microbiological diagnosis of suppurative keratitis in...

British Journal of Ophthalmology, 1987, 71, 315-32 1

Microbiological diagnosis of suppurative keratitis inBangladesh*GERRY WILLIAMS,' FRANK BILLSON,2 RABIUL HUSAIN,3SHAHE ALAM HOWLADER,l NAZRUL ISLAM,3 ANDKATHLEEN McCLELLAN-

From the 'Microbiology Unit, Liverpool Hospital; 2Department of Ophthalmology, University ofSydney; 3Eyeinfirmary and Training Complex of Bangladesh National Society for the Blind

SUMMARY Experience in setting up an inexpensive microbiology laboratory in the BangladeshNational Society for the Blind Eye Hospital and Training Complex at Chittagong is presented,together with the results of a pilot study to identify organisms responsible in 33 consecutive cases ofsuppurative keratitis in the Chittagong area of Bangladesh. Of the 33 cases 21 were positivelyidentified by means of Gram stain and/or culture. Two-thirds of the responsible organisms werebacteria, and one-third were fungi. The bacterial causes included Streptococcus pneumoniae andPseudomonas aeruginosa. The fungi isolated were Aspergillu's fumigatus, Aspergillus ochraceus,and Fusarium solani. Among the causes of failure to diagnose the organism was chronicity ofinfection and previous treatment. The value of the study in the planning of future treatmentregimens, and the implications of setting up similar relatively cheap microbiology laboratoryfacilities in developing countries, are discussed.

Suppurative keratitis is a common problem inBangladesh. From a survey by the InternationalCentre for Diarrhocal Diseases Research, Bangla-desh in the rural Matlab area it has been estimatedthat 230 000 people are blind owing to corneal opacityand that this is mostly due to infection (48%) orinjuries (27-5%) (Khan MU, Haque E, Khan MR,report prepared for publication).Suppurative keratitis in Bangladesh is known to be

caused by both bacteria' and fungi.2 As ophthalmicantifungal medications are not available, fungalkeratitis is inevitably progressive. In order to managethese cases microbiological diagnosis is required.

Microbiological services are frequently under con-siderable pressure in developing countries, bothbecause of the numbers of patients and the problemsof cost and supply of equipment and trained person-nel. Microbiology tests may represent an importantitem of expense for patients who have to pay for testsdone by government hospitals, which normallyrestrict their services to their own patients.

'A pilot study conducted in Chittagong, Bangladesh. in July,August, September, 1983.Corrcspondence to Dr C, Williams, Microbiology Department.Liverpool Hospital, Box 113. Liverpool, New South Wales 2170,Australia.

The Eye Infirmary and Training Complex of theBangladesh National Society for the Blind (BNSB) isa 120-bed eye hospital at Chittagong which has acontinual requirement for microbiological diagnosis,particularly of suppurative keratitis. New patientswith suppurative keratitis are seen every day at theBNSB Eye Hospital in Chittagong and in the sevensmaller BNSB Base Hospitals in different regions ofBangladesh. These infections are serious problemswhich threaten eyesight and require hospitalisationfor periods averaging 10 weeks (Das A, personalcommunication). As a result half the eye hospitalbeds are occupied by patients with frequently intract-able infections, particularly during the monsoonperiod. At harvest time up to 12 patients are seen onsome days with corneal injuries and infections relatedto harvesting or husking rice, and these patientscannot all be admitted. A restricted number of bedsimposes limitations on treatment, and many cases aretreated as outpatients, it being accepted that theoutcome is often hopeless regardless of treatment,particularly in chronic cases suspected of being due tofungi.A pilot study was undertaken in August 1983 and

1984 to study the aetiology and management ofsuppurative keratitis, involving the setting up of a

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Fig. 1 Microbiology Laboratory with some ofthe equipment. From leftto right-candle tin usedfor CO2-dependent culture;cheap plastic ice box modified with heating element to make an incubator; pressure cooker on a portable electric hot plate;glassware; constant temperature water bath on far right.

basic microbiology laboratory using cheap readilytransportable materials costing a total of 2000Australian dollars.

Material and methods

EQUIPMENTThe following equipment was purchased in Australiaand transported to Bangladesh by courtesy of ThaiInternational Airlines, who made no charge for theirservices. Some of the laboratory equipment is illus-trated in Fig. 1.An incubator was constructed from a lightweight

plastic cooling box with a hinged door (Vacationermade by Willow, Australia). The Anax Companyassembled a 300 watt mica heating element con-trolled at 360C by a solid state thermostat andmonitor (PM4 made by RKC) with a second thermo-stat (Robertshaw) set to cut out at 380C as a back-up.A pressure cooker was heated on a portable hot plate(Kambrook) to autoclave culture media and othermaterials. A binocular microscope with built-in illu-mination (Olympus CHB) was used. Other equip-

ment included the following: Bunsen burner andbutane canisters (Pongrass); glass Petri dishesfor agar cultures; McCartney bottles (30 ml) forSabouraud's agar slopes; reagents for Gram,Giemsa, Ziehl-Neelsen, and methenamine silverstains; beam balance and weights; conical flasks;bacteriological wire loops; thermometer; glass slides;Coplin jars; staining bottles; dehydrated culturemedia (Gibco); dehydrated rabbit plasma; antibioticsensitivity test discs (Oxoid Multodiscs).

STAFFA paramedic was selected (author AH) from thehospital staff, and after nine months' experience inthe laboratory was brought to Australia for 12months' training. During the 12 months he attendedfull time the Microbiology Department at LiverpoolHospital, under the direction of author GW andspent time at Sydney Eye Hospital, and completedhis training by spending a month with ProfessorCoster's Department in Flinders Medical Centre,South Australia.During the evening while in Sydney he attended a

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course at the Sydney Technical College earningdistinctions in microbiology and a certificate ofcompetence.

LABORATORY METHODSStains were prepared by standard methods withdistilled water supplied by Glaxo Company in Chitta-gong. Dry media bases were reconstituted with tapwater and autoclaved in the pressure cooker for 15minutes. Trypticase soy agar was used as recom-mended by the manufacturer (Gibco) with theaddition of 2 g of yeast extract per litre (as suggestedby Mr L Taylor, University of Sydney). A beambalance and set of weights was used for weighingmedia and stains. Blood was obtained from sheeppurchased locally and grazed at the hospital. Woolover the neck was clipped and the area swabbed withalcoholic iodine. Blood was aspirated while the veinwas dilated by pressure lower on the neck. Blood wasdefibrinated by mixing with sterile glass beads for fiveminutes. The beads could be separated from thefibrin and reused. Blood was added to sterilisedmedia cooled to about 50'C to a proportion of about10%. For chocolate agar the media were reheated to80'C after addition of blood for about five minutes oruntil chocolate brown. Vitox, a vitamin and aminoacid supplement (Oxoid), was added to some batchesto enhance isolation of Haemophilus influenzae (H.aegyptius) from eye specimens. Poured plates werewrapped in plastic and stored in a refrigerator.The quality of media was checked by culture ofStreptococcus pneumoniae on blood agar andHaemophilus influenzae on chocolate agar. Thesebacteria grew well on the respective media.

Corneal scrapings were plated directly on to agarplates and smeared on to glass slides for staining.Smears were stained routinely by Gram stain.Methenamine silver could be used to restain negativesmears for improved detection of fungi.

Sabouraud's slopes were inoculated by severalseparate strokes to mark but not break the agar sothat subsequent growth from the inoculum could bedistinguished from contaminants not on the mark.3Sabouraud's slopes were sealed with screw caps toprevent dehydration or contamination incubated atroom temperature and were examined daily for threeweeks.Blood and chocolate agar in Petri dishes were

inoculated and then streaked out with a sterile wireloop and incubated at 37°C in a candle tin.

Bacteria and fungi were identified by standardmethods using colony morphology, Gram stain, andbasic tests. Staph. aureus was identified by coagulasetesting using human or rabbit plasma; Str. pyogenesby bacitracin sensitivity, morphology, Gram stain,and catalase; pneumococcus by optochin sensitivity;

and Gram negative rods by decarboxylase, sugarfermentation, Voges-Proskauer, indole, urea andcitrate tests as provided in the API identification kit20E or the Microbact 12E test tray (DiagnosticProducts, Adelaide, SA).Haemophilus influenzae was identified by growth

on chocolate agar and growth around combined Xand V factor discs on nutrient agar. Pseudomonasaeruginosa was identified by characteristic colouringmorphology, pigment production, and oxidase test.Fungi were identified by morphological criteria.Antibiotic disc diffusion sensitivity tests were per-formed by the calibrated dichotomous sensitivitymethod4 on Sensitest agar. Gram stain was usedalone for eight patients, of whom two had pneumo-coccal like bacteria and three had Gram-negativerods in scrapings from corneal ulcers.

PATIENTSThirty-three patients with corneal ulceration wereexamined over a period of three months. Historieswere taken, and the eyes examined with the aid of aslit-lamp. Five of the patients had been in hospital foran average of 3-5 weeks each.

CLINICAL METHODSSpecimens were taken from the cornea by carefullyscraping the ulcer with a Kimura platinum spatula(OPSM) or a sterile Bard-Parker scalpel blade. Thefirst specimen was taken for culture and without localanaesthetic.The patients with pneumococcal infection were

treated with hourly irrigations of a penicillin solution,200 units/ml in normal saline, administered with anintravenous giving set. Chloramphenicol ointmentwas used at night and penicillin 0.5 million units givensubconjunctivally for four days as well as penicillinorally.The Pseudomonas and other Gram-negative rod

infections were treated with 1% gentamicin dropshourly, gentamicin 20 mg subconjunctivally daily forfour days, and polymyxin-oxytetracycline ointmentat night.The fungal ulcers were treated with 1%

miconazole (10 mg/ml) drops second hourly,5 or inthe case of Fusarium solani, which appears resistantto miconazole in vitro, pimafucin (Natamycin) drops2*5% (25 mg/ml) were given second hourly. Micon-azole was also given as subconjunctival injections of10 mg daily for four days, with additional subcon-junctival injections later in some cases.

EPIDEMIOLOGYSixteen rice grains from a batch of stored rice werecultured, eight on Sabouraud's agar and eight onblood agar, in an attempt to detect potential patho-

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gens. Seven samples of pond water and three samplesof canal water from the Chittagong area were cul-tured on blood and Sabouraud's agar before and after10-fold concentration by centrifugation and colonieswere enumerated approximately.

Conjunctival swabs were taken from healthyuntreated eyes of four nursing staff and six cataractpatients and cultured on blood and chocolate agar.Nasal swabs were taken from four doctors, fournursing staff, and eight patients and culturesexamined for staphylococcal colonies. These weretested by slide coagulase to identify Staph. aureus.

Results*

The lightweight incubator functioned well withsteady uniform temperature maintenance at 350C.The media made with tap water grew fastidiouspathogens such as Str. pneumoniae and Haemophilusinfluenza, including strains isolated directly frompatients' eyes. The availability of small quantities ofdistilled water was necessary for preparing stains, butuntreated tap water proved satisfactory for mediaand for washing slides.The Gram stain proved adequate to detect fungi in

those cases from which they were isolated, the mainfactor affecting detection being the adequacy of thescraping taken with the platinum spatula. It wasnoticed particularly with bacterial ulcers thatorganisms were best detected from the scrapings of

*During the period of study four additional patients were notedattending the hospital with recurrent herpes simplex keratitis withtypical dendritic ulcers revealed by fluorescein staining in two casesand a geographic ulcer in a third case previously treated with steroid-containing ointment, which is freely obtainable in the smallest ofvillages throughout Bangladesh. These patients were treated withvidarabine or idoxuridine ointment. Another inpatient, a youngwoman, had bilateral corneal scars with a history of recurrentulceration self treated with steroids which was related to a history ofcold sores on the mouth.

Table 1 Organisms detected

Organism Number of Patients

BacteriaStreptococcus pneumoniae 5Pseudomonas aeruginosa 3Staphylococcus epidermidis 3Enterobacter IProvidencia IUnidentified Gram-ncgativc rods 3Corynebacterium (normal flora) 2

FungiAspergillusfumigatus (Fig. 2) 1Aspergillus ochraceus IFusarium solani (Fig. 3) 1Unidcntificd filamcntous fungi 4

the cornea itself, and were frequently absent from theloose exudate overlying ulcers.

Contamination of agar plates with airbornebacteria or fungi could be distinguished from growthfrom the patient's specimen by the relationship of thecolonies to the inoculating streak. That is, growthalong the streaks was related to the patient, whilegrowth away from the streaks was due to contamina-tion. Airborne bacteria and fungi were relativelymore common in Chittagong than in Sydney.A history of initiating trauma was obtained in 14 of

the 29 patients with suppurative keratitis. The com-monest trauma was due to the husk of the rice grainflying into the eye during the process of beating therice to remove the husks-that is, 'paddy injury'-which was reported in seven cases. Other causes oftrauma included sand (two boys), a date palm, awooden stick, a bamboo splinter, and a needle. In thecase of the pseudomonas keratitis, the cause wasthought to be almost certainly due to contaminatedwater with which the injured eye was initiallywashed.

Scrapings taken from corneal ulcers of the 29patients with suppurative keratitis revealed thefollowing organisms in Gram stains and cultures(Table 1).Gram stains and cultures did not reveal any

significant micro-organisms in the other patients,though two had been treated in hospital for two andfour weeks and others had purchased ointments,which probably included antibiotics in many cases,before coming to the BNSB Eye Infirmary.

Interestingly in three cases Gram stains revealedGram-intermediate (i.e., pinky-blue) curved rods incorneal ulcer scrapings. They were found in cases ofpneumococcal ulcer and in one case where onlysymbiotic Corynebacterium xerosis was isolated inculture. They were also seen in a smear from another

Fig. 2 A typicalpresentation ofsuppurative keratitis. Afungal cause was notsuspected until identified on Gram staincharacterizedd as Aspergillus fumigatus on culture).

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Fig. 3 Culture on Sabaraud's agar ofcolonies ofFusariumsolani.patient who had been in hospital for six weeks fromwhom a fungus was isolated which could not subse-quently be identified owing to overgrowth by acontaminating fungus. No such bacteria could begrown in culture, though anaerobic cultures were notdone and the organisms were reminiscent of theappearance of Mobiluncus, which is a Gram-variableanaerobic curved rod implicated as a cause ofvaginosis.1Also seen in Gram stains of scrapings from three

patients were yeast-like organisms with capsulesstaining Gram-negative which did not grow in cultureand were presumed to be Pityrosporum orbiculare.This is a yeast-type fungus associated particularlywith seborrhoeic dermatitis and probably comingfrom the eyelashes as a harmless saphrophyte.7 Thepatients mostly presented late with advanced cornealulcers, and the appearance of the ulcer was nothelpful in the differential diagnosis of bacterial andfungal infection. The commonest initiating traumafor corneal ulcers was due to husking rice (paddyTable 2 Comparison offungal and bacterial infections

Fungi Bacterial

Total 7 14Injury 1 (paddy grain) 7 (4 paddy grain)Male/female (4/3) 12/2Average duration* 2-3 weeks before presentation 11 daysAverage age 50 years 30 years

*The duration before presentation was estimated from the time ofinjury if the patient could recall it, or was based on the duration ofpatients symptoms. The onset ofsymptoms in the bacterial casestended to be more severe and resulted in early presentation.

injury). Among the patients in whom fungi werefound, only one had a history of paddy injurycompared with four of 14 with bacterial infection.However, paddy grain injury was not associated withpneumococcal infection. Hence there was little in thehistory of patient characteristics to point to fungal orbacterial infections, except a longer history in someof the cases of fungal ulcers. Even the duration ofulceration prior to presentation was often similar inthe fungal and bacterial cases, though four of thepatients with fungal ulcers spent an average of 3*5weeks in hospital receiving antibacterial therapybefore this project started (Table 2).The ulcers showed steady improvement with the

treatment regimens described despite their advancedpathology at presentation, and all patients but one,who had the affected eye enucleated, were dis-charged with the disease apparently arrested.

EPIDEMIOLOGY RESULTSAll eight stored rice grains cultured on Sabouraud'sagar and blood agar grew Bacillus species and six ofthe eight on Sabouraud's agar grew fungi of thefamily Mucorales. Five of the eight also grew a yellowAspergillus species similar to that isolated from onepatient. Two other unidentified fungi were also seenin the cultures.Samples of pond and canal water, which are often

used to wash injured eyes, were cultured. An averageof 103 to 101 bacteria per ml were found, includingGram-negative rods in eight of 10 samples, yeast intwo, and Bacillus species in two.

Conjunctival swabs from four healthy staff and sixuninfected patients revealed occasional Staph.epidermidis in six and Corynebacterium species intwo cases and, interestingly, Haemophilus influenzaein one outpatient presenting for refraction. A surveyfor Staph. aureus carriers revealed positive noseswabs from two of four doctors, one of four nursingstaff or paramedics, and two of eight patients. All fiveisolates of Staph. aureus were resistant to penicillin.

Discussion

Our experience confirms that fungi are an importantcause of suppurative keratitis in Bangladesh,accounting for some 30% of the cases of suppurativekeratitis in this study. Furthermore we have foundthat there is a variety of fungi involved reminiscent ofthe diversity of genera found in (sub) tropical areas inthe USA and elsewhere.' In Florida the commonestcause of fungal ulceration is Fusarium solani, whichhas not previously been well recognised as a causein Bangladesh. Also Aspergillus fumigatus andAspergillus flavus and many other fungi have beenreported in other countries. We found Aspergillus

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fumigatus as previously reported in Bangladesh3and a sulphur yellow species that was probablyAspergillus ochraceus. This yellow species was alsoisolated from rice grains and may be common in theenvironment.The pathogenesis of fungal keratitis is unclear.

Trauma is assumed to be an initiating factor, thoughusually, as in our cases, there is no clear incident ofinjury.8 Bacterial and fungal organisms growing inthe thatch of houses have been implicated as causingallergic pneumonia to people living in warmclimates.9 Environmental cultures from dwellings ofpatients with fungal ulcers might be of interest,though two small random samples of thatch did notreveal fungi in this study.The significance of the finding of fungi as a cause of

suppurative keratitis is important when it is appreci-ated that antifungal therapy for suppurative keratitisis not freely available in Bangladesh. Furthermore,the problem of suppurative keratitis due to fungi isaggravated by the fact that corticosteroid ointmentsare freely available in the villages.We found the Gram stain alone was of value in

making a diagnosis of bacterial or fungal keratitis. Inan extended study to look at this question we foundthat in a series of 66 cultures 58 were culture-positivefor bacterial or fungal organisms, and, of these,47 were anticipated on Gram stain examinationalone.

Establishing the pattern of infective organismscausing suppurative keratitis was fundamental todeveloping appropriate treatment protocols. Theseprotocols are referred to above in clinical methods.The results of these therapeutic regimens was one ofresolution and healing in those cases with provedmicrobiological diagnosis, except for one case ofpseudomonas infection which was too advanced atpresentation and resulted in perforation of thecornea soon after treatment began. The fungal infec-tions were treated with topical and subconjunctivalmiconazole, and this therapy was effective except inone case due to fusarium, which is known to beresistant to miconazole.The frequency of Str. pneumoniae as a cause of

corneal ulcers is of interest. In contrast to somereports" a history of trauma was not obtained frommany of our patients with pneumococcal ulcers. It islikely that many patients have pneumococcal coloni-sation of the throat and/or conjunctiva and that onlyoccasionally the more virulent capsular serotypes ofpneumococci, such as type 4, cause keratitis.The role of the coagulase-negative staphylococci

grouped here as Staph. epidermidis in the cornealulcers from which they were isolated is uncertain.These organisms are now separated into severalspecies of varying degrees of parasitism."I However,

some strains of Staph. epidermidis have been impli-cated in the pathogenesis of keratitis by their produc-tion of toxins.'2The isolation of Providencia species from the eye

of a young child who had measles, gastroenteritis,and keratomalacia may represent an opportunisticrelatively less virulent colonisation of an alreadydamaged eye. The relatively high levels of environ-mental bacteria in canal and pond water suggest thelikelihood of such water, which is needed for wash-ing, as a source of colonisation of the injured cornea.Traditional eye treatments may also contribute.'3The finding of Pseudomonas aeruginosa in a

patient with rapidly progressive keratitis reinforcesthe necessity for vigilance in the case of solutions thatmight be used to wash foreign bodies from eyes. Therapid progression of this infection depends on pro-teinases produced by the organisms, ' though theexotoxin A appears important."' It is of interest thatpseudomonas elastase is a metallo-proteinase, as arecollagenases, including that of neutrophils'6 1' that areinhibited by tetracycline, which binds the metallicradical in the enzyme. Thus the combination ofpolymyxin with oxytetracycline (Terramycin),which is the routine ointment for eye infections atBNSB hospitals, may be appropriate for pseudo-monas keratitis, though the elastase enzyme appearsto be less important than the alkaline protease.'5However, tetracycline is synergistic with ampho-tericin B against Aspergillus species.' Thus theaddition of pimaricin (which is related to ampho-tericin) to treatment with polymyxin-oxytetracyclinemay provide good antifungal prophylactic therapy.Treatment regimens effective in this pilot study,

including antifungal therapy with subconjunctivalmiconazole, yielded results that in our opinion aredeserving of further study.

In conclusion we believe this work confirms thefeasibility of setting up similar relatively cheapmicrobiology laboratory facilities to develop strate-gies for the treatment of suppurative keratitis, and tohelp reduce avoidable blindness in developingcountries. The total cost of equipment, setting up thelaboratory, and training of the microbiologist wasapproximately 10000 Australian dollars. Short ofthis, merely the facilities for doing Gram stainidentification will allow appropriate treatment in themajority of cases, which will subsequently be shownto be positive to culture. The addition of this skill tothe base hospitals in rural areas of Bangladesh isproposed with the Chittagong Eye Hospital andTraining Complex remaining as the reference centrefor problem cases.A further study of treatment regimens will be the

subject of another report when larger numbers ofcases are available. *

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*Following the setting up of this laboratory in 1983-4 Dr A Richardsand Dr E D Wright brought protocols to Chittagong which modifiedthe treatment regimens outlined. These protocols are being filled inby the staff of the laboratory, and this material will be the subject of afuture joint publication.

Foresight Australia funded the microbiology laboratory in Chitta-gong, and the training ofA Howlader in Sydney. Thai InternationalAirlines transported laboratory equipment from Sydney to Bangla-desh without charge. Professor Y E Cossart and Mr L Taylor, of theDepartment of Infectious Diseases, University of Sydney, providedconsiderable assistance and advice. Medications were donated byRoussel Pharmaceuticals, Allergan, and Astra Pharmaceuticals andthe Sydney Eye Hospital.

References

1 Wahed MA. Bacterial corneal ulcer. Trans Ophthalmol SocBangladesh 1981; 9: 9-11.

2 Rahman MD, Mustafizur. Management of fungal corneal ulcer.Trans Ophthalmol Soc Bangladesh 1981; 9: 12-9.

3 Jones DB, Liesegang TJ, Robinson NM. In: Washington JA, ed.Laboratory diagnosis of ocular infections. Cumitech 13.Washington, DC: American Society for Microbiology, 1981.

4 Bell SM. Antibiotic sensitivity testing by the CDS method. In:Hartwig N, ed. Clinical microbiology update programme, N 21.Australian Society for Microbiology, 1984.

5 Foster CS. Miconazole therapy for keratomycosis. Am JOphthalmol 1981; 91: 622-9.

6 Spiegel CA, Eschenbach DA, Amsel R, Holmes KK. Curvedanaerobic bacteria in bacterial (nonspecific) vaginosis and theirresponse to antimicrobial therapy. JInfect Dis 1983; 148: 817-22.

7 Fedukowicz HB. External infections of the eye. New York:Appleton Century Crofts, 1978.

8 Forster RK. Fungal diseases. In: Smolin G, ed. The cornea, basicscience and clinical aspects. Boston: Little, Brown, 1983: 168-77.

9 Blackburn CRB, Green WF. Experimental extrinsic allergicalveolitis induced in the guinea-pig with Streptomyces olivaceus.Clin Allergy 1974; 4: 87-93.

10 Hyndiuk RA, Nassif KF, Burd EM. Bacterial diseases. In:Smolin, G, ed. The cornea, basic science and clinical aspects.Boston: Little, Brown, 1983: 147-68.

11 Klous WE, Schleifer KH. Simplified scheme for routine identi-fication of human Staphylococcus species. J Clin Microbiol 1975;1:82-8.

12 Valenton MJ, Okumoto M. Toxin producing strains of Staphylo-coccus epidermidis. Arch Ophthalmol 1973; 89: 187.

13 Rahman MM, Razzaque MA. A microbiological approach toisolate micro-organisms from paddy grains and paddy leaves,snails, turmeric and rose water. Trans Ophthalmol Soc Bangla-desh 1982; 10: 86-8.

14 Howe TR, Iglewski BH. Isolation and characterisation ofalkaline protease-deficient mutants of Pseudomonas aeruginosain vitro and in a mouse eye model. Infect Immun 1984; 43:1058-63.

15 Ohman DE, Burns RP, Iglewski BH. Corneal infections in micewith toxin A and elastase mutants of Pseudomonas aeruginosa.J Infect Dis 1980; 142: 547-55.

16 Brown SI, Hook CW, Tragakis MP. Presence, significance andinhibition of lysosomal proteoglycanase. Invest Ophthalmol VisSci 1972; 11: 149-52.

17 Hazlett LD, Berk RS. Effect of C3 depletion on experimentalPseudomonas aeruginosa ocular infection: Histopathologicalanalysis. Infect Immun 1984; 43: 783-90.

18 Hughes CE, Harris C, Peterson LR, Gerding DN. Enhancementof the in vitro activity of amphotericin B against Aspergillus spp.by tetracycline analogs. Antimicrob Agents Chemother 1984; 26:837-40.

Acceptedfor publication 30 May 1986.

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