Research ArticleLoop Mediated Isothermal Amplification for Detection ofTrypanosoma brucei gambiense in Urine and Saliva Samples inNonhuman Primate Model

Maina Ngotho1 John Maina Kagira2 Beatrice Muthoni Gachie3 Simon Muturi Karanja4

Maxwell Wambua Waema3 Dawn Nyawira Maranga1 and Naomi Wangari Maina3

1Animal Science Department Institute of Primate Research (IPR) PO Box 24481 Karen Nairobi 00502 Kenya2Animal Health and Production Department College of Agriculture and Natural Resources Jomo Kenyatta University ofAgriculture and Technology (JKUAT) PO Box 62000 Nairobi 00200 Kenya3Biochemistry Department College of Health Sciences Jomo Kenyatta University of Agriculture and Technology (JKUAT)PO Box 62000 Nairobi 00200 Kenya4Public Health Department College of Health Sciences Jomo Kenyatta University of Agriculture and Technology (JKUAT)PO Box 62000 Nairobi 00200 Kenya

Correspondence should be addressed to Naomi Wangari Maina nmainajkuatacke

Received 9 February 2015 Revised 22 April 2015 Accepted 3 May 2015

Academic Editor Malcolm Jones

Copyright copy 2015 Maina Ngotho et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Human African trypanosomiasis (HAT) is a vector-borne parasitic zoonotic disease The disease caused by Trypanosoma bruceigambiense is themost prevalent inAfrica Early diagnosis is hampered by lack of sensitive diagnostic techniquesThis study exploredthe potential of loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) in the detection of T bgambiense infection in a vervet monkey HATmodel Six vervet monkeys were experimentally infected with T b gambiense IL3253andmonitored for 180 days after infection Parasitaemiawas scored daily Blood cerebrospinal fluid (CSF) saliva and urine sampleswere collected weekly PCR and LAMP were performed on serum CSF saliva and urine samples The detection by LAMP wassignificantly higher than that of parasitological methods and PCR in all the samples The performance of LAMP varied betweenthe samples and was better in serum followed by saliva and then urine samples In the saliva samples LAMP had 100 detectionbetween 21 and 77 dpi whereas in urine the detection it was slightly lower but there was over 80 detection between 28 and 91 dpiHowever LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi respectively The findings of thisstudy emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples

1 Introduction

Human African trypanosomiasis (HAT) is a tropical diseasethat is endemic in several countries in sub-Saharan AfricaControl of sleeping sickness relies on passive case detectionand it is considered to be the most cost-effective when com-pared to active case detection [1] Sleeping sickness causedby T b gambiense is currently responsible for over 90 ofall HAT cases [1] Screening of the population at risk is doneby antibody detection with the Card Agglutination Test forTrypanosomiasis (CATT) and confirmed by parasitological

methods Serological tests have varying sensitivities and can-not decisively differentiate between active and cured casesFurthermore cured patients can remain CATT seropositivefor up to three years due to persisting circulating antibodiesthus prohibiting the use of antibody tests for assessmentof treatment success [2] The parasitological detection tech-niques also have limitations The methods are time con-suming tedious and prone to subjectivity In addition lowdetection rates may occur since T b gambiense infection ischaracterized by low parasitemia [3] False negatives (CATTnegative) but parasitemic cases have also been reported [4]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 867846 7 pageshttpdxdoiorg1011552015867846

2 BioMed Research International

These limitations imply the need for more sensitive andspecific diagnosis

The amplification of DNA has emerged as one of thediagnostic techniques used in studies of infectious diseases[5] Species specific genes have been used to characterizetrypanosomes [6] The discovery of the T b gambiense-specific glycoprotein (TgsGP) gene that is specific to the T bgambiense subspecies heralded its use as a probe for diagno-sis It is the only subspecies-specific gene for T b gambienseand encodes a 47 kDa VSG-like receptor protein [7] Ampli-fication of this gene using PCR has successfully been used inclinical samples [8] However challenges of the DNA extrac-tion protocolsmay affect diagnosis of trypanosome infections[9] and requirements of expensive automated thermal cyclersmake PCR impractical for adoption in the field [10]

Loop mediated isothermal amplification (LAMP) is per-formed under isothermal conditions and relies on autocy-cling strand displacement DNA synthesis [11] It requiresa simple heating device and is rapid and results are easilyviewed by several detection formats The autocycling reac-tions lead to accumulation of a large amount of the targetDNA and by-products such as magnesium pyrophosphateallowing for rapid detection using varied formats LAMPuses four to six specially designed primers recognizing six toeight regions of the target DNA sequence resulting in a highspecificity It has been used in detection of the Trypanozoonsubgenus [12] T b rhodesiense [13] and recently Group 1 Tb gambiense [14] The test has high sensitivity and specificityand does not require specialized equipment and thismakes ita suitable diagnostic test in resource poor settings and wouldtherefore be ideal diagnosis of neglected diseases such asHAT

The importance of experimental animal models includescontrolled conditions and planned sampling among otherThe vervet monkey (Chlorocebus aethiops) has been devel-oped as amodel for early stageHAT caused byT b gambiense[15] Using this animal model the performance of LAMPbased on the TgsGP gene was assessed in detection of T bgambiense in serum CSF saliva and urine

2 Materials and Methods

21 Trypanosomes Trypanosoma b gambiense isolate IL3253was used in this study It was isolated from a human HATpatient from Sudan in 1982 The isolate was cryopreserved inliquid nitrogen and for infective purposes the parasites weresubinoculated into immunosuppressed donor Swiss miceAt peak parasitemia heart blood was obtained by cardiacpuncture and parasites harvested and diluted to 105mL usingphosphate saline glucose

22 Experimental Animals Six adult vervet monkeys of bothsexes weighing between 20 and 50 kg were used in thisstudy They were trapped from the wild in an area knownto be nonendemic for human trypanosomiasis The animalsunderwent a 90-day quarantine during which they werescreened for zoonotic diseases and treated for ecto- andendoparasites They were also trained for ease of adapta-tion and maintained on commercial pellets (Unga Feeds

Ltd Nairobi Kenya) supplemented with fresh fruits andvegetables Drinking water was provided ad libitum Themonkeys were housed in stainless steel cages at ambient roomtemperatures of 18ndash25∘C under biosafety level II animalholding conditions At the end of the experiment periodthe animals were euthanized by injection with Euthatal (20sodium pentobarbitone Rotexmedica Trittau Germany)via the femoral vein

23 Study Design Six monkeys were infected intravenouslywith approximately 105 trypanosomes in 1mL of phosphatesaline glucose The infected monkeys were monitored fora total period of 180 days after experimental infectionParasitaemia was estimated daily using methods previouslydescribed using the rapid matching method [16] and haema-tocrit centrifuge technique [17]

24 Sample Collection The monkeys were anaesthetized onweekly basis with ketamine hydrochloride (RotexmedicaTrittau Germany) at a dosage of 10mgkg body weight forsample collection The samples were collected before andafter infection on a weekly basis Three mL of blood fromthe femoral artery and 15mL of cerebrospinal fluid (CSF)via lumbar puncture were collected Saliva samples wereobtained by placing swabs under the animals tongue for tenminutes to allow for adequate wetting Thereafter the swabswere placed in dry cryovial tubesThe urine was obtained viaa collection apparatus placed on the bottom of the monkeycage and stored in 50mL falcon tubes This was done in theearly morning prior to sedation All samples were collectedand stored at minus20∘C

25 DNA Extraction DNAwas extracted from serum urineCSF and saliva samples using genomic DNA isolation kits(Zymo Research USA) as per manufacturerrsquos instructions

26 PCR Amplification of T b gambiense-specific glyco-protein (TgsGP) gene was done using primer sequences aspreviously described [18] The PCR reactions (nested) wereperformed as described [8] using 1 120583L of extracted DNA ina 25 120583L reaction mixture The PCR amplification was per-formed by incubating the samples for 15min at 95∘C followedby 45 cycles of 1min at 94∘C 1min at 63∘C and 1min at 72∘Cand a final extension at 72∘C for 10minThereafter amplifiedproducts were analyzed by electrophoresis in 2 agarosegels Gels were stained with ethidium bromide (05120583gmL)(Sigma USA) and viewed under UV illumination Thenegative controls purified DNA from T b brucei GUTAT1and T b rhodesiense IPR001 and distilled water The positivecontrol was purified T b gambiense IL3253 DNA

27 LAMP The TgsGP primers as previously describedwere used [14] The reaction mixture of 25 120583L consisted of40 pmol of the inner primers 5 pmol of the outer primers20 pmol of the loop primers (Inqaba biotec SA) 08Mbetaine (Sigma-Aldrich St LouisMOUSA) 28mMdNTPsmix 1x Thermopol buffer (20mM Tris-HCl pH 88 10mMKCl 2mM MgSO

4 10mM (NH

4)2SO4 01 Triton X-100)

BioMed Research International 3

1 9 17 25 33 41 49 57 65 73 81 89 97 105

113

121

129

137

145

153

161

169

177

Days after infection

9

8

7

6

5

4

3

2

1

0

Log 1

0pa

rasit

aem

iam

Lof

blo

od

Figure 1 Mean daily parasitaemia of monkeys infected with T bgambiense IL3253 CSF there was no CSF parasitosis observed dur-ing the experimental period

(New England Biolabs UK) and additional 4mM MgSO4

8-unit Bst DNA polymerase (New England Biolabs UK)double distilled water and 2 120583L of the template DNA Thepositive and negative controls were similar to those used inthe PCR reactionThe reactions were carried out in triplicatefor 80 minutes in a Loopamp real-time turbidimeter LA320C(Eiken Chemical Co Japan) Increase in turbidity indicatesDNA amplification After the reaction 120 dilution of SYBRgreen I dye (Sigma-Aldrich St Louis MO USA) was addedto confirm the amplification

28 Data Analysis The percentage detection of the differenttests and sample was determined and significant differencesbetween the tests calculated using the chi-square (1205942) testwere determined The differences were considered statisti-cally significant when 119901 lt 005 The agreement between testswas quantified using Cohenrsquos kappa statistic (k) Epicalc ofEpiInfo 7 was used

29 Ethics All protocols and procedures used in this studywere reviewed and approved by the Institute of PrimateResearch (IPR) Institutional ReviewCommittee which incor-porates Animal Care and Use Committee (IACUC) review(IRC1910)

3 Results

31 Parasitological Methods The prepatent period was twoto three days The parasitaemia rose to a peak of 107trypanosomesmL of blood between 8 and 9 days afterinfection (dpi) Thereafter the parasitaemia declined andwas characterized by fluctuations to a minimum 25 times 105trypanosomesmL of blood by 123 dpi However from 123to 180 dpi the parasitaemia dropped to undetectable levels(Figure 1)

32 PCR The positive control (T b gambiense) gave theexpected 308-base-pair (bp) band The negative controls (Tb brucei and T b rhodesiense) were negative (Figure 2)

M87654321

500bp

300bp308bp

Figure 2 PCR results on gels after electrophoresis Lane 1 (Tbb)Lane 2 (Tbr) Lane 3 (positive control Tbg) Lane 4 (saliva sampleobtained on 14 dpi) Lane 5 (saliva sample obtained 28 dpi) Lane6 (saliva sample obtained 56 dpi) Lane 7 (saliva sample obtained70 dpi) Lane 8 (saliva sample obtained 84 dpi) and M (100 bpmolecular marker)

Turb

idity

06

055

05

045

04

035

03

025

02

015

01

005

0

minus005

minus01

000 2400 4800Time (min)

Serum 7dpiSerum 14dpiSerum 21dpiSerum 35dpiSerum 42dpi

ControlsTbb GUTAT1 (negative)Tbr IPR001 (negative)Tbg IL3253 (positive)

Figure 3 Amplification curves after LAMP reaction in turbidime-ter

33 LAMP Increase in turbidity was noted for the positivecontrol and some samples within 48 minutes of incubationThere was no increase in turbidity in the negative controlsas expected (Figure 3) After addition of SYBR green I dyethe positive LAMP reactions turned green while the negativeones remained orange (Figure 4)

34 Comparison between LAMP PCR and ParasitologicalMethods Parasitological methods detected parasites in theinfected monkeys by day 3 after infection (Figure 1) Thedetection rate gradually dropped and by 119 dpi only 50detection was obtained Thereafter the methods could notdetect the parasites Both PCR and LAMP recorded 100detection in serum samples starting from 7 dpi LAMPdetected trypanosome DNA until 180 dpi but maintained100 detection up to 133 dpi On the other hand PCR could

4 BioMed Research International

1 2 3 4 5 6

Figure 4 Visual appearance of LAMP results after addition of SYBRgreen I dye Green color represents positive reaction while orangerepresents negative reaction Tube 1 Tbg positive control Tube 2negative control (Tbr) Tube 3 serum sample obtained 28 dpi Tube4 CSF obtained 28 dpi Tube 5 saliva sample obtained 28 dpi andTube 6 urine sample obtained 28 dpi

Table 1 Detection () of parasitological methods PCR and LAMPin serum saliva and urine determined at weekly time points invervet monkeys infected with T b gambiense

DPI Parasito PCR LAMPSerum Saliva Serum Saliva Urine

7 100 100 17 100 33 014 83 100 83 100 83 1721 100 100 100 100 100 3328 83 100 100 100 100 8335 67 100 100 100 100 10042 83 100 83 100 100 10049 83 100 33 100 100 10056 83 100 17 100 100 10063 83 100 17 100 100 8370 67 100 0 100 100 10077 83 100 0 100 100 8384 67 100 0 100 83 8391 50 83 0 100 83 8398 67 83 0 100 83 67105 67 83 0 100 83 67112 50 83 0 100 83 33119 50 67 0 100 33 17126 0 83 0 100 33 17133 0 67 0 100 17 0140 0 33 0 83 0 0147 0 33 0 83 0 0154 0 17 0 83 0 0161 0 0 0 83 0 0168 0 0 0 67 0 0175 0 0 0 67 0 0180 0 0 0 33 0 0Key Parasito = parasitological techniques DPI = days after infectionlowastThere was no amplification in any CSF sample with either LAMP or PCRThere was also no amplification noted in urine using PCR

only sustain the 100 detection rate up to 84 dpi Thereafterthe detection dropped and from 161ndash180 dpi PCR did notdetect trypanosome DNA (Table 1)

Table 2 Duration of detection of parasites in blood serum salivaurine and CSF samples using parasitological methods PCR andLAMP in vervet monkeys infected with T b gambiense for 180 daysafter infection (dpi)

Sample Parasitology PCR LAMPBlood 3ndash123 dpi (18) mdash mdashSerum mdash 7ndash154 dpi (4) 7ndash180 dpi (3)Saliva mdash 7ndash63 dpi (11) 7ndash133 dpi (5)Urine mdash mdash 14ndash126 dpi (8)CSF mdash mdash mdashlowast(1) The percentage of negative samples is given in brackets

Posit

ive (

)

90

80

70

60

50

40

30

20

10

0

Blood Serum Saliva UrineSample

Parasitological methodsPCRLAMP

Figure 5 Comparison of LAMP PCR and parasitological methodsin trypanosome mean detection rate in serum saliva and urinesamples obtained from monkeys infected with T b gambienseIL3253 from 3 to 180 days after infectionThere was no amplificationin any CSF sample with either LAMP or PCR There was also noamplification noted in urine using PCR

In saliva samples PCR detected trypanosome DNA from7 to 63 dpi thereafter no trypanosome DNA was detectedBetween 21 and 77 dpi LAMP recorded 100 detection inthe saliva samples The detection dropped thereafter andfrom 140 to 180 dpi there was no trypanosome DNA detec-tion LAMP detected trypanosome DNA in urine samplesbetween days 14 and 126 after infection (Table 1) PCR didnot detect trypanosome DNA in the urine samples NeitherPCR nor LAMP detected trypanosome DNA in the CSFsamples Both PCR and LAMP detected parasites beyondthe period when conventional parasitological methods didTrypanosome DNA was detected longest in serum samplesfollowed by saliva and urine (Table 2) The detection rate ofLAMP was higher than that of PCR in the serum saliva andurine samples (Figure 5)

There was a significant difference in detection betweenLAMP and PCR (119901 lt 005) in all the samples Similarly thedifference in detection between LAMP and parasitologicalmethods was significant (119901 lt 005) There was good agree-ment between PCR and parasitological methods in detection

BioMed Research International 5

Table 3 Comparative analysis of LAMP PCR and parasitological techniques in trypanosome detection in serum saliva and urine samplesobtained from monkeys infected with T b gambiense IL3253

Test Kappa value Level of agreement 1205942 statistic 119901 value

PCR and parasitology 083 (036ndash059) Very good 222 0136LAMP and parasitology 048 (036ndash059) Marginal 2647 lt0001LAMP and PCR (serum) 059 (047ndash072) Marginal 1042 00012LAMP and PCR (saliva) 014 (006ndash021) Poor 7979 lt00001LAMP and PCR (urine) 0 Poor 612 00134

of trypanosomes The difference in detection between bothtests was not significant (119901 gt 005) (Table 3)

4 Discussion

The presence of trypanosome DNA in saliva and urinesamples is of great significance given the need for noninvasivesamples for diagnosis ofHATThe higher sensitivity of LAMPcompared to parasitological methods and PCR is also ofsignificance in regard to the search for new diagnostic testsfor sleeping sickness In the current study the performance ofLAMP and PCR was assessed in an early stage HAT model[15]

The high number of trypanosomes positive serum sam-ples suggests that the trypanosomes were circulating inthe hemolymphatic system The lack of detection of try-panosomeDNA in the CSF samples may mean that the try-panosomes did not cross the blood-brain barrier and hencelate stage disease did not occur Infected monkeys withouttrypanosomes in CSF and having WBC counts of less than 5cellsmm3 are regarded as being in early stage of the disease

PCR performed better than the parasitological methodsin monitoring the presence of the infection but however itcannot be used as a gold standard in diagnosis of trypanoso-miasis because of its challenges to implement in clinicalsettingsThe test was able to detect trypanosome infection fora longer duration compared to the parasitological methodswhich are regarded as the gold standard PCR detected try-panosome DNA in the saliva samples during early infectioncorresponding to the period of high parasitaemia The lackof detection in urine samples could be due to the presenceof inhibitors such as urea and uric acid [19] and elevatedacidic conditions that may inactivate the highly sensitiveTaq DNA polymerase enzyme used in PCR reactions Thisenzyme used is also easily inactivated by tissue and bloodderived inhibitors [20ndash22] Similarly in a previous studyon T b rhodesiense trypanosome DNA was not detectedin urine samples of infected vervet monkeys [unpublisheddata] In this study all the CSF samples were negative fortrypanosome DNA although previous studies have shownthat PCR on CSF samples has high sensitivity in staging ofHAT [23] Optimization of reaction conditions is a majorsetback in development of PCR as a diagnostic tool for HATThis has especially been noted in samples from serologicallypositive but aparasitemic patients [9] In addition PCR iscost restrictive due to the need for specialized equipmentsuch as automated thermal cyclers and the presence of cold

chain to preserve reagents Thus recent studies have focusedon development of tests such as LAMP which can overcomesome of these challenges [10 24]

LAMP performed better than PCR and parasitologicalmethods as demonstrated by its higher detection rate Incontrast to PCR LAMP detected trypanosomeDNA in urinesamples in this study Indeed the test detected trypanosomeDNA during periods of low parasitaemia when both PCRand parasitology were negative The significant differencein detection proves that the performance of LAMP wasmarkedly better than that of both parasitological methodsand PCR This is possibly due to the use of Bst polymerasethat unlike Taq polymerase is hardly inhibited by impurities[25]The detection of trypanosomal DNA in urine and salivasamples is of great value because they are noninvasive samplesand are an improvement from the current blood and CSFsamples [26]

Trypanosomes have been found to be present in manyorgans and body fluids There has been demonstrated local-ization of T b brucei in kidney glomeruli of infected rats[27] and T lewisi in kidney capillaries of infected rats [28]Filtration of the parasite or its DNAmay explain the presenceof trypanosomal DNA in the urine of the infected monkeysused in this study However formation of ammonia inexposed urine causes degradation of DNA andmay lower thesensitivity of the tests

Trypanosome DNA was also detected in the saliva sam-ples of the infected monkeys It is possible that parasitescould have seeped into salivary ducts from either the bloodor lymphatic systems Saliva has a higher pH as compared tourine hence reducing the likelihood of DNA deterioration

In this study we targeted TgsGP gene a single gene whichencodes a protein specific to the T b gambiense subspeciesand hence ideal for specific detection of T b gambiense [29]The LAMP detection rate varied and was 100 in some dura-tions depending on the sample usedThe sensitivity appearedto be affected by parasitaemia and hence the amount of DNAin the sample The concentration of the trypanosomal DNAin the sample was however not assessed The highest meandetection rate (789) over the entire 180 days experimentalperiod was lower than obtained using a repetitive element(repetitive insertionmobile element RIME) [30] A repetitiveDNA like RIMEmeans thatmany copies of DNA are availablefor amplification and hence greater sensitivity is expectedHowever it is important to note that in that study 90 sen-sitivity was obtained with the samples from parasitologicalconfirmed patients We recommend that the sensitivity of

6 BioMed Research International

LAMP targeting the TgsGP gene be further evaluated inclinical samples and especially the noninvasive samples suchas urine and saliva There is also promising development inserological tests A number of rapid tests are under clinicalevaluation in many countries [31] Combined use of the rapidserological tests andmolecular techniques such as LAMPwillenhance early diagnosis of HAT in the rural Africa where thedisease is endemic

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was funded by the Institute of Primate Research(IPR) and JKUAT-RPE program The authors are grateful tothe technical assistance provided by Tom Adino and SamsonMutura of IPR

References

[1] J R Franco P P Simarro A Diarra J A Ruiz-Postigo and J GJannin ldquoThe journey towards elimination of gambiense humanAfrican trypanosomiasis not far nor easyrdquo Parasitology vol141 no 6 pp 748ndash760 2014

[2] V Lejon D M Ngoyi M Boelaert and P Buscher ldquoA CATTnegative result after treatment for human african trypanosomi-asis is no indication for curerdquo PLoS Neglected Tropical Diseasesvol 4 no 1 article e590 2010

[3] W O Inojosa I Augusto Z Bisoffi et al ldquoDiagnosing humanAfrican trypanosomiasis in Angola using a card agglutinationtest observational study of active and passive case findingstrategiesrdquo The British Medical Journal vol 332 no 7556 pp1479ndash1481 2006

[4] A Garcia V Jamonneau E Magnus et al ldquoFollow-up ofcard agglutination trypanosomiasis test (CATT) positive butapparently aparasitaemic individuals in Cote drsquoIvoire evidencefor a complex andheterogeneous populationrdquoTropicalMedicineand International Health vol 5 no 11 pp 786ndash793 2000

[5] A Garcıa-Quintanilla J Gonzalez-Martın G Tudo M Espasaand M T J de Anta ldquoSimultaneous identification of Mycobac-terium genus and Mycobacterium tuberculosis complex in clin-ical samples by 51015840-exonuclease fluorogenic PCRrdquo Journal ofClinical Microbiology vol 40 no 12 pp 4646ndash4651 2002

[6] I Malele L Craske C Knight et al ldquoThe use of specific andgeneric primers to identify trypanosome infections of wildtsetse flies in Tanzania by PCRrdquo Infection Genetics and Evolu-tion vol 3 no 4 pp 271ndash279 2003

[7] M Berberof D Perez-Morga and E Pays ldquoA receptor-likeflagellar pocket glycoprotein specific to Trypanosoma bruceigambienserdquoMolecular and Biochemical Parasitology vol 113 no1 pp 127ndash138 2001

[8] N Maina K J Maina P Maser and R Brun ldquoGenotypic andphenotypic characterization of Trypanosoma brucei gambienseisolates from Ibba South Sudan an area of high melarsoproltreatment failure raterdquo Acta Tropica vol 104 no 2-3 pp 84ndash90 2007

[9] P Solano V Jamonneau P NrsquoGuessan et al ldquoComparisonof different DNA preparation protocols for PCR diagnosis of

HumanAfricanTrypanosomosis inCote drsquoIvoirerdquoActaTropicavol 82 no 3 pp 349ndash356 2002

[10] N Kuboki N Inoue T Sakurai et al ldquoLoop-mediated isother-mal amplification for detection of African trypanosomesrdquoJournal of Clinical Microbiology vol 41 no 12 pp 5517ndash55242003

[11] T Notomi H Okayama H Masubuchi et al ldquoLoop-mediatedisothermal amplification of DNArdquo Nucleic Acids Research vol28 no 12 p e63 2000

[12] Z K Njiru A S J Mikosza E Matovu et al ldquoAfricantrypanosomiasis sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification(LAMP) of parasite DNArdquo International Journal for Parasitol-ogy vol 38 no 5 pp 589ndash599 2008

[13] Z K Njiru A S J Mikosza T Armstrong J C Enyaru J MNdungrsquou and A R C Thompson ldquoLoop-mediated isothermalamplification (LAMP) method for rapid detection of Try-panosoma brucei rhodesienserdquo PLoS Neglected Tropical Diseasesvol 2 no 1 article e147 2008

[14] Z K Njiru R Traub J O Ouma J C Enyaru and EMatovu ldquoDetection of group 1 Trypanosoma brucei gambienseby loop-mediated isothermal amplificationrdquo Journal of ClinicalMicrobiology vol 49 no 4 pp 1530ndash1536 2011

[15] B M Gachie J M Kagira S M Karanja M W Waema J MNgotho and N W N Maina ldquoTrypanosoma brucei gambienseinfection in vervet monkeys a potential model for early-stagediseaserdquo Journal of Medical Primatology vol 43 no 2 pp 72ndash77 2014

[16] W J Herbert and W H Lumsden ldquoTrypanosoma brucei arapid lsquomatchingrsquo method for estimating the hostrsquos parasitemrdquoExperimental Parasitology vol 40 no 3 pp 427ndash431 1976

[17] P T Woo ldquoThe haematocrit centrifuge technique for thediagnosis of African trypanosomiasisrdquoActa Tropica vol 27 no4 pp 384ndash386 1970

[18] M Radwanska F Claes S Magez et al ldquoNovel primersequences for polymerase chain reaction-based detection ofTrypanosoma brucei gambienserdquoTheAmerican Journal of Trop-ical Medicine and Hygiene vol 67 no 3 pp 289ndash295 2002

[19] G Khan H O Kangro P J Coates and R B HeathldquoInhibitory effects of urine on the polymerase chain reactionfor Cytomegalovirus DNArdquo Journal of Clinical Pathology vol44 no 5 pp 360ndash365 1991

[20] A Akane K Matsubara H Nakamura S Takahashi and KKimura ldquoIdentification of the heme compound co-purifiedwith deoxyribonucleic acid (DNA) from bloodstains a majorinhibitor of polymerase chain reaction (PCR) amplificationrdquoJournal of Forensic Sciences vol 39 no 2 pp 362ndash372 1994

[21] W A Al-Soud L J Jonsson and P Radstrom ldquoIdentificationand characterization of immunoglobulin G in blood as a majorinhibitor of diagnostic PCRrdquo Journal of Clinical Microbiologyvol 38 no 1 pp 345ndash350 2000

[22] L Belec J Authier M-C Eliezer-Vanerot C Piedouillet A SMohamed and R K Gherardi ldquoMyoglobin as a polymerasechain reaction (pcr) inhibitor a limitation for pcr from skeletalmuscle tissue avoided by the use of Thermus thermophiluspolymeraserdquo Muscle and Nerve vol 21 no 8 pp 1064ndash10671998

[23] S Deborggraeve V Lejon R A Ekangu et al ldquoDiagnosticaccuracy of pcr in gambiense sleeping sickness diagnosisstaging and post-treatment follow-up a 2-year longitudinalstudyrdquo PLoS Neglected Tropical Diseases vol 5 no 2 articlee972 2011

BioMed Research International 7

[24] Z K Njiru ldquoRapid and sensitive detection of human AfricanTrypanosomiasis by loop-mediated isothermal amplificationcombined with a lateral-flow dipstickrdquo Diagnostic Microbiologyand Infectious Disease vol 69 no 2 pp 205ndash209 2011

[25] O M M Thekisoe N Kuboki A Nambota et al ldquoSpecies-specific loop-mediated isothermal amplification (LAMP) fordiagnosis of trypanosomosisrdquo Acta Tropica vol 102 no 3 pp182ndash189 2007

[26] WHOTDR Report of the Scientific Group Meeting on AfricanTrypanosomiasis WHOTDR Seneva Switzerland 2001

[27] J Simaren and M Ogunnaike ldquoUrinary biochemical changeshistopathologic effect of kidney damage observed in ratsinfected with Trypanosoma b bruceirdquo Tropical Medicine andParasitology vol 11 pp 35ndash46 1989

[28] J O Simaren ldquoUltrastructural demonstration and glomerularcytopathology associated with Trypanosoma lewisirdquo in Proceed-ings of the 3rd International Congress of Parasitology vol 3 pp215ndash233 Springer Paris France 1974

[29] W Gibson L Nemetschke and J Ndungrsquou ldquoConservedsequence of the TgsGP gene in Group 1 Trypanosoma bruceigambienserdquo Infection Genetics and Evolution vol 10 no 4 pp453ndash458 2010

[30] P Mitashi E Hasker D M Ngoyi et al ldquoDiagnostic accuracyof loopamp Trypanosoma brucei detection kit for diagnosisof human African trypanosomiasis in clinical samplesrdquo PLoSNeglected Tropical Diseases vol 7 no 10 Article ID e2504 2013

[31] P Buscher P Mertens T Leclipteux et al ldquoSensitivity andspecificity of HAT Sero-K-SeT a rapid diagnostic test for sero-diagnosis of sleeping sickness caused by Trypanosoma bruceigambiense a case-control studyrdquoThe Lancet Global Health vol2 no 6 pp e359ndashe363 2014

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2 BioMed Research International

These limitations imply the need for more sensitive andspecific diagnosis

The amplification of DNA has emerged as one of thediagnostic techniques used in studies of infectious diseases[5] Species specific genes have been used to characterizetrypanosomes [6] The discovery of the T b gambiense-specific glycoprotein (TgsGP) gene that is specific to the T bgambiense subspecies heralded its use as a probe for diagno-sis It is the only subspecies-specific gene for T b gambienseand encodes a 47 kDa VSG-like receptor protein [7] Ampli-fication of this gene using PCR has successfully been used inclinical samples [8] However challenges of the DNA extrac-tion protocolsmay affect diagnosis of trypanosome infections[9] and requirements of expensive automated thermal cyclersmake PCR impractical for adoption in the field [10]

Loop mediated isothermal amplification (LAMP) is per-formed under isothermal conditions and relies on autocy-cling strand displacement DNA synthesis [11] It requiresa simple heating device and is rapid and results are easilyviewed by several detection formats The autocycling reac-tions lead to accumulation of a large amount of the targetDNA and by-products such as magnesium pyrophosphateallowing for rapid detection using varied formats LAMPuses four to six specially designed primers recognizing six toeight regions of the target DNA sequence resulting in a highspecificity It has been used in detection of the Trypanozoonsubgenus [12] T b rhodesiense [13] and recently Group 1 Tb gambiense [14] The test has high sensitivity and specificityand does not require specialized equipment and thismakes ita suitable diagnostic test in resource poor settings and wouldtherefore be ideal diagnosis of neglected diseases such asHAT

The importance of experimental animal models includescontrolled conditions and planned sampling among otherThe vervet monkey (Chlorocebus aethiops) has been devel-oped as amodel for early stageHAT caused byT b gambiense[15] Using this animal model the performance of LAMPbased on the TgsGP gene was assessed in detection of T bgambiense in serum CSF saliva and urine

2 Materials and Methods

21 Trypanosomes Trypanosoma b gambiense isolate IL3253was used in this study It was isolated from a human HATpatient from Sudan in 1982 The isolate was cryopreserved inliquid nitrogen and for infective purposes the parasites weresubinoculated into immunosuppressed donor Swiss miceAt peak parasitemia heart blood was obtained by cardiacpuncture and parasites harvested and diluted to 105mL usingphosphate saline glucose

22 Experimental Animals Six adult vervet monkeys of bothsexes weighing between 20 and 50 kg were used in thisstudy They were trapped from the wild in an area knownto be nonendemic for human trypanosomiasis The animalsunderwent a 90-day quarantine during which they werescreened for zoonotic diseases and treated for ecto- andendoparasites They were also trained for ease of adapta-tion and maintained on commercial pellets (Unga Feeds

Ltd Nairobi Kenya) supplemented with fresh fruits andvegetables Drinking water was provided ad libitum Themonkeys were housed in stainless steel cages at ambient roomtemperatures of 18ndash25∘C under biosafety level II animalholding conditions At the end of the experiment periodthe animals were euthanized by injection with Euthatal (20sodium pentobarbitone Rotexmedica Trittau Germany)via the femoral vein

23 Study Design Six monkeys were infected intravenouslywith approximately 105 trypanosomes in 1mL of phosphatesaline glucose The infected monkeys were monitored fora total period of 180 days after experimental infectionParasitaemia was estimated daily using methods previouslydescribed using the rapid matching method [16] and haema-tocrit centrifuge technique [17]

24 Sample Collection The monkeys were anaesthetized onweekly basis with ketamine hydrochloride (RotexmedicaTrittau Germany) at a dosage of 10mgkg body weight forsample collection The samples were collected before andafter infection on a weekly basis Three mL of blood fromthe femoral artery and 15mL of cerebrospinal fluid (CSF)via lumbar puncture were collected Saliva samples wereobtained by placing swabs under the animals tongue for tenminutes to allow for adequate wetting Thereafter the swabswere placed in dry cryovial tubesThe urine was obtained viaa collection apparatus placed on the bottom of the monkeycage and stored in 50mL falcon tubes This was done in theearly morning prior to sedation All samples were collectedand stored at minus20∘C

25 DNA Extraction DNAwas extracted from serum urineCSF and saliva samples using genomic DNA isolation kits(Zymo Research USA) as per manufacturerrsquos instructions

26 PCR Amplification of T b gambiense-specific glyco-protein (TgsGP) gene was done using primer sequences aspreviously described [18] The PCR reactions (nested) wereperformed as described [8] using 1 120583L of extracted DNA ina 25 120583L reaction mixture The PCR amplification was per-formed by incubating the samples for 15min at 95∘C followedby 45 cycles of 1min at 94∘C 1min at 63∘C and 1min at 72∘Cand a final extension at 72∘C for 10minThereafter amplifiedproducts were analyzed by electrophoresis in 2 agarosegels Gels were stained with ethidium bromide (05120583gmL)(Sigma USA) and viewed under UV illumination Thenegative controls purified DNA from T b brucei GUTAT1and T b rhodesiense IPR001 and distilled water The positivecontrol was purified T b gambiense IL3253 DNA

27 LAMP The TgsGP primers as previously describedwere used [14] The reaction mixture of 25 120583L consisted of40 pmol of the inner primers 5 pmol of the outer primers20 pmol of the loop primers (Inqaba biotec SA) 08Mbetaine (Sigma-Aldrich St LouisMOUSA) 28mMdNTPsmix 1x Thermopol buffer (20mM Tris-HCl pH 88 10mMKCl 2mM MgSO

4 10mM (NH

4)2SO4 01 Triton X-100)

BioMed Research International 3

1 9 17 25 33 41 49 57 65 73 81 89 97 105

113

121

129

137

145

153

161

169

177

Days after infection

9

8

7

6

5

4

3

2

1

0

Log 1

0pa

rasit

aem

iam

Lof

blo

od

Figure 1 Mean daily parasitaemia of monkeys infected with T bgambiense IL3253 CSF there was no CSF parasitosis observed dur-ing the experimental period

(New England Biolabs UK) and additional 4mM MgSO4

8-unit Bst DNA polymerase (New England Biolabs UK)double distilled water and 2 120583L of the template DNA Thepositive and negative controls were similar to those used inthe PCR reactionThe reactions were carried out in triplicatefor 80 minutes in a Loopamp real-time turbidimeter LA320C(Eiken Chemical Co Japan) Increase in turbidity indicatesDNA amplification After the reaction 120 dilution of SYBRgreen I dye (Sigma-Aldrich St Louis MO USA) was addedto confirm the amplification

28 Data Analysis The percentage detection of the differenttests and sample was determined and significant differencesbetween the tests calculated using the chi-square (1205942) testwere determined The differences were considered statisti-cally significant when 119901 lt 005 The agreement between testswas quantified using Cohenrsquos kappa statistic (k) Epicalc ofEpiInfo 7 was used

29 Ethics All protocols and procedures used in this studywere reviewed and approved by the Institute of PrimateResearch (IPR) Institutional ReviewCommittee which incor-porates Animal Care and Use Committee (IACUC) review(IRC1910)

3 Results

31 Parasitological Methods The prepatent period was twoto three days The parasitaemia rose to a peak of 107trypanosomesmL of blood between 8 and 9 days afterinfection (dpi) Thereafter the parasitaemia declined andwas characterized by fluctuations to a minimum 25 times 105trypanosomesmL of blood by 123 dpi However from 123to 180 dpi the parasitaemia dropped to undetectable levels(Figure 1)

32 PCR The positive control (T b gambiense) gave theexpected 308-base-pair (bp) band The negative controls (Tb brucei and T b rhodesiense) were negative (Figure 2)

M87654321

500bp

300bp308bp

Figure 2 PCR results on gels after electrophoresis Lane 1 (Tbb)Lane 2 (Tbr) Lane 3 (positive control Tbg) Lane 4 (saliva sampleobtained on 14 dpi) Lane 5 (saliva sample obtained 28 dpi) Lane6 (saliva sample obtained 56 dpi) Lane 7 (saliva sample obtained70 dpi) Lane 8 (saliva sample obtained 84 dpi) and M (100 bpmolecular marker)

Turb

idity

06

055

05

045

04

035

03

025

02

015

01

005

0

minus005

minus01

000 2400 4800Time (min)

Serum 7dpiSerum 14dpiSerum 21dpiSerum 35dpiSerum 42dpi

ControlsTbb GUTAT1 (negative)Tbr IPR001 (negative)Tbg IL3253 (positive)

Figure 3 Amplification curves after LAMP reaction in turbidime-ter

33 LAMP Increase in turbidity was noted for the positivecontrol and some samples within 48 minutes of incubationThere was no increase in turbidity in the negative controlsas expected (Figure 3) After addition of SYBR green I dyethe positive LAMP reactions turned green while the negativeones remained orange (Figure 4)

34 Comparison between LAMP PCR and ParasitologicalMethods Parasitological methods detected parasites in theinfected monkeys by day 3 after infection (Figure 1) Thedetection rate gradually dropped and by 119 dpi only 50detection was obtained Thereafter the methods could notdetect the parasites Both PCR and LAMP recorded 100detection in serum samples starting from 7 dpi LAMPdetected trypanosome DNA until 180 dpi but maintained100 detection up to 133 dpi On the other hand PCR could

4 BioMed Research International

1 2 3 4 5 6

Figure 4 Visual appearance of LAMP results after addition of SYBRgreen I dye Green color represents positive reaction while orangerepresents negative reaction Tube 1 Tbg positive control Tube 2negative control (Tbr) Tube 3 serum sample obtained 28 dpi Tube4 CSF obtained 28 dpi Tube 5 saliva sample obtained 28 dpi andTube 6 urine sample obtained 28 dpi

Table 1 Detection () of parasitological methods PCR and LAMPin serum saliva and urine determined at weekly time points invervet monkeys infected with T b gambiense

DPI Parasito PCR LAMPSerum Saliva Serum Saliva Urine

7 100 100 17 100 33 014 83 100 83 100 83 1721 100 100 100 100 100 3328 83 100 100 100 100 8335 67 100 100 100 100 10042 83 100 83 100 100 10049 83 100 33 100 100 10056 83 100 17 100 100 10063 83 100 17 100 100 8370 67 100 0 100 100 10077 83 100 0 100 100 8384 67 100 0 100 83 8391 50 83 0 100 83 8398 67 83 0 100 83 67105 67 83 0 100 83 67112 50 83 0 100 83 33119 50 67 0 100 33 17126 0 83 0 100 33 17133 0 67 0 100 17 0140 0 33 0 83 0 0147 0 33 0 83 0 0154 0 17 0 83 0 0161 0 0 0 83 0 0168 0 0 0 67 0 0175 0 0 0 67 0 0180 0 0 0 33 0 0Key Parasito = parasitological techniques DPI = days after infectionlowastThere was no amplification in any CSF sample with either LAMP or PCRThere was also no amplification noted in urine using PCR

only sustain the 100 detection rate up to 84 dpi Thereafterthe detection dropped and from 161ndash180 dpi PCR did notdetect trypanosome DNA (Table 1)

Table 2 Duration of detection of parasites in blood serum salivaurine and CSF samples using parasitological methods PCR andLAMP in vervet monkeys infected with T b gambiense for 180 daysafter infection (dpi)

Sample Parasitology PCR LAMPBlood 3ndash123 dpi (18) mdash mdashSerum mdash 7ndash154 dpi (4) 7ndash180 dpi (3)Saliva mdash 7ndash63 dpi (11) 7ndash133 dpi (5)Urine mdash mdash 14ndash126 dpi (8)CSF mdash mdash mdashlowast(1) The percentage of negative samples is given in brackets

Posit

ive (

)

90

80

70

60

50

40

30

20

10

0

Blood Serum Saliva UrineSample

Parasitological methodsPCRLAMP

Figure 5 Comparison of LAMP PCR and parasitological methodsin trypanosome mean detection rate in serum saliva and urinesamples obtained from monkeys infected with T b gambienseIL3253 from 3 to 180 days after infectionThere was no amplificationin any CSF sample with either LAMP or PCR There was also noamplification noted in urine using PCR

In saliva samples PCR detected trypanosome DNA from7 to 63 dpi thereafter no trypanosome DNA was detectedBetween 21 and 77 dpi LAMP recorded 100 detection inthe saliva samples The detection dropped thereafter andfrom 140 to 180 dpi there was no trypanosome DNA detec-tion LAMP detected trypanosome DNA in urine samplesbetween days 14 and 126 after infection (Table 1) PCR didnot detect trypanosome DNA in the urine samples NeitherPCR nor LAMP detected trypanosome DNA in the CSFsamples Both PCR and LAMP detected parasites beyondthe period when conventional parasitological methods didTrypanosome DNA was detected longest in serum samplesfollowed by saliva and urine (Table 2) The detection rate ofLAMP was higher than that of PCR in the serum saliva andurine samples (Figure 5)

There was a significant difference in detection betweenLAMP and PCR (119901 lt 005) in all the samples Similarly thedifference in detection between LAMP and parasitologicalmethods was significant (119901 lt 005) There was good agree-ment between PCR and parasitological methods in detection

BioMed Research International 5

Table 3 Comparative analysis of LAMP PCR and parasitological techniques in trypanosome detection in serum saliva and urine samplesobtained from monkeys infected with T b gambiense IL3253

Test Kappa value Level of agreement 1205942 statistic 119901 value

PCR and parasitology 083 (036ndash059) Very good 222 0136LAMP and parasitology 048 (036ndash059) Marginal 2647 lt0001LAMP and PCR (serum) 059 (047ndash072) Marginal 1042 00012LAMP and PCR (saliva) 014 (006ndash021) Poor 7979 lt00001LAMP and PCR (urine) 0 Poor 612 00134

of trypanosomes The difference in detection between bothtests was not significant (119901 gt 005) (Table 3)

4 Discussion

The presence of trypanosome DNA in saliva and urinesamples is of great significance given the need for noninvasivesamples for diagnosis ofHATThe higher sensitivity of LAMPcompared to parasitological methods and PCR is also ofsignificance in regard to the search for new diagnostic testsfor sleeping sickness In the current study the performance ofLAMP and PCR was assessed in an early stage HAT model[15]

The high number of trypanosomes positive serum sam-ples suggests that the trypanosomes were circulating inthe hemolymphatic system The lack of detection of try-panosomeDNA in the CSF samples may mean that the try-panosomes did not cross the blood-brain barrier and hencelate stage disease did not occur Infected monkeys withouttrypanosomes in CSF and having WBC counts of less than 5cellsmm3 are regarded as being in early stage of the disease

PCR performed better than the parasitological methodsin monitoring the presence of the infection but however itcannot be used as a gold standard in diagnosis of trypanoso-miasis because of its challenges to implement in clinicalsettingsThe test was able to detect trypanosome infection fora longer duration compared to the parasitological methodswhich are regarded as the gold standard PCR detected try-panosome DNA in the saliva samples during early infectioncorresponding to the period of high parasitaemia The lackof detection in urine samples could be due to the presenceof inhibitors such as urea and uric acid [19] and elevatedacidic conditions that may inactivate the highly sensitiveTaq DNA polymerase enzyme used in PCR reactions Thisenzyme used is also easily inactivated by tissue and bloodderived inhibitors [20ndash22] Similarly in a previous studyon T b rhodesiense trypanosome DNA was not detectedin urine samples of infected vervet monkeys [unpublisheddata] In this study all the CSF samples were negative fortrypanosome DNA although previous studies have shownthat PCR on CSF samples has high sensitivity in staging ofHAT [23] Optimization of reaction conditions is a majorsetback in development of PCR as a diagnostic tool for HATThis has especially been noted in samples from serologicallypositive but aparasitemic patients [9] In addition PCR iscost restrictive due to the need for specialized equipmentsuch as automated thermal cyclers and the presence of cold

chain to preserve reagents Thus recent studies have focusedon development of tests such as LAMP which can overcomesome of these challenges [10 24]

LAMP performed better than PCR and parasitologicalmethods as demonstrated by its higher detection rate Incontrast to PCR LAMP detected trypanosomeDNA in urinesamples in this study Indeed the test detected trypanosomeDNA during periods of low parasitaemia when both PCRand parasitology were negative The significant differencein detection proves that the performance of LAMP wasmarkedly better than that of both parasitological methodsand PCR This is possibly due to the use of Bst polymerasethat unlike Taq polymerase is hardly inhibited by impurities[25]The detection of trypanosomal DNA in urine and salivasamples is of great value because they are noninvasive samplesand are an improvement from the current blood and CSFsamples [26]

Trypanosomes have been found to be present in manyorgans and body fluids There has been demonstrated local-ization of T b brucei in kidney glomeruli of infected rats[27] and T lewisi in kidney capillaries of infected rats [28]Filtration of the parasite or its DNAmay explain the presenceof trypanosomal DNA in the urine of the infected monkeysused in this study However formation of ammonia inexposed urine causes degradation of DNA andmay lower thesensitivity of the tests

Trypanosome DNA was also detected in the saliva sam-ples of the infected monkeys It is possible that parasitescould have seeped into salivary ducts from either the bloodor lymphatic systems Saliva has a higher pH as compared tourine hence reducing the likelihood of DNA deterioration

In this study we targeted TgsGP gene a single gene whichencodes a protein specific to the T b gambiense subspeciesand hence ideal for specific detection of T b gambiense [29]The LAMP detection rate varied and was 100 in some dura-tions depending on the sample usedThe sensitivity appearedto be affected by parasitaemia and hence the amount of DNAin the sample The concentration of the trypanosomal DNAin the sample was however not assessed The highest meandetection rate (789) over the entire 180 days experimentalperiod was lower than obtained using a repetitive element(repetitive insertionmobile element RIME) [30] A repetitiveDNA like RIMEmeans thatmany copies of DNA are availablefor amplification and hence greater sensitivity is expectedHowever it is important to note that in that study 90 sen-sitivity was obtained with the samples from parasitologicalconfirmed patients We recommend that the sensitivity of

6 BioMed Research International

LAMP targeting the TgsGP gene be further evaluated inclinical samples and especially the noninvasive samples suchas urine and saliva There is also promising development inserological tests A number of rapid tests are under clinicalevaluation in many countries [31] Combined use of the rapidserological tests andmolecular techniques such as LAMPwillenhance early diagnosis of HAT in the rural Africa where thedisease is endemic

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was funded by the Institute of Primate Research(IPR) and JKUAT-RPE program The authors are grateful tothe technical assistance provided by Tom Adino and SamsonMutura of IPR

References

[1] J R Franco P P Simarro A Diarra J A Ruiz-Postigo and J GJannin ldquoThe journey towards elimination of gambiense humanAfrican trypanosomiasis not far nor easyrdquo Parasitology vol141 no 6 pp 748ndash760 2014

[2] V Lejon D M Ngoyi M Boelaert and P Buscher ldquoA CATTnegative result after treatment for human african trypanosomi-asis is no indication for curerdquo PLoS Neglected Tropical Diseasesvol 4 no 1 article e590 2010

[3] W O Inojosa I Augusto Z Bisoffi et al ldquoDiagnosing humanAfrican trypanosomiasis in Angola using a card agglutinationtest observational study of active and passive case findingstrategiesrdquo The British Medical Journal vol 332 no 7556 pp1479ndash1481 2006

[4] A Garcia V Jamonneau E Magnus et al ldquoFollow-up ofcard agglutination trypanosomiasis test (CATT) positive butapparently aparasitaemic individuals in Cote drsquoIvoire evidencefor a complex andheterogeneous populationrdquoTropicalMedicineand International Health vol 5 no 11 pp 786ndash793 2000

[5] A Garcıa-Quintanilla J Gonzalez-Martın G Tudo M Espasaand M T J de Anta ldquoSimultaneous identification of Mycobac-terium genus and Mycobacterium tuberculosis complex in clin-ical samples by 51015840-exonuclease fluorogenic PCRrdquo Journal ofClinical Microbiology vol 40 no 12 pp 4646ndash4651 2002

[6] I Malele L Craske C Knight et al ldquoThe use of specific andgeneric primers to identify trypanosome infections of wildtsetse flies in Tanzania by PCRrdquo Infection Genetics and Evolu-tion vol 3 no 4 pp 271ndash279 2003

[7] M Berberof D Perez-Morga and E Pays ldquoA receptor-likeflagellar pocket glycoprotein specific to Trypanosoma bruceigambienserdquoMolecular and Biochemical Parasitology vol 113 no1 pp 127ndash138 2001

[8] N Maina K J Maina P Maser and R Brun ldquoGenotypic andphenotypic characterization of Trypanosoma brucei gambienseisolates from Ibba South Sudan an area of high melarsoproltreatment failure raterdquo Acta Tropica vol 104 no 2-3 pp 84ndash90 2007

[9] P Solano V Jamonneau P NrsquoGuessan et al ldquoComparisonof different DNA preparation protocols for PCR diagnosis of

HumanAfricanTrypanosomosis inCote drsquoIvoirerdquoActaTropicavol 82 no 3 pp 349ndash356 2002

[10] N Kuboki N Inoue T Sakurai et al ldquoLoop-mediated isother-mal amplification for detection of African trypanosomesrdquoJournal of Clinical Microbiology vol 41 no 12 pp 5517ndash55242003

[11] T Notomi H Okayama H Masubuchi et al ldquoLoop-mediatedisothermal amplification of DNArdquo Nucleic Acids Research vol28 no 12 p e63 2000

[12] Z K Njiru A S J Mikosza E Matovu et al ldquoAfricantrypanosomiasis sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification(LAMP) of parasite DNArdquo International Journal for Parasitol-ogy vol 38 no 5 pp 589ndash599 2008

[13] Z K Njiru A S J Mikosza T Armstrong J C Enyaru J MNdungrsquou and A R C Thompson ldquoLoop-mediated isothermalamplification (LAMP) method for rapid detection of Try-panosoma brucei rhodesienserdquo PLoS Neglected Tropical Diseasesvol 2 no 1 article e147 2008

[14] Z K Njiru R Traub J O Ouma J C Enyaru and EMatovu ldquoDetection of group 1 Trypanosoma brucei gambienseby loop-mediated isothermal amplificationrdquo Journal of ClinicalMicrobiology vol 49 no 4 pp 1530ndash1536 2011

[15] B M Gachie J M Kagira S M Karanja M W Waema J MNgotho and N W N Maina ldquoTrypanosoma brucei gambienseinfection in vervet monkeys a potential model for early-stagediseaserdquo Journal of Medical Primatology vol 43 no 2 pp 72ndash77 2014

[16] W J Herbert and W H Lumsden ldquoTrypanosoma brucei arapid lsquomatchingrsquo method for estimating the hostrsquos parasitemrdquoExperimental Parasitology vol 40 no 3 pp 427ndash431 1976

[17] P T Woo ldquoThe haematocrit centrifuge technique for thediagnosis of African trypanosomiasisrdquoActa Tropica vol 27 no4 pp 384ndash386 1970

[18] M Radwanska F Claes S Magez et al ldquoNovel primersequences for polymerase chain reaction-based detection ofTrypanosoma brucei gambienserdquoTheAmerican Journal of Trop-ical Medicine and Hygiene vol 67 no 3 pp 289ndash295 2002

[19] G Khan H O Kangro P J Coates and R B HeathldquoInhibitory effects of urine on the polymerase chain reactionfor Cytomegalovirus DNArdquo Journal of Clinical Pathology vol44 no 5 pp 360ndash365 1991

[20] A Akane K Matsubara H Nakamura S Takahashi and KKimura ldquoIdentification of the heme compound co-purifiedwith deoxyribonucleic acid (DNA) from bloodstains a majorinhibitor of polymerase chain reaction (PCR) amplificationrdquoJournal of Forensic Sciences vol 39 no 2 pp 362ndash372 1994

[21] W A Al-Soud L J Jonsson and P Radstrom ldquoIdentificationand characterization of immunoglobulin G in blood as a majorinhibitor of diagnostic PCRrdquo Journal of Clinical Microbiologyvol 38 no 1 pp 345ndash350 2000

[22] L Belec J Authier M-C Eliezer-Vanerot C Piedouillet A SMohamed and R K Gherardi ldquoMyoglobin as a polymerasechain reaction (pcr) inhibitor a limitation for pcr from skeletalmuscle tissue avoided by the use of Thermus thermophiluspolymeraserdquo Muscle and Nerve vol 21 no 8 pp 1064ndash10671998

[23] S Deborggraeve V Lejon R A Ekangu et al ldquoDiagnosticaccuracy of pcr in gambiense sleeping sickness diagnosisstaging and post-treatment follow-up a 2-year longitudinalstudyrdquo PLoS Neglected Tropical Diseases vol 5 no 2 articlee972 2011

BioMed Research International 7

[24] Z K Njiru ldquoRapid and sensitive detection of human AfricanTrypanosomiasis by loop-mediated isothermal amplificationcombined with a lateral-flow dipstickrdquo Diagnostic Microbiologyand Infectious Disease vol 69 no 2 pp 205ndash209 2011

[25] O M M Thekisoe N Kuboki A Nambota et al ldquoSpecies-specific loop-mediated isothermal amplification (LAMP) fordiagnosis of trypanosomosisrdquo Acta Tropica vol 102 no 3 pp182ndash189 2007

[26] WHOTDR Report of the Scientific Group Meeting on AfricanTrypanosomiasis WHOTDR Seneva Switzerland 2001

[27] J Simaren and M Ogunnaike ldquoUrinary biochemical changeshistopathologic effect of kidney damage observed in ratsinfected with Trypanosoma b bruceirdquo Tropical Medicine andParasitology vol 11 pp 35ndash46 1989

[28] J O Simaren ldquoUltrastructural demonstration and glomerularcytopathology associated with Trypanosoma lewisirdquo in Proceed-ings of the 3rd International Congress of Parasitology vol 3 pp215ndash233 Springer Paris France 1974

[29] W Gibson L Nemetschke and J Ndungrsquou ldquoConservedsequence of the TgsGP gene in Group 1 Trypanosoma bruceigambienserdquo Infection Genetics and Evolution vol 10 no 4 pp453ndash458 2010

[30] P Mitashi E Hasker D M Ngoyi et al ldquoDiagnostic accuracyof loopamp Trypanosoma brucei detection kit for diagnosisof human African trypanosomiasis in clinical samplesrdquo PLoSNeglected Tropical Diseases vol 7 no 10 Article ID e2504 2013

[31] P Buscher P Mertens T Leclipteux et al ldquoSensitivity andspecificity of HAT Sero-K-SeT a rapid diagnostic test for sero-diagnosis of sleeping sickness caused by Trypanosoma bruceigambiense a case-control studyrdquoThe Lancet Global Health vol2 no 6 pp e359ndashe363 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

1 9 17 25 33 41 49 57 65 73 81 89 97 105

113

121

129

137

145

153

161

169

177

Days after infection

9

8

7

6

5

4

3

2

1

0

Log 1

0pa

rasit

aem

iam

Lof

blo

od

Figure 1 Mean daily parasitaemia of monkeys infected with T bgambiense IL3253 CSF there was no CSF parasitosis observed dur-ing the experimental period

(New England Biolabs UK) and additional 4mM MgSO4

8-unit Bst DNA polymerase (New England Biolabs UK)double distilled water and 2 120583L of the template DNA Thepositive and negative controls were similar to those used inthe PCR reactionThe reactions were carried out in triplicatefor 80 minutes in a Loopamp real-time turbidimeter LA320C(Eiken Chemical Co Japan) Increase in turbidity indicatesDNA amplification After the reaction 120 dilution of SYBRgreen I dye (Sigma-Aldrich St Louis MO USA) was addedto confirm the amplification

28 Data Analysis The percentage detection of the differenttests and sample was determined and significant differencesbetween the tests calculated using the chi-square (1205942) testwere determined The differences were considered statisti-cally significant when 119901 lt 005 The agreement between testswas quantified using Cohenrsquos kappa statistic (k) Epicalc ofEpiInfo 7 was used

29 Ethics All protocols and procedures used in this studywere reviewed and approved by the Institute of PrimateResearch (IPR) Institutional ReviewCommittee which incor-porates Animal Care and Use Committee (IACUC) review(IRC1910)

3 Results

31 Parasitological Methods The prepatent period was twoto three days The parasitaemia rose to a peak of 107trypanosomesmL of blood between 8 and 9 days afterinfection (dpi) Thereafter the parasitaemia declined andwas characterized by fluctuations to a minimum 25 times 105trypanosomesmL of blood by 123 dpi However from 123to 180 dpi the parasitaemia dropped to undetectable levels(Figure 1)

32 PCR The positive control (T b gambiense) gave theexpected 308-base-pair (bp) band The negative controls (Tb brucei and T b rhodesiense) were negative (Figure 2)

M87654321

500bp

300bp308bp

Figure 2 PCR results on gels after electrophoresis Lane 1 (Tbb)Lane 2 (Tbr) Lane 3 (positive control Tbg) Lane 4 (saliva sampleobtained on 14 dpi) Lane 5 (saliva sample obtained 28 dpi) Lane6 (saliva sample obtained 56 dpi) Lane 7 (saliva sample obtained70 dpi) Lane 8 (saliva sample obtained 84 dpi) and M (100 bpmolecular marker)

Turb

idity

06

055

05

045

04

035

03

025

02

015

01

005

0

minus005

minus01

000 2400 4800Time (min)

Serum 7dpiSerum 14dpiSerum 21dpiSerum 35dpiSerum 42dpi

ControlsTbb GUTAT1 (negative)Tbr IPR001 (negative)Tbg IL3253 (positive)

Figure 3 Amplification curves after LAMP reaction in turbidime-ter

33 LAMP Increase in turbidity was noted for the positivecontrol and some samples within 48 minutes of incubationThere was no increase in turbidity in the negative controlsas expected (Figure 3) After addition of SYBR green I dyethe positive LAMP reactions turned green while the negativeones remained orange (Figure 4)

34 Comparison between LAMP PCR and ParasitologicalMethods Parasitological methods detected parasites in theinfected monkeys by day 3 after infection (Figure 1) Thedetection rate gradually dropped and by 119 dpi only 50detection was obtained Thereafter the methods could notdetect the parasites Both PCR and LAMP recorded 100detection in serum samples starting from 7 dpi LAMPdetected trypanosome DNA until 180 dpi but maintained100 detection up to 133 dpi On the other hand PCR could

4 BioMed Research International

1 2 3 4 5 6

Figure 4 Visual appearance of LAMP results after addition of SYBRgreen I dye Green color represents positive reaction while orangerepresents negative reaction Tube 1 Tbg positive control Tube 2negative control (Tbr) Tube 3 serum sample obtained 28 dpi Tube4 CSF obtained 28 dpi Tube 5 saliva sample obtained 28 dpi andTube 6 urine sample obtained 28 dpi

Table 1 Detection () of parasitological methods PCR and LAMPin serum saliva and urine determined at weekly time points invervet monkeys infected with T b gambiense

DPI Parasito PCR LAMPSerum Saliva Serum Saliva Urine

7 100 100 17 100 33 014 83 100 83 100 83 1721 100 100 100 100 100 3328 83 100 100 100 100 8335 67 100 100 100 100 10042 83 100 83 100 100 10049 83 100 33 100 100 10056 83 100 17 100 100 10063 83 100 17 100 100 8370 67 100 0 100 100 10077 83 100 0 100 100 8384 67 100 0 100 83 8391 50 83 0 100 83 8398 67 83 0 100 83 67105 67 83 0 100 83 67112 50 83 0 100 83 33119 50 67 0 100 33 17126 0 83 0 100 33 17133 0 67 0 100 17 0140 0 33 0 83 0 0147 0 33 0 83 0 0154 0 17 0 83 0 0161 0 0 0 83 0 0168 0 0 0 67 0 0175 0 0 0 67 0 0180 0 0 0 33 0 0Key Parasito = parasitological techniques DPI = days after infectionlowastThere was no amplification in any CSF sample with either LAMP or PCRThere was also no amplification noted in urine using PCR

only sustain the 100 detection rate up to 84 dpi Thereafterthe detection dropped and from 161ndash180 dpi PCR did notdetect trypanosome DNA (Table 1)

Table 2 Duration of detection of parasites in blood serum salivaurine and CSF samples using parasitological methods PCR andLAMP in vervet monkeys infected with T b gambiense for 180 daysafter infection (dpi)

Sample Parasitology PCR LAMPBlood 3ndash123 dpi (18) mdash mdashSerum mdash 7ndash154 dpi (4) 7ndash180 dpi (3)Saliva mdash 7ndash63 dpi (11) 7ndash133 dpi (5)Urine mdash mdash 14ndash126 dpi (8)CSF mdash mdash mdashlowast(1) The percentage of negative samples is given in brackets

Posit

ive (

)

90

80

70

60

50

40

30

20

10

0

Blood Serum Saliva UrineSample

Parasitological methodsPCRLAMP

Figure 5 Comparison of LAMP PCR and parasitological methodsin trypanosome mean detection rate in serum saliva and urinesamples obtained from monkeys infected with T b gambienseIL3253 from 3 to 180 days after infectionThere was no amplificationin any CSF sample with either LAMP or PCR There was also noamplification noted in urine using PCR

In saliva samples PCR detected trypanosome DNA from7 to 63 dpi thereafter no trypanosome DNA was detectedBetween 21 and 77 dpi LAMP recorded 100 detection inthe saliva samples The detection dropped thereafter andfrom 140 to 180 dpi there was no trypanosome DNA detec-tion LAMP detected trypanosome DNA in urine samplesbetween days 14 and 126 after infection (Table 1) PCR didnot detect trypanosome DNA in the urine samples NeitherPCR nor LAMP detected trypanosome DNA in the CSFsamples Both PCR and LAMP detected parasites beyondthe period when conventional parasitological methods didTrypanosome DNA was detected longest in serum samplesfollowed by saliva and urine (Table 2) The detection rate ofLAMP was higher than that of PCR in the serum saliva andurine samples (Figure 5)

There was a significant difference in detection betweenLAMP and PCR (119901 lt 005) in all the samples Similarly thedifference in detection between LAMP and parasitologicalmethods was significant (119901 lt 005) There was good agree-ment between PCR and parasitological methods in detection

BioMed Research International 5

Table 3 Comparative analysis of LAMP PCR and parasitological techniques in trypanosome detection in serum saliva and urine samplesobtained from monkeys infected with T b gambiense IL3253

Test Kappa value Level of agreement 1205942 statistic 119901 value

PCR and parasitology 083 (036ndash059) Very good 222 0136LAMP and parasitology 048 (036ndash059) Marginal 2647 lt0001LAMP and PCR (serum) 059 (047ndash072) Marginal 1042 00012LAMP and PCR (saliva) 014 (006ndash021) Poor 7979 lt00001LAMP and PCR (urine) 0 Poor 612 00134

of trypanosomes The difference in detection between bothtests was not significant (119901 gt 005) (Table 3)

4 Discussion

The presence of trypanosome DNA in saliva and urinesamples is of great significance given the need for noninvasivesamples for diagnosis ofHATThe higher sensitivity of LAMPcompared to parasitological methods and PCR is also ofsignificance in regard to the search for new diagnostic testsfor sleeping sickness In the current study the performance ofLAMP and PCR was assessed in an early stage HAT model[15]

The high number of trypanosomes positive serum sam-ples suggests that the trypanosomes were circulating inthe hemolymphatic system The lack of detection of try-panosomeDNA in the CSF samples may mean that the try-panosomes did not cross the blood-brain barrier and hencelate stage disease did not occur Infected monkeys withouttrypanosomes in CSF and having WBC counts of less than 5cellsmm3 are regarded as being in early stage of the disease

PCR performed better than the parasitological methodsin monitoring the presence of the infection but however itcannot be used as a gold standard in diagnosis of trypanoso-miasis because of its challenges to implement in clinicalsettingsThe test was able to detect trypanosome infection fora longer duration compared to the parasitological methodswhich are regarded as the gold standard PCR detected try-panosome DNA in the saliva samples during early infectioncorresponding to the period of high parasitaemia The lackof detection in urine samples could be due to the presenceof inhibitors such as urea and uric acid [19] and elevatedacidic conditions that may inactivate the highly sensitiveTaq DNA polymerase enzyme used in PCR reactions Thisenzyme used is also easily inactivated by tissue and bloodderived inhibitors [20ndash22] Similarly in a previous studyon T b rhodesiense trypanosome DNA was not detectedin urine samples of infected vervet monkeys [unpublisheddata] In this study all the CSF samples were negative fortrypanosome DNA although previous studies have shownthat PCR on CSF samples has high sensitivity in staging ofHAT [23] Optimization of reaction conditions is a majorsetback in development of PCR as a diagnostic tool for HATThis has especially been noted in samples from serologicallypositive but aparasitemic patients [9] In addition PCR iscost restrictive due to the need for specialized equipmentsuch as automated thermal cyclers and the presence of cold

chain to preserve reagents Thus recent studies have focusedon development of tests such as LAMP which can overcomesome of these challenges [10 24]

LAMP performed better than PCR and parasitologicalmethods as demonstrated by its higher detection rate Incontrast to PCR LAMP detected trypanosomeDNA in urinesamples in this study Indeed the test detected trypanosomeDNA during periods of low parasitaemia when both PCRand parasitology were negative The significant differencein detection proves that the performance of LAMP wasmarkedly better than that of both parasitological methodsand PCR This is possibly due to the use of Bst polymerasethat unlike Taq polymerase is hardly inhibited by impurities[25]The detection of trypanosomal DNA in urine and salivasamples is of great value because they are noninvasive samplesand are an improvement from the current blood and CSFsamples [26]

Trypanosomes have been found to be present in manyorgans and body fluids There has been demonstrated local-ization of T b brucei in kidney glomeruli of infected rats[27] and T lewisi in kidney capillaries of infected rats [28]Filtration of the parasite or its DNAmay explain the presenceof trypanosomal DNA in the urine of the infected monkeysused in this study However formation of ammonia inexposed urine causes degradation of DNA andmay lower thesensitivity of the tests

Trypanosome DNA was also detected in the saliva sam-ples of the infected monkeys It is possible that parasitescould have seeped into salivary ducts from either the bloodor lymphatic systems Saliva has a higher pH as compared tourine hence reducing the likelihood of DNA deterioration

In this study we targeted TgsGP gene a single gene whichencodes a protein specific to the T b gambiense subspeciesand hence ideal for specific detection of T b gambiense [29]The LAMP detection rate varied and was 100 in some dura-tions depending on the sample usedThe sensitivity appearedto be affected by parasitaemia and hence the amount of DNAin the sample The concentration of the trypanosomal DNAin the sample was however not assessed The highest meandetection rate (789) over the entire 180 days experimentalperiod was lower than obtained using a repetitive element(repetitive insertionmobile element RIME) [30] A repetitiveDNA like RIMEmeans thatmany copies of DNA are availablefor amplification and hence greater sensitivity is expectedHowever it is important to note that in that study 90 sen-sitivity was obtained with the samples from parasitologicalconfirmed patients We recommend that the sensitivity of

6 BioMed Research International

LAMP targeting the TgsGP gene be further evaluated inclinical samples and especially the noninvasive samples suchas urine and saliva There is also promising development inserological tests A number of rapid tests are under clinicalevaluation in many countries [31] Combined use of the rapidserological tests andmolecular techniques such as LAMPwillenhance early diagnosis of HAT in the rural Africa where thedisease is endemic

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was funded by the Institute of Primate Research(IPR) and JKUAT-RPE program The authors are grateful tothe technical assistance provided by Tom Adino and SamsonMutura of IPR

References

[1] J R Franco P P Simarro A Diarra J A Ruiz-Postigo and J GJannin ldquoThe journey towards elimination of gambiense humanAfrican trypanosomiasis not far nor easyrdquo Parasitology vol141 no 6 pp 748ndash760 2014

[2] V Lejon D M Ngoyi M Boelaert and P Buscher ldquoA CATTnegative result after treatment for human african trypanosomi-asis is no indication for curerdquo PLoS Neglected Tropical Diseasesvol 4 no 1 article e590 2010

[3] W O Inojosa I Augusto Z Bisoffi et al ldquoDiagnosing humanAfrican trypanosomiasis in Angola using a card agglutinationtest observational study of active and passive case findingstrategiesrdquo The British Medical Journal vol 332 no 7556 pp1479ndash1481 2006

[4] A Garcia V Jamonneau E Magnus et al ldquoFollow-up ofcard agglutination trypanosomiasis test (CATT) positive butapparently aparasitaemic individuals in Cote drsquoIvoire evidencefor a complex andheterogeneous populationrdquoTropicalMedicineand International Health vol 5 no 11 pp 786ndash793 2000

[5] A Garcıa-Quintanilla J Gonzalez-Martın G Tudo M Espasaand M T J de Anta ldquoSimultaneous identification of Mycobac-terium genus and Mycobacterium tuberculosis complex in clin-ical samples by 51015840-exonuclease fluorogenic PCRrdquo Journal ofClinical Microbiology vol 40 no 12 pp 4646ndash4651 2002

[6] I Malele L Craske C Knight et al ldquoThe use of specific andgeneric primers to identify trypanosome infections of wildtsetse flies in Tanzania by PCRrdquo Infection Genetics and Evolu-tion vol 3 no 4 pp 271ndash279 2003

[7] M Berberof D Perez-Morga and E Pays ldquoA receptor-likeflagellar pocket glycoprotein specific to Trypanosoma bruceigambienserdquoMolecular and Biochemical Parasitology vol 113 no1 pp 127ndash138 2001

[8] N Maina K J Maina P Maser and R Brun ldquoGenotypic andphenotypic characterization of Trypanosoma brucei gambienseisolates from Ibba South Sudan an area of high melarsoproltreatment failure raterdquo Acta Tropica vol 104 no 2-3 pp 84ndash90 2007

[9] P Solano V Jamonneau P NrsquoGuessan et al ldquoComparisonof different DNA preparation protocols for PCR diagnosis of

HumanAfricanTrypanosomosis inCote drsquoIvoirerdquoActaTropicavol 82 no 3 pp 349ndash356 2002

[10] N Kuboki N Inoue T Sakurai et al ldquoLoop-mediated isother-mal amplification for detection of African trypanosomesrdquoJournal of Clinical Microbiology vol 41 no 12 pp 5517ndash55242003

[11] T Notomi H Okayama H Masubuchi et al ldquoLoop-mediatedisothermal amplification of DNArdquo Nucleic Acids Research vol28 no 12 p e63 2000

[12] Z K Njiru A S J Mikosza E Matovu et al ldquoAfricantrypanosomiasis sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification(LAMP) of parasite DNArdquo International Journal for Parasitol-ogy vol 38 no 5 pp 589ndash599 2008

[13] Z K Njiru A S J Mikosza T Armstrong J C Enyaru J MNdungrsquou and A R C Thompson ldquoLoop-mediated isothermalamplification (LAMP) method for rapid detection of Try-panosoma brucei rhodesienserdquo PLoS Neglected Tropical Diseasesvol 2 no 1 article e147 2008

[14] Z K Njiru R Traub J O Ouma J C Enyaru and EMatovu ldquoDetection of group 1 Trypanosoma brucei gambienseby loop-mediated isothermal amplificationrdquo Journal of ClinicalMicrobiology vol 49 no 4 pp 1530ndash1536 2011

[15] B M Gachie J M Kagira S M Karanja M W Waema J MNgotho and N W N Maina ldquoTrypanosoma brucei gambienseinfection in vervet monkeys a potential model for early-stagediseaserdquo Journal of Medical Primatology vol 43 no 2 pp 72ndash77 2014

[16] W J Herbert and W H Lumsden ldquoTrypanosoma brucei arapid lsquomatchingrsquo method for estimating the hostrsquos parasitemrdquoExperimental Parasitology vol 40 no 3 pp 427ndash431 1976

[17] P T Woo ldquoThe haematocrit centrifuge technique for thediagnosis of African trypanosomiasisrdquoActa Tropica vol 27 no4 pp 384ndash386 1970

[18] M Radwanska F Claes S Magez et al ldquoNovel primersequences for polymerase chain reaction-based detection ofTrypanosoma brucei gambienserdquoTheAmerican Journal of Trop-ical Medicine and Hygiene vol 67 no 3 pp 289ndash295 2002

[19] G Khan H O Kangro P J Coates and R B HeathldquoInhibitory effects of urine on the polymerase chain reactionfor Cytomegalovirus DNArdquo Journal of Clinical Pathology vol44 no 5 pp 360ndash365 1991

[20] A Akane K Matsubara H Nakamura S Takahashi and KKimura ldquoIdentification of the heme compound co-purifiedwith deoxyribonucleic acid (DNA) from bloodstains a majorinhibitor of polymerase chain reaction (PCR) amplificationrdquoJournal of Forensic Sciences vol 39 no 2 pp 362ndash372 1994

[21] W A Al-Soud L J Jonsson and P Radstrom ldquoIdentificationand characterization of immunoglobulin G in blood as a majorinhibitor of diagnostic PCRrdquo Journal of Clinical Microbiologyvol 38 no 1 pp 345ndash350 2000

[22] L Belec J Authier M-C Eliezer-Vanerot C Piedouillet A SMohamed and R K Gherardi ldquoMyoglobin as a polymerasechain reaction (pcr) inhibitor a limitation for pcr from skeletalmuscle tissue avoided by the use of Thermus thermophiluspolymeraserdquo Muscle and Nerve vol 21 no 8 pp 1064ndash10671998

[23] S Deborggraeve V Lejon R A Ekangu et al ldquoDiagnosticaccuracy of pcr in gambiense sleeping sickness diagnosisstaging and post-treatment follow-up a 2-year longitudinalstudyrdquo PLoS Neglected Tropical Diseases vol 5 no 2 articlee972 2011

BioMed Research International 7

[24] Z K Njiru ldquoRapid and sensitive detection of human AfricanTrypanosomiasis by loop-mediated isothermal amplificationcombined with a lateral-flow dipstickrdquo Diagnostic Microbiologyand Infectious Disease vol 69 no 2 pp 205ndash209 2011

[25] O M M Thekisoe N Kuboki A Nambota et al ldquoSpecies-specific loop-mediated isothermal amplification (LAMP) fordiagnosis of trypanosomosisrdquo Acta Tropica vol 102 no 3 pp182ndash189 2007

[26] WHOTDR Report of the Scientific Group Meeting on AfricanTrypanosomiasis WHOTDR Seneva Switzerland 2001

[27] J Simaren and M Ogunnaike ldquoUrinary biochemical changeshistopathologic effect of kidney damage observed in ratsinfected with Trypanosoma b bruceirdquo Tropical Medicine andParasitology vol 11 pp 35ndash46 1989

[28] J O Simaren ldquoUltrastructural demonstration and glomerularcytopathology associated with Trypanosoma lewisirdquo in Proceed-ings of the 3rd International Congress of Parasitology vol 3 pp215ndash233 Springer Paris France 1974

[29] W Gibson L Nemetschke and J Ndungrsquou ldquoConservedsequence of the TgsGP gene in Group 1 Trypanosoma bruceigambienserdquo Infection Genetics and Evolution vol 10 no 4 pp453ndash458 2010

[30] P Mitashi E Hasker D M Ngoyi et al ldquoDiagnostic accuracyof loopamp Trypanosoma brucei detection kit for diagnosisof human African trypanosomiasis in clinical samplesrdquo PLoSNeglected Tropical Diseases vol 7 no 10 Article ID e2504 2013

[31] P Buscher P Mertens T Leclipteux et al ldquoSensitivity andspecificity of HAT Sero-K-SeT a rapid diagnostic test for sero-diagnosis of sleeping sickness caused by Trypanosoma bruceigambiense a case-control studyrdquoThe Lancet Global Health vol2 no 6 pp e359ndashe363 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

1 2 3 4 5 6

Figure 4 Visual appearance of LAMP results after addition of SYBRgreen I dye Green color represents positive reaction while orangerepresents negative reaction Tube 1 Tbg positive control Tube 2negative control (Tbr) Tube 3 serum sample obtained 28 dpi Tube4 CSF obtained 28 dpi Tube 5 saliva sample obtained 28 dpi andTube 6 urine sample obtained 28 dpi

Table 1 Detection () of parasitological methods PCR and LAMPin serum saliva and urine determined at weekly time points invervet monkeys infected with T b gambiense

DPI Parasito PCR LAMPSerum Saliva Serum Saliva Urine

7 100 100 17 100 33 014 83 100 83 100 83 1721 100 100 100 100 100 3328 83 100 100 100 100 8335 67 100 100 100 100 10042 83 100 83 100 100 10049 83 100 33 100 100 10056 83 100 17 100 100 10063 83 100 17 100 100 8370 67 100 0 100 100 10077 83 100 0 100 100 8384 67 100 0 100 83 8391 50 83 0 100 83 8398 67 83 0 100 83 67105 67 83 0 100 83 67112 50 83 0 100 83 33119 50 67 0 100 33 17126 0 83 0 100 33 17133 0 67 0 100 17 0140 0 33 0 83 0 0147 0 33 0 83 0 0154 0 17 0 83 0 0161 0 0 0 83 0 0168 0 0 0 67 0 0175 0 0 0 67 0 0180 0 0 0 33 0 0Key Parasito = parasitological techniques DPI = days after infectionlowastThere was no amplification in any CSF sample with either LAMP or PCRThere was also no amplification noted in urine using PCR

only sustain the 100 detection rate up to 84 dpi Thereafterthe detection dropped and from 161ndash180 dpi PCR did notdetect trypanosome DNA (Table 1)

Table 2 Duration of detection of parasites in blood serum salivaurine and CSF samples using parasitological methods PCR andLAMP in vervet monkeys infected with T b gambiense for 180 daysafter infection (dpi)

Sample Parasitology PCR LAMPBlood 3ndash123 dpi (18) mdash mdashSerum mdash 7ndash154 dpi (4) 7ndash180 dpi (3)Saliva mdash 7ndash63 dpi (11) 7ndash133 dpi (5)Urine mdash mdash 14ndash126 dpi (8)CSF mdash mdash mdashlowast(1) The percentage of negative samples is given in brackets

Posit

ive (

)

90

80

70

60

50

40

30

20

10

0

Blood Serum Saliva UrineSample

Parasitological methodsPCRLAMP

Figure 5 Comparison of LAMP PCR and parasitological methodsin trypanosome mean detection rate in serum saliva and urinesamples obtained from monkeys infected with T b gambienseIL3253 from 3 to 180 days after infectionThere was no amplificationin any CSF sample with either LAMP or PCR There was also noamplification noted in urine using PCR

In saliva samples PCR detected trypanosome DNA from7 to 63 dpi thereafter no trypanosome DNA was detectedBetween 21 and 77 dpi LAMP recorded 100 detection inthe saliva samples The detection dropped thereafter andfrom 140 to 180 dpi there was no trypanosome DNA detec-tion LAMP detected trypanosome DNA in urine samplesbetween days 14 and 126 after infection (Table 1) PCR didnot detect trypanosome DNA in the urine samples NeitherPCR nor LAMP detected trypanosome DNA in the CSFsamples Both PCR and LAMP detected parasites beyondthe period when conventional parasitological methods didTrypanosome DNA was detected longest in serum samplesfollowed by saliva and urine (Table 2) The detection rate ofLAMP was higher than that of PCR in the serum saliva andurine samples (Figure 5)

There was a significant difference in detection betweenLAMP and PCR (119901 lt 005) in all the samples Similarly thedifference in detection between LAMP and parasitologicalmethods was significant (119901 lt 005) There was good agree-ment between PCR and parasitological methods in detection

BioMed Research International 5

Table 3 Comparative analysis of LAMP PCR and parasitological techniques in trypanosome detection in serum saliva and urine samplesobtained from monkeys infected with T b gambiense IL3253

Test Kappa value Level of agreement 1205942 statistic 119901 value

PCR and parasitology 083 (036ndash059) Very good 222 0136LAMP and parasitology 048 (036ndash059) Marginal 2647 lt0001LAMP and PCR (serum) 059 (047ndash072) Marginal 1042 00012LAMP and PCR (saliva) 014 (006ndash021) Poor 7979 lt00001LAMP and PCR (urine) 0 Poor 612 00134

of trypanosomes The difference in detection between bothtests was not significant (119901 gt 005) (Table 3)

4 Discussion

The presence of trypanosome DNA in saliva and urinesamples is of great significance given the need for noninvasivesamples for diagnosis ofHATThe higher sensitivity of LAMPcompared to parasitological methods and PCR is also ofsignificance in regard to the search for new diagnostic testsfor sleeping sickness In the current study the performance ofLAMP and PCR was assessed in an early stage HAT model[15]

The high number of trypanosomes positive serum sam-ples suggests that the trypanosomes were circulating inthe hemolymphatic system The lack of detection of try-panosomeDNA in the CSF samples may mean that the try-panosomes did not cross the blood-brain barrier and hencelate stage disease did not occur Infected monkeys withouttrypanosomes in CSF and having WBC counts of less than 5cellsmm3 are regarded as being in early stage of the disease

PCR performed better than the parasitological methodsin monitoring the presence of the infection but however itcannot be used as a gold standard in diagnosis of trypanoso-miasis because of its challenges to implement in clinicalsettingsThe test was able to detect trypanosome infection fora longer duration compared to the parasitological methodswhich are regarded as the gold standard PCR detected try-panosome DNA in the saliva samples during early infectioncorresponding to the period of high parasitaemia The lackof detection in urine samples could be due to the presenceof inhibitors such as urea and uric acid [19] and elevatedacidic conditions that may inactivate the highly sensitiveTaq DNA polymerase enzyme used in PCR reactions Thisenzyme used is also easily inactivated by tissue and bloodderived inhibitors [20ndash22] Similarly in a previous studyon T b rhodesiense trypanosome DNA was not detectedin urine samples of infected vervet monkeys [unpublisheddata] In this study all the CSF samples were negative fortrypanosome DNA although previous studies have shownthat PCR on CSF samples has high sensitivity in staging ofHAT [23] Optimization of reaction conditions is a majorsetback in development of PCR as a diagnostic tool for HATThis has especially been noted in samples from serologicallypositive but aparasitemic patients [9] In addition PCR iscost restrictive due to the need for specialized equipmentsuch as automated thermal cyclers and the presence of cold

chain to preserve reagents Thus recent studies have focusedon development of tests such as LAMP which can overcomesome of these challenges [10 24]

LAMP performed better than PCR and parasitologicalmethods as demonstrated by its higher detection rate Incontrast to PCR LAMP detected trypanosomeDNA in urinesamples in this study Indeed the test detected trypanosomeDNA during periods of low parasitaemia when both PCRand parasitology were negative The significant differencein detection proves that the performance of LAMP wasmarkedly better than that of both parasitological methodsand PCR This is possibly due to the use of Bst polymerasethat unlike Taq polymerase is hardly inhibited by impurities[25]The detection of trypanosomal DNA in urine and salivasamples is of great value because they are noninvasive samplesand are an improvement from the current blood and CSFsamples [26]

Trypanosomes have been found to be present in manyorgans and body fluids There has been demonstrated local-ization of T b brucei in kidney glomeruli of infected rats[27] and T lewisi in kidney capillaries of infected rats [28]Filtration of the parasite or its DNAmay explain the presenceof trypanosomal DNA in the urine of the infected monkeysused in this study However formation of ammonia inexposed urine causes degradation of DNA andmay lower thesensitivity of the tests

Trypanosome DNA was also detected in the saliva sam-ples of the infected monkeys It is possible that parasitescould have seeped into salivary ducts from either the bloodor lymphatic systems Saliva has a higher pH as compared tourine hence reducing the likelihood of DNA deterioration

In this study we targeted TgsGP gene a single gene whichencodes a protein specific to the T b gambiense subspeciesand hence ideal for specific detection of T b gambiense [29]The LAMP detection rate varied and was 100 in some dura-tions depending on the sample usedThe sensitivity appearedto be affected by parasitaemia and hence the amount of DNAin the sample The concentration of the trypanosomal DNAin the sample was however not assessed The highest meandetection rate (789) over the entire 180 days experimentalperiod was lower than obtained using a repetitive element(repetitive insertionmobile element RIME) [30] A repetitiveDNA like RIMEmeans thatmany copies of DNA are availablefor amplification and hence greater sensitivity is expectedHowever it is important to note that in that study 90 sen-sitivity was obtained with the samples from parasitologicalconfirmed patients We recommend that the sensitivity of

6 BioMed Research International

LAMP targeting the TgsGP gene be further evaluated inclinical samples and especially the noninvasive samples suchas urine and saliva There is also promising development inserological tests A number of rapid tests are under clinicalevaluation in many countries [31] Combined use of the rapidserological tests andmolecular techniques such as LAMPwillenhance early diagnosis of HAT in the rural Africa where thedisease is endemic

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was funded by the Institute of Primate Research(IPR) and JKUAT-RPE program The authors are grateful tothe technical assistance provided by Tom Adino and SamsonMutura of IPR

References

[1] J R Franco P P Simarro A Diarra J A Ruiz-Postigo and J GJannin ldquoThe journey towards elimination of gambiense humanAfrican trypanosomiasis not far nor easyrdquo Parasitology vol141 no 6 pp 748ndash760 2014

[2] V Lejon D M Ngoyi M Boelaert and P Buscher ldquoA CATTnegative result after treatment for human african trypanosomi-asis is no indication for curerdquo PLoS Neglected Tropical Diseasesvol 4 no 1 article e590 2010

[3] W O Inojosa I Augusto Z Bisoffi et al ldquoDiagnosing humanAfrican trypanosomiasis in Angola using a card agglutinationtest observational study of active and passive case findingstrategiesrdquo The British Medical Journal vol 332 no 7556 pp1479ndash1481 2006

[4] A Garcia V Jamonneau E Magnus et al ldquoFollow-up ofcard agglutination trypanosomiasis test (CATT) positive butapparently aparasitaemic individuals in Cote drsquoIvoire evidencefor a complex andheterogeneous populationrdquoTropicalMedicineand International Health vol 5 no 11 pp 786ndash793 2000

[5] A Garcıa-Quintanilla J Gonzalez-Martın G Tudo M Espasaand M T J de Anta ldquoSimultaneous identification of Mycobac-terium genus and Mycobacterium tuberculosis complex in clin-ical samples by 51015840-exonuclease fluorogenic PCRrdquo Journal ofClinical Microbiology vol 40 no 12 pp 4646ndash4651 2002

[6] I Malele L Craske C Knight et al ldquoThe use of specific andgeneric primers to identify trypanosome infections of wildtsetse flies in Tanzania by PCRrdquo Infection Genetics and Evolu-tion vol 3 no 4 pp 271ndash279 2003

[7] M Berberof D Perez-Morga and E Pays ldquoA receptor-likeflagellar pocket glycoprotein specific to Trypanosoma bruceigambienserdquoMolecular and Biochemical Parasitology vol 113 no1 pp 127ndash138 2001

[8] N Maina K J Maina P Maser and R Brun ldquoGenotypic andphenotypic characterization of Trypanosoma brucei gambienseisolates from Ibba South Sudan an area of high melarsoproltreatment failure raterdquo Acta Tropica vol 104 no 2-3 pp 84ndash90 2007

[9] P Solano V Jamonneau P NrsquoGuessan et al ldquoComparisonof different DNA preparation protocols for PCR diagnosis of

HumanAfricanTrypanosomosis inCote drsquoIvoirerdquoActaTropicavol 82 no 3 pp 349ndash356 2002

[10] N Kuboki N Inoue T Sakurai et al ldquoLoop-mediated isother-mal amplification for detection of African trypanosomesrdquoJournal of Clinical Microbiology vol 41 no 12 pp 5517ndash55242003

[11] T Notomi H Okayama H Masubuchi et al ldquoLoop-mediatedisothermal amplification of DNArdquo Nucleic Acids Research vol28 no 12 p e63 2000

[12] Z K Njiru A S J Mikosza E Matovu et al ldquoAfricantrypanosomiasis sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification(LAMP) of parasite DNArdquo International Journal for Parasitol-ogy vol 38 no 5 pp 589ndash599 2008

[13] Z K Njiru A S J Mikosza T Armstrong J C Enyaru J MNdungrsquou and A R C Thompson ldquoLoop-mediated isothermalamplification (LAMP) method for rapid detection of Try-panosoma brucei rhodesienserdquo PLoS Neglected Tropical Diseasesvol 2 no 1 article e147 2008

[14] Z K Njiru R Traub J O Ouma J C Enyaru and EMatovu ldquoDetection of group 1 Trypanosoma brucei gambienseby loop-mediated isothermal amplificationrdquo Journal of ClinicalMicrobiology vol 49 no 4 pp 1530ndash1536 2011

[15] B M Gachie J M Kagira S M Karanja M W Waema J MNgotho and N W N Maina ldquoTrypanosoma brucei gambienseinfection in vervet monkeys a potential model for early-stagediseaserdquo Journal of Medical Primatology vol 43 no 2 pp 72ndash77 2014

[16] W J Herbert and W H Lumsden ldquoTrypanosoma brucei arapid lsquomatchingrsquo method for estimating the hostrsquos parasitemrdquoExperimental Parasitology vol 40 no 3 pp 427ndash431 1976

[17] P T Woo ldquoThe haematocrit centrifuge technique for thediagnosis of African trypanosomiasisrdquoActa Tropica vol 27 no4 pp 384ndash386 1970

[18] M Radwanska F Claes S Magez et al ldquoNovel primersequences for polymerase chain reaction-based detection ofTrypanosoma brucei gambienserdquoTheAmerican Journal of Trop-ical Medicine and Hygiene vol 67 no 3 pp 289ndash295 2002

[19] G Khan H O Kangro P J Coates and R B HeathldquoInhibitory effects of urine on the polymerase chain reactionfor Cytomegalovirus DNArdquo Journal of Clinical Pathology vol44 no 5 pp 360ndash365 1991

[20] A Akane K Matsubara H Nakamura S Takahashi and KKimura ldquoIdentification of the heme compound co-purifiedwith deoxyribonucleic acid (DNA) from bloodstains a majorinhibitor of polymerase chain reaction (PCR) amplificationrdquoJournal of Forensic Sciences vol 39 no 2 pp 362ndash372 1994

[21] W A Al-Soud L J Jonsson and P Radstrom ldquoIdentificationand characterization of immunoglobulin G in blood as a majorinhibitor of diagnostic PCRrdquo Journal of Clinical Microbiologyvol 38 no 1 pp 345ndash350 2000

[22] L Belec J Authier M-C Eliezer-Vanerot C Piedouillet A SMohamed and R K Gherardi ldquoMyoglobin as a polymerasechain reaction (pcr) inhibitor a limitation for pcr from skeletalmuscle tissue avoided by the use of Thermus thermophiluspolymeraserdquo Muscle and Nerve vol 21 no 8 pp 1064ndash10671998

[23] S Deborggraeve V Lejon R A Ekangu et al ldquoDiagnosticaccuracy of pcr in gambiense sleeping sickness diagnosisstaging and post-treatment follow-up a 2-year longitudinalstudyrdquo PLoS Neglected Tropical Diseases vol 5 no 2 articlee972 2011

BioMed Research International 7

[24] Z K Njiru ldquoRapid and sensitive detection of human AfricanTrypanosomiasis by loop-mediated isothermal amplificationcombined with a lateral-flow dipstickrdquo Diagnostic Microbiologyand Infectious Disease vol 69 no 2 pp 205ndash209 2011

[25] O M M Thekisoe N Kuboki A Nambota et al ldquoSpecies-specific loop-mediated isothermal amplification (LAMP) fordiagnosis of trypanosomosisrdquo Acta Tropica vol 102 no 3 pp182ndash189 2007

[26] WHOTDR Report of the Scientific Group Meeting on AfricanTrypanosomiasis WHOTDR Seneva Switzerland 2001

[27] J Simaren and M Ogunnaike ldquoUrinary biochemical changeshistopathologic effect of kidney damage observed in ratsinfected with Trypanosoma b bruceirdquo Tropical Medicine andParasitology vol 11 pp 35ndash46 1989

[28] J O Simaren ldquoUltrastructural demonstration and glomerularcytopathology associated with Trypanosoma lewisirdquo in Proceed-ings of the 3rd International Congress of Parasitology vol 3 pp215ndash233 Springer Paris France 1974

[29] W Gibson L Nemetschke and J Ndungrsquou ldquoConservedsequence of the TgsGP gene in Group 1 Trypanosoma bruceigambienserdquo Infection Genetics and Evolution vol 10 no 4 pp453ndash458 2010

[30] P Mitashi E Hasker D M Ngoyi et al ldquoDiagnostic accuracyof loopamp Trypanosoma brucei detection kit for diagnosisof human African trypanosomiasis in clinical samplesrdquo PLoSNeglected Tropical Diseases vol 7 no 10 Article ID e2504 2013

[31] P Buscher P Mertens T Leclipteux et al ldquoSensitivity andspecificity of HAT Sero-K-SeT a rapid diagnostic test for sero-diagnosis of sleeping sickness caused by Trypanosoma bruceigambiense a case-control studyrdquoThe Lancet Global Health vol2 no 6 pp e359ndashe363 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Table 3 Comparative analysis of LAMP PCR and parasitological techniques in trypanosome detection in serum saliva and urine samplesobtained from monkeys infected with T b gambiense IL3253

Test Kappa value Level of agreement 1205942 statistic 119901 value

PCR and parasitology 083 (036ndash059) Very good 222 0136LAMP and parasitology 048 (036ndash059) Marginal 2647 lt0001LAMP and PCR (serum) 059 (047ndash072) Marginal 1042 00012LAMP and PCR (saliva) 014 (006ndash021) Poor 7979 lt00001LAMP and PCR (urine) 0 Poor 612 00134

of trypanosomes The difference in detection between bothtests was not significant (119901 gt 005) (Table 3)

4 Discussion

The presence of trypanosome DNA in saliva and urinesamples is of great significance given the need for noninvasivesamples for diagnosis ofHATThe higher sensitivity of LAMPcompared to parasitological methods and PCR is also ofsignificance in regard to the search for new diagnostic testsfor sleeping sickness In the current study the performance ofLAMP and PCR was assessed in an early stage HAT model[15]

The high number of trypanosomes positive serum sam-ples suggests that the trypanosomes were circulating inthe hemolymphatic system The lack of detection of try-panosomeDNA in the CSF samples may mean that the try-panosomes did not cross the blood-brain barrier and hencelate stage disease did not occur Infected monkeys withouttrypanosomes in CSF and having WBC counts of less than 5cellsmm3 are regarded as being in early stage of the disease

PCR performed better than the parasitological methodsin monitoring the presence of the infection but however itcannot be used as a gold standard in diagnosis of trypanoso-miasis because of its challenges to implement in clinicalsettingsThe test was able to detect trypanosome infection fora longer duration compared to the parasitological methodswhich are regarded as the gold standard PCR detected try-panosome DNA in the saliva samples during early infectioncorresponding to the period of high parasitaemia The lackof detection in urine samples could be due to the presenceof inhibitors such as urea and uric acid [19] and elevatedacidic conditions that may inactivate the highly sensitiveTaq DNA polymerase enzyme used in PCR reactions Thisenzyme used is also easily inactivated by tissue and bloodderived inhibitors [20ndash22] Similarly in a previous studyon T b rhodesiense trypanosome DNA was not detectedin urine samples of infected vervet monkeys [unpublisheddata] In this study all the CSF samples were negative fortrypanosome DNA although previous studies have shownthat PCR on CSF samples has high sensitivity in staging ofHAT [23] Optimization of reaction conditions is a majorsetback in development of PCR as a diagnostic tool for HATThis has especially been noted in samples from serologicallypositive but aparasitemic patients [9] In addition PCR iscost restrictive due to the need for specialized equipmentsuch as automated thermal cyclers and the presence of cold

chain to preserve reagents Thus recent studies have focusedon development of tests such as LAMP which can overcomesome of these challenges [10 24]

LAMP performed better than PCR and parasitologicalmethods as demonstrated by its higher detection rate Incontrast to PCR LAMP detected trypanosomeDNA in urinesamples in this study Indeed the test detected trypanosomeDNA during periods of low parasitaemia when both PCRand parasitology were negative The significant differencein detection proves that the performance of LAMP wasmarkedly better than that of both parasitological methodsand PCR This is possibly due to the use of Bst polymerasethat unlike Taq polymerase is hardly inhibited by impurities[25]The detection of trypanosomal DNA in urine and salivasamples is of great value because they are noninvasive samplesand are an improvement from the current blood and CSFsamples [26]

Trypanosomes have been found to be present in manyorgans and body fluids There has been demonstrated local-ization of T b brucei in kidney glomeruli of infected rats[27] and T lewisi in kidney capillaries of infected rats [28]Filtration of the parasite or its DNAmay explain the presenceof trypanosomal DNA in the urine of the infected monkeysused in this study However formation of ammonia inexposed urine causes degradation of DNA andmay lower thesensitivity of the tests

Trypanosome DNA was also detected in the saliva sam-ples of the infected monkeys It is possible that parasitescould have seeped into salivary ducts from either the bloodor lymphatic systems Saliva has a higher pH as compared tourine hence reducing the likelihood of DNA deterioration

In this study we targeted TgsGP gene a single gene whichencodes a protein specific to the T b gambiense subspeciesand hence ideal for specific detection of T b gambiense [29]The LAMP detection rate varied and was 100 in some dura-tions depending on the sample usedThe sensitivity appearedto be affected by parasitaemia and hence the amount of DNAin the sample The concentration of the trypanosomal DNAin the sample was however not assessed The highest meandetection rate (789) over the entire 180 days experimentalperiod was lower than obtained using a repetitive element(repetitive insertionmobile element RIME) [30] A repetitiveDNA like RIMEmeans thatmany copies of DNA are availablefor amplification and hence greater sensitivity is expectedHowever it is important to note that in that study 90 sen-sitivity was obtained with the samples from parasitologicalconfirmed patients We recommend that the sensitivity of

6 BioMed Research International

LAMP targeting the TgsGP gene be further evaluated inclinical samples and especially the noninvasive samples suchas urine and saliva There is also promising development inserological tests A number of rapid tests are under clinicalevaluation in many countries [31] Combined use of the rapidserological tests andmolecular techniques such as LAMPwillenhance early diagnosis of HAT in the rural Africa where thedisease is endemic

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was funded by the Institute of Primate Research(IPR) and JKUAT-RPE program The authors are grateful tothe technical assistance provided by Tom Adino and SamsonMutura of IPR

References

[1] J R Franco P P Simarro A Diarra J A Ruiz-Postigo and J GJannin ldquoThe journey towards elimination of gambiense humanAfrican trypanosomiasis not far nor easyrdquo Parasitology vol141 no 6 pp 748ndash760 2014

[2] V Lejon D M Ngoyi M Boelaert and P Buscher ldquoA CATTnegative result after treatment for human african trypanosomi-asis is no indication for curerdquo PLoS Neglected Tropical Diseasesvol 4 no 1 article e590 2010

[3] W O Inojosa I Augusto Z Bisoffi et al ldquoDiagnosing humanAfrican trypanosomiasis in Angola using a card agglutinationtest observational study of active and passive case findingstrategiesrdquo The British Medical Journal vol 332 no 7556 pp1479ndash1481 2006

[4] A Garcia V Jamonneau E Magnus et al ldquoFollow-up ofcard agglutination trypanosomiasis test (CATT) positive butapparently aparasitaemic individuals in Cote drsquoIvoire evidencefor a complex andheterogeneous populationrdquoTropicalMedicineand International Health vol 5 no 11 pp 786ndash793 2000

[5] A Garcıa-Quintanilla J Gonzalez-Martın G Tudo M Espasaand M T J de Anta ldquoSimultaneous identification of Mycobac-terium genus and Mycobacterium tuberculosis complex in clin-ical samples by 51015840-exonuclease fluorogenic PCRrdquo Journal ofClinical Microbiology vol 40 no 12 pp 4646ndash4651 2002

[6] I Malele L Craske C Knight et al ldquoThe use of specific andgeneric primers to identify trypanosome infections of wildtsetse flies in Tanzania by PCRrdquo Infection Genetics and Evolu-tion vol 3 no 4 pp 271ndash279 2003

[7] M Berberof D Perez-Morga and E Pays ldquoA receptor-likeflagellar pocket glycoprotein specific to Trypanosoma bruceigambienserdquoMolecular and Biochemical Parasitology vol 113 no1 pp 127ndash138 2001

[8] N Maina K J Maina P Maser and R Brun ldquoGenotypic andphenotypic characterization of Trypanosoma brucei gambienseisolates from Ibba South Sudan an area of high melarsoproltreatment failure raterdquo Acta Tropica vol 104 no 2-3 pp 84ndash90 2007

[9] P Solano V Jamonneau P NrsquoGuessan et al ldquoComparisonof different DNA preparation protocols for PCR diagnosis of

HumanAfricanTrypanosomosis inCote drsquoIvoirerdquoActaTropicavol 82 no 3 pp 349ndash356 2002

[10] N Kuboki N Inoue T Sakurai et al ldquoLoop-mediated isother-mal amplification for detection of African trypanosomesrdquoJournal of Clinical Microbiology vol 41 no 12 pp 5517ndash55242003

[11] T Notomi H Okayama H Masubuchi et al ldquoLoop-mediatedisothermal amplification of DNArdquo Nucleic Acids Research vol28 no 12 p e63 2000

[12] Z K Njiru A S J Mikosza E Matovu et al ldquoAfricantrypanosomiasis sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification(LAMP) of parasite DNArdquo International Journal for Parasitol-ogy vol 38 no 5 pp 589ndash599 2008

[13] Z K Njiru A S J Mikosza T Armstrong J C Enyaru J MNdungrsquou and A R C Thompson ldquoLoop-mediated isothermalamplification (LAMP) method for rapid detection of Try-panosoma brucei rhodesienserdquo PLoS Neglected Tropical Diseasesvol 2 no 1 article e147 2008

[14] Z K Njiru R Traub J O Ouma J C Enyaru and EMatovu ldquoDetection of group 1 Trypanosoma brucei gambienseby loop-mediated isothermal amplificationrdquo Journal of ClinicalMicrobiology vol 49 no 4 pp 1530ndash1536 2011

[15] B M Gachie J M Kagira S M Karanja M W Waema J MNgotho and N W N Maina ldquoTrypanosoma brucei gambienseinfection in vervet monkeys a potential model for early-stagediseaserdquo Journal of Medical Primatology vol 43 no 2 pp 72ndash77 2014

[16] W J Herbert and W H Lumsden ldquoTrypanosoma brucei arapid lsquomatchingrsquo method for estimating the hostrsquos parasitemrdquoExperimental Parasitology vol 40 no 3 pp 427ndash431 1976

[17] P T Woo ldquoThe haematocrit centrifuge technique for thediagnosis of African trypanosomiasisrdquoActa Tropica vol 27 no4 pp 384ndash386 1970

[18] M Radwanska F Claes S Magez et al ldquoNovel primersequences for polymerase chain reaction-based detection ofTrypanosoma brucei gambienserdquoTheAmerican Journal of Trop-ical Medicine and Hygiene vol 67 no 3 pp 289ndash295 2002

[19] G Khan H O Kangro P J Coates and R B HeathldquoInhibitory effects of urine on the polymerase chain reactionfor Cytomegalovirus DNArdquo Journal of Clinical Pathology vol44 no 5 pp 360ndash365 1991

[20] A Akane K Matsubara H Nakamura S Takahashi and KKimura ldquoIdentification of the heme compound co-purifiedwith deoxyribonucleic acid (DNA) from bloodstains a majorinhibitor of polymerase chain reaction (PCR) amplificationrdquoJournal of Forensic Sciences vol 39 no 2 pp 362ndash372 1994

[21] W A Al-Soud L J Jonsson and P Radstrom ldquoIdentificationand characterization of immunoglobulin G in blood as a majorinhibitor of diagnostic PCRrdquo Journal of Clinical Microbiologyvol 38 no 1 pp 345ndash350 2000

[22] L Belec J Authier M-C Eliezer-Vanerot C Piedouillet A SMohamed and R K Gherardi ldquoMyoglobin as a polymerasechain reaction (pcr) inhibitor a limitation for pcr from skeletalmuscle tissue avoided by the use of Thermus thermophiluspolymeraserdquo Muscle and Nerve vol 21 no 8 pp 1064ndash10671998

[23] S Deborggraeve V Lejon R A Ekangu et al ldquoDiagnosticaccuracy of pcr in gambiense sleeping sickness diagnosisstaging and post-treatment follow-up a 2-year longitudinalstudyrdquo PLoS Neglected Tropical Diseases vol 5 no 2 articlee972 2011

BioMed Research International 7

[24] Z K Njiru ldquoRapid and sensitive detection of human AfricanTrypanosomiasis by loop-mediated isothermal amplificationcombined with a lateral-flow dipstickrdquo Diagnostic Microbiologyand Infectious Disease vol 69 no 2 pp 205ndash209 2011

[25] O M M Thekisoe N Kuboki A Nambota et al ldquoSpecies-specific loop-mediated isothermal amplification (LAMP) fordiagnosis of trypanosomosisrdquo Acta Tropica vol 102 no 3 pp182ndash189 2007

[26] WHOTDR Report of the Scientific Group Meeting on AfricanTrypanosomiasis WHOTDR Seneva Switzerland 2001

[27] J Simaren and M Ogunnaike ldquoUrinary biochemical changeshistopathologic effect of kidney damage observed in ratsinfected with Trypanosoma b bruceirdquo Tropical Medicine andParasitology vol 11 pp 35ndash46 1989

[28] J O Simaren ldquoUltrastructural demonstration and glomerularcytopathology associated with Trypanosoma lewisirdquo in Proceed-ings of the 3rd International Congress of Parasitology vol 3 pp215ndash233 Springer Paris France 1974

[29] W Gibson L Nemetschke and J Ndungrsquou ldquoConservedsequence of the TgsGP gene in Group 1 Trypanosoma bruceigambienserdquo Infection Genetics and Evolution vol 10 no 4 pp453ndash458 2010

[30] P Mitashi E Hasker D M Ngoyi et al ldquoDiagnostic accuracyof loopamp Trypanosoma brucei detection kit for diagnosisof human African trypanosomiasis in clinical samplesrdquo PLoSNeglected Tropical Diseases vol 7 no 10 Article ID e2504 2013

[31] P Buscher P Mertens T Leclipteux et al ldquoSensitivity andspecificity of HAT Sero-K-SeT a rapid diagnostic test for sero-diagnosis of sleeping sickness caused by Trypanosoma bruceigambiense a case-control studyrdquoThe Lancet Global Health vol2 no 6 pp e359ndashe363 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

LAMP targeting the TgsGP gene be further evaluated inclinical samples and especially the noninvasive samples suchas urine and saliva There is also promising development inserological tests A number of rapid tests are under clinicalevaluation in many countries [31] Combined use of the rapidserological tests andmolecular techniques such as LAMPwillenhance early diagnosis of HAT in the rural Africa where thedisease is endemic

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was funded by the Institute of Primate Research(IPR) and JKUAT-RPE program The authors are grateful tothe technical assistance provided by Tom Adino and SamsonMutura of IPR

References

[1] J R Franco P P Simarro A Diarra J A Ruiz-Postigo and J GJannin ldquoThe journey towards elimination of gambiense humanAfrican trypanosomiasis not far nor easyrdquo Parasitology vol141 no 6 pp 748ndash760 2014

[2] V Lejon D M Ngoyi M Boelaert and P Buscher ldquoA CATTnegative result after treatment for human african trypanosomi-asis is no indication for curerdquo PLoS Neglected Tropical Diseasesvol 4 no 1 article e590 2010

[3] W O Inojosa I Augusto Z Bisoffi et al ldquoDiagnosing humanAfrican trypanosomiasis in Angola using a card agglutinationtest observational study of active and passive case findingstrategiesrdquo The British Medical Journal vol 332 no 7556 pp1479ndash1481 2006

[4] A Garcia V Jamonneau E Magnus et al ldquoFollow-up ofcard agglutination trypanosomiasis test (CATT) positive butapparently aparasitaemic individuals in Cote drsquoIvoire evidencefor a complex andheterogeneous populationrdquoTropicalMedicineand International Health vol 5 no 11 pp 786ndash793 2000

[5] A Garcıa-Quintanilla J Gonzalez-Martın G Tudo M Espasaand M T J de Anta ldquoSimultaneous identification of Mycobac-terium genus and Mycobacterium tuberculosis complex in clin-ical samples by 51015840-exonuclease fluorogenic PCRrdquo Journal ofClinical Microbiology vol 40 no 12 pp 4646ndash4651 2002

[6] I Malele L Craske C Knight et al ldquoThe use of specific andgeneric primers to identify trypanosome infections of wildtsetse flies in Tanzania by PCRrdquo Infection Genetics and Evolu-tion vol 3 no 4 pp 271ndash279 2003

[7] M Berberof D Perez-Morga and E Pays ldquoA receptor-likeflagellar pocket glycoprotein specific to Trypanosoma bruceigambienserdquoMolecular and Biochemical Parasitology vol 113 no1 pp 127ndash138 2001

[8] N Maina K J Maina P Maser and R Brun ldquoGenotypic andphenotypic characterization of Trypanosoma brucei gambienseisolates from Ibba South Sudan an area of high melarsoproltreatment failure raterdquo Acta Tropica vol 104 no 2-3 pp 84ndash90 2007

[9] P Solano V Jamonneau P NrsquoGuessan et al ldquoComparisonof different DNA preparation protocols for PCR diagnosis of

HumanAfricanTrypanosomosis inCote drsquoIvoirerdquoActaTropicavol 82 no 3 pp 349ndash356 2002

[10] N Kuboki N Inoue T Sakurai et al ldquoLoop-mediated isother-mal amplification for detection of African trypanosomesrdquoJournal of Clinical Microbiology vol 41 no 12 pp 5517ndash55242003

[11] T Notomi H Okayama H Masubuchi et al ldquoLoop-mediatedisothermal amplification of DNArdquo Nucleic Acids Research vol28 no 12 p e63 2000

[12] Z K Njiru A S J Mikosza E Matovu et al ldquoAfricantrypanosomiasis sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification(LAMP) of parasite DNArdquo International Journal for Parasitol-ogy vol 38 no 5 pp 589ndash599 2008

[13] Z K Njiru A S J Mikosza T Armstrong J C Enyaru J MNdungrsquou and A R C Thompson ldquoLoop-mediated isothermalamplification (LAMP) method for rapid detection of Try-panosoma brucei rhodesienserdquo PLoS Neglected Tropical Diseasesvol 2 no 1 article e147 2008

[14] Z K Njiru R Traub J O Ouma J C Enyaru and EMatovu ldquoDetection of group 1 Trypanosoma brucei gambienseby loop-mediated isothermal amplificationrdquo Journal of ClinicalMicrobiology vol 49 no 4 pp 1530ndash1536 2011

[15] B M Gachie J M Kagira S M Karanja M W Waema J MNgotho and N W N Maina ldquoTrypanosoma brucei gambienseinfection in vervet monkeys a potential model for early-stagediseaserdquo Journal of Medical Primatology vol 43 no 2 pp 72ndash77 2014

[16] W J Herbert and W H Lumsden ldquoTrypanosoma brucei arapid lsquomatchingrsquo method for estimating the hostrsquos parasitemrdquoExperimental Parasitology vol 40 no 3 pp 427ndash431 1976

[17] P T Woo ldquoThe haematocrit centrifuge technique for thediagnosis of African trypanosomiasisrdquoActa Tropica vol 27 no4 pp 384ndash386 1970

[18] M Radwanska F Claes S Magez et al ldquoNovel primersequences for polymerase chain reaction-based detection ofTrypanosoma brucei gambienserdquoTheAmerican Journal of Trop-ical Medicine and Hygiene vol 67 no 3 pp 289ndash295 2002

[19] G Khan H O Kangro P J Coates and R B HeathldquoInhibitory effects of urine on the polymerase chain reactionfor Cytomegalovirus DNArdquo Journal of Clinical Pathology vol44 no 5 pp 360ndash365 1991

[20] A Akane K Matsubara H Nakamura S Takahashi and KKimura ldquoIdentification of the heme compound co-purifiedwith deoxyribonucleic acid (DNA) from bloodstains a majorinhibitor of polymerase chain reaction (PCR) amplificationrdquoJournal of Forensic Sciences vol 39 no 2 pp 362ndash372 1994

[21] W A Al-Soud L J Jonsson and P Radstrom ldquoIdentificationand characterization of immunoglobulin G in blood as a majorinhibitor of diagnostic PCRrdquo Journal of Clinical Microbiologyvol 38 no 1 pp 345ndash350 2000

[22] L Belec J Authier M-C Eliezer-Vanerot C Piedouillet A SMohamed and R K Gherardi ldquoMyoglobin as a polymerasechain reaction (pcr) inhibitor a limitation for pcr from skeletalmuscle tissue avoided by the use of Thermus thermophiluspolymeraserdquo Muscle and Nerve vol 21 no 8 pp 1064ndash10671998

[23] S Deborggraeve V Lejon R A Ekangu et al ldquoDiagnosticaccuracy of pcr in gambiense sleeping sickness diagnosisstaging and post-treatment follow-up a 2-year longitudinalstudyrdquo PLoS Neglected Tropical Diseases vol 5 no 2 articlee972 2011

BioMed Research International 7

[24] Z K Njiru ldquoRapid and sensitive detection of human AfricanTrypanosomiasis by loop-mediated isothermal amplificationcombined with a lateral-flow dipstickrdquo Diagnostic Microbiologyand Infectious Disease vol 69 no 2 pp 205ndash209 2011

[25] O M M Thekisoe N Kuboki A Nambota et al ldquoSpecies-specific loop-mediated isothermal amplification (LAMP) fordiagnosis of trypanosomosisrdquo Acta Tropica vol 102 no 3 pp182ndash189 2007

[26] WHOTDR Report of the Scientific Group Meeting on AfricanTrypanosomiasis WHOTDR Seneva Switzerland 2001

[27] J Simaren and M Ogunnaike ldquoUrinary biochemical changeshistopathologic effect of kidney damage observed in ratsinfected with Trypanosoma b bruceirdquo Tropical Medicine andParasitology vol 11 pp 35ndash46 1989

[28] J O Simaren ldquoUltrastructural demonstration and glomerularcytopathology associated with Trypanosoma lewisirdquo in Proceed-ings of the 3rd International Congress of Parasitology vol 3 pp215ndash233 Springer Paris France 1974

[29] W Gibson L Nemetschke and J Ndungrsquou ldquoConservedsequence of the TgsGP gene in Group 1 Trypanosoma bruceigambienserdquo Infection Genetics and Evolution vol 10 no 4 pp453ndash458 2010

[30] P Mitashi E Hasker D M Ngoyi et al ldquoDiagnostic accuracyof loopamp Trypanosoma brucei detection kit for diagnosisof human African trypanosomiasis in clinical samplesrdquo PLoSNeglected Tropical Diseases vol 7 no 10 Article ID e2504 2013

[31] P Buscher P Mertens T Leclipteux et al ldquoSensitivity andspecificity of HAT Sero-K-SeT a rapid diagnostic test for sero-diagnosis of sleeping sickness caused by Trypanosoma bruceigambiense a case-control studyrdquoThe Lancet Global Health vol2 no 6 pp e359ndashe363 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

[24] Z K Njiru ldquoRapid and sensitive detection of human AfricanTrypanosomiasis by loop-mediated isothermal amplificationcombined with a lateral-flow dipstickrdquo Diagnostic Microbiologyand Infectious Disease vol 69 no 2 pp 205ndash209 2011

[25] O M M Thekisoe N Kuboki A Nambota et al ldquoSpecies-specific loop-mediated isothermal amplification (LAMP) fordiagnosis of trypanosomosisrdquo Acta Tropica vol 102 no 3 pp182ndash189 2007

[26] WHOTDR Report of the Scientific Group Meeting on AfricanTrypanosomiasis WHOTDR Seneva Switzerland 2001

[27] J Simaren and M Ogunnaike ldquoUrinary biochemical changeshistopathologic effect of kidney damage observed in ratsinfected with Trypanosoma b bruceirdquo Tropical Medicine andParasitology vol 11 pp 35ndash46 1989

[28] J O Simaren ldquoUltrastructural demonstration and glomerularcytopathology associated with Trypanosoma lewisirdquo in Proceed-ings of the 3rd International Congress of Parasitology vol 3 pp215ndash233 Springer Paris France 1974

[29] W Gibson L Nemetschke and J Ndungrsquou ldquoConservedsequence of the TgsGP gene in Group 1 Trypanosoma bruceigambienserdquo Infection Genetics and Evolution vol 10 no 4 pp453ndash458 2010

[30] P Mitashi E Hasker D M Ngoyi et al ldquoDiagnostic accuracyof loopamp Trypanosoma brucei detection kit for diagnosisof human African trypanosomiasis in clinical samplesrdquo PLoSNeglected Tropical Diseases vol 7 no 10 Article ID e2504 2013

[31] P Buscher P Mertens T Leclipteux et al ldquoSensitivity andspecificity of HAT Sero-K-SeT a rapid diagnostic test for sero-diagnosis of sleeping sickness caused by Trypanosoma bruceigambiense a case-control studyrdquoThe Lancet Global Health vol2 no 6 pp e359ndashe363 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology