Rebecca L. Penland

Intravitreous Bevacizumab as Anti–Vascular Endothelial Growth Factor Therapy for Retinopathy of Prematurity: A Morphologic Study

O verexpression of vascular endothelial growth factor (VEGF) appears important in the pathogenesis of retinopathy of prematurity (ROP). Bevacizumab (Avastin; Genentech, Inc, South San Francisco, California) is a recombinant humanized monoclonal IgG1 antibody. It binds to and inhibits the biological activity of human VEGF. It has been estimated that more than 10 000 patients worldwide have been treated with intravitreous bevacizumab. We report results of a study in postmortem eyes with intravitreous bevacizumab treatment for zone 1, stage 2 ROP in an extremely low-birth-weight infant.

Comparison of the Etest and the NCCLS-approved agar dilution method to detect metronidazole and clarithromycin resistant Helicobacter pylori.

Although the NCCLS has approved the agar dilution method as the test of choice for antimicrobial susceptibility testing of Helicobacter pylori, a critical evaluation of this method in clinical trials to detect antibiotic resistance has not been performed. This study compares the Etest and agar dilution methods for detection of metronidazole and clarithromycin resistance in clinical isolates of H. pylori. MIC data were gathered from US-based clinical trials. The Etest was performed on Mueller-Hinton sheep blood agar plates following incubation for 4 days under 12% CO(2). The agar dilution test was performed according to the recently approved NCCLS methodology using aged sheep blood in a Mueller-Hinton agar base. Metronidazole resistance as determined by Etest was significantly higher than that determined by agar dilution (39%; 690/1768 vs. 25. 1%; 367/1465)(P<0.01). Clarithromycin resistance as determined by Etest was higher than that determined by agar dilution, but was not significantly different (12.5%; 209/1671 vs. 10.6%; 150/1414)(P>0.5). Inter-patient metronidazole resistance showed that the MIC values for identical isolates tested by both methods were equivalent in 58% (109/188). Of the 42% with a >2log(2) difference in MIC values, 17. 6% had a change in susceptibility pattern. For clarithromycin, 71.4% (237/332) of the MIC values for identical isolates tested by both methods had equivalent MIC values. Of the MIC values with a >2log(2) difference in MIC values, only 3% showed a change in susceptibility pattern. Intra-patient variability, i.e. paired isolates from the same patient, was assessed only for metronidazole. Of the 1393 paired isolates tested by Etest, 38.8% were shown to be resistant. Almost 69% of the Etest MIC determinations were deemed equivalent and 16.7% had a change in susceptibility pattern. Of the 639 paired isolates tested by agar dilution, 23.9% were resistant to metronidazole. Almost 72% of the agar dilution MIC values were equivalent and 11.3% of the determinations had a change in susceptibility pattern. Clarithromycin resistance rates are similar, when determined by either test method. The Etest yields a significantly higher prevalence of metronidazole resistance among H. pylori compared with the agar dilution method and both methods yield discordant results, when isolates from different parts of the same stomach are compared. Neither method is reliable in determining metronidazole resistance in H. pylori.

Effects of Helicobacter pylori infection on the link between regenerating gene expression and serum gastrin levels in Mongolian gerbils

Although regenerating gene (Reg) protein is reported to have a trophic effect on gastric epithelial cells, its involvement in human gastric diseases is not clear. We have recently shown that both gastrin and gastric mucosal inflammation enhance Reg gene expression in the fundic mucosa in rats. This study was designed to clarify whether Reg protein is involved in Helicobacter pylori–induced gastritis and whether Reg gene expression is linked to serum gastrin levels in this condition. Mongolian gerbils were inoculated with an H. pylori strain isolated from a gastric cancer patient. Four weeks later, some of the gerbils with H. pylori infection were eradicated by lansoprazole, amoxicillin, and clarithromycin. The time courses of changes in Reg gene expression, serum gastrin levels, gastric acidity, and histopathologic factors were examined. Four weeks after H. pylori infection, gastritis started spreading to the fundic mucosa, and gastric acidity started reducing. Serum gastrin levels and Reg mRNA expression in the fundus were significantly increased 6 weeks after infection. Reg mRNA expression in the fundus correlated significantly with both serum gastrin levels and the severity of fundic mucosal inflammation. After H. pylori eradication, serum gastrin levels and fundic mucosal inflammation were normalized, and the increase in Reg mRNA expression was abolished. The Reg gene is associated with hypergastrinemia and fundic mucosal inflammation and may be involved in H. pylori–induced gastritis.

Vibrio ocular infections on the U.S. Gulf Coast.

Purpose. To describe the epidemiology of Vibrio eye infections. Method. We reviewed the records of a patient from our institution with V. vulnificus keratitis and conducted a literature search for other cases of ocular infections with Vibrio species. Results. A 39-year-old fisherman was struck in his left eye with an oyster shell fragment, developed suppurative V. vulnificus keratitis, and was successfully treated with combined cefazolin and gentamicin. Including our patient, 17 cases of eye infections with Vibrio spp. have been reported, and 11 (65%) involved exposure to seawater or shellfish. Of the seven cases due to V. vulnificus (six keratitis and one endophthalmitis), six had known exposure to shellfish or seawater along the U.S. coast of the Gulf of Mexico. Of five cases of V. alginolyticus conjunctivitis, three had been exposed to fish or shellfish. Three infections with V. parahaemolyticus (one keratitis and two endophthalmitis) were reported; two of these occurred in people exposed to brackish water on or near the Gulf Coast. Two cases of postsurgical endophthalmitis, one with V. albensis and one with V. fluvialis, also were reported. Conclusions. In addition to septicemia, gastroenteritis, and wound infections, halophilic noncholera Vibrio species can cause sight-threatening ocular infections. Ocular trauma by shellfish from contaminated water is the most common risk factor for Vibrio conjunctivitis and keratitis. Nearly one half of reported Vibrio infections of the eye occurred along the U.S. coast of the Gulf of Mexico.

Microbiologic analysis of bottled water: is it safe for use with contact lenses?

OBJECTIVE To analyze commercially available bottled water as a possible source of microbial contamination of contact lenses. METHODS Two different lots of 23 brands of noncarbonated bottled water were tested for coliforms, total bacteria, fungi, and free-living amebae. A sample consisted of three separate 100-ml aliquots from one lot of each brand (46 samples). Aliquots were vacuum-filtered using a 0.45-microm Nalgene analytical filter unit, and the membrane filter was placed on a filter pad in a Petri dish containing test medium. Plates were examined under a stereomicroscope, and the number of colony-forming units (CFUs) was calculated for each sample. To test for the presence of free-living amebae, three aliquots totaling approximately 3800 ml were concentrated using 8-microm filters, and the filters were placed on non-nutrient agar with live Enterobacter aerogenes. To assess the possibility of contaminating contact lenses, etafilcon lenses were rinsed in 2-ml aliquots of four brands of bottled water and then cultured. RESULTS Seventeen (37%) of 46 samples, representing 11 (48%) of 23 brands, contained viable micro-organisms. Bacteria, including coliforms, were recovered from 12 samples of 8 brands. Yeasts or molds were recovered from seven samples of five brands. Free-living amebae were isolated from two samples, and fresh-water algae were found in both samples of one brand. Nine (20%) of 46 samples, representing 7 (30%) of the 23 brands, had more than 500 CFUs per ml or contained coliforms. Sterile contact lenses became contaminated when exposed for 1 minute to two of four brands of water from which micro-organisms were recovered. CONCLUSION Some bottled waters contain high numbers of potential ocular pathogens. Bottled water is not safe for routine use with contact lenses.

Helicobacter pylori infection in patients with Hashimoto's thyroiditis

Recent studies have suggested a role for some infectious agents, including Cytotoxin-associated gene A (CagA)-positive strains of H. pylori, in the pathogenesis of Hashimoto’s thyroiditis (HT), an autoimmune disorder of the thyroid gland defined by the detection of antibodies against thyroglobulin (anti-Tg) and thyroperoxidase (anti-TPO) [1–5]. To verify the possible role of H. pylori in HT we have designed a pilot study with two complementary aims: 1, to compare the prevalence of H. pylori infection (with Cag-positive and -negative strains) in patients with HT and in subject with no evidence of thyroid disease; and 2, to explore the possibility of a molecular mimicry between either anti-Tg or anti-TPO antibodies and any antigenic constituent of H. pylori. Sixteen patients (two men and 14 women, mean age 43.6 ± 11 years) with HT were evaluated for H. pylori infection by 13C urea breath test. Sera from all patients were tested for specific anti-CagA immunoglobulin G (IgG) using a commercial enzyme-linked immunosorbent assay (ELISA) (Radim, Pomezia, Italy). A control group of 20 blood donors (two men and 18 women, mean age 44.2 ± 12 years) without HT was also evaluated. Levels of anti-Tg and antiTPO antibodies in equal amounts of serum were evaluated through immunofluorescence either at enrollment or after absorption [6] with CagA-positive and CagA-negative H. pylori strains as well as with other Gram-negative bacteria, such as Campylobacter jejuni, Pseudomonas aeruginosa, Klebsiella spp. and Escherichia coli. Overall, 37.5% of the patients (six of 16; five women and one man; mean age 47 ± 12 years) with HT had H. pylori infection, compared to 35% of the controls (p > .05). Among infected subjects, 50% of the patients and 45% of the controls were infected by CagA-positive strains (p > .05). None of the absorbance tests with any of the bacteria induced significant changes in the levels of either anti-Tg or anti-TPO antibodies in the sera of patients with HT. The similar prevalence of H. pylori-infection (with and without CagA-positive strains) in patients and controls, as well as the lack of effect of the in vitro absorbance test on the relevant serum antibody titers, indicates that, in contrast to previous observations, an association between H. pylori infection and HT is unlikely. To further explore this relationship, we are currently conducting a clinical trial based on the evaluation of anti-Tg and anti-TPO antibody titers before and after eradication of H. pylori. Another area that may deserve further investigation is the possible cross-reactivity between anti-CagA antibodies and thyroid antigens other than TPO and Tg.

Colorimetric indicators of microbial contamination in corneal preservation medium.

Purpose. To compare acid-base and oxidation-reduction indicators and to investigate the effect of buffer and temperature on the colorimetric detection of microbial growth in corneal preservation media. Methods. Corneal preservation media containing gentamicin, without or with HEPES buffer, were prepared with either phenol red or AlamarBlue indicators (AccuMed International, Westlake, OH, U.S.A.). Both media were inoculated with Staphylococcus aureus, Streptococcus sanguis, Pseudomonas aeruginosa, Serratia marcescens, or Candida albicans and then incubated at 4°C, 22°C, or 35°C. The pH or percent reduction were determined hourly for eight hours, then daily for one week. Results. The length of time before a confirmed change in pH or reduction occurred varied by microorganism, storage temperature, and buffering capacity. At 4°C, none of the microorganisms caused a detectable pH change in buffered medium within one day after inoculation, although two bacterial species reduced AlamarBlue within four hours. At 22°C and 35°C, all bacteria except P. aeruginosa produced a pH shift within a few hours, and all tested bacterial species reduced AlamarBlue. For bacteria producing detectable pH changes, HEPES-buffered medium took longer to change than medium without HEPES. C. albicans was not detectable in HEPES-buffered medium at any temperature by phenol red and was only detectable by AlamarBlue after 2–3 days at 22°C and 35°C. Conclusion. Acidic shifts in refrigerated corneal preservation medium do not occur during contamination by several microorganisms. AlamarBlue, a redox indicator, is more sensitive than phenol red in detecting some bacteria. C. albicans is not reliably detected by pH or redox indicators.

EMERGENCE OF PENICILLIN-RESISTANT STREPTOCOCCUS PNEUMONIAE OCULAR INFECTIONS

BACKGROUND Approximately 15-20% of Streptococcus pneumoniae clinical isolates in the United States are not susceptible to penicillin. Isolates with a minimum inhibitory concentration (MIC) of 0.12-1.0 mg/ml are intermediately resistant, and those with an MIC > 2.0 microg/ml are classified as having high-level penicillin resistance. PURPOSE We determined the proportion of penicillin-resistant S. pneumoniae recovered from ocular and periocular infections from 1976 through 1995, compared these cases with penicillin-susceptible controls, and evaluated the susceptibility of penicillin-resistant isolates to selected cephalosporins and fluoroquinolones. METHODS MICs for cephalothin, ceftazidime, ciprofloxacin, and ofloxacin were determined for available isolates by the E test. We performed a case-comparison study to evaluate differences between patients with penicillin-susceptible and -nonsusceptible ocular pneumococcal infections. RESULTS Of 173 ocular isolates of S. pneumoniae isolated in the 20-year period, 12 (7%) were not susceptible to penicillin, including eight (5%) intermediate isolates and four (2%) highly resistant isolates. Penicillin-intermediate and -resistant pneumococci were recovered at a rate of none of 46 isolates from 1976 through 1980, one (4%) of 25 from 1981 through 1985, one (2%) of 51 from 1986 through 1990, and 10 (20%) of 51 from 1991 through 1995 (p < 0.0004). We found no significant differences in presenting characteristics between patients with ocular infections due to penicillin-susceptible pneumococci and those caused by nonsusceptible strains. All retested isolates with intermediate susceptibility to penicillin had a cephalothin MIC < or = 1.5 microg/ml, and all retested isolates with high-level penicillin resistance had a cephalothin MIC > or = 4 microg/ml. The ceftazidime MIC for all penicillin-resistant isolates was > or = 24 microg/ml. All penicillin-nonsusceptible isolates had MICs < or = 1.5 microg/ml for ciprofloxacin and < or = 3 microg/ml for ofloxacin. CONCLUSION Penicillin resistance has recently emerged among ocular S. pneumoniae. Cephalothin, ciprofloxacin, and ofloxacin have good activity against some ocular isolates with intermediate penicillin susceptibility, although another agent such as vancomycin may be needed for pneumococci with high-level penicillin-resistance.

Laboratory diagnosis of Acanthamoeba keratitis using buffered charcoal-yeast extract agar

PURPOSE To evaluate the use of buffered charcoal-yeast extract agar for the isolation of Acanthamoeba from clinical specimens. METHODS We retrospectively reviewed laboratory records of patients with ocular acanthamebic infection from October 1993 to September 1997 to compare the recovery of Acanthamoeba from clinical specimens inoculated onto various media. We then compared the experimental recovery of 10 corneal isolates of Acanthamoeba on buffered charcoal-yeast extract and blood agars. RESULTS Paired data for buffered charcoal-yeast extract and blood agars were available from 24 cultures performed in 13 cases of ocular acanthamebic infection. Acanthamebic trails were detected on both buffered charcoal-yeast extract and blood agars in nine cultures, only on buffered charcoal-yeast extract agar in nine cultures, and only on blood agar in one culture (P = .027). In the experimental study, all 10 clinical isolates produced trails on buffered charcoal-yeast extract agar, and the mean recovery after 10 days of incubation ranged from 38% to 95% of the original inoculum number. For seven of the 10 isolates, more than 70% of the original inoculum was recovered on buffered charcoal-yeast extract agar. Only two of the 10 strains produced persistent trails on the blood agar, and the mean recoveries after 10 days of incubation were 0.67% and 1.17%. Recovery was significantly better on buffered charcoal-yeast extract agar than blood agar (P < or = .0003). CONCLUSION Buffered charcoal-yeast extract agar is an excellent commercially available culture medium for the recovery of Acanthamoeba.

Use of an antimicrobial removal device in endophthalmitis cultures.

PURPOSE This study sought to determine whether the use of an antimicrobial removal device (ARD) to process intraocular fluids increases microbial detection compared with conventional cultures. METHODS The authors retrospectively reviewed all cases of endophthalmitis submitted to their laboratory from January 1982 through December 1996. Aqueous or vitreous specimens or both that were cultured on conventional media (blood agar, chocolate agar, anaerobic blood agar, and thiol broth) and by ARD processing were included in the study. Specimens were inoculated into tubes with ARD for 5 to 10 minutes. The fluid was then withdrawn and cultured using conventional media; thioglycolate broth was added to the tube containing the resin beads. The conventional and ARD-processed cultures were incubated at 35 degrees C for at least 7 days. RESULTS Of the 338 endophthalmitis cultures processed using both conventional cultures and a parallel ARD, 166 (49.1%) yielded positive microbial growth on one or more media. Of the 166 culture-confirmed cases, 127 (76.5%) were positive in both the ARD-processed and direct cultures, 17 (10.2%) were positive by conventional culture only, and 22 (13.3%) were positive by the ARD-processed sample alone (P = 0.52). The spectrum of microorganisms was similar among all culture groups. The detection of coagulase-negative staphylococci and micrococci by ARD alone was slightly better than detection by conventional culture only (P = 0.06). Of 93 positive cultures from 179 patients in whom prior antibiotic use was documented, 75 (80.6%) were positive by both methods, 8 (8.6%) by conventional cultures only, and 10 (10.8%) only by the ARD-processed specimen (P = 0.81). CONCLUSION Use of an antimicrobial removal device does not significantly increase the microbial yield of endophthalmitis cultures compared with conventional culture techniques, whether or not antimicrobial therapy is being used.