ARTICLE
Comparison of Validated Polymerase Chain Reaction and Culture Isolation for the Routine Detection of Acanthamoeba From Ocular Samples Regis P. Kowalski,
M.S., M.(A.S.C.P.),
Melissa A. Melan, Ph.D., Lisa M. Karenchak, and Alex Mammen, M.D.
Purpose: Acanthamoeba keratitis should be definitively diagnosed for appropriate therapy. Our institution has validated polymerase chain reaction (PCR) as a routine diagnostic test to detect Acanthamoeba DNA from ocular samples. We compared PCR with culture isolation for detecting Acanthamoeba from ocular samples. Methods: The microbiology records of patients that had specimens submitted (May 2012 to January 2014) for laboratory testing for Acanthamoeba keratitis were reviewed for (1) Acanthamoeba culture isolation, (2) Acanthamoeba DNA detection by PCR, and (3) non-Acanthamoeba culture results. For Acanthamoeba isolation, corneal samples were planted on nonnutrient agar overlaid with Enterobacter aerogenes. Validated PCR (May 2012) for Acanthamoeba DNA was processed at the Division of Molecular Diagnostics, UPMC, Pittsburgh, PA. Additional cultures were obtained for bacteria, fungus, and virus (i.e., herpes simplex virus) using standard techniques. Results: Culture isolation and PCR were processed on 125 patients with a differential diagnosis of Acanthamoeba keratitis. Of these, 104 (83.2%) were culture negative, PCR negative; 14 (11.2%) were culture positive, PCR positive; 4 (3.2%) were culture negative, PCR positive; and, 3 (2.4%) were culture positive, PCR negative. Culture and PCR were statistically equivalent for detecting Acanthamoeba from ocular samples (P¼1.0, McNemar’s test). Nineteen of the culture-negative, PCR-negative corneal samples (18.3%) were positive for other pathogens such as bacteria, fungus, and virus. Conclusions: There is no clear advantage of PCR over culture isolation for detecting Acanthamoeba in ocular specimens. Other pathogens such as bacteria, fungus, and virus must still be considered in severe persistent keratitis. Polymerase chain reaction seems to be a complementary test for the clinical support of Acanthamoeba keratitis.
From the Department of Ophthalmology (R.P.K., L.M.K., A.M.), Charles T. Campbell Eye Microbiology Laboratory, University of Pittsburgh Medical Center (UPMC), University of Pittsburgh, Pittsburgh, PA; and Division of Molecular Diagnostics (M.A.M.), Department of Pathology, University of Pittsburgh Medical Center (UPMC), University of Pittsburgh, Pittsburgh, PA. The authors have no conflicts of interest to disclose. Supported by the Pennsylvania Lions Club, The Charles T. Campbell Foundation, Eye and Ear Foundation of Pittsburgh, PA, National Institutes of Health Core Grant P30 EY008098, and Unrestricted Grant from Research to Prevent Blindness, New York, NY. Presented in part at the 2013 OMIG Meeting, November 15, 2013, New Orleans, LA; 2014 ECCMID Meeting, May 10 to 13, Barcelona, Spain; and the 2014 ISOPT Meeting, June 19 to 22, Reykjavik, Iceland. Address correspondence to Regis P. Kowalski, M.S., M.(A.S.C.P.), The Eye and Ear Institute Building, Ophthalmic Microbiology, Room 642, 203 Lothrop Street, Pittsburgh, PA 15213; e-mail: [email protected] Accepted December 23, 2014. DOI: 10.1097/ICL.0000000000000131
Eye & Contact Lens Volume 41, Number 6, November 2015
B.S., M.(A.S.C.P.),
Key Words: Acanthamoeba—Keratitis—PCR—Laboratory diagnosis. (Eye & Contact Lens 2015;41: 341–343)
T
he diagnosis of Acanthamoeba keratitis is commonly ascertained with a combination of clinical presentation, isolation in culture, observation on stained material, and confocal microscopy (http://eyemicrobiology.upmc.com/Acanthamoeba.htm).1–3 Using sets of true-positive and true-negative ocular specimens, we were able to develop polymerase chain reaction (PCR) to detect Acanthamoeba DNA from ocular samples to complement testing.4 On further refinement, we were able to validate PCR for the detection of Acanthamoeba DNA from ocular samples on a routine clinical basis.5 The detection of Acanthamoeba DNA was based on a technique by Qvarnstrom et al.6 for detecting 40 different Acanthamoeba 18S rRNA sequences and 7 strains from 4 genotypes (T1, T4, T7, and T10). In our laboratory, severe prolonged keratitis is processed for Acanthamoeba detection (culture, PCR, and Giemsa stain), bacteria (culture and smear), fungus (culture and smear), and virus in select cases (culture and PCR). In this study, our primary aim was to compare PCR with culture isolation for the detection of Acanthamoeba under routine laboratory testing. It was our intention to determine whether the detection of Acanthamoeba DNA was more diagnostic than growing Acanthamoeba in culture. We hypothesized that PCR is more diagnostic than culture isolation for detecting Acanthamoeba from ocular samples, and we would support this hypothesis with a review of laboratory data.
METHODS The microbiology records of patients that had specimens submitted from May 2012 to May 2014 for Acanthamoeba keratitis laboratory testing were reviewed, without recording patient identifiers for (1) Acanthamoeba culture isolation, (2) Acanthamoeba DNA detection by PCR, and (3) non-Acanthamoeba culture results. The Institutional Review Board of the University of Pittsburgh determined that informed consent was not required for this study. The suspicion of Acanthamoeba keratitis was presumed to vary from very high to low. Samples were obtained from the university private cornea practice, consults from the local community, emergency department, and outside practices aware of our testing capabilities (note: our testing is available to all who practice 341
Copyright @ Contact Lens Association of Opthalmologists, Inc. Unauthorized reproduction of this article is prohibited.
R. P. Kowalski et al. ophthalmology) (http://eyemicrobiology.upmc.com). The hospital laboratory is part of a medical school setting that includes the Department of Ophthalmology private practice and a residency program. For Acanthamoeba isolation, ocular samples were planted on nonnutrient agar (Difco Agar Noble; Becton Dickinson and Company, Sparks, MD) overlaid with Enterobacter aerogenes and monitored for growth over 7 days. A positive Acanthamoeba culture was determined by observation of spreading trophozoites and presence of cysts transformed from trophozoites (http://eyemicrobiology.upmc.com/Acanthamoeba.htm).3 Isolates from PCR negative samples were also tested (confirmed) for Acanthamoeba DNA using PCR. The validation of PCR for Acanthamoeba DNA was completed in May 2012 by the Division of Molecular Diagnostics, Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA. The sensitivity, specificity, positive predictive value, and negative predictive value of testing were determined to be 100%, 97%, 75%, and 100%, respectively. The limit of detection was determined to be 10 plasmid copies per reaction or 0.5 pg of Acanthamebic genomic DNA. Within-run and between-run reproducibility (coefficient of variance) were 0.75% and 1.1%, respectively. Testing included internal controls to assure that there were no inhibitors of the PCR process in the patient samples. All patient ocular samples for PCR testing were generally obtained using kimura spatulas, jeweler’s forceps, or soft-tipped plastic stem applicators (i.e., dacron, cotton, not calcium alginate), placing the collected specimens in 2 mL tubes of Bartels ChlamTrans Transport Medium (CTM) (Trinity Biotech USA, Jamestown, NY). A few corneal biopsies were homogenized for testing. A minimal sample of 0.4 mL was delivered for PCR testing. If delayed, overnight storage was at 6°C and storage from 24 to 72 hr was at 280°C. To maximize the yield of pathogen isolation, the method of sample collection was at the discretion of the attending physician.
Eye & Contact Lens Volume 41, Number 6, November 2015 In addition to Acanthamoeba testing, culture isolation for bacteria and fungus were plated on chocolate II agar, trypticase soy agar supplemented with 5% sheep blood, and Sabouraud dextrose with gentamicin plates (BBL; Becton Dickinson and Company) (http://eyemicrobiology.upmc.com/Bacteria.htm).3 Polymerase chain reaction for herpes simplex virus (HSV) DNA and HSV isolation were processed from the CTM collected for Acanthamoeba testing from those cases where HSV was part of the differential diagnosis. All excess CTM patient samples were stored at 280°C for future validation testing. McNemar test was used to compare paired proportions of PCR testing and Acanthamoeba culture isolation: (1) PCR negative, culture negative, (2) PCR negative, culture positive, (3) PCR positive, culture negative, and (4) PCR positive, culture positive (http://graphpad.com/quickcalcs/McNemar1.cfm).
RESULTS Figure 1 summarizes the results of laboratory testing. Acanthamoeba culture isolation and PCR were processed on 125 patients with keratitis. Of the 125 patients with keratitis, 104 (83.2%) were culture negative, PCR negative; 14 (11.2%) were culture positive, PCR positive; 4 (3.2%) were culture negative, PCR positive; and 3 (2.4%) were culture positive, PCR negative. Culture isolation and PCR were statistically equivalent for detecting Acanthamoeba from corneal samples (P¼1.0, McNemar test). Two of the three isolates from the culture-positive PCR-negative samples tested positive for Acanthamoeba DNA. Nineteen of the 104 (culture negative, PCR negative) corneal samples (8.3%) were positive for other pathogens (6 Pseudomonas aeruginosa, 2 Aspergillus species, 2 Klebsiella pneumoniae, and 1 each of MRSA, MSSA, Moraxella lacunata, Alcaligenes xylosoxidans, Streptococcus pneumoniae, Nocardia species, Fusarium species, Candida albicans, and HSV) (Fig. 1).
FIG. 1. Laboratory results of 125 patients with a clinical differential of Acanthamoeba keratitis. Positive for non-Acanthamoeba pathogens is based on 19 isolates from the culture-negative polymerase chain reaction–negative group.
342
Eye & Contact Lens Volume 41, Number 6, November 2015
Copyright @ Contact Lens Association of Opthalmologists, Inc. Unauthorized reproduction of this article is prohibited.
Eye & Contact Lens Volume 41, Number 6, November 2015
DISCUSSION The hypothesis that PCR was more diagnostic than culture isolation was rejected in this study. It could be noted that PCR was more diagnostic in four cases, but PCR also missed detection in three cases. Two of the three isolates from culture-positive PCR-negative samples were positive for Acanthamoeba DNA, thus sampling error not poor sensitivity could be reasoned for the lack of detection in the two patient samples. The isolate that tested PCR negative was an unusual phenotypic slow-growing isolate. Otherwise, it seems that Acanthamoeba is hardy and isolates well in culture, but residual DNA for the detection of nonviable Acanthamoeba may not be present. It is interesting that this study supports the pan testing for other etiologic pathogenic agents in cases of severe keratitis with long-term onset. Nearly 20% of Acanthamoeba PCR-negative culture-negative cases were positive for bacteria, fungus, or virus. Validation for detecting Acanthamoeba DNA by PCR can be an extended process because of the lack of true-positive ocular specimens (Acanthamoeba culture positive). At our institution, we initially developed PCR using the Cepheid SmartCycler II system (Cepheid, Sunnyvale, CA),4 but as part of testing consolidation, a new validation with the Roche LightCycler 1.2 (Roche, Nutley, NJ) was performed with a new set of true-positive and true-negative samples over a 3-year period. Our initial PCR development is open as public record,4 but this study is part of our final continuing validation. Validation of testing is mandatory by laboratory certification before using any diagnostic technique for clinical studies. Each laboratory must perform its own validation and cannot use the validation of other laboratories to perform clinical testing. This assures that all laboratories are proficient in performing testing. Wide-spread availability of PCR for Acanthamoeba DNA detection from ocular specimens may be limited to reference laboratories. It is the hope that this study will be constructively critiqued with the routine findings of other clinical laboratories. For example, Mathers et al.7 have provided a correlation of confocal microscopy
© 2015 Contact Lens Association of Ophthalmologists
Detection of Acanthamoeba From Ocular Samples with a nonvalidated method of PCR. The strength of our study was that the testing results were produced under routine continuing laboratory testing conditions and not as an individual study. An inherent weakness of the study was that there was no consistent technique for sampling. Different physicians used different methods such as forceps, soft-tipped applicators, biopsies, etc., for collecting test samples. More studies are required to optimize sample collection. We encourage others to further refine PCR for detecting Acanthamoeba DNA. In summary, Acanthamoeba PCR testing for DNA seems to be a complementary test for supporting the diagnosis of Acanthamoeba keratitis. Culture isolation seems to be concordant with PCR DNA detection. If culture isolation is not available, ophthalmologists will need to locate laboratories that offer validated PCR testing of ocular samples for Acanthamoeba DNA. REFERENCES 1. Rezaei KM, Naghshgar N, Javadi MA, et al. Various confocal scan features of cysts and trophozoites in Acanthamoeba keratitis. Eur J Ophthalmol 2012; 22(Suppl 7):S46–S50. 2. Vaddavalli PK, Garg PS, Sharma S, et al. Role of confocal microscopy in the diagnosis of fungal and Acanthamoeba keratitis. Ophthalmology 2011;118: 29–35. 3. Kowalski RP. Practical ophthalmic microbiology for the detection of corneal pathogens. In: Krachmer JH, Mannis MJ, Holland EJ, eds. Second Edition— Cornea –Volume One—Fundamentals, Diagnostics, and Management. Philadelphia, PA, Elsevier Mosby, 2005, pp. 237–245. 4. Thompson PP, Shanks RMQ, Gordon YJ, et al. Validation of real-time PCR for laboratory diagnosis of Acanthamoeba keratitis. J Clin Microbiol 2008; 46:3232–3236. 5. Melan MA, Oakley GJ, Kowalski RP, et al. Adaptation of a qualitative PCR assay to detect the presence of Acanthamoeba in ophthalmologic samples. J Mole Diagn 2013;14:674. 6. Qvarnstrom Y, Visvesvara GS, Sriram R, da Silva AJ. Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri. J Clin Microbiol 2006;44:3589–3595. 7. Mathers WD, Nelson SE, Lane JL, et al. Confirmation of confocal microscopy diagnosis of Acanthamoeba keratitis using polymerase chain reaction analysis. Arch Ophthalmol 2000;118:178–183.
343
Copyright @ Contact Lens Association of Opthalmologists, Inc. Unauthorized reproduction of this article is prohibited.
Comparison of Validated Polymerase Chain Reaction and Culture Isolation for the Routine Detection of Acanthamoeba From Ocular Samples Regis P. Kowalski,
M.S., M.(A.S.C.P.),
Melissa A. Melan, Ph.D., Lisa M. Karenchak, and Alex Mammen, M.D.
Purpose: Acanthamoeba keratitis should be definitively diagnosed for appropriate therapy. Our institution has validated polymerase chain reaction (PCR) as a routine diagnostic test to detect Acanthamoeba DNA from ocular samples. We compared PCR with culture isolation for detecting Acanthamoeba from ocular samples. Methods: The microbiology records of patients that had specimens submitted (May 2012 to January 2014) for laboratory testing for Acanthamoeba keratitis were reviewed for (1) Acanthamoeba culture isolation, (2) Acanthamoeba DNA detection by PCR, and (3) non-Acanthamoeba culture results. For Acanthamoeba isolation, corneal samples were planted on nonnutrient agar overlaid with Enterobacter aerogenes. Validated PCR (May 2012) for Acanthamoeba DNA was processed at the Division of Molecular Diagnostics, UPMC, Pittsburgh, PA. Additional cultures were obtained for bacteria, fungus, and virus (i.e., herpes simplex virus) using standard techniques. Results: Culture isolation and PCR were processed on 125 patients with a differential diagnosis of Acanthamoeba keratitis. Of these, 104 (83.2%) were culture negative, PCR negative; 14 (11.2%) were culture positive, PCR positive; 4 (3.2%) were culture negative, PCR positive; and, 3 (2.4%) were culture positive, PCR negative. Culture and PCR were statistically equivalent for detecting Acanthamoeba from ocular samples (P¼1.0, McNemar’s test). Nineteen of the culture-negative, PCR-negative corneal samples (18.3%) were positive for other pathogens such as bacteria, fungus, and virus. Conclusions: There is no clear advantage of PCR over culture isolation for detecting Acanthamoeba in ocular specimens. Other pathogens such as bacteria, fungus, and virus must still be considered in severe persistent keratitis. Polymerase chain reaction seems to be a complementary test for the clinical support of Acanthamoeba keratitis.
From the Department of Ophthalmology (R.P.K., L.M.K., A.M.), Charles T. Campbell Eye Microbiology Laboratory, University of Pittsburgh Medical Center (UPMC), University of Pittsburgh, Pittsburgh, PA; and Division of Molecular Diagnostics (M.A.M.), Department of Pathology, University of Pittsburgh Medical Center (UPMC), University of Pittsburgh, Pittsburgh, PA. The authors have no conflicts of interest to disclose. Supported by the Pennsylvania Lions Club, The Charles T. Campbell Foundation, Eye and Ear Foundation of Pittsburgh, PA, National Institutes of Health Core Grant P30 EY008098, and Unrestricted Grant from Research to Prevent Blindness, New York, NY. Presented in part at the 2013 OMIG Meeting, November 15, 2013, New Orleans, LA; 2014 ECCMID Meeting, May 10 to 13, Barcelona, Spain; and the 2014 ISOPT Meeting, June 19 to 22, Reykjavik, Iceland. Address correspondence to Regis P. Kowalski, M.S., M.(A.S.C.P.), The Eye and Ear Institute Building, Ophthalmic Microbiology, Room 642, 203 Lothrop Street, Pittsburgh, PA 15213; e-mail: [email protected] Accepted December 23, 2014. DOI: 10.1097/ICL.0000000000000131
Eye & Contact Lens Volume 41, Number 6, November 2015
B.S., M.(A.S.C.P.),
Key Words: Acanthamoeba—Keratitis—PCR—Laboratory diagnosis. (Eye & Contact Lens 2015;41: 341–343)
T
he diagnosis of Acanthamoeba keratitis is commonly ascertained with a combination of clinical presentation, isolation in culture, observation on stained material, and confocal microscopy (http://eyemicrobiology.upmc.com/Acanthamoeba.htm).1–3 Using sets of true-positive and true-negative ocular specimens, we were able to develop polymerase chain reaction (PCR) to detect Acanthamoeba DNA from ocular samples to complement testing.4 On further refinement, we were able to validate PCR for the detection of Acanthamoeba DNA from ocular samples on a routine clinical basis.5 The detection of Acanthamoeba DNA was based on a technique by Qvarnstrom et al.6 for detecting 40 different Acanthamoeba 18S rRNA sequences and 7 strains from 4 genotypes (T1, T4, T7, and T10). In our laboratory, severe prolonged keratitis is processed for Acanthamoeba detection (culture, PCR, and Giemsa stain), bacteria (culture and smear), fungus (culture and smear), and virus in select cases (culture and PCR). In this study, our primary aim was to compare PCR with culture isolation for the detection of Acanthamoeba under routine laboratory testing. It was our intention to determine whether the detection of Acanthamoeba DNA was more diagnostic than growing Acanthamoeba in culture. We hypothesized that PCR is more diagnostic than culture isolation for detecting Acanthamoeba from ocular samples, and we would support this hypothesis with a review of laboratory data.
METHODS The microbiology records of patients that had specimens submitted from May 2012 to May 2014 for Acanthamoeba keratitis laboratory testing were reviewed, without recording patient identifiers for (1) Acanthamoeba culture isolation, (2) Acanthamoeba DNA detection by PCR, and (3) non-Acanthamoeba culture results. The Institutional Review Board of the University of Pittsburgh determined that informed consent was not required for this study. The suspicion of Acanthamoeba keratitis was presumed to vary from very high to low. Samples were obtained from the university private cornea practice, consults from the local community, emergency department, and outside practices aware of our testing capabilities (note: our testing is available to all who practice 341
Copyright @ Contact Lens Association of Opthalmologists, Inc. Unauthorized reproduction of this article is prohibited.
R. P. Kowalski et al. ophthalmology) (http://eyemicrobiology.upmc.com). The hospital laboratory is part of a medical school setting that includes the Department of Ophthalmology private practice and a residency program. For Acanthamoeba isolation, ocular samples were planted on nonnutrient agar (Difco Agar Noble; Becton Dickinson and Company, Sparks, MD) overlaid with Enterobacter aerogenes and monitored for growth over 7 days. A positive Acanthamoeba culture was determined by observation of spreading trophozoites and presence of cysts transformed from trophozoites (http://eyemicrobiology.upmc.com/Acanthamoeba.htm).3 Isolates from PCR negative samples were also tested (confirmed) for Acanthamoeba DNA using PCR. The validation of PCR for Acanthamoeba DNA was completed in May 2012 by the Division of Molecular Diagnostics, Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA. The sensitivity, specificity, positive predictive value, and negative predictive value of testing were determined to be 100%, 97%, 75%, and 100%, respectively. The limit of detection was determined to be 10 plasmid copies per reaction or 0.5 pg of Acanthamebic genomic DNA. Within-run and between-run reproducibility (coefficient of variance) were 0.75% and 1.1%, respectively. Testing included internal controls to assure that there were no inhibitors of the PCR process in the patient samples. All patient ocular samples for PCR testing were generally obtained using kimura spatulas, jeweler’s forceps, or soft-tipped plastic stem applicators (i.e., dacron, cotton, not calcium alginate), placing the collected specimens in 2 mL tubes of Bartels ChlamTrans Transport Medium (CTM) (Trinity Biotech USA, Jamestown, NY). A few corneal biopsies were homogenized for testing. A minimal sample of 0.4 mL was delivered for PCR testing. If delayed, overnight storage was at 6°C and storage from 24 to 72 hr was at 280°C. To maximize the yield of pathogen isolation, the method of sample collection was at the discretion of the attending physician.
Eye & Contact Lens Volume 41, Number 6, November 2015 In addition to Acanthamoeba testing, culture isolation for bacteria and fungus were plated on chocolate II agar, trypticase soy agar supplemented with 5% sheep blood, and Sabouraud dextrose with gentamicin plates (BBL; Becton Dickinson and Company) (http://eyemicrobiology.upmc.com/Bacteria.htm).3 Polymerase chain reaction for herpes simplex virus (HSV) DNA and HSV isolation were processed from the CTM collected for Acanthamoeba testing from those cases where HSV was part of the differential diagnosis. All excess CTM patient samples were stored at 280°C for future validation testing. McNemar test was used to compare paired proportions of PCR testing and Acanthamoeba culture isolation: (1) PCR negative, culture negative, (2) PCR negative, culture positive, (3) PCR positive, culture negative, and (4) PCR positive, culture positive (http://graphpad.com/quickcalcs/McNemar1.cfm).
RESULTS Figure 1 summarizes the results of laboratory testing. Acanthamoeba culture isolation and PCR were processed on 125 patients with keratitis. Of the 125 patients with keratitis, 104 (83.2%) were culture negative, PCR negative; 14 (11.2%) were culture positive, PCR positive; 4 (3.2%) were culture negative, PCR positive; and 3 (2.4%) were culture positive, PCR negative. Culture isolation and PCR were statistically equivalent for detecting Acanthamoeba from corneal samples (P¼1.0, McNemar test). Two of the three isolates from the culture-positive PCR-negative samples tested positive for Acanthamoeba DNA. Nineteen of the 104 (culture negative, PCR negative) corneal samples (8.3%) were positive for other pathogens (6 Pseudomonas aeruginosa, 2 Aspergillus species, 2 Klebsiella pneumoniae, and 1 each of MRSA, MSSA, Moraxella lacunata, Alcaligenes xylosoxidans, Streptococcus pneumoniae, Nocardia species, Fusarium species, Candida albicans, and HSV) (Fig. 1).
FIG. 1. Laboratory results of 125 patients with a clinical differential of Acanthamoeba keratitis. Positive for non-Acanthamoeba pathogens is based on 19 isolates from the culture-negative polymerase chain reaction–negative group.
342
Eye & Contact Lens Volume 41, Number 6, November 2015
Copyright @ Contact Lens Association of Opthalmologists, Inc. Unauthorized reproduction of this article is prohibited.
Eye & Contact Lens Volume 41, Number 6, November 2015
DISCUSSION The hypothesis that PCR was more diagnostic than culture isolation was rejected in this study. It could be noted that PCR was more diagnostic in four cases, but PCR also missed detection in three cases. Two of the three isolates from culture-positive PCR-negative samples were positive for Acanthamoeba DNA, thus sampling error not poor sensitivity could be reasoned for the lack of detection in the two patient samples. The isolate that tested PCR negative was an unusual phenotypic slow-growing isolate. Otherwise, it seems that Acanthamoeba is hardy and isolates well in culture, but residual DNA for the detection of nonviable Acanthamoeba may not be present. It is interesting that this study supports the pan testing for other etiologic pathogenic agents in cases of severe keratitis with long-term onset. Nearly 20% of Acanthamoeba PCR-negative culture-negative cases were positive for bacteria, fungus, or virus. Validation for detecting Acanthamoeba DNA by PCR can be an extended process because of the lack of true-positive ocular specimens (Acanthamoeba culture positive). At our institution, we initially developed PCR using the Cepheid SmartCycler II system (Cepheid, Sunnyvale, CA),4 but as part of testing consolidation, a new validation with the Roche LightCycler 1.2 (Roche, Nutley, NJ) was performed with a new set of true-positive and true-negative samples over a 3-year period. Our initial PCR development is open as public record,4 but this study is part of our final continuing validation. Validation of testing is mandatory by laboratory certification before using any diagnostic technique for clinical studies. Each laboratory must perform its own validation and cannot use the validation of other laboratories to perform clinical testing. This assures that all laboratories are proficient in performing testing. Wide-spread availability of PCR for Acanthamoeba DNA detection from ocular specimens may be limited to reference laboratories. It is the hope that this study will be constructively critiqued with the routine findings of other clinical laboratories. For example, Mathers et al.7 have provided a correlation of confocal microscopy
© 2015 Contact Lens Association of Ophthalmologists
Detection of Acanthamoeba From Ocular Samples with a nonvalidated method of PCR. The strength of our study was that the testing results were produced under routine continuing laboratory testing conditions and not as an individual study. An inherent weakness of the study was that there was no consistent technique for sampling. Different physicians used different methods such as forceps, soft-tipped applicators, biopsies, etc., for collecting test samples. More studies are required to optimize sample collection. We encourage others to further refine PCR for detecting Acanthamoeba DNA. In summary, Acanthamoeba PCR testing for DNA seems to be a complementary test for supporting the diagnosis of Acanthamoeba keratitis. Culture isolation seems to be concordant with PCR DNA detection. If culture isolation is not available, ophthalmologists will need to locate laboratories that offer validated PCR testing of ocular samples for Acanthamoeba DNA. REFERENCES 1. Rezaei KM, Naghshgar N, Javadi MA, et al. Various confocal scan features of cysts and trophozoites in Acanthamoeba keratitis. Eur J Ophthalmol 2012; 22(Suppl 7):S46–S50. 2. Vaddavalli PK, Garg PS, Sharma S, et al. Role of confocal microscopy in the diagnosis of fungal and Acanthamoeba keratitis. Ophthalmology 2011;118: 29–35. 3. Kowalski RP. Practical ophthalmic microbiology for the detection of corneal pathogens. In: Krachmer JH, Mannis MJ, Holland EJ, eds. Second Edition— Cornea –Volume One—Fundamentals, Diagnostics, and Management. Philadelphia, PA, Elsevier Mosby, 2005, pp. 237–245. 4. Thompson PP, Shanks RMQ, Gordon YJ, et al. Validation of real-time PCR for laboratory diagnosis of Acanthamoeba keratitis. J Clin Microbiol 2008; 46:3232–3236. 5. Melan MA, Oakley GJ, Kowalski RP, et al. Adaptation of a qualitative PCR assay to detect the presence of Acanthamoeba in ophthalmologic samples. J Mole Diagn 2013;14:674. 6. Qvarnstrom Y, Visvesvara GS, Sriram R, da Silva AJ. Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri. J Clin Microbiol 2006;44:3589–3595. 7. Mathers WD, Nelson SE, Lane JL, et al. Confirmation of confocal microscopy diagnosis of Acanthamoeba keratitis using polymerase chain reaction analysis. Arch Ophthalmol 2000;118:178–183.
343
Copyright @ Contact Lens Association of Opthalmologists, Inc. Unauthorized reproduction of this article is prohibited.
