development and validation of a pcr-elisa for the diagnosis of...

Research ArticleDevelopment and Validation of a PCR-ELISA forthe Diagnosis of Symptomatic and Asymptomatic Infection byLeishmania (Leishmania) infantum

Fernanda Alvarenga Cardoso Medeiros1 Luciana Inaacutecia Gomes1

Edward Oliveira1 Carolina Senra Alves de Souza1 Maria Vitoacuteria Mouratildeo1

Glaacuteucia Fernandes Cota1 Letiacutecia Helena dos Santos Marques2

Mariacircngela Carneiro2 and Ana Rabello1

1Centro de Pesquisas Rene Rachou Oswaldo Cruz Foundation (Fiocruz) Belo Horizonte MG Brazil2Departamento de Parasitologia Instituto de Ciencias Biologicas Universidade Federal de Minas Gerais Belo Horizonte MG Brazil

Correspondence should be addressed to Edward Oliveira edwardjocpqrrfiocruzbr

Received 31 August 2016 Accepted 7 December 2016 Published 9 January 2017

Academic Editor Sukla Biswas

Copyright copy 2017 Fernanda Alvarenga Cardoso Medeiros et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

A kDNA PCR enzyme-linked immunosorbent assay (kDNA PCR-ELISA) for the diagnosis of human visceral leishmaniasis (HVL)was developed The detection limit of the reaction precision measurements and cut-off of the kDNA PCR-ELISA were definedin a proof-of-concept phase A reference strain of Leishmania (Leishmania) infantum and a bank of 14 peripheral blood samplesfrom immunocompetent patients with VL were characterized using techniques considered gold standards and 11 blood samplesobtained from healthy individuals of an endemic area were also assessed Phase II evaluation determined the performance of theassay in peripheral blood samples from 105 patients with VL (adults and children) 25 patients with LeishmaniaHIV coinfection40 healthy individuals and 33 asymptomatic individuals living in endemic areas The kDNA PCR-ELISA exhibited satisfactoryprecision with a detection limit of 007 fg of DNA from L (L) infantum and 1 parasitemL blood The overall sensitivity of theassay for all groups studied was 100 (95 confidence interval [CI] 971ndash100) and the specificity was 95 (95 CI 835ndash986)The kDNA PCR-ELISA was shown to be a useful tool for VL symptomatic and asymptomatic individuals diagnosis and its use inendemic countries may help monitor control interventions

1 Introduction

Visceral leishmaniasis (VL) is a major public health problemand has an estimated annual incidence of approximately02 to 04 million cases and a focal distribution with morethan 90 of global cases occurring in six countries IndiaBangladesh Sudan South Sudan Brazil and Ethiopia [1] InBrazil the disease is an endemic anthropozoonosis causedby the parasitic protozoa Leishmania (Leishmania) infantumwhich is an obligate intracellular protozoan belonging tothe Leishmania donovani complex L infantum is primarilytransmitted by the sand fly Lutzomyia longipalpis and dogs

are the main reservoir of these parasites The number ofcases has increased in large Brazilian cities despite Brazilianmeasures to control VL [2 3] A mean of 3771 cases aredetected annually in Brazil [4]

A noninvasive easily implemented high performancetest for the detection of symptomatic and asymptomaticinfection is lacking despite several technological advances inrecent years Parasitological examination which is a refer-ence technique requires invasive sample collection from thespleen or bone marrow which must be performed by trainedprofessionals and often exhibits low and variable sensitivityAntibody-based tests such as immunofluorescence antibody

HindawiJournal of Tropical MedicineVolume 2017 Article ID 7364854 10 pageshttpsdoiorg10115520177364854

2 Journal of Tropical Medicine

test (IFAT) enzyme-linked immunosorbent assay (ELISA)and recombinant antigen-based immunochromatographytest (ICT) support the clinical diagnosis of VL but thesemethods have drawbacks such as cross-reactions in thepresence of other diseases and an inability to distinguishbetween active and past infection Some studies demon-strated that serological tests based on antigens of a singleLeishmania sp exhibited low diagnostic performance inregions with circulating heterologous parasites [5 6] Theserological tests with the exception of the Direct Agglutina-tion Tests (DAT) andWestern blotting exhibit low sensitivityin VL patients coinfected with HIV [7ndash9] because of theirinsufficient immune humoral response

Molecular methods are useful for the detection of lowparasitic loads and exhibit high sensitivity in the diagno-sis of VL [10] PCR-based assays are the most commonlyused approaches in molecular diagnostics One additionaladvantage of PCR is that it is based on the identificationof conserved DNA sequences which allows the detection ofspecific pathogen species in a variety of clinical samples evenarchived materials [11] PCR sensitivity is directly related tothe copy number of the target and the primer and cyclingprotocol used [12] The internal transcribed spacers (ITSITS1 and ITS2) regions gp63 gene HSP-70 gene miniexon(ME) and SSU-rRNA and conserved and variable regionsof kinetoplast DNA (kDNA) minicircles are used for thediagnosis and characterization of Leishmania strains [13]Conventional PCR techniques targeting kDNA exhibit highsensitivity for VL diagnosis [13 14] but this technique isnot automated and is laborious which limits its use to asmall number of samples To overcome this limitation wedeveloped a kDNA PCR-ELISA which allows simultaneoustesting of a large number of blood samples without theuse of commercial kits for the detection of PCR productsvia measurement of absorbance readings for the laboratorydiagnosis of VL

2 Materials and Methods

This work was performed in two phases

(i) In a proof-of-concept phase the assay was standard-ized using a reference strain of L (L) infantum and abank of peripheral blood samples from nonimmuno-suppressed patients with VL that were characterizedusing techniques considered gold standards and fromhealthy individuals in an endemic area

(ii) In a phase II evaluation the assay was validated inclinical samples from varied groups of VL patients ofdifferent ages and in asymptomatic and noninfectedindividuals to certify the wide use of the assay forthe diagnosis of human VL We describe the step-by-step development and internal validation of the kDNAPCR-ELISA below

21 Parasites Promastigotes of L (L) infantum (MHOMBR2002LPC-RPV) L (L) donovani (MHOMET196HU3) L (Viannia) braziliensis (MHOMBR75M2903) L

(V) guyanensis (MHOMBR1975M4147) and L (L) ama-zonensis (IFLABR1967PH-8) were cultured in Schneidermedium (Sigma-Aldrich St Louis MO USA) supplementedwith 10 foetal bovine serum (Invitrogen Carlsbad CAUSA) and counted in aNeubauer chamber An aliquot of 1mLof the suspension of parasites was centrifuged at 2300119892 for 2minutes and stored at minus70∘C

22 Samples

221 Peripheral Blood Samples Peripheral blood samplescollected from 14 patients with VL nonimmunosuppressedliving in endemic areas in Brazil with typical VL symptomsand parasitological confirmation (direct exam andor cul-ture) with a mean age of 127 years (range one month to 768years) were used as positive controls

Peripheral blood samples from 11 healthy individualsfrom the endemic area of Belo Horizonte MG were used asnegative controls

222 Spiked Blood Samples Aliquots of peripheral bloodsamples obtained from 11 healthy individuals were addedto the reference sample L (L) infantum (MHOMBR2002LPC-RPV) to obtain a concentration of 10000 parasitesmLof blood

223 Evaluation of kDNA PCR-ELISA Performance in theDiagnosis of VL VL-HIV Coinfected Asymptomatic andNoninfected Endemic Controls A minimum sample size of38 VL cases was calculated with a mean sensitivity of 975to assess the accuracy of the assay which is consistent withpreviously published molecular tests [15 16] The specificityin these studies was 100 and a minimum sample size of 38noninfected controls was estimated with a level of confidenceof 95

119899 = 1199112 sdot 119901 sdot(1 minus 119901)

1199092 (1)

where 119901 is the sensitivity or specificity and 1199092 is the confi-dence interval (95) [17]

The following groups were included

Children VL Group This group was composed of 81 childrenadmitted to Hospital Joao Paulo II-FHEMIG (FundacaoHospitalar de Minas Gerais) Belo Horizonte Minas Geraiswho presented with VL symptoms and parasitological con-firmation (direct exam andor culture) andor serologicaldiagnosis for VL (indirect immunofluorescence antibodytest-IFAT andor immunochromatographic rK39 antigen-test) associated with clinical improvement after treatmentPatients in this group were aged 3 months to 10 years (mean25 years)

Adult VL Group This group was constituted by 24 adultpatients aged 18 to 68 years (mean of 36 years) who wereadmitted to Hospital Eduardo de Menezes-FHEMIG andpresented VL symptoms with parasitological confirmation(direct exam and or culture) andor serological diagnosis for

Journal of Tropical Medicine 3

VL (indirect immunofluorescence antibody test-IFAT andorimmunochromatographic rK39 antigen-test) associated withclinical improvement after treatment

VLHIV Group This group was composed of 25 HIV-infected patients who were admitted to Hospital Eduardo deMenezes-FHEMIG aged 21 to 61 years (mean of 41 years)presenting VL symptoms and parasitological confirmation(direct exam andor culture) andor serological diagnosis forVL (indirect immunofluorescence antibody test-IFAT andorimmunochromatographic rK39 antigen-test) associated withclinical cure after treatment HIV infection was diagnosedby a positive ELISA or chemiluminescence followed bya confirmatory Western blot performed in two differentsamples

Asymptomatic GroupThis group was composed of 33 asymp-tomatic children aged 3months to 68 years (mean 28 years)living in an endemic area of Belo Horizonte and presentingpositive serology (ELISA using recombinant antigen K39andor DAT) or molecular (SSU-rRNA qPCR) tests forLeishmania infection

Noninfected Group This group consisted of 40 children aged11 to 67 years (mean 35 years) living in an endemic area ofBelo Horizonte and presenting negative serological (ELISAusing recombinant antigen K39 andor DAT) and molecular(SSU-rRNA qPCR) tests for Leishmania spp infection

23 DNA Extraction DNA from parasites and total DNAfrom peripheral blood samples and spiked blood sampleswere extracted using the ldquoQIAamp DNA minirdquo (QIAGENGmbH Hilden Germany) kit according to the manu-facturerrsquos specifications Negative DNA extraction controlswere performed for each experiment by the addition of allreagents except the sample The yield was determined byabsorbance at 260 nm in a spectrophotometer (NanoDropND-1000 Thermo Fisher Scientific Wilmington DE USA)The A260A280 absorbance ratio was analysed to verify thepurity of the DNA obtained

24 kDNA PCR-ELISA

241 kDNA PCR Amplification The reaction was made for afinal volume of 25 120583L containing 2120583L of DNA sample 2 unitsof PlatinumTaqDNAPolymerase (Invitrogen Carlsbad CAUSA) 1x PCR buffer (200mMTris-HCl pH 84 and 500mMKCl) sense primer 150 (51015840-GGGGTAGGGGCGTTCTC-GCGAA-31015840) labelled with biotin at the 51015840 end and antisenseprimer 152 (31015840-CGCGCGATCTATATTTACACCA-ACCCC-51015840) [18] at 06 120583M each 20mMMgCl

2 and 02mM

dNTPs (Promega Madison WI USA) The cycling protocolconsisted of one step at 95∘C for 5min followed by 34 cyclesat 95∘C for 30 seconds 60∘C for 30 seconds and 72∘C for 30seconds A final step of 72∘C for 5 minutes was performedEach PCR assay included negative (PCR mix without DNAcontrol for the process of DNA extraction andDNA extractedfrom samples of healthy volunteers) and positive (genomic

DNA extracted from the reference strain of L (L) infantumMHOMBR2002LPC-RPV) controls

242 ELISA Colorimetric Assay For the detection of theamplified products the 51015840-GATTTCTGCACCCATTTTTC-31015840 probe labelledwith fluorescein at the 51015840 end designed usingthe Primer3 program was used [19] A better site for anneal-ing of the probe was verified to be among 76 to 91 bp of 120 bpLeishmania kDNA fragment amplified using the PCR assayThe search of the lowest homology with genomic sequencesfromLeishmania braziliensisLeishmania amazonensisLeish-mania guyanensis Trypanosoma cruzi Plasmodium vivaxand Mycobacterium tuberculosis for selection and design ofthe probe was performed in the BLAST program (NationalCenter for Biotechnology Information website) using thedatabase nucleotides and MegaBlast option

ELISA followed the parameters described previously[20 21] Briefly wells of MaxiSorp polystyrene microplates(NuncThermo Scientific VernonHills IL USA) were coatedwith 100 120583L of streptavidin (5 120583gmL) (streptavidin fromStreptomyces avidinii Sigma-Aldrich St Louis MO USA) inPBS via incubation at 37∘C for 1 hour plus overnight at 2ndash8∘CCoated plates were washed 4 times with PBS-Tween (PBS-T)005 followed by incubation at 37∘C for 2 hours with 200120583L3 BSA in PBS-TThe plates were washed 4 times with PBS-Tand stored at minus20∘C until use

PCR at a volume of 100 120583L diluted 1 25 in PBS wereperformed in duplicate in microplates at 37∘C for 30minutesThe microplates were washed 3 times with 300 120583L of PBS-T and incubated for 10 minutes at room temperature in01M NaOH 100 120583Lwell The microplates were washed oncewith 01M NaOH (300120583Lwell) and 3 times with 300 120583L of01M Tris-HCl Next 100 120583Lwell of a fluorescein-labelledprobe at the 51015840 end diluted to 02 pM in hybridisationsolution (70 SSPE5x 075M sodium chloride 005Msodium phosphate 0005M EDTA 30 formamide and01 SDS) was added and the microplate incubated for onehour at 37∘C The microplates were washed 3 times with 6xSSC solution (09M NaCl and 009M sodium citrate) andtwice with a 3x SSC solution (045M NaCl and 0045Msodiumcitrate) containing 01 SDS PBS (150120583L) containing1 BSA solution (PBS-BSA) was added to each well andmicroplates were incubated for 30 minutes at 37∘C Anantifluorescein conjugate (antifluoresceinOregon Green-peroxidase Invitrogen-Thermo Scientific Wilmington DEUSA) (150 120583L) was diluted 1 9000 in PBS-BSA solution andadded to each well The microplates were incubated for onehour at 37∘CThemicroplates were washed 4 times with PBS-T (300 120583Lwell) and 100 120583Lwell of a chromogenic mixture(331015840551015840-tetramethylbenzidine + hydrogen peroxide Sigma-Aldrich) was added Plates were incubated for 10 minutes atroom temperature in the dark and the reaction was stoppedby the addition of 100 120583L of 3N sulfuric acid Absorbancereadings were performed in a microplate reader at 450 nm(Model 550 Bio-Rad Hercules CA USA)

Each ELISAwas performedwith the positive and negativecontrols described above for the PCR and all experimentswere performed in duplicate Data represent themean values


243 ACTB PCR-ELISA As a control for the DNA extrac-tion and amplification procedure all samples were amplifiedfor the human beta actin (ACTB) gene using an Aco1 (51015840-ACCTCATGAAGATCCTCACC-31015840) primer labelled withbiotin at the 51015840 end and an Aco2 primer (51015840-CCATCTCTT-GCTCGAAGTCC-31015840) which generated a product of 120-base pairs [22] The PCR was performed in a final volume of20120583L containing 2120583L of DNA sample 2 units of PlatinumTaq DNA Polymerase (Invitrogen) 1x PCR buffer (200mMTris-HCl pH 84 and 500mM KCl) 05 120583M of each primer20mM MgCl

2 and 02mM dNTP (Promega) The cycling

protocol consisted of an initial step of 95∘C for 5min and 35cycles at 95∘C for 20 seconds 60∘C for 30 seconds and 72∘Cfor 1minute A final step at 72∘C for 6minutes was performedEach PCR assay included negative controls (PCRmixwithoutDNA and control for the process of DNA extraction)

The 51015840-TCTCCTTAATGCACGCACG-31015840 probe labelledwith fluorescein at the 51015840 end was used to detect the amplifiedproducts using the ELISA colorimetric assay [21] The assayfollowed the same conditions described above except that100 120583L of the PCR diluted 1 10 in PBS was used andthe antifluorescein conjugate (antifluoresceinOregonGreen-peroxidase Invitrogen) was diluted 1 1000 in PBS-BSA

244 Analytical Precision (Analytical Sensitivity and Speci-ficity) The detection limit was measured in 6 gel poly-acrylamide and kDNA PCR-ELISA for the detection of PCRproducts from DNA L infantum in the following concentra-tions 700 pg 70 pg 7 pg 700 fg 70 fg 7 fg 07 fg 007 fg and0007 fg120583L which were obtained by successive 1 10 dilutionin RNase-free water The detection limit was measured viathe detection of PCR products from DNA extracted fromspiked blood samples with 1 10 100 1000 and 10000 of Linfantum parasitesmL obtained by successive 1 10 dilutionsThe analytical specificity was determined by the analysis ofPCR products from genomic DNA extracted of L infantumL donovani L braziliensis L guyanensis and L amazonensisin 6 gel polyacrylamide and kDNA PCR-ELISA

245 Diagnostic Precision (Repeatability and Reproducibility)The intra-assay (repeatability) was performed in four repli-cates in a single run to detect PCR products amplified fromthe DNA of the reference L infantum (MHOMBR2002LPC-RPV) at 700 fg 70 fg 07 fg and 007 fg120583L Four DNAsamples from the peripheral blood of patients with VL andfour DNA samples from the peripheral blood of noninfectedindividuals were assayed Interassay precision (reproducibil-ity) was assessed using PCR products from genomic DNAextracted from L infantum VL patients and noninfectedindividuals performed in duplicate on consecutive days

25 Data Analysis In the kDNA PCR-ELISA developmentphase the cut-off was determined using Receiver OperatingCharacteristic (ROC) curve analysis in GraphPad Prism50 software (San Diego CA USA) The detection limitand analytical specificity of the kDNA PCR-ELISA werealso assessed The intra-assay (repeatability) and interassay(reproducibility) precision levels were measured using the

variation coefficient (VC) of the results of the retested samplesusing the equation VC = Standard DeviationMean times 100

The sensitivity and specificity were calculated by evalu-ating the absorbance readings of the kDNA PCR-ELISA in130 DNA samples from VL patients (Children VL Group andAdult VL Group and VLHIV Group) and 40 samples fromthe Noninfected Group using ROC curve analyses

Absorbance readings were individually assessed using theShapiro-Wilk normality test (119882 test) and compared usingthe Kruskal-Wallis test The groups that presented normal(Adult VL and VLHIV Groups) or nonnormal distribution(ChildrenVL andAsymptomaticVLGroups)were comparedusing the unpaired 119905-test and MannndashWhitney 119880 test respec-tively All tests were performed in the software GraphPadPrism 60 considering a level of significance of 119901 lt 005

The cut-off value for the ACTB PCR-ELISA was calcu-lated as the mean of the absorbance readings of the negativecontrols plus three standard deviations and was determinedin the development phase of the assay

26 Ethical Considerations Informed consent was obtainedfrom patients individuals or guardians at the time of bloodcollection and all involved centers evaluated and approved theprotocol studies (Protocol Number 142011)

3 Results

31 Analytical Sensitivity Specificity and Precision of thekDNA PCR-ELISA ROC curve analysis using the absorb-ance readings of the control samples (14 peripheral bloodsamples collected frompatientswithVL and peripheral bloodsamples obtained from healthy individuals of an endemicarea) defined a cut-off of 0096 which showed better sensitiv-ity without compromising specificity and an area under thecurve of 10 (95 CI 086 to 10)

This kDNA PCR-ELISA presented a limit of detectionof 007 fg which was defined using a serial dilution ofthe reference L (L) infantum (MHOMBR2002LPC-RPV)DNA in water The detection limit was evaluated using DNAextracted from successive dilutions of blood samples (11 sam-ples evaluated) spiked with an initial concentration of 10000parasites The kDNA PCR-ELISA consistently detected thelowest concentration 1 parasitemL of blood in all samplesevaluated Similar results were observed for electrophoresisanalyses on 6 polyacrylamide gels with products from thesame PCR assay (Figures 1 and 2)

In the evaluation of analytical specificity the kDNAPCR-ELISA showed positive results for species from the Ldonovani complex (L (L) donovani and L (L) infantum) andnegative results for other species (L (V) braziliensis L (V)guyanensis and L (L) amazonensis) Positive results for allspecies of Leishmaniawere observedwhen the same productsof the PCR assay were analysed in 6 polyacrylamide gelelectrophoresis stained with silver nitrate (Table 1)

The obtained variation coefficients (VCs) in the intra-assay tests were 681 778 344 and 1122 for DNAfrom the culture samples at concentrations of 700 fg 70 fg07 fg and 007 fg respectively and ranged from 136 to


(a)

2181614121

080604020

Abso

rban

ce (450

nm)

700

pg120583

L

70

pg120583

L

7pg

120583L

700

fg120583

L

70

fg120583

L

7fg

120583L

07

fg120583

L

007

fg120583

L

0007

fg120583

L

00007

fg120583

L

NC1

NC2

Blan

k

(b)

Figure 1 Analytical sensitivity of the conventional PCR and kDNA PCR-ELISA in L infantum gDNA (a) Analytical sensitivity of 6polyacrylamide gel and (b) kDNA PCR-ELISA for the detection of PCR products from DNA extracted of L infantum at concentrationsof 1 700 pg 2 70 pg 3 7 pg 4 700 fg 5 70 fg 6 7 fg 7 07 fg 8 007 fg and 9 0007 fg120583L NC negative controls and M 100 pb DNALadder (Promega)

(a)

15141312111

0908070605040302010

Abso

rban

ce (450

nm)

0 1 10 100 1000 10000 parasites

(b)

Figure 2 Analytical sensitivity of conventional PCR and kDNA PCR-ELISA in spiked blood samples (a) Analytical sensitivity of 6polyacrylamide gels and (b) kDNA PCR-ELISA for the detection of PCR products from DNA extracted from spiked blood samples withthe addition of 1 parasite (lines 1 and 2) and 10 (lines 3 and 4) 100 (lines 5 and 6) 1000 (lines 7 8 and 9) and 10000 parasites (lines 10 11and 12) PC positive control NC negative control andM 100 pb DNA Ladder (Promega)The absorbance readings correspond to the meanof the duplicate for each parasite concentration

1755 in the positive blood samples and 278 to 881 innegative blood samples The VC values in the reproducibilityanalyses (interassay precision) were calculated from theresults of four different assays performed on consecutive dayswith the samples evaluated in duplicateTheVCs were 536603 944 and 2313 for DNA from the culture samplesat concentrations of 700 fg to 007 fg respectively and rangedfrom 544 to 128 for positive blood samples and 824 to2145 for negative blood samples

32 Diagnostic Performance of the kDNA PCR-ELISA in Dif-ferent Groups of Patients with VL VL-HIVCoinfected Asymp-tomatic and Noninfected Endemic Controls The kDNA PCR-ELISA detected infection by L (L) infantum in 100 ofthe VL Group (children and adult patients) and 100 ofthe VLHIV group The assay detected infection in 100

of asymptomatic individuals living in endemic areas andonly 5 of noninfected individuals (Noninfected Group) hadpositive results (Figure 3) Therefore the kDNA PCR-ELISAproduced an overall sensitivity of 100 (95CI 971 to 100)and specificity of 95 (95 CI 835 to 986)

The levels of Leishmania DNA measured by theabsorbance readings of the PCR-ELISAs were very similarin the Children VL and Adult VL and VLHIV groups(119901 = 018) In contrast the absorbance readings in theAsymptomatic Group varied from 0115 to 1151 (median =0347) which indicates lower levels of circulating LeishmaniaDNA in the children of this group (Figure 3)

All samples of the VL VLHIV asymptomatic andnegative groups were assessed using the PCR-ELISA to detectthe human ACTB gene and produced absorbance readingsabove the previously determined cut-off (0099)


Table 1 Analytical specificity of the PCR detected in 6 polyacrylamide gels and the kDNA PCR-ELISA using DNA extracted from differentLeishmania species

DNA samples Gel electrophoresis (kDNA) kDNA ELISAAbsorbance readings (450 nm) Status

L (L) infantum Positive 2237 PositiveL (L) donovani Positive 1397 PositiveL (V) braziliensis Positive 0056 NegativeL (V) guyanensis Positive 0063 NegativeL (L) amazonensis Positive 0095 Negative

Abso

rban

ce (450

nm)

Child

ren

VL

(n=81

)

Adul

t VL

(n=24

)

Asy

mpt

omat

ic(n

=33)

(n=40)

20

18

16

14

12

10

08

06

04

02

00

Non

infe

cted

(n=25)

VL

HIV

Figure 3 Circulating L infantum DNA levels measured usingthe kDNA PCR-ELISA in different groups kDNA PCR-ELISAabsorbance results for the detection of PCR products from DNAextracted from peripheral blood samples in different groups ofpatients with VL VLHIV asymptomatic and noninfected endemiccontrolsThere was no statistically difference between Adult VL andVLHIV Groups (119901 = 038) In contrast the median (0347) in theAsymptomatic Group was lower than the Children VL Group (119901 lt00001)The dashed line represents the cut-off (0096) Children VLGroup Median = 1157 25 Percentile = 0886 and 75 Percentile= 1345 Adult VL Group Median = 1057 25 Percentile = 0675and 75 Percentile = 1175 VLHIV Group Median = 1099 25Percentile = 0842 and 75Percentile = 1340 AsymptomaticGroupMedian = 0347 25 Percentile = 0255 and 75 Percentile = 0531

4 Discussion

Despite the technological developments of the twentiethcentury VL diagnosis made little progress in recent decadeswith few new or alternative tests There are two main reasonsfor this lack of progress VL is a neglected disease thatprovides businesses small returns on investment in researchand development and it is an infection with high biologicalcomplexity [23]

The present study improves the diagnosis of L infantumin humans by presenting the development and proof-of-concept analysis and phase II evaluation of a kDNA PCR-ELISA This method combines PCR and ELISA into a single

analytical technique and provides an objective interpretationof the results compared to conventional PCR followed byelectrophoresis because it uses a gene-specific probe to detectthe amplified product The PCR-ELISA also allows multiplesample testing and quantitative and qualitative analyses suchas quantitative real-time PCR (qRT-PCR) but using basiclaboratory equipment for the processing ELISA tests that ispresent in most clinical laboratories [20 21 24] PCR-ELISAhas the additional advantage of using a whole blood samplethat is obtained in a minimally invasive manner RecentlyRuiter and colleagues [25] demonstrated that there wasno statistically significant difference between the accuracyof molecular methods using whole blood samples in VLdiagnosis compared with the use of more invasive bonemarrow clinical samples

The kDNA PCR-ELISA is based on the capture of anamplified product with a sense primer labelled with biotin atthe 51015840end in the polystyrene plate coated with streptavidinfollowed by hybridisation with a fluorescein-labelled probeat the 51015840 end and an antifluorescein-peroxidase conjugate fordetection [21] Kinetoplast DNA or kDNA was chosen as theDNA target for the PCR-ELISA because it is present in a largecopy number in the Leishmania cell [26 27] However it doesnot permit precise quantification of the DNA of Leishmaniaspp because of the large variability in copy number ofits minicircle forms (the specific kDNA target) in differentstrains which led us to choose a qualitative type of a PCR-ELISA

Different PCR-ELISAwere developed for the diagnosis ofVL butmost of these assays were evaluated in only one groupof patients [28ndash30] which did not allow broad validationfor clinical diagnosis Other tests were not designed tospecifically detect causative agents of VL [28 29 31] whichdiscourages its use in surveillance and disease control pro-grammes Two tests were described only at the developmentphase [31 32] and many others used commercial kits for thedetection of PCR-amplified products that may suffer fromdiscontinuity of production [28 30 33 34]

The analytical specificity of the kDNA PCR-ELISA forcausative VL agents was only possible because of the use of aprobe designed to complement a specific nucleotide sequencefor the L donovani complex Only the L (L) infantum andL (L) donovani species were detected using the assay in theproposed test (Table 1) The same result was obtained usingonly one platform and a probe specific to L (L) infantumcompared with VL PCR-ELISA previously developed [33]


The lower detection limits of the assay (007 fg of purifiedL (L) infantum DNA and 1 parasitemL of human blood)(Figures 1 and 2) are lower or similar to the detection of otherPCR-ELISA [29 31 33 35]

The kDNA PCR-ELISA exhibited diagnostic precisionbecause it presented ratios of repeatability and reproducibilityfrom 136 to 1755 and 536 to 2313 respectively Theupper limit for the coefficient of variation in molecular testsis not established but our results are similar to other reportedtests based on the PCR-ELISA which revealed intra-assaycoefficients of variation (repeatability) from 19 to 120 andinterassay coefficient of variation (repeatability) from 30 to270 [21 36ndash38] The same protocol was used to developthe human beta actin (ACTB) PCR-ELISA for evaluations ofall samples analysed This test was developed as an internalcontrol of DNA integrity and a control for variation inthe efficiency of DNA extraction and PCR amplificationprimarily in peripheral blood samples that were negativein the kDNA PCR-ELISA test To our knowledge no otherPCR-ELISA that was developed for the diagnosis of VLdemonstrated the same type of control

Phase II evaluation of a kDNA PCR-ELISA used 130 DNAsamples from the peripheral blood of patients (Children VLGroup and Adult VL Group and VLHIV Group) and 40samples from a Noninfected Group The assay exhibited agood performance with a sensitivity of 100 and specificityof 95

The equivalence of kDNA PCR-ELISA performance withvalues demonstrated by other molecular methods for thediagnosis of VL must be highlighted including PCR assayfollowed by electrophoresis (sensitivity of 91 to 100 andspecificity of 87 to 100) [15 16 39] The sensitivity of thekDNA PCR-ELISA was similar to the value of 98 for theassay developed by Costa et al and compared with otherassays that used the same methodology to diagnose VLand evaluated for test performance [28] This study usedperipheral blood and bone marrow aspirate samples for eval-uations and theDNA target was the SSU-rRNA coding regionof L (L) donovani The kDNA PCR-ELISA demonstratedsuperior performance compared to the assay developed byDeDoncker et al [29] which used the target SSU-rRNA that isconserved in Leishmania genus and presented a sensitivity of839 (95 CI 717ndash924) and specificity of 872 (95 CI726ndash957)

The kDNA PCR-ELISA was validated in this study inclinical samples from varied groups of VL patients at differentages and asymptomatic and noninfected endemic controls toensure its widespread use as a method for the diagnosis of thedisease and asymptomatic infectionMolecular tests are espe-cially useful for VL diagnosis in immunosuppressed patientsin whom serological tests tend to be less sensitive because ofimpaired humoral immune responseThe sensitivity of kDNAPCR-ELISA in VLHIV coinfected patients was 100 Thisresult is superior to Piarroux et al [40] (82) and similar toLachaud et al [41] (100) (both of these studies used bonemarrow samples) and Fissore et al [42] with serum samples(sensitivity of 97) and PCR electrophoresis analysis on 2agarose gel

Two samples of noninfected individuals (negative group)that previously presented negative results in qPCR werepositive in the kDNA PCR-ELISA but with low absorbancereadings (Figure 3) This fact may be explained by themolecular target used in the qPCR assay the SSU-rRNA andthese two positive samples may be asymptomatic individualswho were not identified using the reference antibody-basedmethods Lachaud et al [14] compared variousmolecular tar-gets for the detection of LeishmaniaDNA in peripheral bloodsamples the ribosomal DNA target two nuclear targets andthree kinetoplast (minicircle) targets and the kinetoplast wasgenerally the most sensitive Unfortunately the high numberof kDNAcopies increases the risk of carryover contaminationin the handling and processing steps mainly if not handledby trained professionals In the development of kDNA PCR-ELISA all procedures were done by a trained researcher andphysical barriers (eg separation of rooms and materials andthe use of laminar flow hood with ultraviolet light) wereutilized in the sample handling steps to minimize the risk ofcontamination

Currently one of the most important challenges inassessing L infantum transmission in endemic areas is theidentification of asymptomatic infection Serological tests arethe methods of choice for population studies but our resultssuggest that these methods are neither adequate nor accuratefor the diagnosis of the asymptomatic formThe performanceof serological tests to detect L infantum antibodies wasevaluated primarily in patients presenting VL symptoms[43ndash47] Some studies demonstrated that ELISA methodscannot be reliably used as a tool to identify asymptomaticindividuals and it may underestimate the positive ratesespecially when only one antigen is used [48 49]

The kDNA PCR-ELISA in this study is a potential tool toidentify and follow up asymptomatic individuals especiallyin population-based studies in which there are a largernumber of individuals to be evaluated The assay detected allchildren from the Asymptomatic VL Group as positive withabsorbance readings from 0115 to 1151 and a median (0347)that was lower than the Children VL Group (Median 1157119901 lt 00001) which suggests the presence of circulating DNAL InfantumThesefindings corroborate data published by dosSantosMarques et al [50] who found lower circulating DNAlevels in asymptomatic compared to VL children tested withqPCR targeting SSU-rRNA from Leishmania spp Moleculardiagnosis of asymptomatic individuals has great importancebecause it provides monitoring of individuals who are incontact but have not developed the disease and allows themonitoring of transmission areas to develop more effectivestrategies for VL control programmes

5 Conclusions

A PCR-ELISA targeting kDNA from the Leishmania dono-vani complex was developed for human VL diagnosis inperipheral blood samples The assay exhibited good perfor-mance in proof-of-concept analyses and phase II evalua-tion in age-variable groups of L infantum-infected patientsasymptomatic individuals and healthy endemic controls Ahuman beta actin (ACTB) PCR-ELISA was developed as an


internal control for DNA extraction and PCR amplificationTherefore the kDNA PCR-ELISA is safe and suitable for thediagnosis of human L infantum infection in reference lab-oratories in endemic countries and allows the simultaneoustesting of a large number of samples with broad applicationfor disease control programmes It is important to note thatthe present study is a case control evaluation which mayjustify the existence of bias and an exaggerated estimate ofsensitivity and specificity So a large field trial assessing theusefulness of the PCR-ELISA as a confirmatory VL test isstill necessary before the PCR-ELISA implementation bothfor the evaluation of asymptomatic individuals with positiveor negative serology and for the diagnosis of true VL casesamong symptomatic suspected patients in an endemic region

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Fernanda Alvarenga Cardoso Medeiros Luciana InaciaGomes and Ana Rabello conceived and designed the studyAcquisition of the financial support for the study was by Fer-nanda Alvarenga Cardoso Medeiros Luciana Inacia Gomesand Ana Rabello Fernanda Alvarenga Cardoso MedeirosCarolina Senra Alves de Souza and Letıcia Helena dosSantos Marques performed the experiments Luciana InaciaGomes Edward Oliveira Ana Rabello Glaucia FernandesCota and Mariangela Carneiro analysed the data MariaVitoria Mourao Letıcia Helena dos Santos Marques GlauciaFernandes Cota and Mariangela Carneiro proved biologicalsamples Fernanda Alvarenga Cardoso Medeiros LucianaInacia Gomes Edward Oliveira and Ana Rabello wrote thepaper All authors contributed equally to this work

Acknowledgments

The authors thank Eliane de Morais Teixeira for providingLeishmania spp

References

[1] WHO ldquoLeishmaniasis in high-burden countries an epidemio-logical update based on data reported in 2014rdquoWeekly Epidemi-ological Record vol 22 pp 287ndash296 2016

[2] M O Harhay P L Olliaro D L Costa and C H N CostaldquoUrban parasitology visceral leishmaniasis in Brazilrdquo Trends inParasitology vol 27 no 9 pp 403ndash409 2011

[3] V E M de Araujo L C Pinheiro M C M de Almeida etal ldquoRelative risk of visceral leishmaniasis in Brazil a spatialanalysis in urban areardquo PLoS Neglected Tropical Diseases vol7 no 11 article no e2540 2013

[4] SINAN-Sistema de Informacao de Agravos de NotificacaoMinisterio da Saude Brasılia 2015 httpdatasussaudegovbr

[5] K Ritmeijer Y Melaku M Mueller S Kipngetich C OrsquoKeeffeand R N Davidson ldquoEvaluation of a new recombinant K39

rapid diagnostic test for Sudanese visceral leishmaniasisrdquoAmer-ican Journal of Tropical Medicine and Hygiene vol 74 no 1 pp76ndash80 2006

[6] E Abass C Kang F Martinkovic et al ldquoHeterogeneity ofLeishmania donovani parasites complicates diagnosis of vis-ceral leishmaniasis comparison of different serological tests inthree endemic regionsrdquo PLoS ONE vol 10 no 3 Article IDe0116408 2015

[7] R Ter Horst T Tefera G Assefa A Z Ebrahim R N David-son and K Ritmeijer ldquoField evaluation of rK39 test and directagglutination test for diagnosis of visceral leishmaniasis in apopulation with high prevalence of human immunodeficiencyvirus in Ethiopiardquo The American Journal of Tropical Medicineand Hygiene vol 80 no 6 pp 929ndash934 2009

[8] G F Cota M R de Sousa F N Demarqui and A RabelloldquoThe diagnostic accuracy of serologic and molecular methodsfor detecting visceral leishmaniasis in HIV infected patientsmeta-analysisrdquo PLoS Neglected Tropical Diseases vol 6 no 5Article ID e1665 2012

[9] G F Cota M R De Sousa B M De Freitas Nogueira etal ldquoComparison of parasitological serological and moleculartests for visceral leishmaniasis inHIV-infected patients a cross-sectional delayed-type studyrdquo American Journal of TropicalMedicine and Hygiene vol 89 no 3 pp 570ndash577 2013

[10] M Andreadou E Liandris I N Kasampalidis et al ldquoEvalua-tion of the performance of selected in-house and commerciallyavailable PCR and real-time PCR assays for the detection ofLeishmania DNA in canine clinical samplesrdquo ExperimentalParasitology vol 131 no 4 pp 419ndash424 2012

[11] G Srividya A Kulshrestha R Singh and P Salotra ldquoDiagnosisof visceral leishmaniasis developments over the last decaderdquoParasitology Research vol 110 no 3 pp 1065ndash1078 2012

[12] AMMontalvo J Fraga IMaes J-CDujardin andGVanDerAuwera ldquoThree new sensitive and specific heat-shock protein70 PCRs for global Leishmania species identificationrdquo EuropeanJournal of ClinicalMicrobiologyamp Infectious Diseases vol 31 no7 pp 1453ndash1461 2012

[13] I S Koltas F Eroglu S Uzun and D Alabaz ldquoA comparativeanalysis of different molecular targets using PCR for diagnosisof old world leishmaniasisrdquo Experimental Parasitology vol 164pp 43ndash48 2016

[14] L Lachaud S Marchergui-Hammami E ChabbertDereure JDereure J P Dedet and P Bastien ldquoComparison of six PCRmethods using peripheral blood for detection of canine visceralleishmaniasisrdquo Journal Clinical Microbiology vol 40 no 1 pp210ndash215 2002

[15] Z Wu Y Bao Y Ding M Yu L Lu and Y Zhang ldquoAnexperimental study on application of PCR in detection of Kala-azarrdquo The Southeast Asian Journal of Tropical Medicine andPublic Health vol 28 no 1 pp 169ndash172 1997

[16] R Maurya R K Singh B Kumar P Salotra M Rai and SSundar ldquoEvaluation of PCR for diagnosis of Indian kala-azarand assessment of curerdquo Journal of Clinical Microbiology vol43 no 7 pp 3038ndash3041 2005

[17] S Banoo D Bell P Bossuyt et al ldquoEvaluation of diagnostictests for infectious diseases general principlesrdquo Nature ReviewsMicrobiology vol 4 no 9 pp S21ndashS31 2006

[18] A CVolpini VMA Passos G COliveira andA J RomanhaldquoPCR-RFLP to identify Leishmania (Viannia) braziliensis andL (Leishmania) amazonensis causing American cutaneousleishmaniasisrdquo Acta Tropica vol 90 no 1 pp 31ndash37 2004


[19] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in molecularbiology (Clifton NJ) vol 132 pp 365ndash386 2000

[20] E Luneberg J S Jensen and M Frosch ldquoDetection of Myco-plasma pneumoniae by polymerase chain reaction and nonra-dioactive hybridization in microtiter platesrdquo Journal of ClinicalMicrobiology vol 31 no 5 pp 1088ndash1094 1993

[21] L I Gomes L H Dos Santos Marques M J Enk M Cde Oliveira P M Z Coelho and A Rabello ldquoDevelopmentand evaluation of a sensitive PCR-ELISA system for detectionof Schistosoma infection in fecesrdquo PLoS Neglected TropicalDiseases vol 4 no 4 article e664 2010

[22] O Musso P Sommer E Drouet et al ldquoIn situ detection ofhuman cytomegalovirus DNA in gastrointestinal biopsies fromAIDS patients by means of various PCR-derived methodsrdquoJournal of Virological Methods vol 56 no 2 pp 125ndash137 1996

[23] S Antinori L Schifanella and M Corbellino ldquoLeishmaniasisnew insights from an old and neglected diseaserdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 31no 2 pp 109ndash118 2012

[24] M J Sue S K Yeap A R Omar and S W Tan ldquoApplication ofPCR-ELISA in molecular diagnosisrdquo BioMed Research Interna-tional vol 2014 Article ID 653014 6 pages 2014

[25] C M De Ruiter C Van Der Veer M M G Leeflang SDeborggraeve C Lucas and E R Adams ldquoMolecular toolsfor diagnosis of visceral leishmaniasis systematic review andmeta-analysis of diagnostic test accuracyrdquo Journal of ClinicalMicrobiology vol 52 no 9 pp 3147ndash3155 2014

[26] M R Rodgers S J Popper and D F Wirth ldquoAmplificationof kinetoplast DNA as a tool in the detection and diagnosis ofLeishmaniardquo Experimental Parasitology vol 71 no 3 pp 267ndash275 1990

[27] W Degrave O Fernandes D Campbell M Bozza and ULopes ldquoUse of molecular probes and PCR for detection andtyping of Leishmaniamdasha mini-reviewrdquo Memorias do InstitutoOswaldo Cruz vol 89 no 3 pp 463ndash469 1994

[28] J-M Costa R Durand M Deniau et al ldquoPCR enzyme-linkedimmunosorbent assay for diagnosis of leishmaniasis in humanimmunodeficiency virus-infected patientsrdquo Journal of ClinicalMicrobiology vol 34 no 7 pp 1831ndash1833 1996

[29] S De Doncker V Hutse S Abdellati et al ldquoA new PCR-ELISAfor diagnosis of visceral leishmaniasis in blood of HIV-negativesubjectsrdquo Transactions of the Royal Society of Tropical Medicineand Hygiene vol 99 no 1 pp 25ndash31 2005

[30] A Alborzi B Pourabbas F Shahian J Mardaneh G RPouladfar andM Ziyaeyan ldquoDetection of Leishmania infantumkinetoplast DNA in the whole blood of asymptomatic indi-viduals by PCR-ELISA and comparison with other infectionmarkers in endemic areas southern IranrdquoTheAmerican Journalof Tropical Medicine and Hygiene vol 79 no 6 pp 839ndash8422008

[31] T Kobets J Badalova I Grekov H Havelkova M Svobodovaand M Lipoldova ldquoLeishmania parasite detection and quan-tification using PCR-ELISArdquo Nature protocols vol 5 no 6 pp1074ndash1080 2010

[32] N Rolao S Cortes O R Rodrigues and L Campino ldquoQuan-tification of Leishmania infantum parasites in tissue biopsiesby real-time polymerase chain reaction and polymerase chainreaction-enzyme-linked immunosorbent assayrdquo Journal of Par-asitology vol 90 no 5 pp 1150ndash1154 2004

[33] J Martin-Sanchez M C Lopez-Lopez C Acedo-SanchezJ J Castro-Fajardo J A Pineda and F Morillas-Marquez

ldquoDiagnosis of infections with Leishmania infantum using PCR-ELISArdquo Parasitology vol 122 no 6 pp 607ndash615 2001

[34] J Martın-Sanchez J A Pineda M Andreu-Lopez et al ldquoThehigh sensitivity of a PCR-ELISA in the diagnosis of cutaneousand visceral leishmaniasis caused by Leishmania infantumrdquoAnnals of Tropical Medicine and Parasitology vol 96 no 7 pp669ndash677 2002

[35] J Martın-Sanchez and M M Francisco Kit para diagnosticoespecıfico de las leishmaniosis causadas por lsquoLeishmania infan-tumrsquo por la tecnica de PCR-ELISA Espanha patente CI- C12Q168 01 Novmber 2003

[36] P Morata M I Queipo-Ortuno J M Reguera M A Garcıa-Ordonez AAnaCardenas and JDColmenero ldquoDevelopmentand evaluation of a PCR-enzyme-linked immunosorbent assayfor diagnosis of human brucellosisrdquo Journal of Clinical Microbi-ology vol 41 no 1 pp 144ndash148 2003

[37] M I Queipo-Ortuno M A Garcıa-Ordonez R Gil J RojasJ D D Colmenero and P Morata ldquoPCR-DIG ELISA withbiotinylated primers is unsuitable for use in whole bloodsamples from patients with brucellosisrdquoMolecular and CellularProbes vol 18 no 4 pp 243ndash250 2004

[38] M Musiani G Gallinella S Venturoli and M Zerbini ldquoCom-petitive PCR-ELISA protocols for the quantitative and thestandardized detection of viral genomesrdquo Nature Protocols vol2 no 10 pp 2511ndash2519 2007

[39] J Disch F C Maciel M C de Oliveira M Orsini and ARabello ldquoDetection of circulating Leishmania chagasi DNA forthe non-invasive diagnosis of human infectionrdquo Transactions ofthe Royal Society of Tropical Medicine and Hygiene vol 97 no4 pp 391ndash395 2003

[40] R Piarroux F Gambarelli H Dumon et al ldquoComparisonof PCR with direct examination of bone marrow aspirationmyeloculture and serology for diagnosis of visceral leishma-niasis in immunocompromised patientsrdquo Journal of ClinicalMicrobiology vol 32 no 3 pp 746ndash749 1994

[41] L Lachaud J Dereure E Chabbert et al ldquoOptimized PCRusing patient blood samples for diagnosis and follow-up ofvisceral leishmaniasis with special reference to AIDS patientsrdquoJournal of ClinicalMicrobiology vol 38 no 1 pp 236ndash240 2000

[42] C Fissore P Delaunay B Ferrua et al ldquoConvenience of serumfor visceral leishmaniasis diagnosis by PCRrdquo Journal of ClinicalMicrobiology vol 42 no 11 pp 5332ndash5333 2004

[43] R F S Braz E T Nascimento D R A Martins et al ldquoThe sen-sitivity and specificity of Leishmania chagasi recombinant K39antigen in the diagnosis of American visceral leishmaniasis andin differentiating active from subclinical infectionrdquo AmericanJournal of Tropical Medicine andHygiene vol 67 no 4 pp 344ndash348 2002

[44] S F Guimaraes Carvalho E Moreira Lemos R Corey andR Dietze ldquoPerformance of recombinant K39 antigen in thediagnosis of Brazilian visceral leishmaniasisrdquo American Journalof Tropical Medicine and Hygiene vol 68 no 3 pp 321ndash3242003

[45] M Boelaert S Rijal S Regmi et al ldquoA comparative study ofthe effectiveness of diagnostic tests for visceral leishmaniasisrdquoAmerican Journal of Tropical Medicine and Hygiene vol 70 no1 pp 72ndash77 2004

[46] F Chappuis S Rijal A Soto J Menten and M BoelaertldquoA meta-analysis of the diagnostic performance of the directagglutination test and rK39 dipstick for visceral leishmaniasisrdquoBritish Medical Journal vol 333 no 7571 pp 723ndash726 2006


[47] Z Maia M Lırio S Mistro C M C Mendes S R Mehta andR Badaro ldquoComparative study of rK39 Leishmania antigen forserodiagnosis of visceral leishmaniasis systematic review withmeta-analysisrdquo PLoS Neglected Tropical Diseases vol 6 no 1Article ID e1484 2012

[48] L de Gouvea Viana T S M de Assis M Orsini et al ldquoCom-bined diagnostic methods identify a remarkable proportion ofasymptomatic Leishmania (Leishmania) chagasi carriers whopresent modulated cytokine profilesrdquo Transactions of the RoyalSociety of TropicalMedicine andHygiene vol 102 no 6 pp 548ndash555 2008

[49] E C Moreno A V Goncalves A V Chaves et al ldquoInaccuracyof enzyme-linked immunosorbent assay using soluble andrecombinant antigens to detect asymptomatic infection byLeishmania infantumrdquo PLoS Neglected Tropical Diseases vol 3no 10 article no e536 2009

[50] L H dos Santos Marques L I Gomes I C M da Rochaet al ldquoLow parasite load estimated by qPCR in a cohort ofchildren living in urban area endemic for visceral leishmaniasisin Brazilrdquo PLoSNeglected Tropical Diseases vol 6 no 12 ArticleID e1955 2012

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


test (IFAT) enzyme-linked immunosorbent assay (ELISA)and recombinant antigen-based immunochromatographytest (ICT) support the clinical diagnosis of VL but thesemethods have drawbacks such as cross-reactions in thepresence of other diseases and an inability to distinguishbetween active and past infection Some studies demon-strated that serological tests based on antigens of a singleLeishmania sp exhibited low diagnostic performance inregions with circulating heterologous parasites [5 6] Theserological tests with the exception of the Direct Agglutina-tion Tests (DAT) andWestern blotting exhibit low sensitivityin VL patients coinfected with HIV [7ndash9] because of theirinsufficient immune humoral response

Molecular methods are useful for the detection of lowparasitic loads and exhibit high sensitivity in the diagno-sis of VL [10] PCR-based assays are the most commonlyused approaches in molecular diagnostics One additionaladvantage of PCR is that it is based on the identificationof conserved DNA sequences which allows the detection ofspecific pathogen species in a variety of clinical samples evenarchived materials [11] PCR sensitivity is directly related tothe copy number of the target and the primer and cyclingprotocol used [12] The internal transcribed spacers (ITSITS1 and ITS2) regions gp63 gene HSP-70 gene miniexon(ME) and SSU-rRNA and conserved and variable regionsof kinetoplast DNA (kDNA) minicircles are used for thediagnosis and characterization of Leishmania strains [13]Conventional PCR techniques targeting kDNA exhibit highsensitivity for VL diagnosis [13 14] but this technique isnot automated and is laborious which limits its use to asmall number of samples To overcome this limitation wedeveloped a kDNA PCR-ELISA which allows simultaneoustesting of a large number of blood samples without theuse of commercial kits for the detection of PCR productsvia measurement of absorbance readings for the laboratorydiagnosis of VL

2 Materials and Methods

This work was performed in two phases

(i) In a proof-of-concept phase the assay was standard-ized using a reference strain of L (L) infantum and abank of peripheral blood samples from nonimmuno-suppressed patients with VL that were characterizedusing techniques considered gold standards and fromhealthy individuals in an endemic area

(ii) In a phase II evaluation the assay was validated inclinical samples from varied groups of VL patients ofdifferent ages and in asymptomatic and noninfectedindividuals to certify the wide use of the assay forthe diagnosis of human VL We describe the step-by-step development and internal validation of the kDNAPCR-ELISA below

21 Parasites Promastigotes of L (L) infantum (MHOMBR2002LPC-RPV) L (L) donovani (MHOMET196HU3) L (Viannia) braziliensis (MHOMBR75M2903) L

(V) guyanensis (MHOMBR1975M4147) and L (L) ama-zonensis (IFLABR1967PH-8) were cultured in Schneidermedium (Sigma-Aldrich St Louis MO USA) supplementedwith 10 foetal bovine serum (Invitrogen Carlsbad CAUSA) and counted in aNeubauer chamber An aliquot of 1mLof the suspension of parasites was centrifuged at 2300119892 for 2minutes and stored at minus70∘C

22 Samples

221 Peripheral Blood Samples Peripheral blood samplescollected from 14 patients with VL nonimmunosuppressedliving in endemic areas in Brazil with typical VL symptomsand parasitological confirmation (direct exam andor cul-ture) with a mean age of 127 years (range one month to 768years) were used as positive controls

Peripheral blood samples from 11 healthy individualsfrom the endemic area of Belo Horizonte MG were used asnegative controls

222 Spiked Blood Samples Aliquots of peripheral bloodsamples obtained from 11 healthy individuals were addedto the reference sample L (L) infantum (MHOMBR2002LPC-RPV) to obtain a concentration of 10000 parasitesmLof blood

223 Evaluation of kDNA PCR-ELISA Performance in theDiagnosis of VL VL-HIV Coinfected Asymptomatic andNoninfected Endemic Controls A minimum sample size of38 VL cases was calculated with a mean sensitivity of 975to assess the accuracy of the assay which is consistent withpreviously published molecular tests [15 16] The specificityin these studies was 100 and a minimum sample size of 38noninfected controls was estimated with a level of confidenceof 95

119899 = 1199112 sdot 119901 sdot(1 minus 119901)

1199092 (1)

where 119901 is the sensitivity or specificity and 1199092 is the confi-dence interval (95) [17]

The following groups were included

Children VL Group This group was composed of 81 childrenadmitted to Hospital Joao Paulo II-FHEMIG (FundacaoHospitalar de Minas Gerais) Belo Horizonte Minas Geraiswho presented with VL symptoms and parasitological con-firmation (direct exam andor culture) andor serologicaldiagnosis for VL (indirect immunofluorescence antibodytest-IFAT andor immunochromatographic rK39 antigen-test) associated with clinical improvement after treatmentPatients in this group were aged 3 months to 10 years (mean25 years)

Adult VL Group This group was constituted by 24 adultpatients aged 18 to 68 years (mean of 36 years) who wereadmitted to Hospital Eduardo de Menezes-FHEMIG andpresented VL symptoms with parasitological confirmation(direct exam and or culture) andor serological diagnosis for







24 kDNA PCR-ELISA


2 and 02mM




















3 Results






(a)

2181614121

080604020

Abso

rban

ce (450

nm)

700

pg120583

L

70

pg120583

L

7pg

120583L

700

fg120583

L

70

fg120583

L

7fg

120583L

07

fg120583

L

007

fg120583

L

0007

fg120583

L

00007

fg120583

L

NC1

NC2

Blan

k

(b)


(a)

15141312111

0908070605040302010

Abso

rban

ce (450

nm)

0 1 10 100 1000 10000 parasites

(b)











Abso

rban

ce (450

nm)

Child

ren

VL

(n=81

)

Adul

t VL

(n=24

)

Asy

mpt

omat

ic(n

=33)

(n=40)

20

18

16

14

12

10

08

06

04

02

00

Non

infe

cted

(n=25)

VL

HIV


4 Discussion
















5 Conclusions




Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















24 kDNA PCR-ELISA


2 and 02mM




















3 Results






(a)

2181614121

080604020

Abso

rban

ce (450

nm)

700

pg120583

L

70

pg120583

L

7pg

120583L

700

fg120583

L

70

fg120583

L

7fg

120583L

07

fg120583

L

007

fg120583

L

0007

fg120583

L

00007

fg120583

L

NC1

NC2

Blan

k

(b)


(a)

15141312111

0908070605040302010

Abso

rban

ce (450

nm)

0 1 10 100 1000 10000 parasites

(b)











Abso

rban

ce (450

nm)

Child

ren

VL

(n=81

)

Adul

t VL

(n=24

)

Asy

mpt

omat

ic(n

=33)

(n=40)

20

18

16

14

12

10

08

06

04

02

00

Non

infe

cted

(n=25)

VL

HIV


4 Discussion
















5 Conclusions




Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























3 Results






(a)

2181614121

080604020

Abso

rban

ce (450

nm)

700

pg120583

L

70

pg120583

L

7pg

120583L

700

fg120583

L

70

fg120583

L

7fg

120583L

07

fg120583

L

007

fg120583

L

0007

fg120583

L

00007

fg120583

L

NC1

NC2

Blan

k

(b)


(a)

15141312111

0908070605040302010

Abso

rban

ce (450

nm)

0 1 10 100 1000 10000 parasites

(b)











Abso

rban

ce (450

nm)

Child

ren

VL

(n=81

)

Adul

t VL

(n=24

)

Asy

mpt

omat

ic(n

=33)

(n=40)

20

18

16

14

12

10

08

06

04

02

00

Non

infe

cted

(n=25)

VL

HIV


4 Discussion
















5 Conclusions




Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

2181614121

080604020

Abso

rban

ce (450

nm)

700

pg120583

L

70

pg120583

L

7pg

120583L

700

fg120583

L

70

fg120583

L

7fg

120583L

07

fg120583

L

007

fg120583

L

0007

fg120583

L

00007

fg120583

L

NC1

NC2

Blan

k

(b)


(a)

15141312111

0908070605040302010

Abso

rban

ce (450

nm)

0 1 10 100 1000 10000 parasites

(b)











Abso

rban

ce (450

nm)

Child

ren

VL

(n=81

)

Adul

t VL

(n=24

)

Asy

mpt

omat

ic(n

=33)

(n=40)

20

18

16

14

12

10

08

06

04

02

00

Non

infe

cted

(n=25)

VL

HIV


4 Discussion
















5 Conclusions




Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Abso

rban

ce (450

nm)

Child

ren

VL

(n=81

)

Adul

t VL

(n=24

)

Asy

mpt

omat

ic(n

=33)

(n=40)

20

18

16

14

12

10

08

06

04

02

00

Non

infe

cted

(n=25)

VL

HIV


4 Discussion
















5 Conclusions




Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























5 Conclusions




Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















development and validation of a pcr-elisa for the diagnosis of...

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