1. TRANSACTIONS THE ROYALSOCIETY TROPICAL
OF OF MEDICINEAND HYGIENE(2000)
94,377-381
Distribution of Plasmodium vivaxvariants (VK210, VK247 and P. vivax-like) in
three endemic areas of the Amazon region of Brazil and their correlation with
chloroquine treatment
Ricardo L. D. Machado and Marinete M. l%voa* Laboratdrio de Makiria, ServiGo de Parasitologia, Instituw Evandro
ChagaslFUNASA, Av. Almirante Barroso 492, 66 090-000 Be&n, Park, Brazil
Abstract
The present study evaluated the glassfibre membrane (GFM)-polymerase chain reaction (PCR) - enzyme-
linked immunosorbent assay(ELISA) technique for genotyping the Plasmodium vivax variants, to verify the
distribution of l? vivax variants (VK2 10, VK247 and Z? viva-like) in parts of Brazil and their correlation
with levels of parasitaemia, previous malaria experience and clear&e-of parasitaemia linked to different
treatment schedules. The samoleswere taken from individuals living in Macar& Port0 Velho and Belem. all
ofwhich are endemic areasof&ax malaria in the Amazon region of?Brazil. B&d sampleswere collected on
GFMs. The gene that codesfor the circumsporozoite proteins of l? vivaxvariants was amplified by PCRand
the amplified fragments were hybridized to variant-specific, digoxigenin-labelled oligonucleotide probes by
ELISA. The GFM-PCR-ELISA techniaue was shown to be accurate for eoidemiolonical survevs of the
vivax complex. All variants were detected & all 3 areas,but only P. vivax VK2i 0 was fo&d as a sin&e agent
of infection, while the other 2 occurred as mixed infections. The l? vivax-like variant was found to be
associated with low parasitaemia and VK210 with the highest parasitaemia levels; none of the l? vivux
variants was linked with a previous malaria experience. In all casesparasitaemia clearance was identical
regarding the type of treatment and consequently it is not possible to confirm the previously reported
correlation between I? vivax genotype and response to chloroquine.
Keywords: malaria, Plasmodium vivax, variants,VK210, VK247, Plasmodiumv&x-like, epidemiology, chloroquine,
glass fibre membranes, polymerase chain reaction, ELISA, Brazilian Amazon
Introduction to be absolute (COLLIGNON, 1994). Reduction in sus-
Of the 4 known human malaria parasites, Plasmodium ceptibility to chloroquine was reported from Solomon
vivax, I? falciparum, I? malariae, and l? ovale, only the Island (WHITBY et aZ., 1989), Papua New Guinea
first 3 species have been detected in Brazil (QARI et al., (SCHUURKAMPet aZ., 1992; MURPHY et aZ., 1993) and
1993a). During the past 5 years l? vivax has been India (GARG et al., 1995). Studies conducted by KAIN et
responsible for 77~2% of all the reported malaria cases al. (1993a) showed that the responseto chloroquine may
(BRAZILIAN MINISTRY OF HEALTH, 1999). vary depending onthe type of Z?vivaxvariant. However, if
Although the circumsporozoite protein (CSP) of the there is a relationship between CSP genotype and parasite
infective sporozoite has been a major target in the clearance following treatment titi &ioroq&e, the
development of recombinant malaria vaccines, this underlving mechanism is unknown UZAIN et al.. 1993b).
approach has had to be re-evaluated because of the The-development of a sensitive and specific polymei-
c&overy of sequence variation in the CSP gene (QARI ase chain reaction (PCR) method for detection of
et al.. 1993a; GOPINATH et al.. 1994). Based on the CSP parasite DNA in blood samplesneeds to consider criteria
gene; ROSE&BERG al. (1589) described a I? vivax
et such as the selection of specific DNA, suitable DNA
variant form (VK247) in Thailand, and QAFU et al. extraction procedure and the conditions of this extrac-
(1993a) reported the presence in Papua New Guinea of tion (WILSON et al., 199 1). As the PCR technique yields
a human malaria parasite, referred to as ‘l? vivax-like’, amplification of only a single copy of the microiorga-
morphologically resembling l? vivax but with the repe- nism’s genes (FOOTEet al., 1989; WILSON et al.. 1989) it
titive sequence of the central region of the CSP differing is regarded as-anexcellentmethdd to detect low levelsof
from 2 described types of l? vivax. Several studies have DNA (KAIN et al., 1993b). Nevertheless, the major
been conducted to evaluate the global distribution of difficultv in the routine use of PCR amolification for
variant VK247: it was detected in indigenous popula- human blood samples is in obtaining an&purifying the
tions of Brazil (COCHRANE et al., 199%), in endemic DNA. Furthermore, it is known that haemoglobin and
areasof Thailand (WIRTZ et al.. 199 1: KPJN et al.. 1992. the other proteins can inhibit the PCR and that purified
1993a, 1993b), aid in South he&a, Africa (I&N e; DNA can contain traces of denaturing and inhibiting
al., 1991), Mexico, Afghanistan and Papua New Guinea proteins (MERCIER et al., 1990; LONG et al., 1995).
(KAIN et al., 1992). In addition, blood samples from hiACHAD0 et al. (1998) reported an adaptation of a
Panua New Guinea. Indonesia. Brazil and Madagascar method for blood sample collection (WARHURSTet al.,
weie positive for I? &ax-like GNA (QARI et al., 1593b) 1991) for the extraction and amplification of Plasmodium
Serological tests have detected all 3 P. vivax variants in DNA in the diagnosis of malaria infection (O-IRA et
samplesfrom SPoPaula State (CUFZADO al., 1995) and
et aZ., 1995). This new technique involving glass fibre
indigenous communities of the Amazon region of Brazil membrane (GFM), GFM-PCR-ELISA, requires less
(ARRUDAet al., 1996, 1998). expertise, saves time and reduces the cost for specific
Overthepast30years,widespreadresistanceofmalaria malarial gene sequences.
parasites to chloroquine has, so far, been restricted to P. The objective of the present study was to evaluate the
falciparum, and chloroquine still remains the drug of GFM-PCR-ELISA methodology for genotyping the P.
choice for both prophylaxis and treatment of P. vivax vivax variants, to verify their distribution in 3 endemic
infection (RIECM et al., 1989; BALDASSARRE al.,
et areas of the Amazon region of Brazil and to correlate
199 1). The first evidence that P. vivax is develoDinrr them with initial parasitaemia, previous malaria experi-
resistance to chloroquine was reported in Papua hew ence and the time of parasitaemia clearance linked to
Guinea by RIECKMANN et al. (1989). It is difficult to different treatment schedules.
ascertain how common chloroquine resistance is in l?
vivax infection, particularly asresistance does not appear Materials and Methods
Study population
Human blood was collected in Macapi (Amapi
*Author for correspondence: e-mail marinete@iec.pa.gov.br State), Beltm (Pa& State) and Port0 Velho (RondBnia

2. 378 RICARDO L. D. MACHADO AND MARINETE M. POVOA
State). Macapa and BelCm are in the eastern Amazon, amplification was carried out in 50 pL volume utilizing
while Port0 Velho is in the western Amazon (Figure). prepared DNA (5 pL), with freshly prepared master-mix
The patients were randomly recruited to participate in [33.25 pL of double-distilled water, 0.25 pL (1.25U) of
this studv at the National Health Foundation units in Taq polymerase (Bioloine, London, UK), 1 pL of each
each study area, with the assistance of their physicians. dNTP to provide 200 p&i final concentration and 5 uL of
10Xreactionbuffer(10mMtris-HClpH8~3;0~01%(wi
Blood sample collection v) gelatin; 1.5 mM MgCl,; 50 mM KCl)] . The biotiny-
A questionnaire for information on malaria episodes, lated primer and the unlabelled primer designed from
patient’s identification, epidemiological details, and the specific terminal ends of the l? vivax CSP gene (QARI et
mode of treatment was completed for each individual. al., 1993a) were used for amplification of the CSPgene.
The samples from MacapP (n = 42) were collected in The reaction mixture was initiallv denatured at 94°C for
Sentember 1995, from Port0 Velho (n = 33) in Anril 5 min, followed by 30 cycles of amplification (94°C for
1996 and from -BelCm (n = 40) during August and 1 min, 42°C for 50 s and 72°C for 90 s) and a final
September 1996, with informed consent from all indivi- extension at 72°C for 5 min. The PCR products (6 @L)
duals. The blood samples were obtained before therapy were electrophoresed on a 1% agarose gel and stained
was initiated, and only from those individuals who had a with ethidium bromide. The PCR products (5 pL) were
thick blood film and/or Quantitative Buffy Coat (QBC’) hybridized by liquid-phase non-isotopic method (OLI-
microscopically positive for l? vivax parasites and who VERA et al., 1995) for homologous I? vivax variants-
had agreed to participate in the study. Vacutainer tubes specific digoxigenin-labelled probes, designed from the
containing EDTA (Becton Dickinson, UK) were used to CSP repetitive region (QARI et al., 1993a). Primer and
collect 10 mL of blood/individual for PCR typing of l? probe sequences and concentrations were described
vivax and cryopreservation; 100 pL of whole-blood were previously by QAFU et al. (1993a) and the hybridization
used for urenarine the GFM disc (Titertek. ICN Bio- conditions were described by OLIVEIRA et al. (1995). For
medicals &Limited,“UK) following the protocol described negative control we used 2 samples of non-infected
by WARHURST et al. (199 1). human blood and human DNA. Positive controls con-
sisted of DNA from 3 different plasmids containing the
Treatment CSP repetitive region of variants.
Patients from Belem were treated as follows: chlor-
oquine 10 mg/kg in a single dose, plus primaquine Statistical analysis
0.50 mg/kg during 7 days (scheme A). Those from Port0 The data were organized in a database manager
Velho and Macapa were treated with chloroquine 25 mg/ (Dbase III) and later on processed and analysed in an
kg over 3 days (10 mg/kg on day 1 and 75 mg/kg on days epidemiology and statistical program (EpiInfo 6.0). For
2 and 3), plus primaquine 0.25 mg/kg for 14 days testing the significance of the variables we used the
(scheme B) . These were the standard treatments recom- Kruskal-Wallis and Mann-Whitney 2-tailed tests. To
mended by local physicians at each locality. obtain the independence among the proportions, the x2
test was applied with Yates’ correction or Fisher’s exact
DNA template preparation, PCR amplification, test (Ztailed). The adopted significance level for statis-
electrophoresis and hybridization tical inference was P < 0.05.
We used the GFM-PCR-ELISA technique (h&XI-IA-
DO et al., 1998). In brief, blood samples obtained directly Results
from patients were spotted on GFM and prepared for All 115 samples collected were both microscopically
PCR using the method of WARHuRsT et al. ( 199 1). PCR and QBC’ positive for I? vivax. The parasitaemia in the
Figure. Study area for the I? &ax variants. Macape (00” 02’20”s; 51” 03’59”W), Belkm (-01” 27’21”s; 48” 30’16”W) and
Velho (-08” 45’43”s; 63” 54’14”W).

3. BRAZILL4N I? VIVAXVARIANTS AND CHEMOTHERAPY 319
MacapP patients ranged from 40 to 26 500 infected red previous malaria episodes and the infecting P. vivux type
blood cells/mm3 [geometric mean (GM) 12251; in those (P > 0.05) in all 3 study areas.
from Port0 Velho it was from 375 to 6800 (GM 1445);
and from Belem 350 to 23 000 (GM 3126). Discussion
Samples with parasitaemia <500 infected red blood The existence of a new species or subspecies of
cells/mm3 (n = 30) did not produce a result using the Plasmodium causing human malaria would have impor-
PCR-ELISA technique. However, using 5 l.tLofthe first tant implications for diagnosis and vaccine design.
PCR products for re-amplification, it was possible to Furthermore, the P. vivax malaria variants may have
demonstrate the same fragment (1.2 Kb) as that ob- different characteristics in the intensity of symptoms, the
tained from the samples with a parasitaemia >500 response to the drug treatment and to vector preference,
(~1= 85) in agarose gel. which could cause drug resistance and failure of control
The typing of l? vivax indicated that the prevalence measures (GOPINATH et al., 1994). It is important,
and frequency of variants among the study areas were therefore, to determine the prevalence and distribution
not significantly different from one area to another of infection with these l? vivux genotypes.
(P > 0.05), since all types were present in all areas (Table The results of this study have indicated that P. vivax
1). DNA can be detected and genotyped by GFM-PCR-
Correlation of the l? r&ax genotype with the highest ELISA, even when parasitaemia is very low. This tech-
initial parasitaemia was significant (P < 0.05) for l? nique offers advantages over microscopy for the study of
&ax VK210 pure or in mixed infection with VK247, the P. vivax variants because they cannot be distin-
whereas the P. vivax-like parasite was associated with the guished morphologically, and the technique is also
lowest initial parasitaemia (Table 2). suitable for epidemiological surveys on the vivax com-
Table 2 shows that the time required for parasitaemia plex.
clearance determined by thick smear for all l? vivux With regard to the distribution of P. vivax variants we
genotypes was similar, regardless of the treatment used have found the variant VK2 10 to be the most prevalent of
(scheme A or B), and the differences did not reach the single variant infections. KAIN et al. (1991) had
statistical significance. Table 2 also demonstrates that similar results when analysing samples from South
there is no correlation between the mean number of America, West Africa and the Indian subcontinent. This
Table 1. Distribution of P. vivax genotypes in the three study areas in the Amazon region of Brazil
Pure types” Mixed types
Study area 1 2 3 1+2 1+3 2+3 1+2+3
Macapa 1; y;;; 0 0 1 (2.4%) 1 (2.4%) 6 (14.3%) 23 (54.8%)
Port0 Velho 0 0 4 (12.1%) 1 (3.0%) 5 (15.2%) 14 (42.4%)
Belem 15 (37.5;) 0 0 8 (20.0%) 1 (2.5%) 5 (12.5%) 11 (27.5%)
P = 0.23b
Values are numbers (%) in each category.
;T,ype 1, VK2 10; type 2, VK247; type 3, I? v&xx-like.
x.
Table 2. P. vivax genotypes and their correlation with initial parasitaemia,
past history of malaria and the number of days for the parasitaemia
clearance
Parasitaemia Previous Total
clearance after malaria number of
chloroquine and experience’ l? vivux
P. vivux Parasitaemiab primaquine (number of genotypes
genotype” (parasites/mm3) treatmentc (days) episodes) found
1 6122 f 5999 2.2 f 1.35 1.4 zt 1.98 35
1+2 5880 f 5740 2.2 f 1.09 0.8 3~ 1.42 13
1+2+3 1840 f 1597 2.0 f 1.30 1.9 f 1.95 48
1+3 900 f 540 2.7 dr 1.15 1.7 f 2.88 03
2+3 1506 f 1708 2.0 It 1.15 1.7 f 2.88 16
pi 0.01 0.86 0.39
“Type l,VK210; type 2, VK247; type 3, P. vivux-like.
b,cValues are bgeometric means f standard deviation (SD), or cmeans f SD.
dKruskal-Wallis test.
Mann-Whitney 2-tailed test comparing P. &ax genotype with time for parasite clearance after
treatment
P value
Typelvstypes1+2 0.56
Typelvstypes1+2+3 0.25
Typelvstypes1+3 0.69
Typelvstypes2+3 0.52
Types1+2vstypes1+2+3 0.60
Types1+2vstypes1+3 0.82
Types1+2vstypes2+3 0.93
Types1+2+3vstypes1+3 0.95
Types1+2+3vstypes2+3 0.48
Types1+3vstypes2+3 0.81

4. 380 RICARDO L. D. MACHADO AND MARINETE M. POVOA
fact suggests that this variant is well adapted worldwide. tients, but also found no correlation with the genotypes
The VK247 variant has a worldwide distribution and is of I? vivax. As immunity in malaria is directly related to
frequently found as a mixed infection with the VK210 the number of infections, it is likely that our patients had
genotype (COCHRANE et aZ., 1990; WIRTZ et aZ., 1990; not developed immunity and consequently there was no
CURADOetal., lg%?);&RUDAeta~., 1996,1998).While selection of genotypes.
this genotype has also been detected as the only kfecting In order to differentiate better the 3 P. vivax geno-
agent of malaria in Brazil. Afhanistan. Thailand. Panua types, studies focusing on other genes might provide
gew Guinea and Mexicd(Q&u etal., i991,199i; FIN further genetic information, enabling a better under-
et al., 1992, 1993a, 1993b), we did not find it as a pure standing of their phylogeny and relationship with other
infection. Studies have shown that pure infection is more human and non-human malaria parasites.
frequent in Asia than in South America, suggesting a
better adaptation of this genotype in this continent Acknowledgements
(KAIN et al., 1992, 1993a, 1993b; QARI et al., 1992). We thank Josi Maria de Souza Nascimento for technical
QARI et al. (1993a, 1993b) drew attention to the l? support and Professor Ralph Lainson, Dr Alexandre Linhares
v&x-like parasite and demonstrated the global occur- and Dr Jan E. Conn for critical reading of the manuscript. We
thank Dr Shoukat Qari and Altaf La1 (CDC, Atlanta, USA) for
rence of this variant in mixed infection, including in the technical support and suggestions. Financial support was
Brazilian Amazon Region. ARRUDA et al. (1996, 1998) provided by the British Council (BRAS/881/161)/CNPq (Pro-
and CURADO et al. (1997) had demonstrated by serolo- cess 9101191953), CNPq (Process 830381/95.3), PICD/
gical methods the occurrence of this variant co-circulat- CAPES, CDC Atlanta, Wellcome Trust and the Instituto
ing with other types, but do not indicate whether the Evandro Chagas/FUNASA. R.L.D.M. is a PhD student from
infections were mixed or the antibodies were raised from Universidade Federal do Pari supported by CNPq. The proto-
separate single infections. Similar results were obtained col for this study was reviewed and approved by the Research
in the present study. If our results are compared with Board of the Evandro Chagas Institute.
those of D. A. Oliveira (personal communication) (39% References
of the P. z&z-like variant in mixed infections), we note Arruda,M., Dutra, L., Veiga, E., Souza, R. & Clavijo, P. (1996).
that the P. viva-like variant has a significantly wider A sero-epidemiological study on Plasmodium wivax in three
distribution within the studied individuals (x2 test, states of the Amazon Basin of Brazil. Revisru da Sociedude
P < 0.05). The reasons for this wide distribution are still Brasileira de Medicina Tropical, 29 (supplement I)? 383.
unknown, but the I? vivax variants can reflect differ- Arruda, M. de, Souza, R. C., Veiga, M. E., Ferrelra, A. F. &
ences in the transmission intensity in these areas, varia- Zimmerman, R. H. (1998). Prevalence of Plasmodium vivux
bility of the vectorial competence and of the different variants VK247 and I? tivax-like human malaria: a retro-
genotypes of l? vivax, and/or the variation, in time, in spective study in indigenous Indian populations of the
Amazon region of Brazil. Transactions of the Royal Society of
which these genotypes appear or emerge in new areas Tropical Medicine and Hygiene, 92,628.
(COX, 1991). Baldassarre, J. S., Ingerman, M. J., Nansteel, J. & Santoro, J.
The mean parasitaemia indexes were significantly (199 1). Chloroquine resistance in Plasmodium vivax. 3oumd
lower for mixed infection, including l? viva&like, th& of Infectious Diseases, 164,222-223.
for nure infections. and VK2 10 was correlated with the Brazilian Ministry of Health (1999). EpkiemiobgiicaZ Survey of
hidest parasitaemia levels, an observation also reported Malaria in Brazil. Brasilia, Brazil: National Health Founda-
by KAIN et al. (1993a). These authors also showed that tion.
when VK210 was mixed with VK247 the parasitaemia Cochrane, A. H., Nardin, E. H., Arruda, M., Maracic, M.,
Clavijo, P., Collins, W. E. & Nussenzweig, R. S. (1990).
was enhanced, which was not observed in our study. The Widespread reactivity of human sera with avariant repeat of
high prevalence of genotype VK2 10, the oldest type of P. the circumsporozoite protein of Plasmodium vivax. American
viva3c, could be related to a wide distribution over a long Journal of Tropical Medicine and Hygiene, 43,446-45 1.
period of time, while the l? viva-like parasite, which is Collignon, P. (1994). Chloroquine resistance in Plasmodium
phylogenetically linked to P. simtbvale of Old World vivax. Journal of Infectious Diseases, 164,222-223.
monkeys (ESCALANTE et al., 1995), probably causes the Cox, F. E. G. (1991). Variation and vaccination. Nature, 193,
lowest parasitaemia because this parasite is undergoing 349.
adaptation to humans. Curado, I. I., Duarte, A. M. R. C., Lal, A. A., Nussenzweig, R.
S., Oliveira, S. & Kloetzel, J. K. (1995). Serological invesnga-
&YIN et al. (1993a) suagested that the response to tion of human Plasmodium &ax-like malaria in several
chloroquine may vary hep&ling on the type of‘l? vivax localities in the State of SHo Paulo, Brazil. Memdrb do
variant. since VK210 genotwe and mixed infections Institute OswaZdo Cruz, 90(supplement I), 284.
with V&47 took lo@& td’clear parasitaemia while Curado, I., Duarte, A. M. R. C., Lal, A. A., Oliveira, S. G. &
VK247 pure infection tended to be shorter. However, if Kloetzel, J. K. (1997). Antibodies anti bloodstream and
there is a relationship between CSP genotype and circumsporozoite antigens (Plasmodium vivax and Plasmo-
parasite clearance following treatment with chloroquine, dium malariaelI? bruzikznum) in areas of very low malaria
the underlying mechanism is unknown (KAIN et al., endemicity in Brazil. Membrias do kkUt0 Oswald0 Cruz, 92,
235-243.
1993b). The results observed in the present study did not Escalante, A. A., Barrio, E. & Ayala, F. J. (1995). Evolutionary
show any significant difference in-the time of-parasite origin of human and primate malaria: evidence from the
clearance (P > 0.05) for the 2 treatment schedules and circumsporozoite protein gene. Molecular Biology and Evolu-
the small -variation’ was independent to the infective tion, 12, 616-626.
genotype(s). The difference between our results and Foote. S. 1.. Thomuson. 1. K.. Cowman. A. F. & Kemo. D. 1.
those from KAIN et al. (1993a) can be explained by: i) (19i9) .” Arnplifiiation “of &e multi&g resistence g&e h
in the study by Kain et al. parasitaemia clearance was some chloroquine-resistant Plasmodium falcipatum. Cell, 57,
followed-up for shorter intervals of time (hours) than in 921-939.
Garg, M., Gonipathan, N., Bodhe, P. & Kshirsagar, N. A.
ours (days); ii) our patients were treated with chloquine (1995). Vivax malaria resistant to chloroquine: case reports
and primaquine which also has schizonticide action; and from Bombay. Transactions of the Royal Society of Tropical
iii) we included an analysis of a third genotype (I? vivux- Medicine and Hygiene, 89,656-657.
like) that is co-circulating with the other 2. However, our Gopinath, R., Wongsrichanalai, C., Cordbn-Rosales, C., Mir-
results do not exclude the hypothesis that strain-variable abelli, L., Kyle, D. & Kain, K. C. (1994). Failure fo detect a
responses to chloroquine may exist. Plusmodium vivax-like malaria parasite in globally collected
In our study, the mean number of previous episodes of blood samples.Journalof Infect&s Diseases, 170,1630- 1633.
malaria was low (1.7) and most patients were non- Kain. K. C.. Kevstone. I.. Franke. E. D. &Lanar.D. E. (1991).
Global d&it&on by a variani of the circumsporozoke gene
immune. This indicates that no correlation exists be- of Plasmodium vivax. Journal Of InfeChcWsDiseases, 164, 208-
tween previous malaria experience and the P. v&x 210.
genotype. KAIN et al. (1993a) had found a higher number Kain, K. C., Brown, A. E., Webster, H. K., Wirtz, R. A.,
of previous malaria experiences (7.0-7.6) in their pa- Keystone, J. S., Rodriguez, M. H., Kinahan, J., Rowland, M.

5. BRAZILIAN l? VIVAXVARIANTS AND CHEMOTHERAPY 381
& Ianar, D. E. (1992). Circumsporozoite genotyping of ozoite protein of the human malaria parasite Phzsmodium
global isolates of PZasmodium vivax from dried blood speci- vivax. Molecular and Biochemical Parasiw&y, 55, 105 - 114.
mens. Journal of Clinical Microbiology, 30, 1863- 1866. Oari. S. H., Shi. Y. P., Goldman, I. F.. Udhavakumar, V.,
Kain, K. C., Brown, A. E., Lanar, D. E., Ballou, W. R. & -Alpers,M. P., Collins;W. E. 8tLa1,A.A: (1993a). Identifica~
Webster, H. K. (1993a). Response of Plasmodium vivax don of PZusmodium tivax-like human malaria parasite. Lancet,
variants to chloroquine as determined by microscopy and 341,780-783.
quantitative polymerase chain reaction. American Journal of Qari, S. H., Shi, Y. P., Povoa, M. M., Alpers, M. P., Deloron, P.,
Tropical Medicine and Hygiene, 49,478-484. Murphy, G. S., Harjosuwamo, S. & I...& A. A. (1993b).
Kain, K. C., Brown, A. E., Mirabelli, L. & Webster, H. K. Global occurrence of Plasmodium n&z-like human malaria
(1993b). Detection of Plasmodium vivax by polymerase chain parasite. Journal of Infectious Diseases, 168, 1485- 1489.
reaction in a field study. Journal of Infectious Diseases, 168, Rieckmann, K. H., Davis, D. R. & Hutton, D. C. (1989).
1323-1326. Plasmodium vivux resistance to chloroquine? Lancet, ii, 1395.
Long, G. W., Fries, L., Watt, G. H. & Hoflinan, S. L. (1995). Rosenberg, R., Wirtz, R. A., Lanar, D. E., Sattabongkot, J.,
Polymerase chain reaction amplification from Phzsmodium Hall, T., Waters, A. P. 81Prasittisuk, C. (1989). Circumspor-
falcipamm on dried blood spots. American Journal of Tropical ozoite protein heterogeneity in the human malaria parasite
Medicine and Hygiene, 52,344-346. Plasmodium vivax. Science, 245,973-976.
Machado, R. L. D., Garret,D. O., Adagu, I. S., Warhurst, D. C. Schuurkamp, G. J., Spicer, P. E., Kereu, R. K., Bulungol, P. K.
& Povoa, M. M. (1998). Simplified diagnosis of malaria & Rieckmamt, K. H. (1992). Chloroquine-resistant Plasmo-
infection: GFMPCRELISA, a simplified nucleic acid am- dium v&x in Papua New Guinea. Transactions of the Royal
plification technique by PCRiELISA. Revista do Znstituto de Society of Tropical Medicine and Hygiene, 86, 12 1 - 122.
Medicina Tropical de Skio Pa&, 40,333-334. Warhurst, D. C., Awad-el-Karien, F. M. & Miles, M. A. (199 1).
Mercier, B., Gaucher, C., Feugeas, 0. & Mazurier, C. (1990). Simplified preparation of malaria blood samples for polymer-
Direct PCR from whole blood, without DNA extraction. ase chain reaction. Lancet, 337,303-304.
Nucleic Acids Research, 18, 5908. Whitby, M., Wood, G., Veenendaal, J. R.& Rieckmann, K.
Murphy, G. S., Basri, H., Purmono, Andersen, E. M., Bangs, (1989). Chloroquine-resistant Plasmodium vivax. Lancet, ii,
M. J., Mount, D. L., Gorden, J., Lal, A. A.,Purwokusumo, A. 1395.
R., Hatjosuwamo, S., Sorensen, K. & Hoffman, S. (1993). Wilson, C. M., Serrano, A. E. &Washy, A. (1989). Amplifica-
Vivax malaria resistant to treaunent and prophylaxis with tion ofgene related to mammalian mdrgenes in drug-resistant
chloroquine. Lancet, 341,96-100. Plasmodium falcipamm. Science, 244,1184- 1186.
Oliveira, D. A., Holloway, B. I’., Durigon, E. L., Collins, W. E. Wilson, R. J. M., Gardner, M. J., Feagin, J. E. & Willianson, D.
M. & Lal, A. A. (1995). Polymerase chain reaction and liquid- H. (199 1). Have malana parasites three genomes? Parasitol-
phase, nonisotopic hybridization for species-specific and ogy Today, 7, 134-136.
sensitive detection of malaria infection. American Journal of Wirtz, R. A., Rosenberg, R., Sattabonakot, 1. &Webster. H. K.
Tropical Medicine and Hygiene, 52, 139-144. (1990). Prevalence of antibody to heterologous circumspor-
Qari, S. H., Goldman, I. F., Povoa, M. M., Oliveira, S. G., ozoite protein of Plasmodium vivax in Thailand. Lancet, 336,
Alpers, M. I’. & Lal, A. A. (1991). Wide distribution of the 593-595.
variant form of the human malaria parasite PZasmodium vivax.
Journal of Biological Chemistry, 266,16297-16300.
Qari, S. H., Goldman, I. F., Povoa, M. M., Sand, S., Alpers, Received 9 August 1999; revised 19January 2000; accepted
MI’. & Lal, A. A. (1992). Polymorphism in the circumspor- for publication 26 January 2000
1 Announcement ]
The Tropical Health and Education Trust (THET)
Since THET was established in 1988, it has actively supported training for health care in tropical countries, where
resources are limited. THET works with those who are responsible for such training so that their goals can be
reached, as they prepare their staff and students for the tasks that they will be called on to do, relevant to the needs of
their local community.
THET achieves this strategy through collaborative links between a hospital or medical/nursing school in the
United Kingdom and its counterpart overseas. These institutional links are made up of individual links, each of
which is carefully planned over a number of years. Precise goals are set so that progress, which is closely monitored,
can be used as a basis for additional funding. Links cover many disciplines from nursing, clinical medicine,
community health, laboratory and library services, and management, to fieldwork by students.
We respond to requests to establish these links and, while we do not prescribe what the pattern of a link should be,
we are only prepared to seek funding for those that are relevant and needed, and that directly or indirectly contribute
to the health care of poor people. THET does not fund individuals, except when newly acquired personal skills
contribute to an institution and the service that it provides.
Trustees: Professor Eldryd Parry (chairman), Professor Roy Anderson, FRS, Professor Brian Greenwood, FRS,
Miss Thelma Holland, Mrs Helen Parry, Mr John Rennie FRCS, Mr Richard Southwell, QC and Professor David
Warrell.
For further information please contact THET, Euston House, 24 Eversholt Street, London NW1 lAD, UK;
phone +44 (0)20 7611 870516, fax t-44 (0)20 7611 068314, e-mail vpthetl@aol.com