Giovanni Di Per&, Pier0 Olliaro2, Stefano Nardi3, Benedetta AllegranzP,. Roberto ... Lanzafamel, Angelo Cazzadoril, Stefano Bonora' and Ercole Concial.
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(1997) 91,403%405
The ParaSight’” -F rapid dipstick antigen capture assay for monitoring clearance after drug treatment of Plasmodium fafciparum malaria Giovanni Di Per&, Ventol, Massimiliano Immunology
Pier0 Olliaro2, Stefano Nardi3, Benedetta Lanzafamel, Angelo Cazzadoril, Stefano
and Infectious
for Research and Training
Diseases of the University oflerona,Veronaj in Tropical Diseases, Geneva, Switzerland;
parasite
AllegranzP, Roberto Deganello3, Sandro ‘Institute of Bonora’ and Ercole Concial
Italy; 2UNDPlWorld BanklWHO Kiremba Hospital, Ngozi I%vince,
Special Programme Burundi
Keywords:
malaria, Plasmodium fulciparum, diagnosis, dipstick antigen capture assay,faraSightTM-F test
Introduction The role of laboratory diagnosis of malaria is primarily to support clinical care (WHO, 1992). Light microscopical examination is the mainstay of malaria diagnosis. It is relatively simple and has low direct costs, and can theoretically be carried out at the periphery of the health system, but its reliability and cost-effectiveness are questionable (PAYNE 1988; WHO, 1988): it is rather lengthy (thus potentially delaying the initiation of treatment), needs trained personnel, and has significant start LID and maintenance costs. The availabilitv of new, cost-ef?ective techniques is crucial for the implementation of the Global Strateav for Malaria Control (WHO. 1993). To obviate the problems of standard microscopical examination, several other techniques have been tested. Some focus on improved microscopy-e.g., the quantitative huffy coat (QBCTM) method (RICKMAN et al., 1989) and fluorochrome techniques (KAWAMOTO, 199 1). without solvina the eauinment and loaistical problems. The polym&ase cham reaction (PC@ has also been successfully used in the diagnosis of malaria, but has not yet been adapted for routine use in malaria control programmes (SNOUNOU et al., 1993). The rapid dipstick antigen capture assay is based on the detection of the histidine-rich protein 2 of Plasmodium falciparum (PMRP-2). It uses a water-soluble immunoglobulin Gl murine monoclonal antibody, directed against a segment of PfHRP-2, deposited on a nitrocellulose/glass fibre dipstick (SHIFF et al., 1994). There appears to be little evidence of cross reaction with other Plasmodium spp. which infect humans. The test is fast and simple, requires minimal training and equipment, and has potential for use in the management of malaria (WHO, 1995). The objective of the present study was to assess the performance of the dipstick assay in monitoring drug response and low parasitaemias. The study was conducted in patients with uncomplicated falciparum malaria treated with antimalarial drugs and subsequently examined with the dipstick assay and both standard and prolonged microscopical examination of thick blood films. -I
Materials and Methods The study was conducted at Kiremba Hospital, Ngozi Address for correspondence: Giovanni Di Petri, Istituto di Immunologia e Malattie Infenive, Universita diverona, Ospedale Civile Maggiore, 37126 Verona, Italy; phone and fax +39 45 8349314.
Province, Burundi. Patients with thick blood films showing single infections of I? falciparum attending the outpatient clinic of the hospital and willing to participate in the study were enrolled and sequentially treated with oral quinine (10 mg/kg every 8 h for 5 d), chloroquine (25 mg/kg over 3 d), or mefloquine (25 mg single dose). Patients or their guardians gave informed consent. On admission, and daily thereafter, Giemsa-stained (10% in buffered solution) thick and methanol-fixed thin blood films were prepared from each patient and red and white blood cells and platelets were counted by means of a Coulter counter which was calibrated daily. Three examination techniques were compared on each sample on admission and then daily during treatment and follow-up until no parasite was detected on 2 consecutive davs: (i) standard and (ii) prolonged thick blood film examination, and (iii) dipstick at&en capture assav (ParaSiahtTM-F test; Becton Dickinson. Tropical Disease D&agnostics, Cockeysville, Maryland; USA). Thick blood films were examined independently by 3 skilled microscopists. For the standard examination, each one made a continuous observation for 5 min (a timer was used) unless malaria parasites were seen. A thick blood film was considered positive when at least 2 microscopists found at least one asexual form of I? falciparum. Since the thickness of the film could influence the sensitivity, in order to keep the procedure as uniform as possible 1 ~.IL of whole blood was placed on a glass slide by means of a calibrated Eppendorf pipette and a thick film 8-12 mm in diameter was made. For the prolonged examination (the reference procedure), the 3 microscopists each examined a film for 20 min; films were deemed negative only if none of them found a parasite. The dipstick antigen capture assay was carried out on a dron of blood, taken into a calibrated canillarv tube contaimng anticoagulant, from the same sample used to make the thick blood film. as described bv SNIFF et al. (1993), except that, before adding the reagent containing rabbit anti-PfHRP-2 antibodies, any residual lysed blood still present in the plastic well was removed to obviate the otherwise persistent reddish background. Parasitaemia was estimated as the ratio between the numbers of parasites and white blood cells seen in the blood films multiplied by the number of white blood cells per pL determined by the Coulter counter. The geometric mean of each microscopist’s count was used. To qualify for analysis, patients had to remain in the hospital until no parasite was detected on 2 consecutive
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Abstract Three methods for the detection of Plasmodium falciparum infection in peripheral blood were compared during antimalarial treatment and follow-up in 32 Burundian patients: dipstick antigen capture assay, standard (TBF) and prolonged thick blood film examination (PTBF) (3x5 min and 3x20 min examination respectively). Parasitaemia was determined daily by comparison with total white blood cell counts (determined by Coulter counter) until no parasite was detected on 2 consecutive days by PTBF. Cumulatively, 23 1 observations were made with each assay: 64 were negative and 167 positive by PTBF (59 had parasite counts 5 1OO/pL) . Compared to PTBF, the sensitivities ofTBF and the dipstick assay were 1.0 for parasite counts >lOO/pL and 0.458 and 0.966 respectively for counts lOO/l.lL 5 1oo/clr, All Negative samples
108 59 167 64
108(100%) 27 (45.8%) 135 (80.8%) 0
Dipstick Positive
108(100%) 57 (96.6%) 1675(‘-,’ cl
32 (5:.2X) 32(19.2%) 64(100%)
Discussion The result of a review of published and unpublished assessments of the rapid antigen capture test (WHO,
sensitivity, specificity Table 2. Diagnosis of I? falciparum infection: ParaSightT”-F dipstick assay and standard thick blood film examination blood film examination
I
Sensitivity Specificity Predictive value Positive Negative
0.959 0.966
2 (30.4%) 2 (1.2%) 57 (89.1%)
sult was negative at the subsequent assessment, when the 2 tests were concordant. One patient receiving chloroquine gave a positive dipstick result on 2 consecutive occasions when the prolonged blood film examination was negative. The dipstick result was negative the next day, and the patient had no subsequent clinical recurrence. With parasitaemias > 100&L, both the standard blood film examination and the dipstick were 100% sensitive. However, the dipstick was significantly more sensitive and specific with parasitaemias
