Research Article Detection of Bordetella pertussis from ...cination program in [ ]. During , there were casesoranincidenceof. perpeoplereported from European countries [ ]. In the

Hindawi Publishing CorporationInternational Journal of BacteriologyVolume 2013 Article ID 324136 5 pageshttpdxdoiorg1011552013324136

Research ArticleDetection of Bordetella pertussis from Clinical Samples byCulture and End-Point PCR in Malaysian Patients

Tan Xue Ting Rohaidah Hashim Norazah Ahmad and Khairul Hafizi Abdullah

Bacteriology Unit Infectious Disease Research Centre Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia

Correspondence should be addressed to Tan Xue Ting tanxtimrgovmy

Received 29 January 2013 Revised 26 March 2013 Accepted 28 March 2013

Academic Editor Sam R Telford

Copyright copy 2013 Tan Xue Ting et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Pertussis or whooping cough is a highly infectious respiratory disease caused by Bordetella pertussis In vaccinating countriesinfants adolescents and adults are relevant patients groups A total of 707 clinical specimens were received from major hospitalsin Malaysia in year 2011 These specimens were cultured on Regan-Lowe charcoal agar and subjected to end-point PCR whichamplified the repetitive insertion sequence IS481 and pertussis toxin promoter gene Out of these specimens 275 were positive 4by culture only 6 by both end-point PCR and culture and 265 by end-point PCR onlyThemajority of the positive cases were fromle3 months old patients (771) (119875 lt 0001) There was no significant association between type of samples collected and end-pointPCR results (119875 gt 005) Our study showed that the end-point PCR technique was able to pick up more positive cases compared toculture method

1 Introduction

Whooping cough is a major cause of the infant [1] and child-hood mortality [2] World Health Organization (WHO)reported about 16 million pertussis cases worldwide in 2008in which 95 of cases occurred in developing countries andmore than 100000 children died from this disease [1] Per-tussis still remains endemic despite the introduction of vac-cination program in 1974 [3] During 2003ndash2007 there were43482 cases or an incidence of 41 per 100000 people reportedfrom 20 European countries [4] In the United Statesthe incidence of pertussis also increased from 353 in year2007 to 554 in year 2009 [5] Although pertussis is alwaysclassified as infants and children disease an increasing num-ber of cases in adolescents and adults group were also ob-served [2 5]

According to the vaccination schedule of Malaysia everycitizen should be given vaccination at 2 3 and 5 monthsold and a booster at 18 months old [6] This is in line withthe vaccination schedule recommended by WHO Althoughthere are two types of vaccines available whole cell or acel-lular vaccine [6] the whole cell pertussis vaccine is the mostwidely used vaccine against pertussis [7] The immunizationcoverage of three-dose pertussis vaccination is 953 [8]

Pertussis is a notifiable disease in Malaysia [9]The incidenceof pertussis has been less than 1 per 100000 people from 1989to 2010 [10]

The clinical pertussis is defined by cough illness whichlast 2 weeks or more with at least one of the symptomsof paroxysms of coughing characteristic inspiratory whoopand posttussive vomiting without apparent cause [11] How-ever pertussis in adults and adolescents are rarely recognizedbecause they normally have atypical course [12] or asymp-tomatic [13] Hence they become the reservoir of the diseaseand may transmit the infection to infants [2 12] Pertussismay also result in many complications such as pneumoniaapnoea encephalopathy death loss of weight convulsionloss of bladder control and rib fractures [14]

Culture was the gold standard for the diagnosis of pertus-sis due to its specificity however it is slow and not sensitive[15] Detection by real-time polymerase chain reaction hasbeen carried out by many laboratories and was found tobe useful in the diagnosis of B pertussis infection [16ndash18] However this method of detection requires the use ofexpensive instrument Therefore we applied end-point PCRtechnique and compared it with culture method to detect Bpertussis from suspected clinical samples

2 International Journal of Bacteriology

2 Materials and Methods

A total of 707 specimens were received from 57 Malaysianhospitals in year 2011 These samples were obtained fromsuspected pertussis cases as diagnosed by clinicians and sentto our laboratory for B pertussis culture and end-point PCRdetection One sample was obtained per patient The sam-ples received were nasopharyngeal aspirates nasopharyngealswabs tracheal aspirates throat swabs pernasal swabs andendotracheal tube swabs The nasopharyngeal aspirates weretransported in sterile containers The swab samples weretransported in Regan-Lowe medium Amies Stuart or with-out any transport medium The samples were processed onthe same day of arrival

21 Culture All swab samples were rolled onto separateRegan-Lowe charcoal agar Regan-Lowe charcoal agar plateswere prepared fromdehydrated powder obtained fromOxoidand 23120583gmLminus1 cephalexin and 15 vv defibrinated horseblood were added to the medium For nasopharyngeal andtracheal aspirates approximately 01mL of the fluid wasinoculated onto the Regan-Lowe charcoal agar plates Theagar plates were then incubated at 37∘C aerobicallyThe plateswere inspected after 72 hours If no growth was observedthe plates were further reincubated until 120 hours Anysuspected colony was identified using B pertussis-specificantisera (Difco Sparks MD USA) [19]

22 Polymerase Chain Reaction The same swab sampleswere transferred into 15mL Eppendorf tubes containingphosphate buffered saline (PBS) (pH 74) and vortexed for60 s to release cellular material into fluid The swabs werethen removed and the PBS suspensions were ready for DNAextraction Nasal and tracheal aspirates were subjected todirect DNA extraction

DNA extraction was performed using QIAamp DNAblood mini kit (QIAGEN) following the manufacturerrsquosinstructions The DNA was kept at minus20∘C until furthertesting The primers used for the end-point PCR are listedin Table 1 The DNA was first screened for the presence ofIS481 using BP1 and BP2 primers [19] Amplification wasperformed using Biometra TPersonal version 20 (BiometraGmbhGoettingen Germany)with the following parameterspredenaturation at 95∘C for 5min followed by 30 cycles ofdenaturation at 95∘C for 1min annealing at 57∘C for 1minextension at 72∘C for 1min with a final extension at 72∘C for5minThe expected end-point PCRproduct size for fragmentof IS481 is 151 bp

If the screening test was positive the extracted DNA wasthen subjected to end-point PCR amplification of pertussistoxin (PT) promoter using BPTOX F and BPTOX R primers[19]The parameters used were as follows predenaturation at95∘C for 5min followed by 30 cycles of denaturation at 95∘Cfor 1min annealing at 60∘C for 1min extension at 72∘C at1min with a final extension at 72∘C for 5min The expectedend-point PCR product size of fragment of PT promoter is190 bp

The internal control primers Hg1 and Hg2 were used inboth screening and confirmatory test to target a region of

Table 1 Sequences of the primers

Primer SequenceBP1 51015840-GAT TCA ATA GGT TGT ATG CAT GGT T-31015840

BP2 51015840-TGG ACC ATT TCG AGT CGA CG-31015840

BPTOX F 51015840-GCG CAT GCG TGC AGA TTC GTC-31015840

BPTOX R 51015840-CCC TCT GCG TTT TGA TGG TGC C-31015840

HG1 51015840-CAA CTT CAT CCA CGT TCA CC-31015840

HG2 51015840-GAA GAG CCA AGG ACA GGT AC-31015840

human hemoglobin gene as described by [20] The expectedend-point PCR product size for internal control is 268 bp

The end-point PCR products were visualized by 15agarose gel electrophoresis stained with RedSafe TM NucleicAcid Staining Solution (iNtRON Biotechnology Inc Sung-nam Kyungki-Do Republic of Korea) and documented witha ChemiDoc XRS+ system (Bio-Rad Hercules CA USA)

The end-point PCR result was considered positive if bothtarget and internal control bands or target band alone wasobtained However it was considered negative when theinternal control band alone was observed Cases were definedas positive if both screening and confirmatory tests werepositive

3 Results

From the 707 samples processed 275 samples were positivefor Bordetella pertussis Four cases were detected by culturemethod alone 6 were detected by both end-point PCR andculture methods and another 265 were detected by end-point PCR alone Overall 454 samples were positive in IS481screening test and only 271 samples (60) were positive inPT promoter confirmatory test For those negative in con-firmatory test (183 samples) the cases were recognized asBordetella spp

Most of the positive cases were obtained from nasopha-ryngeal aspirates (409) followed by nasopharyngeal swab(356) pernasal swab (333) tracheal aspirate (286)and throat swab (143) and none from endotracheal tubeswab (Table 2)

The majority of positive cases were from Kedah (42275)Terengganu (39275) and Selangor (35275) The lowestnumber of pertussis cases was found in Kuala Lumpur with 2cases only

Positive cases were highest among patients aged le3months old (212275) followed by patients with aged 4ndash12months old (37275) However there were also 12 casesdetected from individuals between 13 to 24months old and 14cases in individuals more than 24 months old (Table 3)

4 Discussion

The end-point PCR assay has been used to detect B pertussisin the routine work [19] It was shown to be rapid andhighly sensitive compared to culture which is slow and haslow sensitivity Although culture has high specificity it canbe affected by several factors such as specimen transportcondition illness duration and antibiotic treatment [21 22]

International Journal of Bacteriology 3

Table 2 Number of positive cases which detected from different specimens

Specimen type Total number of specimens Number of positive specimensCulture only End-point PCR only Culture and end-point PCR

Nasopharyngeal aspirate 521 3 207 3Nasopharyngeal swab 149 0 50 3Tracheal aspirate 7 0 2 0Throat swab 14 1 1 0Pernasal swab 15 0 5 0Endotracheal tube swab 1 0 0 0

Table 3 Age range (month) of positive pertussis cases

Age (month) Total number of specimens Number of pertussis casesCulture only End-point PCR only Culture and end- point PCR

lt1 37 0 13 01ndash3 431 4 189 64ndash6 105 0 20 07ndash9 38 0 7 010ndash12 32 0 10 013ndash15 7 0 4 016ndash18 9 0 3 019ndash21 7 0 1 022ndash24 8 0 4 0gt24 33 0 14 0

In this study we screened the samples for the presenceof IS481 and proceeded to the detection of pertussis toxinwhen IS481 screening was positive The repetitive insertionsequence IS481 was shown to be present in B pertussis with200 copy numbers [23 24] However it was not specificenough as it is also present inB holmesii andB bronchiseptica[25] The other major virulent determinant is pertussis toxinpromoter region which is present as a single-copy geneSingle-target testing using IS481 may miss up to 20 of truepositive cases [17] Therefore we used dual-target PCR assaytargeting IS481 and pertussis toxin promoter region as prac-ticed by other researchers to increase the sensitivity of the test[19 26]The internal control was introduced into the assay todifferentiate between false negative and true negative results[27]

There were four cases that were culture positive butundetectable by our end-point PCR assay We believe thiscould be due to the inhibitory substances which may bepresent in the specimens as described by Douglas et al [28]Mattoo and Cherry [29] have suggested treating the spec-imens with mucolytic agent to remove the inhibitory sub-stances prior to the end-point PCR

There were several factors that could lead to the culturesensitivity in this study Many of the samples were notreceived on the same day of specimen collection except fromthe states of Selangor Kuala Lumpur and Putrajaya whichare geographically near our laboratory Besides we foundthat some of the swabs were not transported in the propertransport media namely Regan-Lowe medium as suggestedby WHO [30] Prior antibiotic treatment before collection of

samples could also affect the growth of B pertussis [31 32]Most of the patients had been on antibiotic treatment beforetheir samples were collected and this could contribute to theinability to recover viable bacteria by culture method

There was no significant association between the types ofsamples collected and end-point PCR results (119875 gt 005) (chi-square test by software SPSS 16) However nasopharyngealaspirate samples had the highest number of positive end-point PCR results (403) compared to nasopharyngeal swab(356) and others (216) Douglas et al [28] described thatthe low detection in the swab specimens was because of theinsufficient cells found on the swabs In contrast Farrell etal [23] obtained high percentage of end-point PCR positivefrom throat swab specimens in their study The detectionfrom throat swab in our study was low that is 04 and10 from end-point PCR and culture respectively The smallnumber of throat swab samples in this study could alsocontribute to the low number of positive results

In our study infant aged le3 months old was the highestgroup to be infected by B pertussis This result was expectedbecause they have not been fully vaccinated The proportionof those in the age group le3months old (453) with positiveresult was significantly higher than that in the age group gt3months old (264) (119875 lt 0001) (chi-square test by softwareSPSS 16) This could be due to the direct transmission fromtheir parents relatives or guardians whomay be the reservoirfor the bacteria This idea was supported by Kenneth et al[33] who stated that immunity against pertussis vaccinationwill wane after 10-year-vaccination and the transmission


rate to infant by household caretaker ranged between 30ndash40 Hence booster immunizations of adolescents in 10ndash19years old are strongly suggested to reduce disease burdenhealthcare setting cost and increase work productivity [3334]

5 Conclusion

In conclusion our study showed that more pertussis caseswere detected by end-point PCR compared with culturemethod Most of the positive samples were from nasopha-ryngeal aspirates However there is no significant associationbetween the type of specimens and the end-point PCR resultsMost of the cases involved le3 months old patients who hadnot completed the scheduled vaccinations Education andawareness are necessary to prevent the disease becomingwidespread

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgment

The authors thank the Director General of Health Ministryof Health Malaysia for permission to publish this paper

References

[1] World Health Organization ldquoPertussis vaccines- WHO posi-tion paperrdquo Weekly Epidemiology Record vol 85 no 40 pp385ndash400 2010

[2] Centers for Disease Control and Prevention ldquoPertussis Epi-demiology and prevention of vaccine- preventable diseaserdquo inVaccines and Immunizations vol 12 pp 215ndash232 12th edition2012

[3] A M Wendelboe A Van Rie S Salmaso and J A EnglundldquoDuration of immunity against pertussis after natural infectionor vaccinationrdquo Pediatric Infectious Disease Journal vol 24 no5 pp S58ndashS61 2005

[4] EUVAC-NET EUVAC-NET pertussis surveillance report2003ndash2007 2009 httpwwweuvacnetgraphicseuvacpdfpertussis2pdf

[5] Centers for Disease Control and Prevention ldquoSummary of noti-fiable diseases United States 2009rdquo Morbidity and MortalityWeekly Report vol 58 no 53 pp 1ndash100 2011

[6] Ministry of Health Malaysia ldquoPertussisrdquo 2012 httpwwwmy-healthgovmyv2indexphpmypertussis

[7] Ministry of Health of Malaysia ldquoChildhood Immunisation-health technology assessmentrdquo 2002 httpwwwmohgovmyattachments745

[8] Ministry of Health of Malaysia and Academy of Medicine ofMalaysia Clinical Practice Guidelines-Childhood Immunisa-tion 2004 httpwwwmohgovmyattachments3934

[9] Ministry of Health of Malaysia Case Definitions for InfectiousDiseases in Malaysia 2nd edition 2006 httpjknsarawakmohgovmyenuploadscase definitions for infectious diseases inmalaysia 2nd edition jan 2006pdf

[10] Ministry of Health of Malaysia ldquoAnnual report 2010rdquo 2012httpwwwmohgovmyvduk

[11] Ministry of Health of Malaysia ldquoCase investigation and out-break management for healthcare personnel pertussisrdquo 2010httpjknkelantanmohgovmyv3uploadsPDdownloadsguide-lines on pertussis case and outoutbr management[1]pdf

[12] World Health Organization ldquoPertussis vaccines- WHO posi-tion paperrdquo Weekly Epidemiological Record vol 80 no 4 pp29ndash40 2005

[13] E Rothstein and K Edwards ldquoHealth burden of pertussis inadolescents and adultsrdquo Pediatric Infectious Disease Journal vol24 no 5 pp S44ndashS47 2005

[14] Centers for Disease Control and Prevention ldquoPertussis(whooping cough)-complicationsrdquo 2013 wwwcdcgovper-tussisaboutcomplicationshtml

[15] F Zepp U Heininger J Mertsola et al ldquoRationale for pertussisbooster vaccination throughout life in Europerdquo The LancetInfectious Diseases vol 11 no 7 pp 557ndash570 2011

[16] L Knorr J D Fox P A G Tilley and J Ahmed-Bentley ldquoEval-uation of real-time PCR for diagnosis of Bordetella pertussisinfectionrdquo BMC Infectious Diseases vol 6 article 62 2006

[17] X Qin E Galanakis E T Martin and J A Englund ldquoMultitar-get PCR for diagnosis of pertussis and its clinical implicationsrdquoJournal of Clinical Microbiology vol 45 no 2 pp 506ndash511 2007

[18] M Riffelmann C H W Von Konig V Caro and N GuisoldquoNucleic acid amplification tests for diagnosis of Bordetellainfectionsrdquo Journal of Clinical Microbiology vol 43 no 10 pp4925ndash4929 2005

[19] D M Dragsted B Dohn J Madsen and J S Jensen ldquoCompar-ison of culture and PCR for detection of Bordetella pertussis andBordetella parapertussis under routine laboratory conditionsrdquoJournal of Medical Microbiology vol 53 no 8 pp 749ndash7542004

[20] J R Lingappa W Lawrence S West-Keefe R Gautom andB T Cookson ldquoDiagnosis of community-acquired pertussisinfection comparison of both culture and fluorescent-antibodyassays with PCR detection using electrophoresis or dot blothybridizationrdquo Journal of Clinical Microbiology vol 40 no 8pp 2908ndash2912 2002

[21] H O Hallander ldquoMicrobiological and serological diagnosis ofpertussisrdquo Clinical Infectious Diseases vol 28 no 6 pp S99ndashS106 1999

[22] R M Wadowsky R H Michaels T Libert L A Kingsleyand G D Ehrlich ldquoMultiplex PCR-based assay for detection ofBordetella pertussis in nasopharyngeal swab specimensrdquo Journalof Clinical Microbiology vol 34 no 11 pp 2645ndash2649 1996

[23] D J Farrell G Daggard and T K S Mukkur ldquoNested duplexPCR to detect Bordetella pertussis and Bordetella parapertussisand its application in diagnosis of pertussis in nonmetropolitansoutheast Queensland Australiardquo Journal of Clinical Microbiol-ogy vol 37 no 3 pp 606ndash610 1999

[24] J Parkhill M Sebaihia A Preston et al ldquoComparative analysisof the genome sequences of bordetella pertussis Bordetellaparapertussis and Bordetella bronchisepticardquo Nature Geneticsvol 35 no 1 pp 32ndash40 2003

[25] A Tizolova N Guiso and S Guillot ldquoInsertion sequencesshared by Bordetella species and implications for the biologicaldiagnosis of pertussis syndromerdquo European Journal of ClinicalMicrobiologyamp InfectiousDiseases vol 32 no 1 pp 89ndash96 2013

[26] K M Tatti K H Wu M L Tondella et al ldquoDevelopment andevaluation of dual-target real-time polymerase chain reactionassays to detect Bordetella spprdquo Diagnostic Microbiology andInfectious Disease vol 61 no 3 pp 264ndash272 2008


[27] P Mastrantonio P Stefanelli and M Giuliano ldquoPolymerasechain reaction for the detection ofBordetella pertussis in clinicalnasopharyngeal aspiratesrdquo Journal of Medical Microbiology vol44 no 4 pp 261ndash266 1996

[28] E Douglas J G Coote R Parton and W McPheat ldquoIdentifi-cation of Bordetella pertussis in nasopharyngeal swabs by PCRamplification of a region of the adenylate cyclase generdquo Journalof Medical Microbiology vol 38 no 2 pp 140ndash144 1993

[29] S Mattoo and J D Cherry ldquoMolecular pathogenesis epidemi-ology and clinical manifestations of respiratory infections dueto Bordetella pertussis and other Bordetella subspeciesrdquo ClinicalMicrobiology Reviews vol 18 no 2 pp 326ndash382 2005

[30] World Health Organization Laboratory Manual For the Diag-nosis of the Whooping Cough Caused By Bordetella PertussisBordetella Parapertussis chapter 3 2007

[31] A Faulkner T Skoff S Martin et al ldquoPertussis Manual forthe Surveillance of Vaccine-Preventable Diseasesrdquo in ManualFor the Surveillance of VaccIne-Preventable Diseases 5th edition2011

[32] L Gustafsson H O Hallander P Olin E Reizenstein and JStorsaeter ldquoA controlled trial of a two-component acellular afive-component acellular and a whole-cell pertussis vaccinerdquoThe New England Journal of Medicine vol 334 no 6 pp 349ndash355 1996

[33] W P Kenneth W H Joel F B Marc and I W Joel ldquoEvaluationof strategies for use of acellular pertussis vaccine in adolescentsand adults a cost-benefit analysisrdquo Clinical Infectious Diseasesvol 39 no 1 pp 20ndash28 2004

[34] J I Ward J D Cherry S J Chang et al ldquoEfficacy of anacellular pertussis vaccine among adolescents and adultsrdquo TheNew England Journal of Medicine vol 353 no 15 pp 1555ndash15632005

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Enzyme Research



Microbiology


2 Materials and Methods

A total of 707 specimens were received from 57 Malaysianhospitals in year 2011 These samples were obtained fromsuspected pertussis cases as diagnosed by clinicians and sentto our laboratory for B pertussis culture and end-point PCRdetection One sample was obtained per patient The sam-ples received were nasopharyngeal aspirates nasopharyngealswabs tracheal aspirates throat swabs pernasal swabs andendotracheal tube swabs The nasopharyngeal aspirates weretransported in sterile containers The swab samples weretransported in Regan-Lowe medium Amies Stuart or with-out any transport medium The samples were processed onthe same day of arrival

21 Culture All swab samples were rolled onto separateRegan-Lowe charcoal agar Regan-Lowe charcoal agar plateswere prepared fromdehydrated powder obtained fromOxoidand 23120583gmLminus1 cephalexin and 15 vv defibrinated horseblood were added to the medium For nasopharyngeal andtracheal aspirates approximately 01mL of the fluid wasinoculated onto the Regan-Lowe charcoal agar plates Theagar plates were then incubated at 37∘C aerobicallyThe plateswere inspected after 72 hours If no growth was observedthe plates were further reincubated until 120 hours Anysuspected colony was identified using B pertussis-specificantisera (Difco Sparks MD USA) [19]

22 Polymerase Chain Reaction The same swab sampleswere transferred into 15mL Eppendorf tubes containingphosphate buffered saline (PBS) (pH 74) and vortexed for60 s to release cellular material into fluid The swabs werethen removed and the PBS suspensions were ready for DNAextraction Nasal and tracheal aspirates were subjected todirect DNA extraction

DNA extraction was performed using QIAamp DNAblood mini kit (QIAGEN) following the manufacturerrsquosinstructions The DNA was kept at minus20∘C until furthertesting The primers used for the end-point PCR are listedin Table 1 The DNA was first screened for the presence ofIS481 using BP1 and BP2 primers [19] Amplification wasperformed using Biometra TPersonal version 20 (BiometraGmbhGoettingen Germany)with the following parameterspredenaturation at 95∘C for 5min followed by 30 cycles ofdenaturation at 95∘C for 1min annealing at 57∘C for 1minextension at 72∘C for 1min with a final extension at 72∘C for5minThe expected end-point PCRproduct size for fragmentof IS481 is 151 bp

If the screening test was positive the extracted DNA wasthen subjected to end-point PCR amplification of pertussistoxin (PT) promoter using BPTOX F and BPTOX R primers[19]The parameters used were as follows predenaturation at95∘C for 5min followed by 30 cycles of denaturation at 95∘Cfor 1min annealing at 60∘C for 1min extension at 72∘C at1min with a final extension at 72∘C for 5min The expectedend-point PCR product size of fragment of PT promoter is190 bp

The internal control primers Hg1 and Hg2 were used inboth screening and confirmatory test to target a region of

Table 1 Sequences of the primers

Primer SequenceBP1 51015840-GAT TCA ATA GGT TGT ATG CAT GGT T-31015840

BP2 51015840-TGG ACC ATT TCG AGT CGA CG-31015840

BPTOX F 51015840-GCG CAT GCG TGC AGA TTC GTC-31015840

BPTOX R 51015840-CCC TCT GCG TTT TGA TGG TGC C-31015840

HG1 51015840-CAA CTT CAT CCA CGT TCA CC-31015840

HG2 51015840-GAA GAG CCA AGG ACA GGT AC-31015840

human hemoglobin gene as described by [20] The expectedend-point PCR product size for internal control is 268 bp

The end-point PCR products were visualized by 15agarose gel electrophoresis stained with RedSafe TM NucleicAcid Staining Solution (iNtRON Biotechnology Inc Sung-nam Kyungki-Do Republic of Korea) and documented witha ChemiDoc XRS+ system (Bio-Rad Hercules CA USA)

The end-point PCR result was considered positive if bothtarget and internal control bands or target band alone wasobtained However it was considered negative when theinternal control band alone was observed Cases were definedas positive if both screening and confirmatory tests werepositive

3 Results

From the 707 samples processed 275 samples were positivefor Bordetella pertussis Four cases were detected by culturemethod alone 6 were detected by both end-point PCR andculture methods and another 265 were detected by end-point PCR alone Overall 454 samples were positive in IS481screening test and only 271 samples (60) were positive inPT promoter confirmatory test For those negative in con-firmatory test (183 samples) the cases were recognized asBordetella spp

Most of the positive cases were obtained from nasopha-ryngeal aspirates (409) followed by nasopharyngeal swab(356) pernasal swab (333) tracheal aspirate (286)and throat swab (143) and none from endotracheal tubeswab (Table 2)

The majority of positive cases were from Kedah (42275)Terengganu (39275) and Selangor (35275) The lowestnumber of pertussis cases was found in Kuala Lumpur with 2cases only

Positive cases were highest among patients aged le3months old (212275) followed by patients with aged 4ndash12months old (37275) However there were also 12 casesdetected from individuals between 13 to 24months old and 14cases in individuals more than 24 months old (Table 3)

4 Discussion

The end-point PCR assay has been used to detect B pertussisin the routine work [19] It was shown to be rapid andhighly sensitive compared to culture which is slow and haslow sensitivity Although culture has high specificity it canbe affected by several factors such as specimen transportcondition illness duration and antibiotic treatment [21 22]
















5 Conclusion




Acknowledgment


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















5 Conclusion




Acknowledgment


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusion




Acknowledgment


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Detection of Bordetella pertussis from ...cination program in [ ]. During , there were casesoranincidenceof. perpeoplereported from European countries [ ]. In the