Hajib N Madhavan

Polymerase chain reaction in the diagnosis of bacterial endophthalmitis

BACKGROUND Microbiological investigations of vitreous fluid (VF) and aqueous humour (AH) specimens have often failed to detect the infecting agent in infectious endophthalmitis, resulting in a clinical dilemma regarding therapy. In this study, the polymerase chain reaction (PCR) was evaluated in the diagnosis of bacterial andPropionibacterium acnes endophthalmitis. METHODS 58 intraocular specimens (30 VF and 28 AH) from 55 cases of endophthalmitis and 20 specimens (14 VF and 6 AH) as controls from non-infective disorders were processed for microbiological investigations. Nested PCR directed at the 16S rDNA using universal primers for eubacterial genome was done. PCR for P acnes was performed on specimens microbiologically negative by conventional techniques but eubacterial genome positive. RESULTS Of the 20 controls from non-infective cases, one (5%) was positive using eubacterial primers and none withP acnes primers. PCR for eubacterial genome showed 100% correlation with 20 (34.5%) bacteriologically positive specimens. Eubacterial genome, was detected in 17 (44.7%) of 38 bacteriologically negative specimens and nine (52.9%) out of the 17 were positive for P acnes genome. Among the 21 eubacterial PCR negative specimens, seven were fungus positive. By inclusion of PCR, microbiologically positive specimens increased from 46.5% to 75.8%. PCR on AH was as sensitive as that on VF for the detection of both eubacterial and the P acnes genome. CONCLUSION PCR performed on AH and VF is a reliable tool for the diagnosis of bacterial and P acnesendophthalmitis particularly in smear and culture negative specimens.

Eales Disease—An Update

Eales disease, first described by Henry Eales in 1880, remains an enigma. The disease, observed more commonly in the Indian subcontinent than in the rest of the world, occurs in young healthy adult males, initially presenting as retinal periphlebitis and later as retinal ischemia that may lead to vascular alterations and neovascularization. Recurrent vitreous hemorrhage with or without retinal detachment is the common sequelae. In recent years, immunological, molecular biological, and biochemical studies have indicated the role of human leukocyte antigen, retinal autoimmunity, mycobacterium tuberculosis genome, and free radical mediated damage in the etiopathogenesis of this disease. However, its etiology appears to be multifactorial. The management depends on the stage of the disease and consists of medical treatment with oral corticosteroids in the active inflammatory stage and laser photocoagulation in the advanced retinal ischemia and neovascularization stages. The results of vitreoretinal surgery have been found to be satisfactory in case of vitreous hemorrhage with or without retinal detachment.

Use of polymerase chain reaction in detection of Mycobacterium tuberculosis complex DNA from vitreous sample of Eales' disease

Editor,—Eales’ disease, first described by Henry Eales in 1880 is a primary retinal perivasculitis that predominantly affects the peripheral retina of young and otherwise healthy adults in the age group 15–40 years. Of the several aetiologies proposed, most favoured are tuberculosis and hypersensitivity to tuberculoprotein.1 Since polymerase chain reaction (PCR) using primers for the insertion sequence of IS6110 consisting of upstream primer: 5′ CCTGCGAGCGTAGGCGT CGG3′ and downstream primer: 5′CTC GTCCAGCGCCGCTTCGG 3′ …

Use of polymerase chain reaction in the diagnosis of fungal endophthalmitis

OBJECTIVE To evaluate the usefulness of polymerase chain reaction (PCR) compared with the conventional mycologic methods in the diagnosis of fungal endophthalmitis. DESIGN Prospective comparative validation of diagnostic testing-case-control study. PARTICIPANTS AND CONTROLS Thirty subjects in whom fungal endophthalmitis was suspected and 20 controls with noninfections. INTERVENTION TESTING: Collection of intraocular specimens and testing for the presence of fungus by PCR and conventional methods. MAIN OUTCOME MEASURES Detection of fungus by microscopy, growth by culture, and fungal DNA by PCR for definitive and rapid diagnosis of fungal endophthalmitis. RESULTS None of the controls was positive by microscopy, culture, or PCR. Among the 43 intraocular specimens from 30 subjects, 24 were positive by conventional mycologic methods and 32 were positive by PCR. PCR increased the sensitivity of detection by 18.6%, which was statistically significant (McNemar test: P = 0.039). CONCLUSIONS PCR was a more sensitive and rapid diagnostic tool compared with the conventional mycologic methods in the diagnosis of fungal endophthalmitis.

In-vitro susceptibility testing by agar dilution method to determine the minimum inhibitory concentrations of amphotericin B, fluconazole and ketoconazole against ocular fungal isolates

PURPOSE To standardize in-vitro antifungal susceptibility testing by agar dilution method to find out the minimum inhibitory concentration (MIC) of amphotericin B, fluconazole and ketoconazole on ocular fungal isolates. METHODS A total of 180 ocular fungal isolates (130 filamentous fungi and 50 yeasts) were included. The antifungal drugs such as amphotericin B (0.0625-8 microg/mL), fluconazole (0.2-819.6 microg/mL) and ketoconazole (0.025-6.4 microg/mL) were incorporated in doubling dilutions in the yeast nitrogen base medium. The MIC was determined as the lowest concentration of the antifungal drug preventing growth of macroscopically visible colonies on drug containing plates when there was visible growth on the drug-free control plates. RESULTS All 50 ocular isolates of yeast were susceptible to amphotericin B, while two (4%) and five (10%) strains were resistant to fluconazole and ketoconazole respectively. Of the 130 filamentous fungi tested, six (4.6%) were resistant to amphotericin B, 49 (37.7%) and 10 (7.6%) were resistant to fluconazole and ketoconazole respectively. Percentile 50 (MIC 50) and Percentile 90 (MIC 90) for all the three antifungal agents were calculated. Aspergillus niger, Aspergillus terreus and Candida krusei were found to be resistant to fluconazole and ketoconazole. CONCLUSION This technique was found to be reliable, cost effective and easy to perform with consistent results.

Antibiotic susceptibility pattern of rapidly growing mycobacteria.

BACKGROUND The rapidly growing mycobacteria (RGM) causing human infections primarily consist of the Mycobacterium fortuitum group, Mycobacterium abscessus and Mycobacterium chelonae. The antibiotic susceptibility testing is important to determine the appropriate therapy as the antibiotics used to treat RGM are different from those used for treating infections caused by slow growers of mycobacteria. AIM To determine antibiotic susceptibility of RGM using Kirby Bauer method and following Clinical and Laboratory Standards Institute (CLSI) guidelines. SETTINGS AND DESIGN Larsen and Toubro Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, Retrospective study. MATERIALS AND METHODS The antibiotic susceptibility testing was performed following CLSI method for the drugs Amikacin, Azithromycin, Tobramycin, Ceftazidime, Cephotaxime, Cefuroxime, Cefaperazone, Ceftriaxone, Ciprofloxacin, Ofloxacin, Norfloxacin, Gatifloxacin and Moxifloxacin. RESULTS AND CONCLUSIONS Out of the 148 RGM isolates 146 (98%) were susceptible to amikacin, 138 (91%) to gatifloxacin, 132 (87%) to moxifloxacin, 122 (76%) to ciprofloxacin and 116 (74%) to Norfloxacin. Majority of the RGM were resistant to Ceftazidime, Cephotaxime and Cefaperazone. All the M. abscessus isolates were resistant to tobramycin. The in vitro antibiotic susceptibility testing by disc diffusion method showed that majority of the RGM were sensitive to Amikacin followed by Gatifloxacin, Moxifloxacin and Ciprofloxacin.

Association of herpesviruses in the aqueous humor of patients with serpiginous choroiditis: a polymerase chain reaction-based study.

Purpose: To determine the presence of herpesvirus DNA in the aqueous humor (AH) of patients with serpiginous choroiditis using polymerase chain reaction (PCR). Methods: AH from nine patients previously diagnosed with serpiginous choroiditis were investigated for herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) by conventional virological methods and PCR. The PCR-positive DNA was gel-purified, extracted, and sequenced using a dye-based Applied Biosystems procedure. The sequences were processed through the National Cancer Institute’s BLAST inquiry for species identification. Results: Culture and cytological examination of AH from all nine patients were negative for HSV,VZV, and CMV. Five were positive for VZV, one was positive for HSV, and three were wholly negative using PCR. Subsequent DNA sequencing of the positive samples authenticated the presence of VZV and HSV DNA in the respective patients. Conclusion: VZV and HSV DNA were detected in a subset of patients with serpiginous choroiditis, suggesting that these viruses may function in the pathogenesis of this disease.

A study on the incidence, microbiological analysis and investigations on the source of infection of postoperative infectious endophthalmitis in a tertiary care ophthalmic hospital: an 8-year study.

Background: The objective of the study was the determination of the incidence of culture-proven postoperative endophthalmitis and probable sources of infection. Materials and Methods: It was a prospective study on the microbiology, incidence and probable sources of infection in patients with postoperative infectious endophthalmitis carried out in a tertiary care eye hospital. Consecutive patients diagnosed with postoperative infectious endophthalmitis during the years 2000-2007 were investigated for the causative infective agent and possible sources of infection. The surgical data and microbiological data including the investigations performed to trace the source were recorded in a specific formatted form and were gathered and compiled for analysis. Results: Data of analysis showed that 98 (0.042%) out of 2,31,259 patients who underwent intra-ocular surgery developed infectious endophthalmitis. Among these, 70 (0.053%) occurred after cataract, 10 (0.5%) after penetrating keratoplasty (PK) and 18 (0.018%) following other types of intra-ocular surgeries. The predominant infectious agents isolated were bacteria (89.7%), with equal proportions of Gram-positive and Gram-negative bacteria. Polymicrobial infection was noted in four and fungi in seven patients. Occurrence of postoperative endophthalmitis was sporadic and not related to any specific part of period in a year. Sources of infection were donor corneal rim in six post-PK patients and phaco probe in one who had postphacoemulsification endophthalmitis Conclusions: Overall incidence of postoperative endophthalmitis over an 8-year period was quite low. The sources of infection could be established in six post-PK endophthalmitis patients and in a postcataract surgery.

Postoperative Mycobacterium chelonae endophthalmitis after extracapsular cataract extraction and posterior chamber intraocular lens implantation

OBJECTIVE To describe a case of postoperative endophthalmitis caused by Mycobacterium chelonae after extracapsular cataract extraction with posterior chamber intraocular lens implantation. DESIGN Interventional case report. METHODS The history and clinical presentation of a 66-year-old female patient, in whom a low-grade delayed-onset endophthalmitis and keratitis developed after extracapsular cataract extraction with posterior chamber intraocular lens implantation, is described. Microbiologic investigations of the scrapings of corneal infiltrate at the cataract incision site, aqueous humor and eviscerated material, and histopathologic study of eviscerated material and an enlarged cervical lymph node were performed. MAIN OUTCOME MEASURES The clinical, histopathologic, and microbiologic findings in a case of low-grade delayed-onset endophthalmitis. RESULTS Analysis of the direct smear of both the corneal infiltrate as well as the eviscerated material revealed acid-fast bacilli. M. chelonae was isolated from these specimens. Direct smear and culture of the aqueous humor were negative for bacteria (including mycobacteria) and fungus. Histopathologic examination of the eviscerated material showed a dense infiltration of polymorphonuclear leukocytes in the uveal tissue, extensive necrosis and hemorrhage, and exudates with hemorrhage in the vitreous cavity. Histopathologic examination of the lymph node revealed granulomatous inflammation with caseation necrosis, but did not reveal acid-fast bacilli. CONCLUSIONS M. chelonae, although infrequent, should be considered an etiologic agent of delayed-onset, postoperative endophthalmitis and early bacterial diagnosis should help in institution of appropriate therapy.

Tuberculous granuloma managed by full thickness eye wall resection

PURPOSE To report the use of eye wall resection in the management of tuberculous granuloma. DESIGN Interventional case report. METHODS In a 26-year-old man with biopsy-proven tuberculous granuloma of the left eye, total eye wall resection and donor scleral grafting was performed for management of tuberculous granuloma involving the sclera, part of the cornea, the iris, the chamber angle, and the ciliary body. Adjuvant therapy included oral antitubercular medication. RESULTS The treatment of the infection was successful. The scleral graft healed well, and the crystalline lens was preserved. CONCLUSIONS Total eye wall resection, a technique described in the management of uveal tumors, can be adopted to manage selected cases of tuberculous granuloma of the eye.