Highly Sensitive Detection of Malaria Parasitemia in a Malaria-Endemic Setting: Performance of a New Loop-Mediated Isothermal Amplification Kit in a Remote Clinic in Uganda

Abstract

Background. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too complex for field deployment. A new commercial molecular assay based on loop-mediated isothermal amplification (LAMP) was assessed for field use.Methods. Malaria LAMP (Eiken Chemical, Japan) was evaluated for samples from 272 outpatients at a rural Ugandan clinic and compared with expert microscopy, nested PCR, and quantitative PCR (qPCR). Two technicians performed the assay after 3 days of training, using 2 alternative blood sample–preparation methods and visual interpretation of results by fluorescence assay.Results. Compared with 3-well nested PCR, the sensitivity of both LAMP and single-well nested PCR was 90%; the microscopy sensitivity was 51%. For samples with a Plasmodium falciparum qPCR titer of ≥2 parasites/µL, LAMP sensitivity was 97.8% (95% confidence interval, 93.7%–99.5%). Most false-negative LAMP results involved samples with parasitemia levels detectable by 3-well nested PCR but very low or undetectable by qPCR.Conclusions. Malaria LAMP in a remote Ugandan clinic achieved sensitivity similar to that of single-well nested PCR in a United Kingdom reference laboratory. LAMP dramatically lowers the detection threshold achievable in malaria-endemic settings, providing a new tool for diagnosis, surveillance, and screening in elimination strategies.

Assessing the impact of next-generation rapid diagnostic tests on Plasmodium falciparum malaria elimination strategies

“…(LAMP), which involves a simple DNA extraction procedure, isothermal amplification and visual examination of positivity -is currently only used in research settings 16 . Two recent MSAT trials, which used RDT screening to identify infections, failed to lead to a sustained reduction in malaria parasite prevalence or disease incidence 17,18 .…”

Diagnosis and Treatment of Plasmodium vivax Malaria

“…Recent evaluation of a commercially available kit (with P. falciparum and pan-genus-specific reagents) showed the technique to be comparable to standard nested PCR technique and superior to expert microscopy. 17,18 The procedure can be completed in about 1 hour at the site of collection. A modified LAMP procedure, as well as standard nested PCR, was performed in the diagnosis of P. vivax in large field surveys in China.…”

Challenges of malaria diagnosis in clinical settings and disease surveillance under reduced malaria burden in Tanzania

“…As malaria prevalence declines, transmission also declines to levels whereby infections become asymptomatic; such infections are estimated to be the source of 20%-50% of all human-to-mosquito transmission [42]. Diagnosing asymptomatic cases of malaria is challenging because asymptomatic people do not seek care and asymptomatic cases are often undetectable by microscopy or RDTs. The changing malaria epidemiology and increased importance of surveillance in malaria elimination settings which is characterized by asymptomatic malaria infections require highly sensitive and robust diagnostic tools that can detect asymptomatic infections in the community by mass screening at low costs and fast turnaround results [71]. The loop-mediated isothermal amplification (LAMP), is a promising new NAT.…”

“…This process uses multiple different primers and a DNA polymerase to create a stem-loop structure of DNA, which serves as a template for amplification. LAMP has a sensitivity (92.7%) similar to PCR at low parasite density (10 parasites/µL) [71]. LAMP has lower costs, faster turnaround time with high-throughput, and less technical requirements compared to PCR for malaria diagnosis; moreover, it can detect malaria DNA from dried blood spots on filter paper, which is commonly used in mass screening and treatment during community surveillance in malaria elimination settings [72].…”