Analysis of DNA extraction methods for detection of Treponema pallidum: A comparison of three methods

Grassi, Vera Mileide Trivellato; Ramos, Maria Eliza Barbosa; Grassi, Liliane Trivellato; Silva, Márcia Susana Nunes; Rossetti, Maria Lúcia Rosa

doi:10.1016/j.mimet.2021.106383

Cited by 3 publications

(1 citation statement)

References 31 publications

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“…Our exploratory study identified T. pallidum DNA using a conventional PCR which targeted tpp47. This technique was validated in a previous study of our group in approximately two thirds of BS as well as in two thirds of CSF samples of the newborns exposed to syphilis during pregnancy [17]. On the other hand, newborns from mothers who had repeated negative serology for syphilis during pregnancy had a positive T. pallidum PCR in one-tenth of the BS and one-fifth of the CSF samples.…”

Section: Discussionmentioning

confidence: 70%

Treponema pallidum PCR with blood and cerebrospinal fluid of newborns exposed and not exposed to syphilis during pregnancy

Melo,

Ramos,

Rossetti

et al. 2024

J Infect Dev Ctries

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Introduction: Congenital syphilis (CS) has severe adverse outcomes, including abortion and death. Diagnosis of CS in asymptomatic newborns remains difficult. This study aims to evaluate an in-house polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) and blood samples (BS) to identify T. pallidum DNA in newborns. Methodology: We performed an exploratory cross-sectional study that included newborns exposed to syphilis during pregnancy (SEG) and non-exposed (SNEG) newborns, between 2019 and 2020. In-house conventional PCR for T. pallidum targeting the tpp47 gene was used to analyze CSFS and dried blood spots. Results: BS was obtained from 54 newborns (33 SEG/21 SNEG) and CSF from 55 newborns (33 SEG/22 SNEG). Twenty-five (71.4%) SEG newborns had reactive BS rapid plasmatic reagins (RPR), and all of them had RPR titers less than or equal to the corresponding maternal titers. All RPR CSF tests were negative. PCR for T. pallidum DNA was positive in 19/33 (57.6%) BS, and in 22/33 CSF. The only SEG newborn with clinical signs of early CS had a positive CSF PCR and a negative BS PCR. Conversely, among SNEG newborns, PCR was positive in 2/21 BS and 5/22 (22.7%) CSF. Conclusions: T. pallidum DNA was identified using our PCR tests. The exposed group did not present abnormalities that would indicate CS. This prevented conclusions regarding sensitivity and specificity. Dried spot permitted bedside collection, easy transportation, and storage. Further research is needed to evaluate and improve the accuracy of CS low-cost PCR tests, especially for limited resource settings.

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Section: Discussionmentioning

confidence: 70%

Treponema pallidum PCR with blood and cerebrospinal fluid of newborns exposed and not exposed to syphilis during pregnancy

Melo,

Ramos,

Rossetti

et al. 2024

J Infect Dev Ctries

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Adenine imprinted beads as a novel selective extracellular DNA extraction method reveals underestimated prevalence of extracellular antibiotic resistance genes in various environments

Yuan

Wang

et al. 2022

Science of The Total Environment

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A Loop-Mediated Isothermal Amplification Assay Utilizing Hydroxy Naphthol Blue (LAMP-HNB) for the Detection of Treponema pallidum Subspp. pallidum

Phurijaruyangkun,

Tangjitrungrot,

Jaratsing

et al. 2024

Pathogens

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Treponema pallidum subspp. pallidum is a spirochaete bacterium that causes syphilis, one of the most common sexually transmitted diseases. Syphilis progresses through four distinct stages, each characterized by specific symptoms, namely primary, secondary, latent, and late (tertiary) syphilis. Serology has been considered the primary diagnostic approach. However, it is plagued by problems such as the limited specificity of nontreponemal tests and the inadequate correlation of treponemal tests with disease activity. In this study, we focused on the development of a loop-mediated isothermal amplification assay utilizing hydroxy naphthol blue (LAMP-HNB) for the diagnosis of T. pallidum subspp. pallidum. Specifically, this study seeks to determine the analytical sensitivity (limit of detection; LOD) and analytical specificity. Four hundred clinical serum samples were analyzed for diagnostic sensitivity, specificity, and predictive value, and each technique’s 95% confidence intervals (95% CI, p < 0.05) were evaluated. The limit of detection for polymerase chain reaction with agarose gel electrophoresis (PCR-AGE), the loop-mediated isothermal amplification assay combined with agarose gel electrophoresis (LAMP-AGE), and LAMP-HNB were 116 pg/µL, 11.6 pg/µL, and 11.6 pg/ µL, respectively. Analytical specificity examinations indicated the absence of cross-reactivity with Leptospira interrogans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, human immunodeficiency virus (HIV), and healthy human serum in PCR-AGE, LAMP-AGE, and LAMP-HNB. The diagnostic sensitivity, diagnostic specificity, positive predictive value (PPV), and negative predictive value (NPV) for PCR-AGE were 100.00 (100.00)%, 94.50 (94.40–94.60)%, 94.79 (94.69–94.88)%, and 100.00 (100.00)%, respectively. While, for LAMP-AGE and LAMP-HNB, they were 100.00 (100.00)%, 91.00 (90.87–91.13)%, 91.74 (91.63–91.86)%, and 100.00 (100.00)%, respectively. The LAMP-HNB test is simple, rapid, highly sensitive, and highly specific, without requiring expensive equipment. In the future, the LAMP-HNB assay may develop into a single-step diagnostic process, enabling the use as point-of-care testing for the diagnosis, prevention, and management of syphilis infection.

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Analysis of DNA extraction methods for detection of Treponema pallidum: A comparison of three methods

Cited by 3 publications

References 31 publications

Treponema pallidum PCR with blood and cerebrospinal fluid of newborns exposed and not exposed to syphilis during pregnancy

Treponema pallidum PCR with blood and cerebrospinal fluid of newborns exposed and not exposed to syphilis during pregnancy

Adenine imprinted beads as a novel selective extracellular DNA extraction method reveals underestimated prevalence of extracellular antibiotic resistance genes in various environments

A Loop-Mediated Isothermal Amplification Assay Utilizing Hydroxy Naphthol Blue (LAMP-HNB) for the Detection of Treponema pallidum Subspp. pallidum

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