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Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

ABSTRACT: Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings.

SUBMITTER: Mahittikorn A

PROVIDER: S-EPMC4379150 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

Mahittikorn Aongart A Mori Hirotake H Popruk Supaluk S Roobthaisong Amonrattana A Sutthikornchai Chantira C Koompapong Khuanchai K Siri Sukhontha S Sukthana Yaowalark Y Nacapunchai Duangporn D

PloS one 20150330 3

Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification product ...[more]

PMID: 25822175

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Project description:Leishmaniasis is a neglected tropical disease that is caused by an obligate intracellular protozoan of the genus Leishmania. Recently, an increasing number of autochthonous leishmaniasis cases caused by L. martiniquensis and the novel species L. siamensis have been described in Thailand, rendering an accurate diagnosis of this disease critical. However, only a few laboratories are capable of diagnosing leishmaniasis in Thailand. To expand leishmaniasis diagnostic capabilities, we developed a simple colorimetric loop-mediated isothermal amplification (LAMP) technique for the direct detection of Leishmania DNA.LAMP was performed for 75 min using four primers targeting the conserved region of the18S ribosomal RNA gene, and the DNA indicator used was malachite green (MG). To simulate crude samples, cultured promastigotes of L. siamensis were mixed with blood or saliva. Also, clinical samples (blood, saliva, and tissue biopsies) were obtained from patients with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). All samples were boiled for 10 min and introduced directly into the LAMP reaction mixture without DNA purification.The use of MG resulted in an unambiguous differentiation of positive and negative controls. For L. siamensis, the detection limit was 10(3) parasites/mL or 2.5 parasites/tube. Saliva, tissue biopsies, and whole blood were indicative of active Leishmania infection, and their direct usages did not adversely affect the detection limit. In addition, this LAMP assay could detect DNA from multiple Leishmania species other than L. siamensis and L. martiniquensis, including L. aethiopica, L. braziliensis, L. donovani and L. tropica.The simplicity and sensitivity of LAMP in detecting active Leishmania infection could enable the rapid diagnosis of leishmaniasis, thereby facilitating the survey and control of leishmaniasis in Thailand. However, our limited number of samples warranted a further validation with a larger cohort of patients before this assay could be deployed.

Project description:BACKGROUND:Schistosoma mansoni is the main species causing hepatic and intestinal schistosomiasis in Sub-Saharan Africa, and it is the only species in South America. Adult stages of the parasite reside in the mesenteric venous plexus of infected hosts, and eggs are shed in feces. Collecting patient stool samples for S. mansoni diagnostic purposes is difficult in large-scale field trials. Urine samples would be an alternative approach for molecular S. mansoni detection since they have several advantages over stool samples, including better handling, management and storage. Additionally, loop-mediated isothermal amplification (LAMP) technology is a powerful molecular diagnostic tool for infectious diseases, particularly under field conditions in developing countries. The present study aimed to assess the effectiveness of our previously developed LAMP assay (SmMIT-LAMP) for S. mansoni-specific detection in clinical urine samples. METHODOLOGY/PRINCIPAL FINDINGS:The sensitivity of SmMIT-LAMP in urine was established in simulated fresh human urine samples artificially spiked with genomic DNA from S. mansoni. LAMP for 120 min instead of 60 min improved the sensitivity, reaching values of 0.01 fg/μL. A set of well-defined frozen stored human urine samples collected from Sub-Saharan immigrant patients was selected from a biobank to evaluate the diagnostic validity of SmMIT-LAMP. The set included urine samples from patients with microscopy-confirmed infections with S. mansoni, S. haematobium and other nonschistosome parasites, as well as urine samples from patients with microscopy-negative eosinophilia without a confirmed diagnosis. The SmMIT-LAMP was incubated for 60 and 120 min. A longer incubation time was shown to increase the LAMP-positive results in patient urine samples. We also tested urine samples from mice experimentally infected with S. mansoni, and LAMP-positive results were obtained from the third week after infection. A real-time LAMP assay was also performed with three individual urine samples. CONCLUSIONS/SIGNIFICANCE:The SmMIT-LAMP could effectively detect S. mansoni DNA in mouse urine samples and produced promising results for human clinical samples. The detection of S. mansoni DNA in mouse urine samples from the third week after infection indicates that early diagnosis of active S. mansoni infection is possible using urine as a source of DNA. Further studies are still needed, but our method could be used as a promising molecular tool applicable to urine samples to diagnose human intestinal schistosomiasis caused by S. mansoni.

Dataset Information

Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

Publications

Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

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