A comparison of four DNA extraction protocols for the analysis of urine from patients with visceral leishmaniasis

Abstract

Introduction: Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplifi cation. Methods: A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplifi cation, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results: With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion: Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most effi cient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

“…However, although many studies have compared the various extraction methods of circulating cfDNA in blood [17][18][19], limited data are available on methods for urinary cfDNA extraction [20,21]. Most studies have been conducted on urinary DNA, regardless of being genomic DNA or cfDNA [22][23][24].…”

Isolation and molecular characterization of Leishmania infantum in urine from patients with visceral leishmaniasis in Brazil

“…Further testing of the best way to preserve and treat urine samples may increase the utility of this method. Treatment of samples to reduce inhibition, such as keeping samples at -70°C or below (Madisen et al, 1987), precipitating suspended solids, using ethanol, and concentrating DNA could increase the success of Leishmania detection in urine samples, as well as modification of amplification techniques that can detect smaller fragments of more degraded DNA (Silva et al, 2014).…”

“…The use of PCR-based techniques has also expanded the types of samples from which L. infantum can be detected, including urine (Lachaud et al, 2001). This has successfully detected the DNA of Leishmania parasites in both humans and canines (Fisa et al, 2008;Mebrahtu et al, 1993;Silva et al, 2014;Solano-Gallego et al, 2007) Here we report the detection of L. infantum in human urine samples. VL is a potentially fatal disease (Desjeux, 1996).…”

Rapid Tests and the Diagnosis of Visceral Leishmaniasis and Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome Coinfection

“…The mean CD4+ T-lymphocyte counts in the seven VL-HIV/AIDS coinfected patients included in this study was 162.21 cells/mm 3 8 ; DAT = direct agglutination test; KAtex = a latex agglutination test; Neg = negative; Pos = positive; qPCR = real-time PCR 9 ; rK39 ICT = rK39-based immunochromatographic test; T CD4+ = CD4+ T lymphocytes; VL-HIV/AIDS = visceral leishmaniasis-human immunodeficiency virus/acquired immunodeficiency syndrome.…”