AmpliVue Is a Practical and Timely Test for the Detection of HSV From Keratitis Specimens.

OBJECTIVE: The laboratory diagnostic detection of herpes simplex virus (HSV) from eye samples must be practical, timely, and definitive for appropriate therapy. Although polymerase chain reaction (PCR) and/or cell culture can be definitive, HSV results can be delayed. Enzyme Linked Virus Inducible System (ELVIS) is a test that can provide results within 24 to 48 hr. We evaluated "AmpliVue HSV 1+2 Assay" as a molecular colorimetric test that can detect HSV (1 or 2) DNA within 1 hr. METHODS: Cornea/conjunctival samples were tested retrospectively with AmpliVue against 53 true-positive and 20 true-negative specimens collected in chlamydial transport medium. All clinical specimens were tested by cell culture isolation, PCR, and ELVIS for routine patient care. RESULTS: The sensitivity of AmpliVue against ocular samples that were both culture-positive and PCR-positive was 84%. The specificity of AmpliVue was 100%. Only one clinical sample was HSV-2 positive, whereas all others tested positive for HSV-1. Based on PCR-positive and cell culture-negative samples, AmpliVue (11 of 17) tested more positive than ELVIS (0 of 17) (P=0.003, Fisher Exact). CONCLUSIONS: AmpliVue is moderately sensitive and highly specific as a practical and timely diagnostic test for detecting ocular HSV. Expertise is readily achieved and the test is straightforward with easy interpretation. Negative AmpliVue testing must be confirmed with PCR. AmpliVue has potential as an office-based diagnostic test.

Comparison of Validated Polymerase Chain Reaction and Culture Isolation for the Routine Detection of Acanthamoeba From Ocular Samples.

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 (P=1.0, 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.

The verification of nucleic acid amplification testing (Gen-Probe Aptima Assay) for chlamydia trachomatis from ocular samples.

OBJECTIVE: Chlamydia trachomatis conjunctivitis may present with extended symptoms, and it can have social ramifications as a sexually transmitted disease. For appropriate therapy, C. trachomatis conjunctivitis should be diagnosed definitively. This study presents the verification of nucleic acid amplification testing (NAAT; Gen-Probe Aptima Combo 2 assay) for detection of C. trachomatis ribosomal RNA (rRNA) from direct ocular samples. DESIGN: Retrospective laboratory verification study. SUBJECTS: Patients with infectious conjunctivitis. METHODS: A battery of 25 true-positive specimens (direct ocular specimens from patients with symptoms consistent with C. trachomatis conjunctivitis and with previously demonstrated positive polymerase chain reaction [PCR] results for C. trachomatis DNA by Roche Amplicor) and 25 true-negative specimens (direct ocular specimens with culture-positive results for herpes simplex virus [n = 5], adenovirus [n = 5], Haemophilus influenzae [n = 5], and Streptococcus pneumoniae [n = 5]), and transport medium (n = 5) were tested for C. trachomatis rRNA by NAAT. These true-negative specimens have differential etiologic agents of infectious conjunctivitis. The 25 C. trachomatis specimens with PCR-positive results (obtained May 1994-May 2012) and 20 true-negative infectious ocular specimens (obtained December 2008-August 2013) were collected with soft-tipped applicators and placed in transport medium. All excess specimens were stored at -80°C. All samples were centrifuged at 13,000 rpm for 1 hour at 6°C. For each sample, using the Aptima Unisex collection blue swab, a specimen was collected from the conical apex of the storage tube where a pellet was formed. The Aptima Unisex collection swab was placed in a tube of Aptima swab transport medium for testing. All samples were tested in duplicate. MAIN OUTCOME MEASURES: Detection of C. trachomatis rRNA. RESULTS: Of 25 true-positive samples, 24 (96%) were positive by NAAT, whereas 25 of 25 true-negative samples (100%) showed negative results. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency were determined to be 96%, 100%, 100%, 96%, and 98%, respectively. CONCLUSIONS: The detection of C. trachomatis in ocular specimens by NAAT was verified for laboratory diagnosis. The test will be evaluated prospectively to determine future test performance precisely.

In vitro comparison of combination and monotherapy for the empiric and optimal coverage of bacterial keratitis based on incidence of infection.

PURPOSE: Cefazolin/tobramycin, cefuroxime/gentamicin, and moxifloxacin were compared using bacterial keratitis isolates to determine whether empiric therapy constituted optimal antibacterial treatment. METHODS: Based on percent incidence of corneal infection, 27 Staphylococcus aureus, 16 Pseudomonas aeruginosa, 10 Serratia marcescens, 4 Moraxella lacunata, 3 Haemophilus influenzae, 9 coagulase-negative staphylococci, 7 Streptococcus viridans, 6 Streptococcus pneumoniae, 7 assorted Gram-positive isolates, and 11 assorted Gram-negative isolates were tested for minimum inhibitory concentrations to cefazolin, tobramycin, cefuroxime, gentamicin, and moxifloxacin using E-tests to determine susceptibility and potency. RESULTS: The in vitro coverage (susceptible to at least one antibiotic) of cefuroxime/gentamicin (97%) was statistically equal to cefazolin/tobramycin (93%) and moxifloxacin (92%) (P = 0.29). Double coverage (susceptible to both antibiotics) was equivalent (P = 0.77) for cefuroxime/gentamicin (42%) and cefazolin/tobramycin (40%). The susceptibilities of individual coverage were moxifloxacin (92%), gentamicin (89%), tobramycin (74%), cefazolin (58%), and cefuroxime (52%). Methicillin-resistant S. aureus was best covered by gentamicin 100% (9 of 9). Tobramycin was more potent (P = 0.00001) than gentamicin for P. aeruginosa, whereas cefazolin was more potent (P = 0.0004) than cefuroxime for S. aureus. CONCLUSIONS: Although there seems to be no in vitro empiric coverage advantage between cefazolin/tobramycin, cefuroxime/gentamicin, and moxifloxacin monotherapy, potency differences may occur and optimal treatment can best be determined with laboratory studies.

An ophthalmologist's guide to understanding antibiotic susceptibility and minimum inhibitory concentration data.

PURPOSE: To address the need to establish appropriate evaluation criteria for analyzing in vitro antibiotic susceptibility based on original data. DESIGN: In vitro laboratory investigation. PARTICIPANTS: Bacterial isolates from patients with conjunctivitis. MAIN OUTCOME MEASURES: Minimum inhibitory concentrations (MICs), descriptive statistics, antibiotic susceptibility, potency, and statistical analysis. METHODS: Minimum inhibitory concentrations were determined for 80 bacterial conjunctivitis isolates to moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin, and ofloxacin. Using the MIC values, descriptive statistics (median, MIC50, MIC90, mode, range), antibiotic susceptibility, and potency of each antibiotic were calculated for each bacterial group. The data were analyzed statistically using appropriate randomization and nonparametric tests. RESULTS: The descriptive statistics of gram-positive bacteria (Staphylococcus aureus, Streptococcus pneumoniae) followed a consistent trend where the median, MIC50, MIC90, and mode demonstrated the lowest values, in all instances, for moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin, and ofloxacin. The descriptive statistics for Haemophilus species (the predominant gram-negative bacteria implicated in conjunctivitis) did not describe any consistent trend. In contrast, antibiotic susceptibility testing did not demonstrate any advantage among the 5 fluorquinolones tested, except for moxifloxacin in the S. aureus fluoroquinolone-resistant group. Potency studies indicated that moxifloxacin and gatifloxacin were the most potent for gram-positive bacteria, whereas gatifloxacin and ciprofloxacin were the most potent for Haemophilus species. CONCLUSION: In the absence of human clinical trial data to guide care, in vitro susceptibility data should be analyzed with a set of descriptive statistics along with a nonparametric statistical analysis. No single parameter or test should be relied upon in all instances to demonstrate the in vitro superiority of one antibiotic over another. In this study, fourth-generation fluoroquinolones did have some potency advantages over second-generation fluoroquinolones against gram-positive conjunctival bacterial isolates, but not for Haemophilus isolates.

Gatifloxacin and moxifloxacin: an in vitro susceptibility comparison to levofloxacin, ciprofloxacin, and ofloxacin using bacterial keratitis isolates.

PURPOSE: We compared the in vitro susceptibility patterns and the minimum inhibitory concentrations (MICs) of gatifloxacin (GAT) and moxifloxacin (MOX) (fourth-generation fluoroquinolones) to ciprofloxacin (CIP) and ofloxacin (OFX) (second-generation fluoroquinolones) and levofloxacin (LEV; third-generation fluoroquinolone) using bacterial keratitis isolates. The goal was to determine whether the fourth-generation fluoroquinolones offer any advantages over the second- and third-generation fluoroquinolones. DESIGN: Experimental laboratory investigation. In contrast to an epidemiologic prevalence study, this study was designed to compare the relative susceptibility of each bacterial group to different fluoroquinolones by deliberate selection of representative isolates that were both susceptible and resistant to second-generation fluoroquinolones. METHODS: In retrospect, the MICs of 177 bacterial keratitis isolates were determined to CIP, OFX, LEV, GAT, and MOX using E tests. A relative susceptibility analysis was performed for each bacterial group that included separate bacterial groups that were resistant to second-generation fluoroquinolones. The NCCLS susceptibility patterns and the MICs were compared statistically. Comparing MICs, the antibiotic with the lower MICs has greater antibacterial activity. RESULTS: For most keratitis isolates, there were no susceptibility differences among the five fluoroquinolones. The fourth-generation fluoroquinolones did, however, demonstrate increased susceptibility for Staphylococcus aureus isolates that were resistant to CIP, LEV and OFX. In general, CIP demonstrated the lowest MICs for gram-negative bacteria. The MICs for fourth-generation fluoroquinolones were statistically lower than the second-generation fluoroquinolones for all gram-positive bacteria tested. Comparing the two fourth-generation fluoroquinolones, MOX demonstrated lower MICs for most gram-positive bacteria, whereas GAT demonstrated lower MICs for most gram-negative bacteria. CONCLUSIONS: Based on in vitro testing, the fourth-generation fluoroquinolones may offer some advantages over those currently available for the treatment of bacterial keratitis. Clinical studies will be required to confirm these results.

Infectious disease: changing antibiotic susceptibility.

The field of ophthalmology is fortunate to have an array of antibiotics to treat bacterial infections. Because many of the older antibiotics are no longer useful for treating systemic infections, their use and associated acquired resistance have been reduced. These antibiotics, therefore, likely will continue to be effective for treating ophthalmic infections. The bacteria that cause recurrent infections (e.g., blepharitis) may acquire antibiotic resistance because of the repeated use of one particular agent for therapy (e.g., erythromycin). Recurrent pathologies and those that are treated with antibiotics that have varied broadspectrum activities should be cultured routinely to confirm infection and to institute appropriate therapy. Resistant trends of Staphylococcus aureus to the second-generation fluoroquinolones have been confirmed, and new trends of resistance for Pseudomonas aeruginosa have emerged. These antibiotics are effective but should be used judiciously to avoid bacterial resistance to them and to ensure their future potency.

ELVIS: a new 24-hour culture test for detecting herpes simplex virus from ocular samples.

OBJECTIVE: To compare ELVIS (Enzyme Linked Virus Inducible System) (BioWhittaker, Walkersville, Md), a new, simple, 24-hour cell culture test for detecting herpes simplex virus (HSV), with standard cell culture and Herpchek (NEN, Boston, Mass) for detecting HSV in ocular specimens. METHODS: Retrospectively, 36 true-positive frozen-stock ocular samples that were cell-culture positive for HSV, and 25 true-negative samples (varicella-zoster virus, adenovirus, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus viridans) were tested with ELVIS. Herpchek was processed at the time of initial clinical laboratory testing. Prospectively, 422 patients were tested for HSV with standard cell culture, ELVIS, and Herpchek. The sensitivity, specificity, positive and negative predictive values, and efficacy of ELVIS based on positive and negative cell cultures were determined. RESULTS: Retrospectively, ELVIS was 86.1% sensitive (31/36), 100% specific (25/25), and 91.8% efficient (56/61). The positive predictive value was 100% (31/31), and the negative predictive value was 83.3% (25/30). The sensitivity of ELVIS was equivalent to Herpchek (80.5%, 29/36) (P =.53). Prospectively, the sensitivity of ELVIS (84.8%, 28/33) was equivalent to that of Herpchek (84.8%, 28/33). CONCLUSIONS: ELVIS is an easy HSV diagnostic test that can provide faster positive culture results than standard cell culture, and it is equally sensitive but less time-consuming than Herpchek.

Fourth generation fluoroquinolones: new weapons in the arsenal of ophthalmic antibiotics.

PURPOSE: Fourth generation fluoroquinolones (FQs) will soon be introduced to ophthalmology. In this in vitro study, differences in the susceptibility patterns and the potencies of fourth generation FQs (gatifloxacin-GAT and moxifloxacin-MOX) were compared with third generation (levofloxacin-LEV) and second generation FQs (ciprofloxacin-CIP and ofloxacin-OFX). DESIGN: Experimental laboratory investigation. METHODS: In retrospect, the minimum inhibitory concentrations (MICs) of 93 bacterial endophthalmitis isolates were determined to CIP, OFX, LEV, GAT, and MOX using E-tests. The National Committee of Clinical Laboratory Standards (NCCLS) susceptibility patterns and the potencies of the MICs were statistically compared. RESULTS: With in vitro tests, Staphylococcus aureus isolates that were resistant to CIP and OFX were statistically most susceptible (P =.01) to MOX. Coagulase negative Staphylococci that were resistant to CIP and OFX were statistically most susceptible (P =.02) to MOX and GAT. Streptococcus viridans were more susceptible (P =.02) to MOX, GAT, and LEV than CIP and OFX. Streptococcus pneumoniae was least susceptible (P =.01) to OFX compared with the other FQs. Susceptibilities were equivalent (P =.11) for all other bacterial groups. In general, MOX was the most potent FQ for gram-positive bacteria (P =.05) while CIP, MOX, GAT, and LEV demonstrated equivalent potencies to gram-negative bacteria. CONCLUSIONS: This in vitro study indicated that fourth generation FQs appear to cover bacterial resistance to the second and third generation FQs, were more potent than the second and third generation FQs for gram-positive bacteria, and are equally potent for gram-negative bacteria. Clinical studies will need to confirm these results.