Parasitology

Longitudinal Survey of Plasmodium falciparum Infection in Vietnam: Characteristics of Antimalarial Resistance and Their Associated Factors

Rie Isozumi, Haruki Uemura, Le Duc Dao, Truong Van Hanh, Nguyen Duc Giang, Ha Viet Vien, Bui Quang Phuc, Nguyen Van Tuan, Shusuke Nakazawa
DOI: 10.1128/JCM.01449-09

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Sequence polymorphism for pfcrt at positions 72 to 76. Each pie graph represents a sample year. The pieces in the pies reflect the positive ratio each year. The data for mixed infections are presented at the pop-up positions.

Tables

  • TABLE 1.

    Primers used to genotype pfcrt, pfmdr1, pfdhfr, and pfdhps

    PrimerSequence (5′ → 3′)Purpose
    Crt1FCATTGTCTTCCACATATATGACATAAAOuter PCR for pfcrt
    Crt4RGATCTCTATACCTTCAACATTATTCCTOuter PCR for pfcrt
    Crt2FTTTCCCTTGTCGACCTTAACAGATGGCNested PCR for the former part of pfcrt
    Crt12RATCCTATTTTACCTCTACGACTGTNested PCR for the former part of pfcrt
    Crt11FTTTCTTATAGGCTATGGTATCCTTTNested PCR for the latter part of pfcrt
    Crt15RTTTTAATTTCACACTTACCAAAGTNested PCR for the latter part of pfcrt
    MDR2FAAAGATGGTAACCTCAGTATCAAAGAAGAOuter and nested PCRs for the former part of pfmdr1
    MDR8RATGATTCGATAAATTCATCTATAGCAGCAAOuter and nested PCRs for the former part of pfmdr1
    MDR10RTTTTTTGGACACATCAACAACATCAGAATCNested PCR for the former part of pfmdr1
    MDR5FAGAAGATTATTTCTGTAATTTGATAGAAAAAGCNested PCR for the former part of pfmdr1
    DHFR1FATGATGGAACAAGTCTGCGACGTTTTCGATPCR for pfdhfr
    DHFR2RTTCATTTAACATTTTATTATTCGTTTTCTTPCR for pfdhfr
    DHPS1FCCATTCCTCATGTGTATACAACACPCR for pfdhps
    DHPS2RGTTTTAATCACATGTTTGCACTTTCPCR for pfdhps
  • TABLE 2.

    Primers used for sequencing

    PrimerSequence (5′→3′)Target geneObjective loci
    CRT2.2FTTCGACCTTAACAGATGGCTCACGTTTA pfcrt 72-76, 97
    CRT9.2FTATTTCTTATGACCTTTTTAGGAACGA pfcrt 144, 148, 160, 163
    CRT10.2FATTTATTTACTCCTTTTTAGATATCAC pfcrt 194, 220
    CRT11.2FATTCCTATAACGCATTATAATTATTTC pfcrt 271
    CRT16RAAACATTCCCATATTTATTTCCTC pfcrt 326, 333
    CRT14FTTATAGATTATCGACAAATTTTCT pfcrt 356
    CRT17FGTACAACGTATCATATTTTATAAT pfcrt 371
    MDRp3FGAGTACCGCTGAATTATTTAGA pfmdr1 86, 147,153, 184
    MDR14FATGTTTATGTTAACAATTATCTTACCA pfmdr1 363, 487
    MDR7RGCACATTAATTTTCCAGCATAACTACCAGT pfmdr1 1033, 1034
    MDR6FAGAATTATTGTAAATGCAGCTTTATGGGGATTC pfmdr1 1042, 1109
    MDR15FATGATCACATTATATTAAAAAATGATAT pfmdr1 1246
    DHFR2RTTCATTTAACATTTTATTATTCGTTTTCTT pfdhfr 16, 50, 51, 59, 108, 140, 164
    DHPS3FTTTGTTGAACCTAAACGTGCTGTTCAAA pfdhps 436, 437
    DHPS4RTTTATTTTCATTTTGTTGTTCATCATG pfdhps 540, 581, 613
  • TABLE 3.

    Characteristics of participants and parasite genotypes

    CharacteristicPositionAmino acidsa20012002200520062007P valuec
    Ageb22.1 ± 15.2 (2-75)19.3 ± 14.0 (0-72)23.1 ± 16.3 (1-65)14.3 ± 11.8 (0-48)9.63 ± 10.8 (2-46)0.05
    % Males52.755.566.738.125.00.07
    pfcrt 74M/I/mix31/42/9 (37.8/51.2/11.0)d67/22/11 (67.0/22.0/11.0)33/0/0 (100/0/0)31/6/1 (81.6/15.8/2.6)11/3/2 (68.8/18.8/12.5)<0.001
    75N/E/D/mix31/28/13/10 (37.8/34.1/15.9/12.2)67/7/15/11 (67.0/7.0/15.0/11.0)33/0/0/0 (100/0/0/0)31/1/5/1 (83.8/2.6/13.2/2.6)11/1/2/2 (68.8/6.3/12.5/12.5)<0.001
    76K/T/mix31/42/9 (37.8/51.2/11.0)67/22/11 (67.0/22.0/11.0)33/0/0 (100/0/0)31/6/1 (81.6/15.8/2.6)11/3/2 (68.8/18.8/12.5)<0.001
    144A/F/mix63/12/7 (76.8/14.6/8.5)75/17/8 (75.0/17.0/8.0)36/0/0/ (100/0/0)35/3/0 (92.1/7.9/0)14/2/0 (87.5/12.5/0)0.07
    148L/I/mix63/12/7 (76.8/14.6/8.5)75/17/8 (75.0/17.0/8.0)36/0/0 (100/0/0)35/3/0 (92.1/7.9/0)14/2/0 (87.5/12.5/0)0.07
    194I/T/mix63/12/7 (76.8/14.6/8.5)75/17/8 (75.0/17.0/8.0)36/0/0 (100/0/0)35/3/0 (92.1/7.9/0)14/2/0 (87.5/12.5/0)0.07
    220A/S/mix31/42/9 (37.8/51.2/11.0)65/24/1 (65.0/24.0/11.0)36/0/0 (100/0/0)31/6/1 (81.6/15.8/2.6)11/4/1 (68.8/25.0/6.3)<0.001
    271Q/E/mix31/42/9 (37.8/51.2/11.0)68/22/10 (68.0/22.0/10.0)36/0/0 (100/0/0)31/5/2 (81.6/13.2/5.3)11/4/1 (68.8/25.0/6.3)<0.001
    326N/S/mix54/23/6 (65.9/28.0/7.3)93/3/4 (93.0/3.0/4.0)36/0/0 (100/0/0)36/2/0 (94.7/5.3/0)15/0/1 (92.1/0/6.3)<0.001
    333T/S/mix59/17/7 (72.0/20.7/8.5)76/16/8 (76.0/16.0/8.0)36/0/0 (100/0/0)35/3/0 (92.1/7.9/0)13/2/1 (81.3/12.5/6.3)0.02
    356I/T/mix55/24/4 (67.1/29.3/4.9)90/6/4 (90.0/6.0/4.0)36/0/0 (100/0/0)36/2/0 (92.1/5.3/0)15/0/1 (93.8/0/6.3)<0.001
    371R/I/mix49/28/6 (59.8/34.1/7.3)89/7/4 (89.0/7.0/4.0)36/0/0 (100/0/0)35/1/2 (92.1/2.6/5.3)13/1/2 (81.3/6.3/12.5)<0.001
    pfmdr1 130E/K66/1 (98.5/1.5)90/1 (98.9/1.1)33/0 (100/0)35/0 (100/0)13/1 (92.9/7.1)
    184Y/F/mix53/8/5 (80.3/12.7/7.9)72/7/12 (79.1/7.7/13.2)33/0/0 (100/0/0)31/3/1 (88.6/8.6/2.9)13/1/0 (92.9/7.1/0)0.32
    1042N/D/mix55/8/4 (83.3/12.1/6.0)80/4/7 (87.9/4.4/7.7)33/0/0 (100/0/0)33/3/0 (91.7/8.3/0)12/1/1 (85.7/7.1/7.1)0.17
    1109V/I/mix62/0/4 (93.9/0/6.1)91/1/0 (98.9/1.1/0)33/0/0 (100/0/0)33/3/0 (91.7/8.3/0)13/0/0 (100/0/0)
    pfdhfr 51N/I/mix4/71/0 (5.3/94.7/0)6/39/5 (12.0/78.0/10.0)1/23/0 (4.2/95.8/0)10/24/0 (29.4/70.6/0)3/8/0 (27.3/72.7/0)<0.001
    59C/R/mix4/70/1 (5.3/93.3/1.3)3/43/4 (6.0/86.0/8.0)0/22/2 (0/91.7/8.3)0/34/0 (0/100/0)0/10/0 (0/100/0)0.39
    108S/N/mix4/71/0 (5.3/94.7/0)1/48/1 (2.0/96.0/2.0)0/24/0 (0/100/0)0/35/0 (0/100/0)0/11/0 (0/100/0)0.38
    164I/L/mix36/28/10 (48.6/37.8/13.5)40/5/5 (80.0/10.0/10.0)21/3/0 (87.5/12.5/0)32/3/0 (91.4/8.6/0)9/2/1 (75.0/16.7/8.3)<0.001
    pfdhps 436S/A/F/mix21/25/10/7 (33.3/39.7/15.9/11.1)44/33/8/10 (46.3/34.7/8.4/10.7)10/19/1/0 (33.3/63.3/3.3/0)25/3/0/8 (69.4/8.3/0/22.3)13/0/1/2 (81.3/0/6.3/12.5)<0.001
    437A/G/mix7/56/0 (11.1/88.9/0)0/92/3 (0/96.8/3.2)1/29/0 (3.3/96.7/0)1/34/1 (2.8/94.4/2.8)0/17/0 (0/100/0)<0.001
    540K/E/N/Y/mix19/27/6/0/11 (30.2/63.5/9.5/0/17.4)32/39/7/1/17 (33.3/40.6/7.3/1.0/17.7)7/19/1/0/3 (23.3/63.3/3.3/0/10.0)6/19/0/0/12 (16.2/51.4/0/0/32.4)1/9/0/0/7 (5.9/52.9/0/0/41.2)0.23
    581A/G/mix47/8/8 (74.6/12.7/12.7)61/20/16 (62.9/19.6/17.4)23/4/3 (76.7/13.3/10.0)8/16/13 (21.6/43.2/35.1)4/9/4 (23.5/52.9/23.5)<0.001
    613A/T/S/mix49/1/8/5 (77.8/1.6/12.7/8.0)87/0/6/3 (90.6/0/6.3/3.1)30/0/0/0 (100/0/0/0)36/0/0/0 (100/0/0/0)15/0/0/0 (100/0/0/0)<0.001

    • ↵ a Boldface type indicates amino acid mutations.

    • ↵ b Values are means ± standard deviations (minimum-maximum).

    • ↵ c P values are for the correlation between each year. Data for mixed infections for each locus were excluded from the statistical analysis.

    • ↵ d The data represent the number (percentage) of patients with the indicated genotype.

  • TABLE 4.

    Correlation between genotype and patient characteristicsa

    CharacteristicAmino acid(s)b or no. of mutationsGendercAgedNo. of peopleP value
    No. (%) maleNo. (%) femaleP valueg
    pfcrt 76K58 (63.7)40 (57.1)17.3 ± 14.598
    76T33 (36.3)30 (42.9)0.425.7 ± 15.063<0.01h
    pfmdr1 NYSNDe63 (86.3)55 (88.7)19.0 ± 15.1118
    NFSND1 (1.4)4 (6.5)10.6 ± 6.55
    NYSDD2 (2.7)0 (0)22.5 ± 7.82
    NFSDD7 (9.6)3 (4.8)0.1622.5 ± 14.5100.1i
    pfdhfr + pfdhps≥5f31 (79.5)27 (81.8)0.8
    ≥618 (46.2)20 (60.0)0.22
    ≥711 (28.2)14 (42.4)0.21

    • ↵ a Data for mixed infections were excluded.

    • ↵ b Boldface type indicates amino acid mutations.

    • ↵ c n = 91 for males, n = 70 for females.

    • ↵ d The data are means ± standard deviations.

    • ↵ e pfmdr1 positions 86, 184, 1034, 1042, and 1246, respectively.

    • ↵ f The data represent the number of mutations for pfdhfr and pfdhps.

    • ↵ g P values were obtained from analysis of relationships between gender and each genotype by the χ2 test.

    • ↵ h Obtained from the t test for correlation between pfcrt genotypes and patient age.

    • ↵ i Obtained by analysis of variance for correlation between pfmdr1 genotypes and patient age.

  • TABLE 5.

    Numbers of mutations for pfdhfr and pfdhps and age of patients

    GeneNo. of mutationsAgeaNo. of peopleP value
    pfdhfr 024.6 ± 12.48
    219.5 ± 24.72
    317.5 ± 15.961
    427.8 ± 14.2310.02
    pfdhps 025.4 ± 9.15
    118.0 ± 18.110
    217.9 ± 15.636
    321.1 ± 13.450
    430.3 ± 15.6140.03
    pfdhfr + pfdhps027.0 ± 9.64
    216.3 ± 13.23
    310.5 ± 12.02
    418.3 ± 21.74
    519.1 ± 15.521
    617.6 ± 12.413
    726.7 ± 13.819
    832.8 ± 12.360.02

    • ↵ a The data are means ± standard deviations.

  • TABLE 6.

    Relationship between different genotypes

    GeneAmino acid(s)apfcrt 76pfdhfrpfdhpspfdhfr + pfdhps
    No. (%) of isolates with:P valueAvg no. of mutations ± SDNo. of samplesP valueAvg no. of mutations ± SDNo. of samplesP valueAvg no. of mutations ± SDNo. of samplesP value
    KT
    pfcrt 76K2.7 ± 1.1512.2 ± 1.1604.6 ± 2.035
    T 3.4 ± 1.043<0.0012.8 ± 0.746<0.0016.3 ± 1.534<0.001
    pfmdr1 NYSNDb69 (93.2)33 (73.3)3.1 ± 0.9662.6 ± 0.9804.0 ± 2.049
    NFSND4 (5.4)1 (2.2)2.0 ± 1.732.3 ± 1.045.6 ± 1.63
    NYSDD1 (1.4)1 (2.2)3100
    NFSDD0 (0)10 (22.2)<.0013.6 ± 0.580.092.5 ± 1.066.0 ± 1.56

    • ↵ a Boldface type indicates amino acid mutations.

    • ↵ b pfmdr1 positions 86, 184, 1034, 1042, and 1246, respectively.

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KEYWORDS

Antimalarials
drug resistance
Malaria, Falciparum
Plasmodium falciparum

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