Evaluation of the accuracy of a multi-infection screening test based on a multiplex immunoassay targeting imported diseases common in migrant populations

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. Re e ences [1] Bogue a D, Ma ino a D, Raphaely T. 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