Enhanced Sensitivity for Detection of Plasmodium falciparum gametocytes by magnetic nanoparticles combined with enzyme substrate system
Graphical abstract
Introduction
Plasmodium falciparum gametocyte stages are important for the transmission of malaria parasites from human host to mosquito vector, repeating the infection over 90% of malaria cases worldwide [1]. The transmission of gametocytes results in the distribution of malaria disease among population, affecting lives, society and economic issues [2], [3], [4]. The significant role of gametocytes in malaria transmission drives certain strategies to focus on early diagnosis and specific treatment for blocking the source of infection, especially in asymptomatic carriers [5], [6], [7]. However, gametocyte carriers are usually misdiagnosed by reasons of asymptomatic profile and low parasite density [7], [8], [9]. Several studies described that microscopic examination, which is generally used to detect malaria parasites in routine laboratory, has inconsistent detection limit depending on blood smear preparation and experience of microscopist [10], [11], [12], [13]. According to the biology of gametocytes that are committed to produce at a small-scale, conventional microscopy possibly neglect or underestimate parasite count in the patient. Development of alternative method for gametocyte detection is required to overcome the lack of sensitivity of the traditional method.
Several studies have reported that molecular-based detection methods, e.g., DNA and RNA amplification using PCR-based method, were invented to overcome limitation of conventional microscopy and offer better sensitivity and specificity of the detection [14], [15], [16], [17]. Within few hours, the PCR-based method can exponentially amplify a billion of DNA copies from a single target DNA, allowing an effective detection from a small amount of sample. To further increase in sensitivity of the technique, functionalized magnetic nanoparticles (MNPs) having a good compatibility with enzymes has been applied for isolation of nucleic acid prior to amplification [18], [19], [20]. The using of the PCR-based detection has been described as a benchmark to evaluate the performance of alternative detection methods [21], [22]. Nevertheless, the PCR-based technique requires post-analytical visualization to analyze PCR products by agarose gel electrophoresis, supplying more sophisticated instrument and carcinogenic reagent [23]. To improve post-analytical process, modified colorimetric procedure including specific separation by MNPs and sensitive signal development by enzyme-linked immunosorbent assay (ELISA) has been widely implemented in biomedical applications [24]. It has been also noted that the combination of MNPs-immobilized forward primer and biotin-immobilized reverse primer can be used in PCR detection for diagnosis of both genetic and infectious diseases [25], [26]. For the detection of malaria parasites, enhanced microscopic examination by MNPs separation was obviously developed [27], [28]. However, the high sensitive detection of malaria gametocyte specific genes using MNPs is still in development.
In this study, Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay (MELGA) was developed to improve sensitivity and specificity of malaria gametocyte detection targeting Pfg377 gene, the specific P. falciparum gametocyte gene for the development of osmiophilic body in gametocyte stage III [29]. Because the presence of osmiophilic body is a highly conserved feature of sexual differentiation, the sequence of Pfg377 gene represents an attractive target for malaria transmission blocking strategy [30]. We aimed to immobilize forward and reverse Pfg377 gene-specific primers with MNPs and biotin, respectively. The presence of MNPs in PCR amplicons allowed the facile enrichment of malaria gametocytes gene by applying external magnetic field while that of biotin in the products provided highly sensitive production of detectable signal using horseradish peroxidase-conjugated streptavidin (SA-HRP) and ABTS substrate. With comparison of conventional PCR, the MELGA results showed that the high sensitive and specific detection of Pfg377 gene was determined. Therefore, we accounted that the detection of Pfg377 gene using MELGA can be an effective method for the diagnosis of malaria gametocytes.
Section snippets
Sample collection
This study was approved by the Mahidol University Ethics Committee, Mahidol University, Bangkok, Thailand. Informed consents were taken from all participants before the blood collection. EDTA-treated blood samples were collected from patients infected with P. falciparum and Plasmodium vivax at malaria clinics located in Mae Sot district, Tak province, Thailand. All samples were examined for parasitemia by expertise using microscopic examination of Giemsa stained blood smear and confirmed the
Immobilization of DNA primer onto MNPs
In the preparation of DNA-immobilized MNPs for MELGA, amino-modified Pfg377-F primer (5 nmol) was covalently bound to the surface of carboxylated MNPs by carbodiimide method using EDC as a coupling agent. The residual concentration of the Pfg377-F primer in the supernatant after immobilization was determined from 3 different experiments. The average amount of residual Pfg377-F primer was 1.78±0.02 nmol/mg, corresponding to the immobilized Pfg377-F primer on the MNPs at 3.22±0.02 nmol/mg. Binding
Discussion
Pfg377 gene is specifically expressed in female gametocyte and its transcript is detectable since the development of stage III gametocyte [29]. The expression of Pfg377 gene has been discussed that it is associated to the maturation of gametocyte, the formation of osmiophilic body and the escape of macrogametocytes [29], [30]. Although the detection of Pfg377 gene has been described for the typing of P. falciparum gametocyte isolates, some studies referred to the undetectable level of certain
Acknowledgements
We thank all staff of the Mae Sot and Mae Kasa Malaria Clinics, Tak province for collection of the samples. We also thank Ms. Kanyarat Suthamlert for technical support. This work was supported by the Royal Golden Jubilee Ph.D. Programme (5TMU57T1.PHD/0007/2557) of the Thailand Research Fund. We are also grateful for the research funding from The Office of Higher Education Commission and Mahidol University under the National Research Universities Initiative, and the Annual Government Grant under
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