Journal Article

Evaluation of SYBR green I based visual loop-mediated isothermal amplification (LAMP) assay for genus and species-specific diagnosis of malaria in P. vivax and P. falciparum endemic regions

Singh, Ruchi, Bhatt, RajendraM, Valecha, Neena, Singh, OmP, Savargaonkar, Deepali, Singh, DhirendraPratap

2017

Overview

Loop-mediated isothermal amplification (LAMP) is an emerging nucleic acid based diag- nostic approach that is easily adaptable to the field settings with limited technical resources. This study was aimed to evaluate the LAMP assay for the detection and identification of Plasmodium falciparum and P. vivax infection in malaria suspected cases using genus and species-specific assay. The 18S rRNA-based LAMP assay was evaluated for diagnosis of genus Plasmodium, and species- specific diagnosis of P. falciparum and P. vivax, infection employing 317 malaria suspected cases, and the results were compared with those obtained by 18S nested PCR (n-PCR). All the samples were confirmed by microscopy for the presence of Plasmodium parasite. The n-PCR was positive in all Plasmodium-infected cases (n=257; P. falciparum=133; P. vivax=124) and negative in microscopy negative cases (n=58) except for two cases which were positive for P. vivax, giving a sen- sitivity of 100% (95% CI: 97.04-100%) and a specificity of 100% (95% CI: 88.45-99.5%). Genus-specific LAMP assay missed 11 (3.2%) microscopy and n-PCR confirmed vivax malaria cases. Considering PCR results as a refer- ence, LAMP was 100% sensitive and specific for P. falciparum, whereas it exhibited 95.16% sensitivity and 96.7% specificity for P. vivax. The n-PCR assay detected 10 mixed infection cases while species-specific LAMP detected five mixed infection cases of P. vivax and P. falciparum, which were not detected by microscopy. Genus-specific LAMP assay displayed low sensitivity. Falciparum specific LAMP assay displayed high sensitivity whereas vivax specific LAMP assay displayed low sensitivity. Failed detection of vivax cases otherwise confirmed by the n-PCR assay indicates exploitation of new targets and improved detection methods to attain 100% results for P. vivax detection.

• Publisher: Medknow Publications and Media Pvt. Ltd,Medknow Publications & Media Pvt. Ltd,Wolters Kluwer Medknow Publications
• Subject: Adolescent / Adult / Aged / Aged, 80 and over / Antigens / Bioassay / Child / Child, Preschool / Dehydrogenases / Deoxyribonucleic acid / DNA / Female / Genetic aspects / Humans / Identification / Infant / Infections / Laboratories / LAMP; malaria; molecular diagnosis; PCR; Plasmodium falciparum; P. vivax / Malaria / Malaria, Falciparum - diagnosis / Malaria, Falciparum - parasitology / Malaria, Vivax - diagnosis / Malaria, Vivax - parasitology / Male / Methods / Microscopy / Middle Aged / Molecular Diagnostic Techniques - methods / Nucleic Acid Amplification Techniques - methods / Organic Chemicals - analysis / Parasites / Plasmodium falciparum - genetics / Plasmodium falciparum - isolation & purification / Plasmodium vivax - genetics / Plasmodium vivax - isolation & purification / Polymerase chain reaction / RNA, Ribosomal, 18S - genetics / Sensitivity and Specificity / Staining and Labeling - methods / Vectors (Biology) / Young Adult