Use of the Polymerase Chain Reaction to Directly Detect Malaria Parasites in Blood Samples from the Venezuelan Amazon

Laserson, Kayla F.; Petralanda, Izaskun; Hamlin, Daniel M.; Almera, Raquel; Fuentes, Manuel; Carrasquel, Aníbal; Barker, Robert H.

doi:10.4269/ajtmh.1994.50.169

Cited by 33 publications

(14 citation statements)

References 2 publications

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“…The ASO-PCR and ASED have been extremely useful for surveillance of common mutations that are known to confer drug resistance. 10,14,15,17,31,[36][37][38][39][40][41][42]57 There are two problems with this approach. First, only alleles that have been previously identified are surveyed.…”

Section: Discussionmentioning

confidence: 99%

“…10 Characterization of P. falciparum mutants by culturing in the laboratory is also a time-consuming strategy, and it has the disadvantage that it selects only those parasites that can adapt to tissue culture. The use of allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) and allele-specific enzyme digestion (ASED) assays has been extremely useful in identifying already known point mutations, 10,1415,17,24,31,[36][37][38][39][40][41][42][43] but these methods are not sensitive enough to detect an allele present at a low level in a mixed population.…”

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confidence: 99%

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Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples.

Mookherjee¹,

Howard²,

Nzila-Mouanda³

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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Abstract. As resistance to chloroquine spreads in sub-Saharan Africa, pyrimethamine plus sulfadoxine (PSD) is increasingly used as a first-line treatment for falciparum malaria. Populations of Plasmodium falciparum (Pf) resistant to PSD have been selected quickly in other regions. The resistance is strongly correlated with point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), the two targets of the drug. It is critical to identify drug-resistant Pf-DHFR alleles that are present at a low frequency in these populations since alleles that confer drug resistance will be quickly selected by PSD use. It is difficult to identify these rare alleles by standard molecular techniques. We have designed a yeast expression system that facilitates the identification and rapid analysis of Pf-DHFR alleles that confer PSD resistance, even when they are present at very low frequency in polyclonal patient samples. We analyzed samples from patients in Kilifi, Kenya collected between 1992 and 1995. We determined the prevalence of the drug-sensitive and drug-resistant alleles in patient samples analyzed in parallel by an allelespecific enzyme digestion (ASED) assay. We identified a pyrimethamine-resistant allele (S108N) present at a frequency of Ͻ 1% in a sample that was scored as only S108 by ASED. In addition, a novel pyrimethamine-resistant allele (I164M) was isolated twice, once each from two different patient samples. This approach will allow determination of the prevalence of Pf-DHFR alleles that confer pyrimethamine resistance in particular regions, and the rapid identification of novel alleles that confer drug resistance.Dihydrofolate reductase (DHFR) catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential step in the production of methyl groups for biosynthesis.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples.

Mookherjee¹,

Howard²,

Nzila-Mouanda³

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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“…When the thick blood smear was compared with a PCR gold standard, the sensitivity of the microscopic technique was only 10.2%. Laserson and others 14 also found that PCR detection was much more sensitive, while Jelinek and others 31 found a multiplex PCR assay to be less sensitive than the thick blood smear. The oligonucleotide primers used here amplify repetitive sequences of high copy number, increasing the sensitivity of the PCR assay.…”

Section: Discussionmentioning

confidence: 99%

“…Patient samples (n ϭ 448) were brought to the University of Puerto Rico in 1997 for processing. The PCRs were carried out as previously described 12,14 with some modifications. Briefly, a 3-mm circle of the sample was excised from the filter using a disposable biopsy punch (Acuderm, Inc., Fort Lauderdale, FL) and transferred to 0.25-ml vials containing 50 l of PCR mixture (70 mM Tris, 20 mM (NH 4 ) 2 SO 4 , 2.5 mM MgCl 2 , 1 mM DTT, 0.1% Triton X-100, 50 pg/ml of bovine serum albumin, 0.2 mM deoxynucleotides, 5 pmol of oligonucleotide primers), and 1.25 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk, CT).…”

Section: Methodsmentioning

confidence: 99%

Epidemiologic tools for malaria surveillance in an urban setting of low endemicity along the Colombian Pacific coast.

Carrasquilla

Banguero²,

Sánchez³

et al. 2000

The American Journal of Tropical Medicine and Hygiene

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Abstract. An evaluation of 3 different methods for malaria diagnosis was carried out in an urban area of low endemicity on the Pacific coast of Colombia. Samples were collected from 833 symptomatic patients at a malaria clinic and examined by the polymerase chain reaction (PCR), quantitative buffy coat (QBC; Becton Dickinson, Franklin Lakes, NJ) method, and the traditional thick blood smear. The prevalence of Plasmodium falciparum malaria was 5.88% by thick blood smear, 7.34% by the QBC method, and 21.87% by PCR. The agreement between microscopists was 99.5%. The agreement between the QBC method and thick blood smear was 96.13% (n ϭ 745). Samples positive by PCR but negative by thick blood smear or conversely negative by PCR and positive by thick blood smear were usually of low-level parasitemias. All 3 methods showed agreement in 76.3% of the samples. Sixty-nine (18.8%) samples were positive by PCR but negative by the other 2 methods. Ten samples were positive by both the QBC method and thick blood smear but negative by PCR; most of them had low-level parasitemias. The use of malaria diagnostic methods for epidemiologic surveillance is discussed.

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“…15,[30][31][32] The occurrence of low-density transient parasitemia has been described as a common phenomenon in P. vivax sporozoite infection in nonhuman primates. 29,33,34 This low density, plus the typical spontaneous resolution of parasitemia, necessitates the use of a sensitive diagnostic method such as PCR 35,36 to study the immune dynamic responses to both the pre-erythrocytic and the blood stage phases of P. vivax infection.…”

Section: Discussionmentioning

confidence: 99%

Aotus Lemurinus Griseimembra Monkeys: A Suitable Model for Plasmodium Vivax Sporozoite Infection

Jordán-Villegas

Zapata²,

Perdomo³

et al. 2005

The American Journal of Tropical Medicine and Hygiene

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Abstract. This study describes a successful Plasmodium vivax sporozoite infection in Aotus lemurinus griseimembra. Twenty-eight naive or previously infected monkeys, either splenectomized or spleen intact, were inoculated intravenously or subcutaneously with Plasmodium vivax sporozoites of the Salvador I strain or with two wild isolates (VCC-4 and VCC-5; Vivax-Cali-Colombia). The monkeys were successfully infected regardless of the parasite strain, spleen presence, or inoculation route and showed prepatent periods that ranged from 16 to 89 days. Only one monkey inoculated intravenously failed to develop parasitemia. Since immune protection against malaria pre-erythrocytic forms is mediated by both helper and cytolytic T cells that may home in the spleen and P. vivax cultures are not yet available; the use of spleen-intact A. lemurinus griseimembra, susceptible to both adapted and non-adapted strains of P. vivax sporozoites, is a valuable model for evaluation of pre-erythrocytic vaccine candidates.

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Use of the Polymerase Chain Reaction to Directly Detect Malaria Parasites in Blood Samples from the Venezuelan Amazon

Cited by 33 publications

References 2 publications

Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples.

Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples.

Epidemiologic tools for malaria surveillance in an urban setting of low endemicity along the Colombian Pacific coast.

Aotus Lemurinus Griseimembra Monkeys: A Suitable Model for Plasmodium Vivax Sporozoite Infection

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