Abstract
AIM
To analyze the lab diagnosis and etiology of infectious endophthalmitis.
METHODS
The medical and microbial records of 36 patients diagnosed with infectious endophthalmitis and 8 patients diagnosed with intraocular lens (IOL)-related inflammation between Nov. 1999 and Dec. 2009 were retrospectively reviewed for lab diagnosis and etiology.
RESULTS
The inflammatory cell counts in all aqueous humor specimens from infectious endophthalmitis patients were more than in all aqueous humor specimens from patients with IOL-related inflammation. Sixteen of the 36 aqueous humor samples (44.4%) and 11 of the 24 vitreous humor samples (45.8%) from infectious endophthalmitis patients showed positive results in smears; while 17 aqueous humor samples (47.2%) and 15 vitreous humor samples (62.5%) from infectious endophthalmitis patients showed positive results in culture.
CONCLUSION
The inflammatory cell count may be an important index for infectious endophthalmitis; while, smears can show etiological information earlier.
Keywords: laboratory diagnosis, infectious endophthalmitis, IOL-related inflammation
INTRODUCTION
Infectious endophthalmitis is an inflammatory disease of the intraocular cavities following the introduction of an infectious agent into the posterior segment of the eye[1]. Infectious endophthalmitis can be acquired by one of the following mechanisms: 1) surgical procedure (post- operative); 2) trauma caused by a penetrating foreign object (post-traumatic); 3) during metastasis of bacteria into the eye from a distant infection site (endogenous); 4) miscellaneous (e.g., secondary to keratitis)[2]. It required urgent medical attention because partial or complete vision loss is a common complication of endophthalmitis.
The causative agents of endophthalmitis mainly consist of bacteria, although a fungal etiology is not uncommon[3]. A rapid and accurate identification of the etiological agent and its antibiotic sensitivity by analysis of vitreous and/or aqueous humor forms the basis for effective treatment in infectious endophthalmitis[4]. The purpose of this study is to investigate the application of polymorphonuclear cell counts and smears in the early diagnosis of infectious endophthalmitis.
MATERIALS AND METHODS
From Nov. 1999 to Dec. 2009, we evaluated intraocular specimens (aqueous humor-AH or vitreous fluid-VF) from 44 patients with a clinical diagnosis of endophthalmitis at Eye Centre of Tianjin Medical University. From all clinical records, 36 patients were diagnosed with infectious endophthalmitis; 8 patients were diagnosed with intraocular lens (IOL)-related inflammation and were considered controls. The infectious endophthalmitis patients included 23 cases with postoperative endophthalmitis and 13 cases with post-traumatic endophthalmitis. A diagnosis of infectious endophthalmitis was made on the basis of clinical features, including periocular pain, loss of visual acuity, diffuse bulbar conjunctival hyperemia, chemosis, marked intraocular inflammation with fibrinopurulent reaction, and other signs suggestive of infectious etiology. The diagnosis of IOL-related inflammation was made by onset time, history of steroid treatment, and cytologic and microbiological examinations.
Intraocular sampling (AH and VF) was performed under absolute aseptic conditions. Test AH samples were obtained from all 44 patients by performing a paracentesis for diagnostic purposes. VF samples were collected from 24 of 36 infectious endophthalmitis patients as a therapeutic or diagnostic aspirate during pars plana vitrectomy. All the specimens were sent to the microbiology laboratory immediately. Cytospin smears of VF and AH were subjected to Gram's stain for detection of bacteria and Wright's Stain for inflammatory cells. The specimens (VF and AH) were processed within 30 minutes of collection for culture of bacteria using standard methods. Standard techniques were used to identify the species in all isolates[5]. VF and AH samples were used to inoculate blood agar plates, which were cultured at 37(C and observed after 18-24 hours. Any blood agar plate showing pathogens was tested by the minimum inhibitory concentration (MIC) method for drug sensitivity.
Statistical Analysis
The positive rate between microbiologic cultures and smears were compared using the χ2 test. For all tests, the level of significance was set at a P value of <0.05. Statistical analysis was done using the Statistical Package for the Social Sciences software version 11.0.
RESULTS
There were more inflammatory polymorph nuclear cells (seen with oil lens field under microscope) in all 36 AH and 24 VF specimens from infectious endophthalmitis patients than that in AH specimens from IOL-related inflammation patients. In IOL-related inflammation patients, 5 of 8 showed no inflammatory corpuscle in their AH, and the main cell exudates in the AH of the other 3 patients were lymphocytes. Sixteen of 36 AH specimens (44.4%) from infectious endophthalmitis patients were positive for the presence of bacteria by smears, including 11 Gram-positive cocci, 4 Gram-negative bacilli and 1 Gram-positive bacillus. Eleven VF specimens (45.8%) were positive for the presence of bacteria by smears, which was in accordance with the results for the AH samples from the same patients. Smears from all 8 IOL-related inflammation patients did not show any microorganisms.
Positive culture results were confirmed in 17 AH (47.2%) and 15 VF (62.5%) specimens from infectious endophthalmitis patients. All 8 AH specimens from IOL-related inflammation patients were negative for the presence of bacteria by culture. The culture isolates from postoperative endophthalmitis patients were mainly Gram-positive cocci, including 4 Staphylococcus epidermidis, 3 Micrococcus, 2 Enterococcus faecalis, 2 chain cocci, and 1 Staphylococcus saprophyticus. One Gram-negative bacillus-Klebsiella oxytoca and 1 Gram-positive bacillus-Bacillus subtilis were also found in culture isolates from postoperative endophthalmitis patients. Gram-negative bacilli were the main bacteria isolated from post-traumatic endophthalmitis patients, including 3 Pseudomonas aeruginosa, 1 Klebsiella pneumoniae, and 1 Morganella morganii. Two Gram-positive cocci, Staphylococcus epidermidis and Enterococcus faecalis, were also found in isolates from post-traumatic endophthalmitis patients.
In a drug sensitivity test, 14 Gram-positive cocci were all sensitive to vancomycin. Other antibiotics that were effective on these Gram-positive cocci were ciprofloxacin (12/14), oxacillin (8/14), ampicillin (6/14), and gentamicin (5/14). For 6 Gram-negative bacilli, the most effective antibiotic was ceftazidime (5/6), cefotaxime (4/6), gentamicin (3/6), and ampicillin (3/6) were also effective on Gram-negative probes. The only Gram-positive bacillus found, Bacillus subtilis, was sensitive to all regular antibiotics (vancomycin, ciprofloxacin, oxacillin, ampicillin and gentamicin).
There was no statistical difference between positive rates obtained between the smear and culture methods used to test all AH and VF specimens (Table 1). The difference in sensitivity between AH and VF samples for detection of bacteria was not statistically significant (P>0.05, Table 2).
Table 1. Comparison of results from culture and smear methods for intraocular specimens (AH & VF) from infectious endophthalmitis patients.
| AH1 (n=36) |
VF2 (n=24) |
|||
| Culture positive | Culture negative | Culture positive | Culture negative | |
| Smear positive | 16 | 0 | 11 | 0 |
| Smear negative | 1 | 19 | 4 | 9 |
| Total | 17 | 19 | 15 | 9 |
1χ2=0, P>0.05; 2χ2=2.25, P>0.05
Table 2. Comparison of results from aqueous humor (AH) and vitreous fluid (VF).
| AH | VF |
|
| positive | negative | |
| positive | 12 | 0 |
| negative | 3 | 9 |
| Total | 15 | 9 |
χ2=1.33, P>0.05
DISCUSSION
When acute inflammatory responses occur in the eye, the blood vessels around the eye dilate and vascular permeability increases. The inflammatory cells infiltrate and accumulate in the aqueous humor and vitreous fluid. The cells are mostly polymorphonuclear leukocytes (PMNs) when the inflammation is infectious; whereas, T leukocytes are the main accumulating cells of non-infectious endophthalmitis. Our results showed polymorphonuclear cells in AH and VF from post-operative or post-traumatic patients were more than in the AH from IOL-related inflammation patients. Therefore, directly counting the number of PMNs in aspirates of AH or VF could be a leading reference for the diagnosis of infectious endophthalmitis.
Smear studies including staining procedures of intraocular specimens (AH and VF) can be finished in 30 minutes after the collection of samples. This rapid report indicates the existence of pathogens and their basic characterization to clinical doctors, and provides information for choosing a suitable antibiotic and therapeutic regimen. Our study showed most postoperative pathogens were Gram-positive cocci sensitive to vancomycin; whereas, Gram-negative bacilli were mainly infectious bacteria in post-traumatic patients that were more sensitive to ceftazidime. This result is consistent with the report of Anijeet et al[6].
Culture methods are still considered the “gold standard” in the diagnosis of infectious diseases. The culture results from our study were the same as described in previous reports[7],[8]: most Gram-positive cocci were Staphylococcus epidermidis, followed by chain coccus and Enterococcus faecalis. Infectious endophthalmitis caused by Gram-negative bacteria often has a rapid progression; therefore, if Gram-negative bacteria were confirmed by smear or culture, a vitrectomy associated with antibiotic injection should be executed as early as possible[9]. Micrococcus and Bacillus subtilis are non-pathogenic bacteria and can cause chronic inflammatory reaction in eyes, so their clinical value in intraocular specimens should be evaluated in a future study.
Previous studies reported that pathogenic bacteria in AH were more easily phagocytized by self inflammatory cells than those in VF. As a consequence, the positive rate for bacteria in AH specimens was higher than that in VF specimens[7]. Our results showed the positive rates by smear and culture of VF specimens were higher than that of AH specimens, but not statistically significant. Our microscopic examination of smears suggested pathogens had been phagocytized by inflammatory cells. The bacteria in anterior chambers are more easily cleared out than those in the vitreous body. In addition, the redundant nutritional conditions in the vitreous body make bacteria grow faster than in the aqueous humor[10]. This might explain why the positive rate of VF samples was higher.
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