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New Malaria Test Combines Loop-Mediated Isothermal Amplification, Nanopore Sequencing


NEW YORK (GenomeWeb) – Researchers in Japan have developed a new technique to diagnose malaria, using a combination of loop-mediated isothermal amplification (LAMP) and Oxford Nanopore's MinIon nanopore sequencing technology.

Conventional methods of diagnosing malaria involve microscopic examination of Giemsa-stained thick blood smears and rapid diagnostic tests (RDTs). Patients usually arrive at a clinic with symptoms such as fever, sweating, and muscle pain. In severe cases, they also develop neurological focal signs, confusion, and other serious symptoms. Physicians perform RDTs or microscopy to measure the concentration of parasites in the blood, typically 200 parasites per microliter of blood in the case of malaria.

While inexpensive and easily implemented in malaria-endemic areas, the sensitivity and specificity of microscopy-based testing often depend on the proficiency of the clinician. An alternative are RDTs, which produce results in 10 to 15 minutes that are straightforward to interpret.

RDTs, however, struggle with sensitivity and accuracy, which usually leads to the tandem usage of RDT and microscopy tests. In addition, there are patients who are infected but asymptomatic for malaria and have very low parasite counts. Clinicians may not be able to detect these cases through RDT and microscopy, and other methods are required.

Researchers are therefore using molecular diagnostics for malaria detection by identifying Plasmodium nucleic acids via PCR and LAMP amplification techniques.

Nested PCR tests are frequently used in malaria diagnosis because of their high sensitivity, usually as an addendum to identify the specific plasmodium species present.

A Japanese research team has now developed a new method, using LAMP amplification and nanopore sequencing, that yields similar results to that of nested PCR. In a recent study, they highlighted that the LAMP/MinIon method is fast, easy to use, and sensitive, which could be useful in resource-limited endemic regions.  

According to the study, published in BMC Infectious Diseases last month, the method's advantages include high detection sensitivity for all Plasmodium species, multiplex sequencing and streaming analysis of real-time sequencing data, and detection of mixed parasite infections.

For the assay, the researchers designed LAMP primers targeting the 18S-rRNA gene of all five Plasmodium species that infect humans, including two P. ovale subspecies. They examined human blood samples collected from 63 malaria patients in Indonesia from August to December 2010, performing amplicon sequencing on the LAMP products using the MinIon nanopore sequencer to identify each Plasmodium species.

According to Kazuo Imai, the study's lead author and a researcher in the Division of Infectious Diseases and Pulmonary Medicine of the Department of Medicine at the National Defense Medical College, using LAMP in tandem with MinIon sequencing has several advantages. Imai presented the method earlier this month during a poster session at the Infectious Disease Week 2017 meeting in San Diego.

For the study, the researchers collected peripheral blood samples from each patient and stored them at room temperature. They then performed LAMP reactions using a Loopamp DNA Amplification Reagent Kit. Imai emphasized LAMP's ability to amplify the amount of DNA at a steady temperature, in contrast to nested PCR, which requires thermal cycling.

The team then performed sequencing of the amplicons with the MinIon to identify the Plasmodium species. According to Imai, the $1,000 cost for the MinIon device was offset by the number of samples the team could analyze in a single run. Using Oxford Nanopore's Rapid Barcoding Sequencing Kit for multiplex sequencing of LAMP amplicons from clinical samples, the team simultaneously sequenced 12 samples on a single flow cell. Each sample, thus, cost about $83 to run on the MinIon sequencer, and another $1 to run the LAMP amplification. Six amplicons harboring partial sequences of 18S-rRNA of each Plasmodium parasite were also used to validate the performance of the MinIon. Imai said that it would be possible to run up to 96 samples per flow cell to make the assay more cost effective.

Of the 63 clinical samples that the researchers collected in the study, 54 were identified as positive by nested PCR and 55 by the team's LAMP/sequencing method.  

According to the researchers, the entire malaria detection process, from DNA extraction to species identification, only required about three hours in total.  

Imai touted the sequencer's portability and easy library preparation method. The MinIon is a lightweight, pocket-sized device that is powered via a USB port, and it generates sequence data in real time, allowing clinicians to perform analyses during the experiment.

While previous tests using LAMP have also been able to detect multiple malaria species, such as Meridian's Bioscience Malaria assay, Imai said they required specific devices, such as the Illumipro-10 Incubator/Reader, an automated isothermal amplification and detection system. He emphasized that his team's approach used sequencing for detection instead.

Imai's team believes it can also use the LAMP/MinIon method to detect other pathogens, or drug resistance genes in pathogens, in tropical regions with limited diagnostic resources. Imai highlighted applications such as the detection of leishmaniasis and Chagas disease in Latin America, as well as Zika/Chikungunya virus in Southeast Asia. In addition, a group led by Yutaka Suzuki at the University of Tokyo reported earlier this year using a combination of LAMP and MinIon to detect Dengue virus.

Imai also addressed certain limitations of the MinIon sequencer, including the device's significant cost and low raw read accuracy. In the study, the team used 1D sequencing reads, which had a high error rate of 10 percent. In future studies, Imai's team plans to use the recently released Litigation Sequencing Kit ID^2, which reportedly produces reads that have an error rate of 5 percent.  

Xavier Ding, senior scientific officer at the Foundation for Innovative New Diagnostics, said he believes that while the method might work well in research, LAMP/MinIon would not translate well to clinical settings in malaria-endemic regions. For example, Ding raised concerns about the overall length of the procedure and the need to multiplex samples to keep the cost per sample down.

"The authors make the point that they can multiplex up to 96 samples using the systems, but if you need to wait for 96 samples to perform the run, that's definitely not usable within the clinical setting," he said.

Ding went on to elaborate that the entire process takes about three hours, from DNA extraction to MinIon sequencing, which he said does not speed up diagnosis.

In addition, the small sample size in the study prevented Imai and his team from highlighting the performance for P. ovale, P knowlesi, and P. malariae infections, and the researchers said that the method needs further clinical evaluations to ensure sufficient and consistent sensitivity and specificity. The team is currently working on evaluating the method in additional clinical samples, collecting samples from patients infected with P. ovale. In addition, Imai said that the team has developed LAMP primers to detect arteminisin resistance in P. falciparum.

Ding acknowledged that the authors identified certain issues in the paper, but fell a bit short of addressing the specific needs of a clinical test.

"I can see some advantage when [they] perform epidemiological studies or [work in] research-based settings," and the method "definitely gives an advantage in terms of sensitivity," Ding said, but the current protocol does not help in making further clinical decisions.

In the future, Imai said, his team will use the LAMP/MinIon method in Southeast Asia, which is endemic to malaria and other mosquito-borne diseases. While the MinIon has been explored as a diagnostic method for pathogens such as Ebola, Zika, and tuberculosis, Imai is especially interested in using it for direct RNA sequencing in order to directly diagnose RNA viruses from clinical samples.