Acanthamoeba Culture Procedures Manual Template

Acanthamoeba culture procedures manual template

Amoebic keratitis causes significant ocular morbidity in contact lens wearers. Current diagnostic methods for amoebic keratitis are insensitive and labor-intensive and have poor turnaround time.

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We evaluated four laboratory methods for detection of acanthamoebae in clinical specimens. Deidentified, delinked consecutive specimens from patients with suspected amoebic keratitis were assayed for acanthamoebae by direct smear analysis, culture, and PCR using two different primer sets specific for Acanthamoeba ribosomal DNA.

The consensus reference standard was considered fulfilled when the results for any two of the four tests were positive, and the outcome measures were sensitivity and specificity. Of specimens assayed over an month period, 20 were positive for acanthamoebae. Nelson primer PCR demonstrated a single-organism level of analytic sensitivity. The performance characteristics of the assays varied by specimen type, with contact lenses and casings showing the highest rates of detectable acanthamoebae and the highest diagnostic sensitivities for direct smear analysis, culture, and JDP primer PCR, though these results are based on small numbers and should be interpreted cautiously.

These findings have important implications for clinicians collecting diagnostic specimens and for diagnostic laboratories, especially in outbreak situations. Amoebic keratitis AK is a potentially blinding ocular infection caused by an Acanthamoeba sp.

Introduction

The overwhelming majority of cases of AK occur in immunocompetent contact lens wearers 14 , and outbreaks have been linked to contact lens solutions contaminated with acanthamoebae or to those that fail to effectively decontaminate lenses. A recent outbreak in the United States affecting people led to the recall of contact lens solutions and products by both the FDA and Health Canada and has resulted in over lawsuits against the manufacturer 2 , 6 , 7.

Plaintiffs in the lawsuits have been left with impaired vision and, in several cases, have required corneal transplants 7.

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Although contaminated contact lens solutions or solutions that facilitate growth are usually implicated in large outbreaks of AK, isolated cases occur in individuals who have corneal trauma or who disinfect contact lenses with tap water or other home-based preparations.

Swimming and showering while wearing contact lenses are also risk factors for AK. Clinically, AK can be easily mistaken for herpes simplex virus infection or fungal keratitis, and secondary bacterial infection is common, thus complicating diagnosis Delayed diagnosis has repeatedly been associated with poor visual outcome and more-severe clinical progression 4 , 5.

Standard laboratory diagnostic procedures include microscopic examination of Giemsa-, periodic acid Schiff-, hematoxylin-and- eosin-, or acridine orange-stained corneal scrapings or contact lens fluids and culture of these specimens on nonnutrient agar overlaid with Escherichia coli or Klebsiella pneumoniae , and all of these procedures are limited by poor sensitivity, the requirement for technical expertise, and, in the case of culture, long turnaround time 4 , 5.

Due to their excellent sensitivity, molecular methods, including PCR, are increasingly being used to detect acanthamoebae in corneal specimens 9 , 10 , 18 , We herein sought to evaluate the performance of PCR for detection of acanthamoebae in clinical specimens from patients suspected of having AK in comparison to traditional methods, such as direct microscopic examination and culture.

Laboratory Studies

Consecutive specimens corneal scrapings, contact lens solutions, and casings from patients with suspected AK that were sent to the Central Public Health Laboratory for diagnosis between January and June were assayed using the standard diagnostic procedures direct examination and culture outlined below.

Per the Code of Federal Regulations, Title 45, Part 46, the use of deidentified diagnostic specimens for verification purposes is not considered human subject research and is therefore exempt from human subject considerations. The evaluators of each of the three assays were blinded to the outcomes of the other tests. With a sterile glass pipette, 2 or 3 drops from each specimen were inoculated onto each of four culture plates: one with nutrient medium NM overlaid with E.

Acanthamoeba culture procedures manual template

Inoculated culture plates were incubated at room temperature for 8 days and observed every 2 days for growth by using an inverted microscope Fig. A Cyst of the Acanthamoeba sp. With a sterile pipette, 2 drops of each specimen were placed onto a glass slide and allowed to air dry for 10 min. The slides were then fixed in methanol and stained with Giemsa for 8 min.

Acanthamoeba Keratitis

Prior to DNA extraction, frozen specimens were thawed at room temperature. One primer set, specific for multicopy Acanthamoeba genomic sites encoding rRNA, was employed for each reaction.

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Amplicons were visualized on 1. In order to establish the lower limit of detection of acanthamoebae for the above-mentioned PCR procedure, serial dilutions of known positive samples were made. Positive-control culture plates were scraped using a scalpel, and material was transferred to a microcentrifuge tube and suspended in RPMI medium Invitrogen Corp. Serial dilutions were then made using RPMI to achieve the following concentrations: 50, 25, We defined a specimen to be positive for the Acanthamoeba sp.

It was this reference standard against which each individual test was compared for sensitivity and specificity analysis.

Statistical analyses were performed using SigmaStat 2. During the study period, clinical specimens were examined for evidence of Acanthamoeba infection.

The remaining specimens were one contact lens casing, one eye swab, one corneal biopsy sample, and one tear fluid sample. Of the specimens evaluated, 20 Twenty-eight Acanthamoebae were undetectable in eye swabs, corneal biopsy specimens, and tears. Comparison of four diagnostic methods used for evaluation of clinical specimens from patients suspected to have AK a.

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Performance characteristics of four methods for diagnosis of AK used for evaluation of clinical specimens by specimen type. Results for specimen culture using four different types of NM were positive for 14 specimens, yielding a sensitivity and specificity of Only 18 of 26 specimens positive by Nelson primer PCR fulfilled the reference standard criteria for a diagnosis of AK, thus, eight specimens were considered to be false positive.

PCR product was detectable at a concentration of 0. Most organisms were not identified to the species level by sequencing, having greatest homology with the Acanthamoeba sp. PCR in general had a particular performance advantage with specimens such as contact lens solutions, where a dilutional effect may be observed. While traditional direct smear analysis and culture of specimens are highly specific diagnostic methods, they are limited by high false-negativity rates, the requirement for significant technical expertise, and, in the case of culture, a very long turnaround time 4 , 10 , The yield of culture using NM reported herein is almost identical to that reported by others using buffered charcoal yeast agar, non-nutrient agar with E.

Our results are also consistent with others in that culture has previously been shown to outperform JDP primer PCR in the diagnosis of AK from clinical specimens That Nelson primer PCR had a high false-positivity rate in this verification may simply reflect the outperformance of this highly sensitive molecular technique compared to that of the comparator methods. It is possible that Acanthamoeba DNA was detectable by the primer set at a concentration below the limit of detection of whole organisms or parasite DNA for the other assays.

This represents an inherent limitation of any diagnostic evaluation in the absence of a well-performing reference standard 1. Corneal specimens for the diagnosis of AK are notoriously difficult to obtain, and few patients tolerate corneal scraping well 5 , Obtaining a sufficient volume of clinical specimen to facilitate decent smear or culture yields is challenging 5 , Similarly, large-volume specimens such as contact lens solutions have a demonstrable dilutional effect and are therefore subject to poor smear and culture results, as demonstrated by our specimen-based performance analysis.

Thus, diagnostic methods that detect very few organisms in a clinical specimen are clearly advantageous.

Imaging Studies

We have demonstrated an analytic sensitivity for Nelson primer PCR to the single-organism level. Such analytic sensitivity has implications not only for routine diagnosis of AK but also for a test of cure, where one would expect the burden of acanthamoebae to be extremely low in clinical specimens.

In addition, Nelson primer PCR could prove to be a rapid, sensitive tool for screening batches of contact lens solutions in outbreak situations.

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In the clinical laboratory setting, care must be taken to balance maximization of Acanthamoeba culture yield through prolonged incubation and production of a timely and clinically useful result.

In the case of AK, prompt initiation of appropriate therapy is necessary to limit ocular morbidity and optimize visual outcome 4 , 5. Thus, employment of a rapid, sensitive screening tool, such as Nelson primer PCR, followed by a rapid, specific confirmatory test, such as JDP primer PCR, may offer benefits beyond those achieved through direct specimen microscopy and culture alone.

Acanthamoeba Infection Workup

Strategies which simplify the procedure for laboratory investigation of AK are likely worthwhile and cost-effective 4. Of particular interest to clinicians is that the kind of specimen most frequently submitted to the laboratory, the corneal scraping, statistically had the lowest rates of detectable whole organisms and Acanthamoeba DNA by all tests but Nelson primer PCR.

While this may simply reflect that patients with actual AK in our sample were more likely to be contact lens wearers and thus have contact lens-related specimens to submit, it may also reflect that the burden of organisms in contact lenses, cases, and fluids is greater than what is seen in a corneal scraping.

This hypothesis is supported by our specimen-based performance analysis of the individual diagnostic assays, though these results should be interpreted cautiously given the low numbers of positive samples by each specimen type evaluated.

While detection of acanthamoebae from contact lenses, fluids, or casings does not strictly confirm the diagnosis, it is virtually diagnostic in the setting of a compatible clinical history 10 , Thus, submission and processing of these types of atypical specimens may be as important as those for corneal scrapings. Other potential explanations for the noted assay performance disparities by specimen type involve the presence of PCR inhibitors in the corneal tissue itself and the low volume of clinical material obtained by corneal scrapings.

Acanthamoeba culture procedures manual template

Future evaluation of the disparity in yield by specimen type where the potential bias of contact lens use can be controlled is warranted. The evaluation herein highlights the limitations of commonly employed diagnostic assays for AK and supports the potential utility of at least one primer set for molecular detection of acanthamoebae in clinical specimens.

PCR had a clear diagnostic advantage over conventional techniques for large-volume specimens, such as contact lens solutions, where a dilutional effect would be expected. PCR is less labor-intensive than culture, requires fewer specialized technical skills, is more sensitive, and offers a much more rapid turnaround time, all of which culminate in the ability of the clinical laboratory to produce a meaningful, clinically relevant result.

Acanthamoeba culture procedures manual template

We would encourage clinicians to consider submission to the laboratory of contact lens-associated materials in addition to corneal scrapings from any patient in whom AK is suspected. All authors contributed to and critically appraised the manuscript. National Center for Biotechnology Information , U.

Journal List J Clin Microbiol v. J Clin Microbiol.

Collecting a Corneal Culture

Published online Mar Andrea K. Low 2, 3, 4. Donald S. Donald E. Author information Article notes Copyright and License information Disclaimer. Phone: Fax: E-mail: ac. This article has been cited by other articles in PMC.

Acanthamoeba culture procedures manual template

Abstract Amoebic keratitis causes significant ocular morbidity in contact lens wearers. Open in a separate window. Direct examination. Isolation of DNA from specimens. Determination of analytic sensitivity of PCR.

Confirmation of PCR products by sequence analysis. Statistical analysis. TABLE 1.