T a el Medicine and In ec ious Disease 57 (2024) 102681
A ailable online 21 Decembe 2023
1477-8939/© 2023 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
E alua ion o he accu acy o a mul i-in ec ion sc eening es based on a
mul iplex immunoassay a ge ing impo ed diseases common in
mig an popula ions
Ru h Aguila
a
,
1
, Angeline C uz
a
,
1
, Al ons Jim´
enez
a
,
b
, Alex Almuedo
a
, Ca me Roca Saumell
c
,
d
,
Ma ina Gigan e Lopez
e
, O iol Gasch
, Gemma Falc´
o
g
, Ana Jim´
enez-Lozano
h
, Angela Ma ínez-
Pe ez
i
, Consol Sanchez-Collado
j
, And ea Tedesco
k
, Manuel Ca los L´
opez
l
, Ma ía
Jesús Pinazo
a
,
m
,
q
, Thais Leonel
n
, Zeno Biso i
k
, Anna F¨
a ne
o
,
p
, Ca lo a Doba˜
no
a
,
m
,
2
,
Ana Requena-M´
endez
a
,
m
,
o
,
p
,
2
,
*
a
Ba celona Ins i u e o Global Heal h (ISGlobal), Hospi al Clínic-Uni e si a de Ba celona, Ca e Rosell´
o 132, 08036, Ba celona, Spain
b
Biomedical Resea ch Ne wo king Cen e o Epidemiology and Public Heal h (CIBERESP), A enida Mon o e de Lemos 3-5, 28029, Mad id, Spain
c
Cen e d’A enci´
o P ima ia El Clo , Ins i u Ca al`
a de la Salu (ICS), Ca e Concilio de T en o 25, 08018, Ba celona, Spain
d
Facul a de Medicina i Ci`
encies de la Salu , Uni e si a de Ba celona (UB), Ca e Casano a, 143, 08036, Ba celona, Spain
e
Cen e d’A enci´
o P ima ia Cen e Num`
ancia, Ins i u Ca al`
a de la Salu (ICS), Ca e Num`
ancia 23, 08029, Ba celona, Spain
In ec ious Diseases Depa men , Hospi al Uni e si a i Pa c Taulí. Ins i u d’In es igaci´
o i Inno aci´
o Pa c Taulí. Uni e si a Au `
onoma de Ba celona, Pa c Taulí, 1,
08208, Sabadell-Ba celona, Spain
g
Cen e d’A enci´
o P ima ia San Miquel, Ins i u Ca al`
a de la Salu (ICS), Ca e F ancesc Maci`
a i Lluss`
a, 154, 08401, G anolle s-Ba celona, Spain
h
Cen e d’A enci´
o P ima ia Ad i`
a 5A Ma c Au eli, Ins i u Ca al`
a de la Salu (ICS), Ca e Vallmajo , 34, 08021, Ba celona, Spain
i
Cen e d’A enci´
o P ima ia Casano a. Conso ci d’A enci´
o P im`
a ia de Salu de l’Eixample (CAPSBE) Casano a. Ca e Rossell´
o 161, 08036, Ba celona, Spain
j
Cen e d’A enci´
o P ima ia To ell´
o, Ins i u Ca al`
a de la Salu (ICS), A enida Pompeu Fab a, 8, 08570, To ell´
o-Ba celona, Spain
k
Depa men o In ec ious T opical diseases and Mic obiology, IRCCS Sac o Cuo e Don Calab ia Hospi al, Via Semp eboni 5, 37024, Neg a di Valpolicella, I aly
l
Spanish Na ional Resea ch Council (IPBLN-CSIC), A enida del Conocimien o 17, Pa que Tecnol´
ogico de Ciencias de la Salud, 18016, G anada, Spain
m
Biomedical Resea ch Ne wo king Cen e (CIBER) o In ec ious Diseases, Ca los III Heal h Ins i u e (CIBERINFEC, ISCIII), Ca e Melcho Fe n´
andez Almag o, 3,
28029, Mad id, Spain
n
Li e Uni , Hospi al Clínic, Uni e si y o Ba celona, Augus Pi i Sunye Biomedical Resea ch Ins i u e (IDIBAPS), Biomedical Resea ch Ne wo king Cen e o Hepa ic
and Diges i e Diseases (CIBEREHD), Ca e Villa oel, 170, 08036, Ba celona, Spain
o
Depa men o Medicine Solna, Ka olinska Ins i u e , Solna ¨
agen 1, 17177, Solna-S ockholm, Sweden
p
Depa men o In ec ious Diseases, Ka olinska Uni e si y Hospi al, Solna ¨
agen 1, 17177, Solna-S ockholm, Sweden
q
D ugs o Neglec ed Diseases Inicia i e (DNDi), Swi ze land
ARTICLE INFO
Pa o he da a om his s udy we e p esen ed
in he Eu opean Con e ence o Clinical Mic o-
biology and In ec ious Diseases in Copenhagen
(Ap il 2023).
Keywo ds:
Mig an s
Sc eening
Luminex
IgG
Sensi i i y
Speci ici y
Impo ed diseases
ABSTRACT
Backg ound: We aimed o e alua e he pe o mance o a no el mul iplex se ological assay, able o simul aneously
de ec IgG o six in ec ions, as a sc eening ool o impo ed diseases in mig an s.
Me hods: Six panels o 40 (n =240) anonymized se um samples wi h con i med in ec ions we e used as posi i e
con ols o assess he mul iplex assay’s sensi i i y. One panel o 40 se a om non-in ec ed subjec s was used o
es ima e he se oposi i i y cu o s, and 32 non-in ec ed se a we e used as nega i e con ols o es ima e each
se ology’s sensi i i y and speci ici y. The mul i-in ec ion sc eening es was alida ed in a p ospec i e coho o
48 mig an s om endemic a eas.
The sensi i i y o he Luminex assay was calcula ed as he p opo ion o posi i e esul s o e all posi i e samples
iden i ied by e e ence es s. The speci ici y was calcula ed using 32 nega i e samples. Unce ain y was quan-
i ied wi h 95 % con idence in e als using ecei e ope a ing cha ac e is ic analyses.
Resul s: The sensi i i y/speci ici y we e 100 %/100 % o HIV (gp41 an igen), 97.5 %/100 % o Hepa i is B i us
(HBV-co e an igen), 100 %/100 % o Hepa i is C i us (HCV-co e an igen), 92.5 %/90.6 % o s ongyloidiasis
* Co esponding au ho . Ka olinska Ins i u e , Depa men o Medicine Solna, Sweden.
E-mail add ess: [email p o ec ed] (A. Requena-M´
endez).
1
Bo h au ho s con ibu ed equally.
2
Bo h au ho s con ibu ed equally.
Con en s lis s a ailable a ScienceDi ec
T a el Medicine and In ec ious Disease
jou nal homepage: www.else ie .com/loca e/ maid
h ps://doi.o g/10.1016/j. maid.2023.102681
Recei ed 4 Augus 2023; Recei ed in e ised o m 8 Decembe 2023; Accep ed 15 Decembe 2023
T a el Medicine and In ec ious Disease 57 (2024) 102681
2
T a el- ela ed illnesses
Hepa i is C i us
S ongyloidiasis
Schis osomiasis
Chagas disease
[31-kDa ecombinan an igen (NIE)], 97.5 %/100 % o schis osomiasis (combined se pin Schis osoma mansoni
and S.haema obium an igens) and 95 %/90.6 % o Chagas disease [combined T ypanosoma c uzi kine oplas id
memb ane p o ein-11 (KMP11) and pa a lagella od p o eins 2 (PFR2) an igens].
In he mig an coho , an ibody esponse o he combina ion o he T.c uzi an igens co ec ly iden i ied 100 %
indi iduals, whe eas HBV-co e an igen co ec ly iden i ied 91.7 % and S ongyloides-NIE an igen 86.4 %.
Conclusions: We de eloped a new, obus and accu a e 8-plex Luminex assay ha could acili a e he imple-
men a ion o sc eening p og ammes a ge ing mig an popula ions.
1. In oduc ion
Mig a ion is a complex and g owing global phenomenon o c i ical
impo ance o Wes e n coun ies [1]. Mos mig an s a e disp opo ion-
a ely a ec ed by ce ain in ec ions being hei p e alence highe in
mig an s compa ed wi h au och honous popula ions [2,3]. In 2020, 44
% o new HIV diagnoses we e a ibu ed o mig an s li ing in he Eu-
opean Union, pa icula ly mig an s om Sub-Saha an A ica (SSA) [4].
A pooled HBV-p e alence o 5.8 % was es ima ed in Eu opean mig an s
[5], being highe in newly-a i ed A ican mig an s; and a pooled
HCV-p e alence o 1.6 % was es ima ed in mig an s esiding in Eu ope,
simila ly highe in mig an s om SSA [3].
O he pa asi ic in ec ions, such as Chagas disease, s ongyloidiasis
and schis osomiasis, which a e no endemic in Wes e n coun ies [1] a e
highly p e alen among mig an s [6–8]. Chagas disease, caused by
T ypanosoma c uzi is highly p e alen (4.2 %) among mig an s om
La in-Ame ican endemic coun ies li ing in Eu ope [8], pa icula ly in
Boli ian mig an s (18 %) [9]; he possibili y o au och honous ans-
mission (congeni ally, by ans usion, o ansplan a ion [10]) o T. c uzi
may cause a subsequen inc ease o ela ed mo bidi y in na ional heal h
sys ems in Eu ope [11]. S ongyloidiasis is also an eme ging in ec ion o
public heal h conce n due o i s high se op e alence (12.2 %) in mi-
g an s esiding in Eu opean coun ies [7], and o he possibili y o being
a li e- h ea ening condi ion when immunosupp ession is es ablished
[12]. Schis osomiasis is a neglec ed disease becoming ele an in low-
and non-endemic coun ies because o he inc eased mig a ion lows
om high-endemic coun ies, and wi h a 18.8 % se op e alence es i-
ma ed in Eu opean mig an s [7]. I ep esen s a managemen challenge
due o he lack o clinical awa eness and knowledge among heal h
p o essionals [13].
Ea ly de ec ion and ea men o hese in ec ions can lowe he
ela ed mo bidi y and mo ali y [13]. The easibili y o inno a i e in-
eg a ed sc eening p og ammes o in ec ious diseases, has been p o en
in di e en se ings including p ima y ca e uni s in Uni ed Kingdom,
Uni ed S a es, Canada and Spain [13–16].
An ibody-based assays a e used o assessing he exposu e o
di e en in ec ions among mig an s including HIV o i al hepa i is
[17], and also pa asi es (S ongyloides s e co alis [18], Schis osoma spp
[19] and T.c uzi [20]). Quan i a i e suspension a ay assays simul a-
neously de ec ing an ibodies o mul iple pa hogens using a single
specimen can suppo a mo e cos -e ec i e implemen a ion o in e-
g a ed disease su eillance sys ems. Acco dingly, Luminex echnology is
gaining ac ion in diagnosis because o he ease, high h oughpu , and
minimal sample olume equi emen s. I has been used o pe o m he
su eillance o opical i al in ec ions [21], en e opa hogens [22], o
mala ia [23]. Mo e ecen ly, ano he new mul iplex- es has been
de eloped o de ec HIV, i al hepa i is, he pes and T eponema pallidum.
[24].
Ou s udy aimed o de elop a new mul iplex se ological assay using
10 an igens o quan i y IgG o six pa hogens ha a e p e alen in
mig an popula ions li ing in Ca alonia (Spain), including HIV, HBV,
HCV, T. c uzi, S. s e co alis and Schis osoma spp., and o es ima e i s
sensi i i y and speci ici y compa ed wi h p ima y e e ence s anda d
es s o each in ec ion.
2. Me hods
2.1. S udy design
The s udy was pe o med using six panels o a chi ed, ully anony-
mized well-cha ac e ized coded se um samples p e iously diagnosed o
he di e en in ec ions, plus a g oup o samples om non-in ec ed
subjec s.
Each panel consis ed o 40 posi i e con ol specimens o each
pa hogen (HIV, HBV, HCV, T. c uzi, S ongyloides and Schis osoma)
(Fig. 1). HIV posi i e con ols we e de e mined wi h he De e mine HIV-
1/2 Rapid Tes (Abbo Labo a o ies) and con i med wi h he Unigold
apid es (T ini y Bio ech). HBV and HCV posi i e con ols we e
de e mined by RNA quan i a i e es ing ha measu es he i al load.
The schis osomiasis posi i e con ols we e posi i e o s ool and/o
u ine es depending on he species. The s ongyloidiasis posi i e con-
ols we e indi iduals diagnosed wi h s ongyloidiasis and included in a
clinical ial ha assessed he e ec i eness o i e mec in [25] and o
which he inclusion c i e ia we e ha ing a posi i e aecal es o S.
s e co alis o ha ing a posi i e se ological es a high i es i espec i e
o he esul o aecal es . High i es we e assessed as a leas 160 i es
o he indi ec luo escen an ibody es (IFAT)- ha is, whole se um
dilu ion 1/160, and a leas 2×no malized op ical densi y (OD) o he
IVD Resea ch ELISA, - ha i , he a io be ween OD o he sample and
ha o he weak posi i e con ol- [26]. The accu acy o hese es s a
hese high i es has been alida ed showing ha hey app oach 100 %
speci ici y, while main aining 70 % sensi i i y [26]. The T. c uzi posi i e
con ols we e ob ained om a se um biobank o Chagas cases de ined by
being posi i e in a leas wo se ological es s using di e en ecombi-
nan an igens. Fo he pa asi ic in ec ions, u he de ails o he se o-
logical i es, and aecal o u ine samples in case o s ongyloidiasis and
schis osomiasis a e epo ed in Annex 1.
F om he panel o 72 se a o heal hy subjec s, 40 samples we e used
o es ima e he se oposi i i y cu o s o he di e en se ologies, and 32
we e used as nega i e con ols o es ima e he speci ici y o each
se ology (Fig. 1). We alida ed he mul i-in ec ion sc eening es in a
p ospec i e coho o 48 mig an s wi h epidemiological exposu e isk o
he selec ed in ec ions depending on he coun y o o igin (Fig. 1). They
we e ec ui ed du ing he implemen a ion o a mul i-disease sc eening
p og am o asymp oma ic mig an s a ended a p ima y ca e uni s in
Ca alonia (Janua y 2019–Decembe 2020). Asymp oma ic mig an s o
mig an s wi h undi e en ia ed symp oms, ≥18 yea s old, who isi ed
any o he pa icipa ing cen es du ing he s udy pe iod we e included in
he coho . Mig an s bo n in Wes e n coun ies [1] we e excluded om
he s udy. Pa icipan s we e es ed o each in ec ion wi h he e e ence
me hod depending on he exposu e isk. In his ega d, all pa icipa ing
mig an s we e es ed o HIV, HBV and HCV, bu only mig an s coming
om Chagas disease’s, s ongyloidiasis’ and schis osomiasis’ endemic
coun ies we e es ed o hese in ec ions [8,27].
The e e ence se ological es s used o assess he accu acy o he
Luminex in he p ospec i e mig an coho we e pe o med acco ding o
each cen e’s e e al labo a o y o HIV and i al hepa i is; o S ong-
yloides, an enzyme-linked immuno-so ben assay (ELISA) based on IVD-
S.s e co alis c ude an igen (SciMedx®) o an in-house immuno luo es-
cence; o Schis osoma, an ELISA o an in-house indi ec -haemagglu i-
na ion es (Schis osomiasis- Fumouze®); o T. c uzi, a comme cial
R. Aguila e al.
T a el Medicine and In ec ious Disease 57 (2024) 102681
3
ELISA wi h ecombinan an igens (BioELISA-Chagas, Bioki ®), and an
in-house ELISA wi h whole epimas igo es an igen [28], wi h diagnosis
de ined by posi i i y in bo h se ological es s.
2.2. Mul i-in ec ion IgG Luminex assay
The 10 an igens ini ially selec ed o he s udy a e lis ed in Table 1.
P24 (co e an igen) and gp41 (en elope an igen) we e selec ed o HIV
se ology, he i s one o being indica i e o acu e in ec ion [29] and he
second one o being highly immunogenic du ing se ocon e sion [30],
he 31-kDa ecombinan an igen (NIE) an igen was selec ed o he
se ology o S. s e co alis, as ELISAs based on his an igen ha e shown
high sensi i i y in he ange o 71–84 % [26,31,32], S.mansoni (Sm) and
S.haema obium (Sh) se pins we e selec ed o he se ology o he co e-
sponding species o being epo ed as p omising species-speci ic diag-
nos ic an igens by Luminex [33], hepa i is C co e (HCV-co e) and
nons uc u al p o ein 3 (NS3) an igens we e es ed o ch onic hepa i is
C in ec ion, he co e an igen is cu en ly used o diagnos ic [34] and he
NS3 an igen has also been epo ed as se o eac i e [35], hepa i is B co e
(an i-HBc) an igen was used o he de ec ion o ch onic hepa i is B
in ec ion as he p esence o IgG indica es ongoing in ec ion, and people
who ha e immuni y o hepa i is B om a accine do no de elop
an i-HBc [36]. Finally, pa a lagella od p o eins 2 (PFR2) and T.c uzi
kine oplas id memb ane p o ein-11 (KMP11) an igens we e selec ed o
being p e iously epo ed as good ma ke s o T.c uzi in ec ion [37].
Each o he 10 an igens included in he Luminex panel was coupled o a
speci ic magne ic mic osphe e egion; he op imal bead-coupling con-
cen a ion was de e mined o each using he same me hodology, as
desc ibed p e iously [38].
A de ailed desc ip ion o he assays is p o ided in Annex 2. B ie ly,
361 samples we e es ed oge he wi h se ial dilu ions o a posi i e
con ol o gene a e a s anda d cu e o assay quali y con ol, plus 3
echnical blanks. An igen-coupled mic osphe es we e added in mul i-
plex o he 384-well pla e (2000 beads/an igen/well) and mixed wi h
Fig. 1. Flowcha o he s udy
HIV: Human Immunode iciency i us, HBV: Hepa i is B i us, HCV: Hepa i is C i us.
R. Aguila e al.
T a el Medicine and In ec ious Disease 57 (2024) 102681
4
es samples, posi i e con ols, and blanks. Final dilu ion o es samples
was 1/250. The pla e was incuba ed o 1 h a oom empe a u e in
agi a ion and p o ec ed om ligh . Then, he pla e was washed and 25
μ
L o goa an i-human IgG-phycoe y h in, (1:400) we e added o each
well and incuba ed o 30 min. The pla e was washed again and mi-
c osphe es esuspended wi h Luminex Bu e , o be acqui ed on he
Flexmap-3D® eade . A leas 50 mic osphe es/analy e/well we e ac-
qui ed, and median luo escence in ensi y (MFI) was epo ed. Annex 3
shows he s anda d cu es made o 20 se ial dilu ions o he posi i e
con ol. Re e ence s anda d es esul s we e blinded o he esea che s
who pe o med he Luminex es .
2.3. Da a analysis
MFI cu o s o se oposi i i y we e calcula ed o each an igen as he
exponen ial unc ion o he mean plus 2 s anda d de ia ions (SD) o 3SD
o log
10
- ans o med MFIs o 40 nega i e con ols andomly selec ed
among he specimens om 72 subjec s wi h no p e ious exposu e o any
in ec ion. The o he 32 nega i e con ols we e used o es ima e he
speci ici y o he Luminex assay s he e e ence es s (Fig. 1). The e-
a e , o each in ec ion, he e alua ion o he Luminex assay pe o -
mance used 72 specimens wi h known se ological s a us (40 posi i e and
32 nega i e samples). The sensi i i y and 95 % con idence in e als
(95%CI) o he Luminex es we e calcula ed as he p opo ion o posi-
i e esul s (based on he cu -o wi h 2SD) o e all posi i e samples by
he p ima y e e ence es . Sensi i i y and 95%CI we e also calcula ed
wi h he cu o wi h 3SD. Simila ly, speci ici y and 95%CI we e calcu-
la ed o e 32 con ol nega i e samples om he g oup o heal hy con-
ols using bo h 2SD and 3SD cu o s. Unce ain y was quan i ied wi h
95%CI using ecei e ope a ing cha ac e is ic (ROC) analyses. Finally,
he sensi i i y and speci ici y wi h i s espec i e 95%CI we e also es i-
ma ed in a panel o samples p ospec i ely collec ed om 48 mig an s a
isk o in ec ions.
2.4. E hics
Posi i e and nega i e con ol samples used o de elop he assay we e
ob ained om collec ions egis e ed a he Ins i u o de Salud Ca los III,
Hospi al Clínic(Ba celona)/ISGlobal biobanks and Sac o-Cuo e Hospi al
T opica-Biobank (Annex 4). The s udy p o ocol and in o med consen
om mig an s whose samples we e p ospec i ely collec ed we e
app o ed by he e hical commi ee a Hospi al Clínic, (HCB/2017/
0847). Resul s a e epo ed acco ding o he STARD-checklis (Annex 5).
3. Resul s
3.1. Accu acy o he mul i-in ec ion Luminex assay on a e e ence panel
o human samples
We e alua ed he pe o mance o he no el mul iplex se ological
assay on a panel o human se a wi h known in ec ion s a us. Fi s , we
es ima ed he se oposi i i y cu o s o each o he an igens in he
Luminex panel using 2 and 3SD, summa ized in Annex 6.
Fig. 2 ep esen s he IgG se ological le els (log
10
MFI) agains each
an igen in he Luminex panel in 32 nega i e and 246 posi i e con ol
samples (2A), and 48 es samples om mig an s exposed o he in-
ec ions (2B).
Using a 2SD cu o , he sensi i i y was gene ally high (>90 %) o he
majo i y o he an igens, whe eas he speci ici y depended on he
pa hogen and an igen (Table 2 and Fig. 3). The sensi i i y and speci ici y
o he Luminex es o de ec ing HIV using he gp41 ecombinan p o-
ein de e mined by ROC analysis we e 100 % (95%CI 91.2–100) and
100 % (95%CI 89.1–100) using bo h 2SD and 3SD cu o s. In con as ,
he p24 p o ein pe o med wo se, wi h a sensi i i y o 87.5 % (95%CI
73.2–95.8) and a speci ici y o 96.9 % (95%CI 83.8–99.9) wi h he 2SD
cu o , and 80.0 % (95%CI 64.4–90.9) sensi i i y a 3SD cu o (Fig. 3A).
The HBV co e an igen showed a sensi i i y o 97.5 % (95%CI
86.8–99.9) a 2SD-cu o and 95 % (95%CI 83.0–99.4) a 3SD-cu o ,
and a speci ici y o 100 % (95%CI 89.1–100) a 2SD and 3SD-cu o s,
(AUC: 0.99, [95%CI: 0.96–1.00]), (Fig. 3B). The HCV co e an igen
showed a 100 % (95%CI 91.2–100) sensi i i y a 2SD-cu o (97.5 %,
95%CI 86.8–99.9 a 3SD-cu o ) and a speci ici y o 100 % (95%CI
89.1–100) a 2SD and 3SD-cu o s, (AUC: 1.00,[95%CI 1.00–1.00]),
(Fig. 3C).
S ongyloides-NIE an igen epo ed a sensi i i y o 92.5 % (95%CI
79.6–98.4) a 2SD and 3SD-cu o s and speci ici ies o 90.6 % (95%CI
75.0–98.0; 2SD-cu o ) and 100 % (95%CI 89.1–100; 3SD-cu o s),
(AUC:0.96, [95%CI 0.91–1.00]), (Fig. 3D). In a es ic ed analysis
Table 1
The 10 an igens es ed used in he mul i-in ec ion Luminex assay.
An igen Pa hogen Sou ce Coupling
concen a ion
Re e ence
Recombinan
HIV1 p24
p o ein
HIV Abcam
ab43037
30
μ
g/mL [37]
Recombinan
HIV1 gp41
p o ein
HIV Abcam
ab49068
250
μ
g/mL [45]
NIE S ongyloides Exp essed in
E. coli by
Sukwan
Handali (CDC)
om a plasmid
clone dona ed
by Thomas
Nu man (NIH/
NIAID), USA
10
μ
g/mL •[46]
Sm se pin Schis osoma
mansoni
Dona ed by
Sa oshi Kaneko
(Nagasaki
Uni e si y,
Japan)
30
μ
g/mL •[47]
Sh se pin Schis osoma
haema obium
Dona ed by
Sa oshi Kaneko
(Nagasaki
Uni e si y,
Japan)
50
μ
g/mL •[47]
Recombinan
Hepa i is C
Vi us NS3
p o ein
HCV Abcam
ab49024
50
μ
g/mL [48]
Recombinan
Hepa i is C
Vi us Co e
An igen
HCV Abcam
ab49017
50
μ
g/mL [48]
Recombinan
Hepa i is B
Vi us Co e
An igen
HBV Abcam
ab49013
30
μ
g/mL [49],[50]
Chagas PFR2 T ypanosoma
c uzi
Recombinan
p o ein
p oduced by
Ca men
Thomas and
Manuel C.
Lopez, IPBLN-
CSIC, G anada,
Spain
50
μ
g/mL [51]
Chagas
KMP11
T ypanosoma
c uzi
Recombinan
p o ein
p oduced by
Ca men
Thomas and
Manuel C.
Lopez IPBLN-
CSIC, G anada,
Spain)
30
μ
g/mL •[51]
HIV: Human Immunode iciency Vi us, HBV: Hepa i is B Vi us, HCV: Hepa i is C
Vi us; Sm: Schis osoma mansoni; Sh: Schis osoma haema obium; NS3: non-
s uc u al p o ein 3; PFR2: pa a lagella od p o ein 2; KMP11: kine oplas id
memb ane p o ein-11.
R. Aguila e al.
T a el Medicine and In ec ious Disease 57 (2024) 102681
5
(Annex 7) o hose samples ha es ed posi i e in aeces [ei he by po-
lyme ase chain eac ion (PCR) es o any o he di ec echnique], he
pe o mance o he NIE an igen in Luminex wi h he 3SD cu o
imp o ed o 100 % (95%CI 83.9–100) sensi i i y (AUC: 1.00, [95%CI
1.00–1.00]).
The sensi i i y and speci ici y o he Luminex es o de ec schis o-
somiasis was 97.5 % (95%CI 86.6–99.9) and 93.8 % (95%CI 79.2–99.2)
a 2SD cu o , (AUC:0.97, [95%CI 0.93–1.00]) using S. haema obium (Sh)
and S. mansoni (Sm) se pin an igens oge he (Fig. 3E). In e es ingly, he
sensi i i y o he se pin-Sh an igen was 100 % (95%CI 78.2–100) o
de ec ing S. haema obium in ec ions whe eas he sensi i i y o he se pin-
Sm an igen was 96 % (95%CI 79.6–99.9) o de ec ing S. mansoni
in ec ions (Annex 7).
The T. c uzi KMP11 an igen pe o med be e (Sen 92.5 %, [95%CI
79.6–98.4]; Spec 96.9 %, [95%CI 83.8–99.9]; AUC:0.96, [95%CI
0.92–1.00]) compa ed o he PFR2 an igen (Sen 77.5 %, [95%CI
61.5–89.2]; Spec 93.8 %, [95%CI 79.2–99.2]; AUC:0.87, [95%CI
0.80–0.94]). Fu he mo e, when combining bo h an igens, he sensi-
i i y inc eased o 95 % (95%CI 83.0–99.4; AUC:0.97, [95%CI
0.93–1.00]) (Fig. 3F). The e we e wo samples in he T. c uzi in ec ion
panel ha esul ed inde e mina e by he e e ence es , and i was
necessa y a hi d se ological es o con i m he in ec ion. A e
excluding hese samples om he analysis (Annex 7), he sensi i i y
inc eased o 97.4 % (95%CI 86.2–99.9) a 2SD cu o (AUC:0.98, [95%
Fig. 2. Do plo s showing he dis ibu ion o IgG le els measu ed agains he 10 mul iplexed an igens in he di e en panels o samples
2A. Panels o posi i e and nega i e con ol samples o each in ec ion
2B. Panel o es samples om he mig an s coho
Legend:
The posi i e samples (2A) we e selec ed based on se oposi i i y in he e e ence me hods. In o al, 40 posi i e samples we e included o he espec i e in ec ions (n
=240).
Log10MFI: IgG se ological le els (log10 mean luo escence in ensi y); HBV: Hepa i is B Vi us, HCV: Hepa i is C Vi us: Sm: Schis osoma mansoni; Sh: Schis osoma
haema obium.
R. Aguila e al.

T a el Medicine and In ec ious Disease 57 (2024) 102681
6
CI 09.95–1.00]).
In o de o assess possible c oss- eac i i ies, we e alua ed o each
in ec ion he alse posi i e a e among he indi iduals om he six
panels o posi i e con ols (Annex 8). O e all, he Luminex se ology
showed a low equency o alse posi i es, excep o S. s e co alis
showing a a e o 66 % (12/18) and 50 % (9/18) among Schis osoma spp.
Table 2
Pe o mance pa ame e s o he 10 an igens es ed in he mul iplex assay on a e e ence panel o human samples.
2 SD cu o 3 SD cu o AUC (95%
CI)
An igens Pos
T/
Pos
GS
Sensi i i y
(95%CI)
Neg
T/
Neg
GS
Speci ici y
(95%CI)
Co ec ly
classi ied
Pos
T/
Pos
GS
Sensi i i y
(95%CI)
Neg
T/
Neg
GS
Speci ici y
(95%CI)
Co ec ly
classi ied
HIV p24 35/
40
87.5
(73.2–95.8)
31/
32
96.9
(83.8–99.9)
91.7 32/
40
80.0
(64.4–90.9)
32/
32
100
(89.1–100)
88.9 0.93
(0.88–0.99)
gp41 40/
40
100
(91.2–100)
32/
32
100
(89.1–100)
100 40/
40
100
(91.2–100)
32/
32
100
(89.1–100)
100 1.00
(1.00–1.00)
Bo h 40/
40
100
(91.2–100)
31/
32
96.9
(83.8–99.9)
98.6 40/
40
100
(91.2–100)
32/
32
100
(89.1–100)
100 1.00
(1.00–1.00)
HBV HBV
co e
39/
40
97.5
(86.8–99.9)
32/
32
100
(89.1–100)
98.6 38/
40
95
(83.0–99.4)
32/
32
100
(89.1–100)
97.2 0.99
(0.96–1.00)
HCV HCV
co e
40/
40
100
(91.2–100)
32/
32
100
(89.1–100)
100 39/
40
97.5
(86.8–99.9)
32/
32
100
(89.1–100)
98.6 1.00
(1.00–1.00)
HCV
NS3
33/
40
82.5
(67.2–92.7)
31/
32
96.9
(83.8–99.9)
88.9 31/
40
77.5
(61.5–89.2)
32/
32
100
(89.1–100)
87.5 0.91
(0.85–0.97)
Bo h 40/
40
100
(91.2–100)
31/
32
96.9
(83.8–99.9)
98.6 39/
40
97.5
(86.8–99.9)
32/
32
100
(89.1–100)
98.6 1.00
(1.00–1.00)
Chagas
disease
PFR2 31/
40
77.5
(61.5–89.2)
30/
32
93.8
(79.2–99.2)
84.7 17/
40
42.5
(27.0–59.1)
32/
32
100
(89.1–100)
68.1 0.87
(0.80–0.94)
KMP11 37/
40
92.5
(79.6–98.4)
31/
32
96.9
(83.8–99.9)
94.4 34/
40
85.0
(70.2–94.3)
32/
32
100
(89.1–100)
91.7 0.96
(0.92–1.00)
Bo h 38/
40
95.0
(83.0–99.4)
29/
32
90.6
(75.0–98.0)
93.1 34/
40
85.0
(70.2–94.3)
32/
32
100
(89.1–100)
91.7 0.97
(0.93–1.00)
S ongyloides NIE 37/
40
92.5
(79.6–98.4)
29/
32
90.6
(75.0–98.0)
91.7 37/
40
92.5
(79.6–98.4)
32/
32
100
(89.1–100)
95.8 0.96
(0.91–1.00)
Schis osoma
spp
Sm -
se pin
36/
40
90.0
(76.3–97.2)
31/
32
96.9
(83.8–99.9)
93.1 31/
40
77.5
(61.5–89.2)
32/
32
100
(89.1–100)
87.5 0.95
(0.90–1.00)
Sh -
se pin
36/
40
90.0
(76.3–97.2)
31/
32
96.9
(83.8–99.9)
93.1 29/
40
72.5
(56.1–85.4)
31/
32
96.9
(83.8–99.9)
83.3 0.92
(0.87–0.99)
Bo h 39/
40
97.5
(86.8–99.9)
30/
32
93.8
(79.2–99.2)
95.8 36/
40
90.0
(76.3–97.2)
31/
32
96.9
(83.8–99.9)
93.1 0.97
(0.93–1.00)
Pos T: posi i e es ; Pos GS: posi i e Gold S anda d; Neg T: Nega i e es ; Neg GS: Nega i e Gold s anda d; 95%CI: 95 % Con idence In e al; HIV: Human Immu-
node iciency Vi us, HBV: Hepa i is B Vi us, HCV: Hepa i is C Vi us; Sm: Schis osoma mansoni; Sh: Schis osoma haema obium; NS3: non-s uc u al p o ein 3; PFR2:
pa a lagella od p o ein 2; KMP11: kine oplas id memb ane p o ein-11.
Fig. 3. Recei e ope a ing cu es o he 10 an igens es ed in he mul iplex assay o de ec he in ec ions.
R. Aguila e al.
T a el Medicine and In ec ious Disease 57 (2024) 102681
7
in ec ions a a cu o o 2SD and 3SD, espec i ely, and 62 % (13/21)
and 38 % (8/21) in Schis osoma spp. and HBV coin ec ions a a cu o o
2SD and 3SD, espec i ely. The e we e 25 % (5/20) alse posi i es o
HIV and 36 % (4/11) o HCV in Schis osoma spp. and HBV coin ec ions
a a cu o o 2SD ha we e no obse ed a 3SD.
3.2. Accu acy o he mul i-in ec ion Luminex assay on mig an ’s coho
The e we e no posi i e e e ence es s o HIV, HCV, and schis oso-
miasis among he mig an ’s coho , he e o e he sensi i i y and AUC o
he Luminex es o hese in ec ions could no be e alua ed. The spec-
i ici ies assessed o each o hese in ec ions a e de ailed in Table 3.
Among he 48 indi iduals es ed o HBV, 11 o hem had a posi i e
an i-HBc es esul . The co e HBV an igen es ed in Luminex showed a
sensi i i y o 100 % (95%CI 71.5–100) and speci ici y o 89.2 % (95%CI
74.6–97.0) a 2SD-cu o . Fo he combina ion o he T.c uzi an igens, as
well as o he KMP11 an igen alone, he Luminex assay co ec ly
iden i ied (2SD and 3SD-cu o s) he 14 posi i e and 16 nega i e cases
de ec ed wi h he e e ence me hod (Sensi i i y 100 %, [95%CI
76.8–100] and Speci ici y 100 %, [95%CI 79.4–100]; AUC: 1.00, [95%
CI 1.00–1.00]). The NIE-an igen showed 83.3 % (95%CI 35.9–99.6)
sensi i i y and 86.8 % (95%CI 71.9–95.6) speci ici y, a 2SD-cu o .
We also assessed o c oss- eac i i ies among he 48 indi iduals om
he mig an ’s coho (Annex 9), and equency o alse posi i es was
o e all low, excep o S. s e co alis in ec ions wi h 20 % (2/10) alse
posi i es among T. c uzi in ec ions, bu only a 2SD, and 28 % (2/7)
among HBV in ec ions a 2SD and 15 % (1/7) a 3SD cu o . HIV showed
32 % (4/12) alse posi i es among he T. c uzi in ec ed indi iduals a
2SD (only 1 a 3 SD), and Schis osoma spp. showed 28 % (2/7) among
HBV in ec ed indi iduals, using he 2SD and 2 SD cu o .
4. Discussion
We ha e de eloped an 8-plex Luminex assay o he simul aneous
de ec ion o IgG agains HIV, HBV, HCV, Schis osoma spp, S. s e co alis
and T. c uzi. To ou knowledge, his is he i s mul iplex es ha
simul aneously de ec s se e al i al and pa asi ic in ec ions based on
se ological es s ha a e p e alen in mig an popula ions. Al hough 10
an igens we e ini ially e alua ed, only eigh o hem showed a good
pe o mance.
Ou 8-plex assay esul s showed a good ag eemen wi h he e e ence
es s, being highly accu a e o de ec mos pa hogens, pa icula ly gp41-
HIV and HCV-co e an igen, (100 % sensi i i y), he combina ion o
Schis osoma se pin an igens (97.5 %), HBV co e an igen (97.5 %) and he
combina ion o T. c uzi an igens (95 %).
Fo he in ec ions o which wo an igens we e es ed, he sensi i i y
o he es inc eased o Chagas disease and schis osomiasis, whe eas o
HIV he combina ion o p24 and gp41 an igens sligh ly dec eased he
speci ici y o he es compa ed wi h he use o he gp41 an igen alone.
P24 an igen ends o nega i ize o e ime a e he in ec ion [39], as i is
only de ec ed in blood du ing ea ly s ages o he p imo-in ec ion, hus i
is no eliable o sc eening excep o he de ec ion o ecen in ec ions
[40]. Simila ly o o he s udies [41], o HCV he combina ion o NS3
wi h he co e an igen dec eased he speci ici y o he es compa ed wi h
he co e an igen alone. The pe o mance o HIV and HBV an igens was
simila compa ed wi h a p e ious Luminex s udy, whe eas he co e HCV
an igen has demons a ed be e pe o mance [24]. Howe e HIV gp41
and HCV-co e an igens showed some alse posi i es in Schis osoma spp.
and HBV coin ec ions, and HIV also in T.c uzi in ec ions, sugges ing
some oom o imp o emen .
As p e iously desc ibed, he pe o mance o he ecombinan
S ongyloides-NIE an igen (sensi i i y 92.5 %) is no as good as o he
comme cially a ailable es s based on c ude an igens [26]. Howe e , he
sensi i i y was 100 % o hose in ec ions de ec ed by mic oscopy o
PCR. The e o e, he lowe sensi i i y o NIE by Luminex can be a ib-
u ed o a poo e diagnos ic capabili y o he ecombinan an igen
compa ed wi h c ude an igens [9]. None heless, he S ongyloides
se ology wi h c ude an igen may gi e alse posi i es due o
Table 3
Pe o mance pa ame e s o he 10 an igens es ed in he mul iplex assay in a p ospec i e coho o 48 mig an indi iduals wi h isk o exposu e o he es ed in ec ions.
2 SD cu o 3 SD cu o AUC (95%
CI)
An igens Pos
T/
Pos
GS
Sensi i i y
(95%CI)
Neg
T/
Neg
GS
Speci ici y
(95%CI)
Co ec ly
classi ied
Pos
T/
Pos
GS
Sensi i i y
(95%CI)
Neg
T/
Neg
GS
Speci ici y
(95%CI)
Co ec ly
classi ied
HIV p24 0/1 0 43/
45
95.6
(84.5–99.5)
93.5 0/1 0 43/
45
95.6
(84.5–99.5)
93.5 0-48 (. -
1.00)
gp41 1/1 100
(25.0–100)
39/
45
86.7
(73.2–94.9)
87.0 0/1 0 44/
45
97.8
(88.2–99.9)
95.7 0.92 (. -
1.00)
HBV HBV
co e
11/
11
100
(71.5–100)
33/
37
89.2
(74.6–97.0)
91.7 7/11 63.6
(30.8–89.1)
35/
37
94.6
(81.8–99.3)
87.5 0.95
(0.90–1.00)
HCV HCV
co e
– – 46/
48
95.8
(85.7–99.5)
– – – 47/
48
97.9
(88.9–99.9)
– –
HCV
NS3
– – 45/
48
93.8
(82.8–98.7)
– – – 45/
48
93.8
(82.8–98.7)
– –
Chagas
disease
PFR2 14/
14
100
(76.8–100)
16/
16
100
(79.4–100)
100 12/
14
85.7
(57.2–98.2)
16/
16
100
(79.4–100)
93.3 1.00
(1.00–1.00)
KMP1 14/
14
100
(76.8–100)
16/
16
100
(79.4–100)
100 14/
14
100
(76.8–100)
16/
16
100
(79.4–100)
100 1.00
(1.00–1.00)
Bo h 14/
14
100
(76.8–100)
16/
16
100
(79.4–100)
100 14/
14
100
(76.8–100)
16/
16
100
(79.4–100)
100 1.00
(1.00–1.00)
S ongyloides NIE 5/6 83.3
(35.9–99.6)
33/
38
86.8
(71.9–95.6)
86.4 4/6 66.7
(22.2–95.7)
37/
38
97.4
(86.2–99.9)
93.2 0.88
(0.70–1.00)
Schis osoma
spp
Sm -
se pin
– – 20/
22
90.0
(70.8–98.9)
– – 21/
22
95.4
(77.2–99.9)
– –
Sh -
se pin
– – 18/
22
81.8
(59.7–94.8)
– – 21/
22
95.4
(77.2–99.9)
– –
Bo h – – 18/
22
81.8
(59.7–94.8)
– – 21/
22
95.4
(77.2–99.9)
– –
Pos T: Posi i e es ; Pos GS: Posi i e Gold S anda d; Neg T: Nega i e es ; Neg GS: Nega i e Gold s anda d; 95%CI: 95 % Con idence In e al; HIV: Human Immu-
node iciency Vi us, HBV: Hepa i is B Vi us, HCV: Hepa i is C Vi us; Sm: Schis osoma mansoni; Sh: Schis osoma haema obium; NS3: non-s uc u al p o ein 3; PFR2:
pa a lagella od p o ein 2; KMP11: kine oplas id memb ane p o ein-11.
R. Aguila e al.
T a el Medicine and In ec ious Disease 57 (2024) 102681
8
c oss- eac i i y wi h o he helmin hs, such as, ila ial in ec ions, Asca is
lumb icoides in ec ion, and acu e schis osomiasis [42], hus hese
specimens could also be ue nega i es w ongly cha ac e ized by he
e e ence es . In ac , when assessing he a e o alse posi i es due o
possible c oss eac i i ies, S. s e co alis showed a high p e alence o alse
posi i es among o he in ec ions sugges ing he need o imp o e he
speci ici y o he NIE an igen.
Conce ning Chagas disease, al hough he combina ion o bo h an i-
gens showed a be e sensi i i y, he KMP11 an igen pe o med much
be e compa ed wi h PFR2 an igen o de ec T. c uzi, imp o ing e en
he accu acy when excluding bo de line esul s de ec ed by he e e -
ence es . This sugges s ha he KMP11 an igen is pe o ming simila
compa ed wi h o he se ological es s de eloped o T. c uzi sc eening
[43]. In any case, he diagnosis o Chagas disease by Luminex should be
con i med wi h ano he se ological es using a di e en ecombinan
p o ein, as s anda d p ocedu e.
In e es ingly, when he Luminex assay was es ed in a p ospec i e
sample o mig an indi iduals coming om coun ies endemic o he
in ec ions es ed, he assay showed a good accu acy o de ec mos in-
ec ions, al hough sensi i i y was lowe o s ongyloidiasis, sugges ing
ha his Luminex panel may be p omising as a sc eening ool. Howe e ,
he mig an s sample size was e y small and he e we e no pa ien s
in ec ed wi h schis osomiasis, HIV, and HCV. In addi ion, he sensi i i y
and speci ici y o a es may a y depending on he p e alence o he
diseases. A sys ema ic e iew and me a-analysis [44], showed a signi -
ican associa ion be ween p e alence and sensi i i y o speci ici y (ac-
cu acy) in one hi d o he e iewed s udies. Acco dingly, The e o e, he
accu acy o ou mul iplex es may be alida ed in a p ospec i e coho
o mig an s.
We ha e es ed he pe o mance o he 10 an igens using wo
di e en cu o s. The 2SD-cu o gi es highe sensi i i ies by losing some
speci ici y, while he 3SD-cu o gi es lowe sensi i i ies bu 100 %
speci ici y o all in ec ions. In he con ex o sc eening o mig an
popula ions, highe sensi i i ies a e mo e impo an han 100 % speci-
ici ies, as ha ing in ec ed undiagnosed indi iduals is no desi able,
he e o e alse nega i e cases should be minimized.
O e all, he use o bead-based mul iplex assays is gaining ecogni-
ion in diagnosis o in ec ions because o he ease, high h oughpu , and
minimal sample/ eagen olume equi emen s [45–47] allowing
measu ing up o 100 analy es/pe sample, and he FlexMap-3D pla o m
( he one used in his s udy) up o 500 analy es pe sample, wi h
comp ehensi e educ ions in ime and cos . Rema kably, mul iplexing
mo e han one an igen may inc ease he sensi i i y o pa hogen de ec-
ion compa ed o single-bioma ke s. In addi ion, Luminex panels a e
lexible and can be adap ed o he epidemiological isk o indi iduals (i.
e., he coun y o bi h). Thus, o example T. c uzi an igen could be only
included in a speci ic Luminex panel o La in Ame ican mig an s.
In his speci ic s udy, we e alua ed he diagnos ic u ili y o a
mul iplex es ocusing on he posi i i y o nega i i y o samples.
Howe e , an ibody le els could be use ul in s udies add essing IgG ki-
ne ics since ime o in ec ion, o IgG le els depending on i al o pa a-
si ic loads.
Al hough he Luminex echnology i sel is no no el, o ou knowl-
edge, his is he i s mul iplex es ha simul aneously de ec s se e al
i al and pa asi ic in ec ions ha a e p e alen in mig an popula ions
based on se ological es s. The simul aneous classi ica ion o he p es-
ence o absence o mul iple pa hogens in a single assay has he po en ial
o e olu ionize and simpli y he public heal h and diseases su eillance
in endemic a eas whe e he in ec ions a e p e alen [47], and also he
implemen a ion o sc eening p og ammes in mig an popula ions.
Mo eo e , i e o he 10 p o eins used in he panel a e no comme cially
a ailable hus ha e been p oduced in house, and o some o hem his is
he i s ime ha hei diagnos ic pe o mance is epo ed as pa o a
mul i-diagnos ic in ec ion es .
In addi ion, he 8-plex assay o e comes many o he labo a o y
in as uc u e equi emen s o he single in ec ion diagnos ic es ing,
he eby simpli ying he logis ics. This makes i a mo e accessible es o
se ings ha may no ha e ei he he echnical knowledge o he in a-
s uc u e capaci y o conduc he single diagnos ic es s using a dedi-
ca ed pla o m o each o hem (i.e., p ima y ca e). In his ega d, he
cu en exis ence o bench op compac easy- o-use mul iplex equipmen ,
would acili a e he de elopmen o a poin o ca e e sion o he assay in
p ima y ca e.
While u he wo k is needed o op imize and alida e his 8-plex
assay, and maybe expand i o o he in ec ions and o o he assays
using non-in asi e samples such as mucosa o sali a, i has he po en ial
o d as ically educe he disease bu den in se ings whe e es ing has
o en been limi ed, pa icula ly o impo ed pa asi ic diseases. A
de ailed compa a i e cos -analysis be ween he mul iplex assay and he
indi idual s anda d con en ional es s o each in ec ion is in p og ess.
This es has subs an ial po en ial o imp o e diagnos ic es ing, based
on his p oo -o -concep s udy.
The main limi a ion o he s udy is he small sample size o he
p ospec i e mig an coho wi h no posi i e cases o some o he in-
ec ions and also o he small numbe o heal hy con ol indi iduals. Fo
his eason, we plan o conduc a mo e obus p ospec i e s udy o he
alida ion o he 8-plex assay. Ano he limi a ion o he s udy is he lack
o a p ope challenge panel made up o samples om in ec ions and
condi ions ha may cause po en ial c oss- eac i i y (i.e., ila iasis in he
case o s ongyloidiasis) [9]. The eason was he lack o samples wi h
known unique in ec ions o his pu pose. Ins ead, we ha e used he da a
om he indi iduals om he six panels o posi i e con ols and he 48
es ed indi iduals wi h in o ma ion on o he in ec ions as challenge
panels. Finally, we a e measu ing IgG, which is indica i e o exposu e,
no dis inguishing be ween cu en o pas in ec ion. Howe e , he
Luminex pla o m allows measu ing any iso ype o IgG subclass, hus
IgM o IgA could be used in combina ion wi h IgG o dis inguish ecen
om pas in ec ions. In addi ion, he use o speci ic an igens indica i es
o ecen exposu e o in ec ions could be help ul o de e mine he
app oxima e ime o in ec ion.
5. Conclusions
We p esen a p oo -o -p inciple o he excellen pe o mance o a
high h ough pu single es using mul iplexed an igens wi h a minimal
sample olume ha can de ec mul iple in ec ions, common in mig an
popula ions. Ou 8-plex assay has shown o be obus and accu a e,
al hough u he p ospec i e s udies should alida e he ep oducibili y
o he assay. This es could acili a e he implemen a ion o sc eening
p og ammes pa icula ly in non-hospi al-based se ings whe e he
de ec ion o single pa hogens can be logis ically challenging and
expensi e.
Funding
This s udy was suppo ed by wo g an s om Ins i u o de Salud
Ca los III (ISCIII), co- inanced by he Eu opean Regional De elopmen
Fund (FEDER) om he Eu opean Union, h ough he “Fondo de
In es igaci´
on pa a la Salud (FIS)”, PI17/02020 and PI20/00866. This
esea ch eam was suppo ed by CIBER-Conso cio Cen o de In es-
igaci´
on Biom´
edica en Red-(CB 2021), Ins i u o de Salud Ca los III,
Minis e io de Ciencia e Inno aci´
on and Uni´
on Eu opea. ISGlobal e-
cei es suppo om he Spanish Minis y o Science and Inno a ion
h ough he “Cen o de Excelencia Se e o Ochoa 2019–2023” P og am
(CEX2018-000806-S), and suppo om he Gene ali a de Ca alunya
h ough he CERCA P og am. Au ho s om IRCCS Sac o Cuo e Don
Calab ia Hospi al we e suppo ed by he I alian Minis y o Heal h
“Fondi Rice ca co en e-L2P4”. ARM ecei es unding om he S a egic
Resea ch P og am in Epidemiology a Ka olinska Ins i u e . MCL was
suppo ed by he P og ama Es a al I +D +I, Spanish Minis y o Science
and Inno a ion and FEDER (PID2019-109090RB-I00/AEI/10.13039/
501100011033).
R. Aguila e al.
T a el Medicine and In ec ious Disease 57 (2024) 102681
9
The unde s o he s udy had no ole in he s udy design, da a
collec ion, da a analysis, da a in e p e a ion o w i ing o he
manusc ip .
CRediT au ho ship con ibu ion s a emen
Ru h Aguila : Fo mal analysis, In es iga ion, Me hodology, Vali-
da ion, W i ing – o iginal d a . Angeline C uz: Da a cu a ion, Fo mal
analysis, In es iga ion, Me hodology, P ojec adminis a ion, Valida-
ion, Visualiza ion, W i ing – o iginal d a . Al ons Jim´
enez: Da a
cu a ion, Fo mal analysis, Me hodology, P ojec adminis a ion, Vali-
da ion, Visualiza ion, W i ing – e iew & edi ing. Alex Almuedo:
Concep ualiza ion, Funding acquisi ion, In es iga ion, Me hodology,
P ojec adminis a ion, W i ing – e iew & edi ing. Ca me Roca Sau-
mell: Da a cu a ion, In es iga ion, P ojec adminis a ion, W i ing –
e iew & edi ing. Ma ina Gigan e Lopez: Da a cu a ion, Fo mal anal-
ysis, Me hodology, P ojec adminis a ion, Resou ces, W i ing – e iew
& edi ing. O iol Gasch: Concep ualiza ion, Funding acquisi ion,
In es iga ion, Me hodology, P ojec adminis a ion, Resou ces, W i ing
– e iew & edi ing. Gemma Falc´
o: Da a cu a ion, Me hodology, P ojec
adminis a ion, Resou ces, W i ing – e iew & edi ing. Ana Jim´
enez-
Lozano: Da a cu a ion, Fo mal analysis, Me hodology, P ojec admin-
is a ion, Resou ces, W i ing – e iew & edi ing. Angela Ma ínez-
Pe ez: Da a cu a ion, Fo mal analysis, Me hodology, P ojec adminis-
a ion, Resou ces, W i ing – e iew & edi ing. Consol Sanchez-Col-
lado: Da a cu a ion, Fo mal analysis, Me hodology, P ojec
adminis a ion, Resou ces, W i ing – e iew & edi ing. And ea
Tedesco: Da a cu a ion, Fo mal analysis, Me hodology, Resou ces,
W i ing – e iew & edi ing. Manuel Ca los L´
opez: Da a cu a ion,
Fo mal analysis, Resou ces, So wa e, W i ing – e iew & edi ing. Ma ía
Jesús Pinazo: Concep ualiza ion, Da a cu a ion, Fo mal analysis,
In es iga ion, Me hodology, W i ing – e iew & edi ing. Thais Leonel:
Da a cu a ion, Fo mal analysis, P ojec adminis a ion, Resou ces,
W i ing – e iew & edi ing. Zeno Biso i: Da a cu a ion, Fo mal anal-
ysis, P ojec adminis a ion, Resou ces, Supe ision, Valida ion, W i ing
– e iew & edi ing. Anna F¨
a ne : Concep ualiza ion, Me hodology,
Resou ces, Supe ision, Valida ion, W i ing – e iew & edi ing. Ca lo a
Doba˜
no: Concep ualiza ion, Funding acquisi ion, In es iga ion, Me h-
odology, Resou ces, Supe ision, Visualiza ion, W i ing – e iew &
edi ing. Ana Requena-M´
endez: Concep ualiza ion, Fo mal analysis,
Funding acquisi ion, In es iga ion, Me hodology, P ojec adminis a-
ion, Resou ces, Supe ision, Valida ion, Visualiza ion, W i ing – o ig-
inal d a .
Decla a ion o compe ing in e es
Non decla ed.
Acknowledgemen s
We a e g a e ul o all pa ien s who pa icipa ed in his s udy, he
gene al p ac i ione s, nu ses, and o he s a om he p ima y ca e
cen es and hospi als who we e commi ed o pe o m he ield wo k
equi ed o he s udy while main aining hei daily asks. Specially, we
acknowledge he wo k ca ied by Ma ia Luisa Machado and Sil ia
Capilla om Hospi al Uni e si a i Pa c Taulí. Special hanks o he
esea ch g oup a he IRCCS Sac o Cuo e Don Calab ia Hospi al o
p o iding he posi i e con ol samples o s ongyloidiasis (pa o hem)
and schis osomiasis, in pa icula o S e ano Tais and Monica Degani, o
Jose Munoz (Hospi al Clinic, Ba celona) o acili a ing posi i e con ol
samples o s ongyloidiasis (pa o hem) and Denise Naniche (ISGlo-
bal) o in o ma ion on he HIV posi i e con ol samples. We a e also
g a e ul o M
a
Ca men Thomas om he Ins i u e o Pa asi ology and
Biomedicine L´
opez Ney a o supplying he Chagas disease an igens,
Thomas Nu man (NIH/NIAID, USA), Sukwan Handali (CDC) o sup-
plying he NIE, Sa oshi Kaneko (Nagasaki Uni e si y, Japan) o
dona ing he Sm and Sh se pins, and Ma a Vidal, Rebeca San ano and
Inocencia Cuamba o suppo in he an igen p ocu emen and
cha ac e iza ion.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j. maid.2023.102681.
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