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RECOMMENDED CITATION
Poirier MJP,a Moss DM,b Feeser KR,b et al. Detection of immunoglobulin G responses in
Haitian children exposed to chikungunya, dengue, and malaria using a multiplex bead assay.
[Submitted]. Bull World Health Organ E-pub: 14 Mar 2016. doi:
http://dx.doi.org/10.2471/BLT.16.173252
Detection of immunoglobulin G responses in Haitian children
exposed to chikungunya, dengue, and malaria using a
multiplex bead assay
Mathieu JP Poirier,a Delynn M Moss,b Karla R Feeser,b Thomas G Streit,a
Gwong-Jen J Changc, Matthew Whitney,c Brandy J Russell,c Barbara W
Johnson,c Alison J Basile,c Christin H Goodman,c Amanda K Barry,b &
Patrick J Lammieb
a
University of Notre Dame Haiti Program, Hopital Ste. Croix, Leogane, Haiti
b
Centers for Disease Control and Prevention, Atlanta, GA 30329 USA
c
Centers for Disease Control and Prevention, Ft. Collins, CO 80521 USA
Correspondence to Mathieu JP Poirier (email: [email protected]).
(Submitted: 12 March 2016 – Published online: 14 March 2016)
1
Abstract
Objective: A solution to the challenge of differentiating the newly introduced pathogen of
chikungunya virus (CHIKV) to Haiti in 2014 from endemic dengue virus (DENV) and other
pathogens was sought.
Methods: A multiplex bead assay (MBA) was used to detect immunoglobulin (Ig)G responses to
one recombinant CHIKV envelope 1 antigen (CHIKV E1), two DENV virus-like particles
(DENV-2 VLP and DENV-3 VLP), and three recombinant Plasmodium falciparum merozoite
surface protein-1 antigens (MSP-1) using blood samples collected longitudinally from 61
children from 2011 to 2014 and single blood spots collected from 127 children in 2014 in
Leogane, Haiti.
Findings: No IgG responses to CHIKV E1 antigen were observed from 2011 to 2013, but in
2014, the prevalence of positive IgG responses was 78.7%. The prevalence of CHIKV E1
responses was high and similar across all age groups; by contrast, the prevalence of responses in
2014 to DENV antigens increased steadily with age. Prevalence of positive IgG responses to the
MSP-1 antigens was < 12%. Cluster analysis by Getis-Ord Gi Hot Spot Analysis showed
increasing IgG responses to CHIKV E1 and DENV-2 VLP antigens in peri-urban areas
compared to households situated closer to the sea shoreline.
Conclusion: Serologic evidence documents the rapid and intense dissemination of CHIKV in
Haiti. The MBA is an efficient and economical method to monitor the prevalence of exposures to
multiple pathogens simultaneously.
2
Introduction
Dengue virus (DENV) and chikungunya virus (CHIKV) can cause similar symptoms in
humans and are thus difficult to differentiate in clinical settings. Chikungunya is a term derived
from the word in the Kimakonde language, meaning "to become contorted", which describes the
stooped appearance of persons exhibiting the distinctive symptom of debilitating polyarthralgia.
CHIKV belongs to the genus Alphavirus and the family Togaviridae, and other symptoms
include fever, rashes, myalgia, nausea, vomiting and diarrhea, headache, fatigue, and
conjunctivitis [1]. DENV can also cause joint pain and is often referred to as "breakbone fever".
DENV belongs to the genus Flavivirus in the family Flaviviridae and human infection can either
lead to dengue with or without warning signs, or to the potentially deadly severe dengue. Both
CHIKV and DENV are transmitted to humans by Aedes aegypti and Ae. albopictus mosquitoes.
The spread of CHIKV has intensified in recent decades. CHIKV appears to have been
first described in the 1820s in both modern day Tanzania and India, but was not isolated until
1952 in Tanzania [1-3]. In the early 1960s, CHIKV infections were reported in Bangkok,
Thailand and from 1963 to 1973 in India [4-6]. The speed at which CHIKV is capable of
spreading was demonstrated in the 2006 epidemic in Réunion, where it was estimated that more
than 40,000 CHIKV cases occurred per week, resulting in more than 225,000 cases by the end of
the epidemic – about one third of the island’s population [7]. It is speculated that the primary
vector in the Réunion outbreak was Ae. albopictus. By early 2013, CHIKV infections had been
reported in Africa, Asia, Europe, and parts of Oceania [8]. CHIKV infections occurred in the
Caribbean basin, including Haiti, in 2014 [9].
Given this history, and the presence of Ae. aegypti and Ae. albopictus, which had been
spreading DENV in Haiti, conditions in Haiti were very favorable for the introduction and
3
explosive dissemination of CHIKV [10,11]. In December of 2013, the World Health
Organization reported the local spread of CHIKV in nearby St. Martin [11]. By the last week of
April 2014, the International Federation of Red Cross and Red Crescent Societies announced the
arrival of CHIKV in Haiti, and on June 6, 2014, the Centers for Disease Control and Prevention
reported 6,318 cases [13].
Because symptoms and epidemiology of CHIKV and DENV infections are similar and
occurring against a backdrop of other infectious diseases, it would be informative to be able to
assess not only IgG responses to these pathogens, but also to antigens from other pathogens. The
multiplex bead assay (MBA) (Luminex, Austin, TX) offers these advantages. This dual-laser,
cytometric-based microsphere technology has proven useful in many serological studies,
spanning a wide range of antigens. From a single serum specimen or blood spot, the MBA can
acquire data on multiple antigens simultaneously [14-20]. The MBA utilizes a 96-well format
with polystyrene microspheres that can be coupled to an antigen of the researcher’s choice. The
microspheres have unique inherent spectral signatures that identify the antigen-coupled beads by
a red laser, and a green laser quantifies the amount of captured analyte, IgG. Using the MBA, we
assessed the IgG responses to DENV, CHIKV, and malaria antigens in a group of Haitian
children who were monitored longitudinally before the introduction of CHIKV in Haiti from
2011 to 2013 and after CHIKV introduction in 2014.
Methods
Study Population and Design. The study was reviewed and approved by the Ethics
Committee of Hôpital Sainte Croix, Leogane, Haiti, by the Centers for Disease Control and
Prevention Institutional Review Board, and by the University of Notre Dame Institutional
Review Board.
4
For a separate filariasis study, whole blood specimens were collected onto circular filter
paper extensions (TropBio Pty Ltd, Townsville, Queensland, Australia) by finger prick with each
protrusion on the filter-paper wheel designed to absorb 10 µl of whole blood. After collection,
the blood spots were allowed to dry, and then stored at -20º C, as previously described [15].
Samples were collected from 61 children before and after the introduction of CHIKV in Ca Ira, a
small coastal community near Leogane, Haiti. There were four community-wide systematic
collections from participating children in December (Dec) 2011, February (Feb) 2013, and Dec
2013. The fourth collection was made in August (Aug) 2014 after learning of the CHIKV spread.
Of the 61 children, all 61 provided a blood spot in each of the first, third, and fourth collections,
and 34 of the 61 (55.7%) also provided a blood spot in the second collection. In addition, single
blood spots were collected from a systematic cross-sectional group of 127 children who lived in
Ca Ira in August 2014. The age range for all children was 2 to 14 years.
Technical Appendix. All laboratory methods and analytical procedures are detailed in
the technical appendix.
Results
MBA Controls and Cutoffs. Median and range of median fluorescence intensity minus
background (MFI-bg) for each antigen are shown in Table 1, along with cutoff values. The
percent coefficient of variation (% CV) from all plates was less than 10.6. Sera known to be
highly reactive to the antigens, and MAbs known to be specific for the DENV-2 VLP and
DENV-3 VLP antigens showed high MFI-bg by MBA and/or high OD by ELISA, indicating
sufficient antigen coupling to the respective beads (data not shown) [21-23].
Prevalence of pathogen exposure over time. Table 2 shows the MBA prevalence of
positive IgG responses to CHIKV, DENV, and MSP-1 by year of specimen collection. There
5
were no responses to the CHIKV E1 antigen observed from 2011 to 2013 (Fig. 1), but, in 2014,
78.7% of samples tested were positive for antibodies to CHIKV E1. The prevalence of DENV
was 62.3% in 2011 and increased to 79.4% in Feb 2013 and was similar in 2014. Prevalence of
positive IgG responses to MSP-1 was less than 12% throughout the study.
Most of the 61 children showed various combinations of exposures to the three pathogens
studied. Only three (5%) showed no exposure to any of the three pathogens.
Prevalence of pathogen exposure in the cross-sectional group of children. Table 2
also shows the results from the 127 children who provided single blood spots in 2014. The
prevalence of positive IgG responses to CHIKV was 75.6% (96/127). In the same group, the
prevalence of positive IgG responses to DENV and MSP-1 was 60.6% (77/127) and 6.3%
(8/127), respectively.
Most of the cross-sectional children showed various combinations of exposures to the
three pathogens studied. Only 17 (13.4%) showed no exposure to the three pathogens.
IgG responses over time. Figure 1 shows the median MFI-bg to the antigens from 2011
to 2014. The median MFI-bg to the CHIKV E1 antigen was low from 2011 to 2013, and then, in
2014, a large increase was observed. Unlike the median IgG responses to the CHIKV E1 antigen,
the median IgG responses to the DENV-2 and DENV-3 VLP antigens were relatively low in
2011 but steadily increased through 2013 with a slight decrease in 2014. For the entire study
period, the median MFI-bg to the MSP-1 antigens remained low.
Complexities of IgG responses in one child. Figure 2 shows the complexities of the IgG
responses for one child who submitted a blood spot in all collection times. High IgG responses to
the CHIKV E1 antigen were only observed in 2014. Low but positive IgG responses to DENV-2
and DENV-3 VLP antigens were observed in 2011 that declined to negative responses in Feb
6
2013, suggesting an initial exposure prior to 2011. Afterwards, in Dec 2013, a tremendous
increase in IgG responses occurred, suggesting a re-exposure to DENV. For this child, IgG
responses to the MSP-1 antigens never exceeded the cutoffs over the period of follow up.
IgG responses of all children in 2014. Figure 3 shows a box and whiskers plot of IgG
responses to each of the six antigens for all the children (61 longitudinal plus 127 cross-sectional
children [Tables 2 and 3]) in 2014. Median responses to CHIK E1 were higher than responses to
DEN antigens whereas median MSP-1 responses were near background.
Prevalence and median IgG responses by age groups. Figure 4 shows the median
MFI-bg responses and prevalence by age for the group of 127 children in the cross sectional
cohort in 2014. The median MFI-bg (IgG responses) and the prevalence to the two DENV
antigens showed similar patterns with increasing prevalence and IgG responses with age (Figs.
4A and B). In contrast, both the median MFI-bg responses and the prevalence of positive IgG
responses to the CHIKV E1 antigen were similar across all age groups.
Hot Spot Analysis. Shown in Figure 5 are the clusters identified by the Getis-Ord Gi Hot
Spot Analysis which is used to define cluster of antibody responses that differ from the median
response. The study area just outside urban Leogane is shown by the black rectangle (Fig. 5A).
For CHIKV E1 and DENV-2 antigens, the clusters showed decreasing IgG responses from red to
blue in the children as one moves away from the city of Leogane, towards the ocean (Figs 5B
and 5D, respectively). Conversely, malaria MSP-1/GST exhibited no discernible pattern, due to
low prevalence (Fig. 5C).
Discussion
This study confirms the explosive transmission of CHIKV in a naïve population. As of
the first week of 2015, the Pan American Health Organization reported 64,695 suspected cases of
7
CHIKV infection in Haiti [13]. By contrast, neighboring Dominican Republic reported over
500,000 cases, a difference which is more likely due to under-reporting in Haiti than differences
in population size or environment. A nationwide household survey conducted by the Igarapé
Institute in May 2014 estimated that 9.2% of Haitian individuals had already been affected, a rate
that would indicate almost 1 million cases within a month of introduction [24]. The results of this
study are the first to provide longitudinal serological evidence of CHIKV introduction in Haiti;
the overall prevalence of antibody to CHIKV increased from 0 in 2013 to 76.6% ([144/188]
Tables 2 and 3) in 2014.
The CHIK envelope 1 antigen used in this study matched the sequence of the virus
involved in the Réunion outbreak that contained a point-mutation in amino acid 226, where
alanine mutated to valine [7]. The A226V strain was believed to be the East/Central/South Africa
(ECSA) genotype and is suspected to be more adaptable and efficient in Ae. albopictus than in
Ae. aegypti [7]. The West African and Asian genotypes are the only other known strains, of
which the latter has been identified as the genotype responsible for the recent Caribbean CHIKV
outbreak [9]. Antibodies from both the Asian and ECSA genotypes are cross-reactive
(unpublished observation) as demonstrated by a MAb-Antigen capture MBA and an IgG ELISA,
using AgSMB [25,26]. The similarity of the prevalence and median MFI-bg responses across all
age groups (Figure 4A and B) is a reflection of the transmission efficiency of CHIKV.
Among children with blood spots collected longitudinally, initial and secondary
anamnestic immune responses could be shown to some of the antigens (e.g, responses to DENV2 and DENV-3 VLPs in Figure 2). No secondary immune responses were observed among
children who had positive MSP-1 responses, consistent with the relatively low exposure to
malaria in this community.
8
The input of MBA and GPS data into GIS software showed an increasing gradient of IgG
responses to CHIKV and DENV antigens from beach to urban areas (Fig. 5). The areas of high
intensity immune responses may help pin point areas of high-intensity mosquito transmission,
which is valuable information for mosquito control. The GPS data may also indicate the
preference of the vectors of these viruses for habitats with lower salinity or less wind. High
salinity decreases early life-stage survival rates in mosquitoes [27]. Human population densities
similarly increase towards urban areas, which could also play a role in transmission dynamics.
Other vectors, such as Anopheles and Culex have received more attention than Ae. Albopictus as
targets of vector control [28].
The prevalence of positive antibody responses to DENV in this study was consistent with
other DENV studies in Haiti. Prior to 1969, DENV had not been reported in Haiti, but a study
conducted from 1969 to 1971 in Port-au-Prince using a hemagglutination inhibition test found
that the prevalence of persons exposed to DENV increased with age, with 43% of children 1 to 5
years of age, 60% of children 6 to 10, and 76% of adults 41 to 50 showing an antibody response
[10]. In a 1996 study in Port-au-Prince, 85% of 6 to 13 year old children were positive for at
least one of the four DENV serotypes by a neutralizing antibody test [29]. Similarly, we found
the prevalence of positive IgG responses to the DENV antigens increased with age in the 127
children who submitted single blood samples in 2014 (Fig. 4B). In Haitian adults, the prevalence
of positive IgG responses to DENV can be as high as 100% [30]. We found 96% prevalence of
positive IgG responses to DENV-2 VLP and DENV-3-VLP in Haitian adults who submitted
serum specimens in a 1996 filariasis study (data not shown).
Consistent with a decline in P. falciparum in Haiti from historically high levels, the
current prevalence in Leogane is extremely low. In a study conducted in communities other than
9
Ca Ira in Leogane, the prevalence of P. falciparum, using the same MSP-119/GST antigen, was
similar to this study [31].
The MBA elucidated exposure for four diverse infectious agents, including members of
two arboviruses families, a protozoan and a metazoan parasite (lymphatic filariasis study, data
not shown). Moreover, it showed not only the ability to detect simultaneously IgG responses to
multiple antigens from multiple pathogens but also, in some cases, the ability to determine when
the child was exposed and if the child was exposed to single, multiple, or no pathogens. Unlike
single blood samples, multiple blood samples on the same child showing fluctuations in IgG
responses to antigens over time could help define exposure histories. The MBA can easily
incorporate relevant antigens from many suspected pathogens.
Our population study was relatively small; however, a large malaria study conducted in
early 2015 that included the same CHIKV antigen showed a median prevalence of 82% CHIKVpositive IgG responses in urban areas throughout Haiti, which is comparable to our findings in
Leogane, while showing a median prevalence of 55% in rural areas (unpublished observation,
Eric Rogier).
The MBA serological platform is not only at least as sensitive as ELISA, but it is also
more economical than ELISA when testing of multiple antigens is needed. This can provide a
wealth of valuable information to clinicians and for sero-surveillance studies, both of which have
become even more important with the confirmed autochthonous transmission of Zika virus in
Haiti and across the Americas [17,32]. Funding for surveillance of a single disease is becoming
increasingly difficult to defend, and the versatility offered by MBA provides a solution for
multiple pathogens in the same study. Longitudinal monitoring of multiple diseases in the
10
challenging context of developing countries is possible, and may become an invaluable tool in
the future of infectious disease research.
Acknowledgments: We wish to thank all participants who took part in this study, as well as the
dedicated team of professionals in Haiti and the United States who made this study possible.
Funding: This work was supported by the Bill & Melinda Gates Foundation [grant number
402.01], Abbott/AbbVie, and a donor who wishes to remain anonymous. No agreement was
entered with funders that may have limited the ability to complete research as planned. The
corresponding author had full control of all study data and had final responsibility for the
decision to submit for publication.
11
Figure 1. Median MFI-bg (IgG responses) over time from 61 children that donated blood
samples. Plots of median MFI-bg against all antigens at December (Dec) 2011, February (Feb)
2013, Dec 2013, and August (Aug) 2014. Antigens shown by color in figure legend.
12
Figure 2. MFI-bg (IgG responses) of one child with samples collected longitudinally. Plot of
MFI-bg over time to all antigens on one child who submitted blood spots in all collections.
Times are month (December [Dec], February [Feb], and August [Aug]) and year. Age of child
(years) shown in parentheses. Antigens shown by color in figure legend.
13
Figure 3. MFI-bg (IgG responses) of all children in 2014. Box and whisker plot showing IgG
responses to all antigens from all children (61 plus 127) in the year 2014. Horizontal bars in box:
low, 25 percentile; middle, median; upper, 75 percentile. Low whisker, 5 percentile; upper
whisker, 95 percentile; and outliers in closed circles.
14
Figure 4. Median MFI-bg (IgG responses) and IgG positive prevalence by age groups for the
127 children with single blood spots collected in 2014. A, plot of median MFI-bg to all antigens
by age groups. B, plot of prevalence of positive IgG responses to all antigens by age groups.
Antigens shown by color in figure legends.
15
Figure 5. Global Positioning Satellite Data analyzed by Getis-Ord Gi* Hot Spot Analysis, using
MFI-bg values from CHIKV E1, DENV-2 VLP, and MSP-119/GST antigens. A, location of
study (black rectangle) relative to the city of Léogâne. Sea coast near upper left corner of
rectangle; B, CHIKV E1 antigen; C, MSP-119/GST antigen; and D, DENV-2 VLP antigen.
Results displayed are Getis-Ord Gi* statistics (GiZ) which plots the relationship of the IgG
response for the cluster relative to the median response for all samples. The enclosed colored
symbols, the GiZ score, from the Getis-Ord Gi* Hot Spot analysis were aggregated and
displaced from their actual locations and show range from blue, statistically significant low MFIbg intensity, to red, statistically significant high MFI-bg intensity.
16
Table 1. Antigen, cutoffs, overall median, and range of IgG responses (MFI-bg) to each antigen
Antigen (Strain)
Cutoff
Median (Range) of Fluorescence Intensities (MFI-bg) *
DENV-2 VLP
982
10,168 (-18 to 28,395)
DENV-3 VLP
3615
15,047 (3 to 30,214)
CHIKV E1
640†
101 (-9 to 30,027)
MSP-119/GST
105
8 (-6 to 21,627)
MSP-142 (3D7)
189
29 (2 to 23,913)
MSP-142 (FVO)
79
20 (0 to 20,489)
GST
--
7 (-4 to 173)
*Possible MFI range is 1 to 32,766 without background (bg) subtracted. Possible fluorescence intensities with bg
subtracted (MFI-bg) can have negative value6.
†
Cutoff determined by all blood spots collected from Haitian children from 2011 to 2013.
Table 2. Prevalence of positive IgG responses to pathogens chikungunya (CHIKV), dengue
(DENV), and Plasmodium falciparum (MSP-1) of 61 Haitian children from 2011 to 2014, and of
the full 127 child cross-sectional cohort in 2014.
Pathogen
December 2011
February 2013
December 2013
August 2014
August 2014
% Prevalence
% Prevalence
% Prevalence
% Prevalence
% Prevalence
(No. Specimens)
(No. Specimens)
(No. Specimens)
(No. Specimens)
(No. Specimens)
CHIKV
0% (0/61)
0% (0/34)
0% (0/61)
78.7% (48/61)
75.6% (96/127)
DENV
62.3% (38/61)
79.4% (27/34)
80.3% (49/61)
78.7% (48/61)
60.6% (77/127)
P. falciparum (MSP-1)
11.5% (7/61)
2.9% (1/34)
8.2% (5/61)
6.6% (4/61)
6.3% (8/127)
17
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21
Technical Appendix
Antigens. The recombinant chickungunya mutant (A226V) envelope 1 antigen (CHIKV
E1) (CTK Biotech, San Diego, CA, Accession No. FR846306.1) was used. Two DENV antigens,
propagated using a eukaryotic plasmid vector which expressed the premembrane/membrane and
envelope proteins that self-assembled into noninfectious virus-like particles (VLP), DENV-2 and
DENV-3 VLP were used [1,2]. DENV-2 VLP has epitopes that react with dengue serotype 2 and
dengue serotype 4 antibodies, and DENV-3 VLP has epitopes that react to dengue serotype 3 and
dengue serotype 1 antibodies [3]. Recombinant Plasmodium falciparum merozoite surface
protein 1, a19-kDa fragment, (MSP-119) (clone 3D7) was obtained from the Zentrum für
Molekulare Biologie der Universität Heidelberg, Universität Heidelberg (Heidelberg, Germany)
and was linked with glutathione-S-transfersase (MSP-119/GST) [4,5]. In addition, MSP-142
(clone FVO) and MSP-142 (clone 3D7) were also included [6,7].
Antigen coupling to beads. The 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
method (Calbiochem, Woburn, MA) was used to convert carboxyl groups on polystyrene
microspheres (SeroMap Beads; Luminex Corporation, Austin, TX) to esters, which reacted with
primary amine groups on the antigens. Per 12.5 million beads, this reaction occurred in
phosphate buffered saline (PBS), pH 7.2, for DENV-2 VLP, DENV-3 VLP, and CHIKV E1; 30
µg each for the former two and 7.5 µg for the latter. For the malaria antigens, coupling was
performed in 50 mM MES, 0.85% NaCl, pH 5.0, using 28 µg MSP-119/GST, 15 µg MSP-142
(clone 3D7), and 15 µg MSP-142 (clone FVO).
Coupling efficiency was determined by monoclonal antibodies (MAb, 4G2 and D6) and
by sera known to be highly reactive to the antigens [8,9].
Blood Spot Elutions. Blood spots were eluted overnight in 500 µl PBS, 0.5% bovine
serum albumin, 0.5% polyvinyl alcohol, 0.8% polyvinylpyrrolidone, 0.5% casein, 0.3% Tween
20, and 0.02% sodium azide (buffer A). Afterwards, the elutions were diluted 1:4 with buffer A
22
that contained sufficient amounts of non-clarified crude Escherichia coli extract for a final 3 µg
per ml (buffer B), which removes IgG reacting to potential minute traces E. coli that could be
coupled to the beads. Serum specimens were diluted in buffer B at 1:400.
Multiplex bead assay. Eluted blood spots and serum specimens in buffer B were
clarified by centrifugation at 20,238 x g for 10 minutes. In a 96-well filtered-bottom plate,
clarified supernatant, 50 µl, was added to each well containing 1500 antigen-coupled beads from
each bead classification and was allowed to incubate with gentle shaking for 1.5 hours. Each
well was washed three times with 100 µl PBS containing 0.05% Tween 20 (PBST), using
vacuum filtration. For 45 minutes, bound IgG was probed with 50 µl per well of PBS containing
0.5% BSA, 0.05% Tween 20, 0.02% sodium azide (buffer C), 50 ng of biotinylated mouse antihuman IgG (clone H2; Southern Biotech, Birmingham, AL), and 40 ng of biotinylated mouse
anti-human IgG4 (clone HP6025; Invitrogen, South San Francisco, CA [clone H2 did not react
well with human IgG4]). Each well was washed three times with PBST as described. For 30
minutes, each well was exposed to 50 µl of buffer C containing 250 ng of streptavidin rphycoerythrin (Invitrogen, Eugene, OR). After washing as described, 50 µl buffer C only was
added to each well and gently shaken for 30 minutes. The beads were then washed once with
PBST and suspended in 100 µl of PBS per well. Data were acquired using a reader (Luminex
Corporation) equipped with Bio-Plex Manager 6.1 software (Bio-Rad, Hercules, CA), and data
was reported as the mean from the median fluorescence intensity (MFI), increasing fluorescence
signals assigned to available channels 1 – 32,766, from duplicate wells minus the background
(bg) from a serum specimen or blood spot blank (MFI-bg). Controls, diluted for mid-range MFIbg, were run on each plate for determination of inter-plate consistency.
Establishment of cutoffs for the MBA. For determination of serum specimen cutoff in
the validation of the CHIKV E1 antigen by MBA, serum specimens from 86 North American
adults who indicated no travel outside the US were used. MFI-bg outliers greater than 3.0
23
standard deviations (SD) above the mean were determined by z score and were eliminated. The
remainder was used to calculate the mean plus 3 SD as cutoff.
For determination of blood spot cutoff to the DENV-2 and DEN-3 VLP antigens, serum
specimens from the 86 North American adults who indicated no travel outside the US were also
used. Outliers greater than 3.0 SD above the mean were eliminated. The remainder was used to
calculate the mean plus 3 SD as cutoff (Table 1).
For determination of blood spot cutoff to the P. falciparum antigens, the mean plus three
SD were also used from the 86 serum specimens from the North American adults (Table 1).
Here, outliers were not excluded.
For establishment of blood spot cutoff to the CHIKV E1 antigen, all blood spots collected
from the 61 children in Dec 2011, Feb 2013, and Dec 2013 were used to calculate the mean plus
three SD (Table 1). No outliers were eliminated.
Validation of CHIKV E1 antigen. Serum specimens were used in the comparison of
IgG responses to CHIKV E1 antigen by MBA with IgG responses to inactivated CHIKV antigen
from suckling mouse brain (AgSMB) in an enzyme-linked immunosorbent assay (IgG ELISA)
[10]. In the comparison, 52 serum specimens collected from Cambodian adults in 2012 and 26
serum specimens collected from Haitian adults in 1996 were used. CHIKV was known to be
present in Cambodia in 2011 but was not present in Haiti in 1996 [11]. Briefly, the IgG ELISA
captured AgSMB by 1A4B-6 MAb, and normal suckling mouse brain (NorSMB) was used as an
antigen control. Bound IgG from the serum specimens was probed with goat anti-human IgG
linked with alkaline phosphatase. Color was developed using disodium p-nitrophenyl phosphate,
and optical density (OD) was read at 405 nm. Criteria for the determination of positive and
negative from the IgG ELISA have been described [10]. Agreement between MBA and ELISA
was assessed.
24
All of the 78 serum specimens were used in the determination of correlation between the
MBA and the IgG ELISA. The OD of the NorSMB was subtracted from the OD of the AgSMB
to yield delta OD.
Validation of CHIKV E1 Assay. Of 52 Cambodian serum specimens and 26 Haitian
serum specimens, only 50 generated results that were interpretable by the IgG ELISA criteria
[10]. If the IgG ELISA is considered the “gold standard”, then the CHIKV E1 antigen in the
MBA generated results that were 90% sensitive and 85% specific.
From all 78 serum specimens from Cambodia and Haiti, there was a relationship between
MFI-bg of the CHIKV E1 antigen from the MBA and the delta OD (OD AgSMB minus OD
NorSMB) from the IgG ELISA, showing a positive correlation (r2 = 0.85, P < 0.001).
One of the 1996 serum specimens from Haiti was just over the threshold for positivity in
both MBA and IgG ELISA, but plaque reduction neutralization test (PRNT) on this specimen
was negative for CHIKV (data not shown). Because of the negative CHIKV PRNT on this
specimen, we believe that five of the 2011 to 2013 blood spots from the 61 children that were
slightly above the 640 MFI-bg cutoff for CHIKV E1 antigen (range, 706 to1052 MFI-bg) would
also be PRNT negative. The IgG ELISA for CHIKV does not contain adequate serum volumes
for PRNT and has yet to be adapted for blood spots.
Definitions. For positivity, at least two of the three P. falciparum antigens (MSP-1) had
to be above the cutoff (Table 1). For positivity to the DENV antigens, either DENV-2 VLP or
DENV-3 VLP or both had to be above their cutoff (Table 1), and for positivity to the CHIKV E1
antigen had to be above its cutoff (Table 1).
Mapping and Hot Spot Analysis. Global position satellite (GPS) data from the
households of 143 children for whom we had GPS data and for whom we had August 2014
specimen collection were mapped using ArcGIS (ESRI, Redlands, CA). A Getis-Ord Gi* Hot
Spot analysis was run on the MFI-bg data using a 400 meter zone of indifference, which
25
represents the average range of female Ae.aegypti mosquitoes (12). Analysis was run for CHIKV
E1, DENV-2 VLP, and MSP-119/GST antigens. To prevent association of results with household
locations, enclosed colored symbols, the z-score (GiZ), from the Getis-Ord Gi* Hot Spot
analysis were aggregated and displaced from their actual locations. The colored GiZ symbols
range from blue, statistically significant low MFI-bg intensity, to red, statistically significant
high MFI-bg intensity.
Statistical Analysis. For determination of inter-plate consistency, the percent coefficient
of variation (% CV) was determined on the positive controls ran on each plate. For relationships
between groups, the correlation coefficient was determined by Spearman rank order.
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