stacks.cdc.gov · Web viewspecies and quantify parasite density of these samples, photo-induced electron transfer (PET)-PCR was performed utilizing both genus- and species-specific

Supplementary Methods

Supplementary Methods 1. Sample collection

Plasma was obtained from 239 anonymized blood samples from travelers returning to the U.S.

after travelling to malaria endemic areas submitted to the U.S. Centers for Disease Control and

Prevention (CDC) for malaria diagnostic support and surveillance purposes. To determine Plasmodium

species and quantify parasite density of these samples, photo-induced electron transfer (PET)-PCR

was performed utilizing both genus- and species-specific primers as previously described [1], and

results confirmed by nested PCR [2]. Blood samples from persons residing in the U.S. with no travel

history were obtained from blood donors to serve as malaria-negative controls.

Dried blood spot samples from a 2016 Angola health facility survey [3] gathered by teams

visiting 89 randomly selected public health facilities in Huambo and Uíge Provinces in Angola were

screened using the bead assay. Randomly selected outpatients were asked about history of fever and,

regardless of clinical suspicion for malaria, provided blood for malaria RDT testing and anonymous

dried blood spot (DBS) collection on Whatman 903 filter paper (GE Healthcare, Chicago, IL).

Supplementary Methods 2. Blood sample preparation and assay protocol

Liquid blood and plasma samples were diluted to a final concentration of 1:10 in a blocking

buffer (Buffer B: 0.5% Polyvinyl alcohol [Sigma; P8136] 0.5% polyvinylpyrrolidine [Sigma; PVP360],

0.1% casein [ThermoFisher; 37528], 0.5% BSA [Sigma; A9418], 0.3% Tween-20, 0.05% sodium azide,

and 0.01% E. coli extract to prevent non-specific binding). The Angolan DBSs were shipped to the CDC

laboratories in Atlanta, GA under ambient conditions, and blood was eluted in a solution of phosphate

buffered saline (PBS), 0.05% Tween20, and 0.02% NaN3 to a final concentration of 1:20 dilution of

whole blood.

The presence and quantification of antigens was performed with similar methodology as

described previously using the bead-based Luminex® platform (Luminex Corp., Austin, TX) [4]. Three

unique bead regions (Bio-Plex COOH bead, BioRad, Hercules, CA; 171506XXX) were individually

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coated by the EDC/Sulfo-NHS intermediate reaction [4] with separate antibodies specific for each

antigen to be captured: Plasmodium aldolase (12.5ug/12.5x106 beads, rabbit IgG anti-aldolase, Abcam,

Cambridge, UK; ab207494), Plasmodium LDH (12.5ug/12.5x106 beads, mouse IgG anti-LDH, BBI

Solutions, Cardiff, UK; BM355-Z8F7), and P. falciparum PfHRP2 (20ug/12.5x106 beads, mouse IgG

anti-HRP2, Abcam; ab9206). For the assay, a mix of the three coupled bead regions was made in 5mL

Buffer A (PBS, 0.5% BSA, 0.05% Tween20, 0.02% NaN3) so that 1,500 of each bead region would be

added per well in the assay plate. Samples were incubated with 50uL of the bead mix in filter bottom

plates (Millipore; MABVN1250) for 90min under gentle shaking and subsequently washed three times

with 100uL wash buffer (PBS, 0.05% Tween20). Beads were incubated for 45min with a 50uL mix of

detection antibodies: anti-pAldo (1:1000x, rabbit anti-aldolase, Abcam; ab207494), anti-pLDH (1:500x

of 2:1:1 mixture [BBI Solutions BM355-P4A2:BioRad Pv-pLDH HCA156:BioRad Pf-pLDH HCA158]),

and anti-HRP2 (1:500x, mouse IgG anti-HRP2, Abcam, ab9203). All detection antibodies were

previously biotinylated by Thermo Scientific EZ-Link Micro Sulfo-NHS-Biotinylation Kit (ThermoFisher

Scientific) according to the manufacturer’s protocol. Plates were washed three times, and subsequently

incubated with streptavidin-phycoerythrin (1:200x, Invitrogen, Carlsbad, CA). Plates were washed three

times, and after a final 30min wash step with reagent diluent, beads were washed once and

resuspended in 100uL PBS and read on a Bio-Plex 200 instrument (BioRad, Hercules, CA) by

generating the median fluorescence intensity (MFI) signal for 50 beads in each unique region, and then

the mean fluorescence intensity of the MFIs among duplicates. The final measure, denoted as MFI-bg,

was reported by subtracting MFI values from beads on each plate only exposed to sample diluent

during the sample incubation step.

To translate between a MFI-bg value and antigen concentration, equations for standard curves

of recombinant antigens were calculated (Fig. 1). Recombinant pLDH and PfHRP2 antigens were

provided by Microcoat Biotechnologie GmbH (Bernried, Germany), and lyophilized preparations were

rehydrated according to the manufacturer’s instructions. The Plasmodium vivax-specific isoform of

aldolase was produced at the CDC as described previously [5].

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Supplementary Methods 3. Detection of Plasmodium 18S rRNA from DBS by qRT-PCR

Dried blood spots were laser-cut from a Whatman 903 Protein Saver Card as described

previously [6] before being lysed in 2 mL NucliSens Lysis Buffer (bioMérieux) and incubated at 55°C for

30 minutes. Nucleic acids were then extracted and quantitatively amplified on the Abbott m2000 sp/rt

system as described previously [6] with modifications as follows. Pan-Plasmodium primers and probes

were as reported and the RT-PCR kit was the SensiFAST Probe Lo-ROX One-Step Kit (Bioline).

Cycling conditions were 10 min 48C, 2 min 95C and then 45 cycles of 5 sec 95C and 35 sec 50C.

Pan-Plasmodium primers were used to quantify Plasmodium 18S rRNA against an absolute Armored

RNA calibrator corresponding to full-length P. falciparum 18S rRNA (Asuragen) and copies were

converted to estimated parasites/mL using the laboratory-validated conversion factor of 7.4x103 copies

per ring-stage P. falciparum parasite. Multiplexed human TATA-Binding Protein (TBP) primers/probe

served as an internal control and P. falciparum primers/probe were used to specifically identify this

species. Estimated parasite densities were reported for samples containing ≥ 20 parasites per mL of

whole blood; concentrations below this were reported as “Not Detected” [6]. The qRT-PCR has been

benchmarked against DNA-based qPCR assays and demonstrated quantitative and qualitative

agreement with minimal quantitative bias (S. Murphy, pers. comm.).

Supplementary Methods 4. Species identification and confirmation of Pfhrp2 and Pfhrp3 genotype

For Angolan samples showing an antigen profile non-indicative of wild-type P. falciparum

infection and warranting further molecular classification, DNA was extracted from DBS using Qiagen

Mini Kit (Qiagen, Germantown, MD) according to the manufacturer’s protocol. Species identification

was confirmed by both PET-PCR [1] and nested PCR [2] methods. To assay for presence of the Pfhrp2

and Pfhrp3 genes, nested PCR was performed. Primers for exon 1-2 of Pfhrp2, (nest 2 base pair

location: 1,375,053 - 1,375,275) were used. Similarly, to amplify Pfhrp3, primers spanning exon 1-2,

(nest 2 base pair location 2,818,448 - 2,818,673) were also used. Amplification was performed as

follows; 95°C for 5 minutes (95°C for 30 seconds, XX for 30 seconds, 68°C for 30 seconds) x 30 cycles,

68°C for 5 minutes, hold at 4°C (see Supplementary Table 4 for annealing temperatures). Products

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were visualized on a 2% agarose gel to verify PCR amplification [7]. As a single-copy gene PCR control

for the DNA samples, the Pfmsp1 and Pfmsp2 genes were amplified and PCR products run on agarose

gel to verify the presence of enough DNA in each sample to amplify a single-copy gene. A sample’s

genotyping for Pfhrp2/3 was only reported upon successful amplification of both Pfmsp1 and Pfmsp2

gene primers [8].

References

1. Lucchi NW, Karell MA, Journel I, et al. PET-PCR method for the molecular detection of malaria parasites in a national malaria surveillance study in Haiti, 2011. Malar J 2014; 13:462.2. Snounou G. Detection and identification of the four malaria parasite species infecting humans by PCR amplification. Methods Mol Biol 1996; 50:263-91.3. Plucinski MM, Ferreira M, Ferreira CMF, et al. Evaluating malaria case management at public health facilities in two provinces in Angola. Malaria Journal 2017; 16.4. Rogier E, Plucinski M, Lucchi N, et al. Bead-based immunoassay allows sub-picogram detection of histidine-rich protein 2 from Plasmodium falciparum and estimates reliability of malaria rapid diagnostic tests. PLoS One 2017; 12:e0172139.5. World Health Organization. Malaria rapid diagnostic test performance. Results of WHO product testing of malaria RDTs: round 6 (2014-2015). Geneva: World Health Organization 2015.6. Murphy SC, Prentice JL, Williamson K, et al. Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials. The American journal of tropical medicine and hygiene 2012; 86:383-94.7. Abdallah JF, Okoth SA, Fontecha GA, et al. Prevalence of pfhrp2 and pfhrp3 gene deletions in Puerto Lempira, Honduras. Malaria journal 2015; 14:19.8. Cheng Q, Gatton ML, Barnwell J, et al. Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3: a review and recommendations for accurate reporting. Malar J 2014; 13:283.

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Supplementary Tables

Supplementary Table 1. Assay Signal Positivity Cutoff Values and Estimated Limit of Antigen

Detection

AntigenPositivity Cutoff Signal

(MFI-bg)Limit of Detection

(pg/mL)PfHRP2(A) 80 6.7PfHRP2(B) 80 8.8PfHRP2(C)   80 15.8Pf-pLDH 938 4,425Pv-pLDH 938 4,435Pv-pAldo 95 128

Supplementary Table 2. Regression Estimates for Patient Antigen Concentration Based on

Parasite Density from Travelers Returning to the U.S. with Malaria Infection 5

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y=a(x)^by: Antigen concentration (pg/mL) a: exp(intercept)x: Parasitemia (parasites/uL)b: slope

P. falciparum Antigen a b R^2

pAldolase 1510 0.323 0.26pLDH 325 0.543 0.35HRP2 10315 0.219 0.11

P. vivax Antigen a b R^2

pAldolase 1122 0.497 0.25pLDH 8451 0.535 0.13

P. ovaleAntigen a b R^2

pAldolase 2475 0.232 0.14pLDH 5478 0.493 0.4

P. malariaeAntigen a b R^2

pAldolase 1302 0.255 0.24pLDH 10463 0.247 0.1

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Supplementary Table 3. Sensitivity, Specificity, Positive Predictive Value and Negative

Predictive Value of Multipex Antigen Detection, Using Ultrasensitive qRT-PCR as Gold Standard

  PCR+ PCR- Sensitivity Specificity PPV NPVHRP2+ 88 58

90% 48% 60% 84%HRP2- 10 53

pLDH+ 20 0 21% 100% 100% 59%pLDH- 77 111

pAldo+ 44 1 45% 99% 98% 67%pAldo- 54 110

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Supplementary Table 4. Primer Sequences and Annealing Temperatures for Pfhrp2/3 nPCR Reactions

Gene ReactionPrimer name Primer sequence 5'-3'

Annealing temp (°C)

Amplicon size (base pairs)

Pfhrp2 primary 2E12F1 GGT TTC CTT CTC AAA AAA TAA AG 55 228

2E12R1 TCT ACA TGT GCT TGA GTT TCG

nested 2E12F GTA TTA TCC GCT GCC GTT TTT GCC 62

2E12R CTA CAC AAG TTA TTA TTA AAT GCG GAA

Pfhrp3 primary 3E12F1 GGT TTC CTT CTC AAA AAA TAA AA 53 225

3E12R1 CCT GCA TGT GCT TGA CTT TA

nested 3E12F ATA TTA TCG CTG CCG TTT TTG CT 62

3E12R CTA AAC AAG TTA TTG TTA AAT TCG GAG

*Adapted from Abdallah et al (2015). Note since Abdallah et al was published, more recent updates of the 3D7 genome have revealed a 1 base pair difference in two primers, in bold and underlined as follows, 3E12R1 (5'-CCTGCATGTGCTTGACTTTC-3' ), 3E12F (5'- ATATTATCCGCTGCCGTTTTTGCT-3')

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Supplementary Figure 1

Figure S1. Detection of Plasmodium Aldolase and LDH from Persons Mono-infected with Human Malaria. Two-by-two tables are separated by Plasmodium species, and show concordance of pan-Plasmodium antigens by the bead assay.

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Supplementary Figure 2

Figure S2. Distribution of Antigenemia for Antigen Positive Angolan Patients. A) Histograms display range of blood antigen concentrations for Plasmodium aldolase and LDH, as well as PfHRP2 for any antigen positive persons. B) Range of HRP2 blood antigen concentrations by different antigen profiles found in the Angolan population.

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Supplementary Figure 3

Figure S3. Relationship between Antigenemia and Age for Persons Attending Angolan Health Facilities. For antigen positive individuals, the relationship between antigen concentration and age was modeled through LOESS regression.

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Supplementary Figure 4

Figure S4. Relationship between Antigen Concentration and qRT-PCR Estimated P. falciparum Parasite Density for Persons from Angola Survey who were Selected for qRT-PCR Analysis. Regression performed with antigen concentrations retained on continuous scale in upper panels. Lower panels display dose-response curves generated by logistic and LOESS regression with parametric estimates shown in Table S3.

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