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Infectivity of Plasmodium falciparum sporozoites determines emerging parasitemia in infected volunteers

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Science Translational Medicine  21 Jun 2017:
Vol. 9, Issue 395, eaag2490
DOI: 10.1126/scitranslmed.aag2490
  • Fig. 1. Prepatent period and kinetics of parasitemia in CHMI with three P. falciparum strains.

    (A) Prepatent periods in the CHMI-a study (n = 5 per group). (B) Prepatent periods in the CHMI-b study (n = 4 per group). All subjects infected with the NF166.C8 strain in the CHMI-b study had prepatent periods of exactly 7.0 days, which is shown as a single line in (B). (C) Combined data from all 10 comparable CHMI studies performed at our center using thick smear microscopy as an end point (n = 56 NF54, n = 13 NF135.C10, and n = 5 NF166.C8). Box-and-whisker plots represent median/interquartile range and range per strain. In the CHMI-a study (A) and all studies in (C), prepatent periods were determined by time to positive thick blood smear; in the CHMI-b study (B) and subsequent studies at our center, prepatent periods were determined by time to positive qPCR. (D to F) Development of submicroscopic parasitemia after infection, as determined retrospectively by qPCR in the CHMI-a study (D) (n = 5 per group), the CHMI-b study (E) (n = 4 per group), and combined data from all 18 CHMI studies at our center using either thick smear microscopy or qPCR as an end point (F) (n = 90 NF54, n = 22 NF135.C10, and n = 14 NF166.C8). Data represent geometric mean ± 95% CI of all pretreatment samples per group per time point. All subjects in the CHMI-a and CHMI-b studies subsequently received a standard course of atovaquone-proguanil treatment and had their parasitemia cleared completely. Brown circles, NF54 strain; blue squares, NF135.C10 strain; red triangles, NF166.C8 strain.

  • Fig. 2. Invasion of human hepatocytes in vitro by three P. falciparum strains.

    Freshly isolated human hepatocytes were co-incubated with freshly isolated NF54 sporozoites (brown circles), NF135.C10 sporozoites (blue squares), or NF166.C8 sporozoites (red triangles) in vitro. The number of infected hepatocytes per well was counted by immunofluorescence microscopy at the given time points after infection. Shown are the results of three independent experiments. Experiment A was performed with 40,000 sporozoites per well harvested from mosquito batches used to infect subjects in the CHMI-a study and assessed at 5 days after infection. Experiments B1 and B2 were performed with 50,000 sporozoites per well harvested from mosquitoes fed on gametocyte cultures used in the CHMI-b study and assessed at 2 and 6 days after infection. Experiment A was performed at a different institute to experiments B1 and B2, and each of the three experiments was performed with human hepatocytes from a different donor. Data represent the percentage of sporozoites achieving successful invasion in each of three triplicate wells per condition (some NF166.C8 strain wells only in duplicate); horizontal lines represent group means. P values by one-way ANOVA with Tukey’s posttest. ns, not significant.

  • Table 1. Characteristics of P. falciparum parasitemia in CHMI subjects in vivo.
    NF54NF135.C10NF166.C8NF54 versus
    NF135.C10
    NF54 versus
    NF166.C8
    NF135.C10 versus
    NF166.C8
    CHMI-a
      No. of subjects per group555
      First detection of
    parasitemia by qPCR
    (days after infection)
    7.5
    (6.5 to 8.5)
    6.5
    (6.5 to 6.5)
    6.5
    (6.5 to 6.5)
    1.0
    (0.1 to 1.9)*
    1.0
    (0.1 to 1.9)*
      Peak parasitemia
    (log10 parasites/ml)
        First wave¤1.96
    (1.23 to 3.52)
    4.28
    (3.41 to 4.79)
    3.89
    (2.83 to 4.79)
    −2.32
    (−3.69 to −0.96)**
    −1.93
    (−3.30 to −0.56)**
    0.39
    (−0.97 to 1.76)
        Overall4.43
    (3.84 to 5.17)
    4.64
    (4.24 to 5.19)
    4.22
    (2.83 to 4.79)
    −0.20
    (−1.19 to 0.78)
    0.21
    (−0.77 to 1.20)
    0.42
    (−0.56 to 1.40)
    CHMI-b
      No. of subjects
    per group
    44
      First detection of
    parasitemia by qPCR
    (days after infection)
    6.5
    (6.5 to 6.5)
    6.5
    (6.5 to 6.5)
      Peak parasitemia
    (log10 parasites/ml)
        First wave¤3.61
    (2.59 to 4.19)
    4.09
    (3.77 to 4.34)
    −0.48
    (−1.63 to 0.66)
        Overall3.94
    (3.82 to 4.19)
    4.09
    (3.77 to 4.34)
    −0.15
    (−0.62 to 0.32)
    All CHMI studies
      No. of subjects
    per group§
    902214
      First detection of
    parasitemia by qPCR
    (days after infection)§
    7.2
    (6.3 to 10.6)
    6.7
    (6.5 to 7.0)
    6.5
    (6.5 to 6.5)
    0.52
    (−0.004 to 1.0)
    0.70
    (0.070 to 1.3)*
    0.18
    (−0.57 to 0.93)
      Peak parasitemia
    (log10 parasites/ml)
        First wave¤§2.72
    (1.00 to 4.67)
    3.67
    (2.59 to 4.79)
    3.78
    (2.78 to 4.79)
    −0.95
    (−1.41 to −0.49)***
    −1.06
    (−1.61 to −0.51)***
    −0.11
    (−0.79 to 0.58)
        Overall◊||4.28
    (3.13 to 5.17)
    4.72
    (3.35 to 5.84)
    4.22
    (2.83 to 4.97)
    −0.44
    (−0.86 to −0.009)*
    0.065
    (−0.58 to 0.71)
    0.50
    (−0.23 to 1.23)

    †Data represent mean per group (range).

    ‡Data represent mean differences between groups (95% CI of difference). Boldface indicates statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA/Tukey’s post hoc test).

    §Data included from all 18 comparable CHMI studies performed at our center using either thick smear microscopy or qPCR as an end point (table S3).

    ||Data included from only those 10 CHMI studies performed at our center in which subjects were treated upon positive standardized thick blood smear (n = 56 NF54-infected, n = 13 NF135.C10-infected, and n = 5 NF166-infected subjects, respectively).

    ¤Peak height of parasitemia during the first wave to emerge from the liver, defined as the highest parasite density measured by qPCR per subject between day 6.5 and 8.0 after infection; data represent geometric mean (range) per group.

    ◊Peak height of parasitemia per subject at any time after infection (generally at start of treatment); data represent geometric mean (range) per group.

    • Table 2. Characteristics of P. falciparum infectivity of human hepatocytes in vitro. ND, not determined.
      NF54NF135.C10NF166.C8
      Experiment B1Day 2#Schizonts per well*1,100 (1,091 to 1,158)2,420 (2,273 to 2,595)2,479 (1,727 to 3,005)
      Schizont Ø (μm)3.0 (2.3 to 4.1)3.4 (0.9 to 4.5)2.8 (1.8 to 3.6)
      #Nuclei per well18,533 (15,187 to 20,077)20,090 (17,823 to 26,446)17,980 (15,731 to 25,785)
      #Nuclei/schizont§16.88.37.3
      Day 6#Schizonts per well*1,383 (1,289 to 1,398)2,840 (2,656 to 3,276)2,843 (2,692 to 2,993)
      Schizont Ø (μm)11.2 (9.0 to 13.5)18.3 (12.5 to 25.0)16.1 (8.9 to 26.8)
      #Nuclei per well56,181 (52,810 to 69,893)216,449 (181,107 to 313,381)248,961 (201,453 to 394,328)
      #Nuclei per schizont§40.676.287.6
      Multiplication rate||2.49.212.1
      Experiment B2Day 2#Schizonts per well*373 (320 to 458)902 (275 to 1,575)1,098 (1,059 to 1,137)
      Schizont Ø (μm)1.7 (0.9 to 3.6)2.8 (1.8 to 5.4)3.4 (1.8 to 7.1)
      #Nuclei per well8,858 (7,226 to 9,585)16,726 (10,228 to 16,726)21,968 (20,742 to 23,195)
      #Nuclei per schizont§23.818.520.0
      Day 6#Schizonts per well*1,061 (826 to 124)813 (492 to 1,134)
      Schizont Ø (μm)11.4 (6.3 to 16.1)18.9 (12.5 to 28.6)12.5 (8.9 to 17.0)
      #Nuclei per well95,295 (94,347 to 11,184)382,508 (364,489 to 451,483)ND
      #Nuclei per schizont§218361ND
      Multiplication rate||11.519.4ND

      *Number of liver-stage schizonts per well, counted by immunofluorescence microscopy after staining with anti-HSP70 MAbs, as in Fig. 2. Data represent median (range) across triplicate wells.

      †Schizont diameters, as measured by immunofluorescence microscopy and digital image analysis. Data represent mean (range) of n = 19 to 49 schizonts per sample. NF135.C10 schizonts were larger than NF54 schizonts at day 2 (P < 0.05) and day 6 (P < 0.001) in both experiments and were also larger than NF166.C8 schizonts (P < 0.05 to P < 0.001 in all comparisons except experiment B2, day 2); NF166.68 schizonts were larger than NF54 schizonts at day 2 in experiment B2 (P < 0.001) and at day 6 in experiment B1 (P < 0.001). P values by one-way ANOVA with Tukey’s posttest.

      ‡Number of parasite nuclei per well, as determined by qPCR; data represent median (range) of triplicate wells.

      §Number of nuclei per schizont, as calculated by dividing the median #nuclei per well by the median #schizonts per well.

      ||Fold increase in number of nuclei per infected human hepatocyte from day 2 to day 6 after infection, as calculated by dividing the median #nuclei per schizont at day 6 after infection by that at day 2.

      Supplementary Materials

      • www.sciencetranslationalmedicine.org/cgi/content/full/9/395/eaag2490/DC1

        Fig. S1. Study flow charts.

        Fig. S2. Genetic characterization of P. falciparum isolates.

        Table S1. Study demographics.

        Table S2. Characteristics of P. falciparum isolates used in CHMI studies.

        Table S3. Overview of all comparable CHMI studies performed at our center.

        Table S4. Adverse events by severity grade during CHMI-a and CHMI-b studies.

        Table S5. Inclusion and exclusion criteria for CHMI studies.

        References (3136)

      • Supplementary Material for:

        Infectivity of Plasmodium falciparum sporozoites determines emerging parasitemia in infected volunteers

        Matthew B. B. McCall, Linda J. Wammes, Marijke C. C. Langenberg, Geert-Jan van Gemert, Jona Walk, Cornelus C. Hermsen, Wouter Graumans, Rob Koelewijn, Jean-François Franetich, Sandra Chishimba, Max Gerdsen, Audrey Lorthiois, Marga van de Vegte, Dominique Mazier, Else M. Bijker, Jaap J. van Hellemond, Perry J. J. van Genderen, Robert W. Sauerwein*

        *Corresponding author. Email: robert.sauerwein{at}radboudumc.nl

        Published 21 June 2017, Sci. Transl. Med. 9, eaag2490 (2017)
        DOI: 10.1126/scitranslmed.aag2490

        This PDF file includes:

        • Fig. S1. Study flow charts.
        • Fig. S2. Genetic characterization of P. falciparum isolates.
        • Table S1. Study demographics.
        • Table S2. Characteristics of P. falciparum isolates used in CHMI studies.
        • Table S3. Overview of all comparable CHMI studies performed at our center.
        • TableS4. Adverse events by severity grade during CHMI-a and CHMI-b studies.
        • Table S5. Inclusion and exclusion criteria for CHMI studies.
        • References (3136)

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