Research ArticleMalaria

A saliva-based rapid test to quantify the infectious subclinical malaria parasite reservoir

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Science Translational Medicine  02 Jan 2019:
Vol. 11, Issue 473, eaan4479
DOI: 10.1126/scitranslmed.aan4479
  • Fig. 1 Study design of the cross-sectional studies of subclinical malaria parasite carriage among children in Cameroon and Zambia.

    Primary schools in the catchment area in Mfou, Cameroon, where screening and sampling performed are listed. School children between the ages of 5 to 16 were enrolled in the study. For Zambia, children <16 years of age who were present in households were sampled. The sampling strategy in Zambia dovetailed on an ongoing Southern Africa International Center of Excellence for Malaria Research (ICEMR) program project in Nchelenge, Zambia, which was conducted in partnership with the Tropical Diseases Research Centre (TDRC) of Zambia. Informed consent for all enrolled children was provided by a parent/guardian. If a child was found to be positive for malaria parasites by blood smear, they were referred for treatment according to the National Malaria Control Program guidelines. *Children who were not approved by their parent or guardian to participate were not included during the screen, and these children were not included in the total number of samples that were used for downstream molecular analyses.

  • Fig. 2 Saliva and matched blood sample collection, stabilization, and analyses.

    (A) Photograph and diagram of saliva collection (drool method) and stabilization for transport for two different collection periods from schools (Cameroon) and households (Zambia). The total number of saliva samples is 364. Saliva samples were stabilized for transport and analysis by LC-MS/MS for marker discovery. The prevalence of the marker PSSP17 in the saliva of children was then measured by LC-MRM MS. (B) Table summary of the LC-MRM results and estimated prevalence of marker carriage across all samples collected from both Cameroon and Zambia. (C) Table summary data of samples comparing the LC-MRM results with the reference molecular method of qPCR and PCR for pfs25, 18S rRNA gene in Cameroon, and cytB in Zambia, respectively. (D) Comparison of estimated female gametocytes/μl of blood (as determined by pfs25 mRNA qPCR, left y axis, blue bars) and PSSP17 (PF3D7_1218800) protein expression (as determined by PAR, right y axis, gray bars) for a subset of 93 samples with paired qPCR (pfs25) and PAR data available. Sample case codes are indicated on the x axis. Red arrows refer to examples of discordant results, as described in the main text.

  • Fig. 3 Prototype LFIA RDT for PSSP17 (PF3D7_1218800).

    (A) Schematic of the gRAD lateral flow platform and the capture and detection of PSSP17 by EuChelate microparticle-conjugated mAbs. (B) Images of LFIA gRAD strips to estimate the LOD of recombinant PSSP17 in spiked-in assays using naïve human saliva as matrix. (C) Initial analyses of frozen, stabilized samples from Cameroon (n = 12). Samples were randomly selected and, upon unblinding, were found to be either positive or negative by MS (LC-MRM), microscopy, or qPCR. A048 and A048b are two independent saliva samples collected from the same child to show consistency in detection by the LFIA. Positive (+) control: gametocyte lysate spiked into naïve saliva; negative (−) control: irrelevant asexual parasite lysate spiked into naïve saliva. Images on the left side of the figure are gRAD platform strips that were captured by mobile phone camera. (D) A second subset of independent Cameroon samples (n = 10), positive by LC-MRM, was selected and compared across the same categories as in (C). Microscopy was subdivided into gametocyte versus asexual trophozoite positivity/negativity, and qPCR/PCR analyses were subdivided into gametocyte-specific transcript (pfs25) or a transcript present in both trophozoites and gametocytes (18S rRNA). For (B) to (D), 10 μl of saliva containing either recombinant PSSP17 or endogenous marker was used with each test. C, control line; T, test line. Vertical dotted line: Demarcation of the end of the sample loading pad of the gRAD strip.

  • Fig. 4 Evaluation of the LOD of the prototype PSSP17 LFIA and potential use in clinical settings.

    (A) Replicate tests using filtered (or unfiltered), axenic supernatant from stage V gametocyte cultures, asexual blood-stage cultures, and complete media (negative control). (B) Estimated LOD of the PSSP17 LFIA based on gametocyte quantification using LM or qPCR for pfs25 transcripts. (C) Schematic of the Sierra Leone study to test the utility of the LFIA in confirmatory diagnosis of malaria parasite infection. Symptomatic individuals (>1 year of age) presenting in the Mercy Hospital in Bo, Sierra Leone, who provided informed consent were enrolled in the study. Matched blood samples were stored either in RNAlater or as DBS on Whatman FTA cards. (D) LFIA test strips showing PSSP17 detection in the saliva of a subset of symptomatic individuals presenting at a clinic in Bo, Sierra Leone. Negative control, uninfected/naive human saliva; positive control, naïve human saliva with lysed P. falciparum gametocytes. (E) Table comparing orthogonal detection approaches, multiplex PCR, LM, and the PSSP17 LFIA for all 34 samples from Sierra Leone. Multiplex PCR score: 0 = negative, 0.5 = weak positive, and 1 = positive. (F) Table estimating the sensitivity of the LFIA in a clinical setting compared to microscopy or multiplex PCR as the reference standard. For all lateral flow tests: C, control line; T, test line.

  • Table 1 The most abundant P. falciparum proteins identified in children aged 5 to 16 years with subclinical parasitemia in Cameroon.

    A total of 35 proteins were identified by LC-MS/MS with a Mascot ions score of ≥25 (table S1). Of the 35, the 19 proteins below were considered the most abundant. Score indicates the Mascot ions scores, as described previously (23). The P. falciparum female gametocyte-specific biomarker, indicated in bold, was found to have the highest Mascot ions score in our pooled sample. Mr, molecular mass in kilodaltons (kDa).

    Gene IDDescriptionMr (kDa)Annotated Gene
    Ontology
    MS*Mascot ions score
    PF3D7_0401900Acyl-CoA synthetase110.8Long-chain fatty acid–
    CoA ligase activity
    A28
    PF3D7_0422300Alpha tubulin 249.7GTP binding, GTPase
    activity, structural
    molecule
    A31
    PF3D7_0507800Conserved Plasmodium
    protein
    177.4NoneA, G40
    PF3D7_0509400RNA polymerase I340.7DNA binding,
    DNA-directed RNA
    polymerase activity
    G47
    PF3D7_0610400Histone H315.5DNA bindingA534
    PF3D7_0705500Inositol-phosphate
    phosphatase, putative
    330.7NoneG31
    PF3D7_0906100Developmental protein,
    putative
    21.9NoneA, G37
    PF3D7_1102400Flavoprotein, putative78.5NoneA27
    PF3D7_1134700DNA-directed RNA
    polymerase 1, subunit
    2, putative
    175.5DNA binding,
    DNA-directed RNA
    polymerase activity,
    ribonucleoside binding
    G37
    PF3D7_1215100Conserved Plasmodium
    protein
    113.0ATP binding, actin
    binding, calmodulin
    binding, motor activity
    IG27
    PF3D7_1216900DNA binding
    chaperone, putative
    111.1DNA binding, heat
    shock protein binding
    A36
    PF3D7_1218800Conserved
    Plasmodium sexual
    stage protein,
    putative
    39.6NoneG949
    PF3D7_1235700ATP synthase subunit
    beta, mitochondrial
    58.4Hydrogen ion
    transporting ATP
    synthase activity,
    rotational mechanism
    A116
    PF3D7_1313500Conserved Plasmodium
    membrane protein
    209.1Hydrolase activity,
    triglyceride lipase
    activity
    G42
    PF3D7_1319200Conserved Plasmodium
    protein
    103.9Flavin adenine
    dinucleotide binding
    G33
    PF3D7_1325900Conserved Plasmodium
    protein
    326.2ATP binding, actin
    binding, calmodulin
    binding, motor activity
    G34
    PF3D7_1337500Conserved Plasmodium
    protein
    393.3Calcium ion binding,
    receptor activity
    IG33
    PF3D7_1353000Tryptophan-rich
    antigen, pseudogene
    96.3NoneA49
    PF3D7_1411400Plastid replication-
    repair enzyme
    235.83′-5′ Exonuclease
    activity, ATP binding,
    DNA binding, DNA
    helicase/polymerase
    activity
    G31

    *MS evidence for mature gametocytes (G), immature gametocytes (IG), and asexual stages (A), based on the following published references (20, 23).

    • Table 2 Comparative microscopy, qPCR/PCR, LC-MRM, and LFIA for 100 samples collected from children with subclinical parasitemia from Cameroon.

      Time to readout for LFIA test line positivity and a maximum of 15 min for negative tests are included (right column).

      Case codeAgeMicroscopyqPCR/PCRPSSP17
      GametocyteTrophozoitePfs2518S rRNALC-MRMLFIATime to
      readout
      A11512++0.05765+15
      A12011+++0.03222+15
      A1878+++0.01779+10
      A193*7+++0.02164+/−15
      A22510+++0.04005+>20
      A22711+++0.02615+10
      A26810+++0.04036+15
      A27010+++0.04381+10
      A27811+++0.01951+15
      A27912+++0.02216+10
      A29010+++0.05321+>20
      A4478+++0.03030+15
      A4507+++0.03537+>20
      A45210+++0.01364+15
      A4608+++0.03212+>20
      A4749+++0.01039++5
      A4758+++0.01483+10
      A49310+++0.01932++5
      B00610++0.02097+10
      B00710+++0.01784+>20
      B0239++++0.05046+>20
      B02413++0.04245++5
      B0266+++0.08123+15
      B0287++++0.06404++5
      B02910+++0.08174+15
      B0349++++0.04904+5
      B04313+++0.01549+10
      B04613+++0.06638+10
      B0928++++0.07328+5
      B0965++++0.03484+15
      B1158+++0.05615+10
      B1246+++0.0123115
      B1267+++0.09663+15
      B12810+++0.07039++5
      B1457++0.02850++5
      B33610++0.07140++5
      B3458+++0.00521++5
      B3498++0.04474+10
      B35014+0.04074+15
      B3608++0.0231315
      B38012++0.04243++5
      B38112++0.0692415
      B39011++0.03683+10
      B39216+++0.04583+15
      C00110+++0.03759+10
      C00910+++0.07004+5
      C01013+++0.08945++5
      C024*12+++0.06621++5
      C026*10+++0.04700+15
      C046*12+++0.02633+>20
      C0759+++0.01026+10
      C08012++0.01808++5
      C084*8++0.04231+>20
      C095*10++0.01362++5
      C100*10++0.06038+/−10
      C10611++0.0297315
      C1078+0.02309+10
      C112*10++0.01808+/−15
      C117*12++0.10120+/−15
      C1238+++0.01950++5
      C3347+++0.04649+5
      C3376+++0.04951+10
      C3397+++0.03356+15
      C3407+++0.02086+10
      C3416++++0.01425+>20
      C3575++0.0561615
      C3588++0.02162+15
      C3638+++0.04584+5
      D03011+++0.03905++5
      D033*13+++0.03652++5
      D04010++0.03692++5
      D04112++0.0147915
      D0467++0.05459+5
      D0499++0.02713+>20
      D05312++0.04751+10
      D0546+++0.0172315
      D0578++0.02074+10
      D0629++0.00967+>20
      D15510++0.03353+15
      D4469++0.02517+10
      D4478++0.04891+15
      D4499++0.01917+>20
      D4539+++0.04835+10
      D4569+++0.01120+15
      D45913+++0.04252+10
      D46113++0.02149++5
      D46211++0.03318+15
      D48210+++0.03762+10
      D4879+++0.0044515
      D4967++0.02504+15
      D4999+0.06422+10
      D5008+0.01736++5
      D5168++0.03949+5
      D517*9+0.02459++5
      N22710++0.04180++5
      N2349++0.01287+10
      N24510+++0.08426+10
      N2559++0.10392+15
      N3118++0.04298+10
      N3186+++0.04264+10

      *Samples included in Fig. 3D.

      Supplementary Materials

      • www.sciencetranslationalmedicine.org/cgi/content/full/11/473/eaan4479/DC1

        Materials and Methods

        Fig. S1. The correlation of PAR and female gametocyte abundance per microliter of blood (based on pfs25 transcript number).

        Table S1. The complete list of P. falciparum proteins identified in the saliva from children with subclinical infection in Yaoundé, Cameroon.

        Table S2. LM analyses of blood samples from children (5 to 12 years old) with subclinical infections in Yaoundé, Cameroon.

        Table S3. Description of samples collected from schools in Cameroon.

        Table S4. Description of samples collected from homes in Zambia.

        Table S5. Quantification of gametocytes per μl of blood in a subset (n = 100) of samples from Cameroon.

        References (3644)

      • The PDF file includes:

        • Materials and Methods
        • Fig. S1. The correlation of PAR and female gametocyte abundance per microliter of blood (based on pfs25 transcript number).
        • Table S1. The complete list of P. falciparum proteins identified in the saliva from children with subclinical infection in Yaoundé, Cameroon.
        • Table S2. LM analyses of blood samples from children (5 to 12 years old) with subclinical infections in Yaoundé, Cameroon.
        • Legends for tables S3 to S5
        • References (3644)

        [Download PDF]

        Other Supplementary Material for this manuscript includes the following:

        • Table S3 (Microsoft Excel format). Description of samples collected from schools in Cameroon.
        • Table S4 (Microsoft Excel format). Description of samples collected from homes in Zambia.
        • Table S5 (Microsoft Excel format). Quantification of gametocytes per μl of blood in a subset (n = 100) of samples from Cameroon.

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