Application of loop-mediated isothermal amplification for malaria diagnosis during a follow-up study in São Tomé

Abstract

BackgroundA reliable and simple test for the detection of malaria parasite is crucial in providing effective treatment and therapeutic follow-up, especially in malaria elimination programmes. A comparison of four methods, including nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were used for the malaria diagnosis and treatment follow-up in São Tomé and Príncipe, during a successful pre-elimination campaign.MethodDuring the period September to November 2009, blood samples from 128 children (five to 14 years old) with temperature ≥38°C (tympanic) in the District of Agua Grande were examined using four different methods, i.e., histidine-rich protein 2 (HRP-2) based rapid diagnostic tests (HRP-2-RDTs), optical microscopy, nested PCR, and LAMP. First-line treatment with artesunate-amodiaquine was given for uncomplicated malaria and intravenous quinine was given for complicated malaria. Children with persistent positivity for malaria by microscopy, or either by nested PCR, or by LAMP on day 7 were given second-line treatment with artemether-lumefantrine. Treatment follow-up was made weekly, for up to four weeks.ResultsOn day 0, positive results for HRP-2-RDTs, microscopy, nested PCR, and LAMP, were 68(53%), 47(37%), 64(50%), and 65(51%), respectively. When nested PCR was used as a reference standard, only LAMP was comparable; both HRP-2-RDTs and microscopy had moderate sensitivity; HRP-2-RDTs had poor positive predictive value (PPV) and a moderate negative predictive value (NPV) for the treatment follow-up. Seventy-one children with uncomplicated malaria and eight children with complicated falciparum malaria were diagnosed based on at least one positive result from the four tests as well as clinical criteria. Twelve of the 79 children receiving first-line treatment had positive results by nested PCR on day 7 (nested PCR-corrected day 7 cure rate was 85%). After the second-line treatment, nested PCR/LAMP-corrected day 28 cure rate was 83% for these 12 children.ConclusionsHRP-2-RDTs have similar sensitivity as microscopy but less specificity. However, as compared to nested PCR, the poor sensitivity of HRP-2-RDTs indicates that low parasitaemia may not be detected after treatment, as well as the low specificity of HRP-2-RDTs indicates it cannot be applied for treatment follow-up. LAMP has similar sensitivity and specificity to nested PCR. With high PPV and NPV, LAMP is simpler and faster as compared to nested PCR with the advantage of detecting low parasitaemia becoming a potential point-of-care test for treatment follow-up.

Sensitivity and Specificity of Nested PCR for Diagnosing Malaria: Cases in Several Areas of Indonesia

“…This policy will be able to minimize false positive results due to contamination. Discrepancy results of microscopy examination and blood smear is difficult to describe and verifiy except for re-examination on each sample by using the two methods and performed by the same technician (expert in the examination of microscopy blood smear and nested PCR) and carried out with strict control in process [15]. Differences in outcome is also possible because of an error in the interpretation of the results of the microscopic examination and the possibility of errors can also occurs in the nested PCR process, from DNA extraction process, mixing with the mastermix, nesting process, and the reading of electrophoresis gel.…”

Potential Biomarkers and Their Applications for Rapid and Reliable Detection of Malaria

“…LAMP can be conducted under isothermal conditions and does not need expensive thermocyclers [120]. However, this method may pose danger of cross-contamination during addition of dye for visualization of results.…”

A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification

“…A commercial kit for malaria LAMP is currently available from Eiken Chemical Co., Ltd., also targeting this genetic region of plasmodia. This LAMP assay has successfully been tested under field conditions for both the18s rRNA[ 16 ] and the mtDNA primers[ 17 , 18 ] and been successful in clinical evaluation of symptomatic and asymptomatic infections[ 10 ]. Under field conditions, the easy endpoint visualization of results such as colourimetric change is most advantageous and, thus, desirable.…”