5 Reasons Why Your Business Needs Chagas Rapid Test？

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2. Standard methods and protocols for CD diagnosis

CD is marked by an acute and chronic phase, each with specific aspects that reflect the diagnostic methods of choice. Given that the onset of infection is characterized by a high parasitemia, the techniques related to direct parasite detection on peripheral blood by light microscopy comprise the gold standard for diagnosis during the acute stage, including in cases of congenital infection [18, 19]. Thus, the methods mainly applied are thin, thick, or fresh blood films. Alternatively, concentration methods, such as Strout and microhematocrit, may be employed [18, 19]. These protocols are especially indicated for the diagnosis of congenital infection and can be performed with either umbilical cord or venous blood from neonates and infants [20]. Polymerase chain reaction (PCR) can also be used, however, it should preferably be carried out within the first and third months of life [20]. In addition, serological follow-up is indicated for infected infants as well as for those with negative direct parasite detection with an infected mother. This analysis should be performed from the eighth month of life, when maternal IgG is no longer detected [20, 21].

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In contrast, the chronic phase features a subpatent parasite load. In this sense, indirect detection methods that rely on parasite amplification, such as hemoculture, xenodiagnosis, and PCR, may be performed [19]. However, these tests show low sensitivity at this stage, so that a negative result has poor conclusive value to discard infection [19]. As the end of the acute phase is accompanied by seroconversion, the chronic stage is characterized by a continuous production of IgG. Ergo, diagnosis of suspected cases of long-lasting infections is predominantly based on immunoassays. Nevertheless, there is no gold standard or reference test since none of the kits commercially available exhibit ideal performance regarding both sensitivity and specificity [19]. Consequently, at least two tests must be performed, so that infection is either confirmed or ruled out only if both assays present the same profile of reactivity (positive or negative, respectively) [18]. Hence, when inconclusive results are obtained, another test must be carried out [18]. The immunoassays applied must necessarily differ in terms of either detection principles or antigenic set and present complementary sensitivity and specificity [18]. Furthermore, in the absence of a biomarker for cure, conversion to non-reactive profile in subsequent tests in an interval of time has been considered a parameter to confirm the parasite elimination by trypanocidal treatment [22].

Although indirect hemagglutination (IHA) and indirect immunofluorescence (IIF) are frequently used, enzyme-linked immunosorbent assay (ELISA) is the most employed method, once (i) it is better suited for large-scale analysis and (ii) presents a considerable variety in terms of antigenic preparations [23]. In addition, chemiluminescent microparticle immunoassay (CMIA) and western are also recommended, the latter mainly employed as a confirmatory assay and/or discriminatory test in inconclusive cases [18, 19].

ELISA kits are produced with either (i) whole parasite extract, (ii) semi-purified fractions, (iii) recombinant proteins (full-length or chimeras), or (iv) synthetic peptides—the latter two being commonly used as multiplexed formulations. Alternatively, combining purified homogenate with recombinant proteins is employed as well [23]. This flexibility is a major advantage given that test reactivity may vary according to sample origin due to host genetic background and/or differences among parasite strains. As a matter of fact, T. cruzi presents a wide genetic variability, so that populations are classified into six groups named discrete typing units (DTU) TcI-VI and Tcbat [24], which exhibit different geographical prevalence [25, 26]. Moreover, the diversity of ELISA kits also covers the necessary arrangement of sensitivity and specificity complementation. In this regard, the implementation of recombinant proteins and synthetic peptide have shown to improve test accuracy by reducing cross-reactivity with other diseases, especially leishmaniasis [19].

Noteworthily, the protocol for the screening process in blood banks and prenatal care—as a health policy measure to reduce T. cruzi infection dissemination—rely on the application of a single immunoassay, which must present a high sensitivity. In this context, ELISA and CMIA are the main recommended methods [18]. Nonetheless, in case of a positive result, another test must be carried out to confirm the diagnosis.

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3. Applicability of rapids tests and its perspectives in CD context

Considering that those most affected by CD are from either remote areas or small towns with low resource in developing countries, the main immunoassays used for diagnosis and screening purposes do not meet the reality of local points of care, which commonly lack the needed infrastructure and the specialized personnel [27]. Furthermore, field studies are also hampered, as these tests are usually performed with plasma or serum obtained from venous blood extraction [28]. Thus, even if the assay is not carried out locally, transport of materials and equipment for sample adequate handling and storage are still required, as well as a trained team, which often translates in logistic complexity and, ultimately, in higher cost. More importantly, depending on the notification system applied, patient acknowledgment regarding their results is not safely guaranteed when the tests are performed latter on. Consequently, there is a patent demand for rapid tests [29], which have as basic concept a cheap and fast assay, with an easy development that ideally eliminates the necessity of specialized technicians, equipment, including in terms of sample manipulation, and cold storage, enabling their use on-site [30].

The World Health Organization and the Pan American Health Organization’s guidelines consider rapid tests as an alternative screening method for ELISA only in the context of seroepidemiological studies [18]. Their use is not recommended for clinical diagnosis or screening in hemotherapy services based on (i) the detection variation, (ii) the increased false negative rate (2–7 per ) in comparison to the association of two conventional serological methods for diagnosis of chronic patients and (iii) the cost-effectiveness, especially when there is a high demand, such as in blood banks [18].

Nonetheless, in , the Chagas National Program of Bolivia incorporated the use of a specific rapid test (Chagas Stat-Pak, Chembio Inc.) as the frontline method in both clinical practice and seroepidemiological surveys [31]. Notably, Chagas Stat-Pak (CSP) has shown sensitivity and specificity ranging from 93.4 to 100.0% and from 97.3 to 99.3%, respectively, among Bolivian municipalities [28, 31, 32, 33]. However, to follow the recommended diagnosis algorithm, positive cases still must be confirmed by a conventional immunoassay [31]. In this sense, studies have shown promising results regarding the synchronous combination of two rapid tests with different antigen composition as an alternative strategy for definitive diagnosis in low-resources settings in Bolivia, Argentina, and Colombia [28, 31, 34, 35, 36, 37]. The association of two rapid tests displayed ≥93.3% of diagnostic efficiency when using results obtained by at least two conventional immunoassays as reference [28, 31, 35, 36]. This alternative protocol has a great advantage in speeding up the process of patient continued medical assistance, especially regarding the offer of trypanocidal drugs and, eventually, other necessary treatments. In line with that, the combination of rapid testing with electrocardiogram (ECG) performed by a mobile device was also evaluated in Bolivia [38]. Interestingly, out of the 25 people with ECG abnormalities compatible with chagasic chronic cardiomyopathy, 22 (88%) presented a positive profile on the rapid test. As per the current protocol, the diagnosis of these patients was later confirmed by ELISA. ECG was carried out in a device connected to a smartphone and processed by a medically certified app; the exams were performed by non-physicians in a remote area, however, within 24 h the data were analyzed, and results were reported by cardiologists located overseas [38]. Taking into account the lack of clinical tools for patient’s progression monitoring toward symptomatic stage [22, 39, 40, 41], the incorporation of already known potential protein markers for such questions, especially those for early cardiac impairment [42, 43], in rapid test is an appealing and strategical approach to improve the assistance of individuals infected and should be addressed in the near future.

In view of congenital transmission, rapid testing has also been evaluated in Latin American pregnant women, mainly at the time of delivery [44, 45, 46, 47, 48]. This practice has a great impact on disease control when the mother was not tested during prenatal care, once trypanocidal efficacy is high and well tolerated by infants [20, 48]. Surprisingly, reported data indicates that a rapid test outperformed ELISA assays [45, 46]. Furthermore, aiming to investigate the recovery time of newborns infected congenitally and submitted to trypanocidal treatment 1 day after birth, Chippaux et al. [21] monitored periodically the level of specific anti-T. cruzi antibodies by ELISA; newborns without infection, but with an infected mother were included as control. From the eighth month, the authors carried out rapid testing in parallel to the conventional assay. At the ninth month, none of the patients showed immunoreactivity in the rapid test, while 12% remained testing positive by ELISA. Within the following 7 months, all of these patients presented antibodies titres below the ELISA cut-off [21]. This delayed seronegativity may be related to the extended set of antigens and/or the different epitopes components of the ELISA kit in comparison to the rapid test used. Moreover, anti-T. cruzi IgG originally from the mother was not detected in the mentioned control group since the fifth month despite the continued breastfeeding [21]. Conversely to the observed in infants, Jackson et al. [49] still detected reactivity in 2 ELISA assays and in the same rapid test when applying sera from adults of endemic regions after 3 years of treatment with nifurtimox.

Up on the released data reporting rapid tests performance on endemic population, a health center in Geneva, Switzerland, sought to study its feasibility regarding the screening of Latin American immigrants [50]. The majority of infected individuals was from Bolivia, and as a result of the rapid testing agreement with the conventional immunoassays and reproducibility, it was incorporated at the hospital as a point-of-care test in both the primary care center and the maternity ward (testing at delivery) [50]. In Italy, a different test has shown high specificity and was used as a third assay for evaluation of samples with discordant results [51], while in Spain, a rapid test was applied to screen co-infection in Latin Americans immigrants diagnosed with HIV [52].

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4. Performance of immunochromatographic rapid tests commercially available

As previously mentioned, parasite and/or host genetic background may be related to reactivity discrepancy. Thereby, it is strongly suggested that the rapid test is field validated in the area of interest before officially incorporating its use [31, 35]. This process should ideally be carried out at a national level by encompassing different sites [31], once circulating strains may vary from one region to another within the endemic countries [24, 25]. Thus, this topic is focused on the main marketed rapid tests with reported performance studies (Table 1).

Rapid testAntigenSampleVol. (μL)RT (min)SS (%)SP (%)Sample originRef.Chagas
Stat-Pak
(Chembio)B13, 1F8 and H49/JL7S or P
WB5
.4 to 100.097.3 to 99.3Bolivia[28, 31, 32, 33]100..0Colombia[35]97.1 to 100.094.8 to 99.9LATAM1[53, 54]87.2 to 96.093.2 to 99.9LATAM2[34, 50, 56]Trypanosoma
Detect
(InBios)ITC8.2S or P
WB10
.897.9LATAM1[62]89.6 to 92.994.0 to 100.0LATAM2[56, 64]Chagas Detect
Plus
(InBios)ITC8.2S or P
WB10
.2 to 100.087.1 to 99.3Bolivia[28, 31, 60]100.099.1Colombia[35]Simple Stick
Chagas
(Operon)Pep2, TcD, TcE and SAPAS.4 to 100.091.6 to 92.4LATAM2[67, 68]Simple Chagas
WB
(Operon)Pep2, TcD, TcE and SAPAS or P
WB10
NS.9 to 92.570.7 to 96.8LATAM2[56, 67, 70]SD Bioline Chagas Ab
(Standard Diognostics)1F8 and H49S, P or WB.6 to 100.093.8 to 97.7Argentina[36, 37]90.794LATAM2[56]WL Check
Chagas
(Wiener Lab)NSS, P or WB–.3 to 93.498.8 to 100Argentina[36, 37, 74]88.797LATAM2[56]OnSite Chagas
Ab Combo
(CTK Biotech)NSS or P
WB20
40–.1 to 95.591.0 to 96.9LATAM2[56, 68]Chagas Instantest
(Silanes)NSS or P–.679.0LATAM2[56]TR Chaga*
(Bio-Manguinhos)IBMP-8.1
IBMP-8.4S..0Brazil[75]

These assays are based on the detection of anti-T. cruzi antibodies circulating in the bloodstream upon recognition of recombinant proteins (Figure 1). In the absence of a gold standard immunoassay, conventional serology methods have been used as reference to evaluate rapid test performance (Table 2). Generally, the tests can be carried out with small volumes (5 to 100 μL) of either plasma, serum, or whole blood (Table 1). However, the latter comprises the best working sample type as it does not require further processing and, in the case of immediate testing, it may be obtained from fingertip puncture, perfect for the context of the field research. Moreover, the results are obtained within 10–35 min (Table 1).

Conventional immunoassayPrincipleAntigenRapid testRef.Bioelisa Chagas (Biokit)ELISATcD, TcE, Pep 2 and TcLo1.2CSP, SSC, OnSite, SCWB, SD Bioline[50, 57, 68, 69, 70, 73]Certest (BiosChile)ELISALysate (Tulahuén and Mn strains)SSC, OnSite[68]Chagas III (BiosChile)ELISAParasite lysateCSP, CDP, TD, TR Chagas[35, 62, 75]Chagas IgG + IgM I (Vircell)ELISAFRA, B13 and MACHaCSP, CDP, TD[35, 64]Chagas HAI-Immunoserum (TIIC)IHANSCSP[53]Chagas Polychaco (Lemos Lab.)IHANSCSP, CDP, TD, WL Check Chagas, SD Bioline[31, 32, 36, 60, 63, 65, 74]Chagatest v.2.0 (Wiener)ELISAParasite lysateCSP, TD, CDP[28, 31, 32, 33, 34, 60, 63]Chagatest v.3.0 (Wiener)ELISAPep 1, 2, 13, 30, 36, and SAPACSP, CDP, TD, SD Bioline[28, 31, 32, 33, 34, 44, 45, 46, 54, 60, 65, 66, 73]Chagatest (Wiener)IHAPep 1, 2, 13, 30, 36, and SAPASD Bioline[73]Chagatek (Lemos Lab.)ELISAPurified antigensCSP, CDP[28]Elecsys Chagas (Roche Diagnostics)E-CLIARecombinant proteinsTD[64]ELISA cruzi (bioMérieux)ELISAParasite lysateCSP, TD, SSC, OnSite[50, 62, 68]Gold ELISA Chagas (Rem Diag.)ELISARecombinant proteins and purified lysateTR Chagas[75]Hemagen Chagas (Hemagen Lab.)ELISAPurified antigensCSP, CDP, TD[34, 44, 66]IFI Chagas (Bio-Manguinhos)IFIStrain not specifiedTR Chagas[75]ID-PaGIA2 (DiaMed)GAAg2 and TcESSC, OnSite[68]ID-PaGIA3 (DiaMed)GAAg2, TcE and TcDSSC, OnSite[68]Ortho Trypanosoma cruzi (Ortho-Clinical Diag.)ELISAParasite lysateCDP, SSC, OnSite, SCWB[66, 68, 69]T. cruzi IgG (BLK Diag.)ELISAParasite lysateSSC, OnSite[68]—IIFParasite Tulahuén strainWL Check Chagas, SD Bioline[36]In-housebELISAParasite lysate (Y strain)CSP[53]ELISAParasite lysate (Peru strain)CSP[54]ELISAParasite lysate (H1 strain)CSP[46]ELISAParasite lysate (Tulahuén strain)TD, WL Check Chagas, SD Bioline[37, 62]ELISAParasite lysate (PM strain)TD[62]ELISAParasite lysate (Mc, T and Dm28 strains)SSC, OnSite, SCWB[67, 68]ELISAParasite lysateSCWB[69, 70]ELISAParasite lysate (MHOM/CO/06/338)SD Bioline[73]IIFParasite Y strainCSP[53]IIFParasite Colombian strainCSP, CDP[35]IIFNSCDP[60]IIFParasite
Tulahuén strainTD, WL Check Chagas, SD Bioline[62, 37, 74]IIFParasite
Mc, T and Dm28 strainSSC, OnSite, SCBW[67, 68]IHANS
(Tulahuén strain)TD[62]IHANS
(PM strain)TD[62]IHANSWL Check Chagas, SD Bioline[37]TESA-blotSecreted/excreted proteins (Y strain)CSP, CDP[34]Western blotParasite lysate (H1 strain)CSP[46]

CSP detection capability relies on the recognition of the recombinant antigens B13, 1F8, and H49/JL7 [53]. Notably, it has been trending as the most evaluated rapid test in both field and clinical seroprevalence studies, including in non-endemic countries. By using serum as testing sample, CSP has shown sensitivity ranging from 97.1 to 100.0%, and specificity from 94.8 to 99.9% among populations from South and Central Americas [53, 54]. Surprisingly, reactivity of samples from Midwest and Northeast regions of Brazil resulted in 98.5 and 94.8% of sensibility and specificity, respectively [53], even though the epitope B13 is derived from a 140 kDa protein detected in the surface of Y strain trypomastigotes [55], which belongs to one of the predominant DTUs in the respective area [24, 25]. Furthermore, sera from El-Salvador displayed the lowest sensitivity (97.1%) [53]. In a multicenter study carried out by Sánchez-Camargo et al. [56] with sera tested in national reference laboratories for Chagas disease diagnosis located in Brazil, Argentina, Colombia, Costa Rica, Mexico, United States of America, France, Spain, and Japan, the CSP exhibited 87.2 and 93.2% of sensibility and specificity, respectively [56]. On average, 50 samples (ca. of 25 positive and 25 negative) per region were analyzed, however, neither the origin of the donors sorted by each facility in non-endemic areas nor the correspondent data per country was discriminated. The reduced performance was accounted to sera selection, which comprised samples stored for averagely 2 years and with moderate to low reactivity profiling. The later aspect is of major importance, once one cannot rule out that methods used for sample reactivity classification could vary among the laboratories enrolled [56]. Curiously, this study also reported a lower sensitivity when using plasma in comparison to serum, while two other studies have demonstrated high level of agreement (99.7–100.0%) among different sample types (sera stored with or without 50% glycerol, plasma, whole blood and/or eluates from filter paper containing dried whole blood) [50, 53]. In this regard, CSP exhibited outstanding results (100.0% for both sensitivity and specificity) in whole blood testing in Colombia [35].

Interestingly, analysis of CSP in surveys of Latin Americans living in non-endemic countries showed a predominant seroprevalence in Bolivian immigrants [34, 50, 57]; sera testing presented low performance [34], while whole blood accused a sensibility and specificity of 95.2 and 99.9%, respectively, being 97.2% of the positive results from Bolivian origin [50]. By the time the CSP was implemented as a diagnostic tool by the Bolivian Ministry of Health, only one of the studies conducted so far had included samples from such region (n = 21) [53]. Therefore, since then, studies have been done to evaluate and field validate CSP performance in different Bolivian sites. Most of these works used whole blood for the CSP testing and either serum or plasma for conventional serological assays taken as reference. As previously mentioned, detection performance varied among municipalities, with sensibility and specificity ranging from 93.4 to 100.0% and from 97.3 to 99.3%, respectively [28, 31, 32, 33]. Noteworthily, lower sensitivities were obtained by Roddy et al. and Chippaux et al. when working with age groups ranging from 9 months to 17.9 years old (93.4%) and from 11 to 20 years old (89.2%), respectively [32, 33]. More worrisome, up to 7.6% of discrepancy between CSP and ELISA results was observed in women pregnant either at the moment or in the preceding 3 years at the time of the respective study [33]. Nevertheless, the same Chagatest v.3.0 (Wiener) failed to detect infection in 29.5% of women PCR-positive at delivery, while the overall CSP rate of false negative was 9.6% [45]. This research enrolled women from Argentina, Honduras, and Mexico, which displayed 97.3, 96.1, and 67.3% of CSP reactivity, respectively [45]. Although the detection in Mexican women was expressively reduced, CSP still outperformed the ELISA assay applied by 39.4% [45]. In a lower-prevalence scenario in Mexico, by taking western blot as confirmatory test, Gamboa-Léon et al. [46] demonstrated similar results between CSP and Chagatest v.3.0 (Wiener), especially in samples collected from umbilical cord.

Both the Trypanosoma Detect (TD) and Chagas Detect Plus (CDP) are based on the multiepitope recombinant protein ITC8.2 [58]. Produced by InBios, their main differences reside in product format and clearance by the U.S. Food and Drugs Administration agency. While TD is presented as a dipstick with application restricted to research [59], the CDP consists of an improved version [60], designed in cassette format—with the gold conjugate maintained in liquid solution—and is marketed for diagnostic use in the USA [60, 61].

TD evaluation in sera samples from Argentina, Ecuador, Mexico, and Venezuela resulted in 82.5, 84.3, 77.5, and 95.0% sensitivity, respectively. On the other hand, specificity was high, with the lower result detected in Ecuador (95.6%) and the greater in both Mexico and Venezuela (100.0%) [62]. In the work by Sánchez-Camargo et al. [56] previously mentioned, it presented an overall sensitivity and specificity of 92.9 and 94.0%, respectively. Similar sensitivity was observed in Bolivian women at delivery (92.7%), however, a higher specificity was shown (99.0%) [63]. In addition, regarding the same study performed with PCR-confirmed infected women with whole blood also collected at delivery in Argentina, Honduras and Mexico, TD testing resulted in more cases of seroreactivity in comparison to ELISA Wiener (v. 3.0) in samples from the latter two countries, while no difference was observed for those from Argentina [45]. Given that the ELISA kit used and the TD share 4 epitopes (peptides 1, 30, 36 and SAPA) between their sets of antigens [58], the TcF and Kmp-11 peptides included in the latter test may be related to the differential detection observed. Nonetheless, CSP still outperformed TD in both Honduras and Argentina [45]. In a survey of immigrants from endemic countries living in Spain that included an electrochemiluminescence immunoassay as reference, a lower sensitivity was obtained (89.6%) with whole blood, while excellent specificity was maintained (100.0%) [64]. Notably, such reference test (Elecsys Chagas, Roche Diagnostics) does not share any antigen with TD.

Aiming to increase sensitivity, the TD was modified, resulting in the CDP assay [60]. Its first performance evaluation was carried out with paired sets of whole blood and sera samples from Bolivian populations encompassing adults (with or without heart disease), pregnant women at delivery and children up to 17 years old. Tests results obtained with each type of sample displayed 90.3% of agreement, with whole blood showing a reduced sensitivity (96.2 vs. 99.3%) and higher specificity (98.8 vs. 96.9%) [60]. Following studies testing whole blood from individuals in different regions of Bolivia reported sensitivity of 92.1–100.0% and specificity of 87.1–99.3%, being the best results obtained in a high seroprevalence area [28, 31, 65]. In Colombia, CDP showed an outstanding performance with whole blood testing as CSP (≥ 99.1% for both parameters) [35]. Interestingly, reported data of Latin American CDP testing in the USA points to difference of performance between plasma and serum, with greater relevance for those from Mexico and Central America (mostly represented by El-Salvador) [34, 66]. More importantly, CDP showed superior sensitivity to samples from these same regions in comparison to three others conventional serological assays (two ELISAs and one IHA), whereas a lower specificity (87.5–92.3%) was observed in the overall analysis [66].

Simple Stick Chagas (SSC) and Simple Chagas WB (SCWB) also comprise rapid tests elaborated in two different formats that are based on the same antigen, a chimeric recombinant protein that englobes the peptide 2, TcD, TcE and SAPA epitopes [67] (Table 1). The former has a dipstick design and can be used with serum, while the latter is displayed as a cassette and can be carried out with either whole blood, plasma, or serum [67]. Studies that include these tests were mainly centered in evaluating their applicability as screening tool of Latin American immigrants and others with epidemiological background of risk living in Spain [67, 68, 69, 70]. Sera testing with SSC showed 92.4–100.0% of sensitivity and 97.9% of specificity, which is reduced to 91.6–92.4% when considering cross-reactivity [67, 68]. As for whole blood testing with SCWB, peripheral samples exhibited a 92.1 and 93.6% of sensitivity and specificity, respectively [67]. Interestingly, capillary samples obtained by finger prick displayed a performance of 86.4 and 95% for the respective parameters [67, 71]. In these works, the onsite results were confirmed latter on by conventional immunoassays carried out with samples collected and stored on filter paper [67, 71]. On the other hand, a 92.5% of sensitivity was observed by Chejade et al. [70] when working with capillary blood and confirmatory assays done with samples frozen until use. Poorer performance was observed by Sánchez-Camargo et al. [56] when testing sera with SCWB, which displayed an overall of sensitivity and specificity of 84.9% and 70.7%, respectively, and a variable response in the quality control evaluation among the laboratories. Authors also called attention to misguiding instructions in the manufacture datasheet, which reflected in the outcome. However, such errors were already corrected [72]. Finally, little to no cross-reactivity was observed for leishmania with SSC and SCWB, however, both assays showed relevant number of false-positives with samples from individuals infected with malaria [67, 68].

The SD Bioline Chagas Ab rapid test relies on the recognition of recombinant antigens H49 and 1F8 (Table 1). Although Sánchez-Camargo et al. [56] reported 90.4 and 94.0% of sensitivity and specificity, respectively, in Colombia, this assay presented a great potential value for diagnosis confirmation when using sera (100.0% specificity), but not as a screening tool [73]. Nonetheless, in Argentinean adult population, sera and whole blood testing presented satisfactory sensitivity (97.6–100%), whereas better specificity was achieved with the latter type of sample (93.8 vs. 97.7%) [36, 37]. WL Check Chagas (WLC) was used in parallel in both studies, which reported similar results [36, 37]. Adding up to other works, WLC is more suitable as a confirmatory test regardless of the type of sample used, especially in Argentina (≥ 98.8% of specificity) [36, 37, 56, 74].

Performance data of Chagas Instantest, OnSite Chagas Ab Combo, and TR Chagas is scarce, beginning with the antigenic formulations (Table 1). Except for the last one, all rapid tests were analyzed by Sánchez-Camargo et al. [56], which—for the best of our knowledge—consists of the only independent source of information for Chagas Instantest. This assay showed sensitivity of 76.6% along with a specificity of 79.0% [56]. A moderate agreement was detected between the obtained and expected results, besides a bad profile and reproducibility on the quality control evaluation [56]. Moreover, authors reported a high frequency of invalid tests and a strong background color, making a clearer interpretation difficult [56]. As for the OnSite Chagas Ab Combo, sera testing presented 90.1–95.5% of sensitivity and 91.0–96.9% of specificity and displayed cross-reactivity with samples from individuals with either leishmaniasis or malaria [56, 68]. At last, a prototype version of TR Chagas was evaluated in a small sampling group formed by sera from Brazilians (n = 32). In this context, densitometry analysis of bands signaling reactivity toward the chimeric recombinant proteins IBMP-8.1 and/or − 8.4 led to excellent results (100.0% for both parameters) [75]. Apart from the small quantity of samples tested, the study also portraits an analysis assessment opposite to reality in point-of-care settings and field study. In addition, there are no surveys done with the final formulation. Finally, we emphasize that TR Chagas is currently only at disposal of Brazilian Ministry of Health.

Introduction

In recent years, the diagnosis of infectious diseases has broken into a new and interesting phase. The goal is to implement simple, low-cost, and affordable technologies that complement a traditional central laboratory which has large, expensive, bench-top analyzers that need highly trained personnel. Commonly named point-of-care (POC) devices, these technologies are mainly developed to be used in emergent countries, where difficult living conditions and limited health care resources permit diseases to propagate more swiftly [1].

The following rigorous requirements are set for POC diagnosis devices: (a) rapid test results are needed in order to allow the patient to receive follow-up treatment; (b) to avoid false diagnostics, quantitative results should be accurate and comparable to the results from bench-top analyzers at central laboratories; and (c) they should be easy-to-use systems that could be run by non-experts with minimum user intervention [2]. Sensitive and accurate pathogen detection can be done by means of nucleic acid amplification analysis. As a way to detect a pathogen, DNA is frequently amplified by either the polymerase chain reaction (PCR) or by the loop mediated isothermal amplification reaction (LAMP). The latter is theoretically a better method for POC diagnosis for several reasons. LAMP can amplify a limited amount of DNA copies into millions of copies within an hour, it does not require a thermal cycling device since it operates isothermally at 60–65°C, it does not require highly trained personnel to conduct the reaction, and it shows remarkable performance in terms of sensitivity and stability [3].

As a result, LAMP has been efficiently implemented in paper-based platforms, because miniaturization typically enables shorter analysis times, reduces reagent consumption, minimizes risk of sample contamination, and often enhances assay performance [4,5]. A wide range of paper-based microfluidic designs have been developed. This is mostly related to the practicality of using these as POC diagnosis devices. Several studies have reported isothermal amplification in paper-based devices with endpoint optical detection by means of Au nanoparticles [6,7], fluorescent labels [8–11], or the color change of a colorimetric indicator [12,13]. In particular, visual interrogation performed using the naked eye leads to inaccurate quantification with low sensitivity [1,14].

Lateral flow tests are already used as POC devices to detect antigens or antibodies, and they have been recently adapted to detect the nucleic acid amplification products. Lateral flow tests based on Au nanoparticle aggregation are easy to read with the naked eye, but the main problem is that the reaction tube must be opened to place the strips inside, increasing the risk of cross-contamination with amplimers and obtaining false positives [15]. Paper-based diagnosis devices using molecular fluorescent labels usually increase the cost, add disposal problems (most of them are toxic), and present instability problems (photo bleaching). These are some common drawbacks previously reported elsewhere [16,17].

Electrochemical methods solve some of these aspects, and they can be applied in a miniaturized format more readily than optical methods [18]. They are based on the detection of electrochemically active species that bind to dsDNA and are measured through voltammetry-based techniques [19]. As the reaction progresses, the redox indicator binds to the dsDNA; the concentration of the free molecule in solution decreases and thus the redox current. Alternatively, other electrochemical methods add molybdate ions in solution to detect the phosphate ions generated during the LAMP reaction [20].

While electrochemical detection seems to be interesting for POC applications, electrode fouling with the components of the LAMP mixture and their biological components could be a problem that demands the use of disposable electrochemical cells; thus increasing the cost of the diagnosis test [21]. During the LAMP reaction, the conductivity of the solution changes, and this can be an indirect indicator of a positive result. Impedance detection can be used to monitor the amplification reaction with high sensitivity [22] using microelectrodes and microfluidic devices. The conductivity measurements show several advantages over optical and traditional electrochemical approaches for POC diagnosis since they are label-free. However, the electrodes are susceptible to the fouling effect, so they need to be disposed after one determination.

The capacitively coupled contactless conductivity detector (C4D) is based on the detection of the conductivity of a medium without physical contact between the electrodes and the measuring solution [23]. Since the electrodes are separated from the solution by an insulating material, unwanted side reactions such as electrolysis and corrosion, or fouling effects, are precluded [24,25]. Moreover, portable C4D analytical instruments have undergone significant development recently [26], showing the advantage to be suited for miniaturized analytical systems compared to optical-based detection methods. A few years ago, Gooding et al. [27] employed a C4D detector to monitor the LAMP reaction over time and measure changes in the sample’s conductivity. However, they used a pair of ring electrodes, placed outside glass tube walls, in an axial configuration which limit their application in planar analytical systems, as paper-based devices are [28]. Recently, planar C4D electrodes coupled to paper-based analytical devices have been studied for the detection of carbon dioxide gas [29] and soil conductivity [23], showing the potential for in field, portable and low cost applications.

In this study, we designed a POC paper-based diagnosis device based on isothermal amplification (LAMP) of Trypanosoma cruzi DNA using a C4D system to measure changes in the conductivity signal and thus obtain the endpoint result. T. cruzi is a protozoan parasite that causes Chagas Disease, a neglected tropical infection with a public health impact. It affects 8 million people worldwide; so, it is one of the biggest public health problems [30].

Material and methods

Reagents and biocomponents

Hydroxynaphthol blue (HNB) was obtained from Fluka; tris-HCl, KCl, MgSO4, (NH4)2SO4, Betaine, Triton X-100, Tween 20 and salmon sperm DNA (control DNA template) were purchased from Sigma-Aldrich. Bst DNA Polymerase Large Fragment was from New England Biolabs (USA), dNTPs was from INBIO Highway SA (Argentina), and the set of primers was from Macrogen (Korea). The LAMP reaction mix was prepared with tris-HCl (pH 8.8, 20 mmol L-1), KCl (10 mmol L-1), (NH4)2SO4 (10 mmol L-1), MgSO4 (8 mmol L-1), Betaine (800 mmol L-1), Tween 20 (0.1% p v-1), dNTP’s (1.4 mmol L-1), Bst DNA Polymerase (320 U mL-1), primers F3 and B3 (0.2 μmol L-1), LoopF and LoopB (0.8 μmol L-1), and FIP and BIP (1.6 μmol L-1).

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POC paper-based device fabrication

The analytical assay was performed with a single use disposable device and a reusable case, containing the electrodes. The disposable device was fabricated by placing a disc of Whatman grade 1 paper with a diameter of 10 mm, and a polypropylene film (PP), over a 150 μm poly(ethylene terephthalate) (PET) layer, with the aid of a double sided adhesive tape (Fig 1). For the reusable case fabrication, a 4 mm thickness poly(methyl methacrylate) (PMMA) sheet was engraved with a CNC machine (Modela MDX-20 Roland, Shizuoka-ken, Japan) to produce the 30 x 15-mm PMMA case designed as a pocket that hold the disposable device. Two sensing electrodes made with copper adhesive tape were at the base of the PMMA case. The electrodes (2 mm wide and 14 mm long) were arranged in an antiparallel configuration keeping a gap of 1 mm between them (Fig 1). After the disposable device was inserted inside the PMMA case pocket, the resulting distance between the electrodes layer and the paper disc layer was 150 μm. A PMMA cap with a sealing rubber film was fabricated to close the analytical device that was sealed the aid of two clamps.

Schematic representation showing the materials comprising the single use disposable device and the reusable case. Inset pictures display the top and the lateral view of the assembled POC analytical device showing the electrode and paper disc disposition in detail.

https://doi.org/10./journal.pntd..g001

LAMP reaction in paper disc and HNB detection

HNB, a colorimetric reagent previously used to follow LAMP reactions [31,32], was used at a final concentration of 120 μmol L-1 in the reaction mixture containing the DNA template or without it (called non-template control or NTC). The reaction mix with HNB was loaded on the paper disc inside the analytical device and, once closed, it was heated to 65°C for 5, 15, 30, and 60 min of incubation. At each time, the paper disc was irradiated at 540 nm, and the fluorescence emission signal was acquired and analyzed with an image acquisition system and software (Amersham Imager 600, GE Healthcare, Tokyo, Japan). The signal intensity of the paper disc was measured at zero-point signal (I0) and at each detection time (I), the signal intensity ratio was calculated following a previously reported procedure (10).

Contactless conductivity measurement (C4D)

DNA amplification reactions (in paper discs) were detected using a homemade C4D module comprising both the current-to-voltage signal converter and signal treatment circuits. It was constructed according to Silva et al. [33]. The entire homemade C4D detection system uses a sinusoidal wave provided by a function generator (model DS335, SRS Stanford Research Instruments, California, USA) that goes through the excitation electrode. The receiver electrode is connected to the C4D detection module, and then to an ADC converter (National Instruments, NI USB-) allowing digitalization and computer data acquisition by means of a LabVIEW-based software with a time resolution of 1 ms. Optimization experiments were performed by applying a 400, 500, or 600 kHz sinusoidal wave with 1 Vpp amplitude to the excitation electrode.

LAMP reaction in paper disc and C4D detection

In this study, we used LAMP solution reagents containing primers whose design was based on the repetitive satellite DNA sequences (SatDNA) of T. cruzi. SatDNA is the most abundant repetitive sequence in the T. cruzi genome, composed by 105 copies of a 195-nucleotide repeat [34]. The primers were designed in silico and tested performing LAMP reactions using 1 pg of genomic DNA from T. cruzi CL Brener. As we expected due to the use of conserved sequences for the design of the primers, the reaction amplified with the template of all of the Discrete Typing Units (DTU) from I to VI (provided by Dr. Schijman, INGEBI–CONICET, Buenos Aires, Argentina), and the typical pattern of stepped bands were visualized with agarose gel electrophoresis technique. All the experiments performed in this study were done with CL Brener DTU VI. The nucleotide sequence of the primers used in the study (F3, B3, LoopB, LoopF, FIP and BIP) are not provided, since the trial is being developed into a commercial product. In a typical assay, 11 μL of the LAMP reaction mix was loaded at the center of a paper disc (Fig 1, single use disposable device), as the negative control (non-template control, NTC). On the other hand, the device was loaded with 11 μL of the LAMP reaction mix but containing 3.2, 0.32, or 0.032 fg of the DNA template (genomic DNA of T. cruzi). Then, the single use disposable device was immediately folded and placed inside the PMMA case pocket and closed with the PMMA cap (Fig 2). The closed PMMA case containing the disposable device inside was connected to C4D electronics, and the signal was acquired in real time until stabilization (signal slope < 1 mV min-1). Then, the entire device was placed at 65°C for 60 min. After that, it was connected again to C4D electronics, and the signal was acquired following the same criteria used before. The result of the assay arises from a comparison between the signals obtained before and after incubation as follows:

Schematic illustration showing the steps required to perform the LAMP amplification and the conductivity detection for NTC and samples containing T. cruzi DNA template.

https://doi.org/10./journal.pntd..g002

(1) Where “C4D signal (after incubation)” is the signal value obtained with the C4D after incubation and “C4D signal (before incubation)” is the signal value obtained with C4D before incubation.

LAMP reaction in solution

The LAMP reaction was simultaneously performed in solution using the same reagents and conditions employed in this study. Microtubes containing 60 μL of the sample mix, NTC, or DNA template were incubated for 60 min at 65°C (n = 3). After the reaction, the loading buffer (0.05% w v-1 bromophenol blue, 5% w v-1 glycerol) and 10 μL of the LAMP amplification product were mixed, placed in a standard TAE-1.5% agarose gel electrophoresis containing Sybr Safe, and run for 50 min at 50 V (Mupid EXU). The gel was revealed under a UV analyzer (Gel Doc EZ Imager, Bio-Rad). In another set of experiments, LAMP assays were performed using the same conditions described here but adding HNB (120 μmol L-1) to the sample mix. The results were observed by the naked eye and recorded with a digital camera.

LAMP reaction in paper disc analyzed with gel electrophoresis

Gel electrophoresis analysis techniques were employed to detect the LAMP amplification products obtained after a typical reaction the POC analytical device. DNA was eluted from paper discs employing the protocol described here, which was based on previously reported protocols [35,36]. Immediately after the amplification reaction, the paper disc was immersed in 60 μL of ultrapure water (resistivity greater than 18.2 MΩ) and incubated for 2 min at 100°C; then the paper disc was squeezed with a pipette tip and the entire volume was analyzed with gel electrophoresis using the methodology described before (see section: “LAMP reaction in solution” in Material and methods).

Results

LAMP reaction in paper discs and HNB detection

HNB has been used as a colorimetric dye for isothermal amplification [32], and it is analyzed with the naked eye. Kim et al. [10] reported that the fluorescence patron of HNB changes in a Mg2+-dependent way: when LAMP reaction makes progress, pyrophosphate ion is generated as a by-product and reacts with Mg2+; then the HNB fluorescence signal decreases. In the LAMP reaction performed here using 3.2 fg of DNA template, the highest fluorescence signal for HNB was obtained at the initial reaction time (0 min), and it decreased progressively to the minimal signal at the end of the assay (Fig 3). With the naked eye, differences in HNB color were not observed on the paper disc. Fluorescence changes were easily noted for up to 30 min; however, the quantified signal intensity curve reached the highest value at 60 min, indicating the reaction progressed until this time, similar to the results obtained by Kim et al. [10].

The LAMP reaction containing 3.2 fg of T. cruzi DNA template was incubated at 65°C. Paper discs were extracted from the device at different incubation times (0, 5, 15, 30, and 60 min), irradiated at 540 nm, and pictures were taken using a 620 nm emission filter. The plot shows the analytical curve obtained from the signal intensity ratio as function of reaction time (n = 3). Pictures included in the plot show the results obtained for non-template control (NTC) and samples containing 3.2 fg of template, after 60 min of incubation.

https://doi.org/10./journal.pntd..g003

Contactless conductivity measurement (C4D) in paper discs

To find the best instrumental conditions (basically good sensitivity and low noise), we performed conductivity measurements using our paper-based set-up, and KCl solutions (0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mol L-1) were normally used as conductivity standards. The POC analytical device was characterized using frequencies of 500, 600, and 700 kHz and 1 Vpp. Experiments were done at 20–25°C using one paper disc for each KCl concentration. For the three frequencies used, there was a positive relationship between solution ionic strength and C4D (V) output (Fig 4). The linear regression fit the experimental data well with R2 values of 0.973, 0.981, and 0.836 for frequencies of 500, 600, and 700 kHz, respectively. However, the 600 kHz frequency showed better characteristics, higher sensitivity (higher slope) and lower deviations (small error bars). So, this frequency was selected for further experiments.

The conductivity was recorded at room temperature and frequencies of 500, 600, and 700 kHz. The lines represent the lineal regression of each data set (n = 3).

https://doi.org/10./journal.pntd..g004

LAMP reaction in paper disc and C4D detection

Once the LAMP reaction was performed, the first C4D determinations were done using a non-template control (NTC) containing all of the LAMP reagents except the T. cruzi DNA template. The C4D signal was recorded in real time for 20 min at room temperature (20–25°C) before and then after the incubation step (60 min at 65°C). In these conditions, the C4D signal recorded after the incubation was higher than that observed before incubation (Fig 5A). As an additional internal control, similar experiments were done by replacing the LAMP mix with a KCl solution, and no changes in the C4D signal were detected between samples before and after the incubation (RSD < 5%).

The real time C4D signal was measured at room temperature before incubation and after incubation at the indicated temperatures and times. (a) C4D signal displayed by NTC samples, before and after incubation at 65°C for 60 min. (b) Differential conductivity signal (ΔC4D) of NTC samples before and after incubation at different temperatures for 15 min. (c) C4D signal displayed by samples containing 3.2 fg of T. cruzi DNA template before and after incubation at 65°C for 60 min. (d) ΔC4D curves of (□) NTC and (■) samples containing 3.2 fg of T. cruzi DNA template measured before and after different times of incubation at 65°C.

https://doi.org/10./journal.pntd..g005

Aiming to investigate the effect of incubation temperature on the acquired C4D signal, we performed short experiments (15 min) using NTC samples at 25, 35, 45, 55 and 65°C. In all cases, the C4D signal was recorded at room temperature (20–25°C) and the differential C4D signal (ΔC4D signal) was determined (see section: “LAMP reaction in paper disc and C4D detection” in Material and methods). A direct relationship was observed between the incubation temperature and the ΔC4D signal; this behavior is independent from any amplification process, since the assay was performed in NTC (Fig 5B). In the assay, higher incubation temperatures seemed to favor better desorption of the biomolecules from the paper fiber to the mobile phase, showing higher C4D conductivity values. Having tested the effect of incubation temperature on the C4D signal, the temperature was set at 65°C for next experiments. Thereafter, the C4D signal was recorded in a complete LAMP reaction. The amplification was performed at 65°C for 60 min with 3.2 fg of DNA template, and the C4D signal before and after the incubation was taken by real time curves at room temperature (Fig 5C). The C4D signal of the positive LAMP sample after the amplification was higher than before but notably lower than the signal obtained after incubation in NTC conditions (Fig 5A vs. 5C). In fact, samples containing DNA template showed conductivity values similar to NTC controls during the first 5 min of incubation, but later, as amplification proceeded, the conductivity signal showed clear differences between both conditions (Fig 5D). In this sense, the ΔC4D signal was progressively decreasing in the positive LAMP reaction. This effect can be attributed to the consumption of high conductivity reagents (dNTPs and Mg2+) and the generation of low conductivity products such as (DNA)n and magnesium pyrophosphate [21,27], as shown in these equations: (2) (3) (4)

Considering the ΔC4D values obtained in NTC and positive samples, we defined 60 min as the LAMP incubation time required for an accurate read-out, since value differences were clearer than those at shorter incubation times (Fig 5D).

Then we assayed the detection sensitivity by measuring the ΔC4D signal before and after incubation at 65°C for 60 min using serial dilutions of T. cruzi DNA as template (Fig 6A). Besides the NTC reaction, a LAMP reaction with heterologous DNA (salmon sperm DNA, “Salmon DNA”) was prepared as an additional control to check the possible effect in the ΔC4D signal (Fig 6B). The developed analytical device could clearly detect 3.2 fg and 0.32 fg of the specific T. cruzi DNA template (n = 3), which represents 1–5 copies of the target gene per reaction [37,38]. Samples containing 0.032 fg of T. cruzi DNA template were not detected, they showed ΔC4D values similar to those obtained for NTC and heterologous DNA (Salmon DNA) samples, where amplification did not succeed.

(a) Representative C4D raw signals obtained before and after incubation with NTC and samples containing 0.032, 0.32 and 3.2 fg of T. cruzi DNA template. (b) ΔC4D signal obtained for 3.2 fg of a heterologous template (salmon DNA), NTC, and for 0.032, 0.32 and 3.2 fg of T. cruzi DNA template (n = 3).

https://doi.org/10./journal.pntd..g006

LAMP reaction in solution

LAMP reactions in solution were done maintaining the same conditions and using the same reagent employed for the analytical device designed and studied in this article. Amplification reactions were conducted with solutions containing all of the sample mix reagents and adding 1 pg of DNA template. The results of the electrophoresis gel are presented in Electronic Supplementary Material (ESM) S1A Fig. When the electrophoresis gel was analyzed, we observed the typical stepped band pattern of the LAMP reaction (n = 3) for the positive reaction (1 pg of genomic DNA template), while no bands were seen in negative controls. Further analysis was done when HNB was added to the reaction solution; the typical blue color was obtained for positive samples and the violet color was obtained for NTC samples (ESM S1B Fig). These results allowed us to confirm that the LAMP reaction components (enzymes, dNTP´s and primers) are working, and the amplification reaction occurs.

LAMP reaction in paper disc analyzed with gel electrophoresis

LAMP reaction was conducted in paper discs similarly to experiments performed in this article. At the end of the assay, DNA was eluted and agarose gel electrophoresis was used to reveal the amplification product. However, the typical stepped band pattern was not observed in gel electrophoresis when positive samples (containing 3.5 fg of T. cruzi DNA template) were analyzed. While gel electrophoresis is a gold standard technique to reveal amplification products, the limit of detection of the technique is probably not enough to detect the amount of amplimers produced in the paper disc. Then, LAMP reactions in solution containing different amounts of template were conducted to evaluate the gel electrophoresis sensitivity as a reference technique. The results showed that when 10 fg or even lower amounts of DNA template were used to perform the reaction in solution, it was not possible to appreciate the typical LAMP pattern (as shown in ESM S1A Fig). Therefore, if we extrapolate the results obtained in 60 μL solution with 10 fg of T. cruzi DNA template to those results obtained for paper-disc containing 11 μL solution with 3.5 fg of T. cruzi DNA, we conclude that the absence of a band is mainly because the limit of detection was not good enough for detecting the amplimers generated with the reagents and conditions employed in this article.

Discussion

The amplification reactions in paper-based analytical devices were studied employing HNB and fluorescence analytical methods to detect the reaction product. As seen in the results obtained, the entire analytical device can be successfully used for the amplification of T. cruzi DNA template. When the analytical device was coupled with the C4D detector, the conductivity signals were studied. First, we used KCl solutions to evaluate which frequency provide higher sensitivity and lower deviation. Once this parameter was set (600 kHz), amplification assays were performed. Preliminary experiment showed that the incubation temperature affect in some way the conductivity signal obtained at room temperature. The results showed that independently from any amplification reaction, as is seen for NTC, after the incubation step, the conductivity signal increase. This behavior can be explained by an unspecific adsorption of sample biomolecules such as primers, dNTPs, and enzymes on paper fibers at room temperature. Cellulose fibers contain hydroxyl groups which are capable of forming strong hydrogen bonds which result in powerful intermolecular attractions [35,39] The intermolecular forces between cellulose fibers and biomolecules diminish when they are subject to heat treatment, which acts as a physical elution agent. This has been shown particularly for nucleic acid samples in FTA-cards or cellulose discs [36,40]. Moreover, the heat-desorbed biomolecules are still retained in the mobile phase even when the paper disc drops to room temperature, as shown by C4D conductivity measurements for NTC samples in Fig 5B. These explain why the conductivity signal are always higher after the incubation step. Meanwhile the temperature effect was studied to understand the results obtained in a typical amplification assay, the LAMP reaction should be perform at temperatures of 65°C to achieve the best amplification efficiency. Consequently, the following experiments were done at 65°C.

The real-time PCR (qPCR) is one of the most versatile and widely used methods in molecular biology and clinical diagnosis, mainly because of its high sensitivity, and good accuracy. In particular, for T. cruzi detection, there is a lack of well stablished experimental procedures associated to qPCR detection. However, among the previously reported studies, the minimum quantity of T. cruzi DNA detected with qPCR was between 0.13 and 3 fg per reaction [41,42], with variation associated mainly to the experimental procedure employed, as was recently reviewed and discussed [43]. In this study, we presented a POC analytical assay that can be employed to detect 0.32 fg of T. cruzi DNA, which is as low as those DNA amounts detected with the qPCR, but with several advantages associated mainly to POC assays. The contactless conductivity assay presented here is reagent-less (additional reagent for the detection are not needed), label-free (primers do not need to be labelled), and the incubation and detection is performed in the same disposable device avoiding any possibility of cross contamination. The amount of DNA template that the contactless conductivity assay of this study detected is similar to that reported by Zhang et al. [27], who could detect 0.51 fg of DNA template in 30 min with a C4D system, in a greater volume of sample (200 μL), and glass tubes with very thin walls commonly used for nuclear magnetic resonance studies. Further, the proposed assay can detect DNA concentrations lower than other POC devices based on LAMP used for the detection of other analytical targets (such as bacteria). These comparisons applies to those paper-based devices using HNB fluorescence [10] or color [44] change detection, and lateral flow devices [7]. In fact, in previous works, HNB fluorescence change detection of the positive LAMP reaction in paper-based devices could detect 0.7 pg (4.1 x 102 copies) of Streptococcus pneumoniae genomic DNA [10], while a paper-plastic hybrid device detected 18 fg of Staphylococcus aureus DNA with HNB color change detection by the naked eye [44].

Conclusion

A novel POC device was developed using simple and inexpensive materials. The feasibility of the proposed device to diagnose Chagas Disease employing a nucleic acid amplification procedure was successfully demonstrated, thus revealing great potential for POC molecular diagnostics. As the detection principle is generic for most nucleic acid amplification tests (the change of solution conductivity arising from the enzymatic polymerization reaction), the POC device could probably be used to run other diagnosis reactions. To fully corroborate the device performance, Salmon DNA was used as a control, and a low concentration of target nucleic acid template was assayed, showing excellent sensitivity. The detection method proposed was compared to colorimetric detection, which is typically used to develop POC devices, showing good agreement. We demonstrated that sensitive conductivity measurements using disposable single use devices allowed us to develop a POC molecular assay useful for diagnosing T. cruzi parasitosis, given the detection limit obtained of 0.32 fg with the T. cruzi DNA template, which represents 1–5 copies of the target gene/s per reaction. As we use simple low cost materials, and minimal reagent volume, the proposed POC device can overcome other POC methods when cost, easy disposability and biosafety aspects are considered, as the device did not include sharp or glass materials, and is designed to be disposed closed. In field applications, where temperatures can reach 30–35°C, the baseline signal coming from the C4D can be potentially affected. However, as control samples must be run simultaneously with the suspected sample, background variations will be most probably cancelled. Meanwhile this is an approximation, we believe that any potential scenario related to field conditions need to be tested further. To really become an integrated POC system, the device should work with a drop of human blood, and designed as an entire portable detection system including the analytical device, the detection system and the incubator. Future investigations should be performed to study the possibility to use blood samples as such, and evaluate if any type of sample treatment need to be done.

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