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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 1  |  Page : 90-94
 

Evaluation of loop-mediated isothermal amplification assay for rapid diagnosis of Acanthamoeba keratitis


1 Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Ophthalmology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Web Publication16-Mar-2017

Correspondence Address:
Sumeeta Khurana
Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_16_227

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 ~ Abstract 


Background: The clinical features of Acanthamoeba keratitis (AK) are non-specific and closely resemble bacterial, viral and fungal keratitis. Materials and Methods: We compared loop-mediated isothermal amplification (LAMP) with microscopy, non-nutrient agar (NNA) culture and polymerase chain reaction (PCR) in clinical suspects of AK. Results: Of 52 clinical samples (42 AK suspects and 10 proven bacterial, viral or fungal keratitis), 3 were positive by direct microscopy (sensitivity 60%, confidence interval [CI]: 17%–92.7%), and 5 by NNA culture, 18S rDNA PCR and LAMP (sensitivity 100%, CI: 46.3%–100%). The limit of detection of Acanthamoeba DNA was 1 pg/μl by both LAMP and PCR. Conclusion: PCR and LAMP assays targeting 18S rDNA gene were found particularly suitable for a rapid and accurate diagnosis of AK. LAMP assay takes 2–3 h lesser than PCR, and thus offers a rapid, highly sensitive and specific, simple and affordable diagnostic modality for patients suspected of AK, especially in resource limited settings


Keywords: Acanthamoeba, diagnosis, free living amoebae, keratitis, loop-mediated isothermal amplification assay, parasite, polymerase chain reaction


How to cite this article:
Mewara A, Khurana S, Yoonus S, Megha K, Tanwar P, Gupta A, Sehgal R. Evaluation of loop-mediated isothermal amplification assay for rapid diagnosis of Acanthamoeba keratitis. Indian J Med Microbiol 2017;35:90-4

How to cite this URL:
Mewara A, Khurana S, Yoonus S, Megha K, Tanwar P, Gupta A, Sehgal R. Evaluation of loop-mediated isothermal amplification assay for rapid diagnosis of Acanthamoeba keratitis. Indian J Med Microbiol [serial online] 2017 [cited 2017 Mar 29];35:90-4. Available from: http://www.ijmm.org/text.asp?2017/35/1/90/202336





 ~ Introduction Top


Acanthamoeba are opportunistic free living protozoans that are found in natural environmental sources such as soil, air, sea water, freshwater and artificial water supplies such as bottled water tap, wash stations and drinking water fountains.[1]Acanthamoeba keratitis (AK) is a rare but severe and painful ocular corneal infection which leads to visual loss.[2],[3] AK cases are increasing in numbers and the common risk factors include ocular trauma, contact lens wear, and exposure to contaminated water, amongst others.[4] AK is usually not suspected and remains undiagnosed as a cause of keratitis and often is misdiagnosed as viral, fungal or bacterial keratitis, which leads to treatment delays.[5] In many cases, the disease may thus easily progress to cause significant visual loss or perforating keratopathy.[6],[7] Therefore, early diagnosis of AK is required for effective treatment and control.

The conventional approach for diagnosis of AK is light microscopy of a direct smear of corneal scraping or of organisms cultured onto the non-nutrient agar (NNA) plate surface seeded with Escherichia coli, however, this is a time consuming method.[8] The molecular diagnostic methods such as polymerase chain reaction (PCR) and real-time PCR assays have been developed for Acanthamoeba DNA detection,[9],[10] and have proven reliable, sensitive and specific.[11] These assays are still not widely used in resource constrained settings because of the need of highly trained personnel and expensive laboratory equipment. Hence, the development of less time consuming, cost-effective, simple, sensitive and rapid detection methods is necessary.

Loop-mediated isothermal amplification (LAMP) is a newly developed highly specific, efficient, and less time consuming DNA amplification technique which rapidly amplifies target DNA sequences under isothermal conditions, i.e., in the temperature range of 58°C–65°C, without requiring a thermal cycler.[12],[13] In the present work, we evaluated LAMP assay targeting 18S rDNA gene for specific detection of Acanthamoeba from corneal scraping samples and compared it with microscopy, NNA culture, and conventional 18S rDNA PCR.


 ~ Materials and Methods Top


Clinical samples

A total of 52 corneal scrapings from patients with keratitis attending the eye out-patient department of the hospital were collected by experienced ophthalmologists. Of these, 42 patients were suspected of AK with signs and symptoms such as redness in eyes, photophobia, watering, blurring of vision, pain, and ring infiltrates, irritation. As controls, ten corneal scrapings collected from patients with keratitis of aetiology other than Acanthamoeba, including four patients with proven bacterial keratitis (two Pseudomonas aeruginosa, two Staphylococcus aureus), three with viral (three herpes simplex virus [HSV]), and three with fungal keratitis (two Fusarium spp., one Aspergillus spp.), were included; in these ten samples, Acanthamoeba was excluded on the basis of laboratory tests. The study was approved by the Institute Ethics Committee and written informed consent was obtained from all the patients before collecting the corneal scrapings. After collection, the corneal scrapings were transferred into Eppendorf tubes containing 200 µl sterile normal saline and transported to the laboratory.

Laboratory maintained and environmental isolates

Eight Acanthamoeba isolates from patients with AK or acute granulomatous encephalitis being maintained in the department and five Acanthamoeba isolates from environmental samples were included in the study for LAMP standardisation.

Microscopy and culture

Methanol fixed smears of corneal scrapings were allowed to air dry and stained with Giemsa stain and observed under light microscope. The samples were also inoculated on NNA culture plate. The samples were simultaneously processed for bacterial, viral and fungal agents. All the samples were subjected to DNA extraction on the same day.

DNA extraction

DNA was extracted by phenol-chloroform-isoamyl alcohol method using UNSET lysis buffer.[14] Corneal scrapings and scraping from NNA culture plates were washed with phosphate buffered saline (PBS) thrice, following which 500 µl UNSET lysis buffer and 50 µl proteinase K was added and incubated at 56°C for 4 h. The aqueous lysate was repeatedly extracted with 500 µl volumes of phenol-chloroform-isoamyl alcohol (25:24:1) until the protein interface disappeared. DNA was precipitated from the aqueous lysate with 1 ml of absolute ethanol and kept at −20°C overnight, after which it was resuspended in 20 µl of either double distilled water or Tris-EDTA buffer. For calculation of limit of detection for LAMP and PCR assays, the laboratory and environmental isolates were maintained in axenic culture.[15] The rest of DNA extraction procedure was similar as above.

18S rDNA polymerase chain reaction

18S rDNA PCR was carried out using Acanthamoeba specific primers (JDP1 and JDP2) as described previously [Table 1].[10] In breif, PCR amplification cycle included initial denaturation at 95°C for 10 min followed by 39 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min and final extension at 72°C for 7 min. Amplified DNA products (423–551 bp) were separated by 1.5% agarose gel electrophoresis and visualised under ultraviolet light using an image analyser (AlphaImager EC, Protein-Simple, CA, USA). PCR grade water was used as negative control.
Table 1: Primers used to amplify Acanthamoeba 18SrDNA gene by polymerase chain reaction and loop-mediated isothermal amplification assays

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18S rDNA loop-mediated isothermal amplification assay

The Acanthamoeba specific LAMP primer set targeting 18S rDNA consisted of F3 (forward outer primer), B3 (backward outer primer), forward inner primer (FIP), backward inner primer (BIP), loop forward primer (LF) and loop backward primer (LB), as described previously [Table 1].[16] In breif, each reaction was performed using 25 µl of prepared reaction mixture containing 40 pmol each of FIP and BIP, 5 pmol each of F3 and B3, 20 pmol each of LF and LB and 15 µl of isothermal master mix (Ampligene, Ahmedabad, India) as per the manufacturer's instructions, and 2 µl of denatured genomic DNA (50 ng/µl). The reaction mixture was incubated in a water bath (Major Science, Hyderabad, India) at 62°C for 15 min and then 80°C for 2 min to terminate the reaction. PCR grade water was used as negative control in all the reactions. The amplified products were visually inspected for turbidity and confirmed by 1.5% agarose gel electrophoresis.

Analytical sensitivity and gene specificity

The analytical sensitivity of PCR and LAMP assays was assessed by using different Acanthamoeba genomic DNA concentrations (1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg/µl) from culture isolates from laboratory maintained clinical and environmental isolates. DNA concentrations were measured using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Thermo Scientific Instruments). To determine the specificity of the LAMP assay for Acanthamoeba, DNA from other microorganisms including Toxoplasma gondii, Plasmodium falciparum, S. aureus and Aspergillus spp. were also tested.

Statistical analysis

The diagnostic sensitivity, specificity, positive predictive value, negative predictive value, and 95% confidence intervals (CIs) were calculated with reference to samples positive by either of the three tests (microscopy-positive and/or culture-positive and/or LAMP-positive) using the standard formulae. The statistical analysis was carried out using the software SPSS, v. 17.0 (SPSS South Asia Pvt. Ltd., Bengaluru, India).


 ~ Results Top


A total of 52 corneal scrapings, of which 42 were from patients suspected of AK and 10 from patients with keratitis from bacterial, viral and fungal causes were analysed. The clinical specimens were processed on the same day of collection. The microscopy results were available within 24 h and NNA culture results were obtained within 6–7 days. The PCR and LAMP assays were performed within 24 h thus obtaining the results either on the same day or latest by the next day.

Microscopy and non-nutrient agar culture

Of the total 52 samples, 3 were positive by direct microscopy, whereas 5 out of 52 clinical samples were found positive by NNA culture [Table 2].
Table 2: Comparison of results of various laboratory techniques in diagnosis of Acanthamoeba keratitis

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18S rDNA polymerase chain reaction assay

Of the total 52 clinical samples, 5 were positive for Acanthamoeba DNA by PCR assay, while 47 were found negative [Table 2]. 18S rDNA PCR assay on serially diluted genomic DNA extracted from in vitro cultured samples revealed that the minimum genomic DNA concentration required for the detection of Acanthamoeba was 1 pg/µl [Figure 1].
Figure 1: Gel electrophoresis of 18S rDNA polymerase chain reaction assay for Acanthmoeba. Lanes 1, 9: molecular ladder; Lane 2: negative control (no DNA); Lanes 3, 4, 5, 6, 7, 8: results of polymerase chain reaction for DNA concentrations 1 ng, 100 pg, 10 pg, 1 pg, 100 fg and 10 fg, respectively.

Click here to view


18S rDNA loop-mediated isothermal amplification assay

Of the total 52 clinical samples, Acanthamoeba DNA was detected in 5 samples by LAMP assay, whereas 47 samples were found negative [Table 2]. 18S rDNA LAMP assay on serially diluted genomic DNA extracted from in vitro cultured samples revealed that the minimum genomic DNA concentration required for the detection of Acanthamoeba was 1 pg/µl [Figure 2]a. The analytical sensitivity of LAMP assay was evaluated using the genomic DNA of laboratory maintained clinical and environmental Acanthamoeba isolates. A conspicuous step ladder pattern of amplified LAMP products on gel electrophoresis was observed for positive samples. The specificity was evaluated using genomic DNA of other microorganisms such as T. gondii and P. falciparum (unrelated protozoans), S. aureus (bacteria) and Aspergillus spp.(fungus), for which no amplification was detected in LAMP assays [Figure 2]b. Thus, no false-positive amplifications were observed with DNA of the above heterogeneous microorganisms in the assay.
Figure 2: Gel electrophoresis of 18S rDNA loop-mediated isothermal amplification assay for Acanthmoeba.(a) Lane M: molecular ladder; Lane 1: negative control (no DNA); Lanes 2, 3, 4, 5, 6, 7: results of loop-mediated isothermal amplification assay for DNA concentrations 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg, respectively. (b) Lane M: molecular ladder; Lanes 1, 2, 3: corneal scraping samples positive for Acanthmoeba DNA; Lane 4: Plasmodium falciparum DNA; Lane 5: Toxoplasma gondii DNA; Lane 6: negative control (clinical sample with aetiology other than Acanthamoeba); Lane 7: negative control (no DNA).

Click here to view


Overall, three samples were positive by microscopy with a sensitivity of 60% (95% CI: 17.0%–92.7%), whereas the sensitivity was 100% (95% CI: 46.3%–100%) for NNA culture, PCR and LAMP assays. Both PCR and LAMP were negative for DNA of other microorganisms as well as for ten clinical controls with keratitis due to other aetiology, giving the tests a specificity of 100% for DNA detection (95% CI: 90.6%–100%). The positive and negative predictive values of LAMP were 100% (95% CI: 46.3%–100% and 90.6%–100%, respectively) [Table 2].


 ~ Discussion Top


AK is a painful and progressive sight-threatening corneal disease, the increasing incidence of which is attributed to the increased use of contact lenses with poor lens hygiene. Other risk factors are foreign body trauma and exposure to contaminated water.[17] In the absence of a high index of clinical suspicion, AK can be difficult to diagnose and treat as the signs and symptoms closely resemble HSV or fungal keratitis, and often there maybe secondary bacterial infections.[18],[19],[20] Further, the amoebic trophozoites can infiltrate the surrounding structures of the eye and cause neuritis, necrosis, or chorioretinitis, and in severe cases, may eventually require enucleation of the eye.[21],[22] The diagnosis may further be complicated by concurrent antibacterial, antiviral, antifungal or corticosteroid treatment because there is initial clinical improvement on institution of these drugs which is followed by a worsening of the disease.

In the present work, we subjected corneal scrapings from keratitis patients suspected of AK and other proven bacterial/viral/fungal keratitis, to conventional diagnostic modalities such as light microscopy, culture, and nucleic acid detection assays such as PCR and LAMP assays targeting 18S rDNA gene for specific detection of Acanthamoeba. Light microscopy had a low sensitivity (60%) in detecting AK. Other workers have reported similar sensitivity of direct microscopy to the tune of 55% (95% CI: 33.2%–76.8%);[23] however, the sensitivity varied with different samples, being maximum for contact lens and castings (100%), followed by contact lens solution (50%), and least for corneal scrapings (46.2%). The reasons for lower sensitivity of light microscopy could be lesser quantity of corneal scraping sample subjected to microscopy which is a typical situation for such invasive sample as the little sample received is divided into several parts for diagnosis of parasitic, bacterial, viral, fungal infections, and for various assays including microscopy, culture and DNA detection. Another possible reason could be the less sample size in this study and that the microscopy technique used was light microscopy. Other techniques such as immunofluorescence (IF) and confocal microscopy has been found to have sensitivity up to 90% in the diagnosis of AK;[24],[25] however, IF and confocal microscopy are costly procedures and light microscopy still remains the most commonly used microscopy technique in developing nations.[26]

The sensitivity and specificity of NNA culture, PCR and LAMP assays were found to be equivalent and none of the techniques detected any additional positives over and above the other two techniques. Boggild et al. have reported a sensitivity of culture ranging from 50 to 100% in different ocular sample types, the maximum being 100% in contact lenses and casings, followed by 75% with corneal scrapings and 50% for contact lens solution. The sensitivity of PCR reported by the authors ranged between from 46% in corneal scrapings to 100% in contact lenses, casings and lens solutions using different primer sets.[23] The sensitivity of LAMP is in agreement with previous studies which have reported 100% sensitivity and specificity with respect to the standard culture technique.[27],[28] Both LAMP and PCR assays had an analytical sensitivity of 1 pg/µl which is almost equivalent to the amount of DNA in 1 amoeba. Moreover, LAMP is a rapid isothermal reaction which completes in as early as 15 min compared to 3–4 h cycle of PCR, and does not require expensive equipment like a thermal cycler and thus has an edge over PCR in terms of reduced overall turn-around-time as well as feasibility of set-up in resource poor settings such as in developing countries. We performed the LAMP assay in a water bath which is commonly available in almost all laboratories. LAMP assay can further be standardised with colorimetric or fluorometric indicators thus eliminating the need for post-amplification handling.

NNA culture remains the cheapest amongst these assays; however, the turn-around time was 6–7 days. On several occasions, the culture may require extended incubation up to 14 days to ensure excystation. The delayed results of NNA culture prohibit it to be useful for rapid diagnosis of AK. An early and accurate diagnosis is critical to the clinical outcome of AK, since amoebic cysts, once formed are resistant to many drugs.[17] Both the nucleic acid detection assays were found to be suitable for a rapid, highly sensitive and specific diagnosis. The results for both nucleic acid detection assays were obtained within 24 h including the time taken for DNA extraction and for amplification wherein LAMP assay took 2–3 h lesser than PCR for the final results.





In this study, we evaluated culture, PCR and LAMP assays targeting 18S rDNA gene for detection of AK and found both the molecular tests to have good sensitivity and specificity. LAMP and PCR were suitable for rapid diagnosis. LAMP assay took much lesser time and had an equal analytical sensitivity with respect to PCR. Hence, LAMP assay can be utilised as a valuable rapid, highly sensitive and specific, simple to perform, affordable diagnostic modality for patients suspected of AK, especially in resource limited settings.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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