A COMPARISON OF THICK SMEARS, QBC MALARIA, PGR AND PATH FALCIPARUM MALARIA® T E S T T R I P
IN PLASMODIUM FALCIPARUM DIAGNOSIS
G A Y E O.*, D I O U F M.* & DIALLO S.*
Summary:
Blood samples from 182 patients presenting at the out-patient clinic in Richard-Toll, Senegal were analysed by Thick smear microscopy, the QBC, PCR and the new dipstick PATH Malaria®
assay which detects the histidine rich protein II antigen of
Plasmodium falciparum. Thick smear microscopy was used as the reference method.
Sensitivity, specificity, predictive positive and negative values were 100 %, 8 3 . 6 %, 9 3 . 4 % and 100 % for QBC respectively;
100 %, 7 2 . 7 %, 8 9 . 4 % and 100 % for PCR; 9 6 %, 9 2 . 7 %, 96.8 % and 9 1 % for the PATH assay. PATH assay failed to detect one positive sample with Plasmodium malariae.
Assays were also compared with regard to the expense of equipment and reagents and speed and ease of use. The rapid PATH assay can be performed with minimal training and may be specially useful in areas where P. falciparum is the predominant malaria species, in epidemic malaria regions, and where skilled microscopy is not readily available.
KEY W O R D S : thick smear, QBC, PCR, PATH falciparum malaria® assay, Histidine Rich Protein 2, Plasmodium falciparum, malaria, Senegal.
T
h e w o r s e n i n g p r o b l e m s o f antimalarial drugs resistance in many settings has led to facilitate early diagnosis and prompt treatment. Micro- scopic examination o f blood smears remains the method o f c h o i c e for diagnosing malaria. Newer technologies that have b e e n evaluated include the polymerase chain reaction PCR (Barker et al, 1992; Mc Laughlin et al, 1 9 8 7 ) , the Q B C malaria assay (Levine et al, 1 9 8 9 ) , assays detecting the Plasmodium falciparum histidine- rich protein 2 ( P f H R P - 2 ) , a w a t e r s o l u b l e p r o t e i n released from parasitized erythrocytes (Howard et al., 1986; Parra et al, 1 9 9 D - T h e s e related assays include the PATH falciparum malaria®, a dipstick antigen cap- ture assay d e v e l o p e d by PATH (Seattle, USA).T h e purpose o f this study was to evaluate the perfor- m a n c e and use o f the PATH Falciparum Malaria® Test
Trip for the d i a g n o s i s o f Plasmodium falciparum malaria c o m p a r e d to three other P. falciparum dia-
gnostic assays, Thick smear microscopy, Q B C and PCR.
* Department of Parasitology, Faculty of Medicine, UCAD, Dakar, Senegal.
Correspondence: Oumar Gaye.
Tel. (221) 825 19 98 - Fax (221) 825 29 52.
E-mail: ogaye@ucad.refer.sn
Résumé : ÉTUDE COMPARATIVE DE LA GOUTTE ÉPAISSE, DU Q B C , DE LA PCR ET DU PATH FALCIPARUM MALARIA® TEST TRIP POUR LE DIAGNOSTIC DU PALUDISME
1 82 prélèvements de sang effectués chez des malades vus en consultation au centre de santé de Richard-Joli au Sénégal ont été analysés par la goutte épaisse, le QBC, la PCR et le PATH Falciparum Malaria9 Test Trip, ce dernier détectant la protéine Histidine II de Plasmodium falciparum. La goutte épaisse est prise comme méthode de référence.
La sensibilité, la spécificité, les valeurs prédictives positive et négative étaient respectivement de 100 %, 83,6 %, 93,4 % et
100 % pour le QBC; 100 %, 72,7 %, 89,4 % et 100 % pour la PCR; 96 %, 92,7 %, 96,8 % et 91 % pour le PATH. Ce dernier n'a pas diagnostiqué l'infection due à Plasmodium malariae.
íes tests ont été comparés également selon les équipements et matériels utilisés, leur rapidité et la facilité de réalisation. Le PATH peut être réalisé après une rapide formation et peut être utile dans les zones où P. falciparum est l'espèce dominante, et où un examen microscopique de qualité n'est pas possible.
MOTS CLÉS : goutte épaisse, QBC, PATH falciparum malaria®, Histidine Rich Protein 2, Plasmodium falciparum, paludisme, Sénégal.
MATERIALS AND METHODS
PATIENTS
F
rom November 1997 to January 1998, 182 patients with malaria symptoms presenting at the out patient clinic in Richard-Toll, in the north o f Senegal, w e r e enrolled in the study. T h e a g e distribu- tion w a s 1-55 years.T h i c k b l o o d e x a m i n a t i o n s w e r e m a d e using b l o o d from a digital puncture. For PCR, PATH assay and Q B C , additional b l o o d (5 m l ) was c o l l e c t e d b y veni- puncture into 10 ml heparinized tubes.
M I C R O S C O P Y
Thick smears were lysed in water and were stained with Giemsa. Numbers o f malaria parasites per 1,000 white cells w e r e c o u n t e d and the average parasitemias w e r e calculated assuming 8,000 white cells per microliter o f blood. For the Q B C malaria analysis, b l o o d was col- lected in acridine orange-coated microhematocrit tubes ( B e c t o n D i c k i n s o n ) , centrifuged in a microhematocrit centrifuge, e x a m i n e d using a 6 0 x oil immersion o b j e c - tive, and samples w e r e scored as positive or negative.
Parasite, 1999, 6, 273-275
Note de recherche 273
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1999063273
G A Y E O., D I O U F M. & DIALLO S.
POLYMERASE CHAIN REACTION: P C R
DNA w a s isolated from 100 pi o f b l o o d by p h e n o l e x t r a c t i o n a n d i s o p r o p a n o l precipitation ( I s o q u i c k m e t h o d ) , then dissolved in 100 pi o f 0.1 T E solution, then diluted in water 1/10. Amplification was per
formed using 3 pi o f diluated DNA in 3 0 pi reaction v o l u m e s , using Perkin Elmer PCR kit with 2.5 mM m a g n e s i u m and a pair o f specific primers from the small subunit ribosomal DNA g e n e . After an initial hea
ting step for two minutes at 9 4 ° , 15 thermal cycles with o n e minute denaturation at 94°, o n e minute annealing at 58° and o n e minute e x t e n s i o n at 7 2 ° followed by 22 additional thermal cycles with 54° hybridization.
Amplification products w e r e resolved using 2 % aga
rose gels and visualised and p h o t o g r a p h e d after stai
ning in ethidium b r o m i d e ( B a r k e r et al., 1 9 9 2 ) . PATH FALCIPARUM MALARIA® ASSAY
T h e test d e v e l o p e d by the Program for Appropiate T e c h n o l o g y in Health (Seattle, USA) is an antigen-cap
ture assay which detects P. falciparum specific histidine- rich protein II (PfHRP-II). T h e assay uses an IgG cap
ture m o n o c l o n a l antibody which is immobilized o n the nitrocellulose m e n b r a n e . O n c e the test strips identified with patients numbers, four drops o f sample buffer were added into e a c h reaction tube and the tubes w e r e placed in reaction stand. For e a c h sample, 5 µl o f w h o l e b l o o d w e r e added onto the test strip, just b e l o w the blue arrows. T h e n the test strips w e r e d r o p p e d into the reaction tube. After 10 minutes 1 ml o f clearing buffer was added. T h e n the test tube was capped, inverted several times. For negative samples only a line appears in the control region. For positive samples a n o ther line a p p e a r e d at the strip's capture region.
D A T A ANALYSIS
Sensitivity, specificity, positive and negative predictive values w e r e calculated using m i c r o s c o p y as the stan
dard method.
RESULTS
I
n this study 182 patients with clinical symptoms o f malaria w e r e e x a m i n e d using thick smear and Q B C m i c r o s c o p i c analyses, the PCR and the n e w PATH Falciparum Malaria® assay. O n e h u n d r e d and twenty s e v e n samples w e r e d e t e c t e d as positive b y e x a m i n a tion o f stained smears at relative parasitemia ranging from 5 0 0 to 1 8 6 , 2 8 6 parasites p e r microliter o f b l o o d . 126 w e r e P. falciparum. O n e w a s P. malariae.Q B C MALARIA ASSAY
O n the 182 samples analyzed b y Q B C , 127 w e r e posi
tive b y b o t h thick s m e a r m i c r o s c o p y and Q B C . Nine
samples w e r e positive b y Q B C and negative b y micro
scopy, and these samples w e r e also positive by PCR analysis. Relative to m i c r o s c o p y , sensitivity o f Q B C assay w a s 100 %, specificity w a s 8 3 . 6 %, the positive predictive value was 93-4 % and the negative predic
tive value was 100 % ( T a b l e I ) .
M i c r o s c o p y * M i c r o s c o p y " T o t a l
Q B C * 127 9 1 3 6
Q B C - 0 4 6 4 6
T o t a l 1 2 7 5 5 1 8 2
T a b l e I. - C o m p a r i s o n o f Q B C t o t h i c k s m e a r m i c r o s c o p y .
P C R
O n e h u n d r e d and twenty s e v e n ( 1 2 7 ) samples w e r e b o t h positive b y m i c r o s c o p y and b y PCR. Fifteen w e r e negative b y m i c r o s c o p y but positive b y PCR w h i c h detects six m o r e positive samples than Q B C .
Using thick smear m i c r o s c o p y as the standard method, the sensitivity o f PCR was 100 %, the specificity 72.7 %, positive predictive value 8 9 - 4 %, negative predictive value 1 0 0 % ( T a b l e I I ) .
M i c r o s c o p y * M i c r o s c o p y- T o t a l
PCR* 1 2 7 15 1 4 2
P C R - 0 4 0 4 0
T o t a l 1 2 - 5 5 1 8 2
T a b l e II. - C o m p a r i s o n o f P C R t o t h i c k s m e a r m i c r o s c o p y .
P A T H ASSAY
Each assay run w a s performed and s c o r e d within ten minutes. Strong positives w e r e identified near the top o f the Test Strip. A positive control dash w a s s e e n at the control region o f all strips. T h e assay w a s e a s y to perform following the manufacturer's instructions, and requires n o e q u i p m e n t . O n e h u n d r e d and twenty t w o samples w e r e d e t e c t e d as positive b y PATH assay. For the five missed samples, all positive b y Q B C and PCR, o n e had parasitemia at 5 0 0 p/pl b l o o d , a n o t h e r o n e was P. malariae and the last three w e r e samples which h a v e b e e n f r e e z e d a n d t h a w e d r e p e a t e d l y . F o u r samples positive b y PATH assay w e r e negative with PCR, Q B C and m i c r o s c o p y using thick smear micro
s c o p y as the standard method. T h e sensitivity o f PATH assay was 9 6 %, specificity was 9 2 . 7 %, the positive predictive value w a s 9 6 . 8 % and the negative predic
tive value w a s 9 1 % ( T a b l e III).
T h e intensities o f the positive PATH reactions w e r e generally correlated with the densities o f parasitemias determined b y thick s m e a r m i c r o s c o p y .
274 Note de recherche Parasite, 1999, 6, 273-275
PLASMODIUM FALCIPARUM DIAGNOSIS
M i c r o s c o p y * M i c r o s c o p y- T o t a l
P A T H * 1 2 2 4 1 2 6
P A T H - 5 51 5 6
T o t a l 127 5S 182
Table III. - Comparison of PATH assay to thick smear microscopy.
DISCUSSION
A
battery o f 182 b l o o d samples from Senegal was analyzed with four different malaria diagnosis assays. T h i s a l l o w e d c o m p a r i s o n s b e t w e e n t e c h n o l o g i e s a n d illustrates features t o b e c o n s i d e r e d w h e n evaluating diagnostic assays.T h e PCR results w e r e repeated twice and consistently d e t e c t e d 15 additional positive samples c o m p a r e d t o thick smear microscopy. It is plausible that, as reported, the PCR is a maximally sensitive assay, but false posi- tive a r e also c o m m o n p r o b l e m s with PCR ( B a r k e r et al, 1 9 9 2 ) . T h r e e positive samples freezed and t h a w e d repeatedly positive b y m i c r o s c o p y w e r e negative with PATH a s s a y ; although histidine II antigene is appa- rently stable, multiple f r e e z e - t h a w e d s a m p l e s m a y d e c r e a s e the sensitivity o f the assay. T h e b l o o d sample with P. malariae w a s s c o r e d negative. This confirms the antigen histidine-rich protein II is specific o f P. fal- ciparum ( H o w a r d et al, 1 9 8 6 ; Parra et al, 1 9 9 1 ) T h e correlation b e t w e e n parasite densities b y thick s m e a r m i c r o s c o p y a n d signal intensity s c o r e s o f t h e PATH assay suggests that this t e c h n i q u e provided a s e m i q u a n t i t a t i v e e s t i m a t i o n o f p a r a s i t e m i a s . E v e n though the detection limit was not determined during this study, w e might consider PATH assay can detect at least 1000 p/ul, a sample with 5 0 0 p/ul having b e e n not detected.
F o u r samples s c o r e d positively b y PATH assay w e r e negative with the other assays. T h e s e patients reported having h a d malaria a n d having b e e n treated. It is k n o w n that histidine II antigen may b e detectable fol- lowing drug treatment e v e n w h e n parasites are n o longer visible in the b l o o d b y m i c r o s c o p y .
Transportation, speed, c o n v e n i e n c e , equipment, labor, and supply costs are often critical factors in malaria dia- gnosis. T h i c k smear m i c r o s c o p y w h e n correctly d o n e is i n e x p e n s i v e with regard to supply costs, a n d c a n detect as low as ten parasites per microliter o f b l o o d , but it requires a g o o d m i c r o s c o p e and a skilled tech- nician. Q B C a n d PCR can provide additional sensiti- vity, w h i c h may b e useful w h e n asymptomatic carrier d e t e c t i o n is important, b u t e q u i p m e n t , l a b o r a n d supply costs are relatively high.
T h e PATH assay d o e s not require the initial e q u i p m e n t cost. T h e antigen detection is simple, rapid, suitable for use b y relatively less-trained personnel and the rea-
gents are relatively stable and portable. Its sensitivity is a little higher than ICT a n d Parasight. A previous study w e carried out n o t e d 8 9 % and 8 6 % respecti- vely for ICT and Parasight ( G a y e et al, 1 9 9 8 ) .
W h e n malaria diagnosis is not readily available as in peripheral areas, and w h e r e e p i d e m i c outbreak is pos- sible as in t h e c a s e with h y p o e n d e m i c a n d seasonal malaria, PATH assay may b e specially useful ( G a y e et al, 1989, 1 9 9 1 , 1 9 9 7 ) . H o w e v e r it is an indirect tool and final diagnosis should b e m a d e with correlation with clinical a n d e p i d e m i o l o g i c a l criterias. S o far, d e p e n d i n g the ratio cost and efficacy, microscopy e x a - mination remains t h e best tool for malaria diagnosis.
ACKNOWLEDGEMENTS
W e thank Dr D.Burgess, Program officer at PATH cor- poration for providing the kit's test.
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Reçu le 4 janvier 1999 Accepté le 18 juin 1999
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