RESUMO
BACKGROUND: Vertical transmission of Trypanosoma cruzi represents approximately 20% of new Chagas disease cases. Early detection and treatment for women of childbearing age and newborns is a public health priority, but the lack of a simple and reliable diagnostic test remains a major barrier. We aimed to evaluate the performance of a point-of-care loop-mediated isothermal amplification (LAMP) assay for the detection of T cruzi. METHODS: In this proof-of-concept study, we coupled a low-cost 3D printer repurposed for sample preparation and amplification (PrintrLab) to the Eiken T cruzi-LAMP prototype to detect vertically transmitted T cruzi, which we compared with standardised PCR and with the gold-standard algorithm (microscopy at birth and 2 months and serological study several months later). We screened pregnant women from two hospitals in the Bolivian Gran Chaco province, and those who were seropositive for T cruzi were offered the opportunity for their newborns to be enrolled in the study. Newborns were tested by microscopy, LAMP, and PCR at birth and 2 months, and by serology at 8 months. FINDINGS: Between April 23 and Nov 17, 2018, 986 mothers were screened, among whom 276 were seropositive for T cruzi (28·0% prevalence, 95% CI 25·6-31·2). In total, 224 infants born to 221 seropositive mothers completed 8 months of follow-up. Congenital transmission was detected in nine of the 224 newborns (4·0% prevalence, 1·9-7·5) by direct microscopy observation, and 14 more cases were diagnosed serologically (6·3%, 3·6-10·3), accounting for an overall vertical transmission rate of 10·3% (6·6-15·0; 23 of 224). All microscopy-positive newborns were positive by PrintrLab-LAMP and by PCR, while these techniques respectively detected four and five extra positive cases among the remaining 215 microscopy-negative newborns. INTERPRETATION: The PrintrLab-LAMP yielded a higher sensitivity than microscopy-based analysis. Considering the simpler use and expected lower cost of LAMP compared with PCR, our findings encourage its evaluation in a larger study over a wider geographical area. FUNDING: Inter-American Development Bank.
Assuntos
Doença de Chagas , Transmissão Vertical de Doenças Infecciosas , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Trypanosoma cruzi , Humanos , Doença de Chagas/diagnóstico , Doença de Chagas/transmissão , Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/isolamento & purificação , Recém-Nascido , Bolívia/epidemiologia , Feminino , Gravidez , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Estudo de Prova de Conceito , Sensibilidade e Especificidade , AdultoRESUMO
A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected-namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen's kappa index and Bland-Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = -0.58-0.62] and -0.04 [CI = -0.45-0.37] and a Cohen's kappa coefficient of 0.78 [CI = 0.60-0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.
RESUMO
Vertical transmission of Trypanosomacruzi is the cause of congenital Chagas disease, a re-emerging infectious disease that affects endemic and nonendemic regions alike. An early diagnosis is crucial because prompt treatment achieves a high cure rate, precluding evolution to symptomatic chronic Chagas disease. However, early diagnosis involves low-sensitive parasitologic assays, making necessary serologic confirmation after 9 months of life. With the aim of implementing early diagnostic strategies suitable for minimally equipped laboratories, a T. cruzi-loop-mediated isothermal amplification (LAMP) prototype was coupled with an automated DNA-extraction device repurposed from a three-dimensional printer (PrintrLab). The whole process takes <3 hours to yield a result, with an analytical sensitivity of 0.1 to 2 parasite equivalents per milliliter, depending on the T. cruzi strain. Twenty-five blood samples from neonates born to seropositive mothers were tested blindly. In comparison to quantitative real-time PCR, the PrintrLab-LAMP dual strategy showed high agreement, while both molecular-based methodologies yielded optimal sensitivity and specificity with respect to microscopy-based diagnosis of congenital Chagas disease. PrintrLab-LAMP detected all 10 congenitally transmitted T. cruzi infections, showing promise for point-of-care early diagnosis of congenital Chagas disease.