Field evaluation of rapid diagnostic tests for malaria in Yaounde, Cameroon

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  • Acta Tropica 125 (2013) 214 219

    Contents lists available at SciVerse ScienceDirect

    Acta Tropica

    jo ur nal homep age : www.elsev ier .com/ l

    Field evaluation of rapid diagnostic tests for mala

    Rachida Tahara,b,1, Collins Sayangb,c, Vincent Ngane FoumaneRoger Moyou-Somod, Jean Delmontc, Leonardo K. Bascoa,b,,2

    a Institut de Recherche pour le Dveloppement (IRD), Unit Mixte de Recherche 198, Facult de Mdecine La b Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la lutte contre les Endmiesc Centre de Formation et de Recherche en Mdecine et Sant Tropicale, Facult de Mdecine Nord, Boulevardd Department o d I, Y

    a r t i c l e i n f o

    Article history:Received 6 December 2011Received in revised form29 SeptemberAccepted 9 OcAvailable onlin

    Keywords:MalariaPlasmodium faPlasmodium mDrug resistancDiagnosisAtovaquone

    a b s t r a c t

    Rapid diagnostic tests (RDTs) are affordable, alternative diagnostic tools. The present study aimed toevaluate RDTs available in Cameroon and compare their characteristics to follow the parasitological

    Abbreviatiorich protein 2;WBC, white bl

    CorresponBoulevard ChaTel.: +33 4911

    E-mail add1 Present ad

    le DveloppemFacult de Pha

    2 Present addes Armes (IR(IMTSSA), AllMarseille, Fran

    0001-706X/$ http://dx.doi.o 2012tober 2012e 17 October 2012

    lciparumalariaee

    response of patients for 28 days. Malaria diagnosis was assessed in 179 febrile patients using conventionalmicroscopy as the reference method. Parascreen detects both Plasmodium falciparum-specic histidine-rich protein 2 (Pf HRP-2) and Pan-specic plasmodial lactate dehydrogenase (pLDH) in all four humanPlasmodium spp. Diaspot is based on the detection of Pf HRP-2. OptiMAL-IT (pLDH specic for P. falciparumand pLDH for all four human Plasmodium spp.) was assessed for comparison. The reliability of RDTs wasevaluated by calculating the sensitivity, specicity, positive predictive value, negative predictive value,false-positive rate, false-negative rate, and likelihood ratio. The clinical outcome of 18 children treatedwith atovaquoneproguanil and followed for 28 days was evaluated using microscopy and RDTs. Of179 samples, 133 (74.3%) were pure P. falciparum-positive smears, 4 (2.2%) pure P. malariae-positivesmears, and 42 (23.5%) negative smears. Parascreen and Diaspot had high sensitivity (>92%) and positivepredictive values (>94%). The specicities for Parascreen and Diaspot were 81.0% and 90.5%, respectively.The false-positive rates and the false-negative rates were 19.0% and 4.5% for Parascreen and 9.5% and 8.3%for Diaspot, respectively. Most false-negatives occurred in samples with low parasitaemia (500 asexual parasites/L).Four pure P. malariae were only detected by the pan-Plasmodium bands of Parascreen and OptiMAL-IT. Inblood samples from patients treated and followed-up for 28 days, HRP2-based RDTs remained positivein most samples until Day 28. Despite negative smears, OptiMAL-IT remained positive in several patientsuntil Day 7 but was negative in all patients from Day 14 onwards. RDTs can improve the management offebrile patients. The validity, ease of use, and cost of HRP2-based tests were comparable. However, oneof the current weaknesses of the RDT-based strategy using the tests available in Cameroon is inadequatesensitivity for low parasitaemia. In some cases, RDT results may require correct interpretation based onclinical history, clinical examination, and microscopic diagnosis.

    2012 Elsevier B.V. All rights reserved.

    ns: ACT, artemisinin-based combination therapy; HRP2, histidine- pLDH, plasmodial lactate dehydrogenase; RDT, rapid diagnostic test;ood cell.ding author at: Institut de Recherche Biomdicale des Armes (IRBA),rles Livon, Parc du Pharo, 13262 Marseille cedex 07, France.50149; fax: +33 491150164.ress: lkbasco@yahoo.fr (L.K. Basco).dress: Unit Mixte de Recherche 216, Institut de Recherche pourent (IRD) Universit Paris Descartes, Laboratoire de Parasitologie,rmacie, 4 avenue de lObservatoire, 75270 Paris cedex 06, France.dress: Laboratoire de Parasitologie, Institut de Recherche BiomdicaleBA), Institut de Mdecine Tropicale du Service de Sant des Armese du mdecin colonel Eugne Jamot, Parc du Pharo, BP60109, 13262ce.

    1. Introduction

    The attainment of the global goal to reduce morbidity andmortality due to malaria depends on prompt diagnosis andearly treatment of patients (World Health Organization [WHO],2000a). At present, a large majority of patients in Africa arediagnosed to have malaria on the basis of clinical signs and symp-toms. Presumptive clinical diagnosis is highly unreliable sincethe differential diagnosis of malaria includes many febrile ill-nesses of viral and bacterial origin. Moreover, as the majorityof sub-Saharan African countries, including Cameroon, resort toartemisinin-based combination therapy (ACT), which are muchmore expensive than synthetic compounds like chloroquine andsulfadoxinepyrimethamine and limited in quantity due to thenecessity to extract artemisinin from Artemisia annua, a reliable

    see front matter 2012 Elsevier B.V. All rights reserved.rg/10.1016/j.actatropica.2012.10.002f Infectious Diseases, Faculty of Medicine and Biomedical Sciences, University of Yaounocate /ac ta t ropica

    ria in Yaounde, Cameroonb, Georges Soulac,

    Timone, Universit de la Mditerrane, 13385 Marseille, France en Afrique Centrale (OCEAC), B. P. 288, Yaound, Cameroon

    Dramard, 13015 Marseille, Franceaound, Cameroon

  • R. Tahar et al. / Acta Tropica 125 (2013) 214 219 215

    and rapid laboratory diagnosis of malaria will improve the costand effectiveness of the management of malaria-infected patients(Sayang et al., 2009).

    Microscopic examination of Giemsa-stained blood smears isthe gold staintensive anwith numetion to resupossess a mworking conscarce. Highlacking in diagnostic tin the abserequire a drmalaria-spePlasmodiumothers are srich proteinpv pLDH byRDTs detecspecies, andiae).

    Our prevtreatment wYaounde (Simportanceconducted local commcompared tsary and coof patients

    2. Patients

    2.1. Patient

    2.1.1. GrouAll febril

    within the ary dispenswere enrollconsent. Gipricked capparasite desites per L8000/L. In200 WBC), pwas considagainst 500scopists, anfrom two cthe parasitwith a posiartesunate10 mg/kg/dsecond-linemalarial tre

    2.1.2. Groufollow-up

    Eighteenan informeThe inclusitocol were

    parasitaemia between 2000 and 200,000 asexual parasites/L, easyaccess to the dispensary, absence of malnutrition and danger signsof severe and complicated falciparum malaria, and absence ofhistory of allergic reactions to antimalarial drugs (WHO, 2003).

    18 pquonays)

    undtain

    andent fquat03 Wd tother

    Caminist

    T

    o RDoon dapid 5003raphrum

    P. viv Bioy Asmmudiumae) pthe Pias) oase int buons waddi

    Camessiempauoned ines/at dep. LDono

    threetrucacturd tesring

    as ev was

    ata in

    gold thihaved. P) or sult visibban% condard for malaria diagnosis. The procedure is labour-d requires at least 1 h (usually more in a health centrerous suspected cases of malaria) from blood collec-lts. Moreover, many health facilities in Africa do noticroscope, and even if there is a microscope in gooddition, well-trained and experienced microscopists are-quality Giemsa stain and immersion oil may also be

    peripheral health centres. Within this context, rapidests (RDTs) are affordable alternative diagnostic toolsnce of a microscope (WHO, 2000b; Long, 2009). RDTsop of ngerpricked capillary blood (520 L) to detectcic proteins, some of which are specic to the genus

    (pan plasmodial lactate dehydrogenase, pLDH), whilepecically expressed by a given species (e.g. histidine-

    2 [HRP2] or pf pLDH by Plasmodium falciparum and P. vivax) by immunochromatographic methods. Somet P. falciparum, with or without the other Plasmodium

    non-P. falciparum (i.e. P. vivax, P. ovale, and/or P. malar-

    ious study showed the effectiveness of the RDT-basedith ACT over the presumptive treatment approach in

    ayang et al., 2009). This survey also demonstrated the of the characteristics of RDTs. The present study waswith the aim to evaluate two RDTs available throughercial suppliers and one RDT imported from Europe. Wehe diagnostic characteristics of these RDTs in a dispen-mpared them to follow-up the parasitological responsefor 28 days.

    , materials and methods

    s

    p 1, patients enrolled for RDT onlye adults (no age limits) and children (or history of feverpast 48 h) attending the Nlongkak Catholic mission-ary, Yaounde, for presumptive uncomplicated malaria,ed in the study in 20082009 after a written informedemsa-stained blood smear was prepared from nger-illary blood and examined under light microscope. Thensity was expressed as the number of asexual para-

    of blood, assuming white blood cell (WBC) density of case of low parasitaemia (i.e. 15% discordant results. Patientstive blood smear were treated with a 3-day regimen ofamodiaquine (artesunate, 4 mg/kg/day + amodiaquine,ay) as the rst-line and artemetherlumefantrine as

    treatment, according to the current Cameroonian anti-atment guidelines.

    p 2, patients enrolled for RDT evaluation for clinical

    children aged less than 5 years were enrolled afterd written consent of the parents or legal guardians.on criteria as recommended by the 2003 WHO pro-as follows: rectal temperature 38.0 C, P. falciparum

    These (atovafor 3 disteredwas obinationtreatmor adethe 20responartemeby thenian M

    2.2. RD

    TwCamerarum rMAL70matogfalcipadetectZephyrpensaris an iPlasmomalarieither sitaemlatter cis abseinfecti

    In able inAG, Crfor coatovaqassesseparasittest thdium susing m

    All ers insmanufnel, anRDT duRDT wresults

    2.3. D

    Thestainedstudy is vali(HRP2the rewere single ing 95atients were treated with oral atovaquoneproguanile, 20 mg/kg/day, +proguanil, 8 mg/kg/day, once daily

    and followed-up for 28 days. Each dose was admin-er medical supervision. Fingerpricked capillary blooded on Days 0, 2, 3, 7, 14, 21, and 28 for blood exam-

    RDTs. The clinical outcomes were classied as earlyailure, late clinical failure, late parasitological failure,e clinical and parasitological response, according toHO denitions (WHO, 2003). Patients who failed to

    atovaquoneproguanil treatment were treated withlumefantrine. This study was reviewed and approvederoonian national ethics committee and the Cameroo-ry of Public Health.

    Ts that are commercially available in Yaound,uring the study period were assessed. DiaSpot P. falcip-

    test device (batch numbers MAL6090104, MAL6110020,0; Acumen Diagnostics Inc., Livermore, CA) is a chro-

    ic immunoassay for the qualitative detection of the P.HRP2 in whole blood within 15 min. This RDT does notax, P. malariae, or P. ovale. Parascreen (batch no. 101098;medicals, Goa, India; distributed by International Dis-sociation Foundation, Amsterdam, The Netherlands)noassay that detects P. falciparum-specic HRP2 and

    genus-specic (P. falciparum, P. vivax, P. ovale, and P.LDH within 15 min. In the presence of P. falciparum,f HRP2 line alone (usually in the presence of low para-r two bands on the Pf and Pan lines may appear. The

    ndicates P. falciparum or mixed infection. If P. falciparumt one of the three other Plasmodium species (or mixedithout P. falciparum) is present, the Pan band is visible.tion to these two RDT devices that are avail-eroon, OptiMAL-IT (batch no. 46110.23.01; DiaMedr sur Morat, Switzerland) was ordered in Europerison, mostly in patients who were treated withproguanil and followed for 28 days. This RDT was also

    blood samples with low parasitaemia 500 asexualL of blood. OptiMAL-IT is an immunochromatographictects P. falciparum-specic LDH (Pf band) and Plasmo-H (P band for all 4 human Plasmodium spp. LDH species)clonal antibodies within 20 min.

    RDTs were performed according to the manufactur-tions. Two- to three-page users guides provided by theers in French were distributed to the health person-t procedures were explained and demonstrated for each

    a 1-h training session. The ease of application of eachaluated during a feedback session. The interpretation of

    controlled by one or two experienced researchers.

    terpretation and analysis

    standard was microscopic examination of Giemsa-ck blood smears. All RDTs assessed in the present

    an internal control that ensures that the testarascreen RDT was interpreted as positive if PfPan band (LDH) (or both) was visible. Likewise,of OptiMAL-IT was positive if Pf and/or P band(s)le. Diaspot was interpreted as positive when ad was visible. The performance of RDTs, includ-ndence intervals, was assessed by calculating the

  • 216 R. Tahar et al. / Acta Tropica 125 (2013) 214 219

    Table 1Comparison of results obtained with rapid diagnostic tests (RDT).

    RDT Microscopy

    Positive P.falciparum

    Positive pure P.malariae

    Negative

    DiaspotPositive 122 0 4Negative 11 4 38

    ParascreenPositive 127 4 (Pan positive, Pf

    negative)8

    Negative 6 0 34OptiMAL-IT

    Positive 15 1(Pan positive, Pfnegative)

    1

    Negative 9 0 13

    All 179 blood samples were tested in parallel with Diaspot and Parascreen. Of thesesamples, 39 were tested with OptiMAL-IT in samples with low parasitaemia. Pf, P.falciparum; Pa

    sensitivity, tive value, s [positive tpatients witnegative raperson whomalaria), anInStat (Grapof parasitaeunpaired t-tfrom the panicance w

    3. Results

    3.1. Compa

    A total performancfalciparum-smears, anfalciparum-9420 asexuparasites/ranged fromwere not denever been

    Parascrelar positivemalariae samsamples wi430, 3890,

    yielded a positive P band and a negative Pan band with Parascreen(33, 126 [presence of gametocytes], 250, 3890, and 5870 asexualparasites/L), while six showed negative P and Pan bands withParascreen (45, 48, 61, 155, 212, 430 asexual parasites/L). Threefalse-positives were observed with both Diaspot and Parascreenin the same samples. Five additional false-positives were obtainedwith Parascreen (positive P band and negative Pan band), butnot with Diaspot. One of these ve patients with a false-positiveresult started self-medication with artesunateamodiaquine 24 hbefore consultation. Another patient had a negative smear for asex-ual parasites but carried P. falciparum gametocytes (Diaspot andOptiMAL-IT negative). For the remaining patients, the false-positiveresult was unexplained. Three patients carried P. falciparum game-tocytes and asexual parasites: (i) 668 asexual parasites/L, Diaspotpositive, Parascreen Pan and Pf bands positive, OptiMAL-IT notdone; (ii) 126 asexual parasites/L, Diaspot negative, ParascreenPan band negative, Parascreen Pf band positive, OptiMAL-IT neg-ative; and (iii) 92 asexual parasites/L, Diaspot positive, Parascreen

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