from the age where they have received at least three doses of acvs .
the herd immunity against b.  pertussis would be well maintained in the age group of 1 to 4 years .
the lack of detailed clinical information ( e.g. , duration of cough , time of specimen collection and time of onset of symptoms ) makes it difficult to explain why the b.  pertussis  positive rate is equally low ( 18% ) in the  20 years age group .
recent genomic analyses revealed that some bordetella bronchiseptica isolates ( a broad - host - range pathogen ) harbor is481 and/or is1001 
 .
this implies that b.  bronchiseptica can not be discriminated from b.  pertussis and b.  parapertussis using 4plex rt - pcr .
however , b.  bronchiseptica infection in humans is rare , ; are only two case reports exist from japan .
therefore , we believe that b.  bronchiseptica infection is negligible in our results . in conclusion , we successfully used 4plex rt - pcr for the surveillance of pertussis in japan .
additionally , we confirmed that , similar to infants , preteens and teens are at high risk of b.  pertussis infection .
the 4plex rt - pcr described here has become a practical tool for detection of pertussis , and therefore we have started supplying the 4plex rt - pcr kit to japanese prefectural and municipal public institutes for regional surveillance .
between january 2013 and december 2014 , we conducted laboratory - based surveillance of pertussis using multitarget real - time pcr , which discriminates among bordetella pertussis , bordetella parapertussis , bordetella holmesii and mycoplasma pneumoniae . of 355 patients 
 clinically diagnosed with pertussis in japan , b.  pertussis , b. parapertussis and m.  pneumoniae were detected in 26% ( n  =  94 ) , 1.1% ( n  =  4 ) and 0.6% ( n  =  2 ) , respectively , whereas b.  holmesii was not detected . it was confirmed that b.  parapertussis and m.  pneumoniae are also responsible for causing pertussis - like illness . 
 the positive rates for b.  pertussis ranged from 16% to 49% , depending on age . 
 infants aged  3 months had the highest rate ( 49% ) , and children aged 1 to 4 years had the lowest rate ( 16% , p  <  0.01 vs. infants aged  3 months ) . 
 persons aged 10 to 14 and 15 to 19 years also showed high positive rates ( 29% each ) ; the positive rates were not statistically significant compared with that of infants aged    3 months ( p    0.06 ) . 
 our observations indicate that similar to infants , preteens and teens are at high risk of b.  pertussis infection .