Molecular Epidemiology of Adenoviral Conjunctivitis in Hanoi, Vietnam

TABLE. Effect of Climatic Conditions at Procurement on Relative Risk of Candidal Postkeratoplasty Endophthalmitis Compared With Other Microbial Results

Weather at Eye Donation

Outdoor temperature ⬍45 F 45–60 F 61–75 F ⬎75 F Relative humidity Low (⬍40%) Medium (40%–70%) High (⬎70%)

Endophthalmitis (n)*

Age-Adjusted Odds Ratio (95% CI)†

P Value

33 (21.9%) 45 (29.8%) 41 (27.2%) 32 (21.2%)

1.0 (Referent) 3.42 (0.67, 17.4) 4.31 (0.86, 21.6) 17.2 (3.51, 84.7)

— .14 .08 ⬍.001

32 (21.2%) 83 (55.0%) 36 (23.8%)

1.0 (Referent) 0.80 (0.32, 2.00) 0.66 (0.21, 2.06)

— .63 .47

CI ⫽ confidence interval. *Excluding one case of filamentous fungal endophthalmitis with an afternoon temperature of 85 F at donation. † Hosmer-Lemeshow goodness-of-fit probability value of .87 for temperature comparison.

To investigate the serotypes of adenovirus causing conjunctivitis in Hanoi, Vietnam. DESIGN: Clinical diagnosis of adenoviral conjunctivitis and laboratory-based experimental study. METHODS: We collected 21 conjunctival swabs from 21 different patients with a clinical presentation compatible with adenoviral conjunctivitis, in Hanoi, Vietnam. Immunochromatography and real-time polymerase chain reaction (PCR) were performed to detect human adenovirus (HAdV). The sequence of PCR products was analyzed to determine the serotype of HAdV. RESULTS: Of 21 samples, HAdV DNA was detected in 14 samples (66.7%) by real-time PCR. The serotype analysis showed HAdV-8 in 11 samples (78.6%), HAdV-3 in two samples (14.3%), and HAdV-37 in one sample (7.1%). Of 11 HAdV-8 samples, one sample (9.1%) was prototype, and the other 10 samples (90.9%) had identical nucleotide sequence and were identified as a variant of HAdV-8. CONCLUSIONS: HAdV-8 was found to be the predominant serotype in Hanoi, Vietnam. Most of the HAdV-8 samples were a variant of HAdV-8. (Am J Ophthalmol 2006;142:1064 –1066. © 2006 by Elsevier Inc. All rights reserved.)

REFERENCES

PURPOSE:

1. Andrew SE, Nguyen A, Jones GL, Brooks DE. Seasonal effects on the aerobic bacterial and fungal conjunctival flora of normal thoroughbred brood mares in Florida. Vet Ophthalmol 2003;6: 45–50. 2. Rubio EF. Climatic influence on conjunctival bacteria of patients undergoing cataract surgery. Eye 2004;18:778 –784. 3. Wilhelmus KR. Climatology of dematiaceous fungal keratitis. Am J Ophthalmol 2005;140:1156 –1157. 4. Hassan SS, Wilhelmus KR. Eye-banking risk factors for fungal endophthalmitis compared with bacterial endophthalmitis after corneal transplantation. Am J Ophthalmol 2005;139:685– 690. 5. Merchant A, Zacks CM, Wilhelmus K, Durand M, Dohlman CH. Candidal endophthalmitis after keratoplasty. Cornea 2001;20:226 –229. 6. Al-Assiri A, Al-Jastaneiah S, Al-Khalaf A, Al-Fraikh H, Wagoner MD. Late-onset donor-to-host transmission of Candida glabrata after corneal transplantation. Cornea 2006;25:123–125.

Molecular Epidemiology of Adenoviral Conjunctivitis in Hanoi, Vietnam Xue-Hai Jin, MD, Hiroaki Ishiko, PhD, Nguyen Thanh Ha, MD, PhD, Takeshi Ohguchi, MD, Masataka Akanuma, MD, Koki Aoki, MD, PhD, and Shigeaki Ohno, MD, PhD

E

Accepted for publication Jul 18, 2006. From the Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Sapporo, Japan (X.-H.J., T.O., M.A., K.A., S.O.); Research and Development Department, Mitsubishi Kagaku Bio-Clinical Laboratories, Inc, Tokyo, Japan (H.I.); and Department of Ophthalmology, Friendship Hospital, Hanoi, Vietnam (N.T.H.). Inquiries to Xue-Hai Jin, MD, Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan; e-mail: xhjin@med. hokudai.ac.jp

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PIDEMIC OUTBREAK OF CONJUNCTIVITIS OFTEN AP-

pears at different locations of the world. Human adenoviruses (HAdV) are one of the leading causes of acute conjunctivitis seen in daily practice.1 HAdV are divided into six major subtypes, and 51 different serotypes have been identified. HAdV-4, 8, 19, and 37 are commonly responsible for the epidemic keratoconjunctivitis. Rapid identification of these serotypes is helpful in the prevention and control of the epidemics.

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TABLE 1. Result of Real-Time PCR and Immunochromatography Test for Detection of HAdV in the Patients With Adenoviral Conjunctivitis Presentation in Hanoi, Vietnam (n ⫽ 21)

TABLE 2. Copy Number and Serotype of HAdV in Adenoviral Conjunctivitis Samples Collected in Hanoi, Vietnam Sample Number

Copy Number/ml

Serotype

⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻

8 8v 8v 8v 37 8v 3 8v 8v 8v 8v 8v 8v 3

Immunochromatography Test Real-Time PCR

Positive (%)

Negative (%)

Positive (%) Negative (%)

13 (61.9) 0 (0)

1 (4.8) 7 (33.3)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

PCR ⫽ polymerase chain reaction; HAdV ⫽ human adenovirus.

Between June and November 2003, 21 conjunctival swabs were collected from 21 different patients with a clinical presentation compatible with adenoviral conjunctivitis in Hanoi, Vietnam. All patients had given informed consent, according to the Declaration of Helsinki. The swabbed samples were placed in transport media for immunochromatography and deoxyribonucliec acids (DNA) extraction. The immunochromatography test (Adenocheck; Santen, Inc, Osaka, Japan) was carried out according to the manufacturer’s instructions. Viral DNA was extracted by using the Sumitest EX-R&D kit (Genome Science Laboratories Co, Ltd, Fukushima, Japan). To quantify the adenoviral gene, real-time polymerase chain reaction (PCR) was performed on LightCycler instrument (Roche Diagnostics, Mannheim, Germany). The primers were designed to amplify a 554-bp fragment of adenoviral hexon gene. The sequence of PCR products was analyzed and part of it (350 bp) was compared with the database as described.2 Reverse transcription-PCR was performed to amplify enterovirus genome including the entire VP4 region as described.3 Of 21 samples, HAdV DNA was detected by real-time PCR in 14 samples (66.7%), in which 13 samples (61.9%) were also positive for adenovirus by immunochromatography test (Table 1). All the samples were negative for enterovirus by reverse transcription-PCR. The HAdV gene in real-time PCR positive samples was quantified and sequenced. The copy number and serotype of HAdV in conjunctival swabs are shown in Table 2. The copy number of HAdV in immunochromatography test positive samples ranged from 9.6 ⫻ 106 to 1.7 ⫻ 109/ml. The copy number of HAdV in that immunochromatography test was negative but real-time PCR was positive was 3.1 ⫻ 105/ml. The serotype analysis of 14 real-time PCR-positive samples showed HAdV-8 in 11 samples (78.6%), HAdV-3 in two samples (14.3%), and HAdV-37 in one sample (7.1%). Of 11 HAdV-8 samples, one sample (9.1%) was prototype, and the other 10 samples (90.9%) had identical nucleotide sequence and were identified as a variant of HAdV-8. Adenoviral conjunctivitis and enteroviral conjunctivitis are under the National Epidemiological Surveillance of Infectious Diseases, Japan, and are common in Japan. VOL. 142, NO. 6

1.8 1.7 9.6 1.7 4.9 8.9 4.6 4.7 6.8 2.2 1.6 9.4 7.6 3.1

107 108 106 109 107 108 107 107 108 108 109 108 108 105

8v ⫽ a variant of HAdV-8; HAdV ⫽ human adenovirus.

Because of recent active interchange of people among countries, it is important to obtain information on viral conjunctivitis in various foreign countries, especially in the Asian area.4 In this study, the epidemiology of adenoviral conjunctivitis in Hanoi, Vietnam, was investigated. Of 21 samples, HAdV DNA was detected in 14 samples (66.7%) and HAdV-8 was found to be the predominant serotype in this region. This result was different from that of Japan where HAdV-37 was a dominant serotype in recent years. The result shows that dominant serotype of HAdV changes geographically. Of 11 HAdV-8 samples, 10 samples (90.9%) showed the same mutation and were identified as a variant of HAdV-8. Further phylogenetic analysis is needed to identify whether it is a new genomic variant. Immunochromatography has been regarded as a rapid and easy test to detect HAdV antigen with high specificity. HAdV DNA was detected in all the immunochromatography positive samples, which testified the high specificity of immunochromatography. HAdV DNA was detected only in one immunochromatography-negative sample. The result of real-time PCR showed the copy number of HAdV in this sample was far less than that of immunochromatography-positive samples. It seems that the sensitivity of immunochromatography is related to the quantity of HAdV in the sample. In this study, we investigated the molecular epidemiology of adenoviral conjunctivitis in Hanoi, Vietnam, by the combination of real-time PCR and sequencing. HAdV-8 was found to be the dominant serotype in this region. Most of the HAdV-8 samples were identified as a variant of HAdV-8, which needs further phylogenetic analysis.

BRIEF REPORTS

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REFERENCES

1. Kinchington PR, Turse SE, Kowalski RP, Gordon YJ. Use of polymerase chain amplification reaction for the detection of adenoviruses in ocular swab specimens. Invest Ophthalmol Vis Sci 1994;35:4126 – 4134. 2. Shimada Y, Ariga T, Tagawa Y, et al. Molecular diagnosis of human adenoviruses d and e by a phylogeny-based classification method using a partial hexon sequence. J Clin Microbiol 2004;42:1577–1584. 3. Ishiko H, Shimada Y, Yonaha M, et al. Molecular diagnosis of human enteroviruses by phylogeny-based classification by use of the VP4 sequence. J Infect Dis 2002;185:744 –754. 4. Sawada H, Aoki K, Kawana R, et al. Molecular epidemiology of adenoviral conjunctivitis in Sapporo, Japan, and Manila, the Philippines. Jpn J Ophthalmol 1987;31:538 –546.

Analysis of Bacterial Cultures in Infectious Keratitis, 1997 to 2004 David L. Yeh, MD, Sandra S. Stinnett, DrPH, and Natalie A. Afshari, MD PURPOSE: To review bacterial culture results in infectious keratitis at a tertiary referral center. DESIGN: Retrospective case series. METHODS: Analysis of Gram stain, bacterial culture, and targeted antibiotic sensitivities of all cases of presumed infectious keratitis at Duke University Eye Center from 1997 to 2004 (n ⴝ 453). RESULTS: Cultures were positive in 307 cases (68%); 21% demonstrated polymicrobial growth. Among 388 isolates, 81% were Gram-positive. Gram stain results were available in 334 cases, demonstrating 18% sensitivity and 94% specificity. Antibiotic sensitivities revealed 24% resistance of tested S. aureus isolates to cefazolin, and <5% resistance of tested gram-negative isolates to various antibiotics, with no statistically significant increase from 1997 to 2004. CONCLUSIONS: In this study, the microbial profile has remained stable from 1997 to 2004. Gram stain studies were of poor sensitivity, while culture yields were significantly higher and demonstrated a relatively high polymicrobial infection rate. Increasing antibiotic resistance was not clearly demonstrated. (Am J Ophthalmol 2006;142: 1066 –1068. © 2006 by Elsevier Inc. All rights reserved.)

Accepted for publication Jun 20, 2006. From the Department of Ophthalmology, Duke University Eye Center, Duke University Medical Center, Durham, North Carolina. Supported by a Research to Prevent Blindness, New York, New York (N.A.A.). Inquiries to Natalie A. Afshari, MD, Associate Professor of Ophthalmology, Duke University Eye Center, DUMC Box 3802, Durham, NC 27710; e-mail: [email protected]

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tious keratitis, including empiric treatment with monotherapy or multiple fortified antibiotics, and Gram stain/culture guided treatments.1,2 Because of potential rapid progression of disease and devastating visual outcomes, early and effective treatment is recommended. To guide therapy, it is important to be familiar with current microbial patterns, yields of laboratory studies, and emerging antibiotic resistance of common pathogens. Following institutional review board (IRB) approval, a database search was obtained for all cases of presumed infectious keratitis at Duke University Eye Center from 1997 to 2004 for which bacterial culture results were available (n ⫽ 453). No exclusions were made for prior eye surgery, contact lens wear, or prior initiation of antibiotic therapy. Laboratory results were recorded, including, when available, Gram stain, bacterial culture, and antibiotic sensitivities by the Kirby-Bauer disk diffusion method for recovered organisms. Cultures were obtained with calcium alginate swabs with inoculation of blood, chocolate, and Sabouraud’s agar and thioglycolate broth. Any growth was considered an isolate and no exclusions were made for possible contaminants. Statistical analysis was performed using the Cochran-Mantel-Haenszel ␹2 test with modridit scoring. The data were grouped by pairs of years (Tables 1, 2, and 3). Cultures were positive in 307 of 453 (68%), with no significant difference in this percentage across the years (P ⫽ .86). Of note, among positive cultures, 66 of 307 (21%) demonstrated polymicrobial growth. Of 388 total isolates, 81% were Gram-positive and 19% were Gramnegative, with no significant difference across the years (P ⫽ .81). Common isolates were: coagulase-negative Staphylococcus (39%), S. aureus (12%), Pseudomonas species (10%), nonpneumococcal Streptococcus species (10%), Propionibacterium species (9%), and S. pneumoniae (5%). Although distributions fluctuated from year to year, there were no significant trends (P ⫽ .45). Gram stain results, when available (334 of 453 cases), were compared with culture results to determine their yield, with the following definitions: true positive: positive Gram stain correlating with cultured organisms; false positive: positive Gram stain not correlating with cultured organisms; true negative: negative Gram stain correlating with negative culture; and false negative: negative Gram stain despite positive culture. Based on these, an overall sensitivity of 18%, specificity of 94%, positive predictive value of 85%, and negative predictive value of 36% were calculated. Targeted antibiotic sensitivity testing of select organisms was performed according to Duke University Microbiological Laboratory protocol. Among S. aureus isolates tested against cefazolin, 11 of 45 (24%) were resistant. Although there was a trend of increasing resistance (23% in 1997 to 1998 vs 40% in 2003 to 2004), this was not significant (P ⫽ .32). Among cefazolin resistant isolates, OF

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