Selecting PCR for the Diagnosis of Intestinal...

Research ArticleSelecting PCR for the Diagnosis of IntestinalParasitosis Choice of Targets Evaluation of In-House Assaysand Comparison with Commercial Kits

G N Hartmeyer12 S V Hoegh2 M N Skov12 R B Dessau3 andM Kemp12

1Research Unit of Clinical Microbiology Institute of Clinical Research Faculty of Health ScienceUniversity of Southern Denmark Odense Denmark2Department of Clinical Microbiology Odense University Hospital Odense Denmark3Department of Clinical Microbiology Slagelse Hospital Slagelse Denmark

Correspondence should be addressed to G N Hartmeyer gittehartmeyerrsyddk

Received 9 May 2017 Accepted 31 July 2017 Published 30 August 2017

Academic Editor Bernard Marchand

Copyright copy 2017 G N Hartmeyer et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Microscopy of stool samples is a labour-intensive and inaccurate technique for detection of intestinal parasites causing diarrhoeaand replacement by PCR is attractive Almost all cases of diarrhoea induced by parasites over a nine-year period in our laboratorywere due to Giardia lamblia Cryptosporidium species or Entamoeba histolytica detected by microscopy We evaluated and selectedin-house singleplex real-time PCR (RT-PCR) assays for these pathogens in 99 stool samples from patients suspected of havingintestinal parasitosis tested by microscopy The strategy included a genus-specific PCR assay for C parvum and C hominis withsubsequent identification by a PCR that distinguishes between the two species G lamblia was detected in five and C parvum inone out of 68 microscopy-negative samples The performance of the in-house RT-PCR assays was compared to three commerciallyavailable multiplex test (MT-PCR) kit systems in 81 stool samples collected in 28 microscopy-positive and 27 microscopy-negativesamples from individuals suspected of intestinal parasitosis and in 26 samples from individuals without suspicion of parasiticinfectionThe in-house assays detected parasites in more samples from patients suspected of having parasitosis than did any of thekits We conclude that commercial kits are targeting relevant parasites but their performance may vary

1 Background

Correct identification of microbial agents causing diarrhoeain humans is crucial for optimal treatment Detection ofdisease-causing intestinal parasites is traditionally done bymicroscopic examination of stool samples Over the last yearsthis has been changed in favour of using PCR Studies haveshown that both sensitivity and specificity of PCR are bettercompared to microscopy [1ndash5] Moreover microscopy canlead to false conclusions with harmless parasites being inter-preted as disease-causing while life-threatening parasitesmay not be detected This has in particular been demon-strated for intestinal amoeba [6ndash10] For estimating the trueimpact of parasitic intestinal infections it is important toestablish valid and reliable laboratory techniques for testing

stool samples from patients Use of optimized laboratorymethods will improve patient safety through rapid andcorrect diagnosis which leads to timely start of appropriatetreatment

The aim of this study was to evaluate the consequencesof replacing microscopy by real-time PCR (RT-PCR) fordetection of intestinal parasites causing diarrhoea In orderto do so we first established which parasites were detectedby microscopy in our laboratory over a period of nine yearsto determine which parasites were relevant in our patientpopulation We determined which previously detected par-asites would be missed by introducing a limited numberof species-specific PCR assays and how many cases theyrepresented We then evaluated the performance of in-housesingleplex RT-PCR assays for the three most important

HindawiJournal of Parasitology ResearchVolume 2017 Article ID 6205257 6 pageshttpsdoiorg10115520176205257

2 Journal of Parasitology Research

intestinal parasitic pathogens Finally the performance ofthree selected in-house RT-PCR assays for detection ofGiardia lamblia Cryptosporidium parvumCryptosporidiumhominis and Entamoeba histolyticawas compared to those ofthree commercial multiplex real-time PCR (MT-PCR) kits

Two specific objectives were defined (1) evaluation ofperformance of species-specific in-house RT-PCR assaysfor detection of G lamblia C parvumC hominis and Ehistolytica in stool samples submitted for examination forparasites (2) comparison of the performance of the in-houseRT-PCR assays with the performance of three commercialMT-PCR kits for detection of the same parasites

2 Methods

21 Data Collection from the Laboratory Information System(LIS) Data on faecal samples examined for parasites fromOctober 2005 to January 2015 was extracted from the elec-tronic LIS The total number of samples and patients andresults of microscopy were registered

22 Stool Samples In total 125 stool samples of which 99were examined by microscopy on suspicion of parasitosiswere randomly collected from individuals with gastroin-testinal complaints between June 2010 and January 2015Ninety-nine of these samples were included for objectiveone (31 microscopy-positive and 68 microscopy-negative)and eighty-one (28 microscopy-positive and 27 microscopy-negative) were included for testing objective two In addition26 samples from individuals without suspicion of parasitosiswere included without microscopy for objective two

For objective 1 a total of 99 samples were analysed byin-house RT-PCR For objective 2 a total of 81 samples wereanalysed by in-house RT-PCR and by three commercial MT-PCR kits All samples were kept at minus80∘C until PCR wereperformed

23 Microscopy for Intestinal Parasites Microscopic exam-ination for the presence of ova and cysts was routinelyperformed by examination of iodine-stained wet-mountpreparations after formalin-ethyl acetate concentration at amagnification of times400 [11] On specific request and whenCryptosporidium species Cyclospora species orCystoisosporaspecies was suspected from routine microscopy a smearstained by modified Ziehl-Neelsen technique was also exam-ined [12]

24 In-House RT-PCR For the in-house PCR assays DNAwas extracted by using NucliSENS easyMAG system (bi-oMerieux France) in accordance with the manufacturerrsquosinstructions Prior to DNA extraction a cotton swab wassubmerged into the stool sample and suspended in 4mlphysiological NaCl solution An internal extraction and PCRcontrol phocine herpesvirus (PhHV laboratory strain) wasadded to each sample prior to DNA extraction [13] Thenucleic acids were eluted in 100 120583l and processed for PCRimmediately

Detection of the three intestinal parasites (G lambliaC parvumC hominis and E histolytica) was performed

as singleplex RT-PCR in 99 samples analysed as duplicateOne assay was tested for detection of G lamblia three weretested for C parvumC hominis and one assay (assay 3) wasincluded to distinguish between C parvum and C hominisFinally one assay for E histolytica was tested using primersand probes (Table 1) described previously [1 4 13ndash16]

The 25 120583l reactions mixture contained 1x TaqMan FastUniversal PCRMasterMix 2xNoAmpEraseUNG(ThermoFisher Scientific Waltham MA USA) 1000 nM of theprimers and 200 nM of the probes and 5 120583l DNA eluate

The real-time PCR was performed using an AppliedBiosystems 7500 Fast Real-Time PCRThermocycler (ThermoFisher Scientific) with the following cycling conditions 95∘Cfor 20 sec followed by 45 cycles of 95∘C for 3 sec and 60∘C for30 sec

The PCR products were analysed using Sequence Detec-tion Software v14 (Thermo Fisher Scientific) Amanual cyclethreshold was set to 01 with an automatic baseline Thesample was regarded as positive if the Ct-value was le42 andhad an exponential curve Negative and positive extractionand PCR controls were included in all PCR analysis

The singleplex in-house RT-PCR assays for G lamblia CparvumC hominis (assay 1) and E histolyticawere collectivecalled kit A and compared to three commercialMT-PCR kitsused in objective 2 on 79 samples

25 Diagnostic Test Kits Three different commercial kitsavailable at the market were tested RIDAGENE ParasiticStool Panel (PG1705) from R-Biopharm AG DarmstadtGermany (kit B) LightMixModular Gastroenteritis Assaysfrom TIB MOLBIOL Berlin Germany (kit C) and BDMAX Enteric Parasite Panel from BD Diagnostic FranklinLakes NJ USA (kit D) DNA extraction for kits B and C wasdone as described for the in-house assays For kit D DNAextraction was done according BD MAX enteric parasitepanel instructions on the BDMAX system In all commercialkits we used the internal control DNA which was recom-mended and included in the kits Seventy-nine samples weretested in duplicate in all three kits in accordance with themanufacturerrsquos instructions For kit B the PCR assays werecarried out using an Applied Biosystems 7500 Fast Real-TimePCR Thermocycler with the following cycling conditions1min at 95∘C followed by 45 cycles of 95∘C for 15 sec and 60∘Cfor 30 sec For kit C the PCR assays were done on a RocheLightCycler 480 II real-time instrument with the followingcycling conditions 10min at 95∘C followed by 50 cycles of95∘C for 5 sec 62∘C for 5 sec and 72∘C for 15 sec For kitD analysing was done according BD MAX enteric parasitepanel instructions on the BD MAX system A positive resultin kits was regarded positive if one out of two duplicates waspositive used in objective 2

26 Analysis McNemarrsquos test was used for the statisticalcomparison of the paired data in objective 1

27 Ethics Biobank and Data Storage The study is part of aPhD project and approved by the Danish Data ProtectionAgency (jnr 2008-58-0035) All samples were stored atminus80∘C in an approved research biobank established for the

Journal of Parasitology Research 3

Table1Prim

erandprob

esused

forin-ho

user

eal-tim

ePCR

assays

instu

dy

Parasites

Forw

ardprim

ersequ

ence51015840rarr31015840

Target

Reference

Reversep

rimer

sequ

ence51015840rarr31015840

Prob

esequence(FA

M)51015840rarr31015840

Giardialamblia

GAC

GGCTC

AGGACA

ACG

GTT

TTGCC

AGCG

GTG

TCCG

CCCGCG

GCG

GTC

CCTGCT

AGssu-rRNA

Verw

eijetal

(200

4)[1]

Cparvum

Chom

inis

(assay

1)

CTTTT

TAC

CAAT

CACAG

AAT

CAT

CAG

ATG

TGTT

TGCCA

ATG

CAT

ATG

AA

TCGAC

TGGTAT

CCC

TAT

AA

DNAJ-lik

eprotein

gene

Bruijnesteijn

vanCop

penraete

tal

(200

9)[4]

Cparvum

Chom

inis

(assay

2)

CGCTT

CTC

TAG

CCT

TTC

ATG

ACT

TCA

CGTG

TGTTT

GCC

AAT

CCAAT

CAC

AGAATC

ATC

AGAATC

GAC

TGGT

ATC

DNAJ-lik

eprotein

gene

Fontaine

andGuillo

t(2002)

[14]

Cparvum

Chom

inis

(assay

3)

GAACT

GTA

CAG

ATG

CTT

GGGAGAAT

CCTT

CGTTA

GTT

GAAT

CCTCT

TTC

CA

TTGGAG

CTCAT

ATC

AG

Chominisprob

eCT

TGGAGCT

CGTAT

CAG

Cparvum

prob

e

Specificp

rotein-cod

inggene

Yang

etal

(2013)

[15]

Entamoeba

histo

lytica

ATTGTC

GTG

GCA

TCCTA

ACT

CA

GCG

GAC

GGCTC

ATT

ATA

ACA

CATTG

AAT

GAAT

TGGCC

ATT

ssu-rRNA

Verw

eijetal

(200

4)[1]

Stensvoldetal

(2010)

[16]

Internalcontrol

Phocineh

erpesviru

s(PhH

V)

GGGCG

AAT

CAC

AGAT

TGAAT

CGCG

GTT

CCAAAC

GTA

CCAA

TTTTT

ATG

TGTC

CGCCA

CCA

TCT

GGAT

CGlycoproteinB

Nieste

rs(2001)[13]


Table 2 Samples with discordant results obtained from different test kits

Samplenumber Mic

Giardia CryptosporidiumKitA

KitB

KitC

KitD

KitA

KitB

KitC

KitD

6 Giardia 00 00 00 00 00 00 00 007 Giardia ++ 00 +0 ++ 00 00 00 008 Giardia ++ ++ ++ 00 00 00 00 +09 Giardia ++ ++ ++ +0 00 00 00 0012 Giardia ++ +0 +0 00 00 00 00 0015 Crypto 00 00 00 00 ++ 00 ++ ++18 Crypto 00 00 00 00 ++ ++ +0 ++23 Crypto 00 00 00 00 00 00 00 0024 Crypto 00 00 00 00 ++ ++ 00 ++32 undet ++ 00 00 00 00 00 00 0033 undet +0 00 00 00 00 00 00 0034 undet ++ ++ ++ ++ 00 00 00 0035 undet ++ ++ ++ ++ 00 00 00 0042 undet 00 00 00 00 ++ ++ ++ ++49 undet ++ ++ ++ +0 00 00 00 00Total positive 9 6 7 5 4 3 3 5Mic = microscopy undet = undetected + = positive 0 = negative Results from duplicate tests are shown as ++ +0 and 00 Discordant results are in boldKit A = in-house RT-PCR assays kit B = RIDA GENE Parasitic Stool Panel (PG1705) kit C = LightMix Modular Gastroenteritis Assays and kit D = BDMAXEnteric Parasite Panel

study Data were stored in an approved and secure portalbelonging to Region of Southern Denmark

3 Results

31 LIS Data In the period of October 2005 to January 201510593 samples from 4887 patients were examined for intesti-nal parasites Based on microscopy the reported diarrhoea-causing parasites were G lamblia (500 samples237 patients)Cryptosporidium species (78 samples41 patients) and Ehistolytica or E histolyticadispar (159 samples62 patients)and Cyclospora species (25 samples12 patients) As pre-viously described [10] the majority of parasites reportedas E histolytica after microscopy were most probably infact E dispar which is considered nonpathogenic On thebasis of these data we decided in the study to test for Glamblia and C parvumC hominis because of the frequenciesby which they cause disease For Cryptosporidium sp thesevere illness it causes in immunocompromised patients andthe importance to public health as an agent of food andwater borne outbreaks also contributed to the decision Inaddition a test forE histolyticawas included because amoebicdysentery and amoebic liver abscess are severe conditionsrequiring immediate and appropriate treatment Thus onlythe rare cases of cyclosporiasis (approximately one per yearin our diagnostic laboratory) would be missed because of theselection of targets

32 Objective 1 The in-house RT-PCR assay for G lambliawas positive in 13 of the 14 microscopy-positive samples

and in 5 microscopy-negative samples All three tested RT-PCR assays for C parvumC hominis were positive in tenof the eleven microscopy-positive samples as well as in onemicroscopy-negative sampleThe selected species-specific in-house RT-PCR assays detected the expected pathogen inall the microscopy-positive samples except for one sample(number 6 Table 2) which based onmicroscopywas reportedwith G lamblia and one sample (number 23 Table 2) withCryptosporidium spp As none of the RT-PCR assays or MT-PCRkits detected the expected parasites in these samples theymay represent false positive microscopy reporting (Table 2)The Cryptosporidium assay 3 identified C hominis in fourand C parvum in eight samples The E histolytica RT-PCR assay was only positive in 1 of 8 microscopy-positivesamples and none of the microscopy-negative samples Theseven microscopy-positive samples that were negative for Ehistolytica by PCR were all positive when analysed for Edispar by species-specific PCR

There was no statistical difference in the number ofpositive samples when tested by microscopy and PCR for thethree selected parasites (119901 = 022)

33 Objective 2 Kit A detected G lamblia in three samplesmore than kit B two samples more than kit C and foursamples more than kit D For C parvumC hominis kit Adetected one positive samplemore than kit B and one positivesample more than kit C but one less than kit D Only kit Dwas positive for C parvumC hominis in sample (number 8)in which the other kits detected G lamblia (Table 2) All kitsdetected E histolytica in the same sample


Of all samples tested discordant results were obtainedfrom duplicate determinations in one sample using in-houseassays (kit A) two using kit B two using kit C and three usingkit D (Table 2)

None of the MT-PCR kits confirmed the presence of Glamblia in sample numbers 32 and 33 which were positivein two out of two and one of two replicates respectively bythe in-house RT-PCRThese two samples were from the samepatient A sample three days later was positive when tested atthe National Reference Laboratory at Statens Serum InstitutSample numbers 32 and 33 were therefore considered to betrue positive but weak

Sample number 86 (not shown in Table 2) was only testedin kits A B and D and therefore not included in the totalnumber In this sample kit B was positive for bothG lambliaand E histolytica in one out of two duplicates This was notconfirmed by any of the other test kits

Inhibition by faecal constituents was not a problem in thisstudy as it has been reported previously [17]

4 Discussion

In this study we have evaluated PCR assays for replacementof microscopy for routine detection of diarrhoea-causingparasites In contrast tomicroscopy PCR only detects specificparasites Careful selection of targets for the PCR assays isthereforemandatory It is also important to be aware of whichdiarrhoea-causing parasites present in the population thatare not targeted by the selected PCR assays and the numberof patients affected by exclusion of assays for particular rareparasites [18 19]

Based onprevious frequencies of detection bymicroscopyand severity of disease we decided to establish PCR assaysfor G lamblia C parvumC hominis and E histolytica Theonly diarrhoea-causing parasite previously detected and nottargeted by the PCR assays was Cyclospora spp with a littlemore than one case on average each year

The three different assays for C parvumC hominisperformed equally but assay 1 resulted in the lowest CT valuesand was used for objective 2 Assay 3 distinguished betweenC hominis and C parvum and was used for subsequentspecies identification in positive samples Rapid speciesidentification is valuable for epidemiological investigations

The in-house RT-PCR assays detected G lamblia andCryptosporidium spp in microscopy-negative samples frompatients suspected of suffering from intestinal parasitosis andthus appeared more sensitive than microscopy PCR has pre-viously been reported to be more sensitive than microscopyfor detection of specific parasites In ten Hoversquos study in2007 PCR showed 36 better sensitivity than microscopyfor Giardia in clinical stool samples [3] and in Starks studyin 2011 PCR had 29 better sensitivity for Giardia and 2better sensitivity for Cryptosporidium than microscopy [5]We found PCR detected 41 more Giardia than microscopybut did not find any difference for Cryptosporidium in thisstudy A major advantage of PCR over microscopy is thespecificity obtained from the discrimination between Ehistolytica and E dispar [7ndash10] Seven of the eight samplesoriginally reported with E histolytica by microscopy were

negative in the in-house RT-PCR and were subsequentlyidentified as E dispar

The comparison of four test kits including the in-houseassay based kit A showed varying results from replicate testsTesting in single determinations may lead to false results in aminority of cases Future use of these assaysmay be improvedby running tests in duplicate

The limitation of this study was first of all the samplesize which does not allow statistical analysis of differencesin performance of microscopy and RT-PCR in-house assaysTendencies in favour of the in-house assays were seenwhen comparing variation of replicates and sensitivities tocommercial test kits

As indicated by the numbers of cases of intestinal para-sitosis registered in our LIS over the years collection of largernumber of samples will take time The detection of parasitesin microscopy-negative samples suggests that replacementof microscopy with PCR will increase the positive rates andthereby shorten the time needed to establish large samplecollections

5 Conclusion

In our setting it is relevant to test forG lambliaC parvumChominis and E histolytica We expect that replacement ofmicroscopy with in-house RT-PCR assays for these para-sites will result in higher positive rates for G lamblia andC parvumC hominis while false positive results for Ehistolytica will be avoided Addition of a secondary testdifferentiating between C parvum and C hominis will be ofvalue for early discovery of outbreaks

All the commercialMT-PCRkits evaluated here tested forthe relevant targets However some variation in performancewas seen when using the kits The choice of method fordetection of intestinal protozoa may depend on the settingCompared to PCR microscopy is less sensitive and lessspecific more time consuming andmore dependent on indi-vidual skills Use of commercial PCR kits may be attractive inlaboratories handling moderate numbers of samples whilein-house PCR assays can be established and maintained forlarge-scale throughput analyses mainly due to lower costs

Disclosure

The sponsors did not have any role in the study designcollection analysis and interpretation of data

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

The study is a part of a PhD project and supported by grantfrom following Danish organizations AP Moslashller Founda-tion for the Advancement of Medical Science ldquoFonden forLaeligge Else Poulsens Mindelegatrdquo Beckett-Fonden RegionSyddanmarks and Region Sjaeligllands Faeliglles ForskningspuljeUniversity of Southern Denmark and the Department for


Clinical Microbiology at Odense University Hospital Theauthors thank the laboratory staff of the Sections of Parasitol-ogy in the Department for Clinical Microbiology at OdenseUniversity Hospital Odense Denmark for their help incollecting the stool samples and the Department for ClinicalMicrobiology at Slagelse Hospital Slagelse Denmark for useof their BD MAX system specially Tina Vasehus MadsenPhD for assisting with the testingThey also thankDrMingChen in the Department for Clinical Microbiology Hospitalof Southern Jutland Soslashnderborg Denmark for criticallyrevising the paper

References

[1] J J Verweij R A Blange K Templeton et al ldquoSimultaneousdetection of Entamoeba histolytica Giardia lamblia and Cryp-tosporidium parvum in fecal samples by using multiplex real-time PCRrdquo Journal of Clinical Microbiology vol 42 no 3 pp1220ndash1223 2004

[2] T Schuurman P Lankamp A van Belkum M Kooistra-Smidand A van Zwet ldquoComparison of microscopy real-time PCRand a rapid immunoassay for the detection of Giardia lambliain human stool specimensrdquo Clinical Microbiology and Infectionvol 13 no 12 pp 1187ndash1191 2007

[3] R ten Hove T Schuurman M Kooistra L Moller L VanLieshout and J J Verweij ldquoDetection of diarrhoea-causingprotozoa in general practice patients in The Netherlands bymultiplex real-time PCRrdquo Clinical Microbiology and Infectionvol 13 no 10 pp 1001ndash1007 2007

[4] L E S Bruijnesteijn van Coppenraet J A Wallinga G J H MRuijs M J Bruins and J J Verweij ldquoParasitological diagnosiscombining an internally controlled real-time PCR assay forthe detection of four protozoa in stool samples with a testingalgorithm for microscopyrdquo Clinical Microbiology and Infectionvol 15 no 9 pp 869ndash874 2009

[5] D Stark S E Al-Qassab J L N Barratt et al ldquoEvaluation ofmultiplex tandem real-time PCR for detection of cryptosporid-ium spp Dientamoeba fragilis Entamoeba histolytica andGiardia intestinalis in clinical stool samplesrdquo Journal of ClinicalMicrobiology vol 49 no 1 pp 257ndash262 2011

[6] A Kebede J J Verweij B Petros and A M PoldermanldquoShort communication misleading microscopy in amoebiasisrdquoTropical Medicine and International Health vol 9 no 5 pp 651-652 2004

[7] B S Ayed B R Abdallah M Mousli K Aolin M Thellierand A Bouratbine ldquoMolecular differentiation of entamoebahistolytica and entamoeba dispar from Tunisian food handlerswith amoeba infection initially diagnosed by microscopyrdquoParasite vol 15 no 1 pp 65ndash68 2008

[8] K Khairnar S C Parija and R Palaniappan ldquoDiagnosisof intestinal amoebiasis by using nested polymerase chainreaction-restriction fragment length polymorphism assayrdquoJournal of Gastroenterology vol 42 no 8 pp 631ndash640 2007

[9] J J Verweij F Oostvogel E A T Brienen A Nang-BeifubahJ Ziem and A M Polderman ldquoShort communication preva-lence of entamoeba histolytica and entamoeba dispar in north-ern Ghanardquo Tropical Medicine and International Health vol 8no 12 pp 1153ndash1156 2003

[10] G N Hartmeyer S V HoslashghM Chen H Holt M N Skov andM Kemp ldquoNeed for species-specific detection for the diagnosisof amoebiasis in a non-endemic settingrdquo Scandinavian Journalof Infectious Diseases vol 45 no 11 pp 868ndash871 2013

[11] A V Allen and D S Ridley ldquoFurther observations on theformol-ether concentration technique for faecal parasitesrdquoJournal of Clinical Pathology vol 23 no 6 pp 545-546 1970

[12] S A Henriksen and J F Pohlenz ldquoStaining of cryptosporidiaby a modified Ziehl-Neelsen techniquerdquo Acta veterinaria Scan-dinavica vol 22 no 3-4 pp 594ndash596 1981

[13] H G M Niesters ldquoQuantitation of viral load using real-timeamplification techniquesrdquo Methods vol 25 no 4 pp 419ndash4292001

[14] M Fontaine and E Guillot ldquoDevelopment of a TaqMan quanti-tative PCR assay specific for Cryptosporidium parvumrdquo FEMSMicrobiology Letters vol 214 no 1 pp 13ndash17 2002

[15] R Yang C Murphy Y Song et al ldquoSpecific and quantitativedetection and identification of Cryptosporidium hominis andC parvum in clinical and environmental samplesrdquo Experimen-tal Parasitology vol 135 no 1 pp 142ndash147 2013

[16] C R Stensvold M Lebbad J J Verweij et al ldquoIdentificationand delineation of members of the entamoeba complex bypyrosequencingrdquo Molecular and Cellular Probes vol 24 no 6pp 403ndash406 2010

[17] L Monteiro D Bonnemaison A Vekris et al ldquoComplexpolysaccharides as PCR inhibitors in feces helicobacter pylorimodelrdquo Journal of Clinical Microbiology vol 35 no 4 pp 995ndash998 1997

[18] J J Verweij ldquoApplication of PCR-based methods for diagnosisof intestinal parasitic infections in the clinical laboratoryrdquoParasitology vol 141 no 14 pp 1863ndash1872 2014

[19] L van Lieshout and J J Verweij ldquoNewer diagnostic approachesto intestinal protozoardquo Current Opinion in Infectious Diseasesvol 23 no 5 pp 488ndash493 2010

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intestinal parasitic pathogens Finally the performance ofthree selected in-house RT-PCR assays for detection ofGiardia lamblia Cryptosporidium parvumCryptosporidiumhominis and Entamoeba histolyticawas compared to those ofthree commercial multiplex real-time PCR (MT-PCR) kits

Two specific objectives were defined (1) evaluation ofperformance of species-specific in-house RT-PCR assaysfor detection of G lamblia C parvumC hominis and Ehistolytica in stool samples submitted for examination forparasites (2) comparison of the performance of the in-houseRT-PCR assays with the performance of three commercialMT-PCR kits for detection of the same parasites

2 Methods

21 Data Collection from the Laboratory Information System(LIS) Data on faecal samples examined for parasites fromOctober 2005 to January 2015 was extracted from the elec-tronic LIS The total number of samples and patients andresults of microscopy were registered

22 Stool Samples In total 125 stool samples of which 99were examined by microscopy on suspicion of parasitosiswere randomly collected from individuals with gastroin-testinal complaints between June 2010 and January 2015Ninety-nine of these samples were included for objectiveone (31 microscopy-positive and 68 microscopy-negative)and eighty-one (28 microscopy-positive and 27 microscopy-negative) were included for testing objective two In addition26 samples from individuals without suspicion of parasitosiswere included without microscopy for objective two

For objective 1 a total of 99 samples were analysed byin-house RT-PCR For objective 2 a total of 81 samples wereanalysed by in-house RT-PCR and by three commercial MT-PCR kits All samples were kept at minus80∘C until PCR wereperformed

23 Microscopy for Intestinal Parasites Microscopic exam-ination for the presence of ova and cysts was routinelyperformed by examination of iodine-stained wet-mountpreparations after formalin-ethyl acetate concentration at amagnification of times400 [11] On specific request and whenCryptosporidium species Cyclospora species orCystoisosporaspecies was suspected from routine microscopy a smearstained by modified Ziehl-Neelsen technique was also exam-ined [12]

24 In-House RT-PCR For the in-house PCR assays DNAwas extracted by using NucliSENS easyMAG system (bi-oMerieux France) in accordance with the manufacturerrsquosinstructions Prior to DNA extraction a cotton swab wassubmerged into the stool sample and suspended in 4mlphysiological NaCl solution An internal extraction and PCRcontrol phocine herpesvirus (PhHV laboratory strain) wasadded to each sample prior to DNA extraction [13] Thenucleic acids were eluted in 100 120583l and processed for PCRimmediately

Detection of the three intestinal parasites (G lambliaC parvumC hominis and E histolytica) was performed

as singleplex RT-PCR in 99 samples analysed as duplicateOne assay was tested for detection of G lamblia three weretested for C parvumC hominis and one assay (assay 3) wasincluded to distinguish between C parvum and C hominisFinally one assay for E histolytica was tested using primersand probes (Table 1) described previously [1 4 13ndash16]

The 25 120583l reactions mixture contained 1x TaqMan FastUniversal PCRMasterMix 2xNoAmpEraseUNG(ThermoFisher Scientific Waltham MA USA) 1000 nM of theprimers and 200 nM of the probes and 5 120583l DNA eluate

The real-time PCR was performed using an AppliedBiosystems 7500 Fast Real-Time PCRThermocycler (ThermoFisher Scientific) with the following cycling conditions 95∘Cfor 20 sec followed by 45 cycles of 95∘C for 3 sec and 60∘C for30 sec

The PCR products were analysed using Sequence Detec-tion Software v14 (Thermo Fisher Scientific) Amanual cyclethreshold was set to 01 with an automatic baseline Thesample was regarded as positive if the Ct-value was le42 andhad an exponential curve Negative and positive extractionand PCR controls were included in all PCR analysis

The singleplex in-house RT-PCR assays for G lamblia CparvumC hominis (assay 1) and E histolyticawere collectivecalled kit A and compared to three commercialMT-PCR kitsused in objective 2 on 79 samples

25 Diagnostic Test Kits Three different commercial kitsavailable at the market were tested RIDAGENE ParasiticStool Panel (PG1705) from R-Biopharm AG DarmstadtGermany (kit B) LightMixModular Gastroenteritis Assaysfrom TIB MOLBIOL Berlin Germany (kit C) and BDMAX Enteric Parasite Panel from BD Diagnostic FranklinLakes NJ USA (kit D) DNA extraction for kits B and C wasdone as described for the in-house assays For kit D DNAextraction was done according BD MAX enteric parasitepanel instructions on the BDMAX system In all commercialkits we used the internal control DNA which was recom-mended and included in the kits Seventy-nine samples weretested in duplicate in all three kits in accordance with themanufacturerrsquos instructions For kit B the PCR assays werecarried out using an Applied Biosystems 7500 Fast Real-TimePCR Thermocycler with the following cycling conditions1min at 95∘C followed by 45 cycles of 95∘C for 15 sec and 60∘Cfor 30 sec For kit C the PCR assays were done on a RocheLightCycler 480 II real-time instrument with the followingcycling conditions 10min at 95∘C followed by 50 cycles of95∘C for 5 sec 62∘C for 5 sec and 72∘C for 15 sec For kitD analysing was done according BD MAX enteric parasitepanel instructions on the BD MAX system A positive resultin kits was regarded positive if one out of two duplicates waspositive used in objective 2

26 Analysis McNemarrsquos test was used for the statisticalcomparison of the paired data in objective 1

27 Ethics Biobank and Data Storage The study is part of aPhD project and approved by the Danish Data ProtectionAgency (jnr 2008-58-0035) All samples were stored atminus80∘C in an approved research biobank established for the


Table1Prim

erandprob

esused

forin-ho

user

eal-tim

ePCR

assays

instu

dy

Parasites

Forw

ardprim

ersequ

ence51015840rarr31015840

Target

Reference

Reversep

rimer

sequ

ence51015840rarr31015840

Prob

esequence(FA

M)51015840rarr31015840

Giardialamblia

GAC

GGCTC

AGGACA

ACG

GTT

TTGCC

AGCG

GTG

TCCG

CCCGCG

GCG

GTC

CCTGCT

AGssu-rRNA

Verw

eijetal

(200

4)[1]

Cparvum

Chom

inis

(assay

1)

CTTTT

TAC

CAAT

CACAG

AAT

CAT

CAG

ATG

TGTT

TGCCA

ATG

CAT

ATG

AA

TCGAC

TGGTAT

CCC

TAT

AA

DNAJ-lik

eprotein

gene

Bruijnesteijn

vanCop

penraete

tal

(200

9)[4]

Cparvum

Chom

inis

(assay

2)

CGCTT

CTC

TAG

CCT

TTC

ATG

ACT

TCA

CGTG

TGTTT

GCC

AAT

CCAAT

CAC

AGAATC

ATC

AGAATC

GAC

TGGT

ATC

DNAJ-lik

eprotein

gene

Fontaine

andGuillo

t(2002)

[14]

Cparvum

Chom

inis

(assay

3)

GAACT

GTA

CAG

ATG

CTT

GGGAGAAT

CCTT

CGTTA

GTT

GAAT

CCTCT

TTC

CA

TTGGAG

CTCAT

ATC

AG

Chominisprob

eCT

TGGAGCT

CGTAT

CAG

Cparvum

prob

e

Specificp

rotein-cod

inggene

Yang

etal

(2013)

[15]

Entamoeba

histo

lytica

ATTGTC

GTG

GCA

TCCTA

ACT

CA

GCG

GAC

GGCTC

ATT

ATA

ACA

CATTG

AAT

GAAT

TGGCC

ATT

ssu-rRNA

Verw

eijetal

(200

4)[1]

Stensvoldetal

(2010)

[16]

Internalcontrol

Phocineh

erpesviru

s(PhH

V)

GGGCG

AAT

CAC

AGAT

TGAAT

CGCG

GTT

CCAAAC

GTA

CCAA

TTTTT

ATG

TGTC

CGCCA

CCA

TCT

GGAT

CGlycoproteinB

Nieste

rs(2001)[13]



Samplenumber Mic


KitB

KitC

KitD

KitA

KitB

KitC

KitD



3 Results











4 Discussion









5 Conclusion



Disclosure




Acknowledgments




References



























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Prim

erandprob

esused

forin-ho

user

eal-tim

ePCR

assays

instu

dy

Parasites

Forw

ardprim

ersequ

ence51015840rarr31015840

Target

Reference

Reversep

rimer

sequ

ence51015840rarr31015840

Prob

esequence(FA

M)51015840rarr31015840

Giardialamblia

GAC

GGCTC

AGGACA

ACG

GTT

TTGCC

AGCG

GTG

TCCG

CCCGCG

GCG

GTC

CCTGCT

AGssu-rRNA

Verw

eijetal

(200

4)[1]

Cparvum

Chom

inis

(assay

1)

CTTTT

TAC

CAAT

CACAG

AAT

CAT

CAG

ATG

TGTT

TGCCA

ATG

CAT

ATG

AA

TCGAC

TGGTAT

CCC

TAT

AA

DNAJ-lik

eprotein

gene

Bruijnesteijn

vanCop

penraete

tal

(200

9)[4]

Cparvum

Chom

inis

(assay

2)

CGCTT

CTC

TAG

CCT

TTC

ATG

ACT

TCA

CGTG

TGTTT

GCC

AAT

CCAAT

CAC

AGAATC

ATC

AGAATC

GAC

TGGT

ATC

DNAJ-lik

eprotein

gene

Fontaine

andGuillo

t(2002)

[14]

Cparvum

Chom

inis

(assay

3)

GAACT

GTA

CAG

ATG

CTT

GGGAGAAT

CCTT

CGTTA

GTT

GAAT

CCTCT

TTC

CA

TTGGAG

CTCAT

ATC

AG

Chominisprob

eCT

TGGAGCT

CGTAT

CAG

Cparvum

prob

e

Specificp

rotein-cod

inggene

Yang

etal

(2013)

[15]

Entamoeba

histo

lytica

ATTGTC

GTG

GCA

TCCTA

ACT

CA

GCG

GAC

GGCTC

ATT

ATA

ACA

CATTG

AAT

GAAT

TGGCC

ATT

ssu-rRNA

Verw

eijetal

(200

4)[1]

Stensvoldetal

(2010)

[16]

Internalcontrol

Phocineh

erpesviru

s(PhH

V)

GGGCG

AAT

CAC

AGAT

TGAAT

CGCG

GTT

CCAAAC

GTA

CCAA

TTTTT

ATG

TGTC

CGCCA

CCA

TCT

GGAT

CGlycoproteinB

Nieste

rs(2001)[13]



Samplenumber Mic


KitB

KitC

KitD

KitA

KitB

KitC

KitD



3 Results











4 Discussion









5 Conclusion



Disclosure




Acknowledgments




References



























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Samplenumber Mic


KitB

KitC

KitD

KitA

KitB

KitC

KitD



3 Results











4 Discussion









5 Conclusion



Disclosure




Acknowledgments




References



























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






4 Discussion









5 Conclusion



Disclosure




Acknowledgments




References



























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References



























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Selecting PCR for the Diagnosis of Intestinal...

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