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Keywords:

  • Chagas’ disease;
  • Trypanosoma cruzi;
  • diagnosis;
  • test;
  • screening;
  • Bolivia
  • maladie de Chagas;
  • Trypanosoma cruzi;
  • diagnostic;
  • test de dépistage;
  • Bolivie
  • enfermedad de Chagas;
  • Trypanosoma cruzi;
  • diagnóstico;
  • prueba;
  • tamizaje;
  • Bolivia

Summary

  1. Top of page
  2. SummarySensibilité et spécificité du test Chagas Stat-Pak en BolivieSensibilidad y especificidad de la prueba del Chagas Stat-Pak en Bolivia
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Objective  To compare the results of an immunochromatographic test performed on whole blood, Chagas Stat-Pak®, with those of an ELISA test using recombinant antigens.

Method  We tested 995 subjects of a rural population of all ages in the south of Bolivia, 459 pregnant women of the same population and 1030 urban women giving birth from the east of Bolivia.

Results  The sensitivity of the CSP test for the entire studied population (n = 2484) was 94.73% [93.35–96.10]; the specificity was 97.33% [96.50–98.15]. However, the specificity differed significantly between rural pregnant and urban birthing women, which could be attributed either to differences of parasite strain or Chagas prevalence.

Conclusion  The test is simple of use, reliable, relatively inexpensive (<2 US$ each test) and its performances are compatible with a field use for large-scale screenings.

Sensibilité et spécificité du test Chagas Stat-Pak® en Bolivie

Objectif:  Comparer les résultats d’un test immunochromatographique effectué sur le sang total, le Chagas Stat-Pak®, à ceux d’un test ELISA utilisant des antigènes recombinants.

Méthode:  Nous avons testé 995 sujets d’une population rurale, de tous les âges, dans le sud de la Bolivie, 459 femmes enceintes de la même population et 1030 femmes urbaines accouchant dans l’est de la Bolivie.

Résultats:  La sensibilité du test Chagas Stat-Pak® pour l’ensemble de la population étudiée (n = 2484) était de 94,73% [93,35-96,10], la spécificitéétait de 97,33% [96,50-98,15]. Cependant, la spécificité différait considérablement entre les femmes enceintes des zones rurales et celles accouchant dans les zones urbaines, ce qui pourrait être attribué soit à des différences de souches du parasite ou à la prévalence de la maladie de Chagas.

Conclusion:  Le test est d’utilisation simple, fiable et relativement peu coûteux (moins de 2$ US par test) et ses performances sont compatibles avec une utilisation sur le terrain pour des dépistages à grande échelle.

Sensibilidad y especificidad de la prueba del Chagas Stat-Pak® en Bolivia

Objetivo:  Comparar los resultados de la prueba inmunocromatográfica Chagas Stat-Pak®, realizada en sangre, con los de una prueba de ELISA utilizando antígenos recombinantes.

Método:  Hemos analizado a 995 sujetos de una población rural de todas las edades en el sur de Bolivia, 459 mujeres embarazadas de la misma población y 1030 mujeres urbanas que habían dado a luz en el este de Bolivia.

Resultados:  La sensibilidad del Chagas Stat-Pak® para toda la población de estudio (n = 2484) fue del 94.73% [93.35-96.10]; la especificidad fue del 97.33% [96.50-98.15]. Sin embargo, la especificidad difería significativamente entre las mujeres rurales embarazadas y las puérperas urbanas, lo cual podría atribuirse a diferencias en la cepa del parásito o a la prevalencia del Chagas.

Conclusión:  La prueba es fácil de usar, fiable, relativamente barata (menos de 2 US$ por prueba) y su desempeño es compatible con el uso en el campo para tamizajes a gran escala.


Introduction

  1. Top of page
  2. SummarySensibilité et spécificité du test Chagas Stat-Pak en BolivieSensibilidad y especificidad de la prueba del Chagas Stat-Pak en Bolivia
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Chagas disease, caused by Trypanosoma cruzi, affects 9 million people in Latin America (Schofield et al. 2006). In Bolivia, Chagas disease prevalence is the highest in Latin America, with considerable geographical variations (Guillen et al. 1997). The National Programme of Chagas Control recommends testing pregnant women for Chagas in order to manage congenital disease and testing children aged 1–15 years in order to treat infected children. Screening is based on two different tests for diagnostic assessment (Blanco et al. 2000).

To be useful for screening, rapid diagnosis field tests should give a quick response, and should be robust, reliable and inexpensive in order to respond to the requirements of large-scale investigations and field conditions. The better the sensitivity (SS), the smaller the number of false negatives and missed infected subjects. Specificity (SP) is also socially important as it avoids the false positives leading to trypanocid treatment and the risk of associated adverse effects. Consequently, the intended use of the test was to detect T. cruzi infection rapidly during a screening campaign in order to estimate the risk of Chagas disease in pregnant women and children; and to manage appropriate monitoring, e.g. confirmation test in the mother, parasitological diagnosis and immediate treatment of the newborn if necessary. We evaluated the performance of the Chagas Stat-Pak® (CSP; Chembio Diagnostic System, Medford, NY, USA), an immunochromatographic test recently marketed.

Methods

  1. Top of page
  2. SummarySensibilité et spécificité du test Chagas Stat-Pak en BolivieSensibilidad y especificidad de la prueba del Chagas Stat-Pak en Bolivia
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

We conducted prospective studies in the rural community of Caraparí (Department of Tarija, Province of Gran Chaco) in south Bolivia, and the town of Santa Cruz (Department and Province of Santa Cruz), in the east of Bolivia.

In Caraparí, after a general census of the population, we allotted a number to each charted house of the municipality (Chippaux et al. 2008). We counted 78 communities, 2078 houses (306 abandoned and 1772 inhabited) and 8810 residents. Mean age was 25.4 years; 40% of the population (3615) were younger than 16 years. There were 4161 men and 4651 women (sex-ratio M/F = 0.89). We randomly selected houses from a computer list of random numbers on two levels, first at community level according to their frequency, then at habitation level in randomized communities. The survey took place in May and June 2007 and included 1100 people in the sample, i.e. 15% of the population from all age groups. We also included all women of the municipality who were pregnant at the time or had been pregnant in the preceding 3 years (2004–2007). All participants from Caraparí were apparently healthy at the time of the survey.

In Santa Cruz, all women consulting at the Percy Boland reference municipal maternity unit in February and March 2007 were included in the study. This maternity unit assisted at 8788 childbirths in 2007, about half of all deliveries of Santa Cruz. All pregnant women were apparently healthy at the time of the survey.

After obtaining signed informed consent, we collected a finger-prick blood sample into a 600-μl Microtainer® tube with lithium heparin and plasma separator (Becton Dickinson, Franklin Lakes, NJ, USA). The CSP test was performed on whole blood immediately (excepted for pregnant women in Caraparí for which it was completed in the Caraparí hospital laboratory) and the result read after 15 min. After centrifugation, the Microtainer tube was frozen at −20 °C until a second serological test for confirmation.

We used a third-generation ELISA (Chagatest®; Wiener, Rosario, Argentina) as a confirmation test. It was chosen as the reference test because of its accessibility and excellent performance (SS = 99% [96.9–100]; SP = 99% [97.6–100]) shown through latent class analysis compared with six other tests (Pirard et al. 2005). The ELISAs were performed in the reference laboratory of parasitology of Instituto Nacional de Laboratorios de Salud in La Paz within 1 month after sampling Caraparí’s general population and the pregnant women of Santa Cruz, and within 2–3 months after sampling the pregnant women of Caraparí, according to the number of samples sufficient to fill an ELISA plate. Plasma samples were tagged with a number and sample date. Name, age, origin of the sample and CSP result were blinded until the final result of the ELISA test. The cutoff was determined after three negative controls supplied by the manufacturer. Samples with optical density higher than the cutoff ±2 SDs were considered positive, according to the manufacturer’s recommendation. Internal control used a pool of positive plasmas from our laboratory serological bank. Tests giving indeterminate results, i.e. those within 2 SDs around the cutoff, were repeated. In case of discordant CSP and ELISA results, both tests were repeated.

Sensitivity and SP were calculated according to the method of Grimes and Schulz (2002); the agreement between CSP and ELISA tests was measured with Cohen’s kappa tests (κ). For comparisons, we used Fisher’s exact test for the prevalence and chi-square for performance according to age and geographical area; the level of significance was P = 0.05. Calculations were performed using Epi-Info® 6 (Centers for Disease Control, Atlanta, GA, USA).

All persons executing the test were trained professionals experienced in such studies and laboratory techniques. We ensured that presentation of the results conformed with the STARD statement (Bossuyt et al. 2003).

Results

  1. Top of page
  2. SummarySensibilité et spécificité du test Chagas Stat-Pak en BolivieSensibilidad y especificidad de la prueba del Chagas Stat-Pak en Bolivia
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

In Caraparí, we collected 1057 blood samples from 1100 randomized people. There were no refusals, but 43 randomized people were absent at the time of the survey. For various reasons (insufficient quantity of blood collected, uncertain identification, lack of information), we analysed by ELISA only 995 blood samples.

According to CSP and ELISA, the seroprevalence was 45% and 48.4%, respectively (Fisher’s exact test = 0.14). The total number of discordances between the two tests was 42 (4.2%; Table 1). We observed a significant difference of discordances between CSP and ELISA tests according to age (χ2 = 9.39; df = 3; P = 0.02; Table 2) but not according to geographical location inside the municipality (χ2 = 10.72; df = 6; P = 0.10). Among 459 pregnant women according to CSP and ELISA, the prevalence was 71.1% and 70.5%, respectively (Fisher’s exact test = 0.87). SS was 97.7% [95.99–99.35], SP was 82.4% [76.23–88.55] and κ was 0.83 [0.77–0.88]. There were 35 discordances (7.6%; Table 3).

Table 1.   Comparison between Chagas Stat-Pak® and ELISA regarding Caraparí population
Chagas Stat-Pak®ELISATotal
(+)(−)
  1. Sensitivity (SS) = 92.1% [89.36–94.2]; specificity (SP) = 99.2% [98.01–99.7].

  2. κ = 0.92 [0.85–0.98].

(+)4444448
(−)38509547
Total482513995
Table 2.   Comparison between Chagas Stat-Pak® and ELISA according to age
Age (years)nSensitivitySpecificityκ
1–1033392% [81.2–96.9]99.7% [98–99.9]0.94 [0.83–1.05]
11–2023089.2% [81.7–93.9]99.2% [95.7–99.9]0.89 [0.77–1.02]
21–4930092.1% [87.7–95]98.8% [93.7–99.8]0.86 [0.75–0.97]
≥5013294.8% [89.2–97.6]93.8% [71.7–98.9]0.78 [0.61–0.95]
Table 3.   Comparison between Chagas Stat-Pak® and ELISA in pregnant women of Caraparí
Chagas Stat-Pak®ELISATotal
(+)(−)
  1. Sensitivity (SS) = 97.7% [95.99–99.35]; specificity (SP) = 82.4% [76.23–88.55].

  2. κ = 0.83 [0.77–0.88].

(+)29328321
(−)7131138
Total300159459

In Santa Cruz, we obtained 1030 samples from women at time of delivery. According to CSP and ELISA, the seroprevalence was 23.3% and 23.5%, respectively (Fisher’s exact test = 0.94). SS was 96.3% [93.08–98.03], SP was 99.1% [98.18–99.57] and κ was 0.96 [0.90–1.02]. There were16 discordances between CSP and ELISA (1.6%; Table 4).

Table 4.   Comparison between Chagas Stat-Pak® and ELISA in pregnant women of Santa Cruz
Chagas Stat-Pak®ELISATotal
(+)(−)
  1. Sensitivity (SS) = 96.3% [93.08–98.03]; specificity (SP) = 99.1% [98.18–99.57].

  2. κ = 0.96 [0.9–1.02].

(+)2337240
(−)9781790
Total2427881030

Overall, we found a SS of 94.73% [93.35–96.10] and a specificity of 97.33% [96.50–98.15]. The difference in the number of discordances among pregnant women from Caraparí and Santa Cruz (χ2 = 33.58; df = 1; P = 10−4) was dramatic and significant.

Discussion

  1. Top of page
  2. SummarySensibilité et spécificité du test Chagas Stat-Pak en BolivieSensibilidad y especificidad de la prueba del Chagas Stat-Pak en Bolivia
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

To validate the CSP test in very different situations, we performed our surveys both in a rural area highly endemic for T. cruzi and in an urban area with lower prevalence of Chagas disease. Thus, we could confirm the test’s feasibility under field conditions and its accuracy in diverse epidemiological situations.

Three recombining antigens are used in the CSP in order to optimize its performance (Umezawa et al. 2003). Luquetti et al. (2003) using sera from Central America observed higher SS (ranged 98.5–100%) than SP (94.8–98.6%). These results were confirmed by Ponce et al. (2005), also on Central American sera, showing SS and SP at 99.6 and 99.9, respectively, with a very high agreement between CSP and ELISA. In a recent study carried out in Bolivian youngs from 9 months to 17 years (Roddy et al. 2008), the CSP performed on whole blood showed a very similar performance to those in our survey (SP = 99%, SS = 93.4%). The lower SS of CSP in Bolivia could be explained either by an antigenic geographical variability of the parasitic strains (Luquetti et al. 2003) or by using whole blood instead of serum or plasma.

A recent multicentric study performed in pregnant women from four Latin America countries using umbilical cord whole blood samples (Sosa-Estani et al. 2008) showed very similar SS and SP compared with our results but wide geographical variations of the CSP performances. These were attributed to (a) differences in the characteristics of study area and population, (b) dynamics of transmission and history of control programs, (c) strain variability and (d) differences in antibody concentrations in umbilical cord.

However, the significant differences in discordance between CSP and ELISA tests according to age and geographical origin among pregnant women are alarming. The difference according to the age cannot be explained by different parasitic strains, as all samples came from the same area. We do not think that the discrepancy between pregnant women from Caraparí and Santa Cruz could be attributed to the delay in performing the test in Caraparí. Although parasitic strain differences may explain the difference, the higher prevalence according to the age and in pregnant women from Caraparí (70.5%) compared with those from Santa Cruz (23.5%) could be the cause.

Conclusion

  1. Top of page
  2. SummarySensibilité et spécificité du test Chagas Stat-Pak en BolivieSensibilidad y especificidad de la prueba del Chagas Stat-Pak en Bolivia
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Although quite expensive (1.5–2 US$), this test is simple and to use in the field and provides an answer in 15 min, which is an advantage during large-scale campaigns. The purpose of the CSP test is not a definitive diagnosis of Chagas disease but to detect T. cruzi infection for population screening. Although performance of the test should be considered in each particular case accordingly to the aim of the survey, its overall SP (99.3%) and SS (94.7%) appeared to be sufficient for this objective. However, it to confirm a diagnosis of Chagas disease, another test is indicated.

Acknowledgements

  1. Top of page
  2. SummarySensibilité et spécificité du test Chagas Stat-Pak en BolivieSensibilidad y especificidad de la prueba del Chagas Stat-Pak en Bolivia
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The authors thank Susana Montaño, Eddy Espinoza, Tatiana Rios, José Melgarejo and the personnel of the hospital of Caraparí for their contribution; Mercedes Galarza who participated to the survey in Santa Cruz; and Patricia Oporto for her help in ELISA confirmation. This work was supported by Grants from Total E&B Bolivia and IRD (France). The authors thank Chembio for providing the Stat-Paks® tests used in this study.

References

  1. Top of page
  2. SummarySensibilité et spécificité du test Chagas Stat-Pak en BolivieSensibilidad y especificidad de la prueba del Chagas Stat-Pak en Bolivia
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  • Blanco SB, Segura EL, Cura EN et al. (2000) Congenital transmission of Trypanosoma cruzi: an operational outline for detecting and treating infected infants in north-western Argentina. Tropical Medicine and International Health 5, 293301.
  • Bossuyt PM, Reitsma JB, Bruns DE et al. (2003) The STARD statement for reporting studies of diagnostic accuracy: explanation and elaboration. Croatian Medical Journal 44, 639650.
  • Chippaux JP, Postigo JR, Santalla JA, Schneider D & Brutus L (2008) Epidemiological evaluation of Chagas disease in a rural area of southern Bolivia. Transactions of the Royal Society of Tropical Medicine and Hygiene 102, 578584.
  • Grimes D & Schulz K (2002) Uses and abuses of screening tests. Lancet 359, 881884.
  • Guillen G, Diaz R, Jemio A, Cassab JA, Pinto CT & Schofield CJ (1997) Chagas disease vector control in Tupiza, southern Bolivia. Memórias do Instituto Oswaldo Cruz 92, 18.
  • Luquetti A, Ponce C, Ponce E et al. (2003) Chagas’ disease diagnosis: a multicentrix evaluation of Chagas Stat-Pak, a rapid immunochromatographic assay with recombinant proteins of Trypanosoma cruzi. Diagnostic Microbiology and Infectious Disease 46, 265271.
  • Pirard M, Iihoshi N, Boelaert M, Basanta P, López F & Van der Stuyft P (2005) The validity of serologic tests for Trypanosoma cruzi and the effectiveness of transfusional screening strategies in a hyperendemic region. Transfusion 45, 554561.
  • Ponce C, Ponce E, Vinelli E et al. (2005) Validation of a rapid and reliable test for diagnosis of chagas’ disease by detection of Trypanosoma cruzi-specific antibodies in blood of donors and patients in Central America. Journal of Clinical Microbiology 43, 50655068.
  • Roddy P, Goiri J, Flevaud L et al. (2008) Field evaluation of a rapid immunochromatographic assay for detection of Trypanosoma cruzi infection by use of whole blood. Journal of Clinical Microbiology 46, 20222027.
  • Schofield CJ, Jannin J & Salvatella R (2006) The future of Chagas disease control. Trends in Parasitology 22, 583588.
  • Sosa-Estani S, Gamboa-Leon R, Del Cid-Lemus J et al. (2008) Use of rapid-test on umbilical cord blood to screen for Trypanosoma cruzi infection in pregnant women in Argentina, Bolivia, Honduras, and México. The American Journal of Tropical Medicine and Hygiene 79, 755759.
  • Umezawa ES, Bastos SF, Coura JR et al. (2003) An improved serodiagnostic test for Chagas’ disease employing a mixture of Trypanosoma cruzi recombinant antigens. Transfusion 43, 9197.