Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia

Introduction: Toxoplasmosis is the disease which is caused by the protozoan parasite Toxoplasma gondii (T. gondii), which has the ability to infect all warm-blooded animals. Several molecular techniques for the diagnosis of Toxoplasma gondii includes normal and Real Time Polymerase Chain Reaction (RT-PCR), and gene sequencing. Aim: To apply Loop-Mediated Isothermal Amplification (LAMP) for the diagnosis of T. gondii infections in urine and blood samples from female population in Saudi Arabia. Materials and Methods: In this cross-sectional study, genomic DNA was extracted from 22 blood and urine samples (11 each), two LAMP assays based on B1 and Surface Antigen 2 (SAG2) genes of T. gondii was performed. Conventional PCR was done for the LAMP product followed by sequencing to confirm the specificity of LAMP method. Statistical Package for the Social Sciences (SPSS) version 20.0 was used to summarise continuous and categorical variables. Results: From 22 samples, 17 samples were LAMP positive in both urine and blood samples, four were negative in blood and positive in urine samples, and one was positive in blood and negative in urine. Sequencing of PCR product confirm the specificity of the method used. Conclusion: LAMP detection of T. gondii DNA is an appropriate, sensitive and specific method for diagnosis of toxoplasmosis from urine and blood specimens in humans.

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Development and Clinical Evaluation of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Brazil

BioMed Research International ◽

10.1155/2019/8240784 ◽

2019 ◽

Vol 2019 ◽

pp. 1-7 ◽

Cited By ~ 5

Author(s):

Daniel Moreira de Avelar ◽

Débora Moreira Carvalho ◽

Ana Rabello

Keyword(s):

Visceral Leishmaniasis ◽

Lamp Assay ◽

Isothermal Amplification ◽

Health Concern ◽

Serological Tests ◽

Blood Samples ◽

Loop Mediated Isothermal Amplification ◽

Human Visceral Leishmaniasis ◽

Highly Sensitive ◽

Sensitivity Specificity

Visceral leishmaniasis (VL) is considered a major public health concern in Brazil and several regions of the world. A recent advance in the diagnosis of infectious diseases was the development of loop-mediated isothermal amplification (LAMP). The aim of this study was to develop and evaluate a new LAMP assay for detection of K26 antigen-coding gene of L. donovani complex. A total of 219 blood samples of immunocompetent patients, including 114 VL cases and 105 non-VL cases, were analyzed for the diagnosis of VL in the present study. Diagnostic accuracy was calculated against a combination of parasitological and/or serological tests as a reference standard. The results were compared to those of kDNA Leishmania-PCR. The detection limit for the K26-Lamp assay was 1fg L. infantum purified DNA and 100 parasites/mL within 60 min of amplification time with visual detection for turbidity. The assay was specific for L. donovani complex. Sensitivity, specificity, and accuracy were 98.2%, 98.1%, and 98.2%, respectively, for K26-LAMP and 100%, 100%, and 100%, respectively, for kDNA Leishmania-PCR. Excellent agreement was observed between K26-LAMP and kDNA Leishmania-PCR assays (K = 0.96). A highly sensitive and specific LAMP assay targeting K26 antigen-coding gene of L. donovani complex was developed for diagnosis in peripheral blood samples of VL patients.

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