Simultaneous use of two rapid diagnostic tests for the diagnosis of Chagas disease

Introduction

Chagas disease (CD) is a blood and tissue parasitic disease caused by the protozoan Trypanosoma cruzi (T. cruzi), endemic to the American continent. It is prevalent in impoverished rural areas where the insect vector is present. Moreover, it has extended to urban areas within and outside the continent owing to population migrations. ¹

Diagnosis in the chronic phase of infection requires detection of specific antibodies by two different serological tests which detect antibodies against different antigens; these must yield concordant positive results. The most commonly used conventional serology (CS) tests are indirect haemagglutination (IHA) and enzyme-linked immunosorbent assay (ELISA). When results are discordant, a third assay is performed, usually indirect immunofluorescence (IFI). ² All these tests need to be performed in healthcare centres (HCC) that have, at minimum, laboratory facilities for clinical analyses, which is not always possible in regions where CD has the greatest impact.

Immunochromatograpic tests with colloidal gold, commonly named rapid diagnostic tests (RDTs), have been recently developed; these assays have some advantages, including technical simplicity, conservation of the kits at room temperature, possibility of individual application and rapid reading of results with no need for an equipped laboratory. These characteristics make RDT very useful as screening tests in distant locations; however, a positive result should be confirmed later in a laboratory by CS. The low sensitivity values obtained with RDT by different authors suggest that they are useful only if used in parallel with a CS test, rendering them less useful in the field.^3–8 This low sensitivity is observed mainly when a whole blood sample is used, with test performance being improved when applied to serum samples. Diagnostic sensitivity can be enhanced by performing tests simultaneously on a single sample; however, to date, this option has not been assayed with RDT for CD.

Methods

Two RDT detecting antibodies against different T. cruzi antigens were applied individually and simultaneously to serum samples processed also by CS and their results were compared with CS reference tests.

Serum samples were obtained from patients aged >18 years who attended HCCs in localities from a northern Santa Fe province, located in the geographic zone called Gran Chaco, Argentina, of intermediate endemicity for CD. ⁹ None of the patients had received aetiological treatment for CD before the study. All the patients were free from immunologically mediated diseases. Blood samples were collected by venipuncture using disposable sterile materials; sera were separated by centrifugation at the local HCC, preserved in the refrigerator and transported to the laboratory of the Centre for Research on National Endemic Diseases of the National University of Litoral (CIEN-UNL), where they were processed.

All samples were subjected to IHA (IHA Chagas Polychaco, Lemos Laboratory SRL) and ELISA (Chagatest ELISA, Wiener Lab SAIC), following the manufacturer’s instructions. When results were discordant, IFI was performed with commercial conjugates and smears prepared with T. cruzi epimastigotes of Tulahuen 0 strain. ¹⁰ The samples were processed at the laboratory of CIEN-UNL, which complies with the external quality controls by the National Parasitology Institute ‘Dr. Mario Fatala Chabén’, the national-level reference organisation.

To evaluate the RDTs, only those samples with concordant results by IHA and ELISA were used. Negative (n = 64) and positive (n = 42) samples by CS were selected. For IHA, the cut-off titre of 1/16 was taken, following the manufacturer’s instructions. The titre of positive samples was within 1/16 and 1/512, with a mode value of 1/128. For ELISA, the cut-off value was calculated in each run and the optical density (OD) index was calculated by dividing the optical density of the sample by the cut-off of the run. The mean OD index of negative and positive samples was 0.490 and 4.67, respectively. The following commercial kits were used, following the manufacturers’ instructions:

RDT A: WL Check Chagas (Wiener Lab SAIC, Argentina);

RDT B: SD BiolineChagasAb Rapid (Standard Diagnostics Inc., Korea)

Two researchers performed the RDTs using a double-blind procedure; neither of them knew the previous results by CS. All the results were read and interpreted by both researchers.

To evaluate the reliability of the RDT, the percentage of overall agreement between RDTs, the Chamberlain’s percent positive agreement and the κ statistic were calculated. Both RDTs were validated by comparing their results with those obtained by CS, which was considered the reference result. Sensitivity and specificity of each RDT were estimated individually and for both tests as unique result.

The patients signed an informed consent document especially elaborated for this study. CS results were delivered to each patient separately and their significance was explained. Seropositive patients were referred to the HCC for treatment, following national and provincial regulations. This study was evaluated and approved by the Bioethics Committee of the Ministry of Health of Santa Fe Province.

Results

In all cases, there was agreement in the readings of the results of both RDTs between observers. Results of the comparison of both RDTs with those of the CS, as reference tests, are shown in Table 1.

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Table 1.

Results of CS vs. RDT A and RDT B.

	CS		Total	Sensitivity (%)	Specificity (%)
	Positive	Negative
RDT A
Positive	38	0	38	90.5	100
Negative	4	64	68
RDT B
Positive	41	4	45	97.6	93.8
Negative	1	60	61
Total	42	64	106

Table 2 shows the comparison of the results of the RDTs, indicating the percentage of overall agreement, the Chamberlain’s percent positive agreement and the κ statistic in the footnote.

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Table 2.

Results of serology for CD by RDT A and B.

	RDT A		Total
	Positive	Negative
RDT B
Positive	38	7	45
Negative	0	61	61
Total	38	68	106

Table 3 shows the comparison of the results of both RDTs simultaneously, considering positives or negatives when these were detected by both tests, with CS results.

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Table 3.

Results of serology for CD using simultaneous RDTs vs CS.

	CS*		Total	Sensitivity (%)	Specificity (%)
	Positive	Negative
RDTs †
Positive	38	0	39	97.4%	100%
Negative	1	60	61
	39	60	99

Results of both RDTs were discordant in 7/106 samples (6.6%).

The seven samples showing discordance between both RDTs were submitted to CS, with three positive and four negative results. Table 4 shows the sensitivity and specificity, when the three positives are added to the 38 by RDT and the four negatives to the 60 by RDT.

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Table 4.

Results of serology for CD using simultaneous RDTs + CS in discordant samples vs. CS.

	CS*		Total	Sensitivity (%)	Specificity (%)
	Positive	Negative
RDTs + CS †
Positive	41	0	41	97.6	100
Negative	1	64	65
	42	64	106

Discussion

In this study, the validity of two RDTs (A and B) for the diagnosis of CD at the chronic stage was evaluated individually in serum samples and the agreement of their results was assessed. Moreover, validity of the use of both simultaneous RDTs was assessed and the results were compared with the reference CS test.

RDT A exhibited greater specificity than RDT B, yielding a lower number of false-positive results, whereas RDT B had greater sensitivity; however, neither RDT reached 100% sensitivity and are therefore not considered suitable for use as a single screening test. Sensitivity and specificity values of these RDTs are similar to those reported by Sánchez-Camargo et al., who evaluated 11 tests individually, and to values reported in other studies using serum samples, but better than values reported using whole blood.^3–7

An excellent correlation was observed between the results of both RDTs, as shown by the high percentage of overall agreement, the Chamberlain’s percent positive agreement and the κ statistic, with the latter value minimising the influence of agreements due to chance.

International organisations recommend the simultaneous use of two tests for the diagnosis of CD at the chronic stage: one of high specificity and another of high sensitivity. ¹¹ Several alternatives have been proposed to perform CS in areas distant from HCCs, such as collecting blood samples on filter paper,^12,13 preserving blood in glycerin ¹⁴ and collecting oral mucosal transudate specimens. ¹⁵ Although these studies yielded good results, they required transport of all samples to the laboratory and the need to return to the field for venous blood sampling of positive patients, with inherent expenses, loss of patient follow-up and delay in results.

Based on the present results, we propose using a RTD with high specificity and another RTD with high sensitivity. The simultaneous use of both RDTs with serum samples allows us to obtain a very reliable diagnosis of CD at the chronic stage in remote areas and with minimum equipment (a centrifuge), thereby complying with international recommendations. Given the short time involved in the whole process, including blood sampling, centrifugation, processing and RDT reading, the results can be delivered almost immediately. Thus, only discordant samples, which represent a very low percentage, would have to be transported to a laboratory in a HC to be resolved using a third method (IHA, ELISA or IFI). Efforts should be focused in optimising RDT with whole blood so that there will be no need for sampling centrifugation in the future.