Literature Introduction | Rainsure Digital PCR Technology and Multitype Plasmodium Typing and Treatment Quantitative Monitoring

_{Title: A rapid multiplex assay of human malaria parasites by digital PCR}

_{DOI: 10.1016/j.cca.2022.12.001}

_{Journal: Clinica Chimica Acta}

_{Unit: Huashan Hospital Affiliated to Fudan University}

_{Published: December 2022}

_{Research Platform: Rainsure Scientific DropDx-2044 Digital PCR System}

_{In December 2022, the team of Ming Guan and Haoqin Jiang from Huashan Hospital Affiliated to Fudan University published the article "A rapid multiplex assay of human malaria parasites by digital PCR" in the journal "Clinica Chimica Acta".}

_{This study established a novel multiplex digital PCR system focused on rapid molecular detection of low-concentration malaria infection. It is suitable for the diagnosis of mixed malaria infection and continuous monitoring of multiple samples, and as an important clinical detection method for the absolute quantification of pathogens, to ensure rapid and accurate diagnosis of patients suspected of malaria infection. The analytical sensitivity of the multiplex PCR is as low as 0.557copies/μL, the sensitivity is 98.0%, and the specificity is 100%, which is especially suitable for the diagnosis of low-concentration malaria infection and the monitoring of curative effect during treatment.}

_{Research background} 

_{Malaria, AIDS, and tuberculosis are recognized worldwide as one of the three major public problems that endanger human life and health. They are a parasitic disease caused by Plasmodium and transmitted by Anopheles mosquitoes. According to WHO's latest World Malaria Report, there are approximately 241 million malaria cases worldwide and 627,000 deaths from malaria. Compared with the previous year, there were about 14 million new cases and 69,000 new deaths.}

_{Plasmodium is a spore transmitted by Anopheles mosquito and is the causative agent of malaria. Four main kinds of Plasmodium that parasitize the human body, including Plasmodium vivax, Plasmodium malariae, Plasmodium falciparum and Plasmodium ovale. These malaria parasites have two hosts, Anopheles mosquitoes and humans. They parasitize in liver cells and red blood cells successively in humans. In erythrocytes, in addition to schizophyte proliferation, some merozoites form gametophytes and begin the initial development of sexual reproduction.}

_{In 2021, with zero indigenous malaria cases in four years, China was successfully recognized by the World Health Organization (WHO) as a malaria-free country. At present, the cases of malaria infection mainly imported from abroad.}

_{Research methods}

_{A total of 39 malaria-positive patients diagnosed by microscopy were selected from Huashan Hospital from 2016 to 2021; 70 blood samples were collected from these patients. In addition, 20 healthy people, 20 febrile patients and 6 patients with other types of blood-infected parasites were included in this study. Each blood sample was tested in parallel by blood smear and dPCR. By optimizing the single-system quadruple fluorescent-labeled probe detection system, the accurate quantitative ability of dPCR was verified, and the accurate copy number per unit volume of clinical malaria blood samples was output. A rapid diagnostic test was also used to validate the results obtained by dPCR.}

_{Research results}

_{The dPCR system enables rapid diagnosis and quantification of malaria DNA samples. The analytical sensitivity of multiplex dPCR was as low as 0.557 copies/μL (95% CI 0.521-0.607), and its sensitivity for diagnosis of clinical specimens was 98.0%, and its specificity was 100%. In addition, three multiple Plasmodium co-infection samples were detected by the dPCR system, including one case of triple Plasmodium co-infection. By continuously testing daily blood samples from the study's 39 patients, dPCR can monitor the efficacy of drug treatments. The results show that the concentration range of Plasmodium falciparum DNA after medication is 0 copy/μL-5474 copy/μL, realizing real-time monitoring of the curative effect of antimalarial drugs. The study also found that Plasmodium was more easily and more positively detected using plasma-depleted blood cell samples than whole blood.}

_{The multiplex digital PCR system can clearly detect two or three kinds of malaria mixed infection whole blood samples, to effectively diagnose multiple malaria mixed infections. This is an ideal supplement to the traditional blood smear microscopic examination method, which can timely make up for the missed detection of atypical malaria in blood smears and low-concentration mixed infection patients, thereby helping clinicians to adjust medication regimens in a timely manner.}

_{Continuous monitoring of a patient for up to 20 days showed that digital PCR can accurately quantify the concentration of malaria in the patient's whole blood, visually show the real infection status of the patient, and reflect the change of malaria infection rate in the patient in time, achieving the purpose of real-time monitoring the efficacy of antimalarial drugs, is to provide more reliable laboratory evidence for the elimination of malaria in patients.}