detection-plasmodium-sporozoites-anopheles-mosquitoes-using-an-enzyme

Detection of <em>Plasmodium</em> Sporozoites in <em>Anopheles</em> Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female ...

Mahidol University

Plasmodium falciparum gametocytes are usually present in peripheral blood at a very low level, thus requiring a sensitive assay detection method. In this study, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was developed for clinical detection of P. falciparum gametocytes. Transcripts of Pfs16 of sexually committed ring and Pfs25 of mature gametocytes were detected by RT-LAMP in 82 clinical blood samples using nested RT-PCR as a gold standard. RT-LAMP demonstrated a detection limit of 1 parasitized red blood cell (RBC)/500microl of blood for both Pfs16 and Pfs25 transcripts. For Pfs16 transcript, RT-LAMP detected all 30 samples positive by nested RT-PCR (100% sensitivity) and 1 in 52 samples negative by nested RT-PCR (98.1% specificity). For Pfs25 transcript, RT-LAMP detected all 15 samples positive by nested RT-PCR (100% sensitivity) and none of 67 samples negative by nested RT-PCR (100% specificity). Negative predictive value (NPV) and positive predictive value (PPV) of RT-LAMP for detection of Pfs16 transcript were 100% and 96.8%, respectively, and 100% for both when employing Pfs25 transcript. Detection rate of Pfs16 and Pfs25 transcripts by RT-LAMP in microscopically gametocyte-negative samples was 91.7% and 29.2%, respectively. Compared with nested RT-PCR, RT-LAMP had a higher sensitivity but similar specificity, with the advantage of a shorter assay time. As RT-LAMP requires very basic instruments and the results can be obtained by visual inspection, this technique provides a simple and reliable tool for epidemiological studies of malaria transmission and in gametocyte-targeted control programmes.

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for clinical detection of Plasmodium falciparum gametocytes.

The effect of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) against newly excysted juveniles (NEJs) and 4-weeks-old immature parasites of Fasciola gigantica were compared with triclabendazole (TCZ). The anthelmintic efficacy of 1, 10 and 100μg/ml of PB or TCZ following incubation in vitro for 1-24h was compared using a combination of relative motility (RM), survival index (SI) and larval migration inhibition (LMI) assays for parasite viability. The RM and SI values of the PB-treated group decreased at a more rapid rate than the TCZ-treated group. For NEJs, the decreased RM values were first observed at 1h incubation with 1μg/ml PB, and 90% of flukes were killed at 24h. In contrast, in TCZ-treated groups a 10-fold higher concentration of TCZ (10μg/ml) resulted in only 9% dead parasites after 24h incubation. In 4-weeks-old juvenile parasites, PB reduced the RM value at 10μg/ml with 100% of flukes dead after 3h, while TCZ decreased RM values at the concentration of 100μg/ml but with only 5% of flukes killed at 24h. NEJs treated with PB exhibited 88%, 99% and 100% of LMIs at the concentrations of 1, 10 and 100μg/ml, respectively. NEJs incubated with TCZ have an LMI of only 32% at the highest concentration of 100μg/ml. Similarly PB had a significantly greater killing of immature 4weeks juvenile stages than TCZ at all concentrations; however, 4-weeks-old juvenile parasites were more resistant to killing by PB or TCZ at all concentrations when compared to NEJs. Further studies were carried out to investigate the alterations of the parasite tegument by scanning electron microscope (SEM). PB caused similar tegumental alterations in 4-weeks-old juveniles as those observed in TCZ treatment but with greater damage at comparative time points, comprising of swelling, blebbing and rupture of the tegument, loss of spines, and eventual erosion, lesion and desquamation of the total tegument. These data indicate that PB had a greater fasciolicidal effect against immature stages of F. gigantica parasites than TCZ and warrant further studies for use as a potential new anthelmintic against Fasciola infections. 

The in vitro anthelmintic effects of plumbagin on newly excysted and 4-weeks-old juvenile parasites of Fasciola gigantica.

The loop-mediated isothermal amplification (LAMP) method, developed by our group for diagnosis of four human malaria parasites, was evaluated on a large scale at a remote clinic in Thailand where malaria is endemic. A total of 899 febrile patients were analyzed in this study. LAMP was first evaluated in 219 patients, and the result was compared to those of two histidine-rich protein (HRP)-2 rapid diagnostic tests (RDTs) and microscopy as a gold standard. LAMP DNA extraction was conducted by a simple boiling method, and the test results were assessed visually. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 95.7%, 100%, 100%, and 98%, respectively, for LAMP and 98.6%, 98%, 95.8%, and 99.3%, respectively, for RDTs. Since RDT-positive results were based on one out of two RDTs, the sensitivity of RDTs was slightly higher than that of LAMP. However, LAMP tended to be more specific than RDTs. LAMP next was evaluated in 680 patients, and the result was compared to that of microscopy as a gold standard. Sensitivity, specificity, PPV, NPV, and diagnostic accuracy of LAMP were 88.9%, 96.9%, 92.2%, 95.5%, and 94.6%, respectively. Nested PCR was used to confirm the discrepant results. Malaria LAMP in a remote clinic in Thailand achieved an acceptable result, indicating that LAMP malaria diagnosis is feasible in a field setting with limited technical resources. Additionally, the rapid boiling method for extracting DNA from dried blood spots proved to be simple, fast, and suitable for use in the field. 

Loop-mediated isothermal amplification assay for rapid diagnosis of malaria infections in an area of endemicity in Thailand.

Low-density asymptomatic infections of Plasmodium spp. are common in low endemicity areas worldwide, but outside Africa, their contribution to malaria transmission is poorly understood. Community-based studies with highly sensitive molecular diagnostics are needed to quantify the asymptomatic reservoir of Plasmodium falciparum and P. vivax infections in Thai communities.

Article	Year
Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for clinical detection of Plasmodium falciparum gametocytes. Parasitology international\| PubMed ID: 20542143	2010
The in vitro anthelmintic effects of plumbagin on newly excysted and 4-weeks-old juvenile parasites of Fasciola gigantica. Experimental parasitology\| PubMed ID: 24157317	2014
Loop-mediated isothermal amplification assay for rapid diagnosis of malaria infections in an area of endemicity in Thailand. Journal of clinical microbiology\| PubMed ID: 24574279	2014
Very high carriage of gametocytes in asymptomatic low-density Plasmodium falciparum and P. vivax infections in western Thailand. Parasites & vectors\| PubMed ID: 29065910	2017

Sirasate Bantuchai

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