612
tally amplified DNA froni filter pa r disks impregnated with whole blood. Journal of Clinical E. rcrobwlogy, 29, 1171-
xJ. ribonucleic. acid probe. Transactions of the Royal Society of . roptcal Medtctne and Hygiene, 84,630-634.
_-, .
Rosenberg, RF, Wirtz, R. A., Lanar, D. E., Sattabongkot, J., Hall, T., Waters, A. P. & Prasittisuk, C. (1989). Circumsporozoite rotein heterogeneity in the human malaria parasite
1174.
Kain, K. C., Keystone, J., Franke, E. D. & Lanar, D. E. (1991). Global distribution of a variant of the circumsporozoite gene of Plasmodium vivax. Tournal of_ Infectious Diseases, _ 164,2&-210. Kain. K. C.. Brown. A. E.. Webster. H. K.. Wirtz. R. A..Kevstone, J, S., Rodr&uez, M. H., K&&uur,‘J., Rowland, ‘M. -& Lanar, D. E. (1992): Circumsporozoite genotyping of global isolates of Plasmodium vivax from dried blood specimens. 3ownal of Clinuzal Microbiology, in press. Looareesuwan S., White, N. J., Chittamas, S., Bunnag, D. & Harinasutr, 9. (1987). High rate of Plasmodium oivax relapse followinn treatment of falcioarum malaria in Thailand. Lancet, ii, 1652-1055. Martelo. 0. 1.. Smaller. M. & Saladin. T. A. (19691.Malaria in American Vsoldiers.Atchives of Internal Medicine, 123, 383387. Relf, W. A., Boreham, R. E., Tapchaisri, I’,, Khusmith, S., Healey, A., Upcroft, P., Tharavanij, S. & Kidson, C. (1990).
Plasmotkm vivax. Science 245 973-976
Sabchareon, A., Burnouf, T: , Gdattara, D: , Attanath, P., Bouharoun-Tayoun, H., Chantavanich, P., Fducault, C., Chongsuphajaisiddhi, T. & Diuilhe, P. (1991). Parasitologic and clinical human responseto imrnunoglobulin administration in falciparum malaria. AmericanJournal of Tropical Medicine and Hygiene, 45,297-308.
Shute, P. G. (1951). Mos uito infection in artificially induced malaria. British Medical 1 ulletm, 8,56-63. Takagi, T., Kano, S., Masuda, G. & Suzuki, M. (1988). Submicroscopic double infection of Plasmodium falci arum in P. vivax malaria, Transactions of the Royal Society of 4 roprcal Medlcine and Hygiene, 82,384. Received accepted
Diagnosis of Plasmodium vivax malaria using a specific deo-
30 Januasy
for publication
1992; revised 26 March I992
25 March
1992;
TRANSACTIONSOF THE ROYAL SOCIETYOFTROPICAL MEDICINEAND HYGIENE(~~~~)86,612-613
Detection of parasite nucleic acid is among the mqre promising alternatives to Giemsa-stained blood films for the detection of Plasmodium falcifiatitn, but dedxyribonucleic acid (DNA) hybridization probes lacked sensitivity in a recent clinical trial (LANAR et al., 1989). Methods based on ribosomal ribdnucleic acid @RNA) should theoretically be several orders of magnitude more sensitive than DNA probes (LAL et al., 1989). Not only is the quantity of RNA per parasite greater (0.2-1.0 pg RNA vs 0.02 pg DNA) but a higher proportion of RNA than DNA is suitable as a probe target; less than 10% of total DNA has suitable repetitive sequences (LAL et al., 1989). An rRNA probe detected 0.00046% of parasitized red blood cells iti malaria cultures (LAL et al., 1989), but has not been field tested. We therefore evaluated the
RNA probe detection of Plasmodium falciparum parasitaemia G.
Watt’,
K.
Jogsakull
and
Gary
W.
Long223
iDepartment of Medicine, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand; 2Naval
Medical Research Institute, Bethesda, Maryland, USA; 3Johns Hopkins University, School of Hygiene and Publzc Health, Baltimore, Mdi#ind, USA
b .I2
1-r
-10
.OO
. 0.6
-0.4
-0.2
Fig. 1. Sequential probe densitometry readings and thick blood film malaria couots in the 3 patients from whom most specimenswere obtained (a, b, and c). Parasitaemia expressed as the number of asexual stage parasites per ktl of blood is plotted on the left y axes and probe results on the right y axes. Note that the probe results remained positive in patient c, in whom patent parasitaemiareappeared on day 27.
Address for correspondence, proofs and offprint requests: Dr George Watt, Chief, Department of Medicine, AFRIMS, APO A,P 96546, USA. This study was supported by the United States Army Research and Develooment Command and the Naval Medical Research and Development Command. The o inions or assertions contained in this paper are private views o P the authors and are not to be construed as official or as reflecting the views of the United States Army or the United States Navy.
rRNA probe in Thai patients with falciparum malaria participating in chemotherapeutic trials on the ThaiCambodian border. Thick films were made from 10 ul of blood obtained by fingerprick and the number of parasites per ul of blood was determined by the method-of EARLE~& PEREZ (1932). Blood for the RNA probe was collected into heparinized centrifuge tubes. Ten ul were mixed with 1 ml
613
N=
32
I5
27
. I . 1.00 . 0.80 Y & a
.
0.80
f 0.40
0.20
C
$!& 0
. I l-100
l
lo,00
PARASITEMIA Fig. 2. Correlation between rRNA densitometry readings and different levels of parasitaemia for 95 specimens. The median probe result is the horizontal line within the shaded box, the upper and lower limits of which correspond to 95% confidence intervals. The median probe readings for the 4 levels of parasitaemia are significantly different (P
tally amplified DNA froni filter pa r disks impregnated with whole blood. Journal of Clinical E. rcrobwlogy, 29, 1171-
xJ. ribonucleic. acid probe. Transactions of the Royal Society of . roptcal Medtctne and Hygiene, 84,630-634.
_-, .
Rosenberg, RF, Wirtz, R. A., Lanar, D. E., Sattabongkot, J., Hall, T., Waters, A. P. & Prasittisuk, C. (1989). Circumsporozoite rotein heterogeneity in the human malaria parasite
1174.
Kain, K. C., Keystone, J., Franke, E. D. & Lanar, D. E. (1991). Global distribution of a variant of the circumsporozoite gene of Plasmodium vivax. Tournal of_ Infectious Diseases, _ 164,2&-210. Kain. K. C.. Brown. A. E.. Webster. H. K.. Wirtz. R. A..Kevstone, J, S., Rodr&uez, M. H., K&&uur,‘J., Rowland, ‘M. -& Lanar, D. E. (1992): Circumsporozoite genotyping of global isolates of Plasmodium vivax from dried blood specimens. 3ownal of Clinuzal Microbiology, in press. Looareesuwan S., White, N. J., Chittamas, S., Bunnag, D. & Harinasutr, 9. (1987). High rate of Plasmodium oivax relapse followinn treatment of falcioarum malaria in Thailand. Lancet, ii, 1652-1055. Martelo. 0. 1.. Smaller. M. & Saladin. T. A. (19691.Malaria in American Vsoldiers.Atchives of Internal Medicine, 123, 383387. Relf, W. A., Boreham, R. E., Tapchaisri, I’,, Khusmith, S., Healey, A., Upcroft, P., Tharavanij, S. & Kidson, C. (1990).
Plasmotkm vivax. Science 245 973-976
Sabchareon, A., Burnouf, T: , Gdattara, D: , Attanath, P., Bouharoun-Tayoun, H., Chantavanich, P., Fducault, C., Chongsuphajaisiddhi, T. & Diuilhe, P. (1991). Parasitologic and clinical human responseto imrnunoglobulin administration in falciparum malaria. AmericanJournal of Tropical Medicine and Hygiene, 45,297-308.
Shute, P. G. (1951). Mos uito infection in artificially induced malaria. British Medical 1 ulletm, 8,56-63. Takagi, T., Kano, S., Masuda, G. & Suzuki, M. (1988). Submicroscopic double infection of Plasmodium falci arum in P. vivax malaria, Transactions of the Royal Society of 4 roprcal Medlcine and Hygiene, 82,384. Received accepted
Diagnosis of Plasmodium vivax malaria using a specific deo-
30 Januasy
for publication
1992; revised 26 March I992
25 March
1992;
TRANSACTIONSOF THE ROYAL SOCIETYOFTROPICAL MEDICINEAND HYGIENE(~~~~)86,612-613
Detection of parasite nucleic acid is among the mqre promising alternatives to Giemsa-stained blood films for the detection of Plasmodium falcifiatitn, but dedxyribonucleic acid (DNA) hybridization probes lacked sensitivity in a recent clinical trial (LANAR et al., 1989). Methods based on ribosomal ribdnucleic acid @RNA) should theoretically be several orders of magnitude more sensitive than DNA probes (LAL et al., 1989). Not only is the quantity of RNA per parasite greater (0.2-1.0 pg RNA vs 0.02 pg DNA) but a higher proportion of RNA than DNA is suitable as a probe target; less than 10% of total DNA has suitable repetitive sequences (LAL et al., 1989). An rRNA probe detected 0.00046% of parasitized red blood cells iti malaria cultures (LAL et al., 1989), but has not been field tested. We therefore evaluated the
RNA probe detection of Plasmodium falciparum parasitaemia G.
Watt’,
K.
Jogsakull
and
Gary
W.
Long223
iDepartment of Medicine, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand; 2Naval
Medical Research Institute, Bethesda, Maryland, USA; 3Johns Hopkins University, School of Hygiene and Publzc Health, Baltimore, Mdi#ind, USA
b .I2
1-r
-10
.OO
. 0.6
-0.4
-0.2
Fig. 1. Sequential probe densitometry readings and thick blood film malaria couots in the 3 patients from whom most specimenswere obtained (a, b, and c). Parasitaemia expressed as the number of asexual stage parasites per ktl of blood is plotted on the left y axes and probe results on the right y axes. Note that the probe results remained positive in patient c, in whom patent parasitaemiareappeared on day 27.
Address for correspondence, proofs and offprint requests: Dr George Watt, Chief, Department of Medicine, AFRIMS, APO A,P 96546, USA. This study was supported by the United States Army Research and Develooment Command and the Naval Medical Research and Development Command. The o inions or assertions contained in this paper are private views o P the authors and are not to be construed as official or as reflecting the views of the United States Army or the United States Navy.
rRNA probe in Thai patients with falciparum malaria participating in chemotherapeutic trials on the ThaiCambodian border. Thick films were made from 10 ul of blood obtained by fingerprick and the number of parasites per ul of blood was determined by the method-of EARLE~& PEREZ (1932). Blood for the RNA probe was collected into heparinized centrifuge tubes. Ten ul were mixed with 1 ml
613
N=
32
I5
27
. I . 1.00 . 0.80 Y & a
.
0.80
f 0.40
0.20
C
$!& 0
. I l-100
l
lo,00
PARASITEMIA Fig. 2. Correlation between rRNA densitometry readings and different levels of parasitaemia for 95 specimens. The median probe result is the horizontal line within the shaded box, the upper and lower limits of which correspond to 95% confidence intervals. The median probe readings for the 4 levels of parasitaemia are significantly different (P
