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Year : 2003  |  Volume : 21  |  Issue : 1  |  Page : 25-30

16S rRNA PCR for differentiation of pathogenic and non-pathogenic leptospira isolates

Division of Microbiology, Defence R & D Establishment, Jhansi Road, Gwalior-474002, India

Correspondence Address:
Division of Microbiology, Defence R & D Establishment, Jhansi Road, Gwalior-474002, India

 ~ Abstract 

PURPOSE: To determine the risk factors, microbiological features, clinical features and other epidemiological characteristics of Nocardia keratitis seen at a tertiary eye care centre in south India. METHODS: We evaluated 31 patients with Nocardia keratitis seen over two years, from September 1999 to September 2001. Corneal scrapings were subjected to microscopy and culture using standard protocols. RESULTS: Out of 2184 corneal ulcers cultured, 31(1.42%) were found to be Nocardia asteroides. All 31(100%) were detected correctly by 10% potassium hydroxide wet mount preparation. The highest percentage of isolates was susceptible to gentamicin (100%) followed by ciprofloxacin(93.55%). Twenty four (77.42%) patients were from rural areas; 22(70.97%) were agricultural workers; 29(93.55%) had history of trauma; 2(6.45%) had previous ocular surgery; 28(90.32%) had ocular injury with soil and sand; and 22(70.97%) had ocular injury while working in the agricultural fields. Ten (32.26%) patients presented at our institute between 15 to 35 days of onset of illness, 26(83.87%) had previous medical treatment, and 15(48.39%) patients had used traditional eye medicines. The average age of the patients was 46.16 years, with a range of 11 to 75 years. No seasonal variation was observed. CONCLUSIONS: A high index of suspicion of Nocardia infection should exist in patients with a history of trauma to the eye by soil or sand. The organisms are sensitive to commonly used topical ocular antibiotics.

How to cite this article:
Shukla J. 16S rRNA PCR for differentiation of pathogenic and non-pathogenic leptospira isolates. Indian J Med Microbiol 2003;21:25-30

How to cite this URL:
Shukla J. 16S rRNA PCR for differentiation of pathogenic and non-pathogenic leptospira isolates. Indian J Med Microbiol [serial online] 2003 [cited 2020 Nov 28];21:25-30. Available from:

All patients with infectious microbial keratitis who presented from 20th September 1999 to 30th September 2001 were included in this study. All patients were examined with a slit lamp biomicroscope by an ophthalmologist. The location and size of infiltrate was measured with the variable slit on the biomicroscope and recorded in millimeters. A standardized form was filled out on each patient documenting socio demographic information as well as clinical findings including predisposing ocular conditions, associated systemic diseases, duration of symptoms, previous treatment, visual acuity at the time of presentation and other personal data.
After a detailed ocular examination, corneal scrapings were performed using standard techniques, 15 under aseptic conditions from each ulcer by an ophthalmologist after instillation of 4% lignocaine (lidocaine) with sterile blade No.15 on Band Parker handle. Materials obtained were inoculated directly onto sheep blood agar, chocolate agar, non-nutrient agar, Sabouraud dextrose agar, thioglycollate, and brain-heart infusion broth. The inoculated Sabouraud dextrose agar was incubated at 25 C to enhance the growth of fungi and examined daily for upto 3 weeks. The remainder were incubated at 37 C. Inoculated blood agar plates were incubated under aerobic and anaerobic conditions, chocolate agar was incubated with 5% carbon dioxide, and non-nutrient agar was incubated with an added  Escherichia More Details coli suspension overlay.
The material obtained by scraping was also spread onto labelled slides in a thin, even manner for 10% potassium hydroxide (KOH) wet mount, Gram staining and Giemsa staining. Acid fast stains (Ziehl-Neelsen and Kinyoun's) were performed in suspected cases. A culture was considered positive when there was growth of the same organism on two or more media on 'C' streak (inoculum site), confluent growth at the site of inoculation on one solid medium, growth in one medium with consistent direct microscopy findings, growth of the same organism on repeated corneal scraping and growth consistent with clinical signs. The isolated Nocardia species were identified by standard bacteriological methods.[15],[16] All isolates were tested for susceptibility to antibacterial agents such as, gentamicin, ciprofloxacin, vancomycin, chloramphenicol, cefazolin, norfloxacin and co-trimoxazole by Kirby Bauer disc diffusion method.

 ~ Results Top

Microbiological Profile
During the study period of two years, 2184 patients with the clinical diagnosis of corneal ulceration were evaluated at our institute. Out of 2184 corneal ulcers cultured, 31(1.42%) cases were found to be culture positive for  Nocardia asteroides   [Figure:1], [Figure:2]. Twenty nine out of 31 (93.55%) were pure isolates, while 2(6.45%) were mixed with Staphylococcus epidermidis.
Results of in vitro antibacterial susceptibility testing are shown in [Figure:3].
Correlation of smear based diagnosis and culture based diagnosis
Of the 31 patients of culture-proven N.asteroides keratitis, all 31(100%) were detected correctly by 10% potassium hydroxide wet mount preparations [Figure:4], and 28(90.32%) were detected correctly by Gram staining and acid-fast Kinyoun's method of staining.
Out of 31 Nocardia corneal ulcers evaluated [Figure:5], 18(58.06%) were males and 13(41.94%) were females. There were 17(54.84%) left eyes and 14(45.16%) right eyes involved. The average age of the patients was 46.16 years, with a range of 11 to 75 years. The seasonal distribution of 31 cases is shown in the [table]. No seasonal variation was observed.
Majority (24; 77.42%) of the patients were from rural areas; 22(70.97%) patients were agricultural workers; 29(93.55%) had history of trauma; 2(6.45%) had previous ocular surgery with infection associated with a loose suture; 28(90.32%) had ocular injury with soil and sand; 22(70.97%) had corneal injury while working in the agricultural fields. Ten (32.26%) patients presented at our institute between 15 to 35 days of onset of illness. Twenty six (83.87%) patients had previous medical treatment. 15(48.39%) patients had used traditional eye medicines (human milk - 5, goat milk-3, oil - 2, onion juice - 1, mixed - 4).

 ~ Discussion Top

Nocardia and Actinomyces are genera of the order actinomycetaceae. Nocardia were originally classified as fungi and are now recognized as true bacteria.[9],[16] Cell wall analysis shows the characteristic bacterial muramic acid, which, along with the lack of a membrane-bound nucleus, lack of mitochondria, typical bacterial size, and sensitivity to antibacterial agents, defines these organisms as bacteria.[9],[16]
Nocardia are gram positive organisms, which in smears appear as highly-branched and thin (<1.5m) beaded filaments.[9], [16] They are acid fast to 1% sulphuric acid and are stained by Kinyoun's technique, a modification of Ziehl-Neelsen's method of acid-fast stain.[9], [16] The Nocardia do not have complex growth requirements.[9], [16] They grow well on most commonly used routine bacteriologic media. They usually require a minimum of 48 to72 hours before colonies become visible. They may manifest extremely variable colonial morphologies on different culture media. They are dry, chalky, rough, folded, irregular and powdery. The colour of the colony may be creamy, yellow or orange, pink or red.[1], [9], [16]
Keratitis caused by Nocardia is believed to be a rare condition.[8],[9],[10],[11],[12] Except for few large series, the literature mainly comprises of case reports.[10], [17] The incidence of keratitis caused by N. asteroides in this series was 1.42%. These findings are similar to a study from Madurai, Tamil Nadu in 1997, where N.asteroides accounted for 1.61% of a total of 434 corneal ulcers that were cultured.18 Another study from Hyderabad in south India reported that Nocardia contituted 1.7% of 1,689 bacterial isolates from cases of keratitis examined between January 1991 and December 1998.19 Upadhyay et al from Nepal reported that Nocardia constituted 0.25% of total 405 corneal ulcers that were cultured.20 In our previous study in 1999, the incidence of keratitis caused by pure isolates of gram positive cocci were 22. 87%, gram negative bacilli were 7.42%, aerobic actinomycetes (Nocardia species) were 1.24%, gram positive bacilli were 1.11%, gram negative cocci and coccobacilli were 0.49%, and fungal keratitis accounted for 32.26% of 1618 corneal ulcers cultured.21 In this study, from 31 cases, dry, chalky, raised, folded or irregular, white and orange N.asteroides were grown [Figure:1], [Figure:2]. The 10% potassium hydroxide wet mount of all 31 cases of culture proven Nocardia asteroides revealed very fine, intertwined, narrow, delicate, branching filaments of Nocardia [Figure:4]. Of the 31 culture positive Nocardia, 28(90.32%) were detected correctly by Gram staining as gram positive, beaded, coccoid and thin branching filaments and also by Kinyoun's method of acid-fast staining as thin, pink branching filaments in a blue background.
In this study, the highest percentage of Nocardia asteroides isolates were susceptible to gentamicin(100%) followed by ciprofloxacin(93.55%), vancomycin(77.42%) and chloramphenicol(58.06%). Sharma et al in 1999 reported that the highest percentage of Nocardia species were susceptible to gentamicin(100%) followed by vancomycin (75%), ciprofloxacin(68.4%) and chloramphenicol (57.9%).[22] Sridhar et al reported the drug sensitivity pattern of 16 isolates of Nocardia keratitis by Kirby-Bauer technique that all isolates were susceptible to gentamicin.10 Nocardia were susceptible to ciprofloxacin(100%) and gentamicin(95%).[23]
Male predominance was noted (56.06%) in the patients seen in this series. The male preponderence has also been described in earlier reports.[10], [11], [17] There were no seasonal variations, but the cases were often seen during onion and groundnut harvesting season, as corneal trauma by soil or sand is more common during this activity. No seasonal variations were reported in other forms of bacterial as well as fungal keratitis in other studies.[18],[19],[20] However, the incidence of fungal keratitis was found to be slightly higher during the time of year when there is more agricultural activity due to vegetative corneal injuries. Kotigadde et al found higher number of fungal keratitis in the month of October, June and January in 1995.[24]
Majority of the patients in this study were from rural areas (77.42%) and most of them were agricultural workers (70.97%). Agarwal et al have reported 85% of the fungal keratitis in rural area.[25] Traumatic corneal injury is the leading cause of microbial keratitis,18-20 and Nocardia keratitis following ocular trauma has been documented in many reports.[4], [10], [11], [17] Corneal trauma was the most common predisposing factor in this series (93.55%) and the principal traumatic agents were soil and sand. N.asteroides are usually saprophytes in soil and live free in nature,[1],[8],[9] thus the source of infection could be from soil and sand.[1],[10],[11],[26] Non-traumatic predisposing condition associated with development of N.asteroides keratitis identified in this series constituted 2(6.45%) patients who had previous ocular surgery with infection associated with loose sutures in the eye. N. asteroides keratitis has also been diagnosed following ocular surgeries.[27],[28],[29] In comparison, many studies have reported the occurrence of fungal keratitis predominantly in patients with vegetative corneal injuries.
The majority of the patients in south India appear to have access, although limited, to relatively sophisticated eye care. Before their initial examination at our institute, 83.87% of all patients with N.asteroides keratitis had undergone medical treatment of some kind.[18], [20]
Despite the presence of typical clinical features, the diagnosis of Nocardia infection is often delayed, probably because it is a common practice among primary care physicians to start empirical treatment without obtaining material for diagnostic purposes.[9] Although Nocardia keratitis is believed to be a rare entity, a high index of clinical suspicion of this infection should exist in patients with a history of trauma by soil or sand and presence of typical clinical pictures. Detection of thin, delicate, intertwined, narrow, branching filaments, which are gram positive, often appearing as beaded or coccoid forms and are also 1% acid-fast staining positive, should prompt the treating physician to initiate the appropriate therapy for Nocardia infection.
In-vitro susceptibility testing shows the highest percentage of N.asteroides isolates to be susceptible to gentamicin followed by ciprofloxacin and vancomycin. Early diagnosis can help to effectively manage Nocardia keratitis, but requires a high index of clinical suspicion and microbiology laboratory support.  

 ~ References Top

1.Srinivasan M, Reddy J. Ocular Nocardiosis. Journal of TNOA 1998;38:25-29.  Back to cited text no. 1    
2.Curry WA. Human Nocardiosis: a clinical review with selected case reports. Arch intern 1980;140:818-826.  Back to cited text no. 2    
3.Bruce GM, Locatcher-Khorazo D. Actinomyces: Recovery of the Streptothrix in a case of superficial punctate keratitis. Arch Ophthalmol 1942;27:294-98.  Back to cited text no. 3    
4.Perry HD, Nauheim JS, Donnenfeld ED. Nocardia keratitis presenting as a persistent epithelial defect. Cornea 1989;8:41-43.  Back to cited text no. 4    
5.Benedict WL, Lverson HA. Chronic keratoconjunctivitis associated with Nocardia. Arch Ophthalmol 1944;32:84-92.  Back to cited text no. 5    
6.Peniket EJK, Rees DL. Nocardia asteroides infection of the nasolacrimal system. Am J Ophthalmol 1962;27:294-98.  Back to cited text no. 6    
7.Basti S, Gopinathan U, Gupta S. Nocardia necrotizing scleritis after trauma- successful outcome using cefazolin. Cornea 1994;13:274-76.   Back to cited text no. 7    
8.Rao SK, Madhavan HN, Sitalakshmi G, Padmanabhan P. Nocardia asteroides keratitis: report of seven patients and literature review. Indian J Ophthalmol 2000;48:217-21.  Back to cited text no. 8  [PUBMED]  
9.Sridhar MS, Gopinathan U, Garg P, Sharma S, Rao GN. Ocular Nocardia infections with special emphasis on the cornea. Surv Ophthalmol 2001;45:361-378.   Back to cited text no. 9    
10.Sridhar MS, Sharma S, Reddy MK, Mruthyunjay P, Rao GN. Clinicomicrobiological review of Nocardia keratitis. Cornea 1998;17:17-22.  Back to cited text no. 10  [PUBMED]  
11.Hirst LW, Harrison GK, Merz WG, Stark WJ. Nocardia asteroides keratitis. Br J Ophthalmol 1979;63:449-54.  Back to cited text no. 11  [PUBMED]  
12.Parsons MR, Holland EJ, Agapitos PJ. Nocardia asteroides keratitis associated with extended-wear soft contact lenses. Can J Ophthalmol 1989;24:120-22.  Back to cited text no. 12    
13.Henderson JW, Wellman WE, Weed LA. Nocardiosis of the eye - report of a case. Mayo Clin Proc 1960;35:614-18.  Back to cited text no. 13    
14.Zimmerman PL, Mamalis N, Alder JB, Teske MP, Tamura M, Jones GR. Chronic Nocardia asteroides endophthalmitis after extracapsular cataract extration. Arch Ophthalmol 1993;111:837-40.  Back to cited text no. 14  [PUBMED]  
15.Jones DB, Leisegang TJ, Robinson NM. Laboratory diagnosis of ocular infections. (American Society for Microbiology, Washington DC) 1981.   Back to cited text no. 15    
16.Gordon MA. Aerobic pathogenic actinomycetaceae, Chapter 23. In: Manual of Clinical Microbiology, 4th ed. Lennette EH, Balows A, Hausler WJ, Shadomy HJ, Eds. (American Society for Microbiology, Washington DC) 1985:249-62.  Back to cited text no. 16    
17.Srinivasan M, Sharma S. Nocardia asteroides as a cause of corneal ulcer. Arch Ophthalmol 1987;105:464.  Back to cited text no. 17    
18.Srinivasan M, Gonzales CA, George C, Cevallus V, Mascarenhas JM, Asokan B, Wilkins J, Smolin G, Whitcher JP. Epidemiology and aetiological diagnosis of corneal ulceration in Madurai, south India. Br J Ophthalmol 1997;81:965-971.  Back to cited text no. 18    
19.Garg P, Rao GN. Corneal ulcer: diagnosis and management. Community Eye Health 1999;12:21-24.  Back to cited text no. 19    
20.Upadhyay MP, Karmacharya PC, Koirala S, Tuladhar N, Bryan LE, Smolin G, et al. Epidemiologic characteristics, predisposing factors, and etiologic diagnosis of corneal ulceration in Nepal. Am J Ophthalmol 1991;111:92-99.  Back to cited text no. 20    
21.Bharathi MJ, Ramakrishnan R, Vasu S, Meenakshi R, Palaniappan R. Aetiological diagnosis of microbial keratitis in South India. Indian J Med Microbiol 2002;20:19-24.  Back to cited text no. 21    
22.Sharma S, Kunimoto DY, Garg P, Rao GN. Trends in antibiotic resistance of corneal pathogens: Part I. An analysis of commonly used ocular antibiotics. Indian J Ophthalmol 1999;47:95-100.  Back to cited text no. 22    
23.Bharathi MJ, Ramakrishnan R, Vasu R, Meenakshi R, Palaniappan R. In-vitro efficacy of antibacterials against bacterial isolates from corneal ulcers. Indian J Ophthalmol 2002;50:109-114.  Back to cited text no. 23    
24.Kotigadde S, Ballal M, Jyothiralatha, Kumar A, Rao SPN, Shivananda PG. Mycotic keratitis: a study in coastal Karnataka. Indian J Ophthalmol 1992;40:31-33.   Back to cited text no. 24    
25.Agarwal PK, Roy P, Das A, Banerjee A, Maity PK, Banerjee AR. Efficacy of topical and systemic itraconazole as a broad-spectrum antifungal agent in mycotic corneal ulcers - a preliminary study. Indian J Ophthalmol 2001;49:173-76.  Back to cited text no. 25    
26.Newmark E, Polack FM, Ellison AC. Report of a case of Nocardia asteroides keratitis. Am J Ophthalmol 1971;72:813-5.  Back to cited text no. 26  [PUBMED]  
27.Perez-Santonja JJ, Sakla HF, Abad JL, et al. Nocardia keratitis after laser in situ keratomileusis. J Refract Surg 1997;13:314-7.  Back to cited text no. 27  [PUBMED]  
28.Colomina J, Esparza L, Buesa J, Mari J. Corneal ulcer caused by Nocardia asteroides after penetrating keratoplasty. Med Clin (Barc) 1997;108:424-5.  Back to cited text no. 28  [PUBMED]  
29.Sridhar MS, Sharma S, Garg P, Rao GN. Broken suture - predisposing factor for Nocardia asteroides keratitis (letter). Eye 2000;14:112-4.  Back to cited text no. 29  [PUBMED]  
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