1. 106 S.BONNETETAL
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Bensink, M. P. E., Bolmer, M. & Meuwissen, J. H. E. T.
(1989). Infectivity of cultured Plasmodium falciparum game- Received 25 May 1999; revised 18 3;11y 1999; accepted for
tocytes to mosquitoes. Parasitology,98, 165-173. publication 3 August 1999
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (2000) 94,106-107
Amapi, Rondbnia and Roraima States). The same num-
1 Short Report 1 ber of mosquitoes known to be negative for human
I I malaria parasites was also tested.
The source of PZusmodium DNA was the same material
Infectivity of malaria vector used for the ELISA test [triturated mosquitoes-head
and thorax ground in a blocking buffer containing 0.05%
mosquitoes: correlation of positivity nonidet P-40 (WIRTZ et al., 1987)], either 20 @spotted
between ELBA and PCR-ELISA tests on a glass fibre membrane (GFM) prepared for PCR,
using l/8 of the spot as DNA source directly into the PCR
mixture as described by WARHURST et al. (1991), or
Marinete M. l%voa’*, Ricardo L. D. Machado’, extracted as follows: 40 & of ELISA solution was
Maria N. 0. Segura’, Giselle M. R. Vianna’, centrifuged for 10 min at 22 500 g, the pellet was lysed
Adenildo S. Vasc&celds’ and Jan E. Conn3 ‘Pro: by adding 25 &of lysis buffer and incubating at 65°C for
wama de Malhia. Instituto Evandro ChaPaslFAWMS. 30 min, afterwards adding potassium acetate and placing
k. Almirante Bakroso, 492, 66.090-000, -Bel&m, Park, on ice for at least 60 min. The isolated DNA (obtained by
Brazil; ‘Institute Oswald0 Cruz, FIOCRUZ, Av. Brasil, ethanol precipitation) was dissolved in 15 pL of TE-
436.5, 210 45-900, Rio deyaneiro, RJ, Brazil; 3Department buffer containing ribonuclease WILSON et al., 1998).
of Biology, Marsh Life Sciences Building, University of The DNA was amplified as described by MACHADO et al.
Vermont, Burlington, VT, 054050460, USA (1998) using primer sequence, concentrations and reac-
tion conditions indicated by OLIVEIRA et al. (1995). For
the identification of the human malaria parasites we used
Keywords: malaria transmission, anopheline mosquitoes, in- the liquid-phase, non-isotopic hybridization ELISA
fectivity, ELISA, PCR-ELISA, Brazil technique, following the protocol of OLIVEIRA et al.
(1995). For negative controls we used distilled water,
Studies on the infectivity of malaria vector mosquitoes male anopheline and culicine mosquitoes, and human
using the enzyme-linked immunosorbent assay (ELISA) DNA. Positive controls included strain Kl of I? fulcipar-
described by WIRTZ et al. (1987) have been carried out urn, and mosquitoes experimentally infected with l?
worldwide for several years. SOMBOON et al. (1993) falciparum and l? vivax.
reported false-positive results for the ELISA associated Our PCR results confirmed the ELISA test results for
with bovine and swine blood. In order to avoid these all positive and all negative mosquitoes, and in 5 (15.6%)
false-positive results it is advisable either to use only the of the positive mosquitoes (3 An. albitursis and 2 An.
anterior part of the mosquito (head and thorax) which, darlingi) the PCR-ELISA technique detected other
normally, is not contaminated by the ingested animal species of human Plasmodium that were not found by
blood (WIRTZ et al., 1987). or to confirm the ELISA the ELISA test alone (Table). The DNA source was
result by another method’such as polymerase chain obtained only by DNA extraction, which means that the
reaction (PCR). GFM technique is not applicable for this tvpe of material.
We have carried out the PCR-ELISA to confirm the These results indicate-&at the PCR-&ISA is more
detection of human malaria parasites in mosquitoes sensitive than a simnle ELISA test which. however. still
already recorded as positive by ELISA alone. Thirty remains a very goodand useful tool for testing mo&ito
two such mosquitoes were tested, belonging to different infectivity.
species of the genus Anopheles and collected during field
trips to different areas of the Amazonia Region (Pari, Acknowledgements
We thank the staff of the Malaria Entomology Laboratory at
the Evandro Chagas Institute for technical assistance and
*Author for correspondence; fax +55 91226 1284 or 2114417. Professor Ralph Lainson for reviewing the manuscript. This

2. lXA.SMODIUM DETECTION IN MOSQUITOES 107
Table. Comparison between ELISA and PCR-ELISA results for human
malaria parasites in Anopheles mosquitoes from several areas ofthe Amazon
region in Brazil
Species State ELISA PCR-ELISA
An. (NY.) albitarsis Roraima PVIPf PvllwPm
An. (Nys.) albitarsis PvlPj
An. (Nys.) albitarsis Roraima filpf
An. (N&.j albitarsis Roraima PvlPf
An. fNvs. ) braziliensis Roraima PvlPf
An. &$.j nuneztovari Roraima Pvlps
An. (Nys.) darlingi Rondbnia
An. (Nys.) darlingi Rondhnia g
An. (Nys.) albitarsis Amap y&k
An. (Nys.) albitarsis Amapi
An. (Nys.) albitarsis Amapi rym
An. (Nys.) albitarsis Amaph Pv
An. (Nys.) albitarsis AmapL l%
An. (Nys.) albitarsis Amapk Pm
An. (Nys.) albitarsis Amapi Pv
An. (Nys.) braziliensis Amapi Pv
An. (Nys.) darlingi Amapi
An. (Nys.) darlingi Amapi x
An. (Nys.) aquasalis ParP
An. (Nys.) aquasalis Pari
An. (Nys.) aquasalis Pari
An. (Nys.) darlingi Pari Pv
An. (Nys.) darling’ Pari PV
An. (Nys.) darlingi Pari
An. (Nys.) darlingi Pari
An. (Nys.) nuneztovari Pari
An. (Nys.) nuneztovari Pari
An. (Nys.) nuneztovari Pari
An. (Nys.) nuneztovan’ Pari
An. (Nys.) nuneztovari Pa&
An. (Nys.) nuneztovari Pari Pm
An. (Nys.) nuneztovari Pari Pm
Pv, Plasmodium vivax; pf, l? falciparum; Pm, I? malariae.
study received financial support from a National Institute of Warhurst, D. C., Awad-el-Karien, F. M. & Miles, M. A. (199 1).
Health Grant (A140116) to J.E.C. and Evandro Chagas In- Simplified preparation of malaria blood samples for polymer-
stitute/FNS. ase chain reaction. Lancet, 337, 303-304.
Wilson, M. D., Ofosu-Okyere, A., Okoli, A. U., McCall, P. J. &
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