Microbiological spectrum and probability of transmission of infection from ocular tissues of enucleated eyes harvested from septicemic donors

Abstract

BACKGROUND

Tissues from septicemic donor are considered contraindication for transplantation. However, a definite evidence is lacking to link these corneas as a potential source of post-transplantation infection.

AIM

To assess corneal tissue microbiology in septicemic donors and correlate postmortem blood cultures with ocular isolates to determine transplant suitability.

METHODS

A bicentric cross-sectional study evaluated 264 eyes from 136 septicemic and non-septicemic donors at two eye banks of tertiary eye centers. Culture report of tissue samples (conjunctival swab, aqueous and vitreous tap, cornea, sclera, and post- mortem blood sample) was compared with the culture results of post-mortem blood samples. Additionally, correlation if any was evaluated between culture positivity of harvested tissue with death to retrieval time. Statistical results were analyzed using Pearson χ² and Fisher’s exact tests.

RESULTS

Among 21 septicemic donors, microbial growth was observed in 28.6%, 7.1%, 9.5%, 4.8%, and 2.4% of non-utilized conjunctival, corneal, scleral, aqueous, and vitreous samples respectively as opposed to 16.7 [odds ratio (OR) = 10.6, 95% confidence interval (CI): 0.938-4.263, P = 0.058], 9.0 (OR = 0.777, 95%CI: 0.141-2.815, P = 0.485), 10.8 (OR = 0.868, 95%CI: 0.207-2.739, P = 0.530), 2.2 (OR = 2.162, 95%CI: 0.199-13.78, P = 0.308) and 1.3% (OR = 1.776, 95%CI: 0.033-22.74, P = 0.502) of 115 non-septicemic donors. Only one tissue of cornea and sclera, yielded the same organism as the blood culture. The microbiological spectrum of pathogens from culture-positive tissues was reflective of commensal pathogens. A strong positive correlation was seen between death to retrieval time in relation to microbial growth.

CONCLUSION

These findings suggest that sepsis does not significantly impact ocular tissue sterility. Thus, current transplantation guidelines may benefit from further review, particularly in light of the potential to increase the availability of transplantable tissue, consequent to reduced rejection and wastage.

Key Words: Corneal transplantation; Septicemia; Corneal blindness; Donor tissue contamination; Eye banking

Core Tip: Corneal transplantation remains an important sight restoring procedure, but has major demand-supply gap of donor corneas, worsened by exclusion of septicemic donors due to presumed infection risk. This bicentric cross-sectional study evaluated the microbiological spectrum of ocular tissues harvested from septicemic donors and their potential risk of infection transmission. Cultures from conjunctiva, cornea, sclera, aqueous, vitreous, and post-mortem blood were assessed and correlated. Microbial growth rates were comparable between groups, with organisms largely reflecting commensal flora. Only one corneal and scleral sample matched blood culture isolates. A strong association was found between longer death-to-retrieval time and contamination. Overall, septicemia did not significantly compromise tissue sterility, suggesting potential reconsideration of transplantation guidelines to reduce tissue wastage.

INTRODUCTION

Corneal blindness remains a significant cause of preventable blindness worldwide[1]. To mitigate the high incidence of corneal blindness, corneal transplantation has emerged as the primary vision-restoring surgical procedure, which relies heavily on voluntary eye donation from suitable donors. In India, corneal opacities consequent to trauma and infective keratitis account for approximately 7.4% of the total burden of blindness, creating a substantial and growing demand for transplantable corneal tissue. An estimated 200000 donor corneas are required annually, yet only about 65000 are procured each year, resulting in a persistent shortfall of nearly 135000 corneas[2-4].

Hospital Cornea Retrieval Programmes represent a vital strategy for eye banks to enhance corneal tissue procurement. In hospitalized patients, septicemia is one of the leading causes of death[5,6]. Regulatory guidelines classify corneal tissue from septicemic donors as a contraindication for transplantation due to concerns regarding potential donor-to-recipient transmission of infection (standards of eye banking in India, National Program for Control of Blindness, 2020)[7]. Studies have suggested the possibility of such transmissions from septicemic donors through the corneal button, implicating a direct transfer of the pathogen from donor to recipient[8-11]. Some studies have reported a positive correlation between positive corneal rim culture and higher odds of post-corneal transplantation infection rate in the host eye[12-14]. It is postulated that during septicemia, microorganisms may circulate within ocular blood vessels; however, given the avascular nature of the cornea, it is theoretically expected to remain free of organisms[15]. Several studies suggest that current stringent exclusion criteria related to septicemia lack direct evidence of transmission of infection[15-19]. This may lead to the loss of potential eye donors and contribute to the persistent shortage of transplantable corneal tissue[20-22].

This emphasizes the need for large database studies to define any true association and to re-evaluate current contraindications using evidence-based criteria, thereby potentially expanding the donor pool and increasing the availability of good-quality corneal tissue. Given that a positive blood culture is a definitive marker of active septicemia[23], this study evaluates the microbiological spectrum and risk of infection transmission to donor ocular tissues from septicemic patients, as well as the impact of death-to-retrieval time and post-mortem blood culture positivity on transmission risk.

MATERIALS AND METHODS

The study was undertaken as a bicentric cross-sectional study conducted at two eye banks of tertiary eye care centers over a period of three years. The study groups I and II comprised ocular tissues retrieved from septicemic and non-septicemic donors, respectively. Immediate relatives of the deceased who were in legal possession of the body were approached for eye donation. They were informed about the possible utilization of donated eyes for therapeutic or research purposes in their own language, and their consent, if available, was documented on standardized tissue retrieval consent form. Relatives who refused to participate in the study or refused to donate their eyes for research purposes were excluded from the study. The research adheres to the tenets of the Declaration of Helsinki and was approved by the Institutional Ethics Committee of both the study centers (approval No. IHEC-LOP/2018/IM0200). Written informed consent was obtained from the next of kin of the deceased who consented to eye donation.

Inclusion criteria

The study comprised of two groups. Group I (septicemic donors), which included consecutive ocular tissues harvested from donors diagnosed with ‘active septicemia’ based on the documentation available from hospital records and as documented in death certificate/summary. Active septicemia at the time of death was defined as per the following criteria by the Standard of Eye Banking Guidelines, India[7]: (1) Clinical evidence of sepsis (including, but not limited to, bacteremia, viremia, fungemia, septicemia, sepsis syndrome, systemic infection, systemic inflammatory response syndrome, or septic shock; (2) Clinical evidence of infection; (3) Two or more of the following systemic features if highly suggestive of active infection, unexplained by other disease processes: Temperature of > 38 °C (100.4 °F); heart rate > 90/minute; respiratory rate > 20/minute or pressure of arterial carbon dioxide < 32 mmHg (4.3 kPa); white blood cell > 12000 cells/mm³, < 4000 cells/mm³, 114 or > 10% immature (band) forms; and (4) Positive (pre-mortem) blood cultures.

Group II comprised of non-septicemic donors based on death summary/certificate where the cause of death was other than septicemia. The donor tissues harvested in this group specifically comprised of any cause of death rendering the tissue non-utilizable, like systemic/Local malignancies, death of unknown cause, recent history of systemic or ocular inflammation, poor quality tissues (grade poor or less as per Eye Bank Association of India grading system)[24]. Specific cause of death were road traffic accidents with organ failure and on ventilator for > 7 days, hepatitis B related encephalopathy in chronic liver disease, cerebrovascular accident with brain stem infarction with severe exposure keratopathy, active viral encephalitis with respiratory failure etc. This group served as the control group.

Exclusion criteria

Refusal to participate in the study, by immediate relatives of the deceased in possession of body. Refusal of eye donation consent for research purpose, by immediate relatives of deceased in possession of body.

Tissue retrieval and microbiological evaluation

In compliance with the inclusion criteria, donors were registered for study as exhibited in Figure 1. Detailed history with reference to history of ventilator support, blood transfusion in previous three months, use of immunosuppressive drugs and history of high-risk behavior was elucidated. Detailed pre-mortem blood counts and culture reports, if available, were abstracted from medical records.

Figure 1 Clinical workflow of eye donation calls, tissue retrieval and constitution of study groups. ¹74 donors - single eye, 40 donors - both eye; ²21 donors - both eyes; ³4 donors - single eye 91 donors - both eyes; ⁴12 donors - single eye, 2 donors - both eyes; ⁵12 donors - single eye, 7 donors - both eyes; ⁶49 donors - single eye, 6 donors - both eyes; ⁷45 donors - single eye, 40 donors - both eye; ⁸22 donors - single eye, 7 donors - both eyes; ⁹52 donors - single eye, 33 donors - both eyes. N: Number of donors; n: Number of ocular tissue; OT: Ocular tissue.

At the time of tissue retrieval balanced salt solution was used to clean the ocular surface and to keep it moist. Topical povidone iodine and antibiotics were not administered in both groups during harvesting to prevent organismal suppression. Project in-charge of both study centers harvested the tissues after isolating the surgical field with plastic drape/Linen towel. First conjunctival swab sample was taken, followed by whole globe enucleation, preservation in moist chamber and transfer to eye bank. After proper documentation, eyeballs were released for research purpose, clearly mentioning the contraindication for therapeutic use in corneal transplant.

Tissue samples as harvested from two groups comprised of cornea, sclera, aqueous tap, vitreous tap in addition to conjunctival swab. Conjunctival swab sample was obtained by using a sterile swab along the inferior fornix. Whole eye ball was enucleated from orbital socket with aseptic precautions and placed in moist chamber to be transferred to eye bank. The ocular tissues were sectioned in operation theatre under microscope with standard aseptic precautions. 0.2 mL aqueous tap was obtained using a 30G needle through temporal limbus. Vitreous tap was obtained using a 27G cannula mounted on a tuberculin syringe, 4 mm behind the limbus in supero-temporal quadrant. Subsequently corneal and scleral tissue were retrieved.

All samples were obtained separately for both the eyes with clear identification (coding) and documentation to avoid any ambiguity related to tissue type and laterality in microbiological assessment. The labeling of ocular tissue samples was done using tissue codes as ocular tissue right (OTR) and ocular tissue left (OTL), denoting ocular tissue from right and left eye respectively for each donor. While numerical coding was done for each tissue to avoid any culture analysis related equivocation. Numerical codes from 1 to 4 were allotted to each tissue, where 1, 2, 3 and 4 denote corneal, scleral, aqueous and vitreous samples respectively. Blood sample (10 mL) was collected from subclavian or jugular vein or directly through intracardiac route, under aseptic conditions to be further evaluated for post-mortem blood culture. For each donor, as a part of standard protocol, death to retrieval time was noted.

All the samples (conjunctival swab, corneal and scleral tissue, aqueous tap and vitreous tap) were inoculated on the culture media namely blood agar, chocolate agar, thioglycolate broth and Sabouraud’s dextrose agar plates under aseptic conditions. The plates thus inoculated were sent to the department of microbiology for further processing and identification of microbial growth if any.

For culture of corneal and scleral tissues (2 mm away from limbus), a 3 mm × 3 mm section was taken. All samples were sent for culture analysis in both the groups. Microbiological culture of post-mortem blood samples was done for all the donors, instead of relying only on the available pre-mortem blood culture reports. Of 5-10 mL blood sample was retrieved in the collection vial for blood culture and incubated up to seven days or earlier in case of positivity, flashed in automated blood culture system - BacT-Alert 480 (BioMerieux, France). The samples were processed as per standard microbiological techniques and identification of bacterial isolate was done by Vitek2^® and few from conventional culture method of identification. Antimicrobial susceptibility test of the clinical isolates was determined by the Gram-negative, Gram-positive, Vitek2^® antimicrobial susceptibility test cards (N280, N281, P628) (bioMerieux, France), as per manufacturer’s instructions. Post-mortem blood culture analysis was performed for all the donors in study groups. Microbiological culture reports were evaluated for growth of organisms in all tissue samples of both groups. Additionally, correlation if any was assessed between positivity of culture samples with death to preservation time (DPT).

Interpretation of culture reports

Inoculated culture media were evaluated for growth of organisms if any, in tissue samples of study groups in addition to post-mortem blood culture analysis for the donors of both groups. Interpretations were drawn as follows: (1) ‘Negative’ - when no growth was detected after 48 hours of aerobic incubation at 37 °C on any of the culture media inoculated with donor ocular tissues; (2) ‘Positive’ - if the culture plate exhibited growth of organism; (3) ‘Contamination’/‘commensal growth’ - if the organism isolated from ocular tissue was different from the organism isolated from donor blood; and (4) ‘True positive’ - if the organism isolated from the ocular tissue was identical to that isolated from the blood, it was interpreted that the organism causing sepsis was transmitted to donor ocular tissues.

Statistical analysis

Data collected was abstracted and recorded in a pre-designed proforma with subsequent analysis in a standardized format using Microsoft excel. Analysis was performed using IBM-SPSS Statistics v 24.0.0 software with Pearson χ² test and Fisher’s exact depending on the type of variables. P < 0.05 [95% confidence interval (CI)] was considered statistically significant. Recommendations as per Cochran’s criteria were used to assess the level of significance for χ² test results. Post-mortem blood culture analysis was performed for all the donors in study groups. To simplify analysis, amongst the samples tested for different tissues in both the groups, we counted each eye as single sample and hence for blood culture obtained from same donor with two enucleation tissues retrieved, we considered same blood sample twice in statistical analysis.

RESULTS

Of 264 tissues from 136 donors were included in the study as per the inclusion criteria and summarized in Figure 1. Bilateral ocular tissues were retrieved from 128 donors, while single eye retrieval was performed in eight donors to a total of 134 OTR and 130 OTL. Group I consisted of 42 ocular tissues (21 OTRs and 21 OTLs) from 21 donors, while group II consisted of 222 tissues (113 OTRs and 109 OTLs) from 115 donors. The mean age of donors in group I was 51 ± 3 years while in group II it was 48 ± 5 years. In septicemic group, 61.9% corneas were of optical grade as opposed to 56.3% in non-septicemic group.

Post-mortem blood culture analysis

In group I, out of total 42 samples, 16 samples tested positive to reflect a positivity rate of 38.10%. While, among 222 samples of group II donors, despite being part of non-septicemic group, 12 samples tested positive to reflect a positivity rate of 5.40% (odds ratio: 10.6; 95%CI: 4.2-27.63, P = 0.000) results being statistically significant (Table 1). If the organism isolated from the ocular tissue was identical to that isolated from the blood it was considered as true positive, with interpretation that the organism causing sepsis was transmitted to donor ocular tissues. True positive result was analyzed between both study groups. It was seen that in group I, single subject was true positive, with cornea and sclera, showing growth of same organism as the blood culture (Klebsiella pneumoniae), while in group II none of the results showed true positive.

Table 1 Microbiological culture analysis in group I and II.

Group	Blood culture			Conjunctival culture			Corneal culture			Scleral culture			Aqueous culture			Vitreous culture
	Positive		Negative	Positive		Negative	Positive		Negative	Positive		Negative	Positive		Negative	Positive		Negative
Group I (septicaemic), n = 42	16	38.10%	26	12	28.57%	30	3	7.14%	39	4	9.52%	38	2	4.76%	40	1	2.38%	41
Group II (non-septicaemic), n = 222	12	5.40%	210	37	16.66%	185	20	9.01%	202	24	10.81%	198	5	2.25%	217	3	1.35%	219
P value	0.000			0.058			0.485			0.530			0.308			0.502
Odds ratio	10.6			1.99			0.777			0.868			2.162			1.776
95% confidence interval	4.2-27.63			0.938-4.263			0.141-2.815			0.207-2.739			0.199-13.78			0.033-22.74

Among group I most common organism detected was Enterobacter species (9.52%), followed by other organisms in almost equal proportion. Staphylococcus aureus and Pseudomonas aeruginosa were the most common pathogens in non-septicemic group, accounting for 1.80% of the total (Table 2).

Table 2 Microbiological spectrum in ocular tissues of study groups.

Isolated species	Conjunctival swab		Corneal		Scleral		Aqueous		Vitreous		Blood
	Group I (n = 42)	Group II (n = 222)	Group I (n = 42)	Group II (n = 222)	Group I (n = 42)	Group II (n = 222)	Group I (n = 42)	Group II (n = 222)	Group I (n = 42)	Group II (n = 222)	Group I (n = 42)	Group II (n = 222)
Klebsiella pneumoniae	1	-	1	-	1	1	-	-	-	-	2	-
Streptococcus viridans	-	2	1	1	-	-	-	-	-	-	2	-
Streptococcus pneumoniae	-	-	-	-	-	1	-	-	-	-	-	-
Staphylococcus aureus	3	8	-	6	1	4	-	-	-	-	-	4
Coagulase-negative Staphylococcus	6	19	1	13	2	17	-	5	-	3	2	-
Enterobacter species	-	2	-	-	-	-	-	-	-	-	4	-
Haemophilus influenza	-	-	-	-	-	-	2	-	1	-	-	-
Propionibacterium acnes	1	-	-	-	-	-	-	-	-	-	-	-
Candida albicans	-	-	-	-	-	-	-	-	-	-	2	2
Escherichia coli	-	3	-	-	-	1	-	-	-	-	2	2
Pseudomonas aeruginosa	-	-	-	-	-	-	-	-	-	-	-	4
Mixed1	1	3	-	-	-	-	-	-	-	-	2	-

¹Mixed isolates consist of: Group I: (1) Coagulase-negative Staphylococcus and Streptococcus viridans; group II: From 3 different eyes of three donors; Staphylococcus aureus and Streptococcus viridans; (2) Coagulase-negative Staphylococcus and Streptococcus viridans, and (3) Coagulase-negative Staphylococcus and Enterobacter species.

Culture analysis of corneal tissue in group I and II

In group I, microbial growth was seen in three (7.14%) ocular tissue 1, that included Klebsiella pneumoniae, Streptococcus viridans and Staphylococcus epidermidis respectively in each of the positive tested donor tissue (Table 2). In Group II, 20 (9.01%) ocular tissue 1 showed evidence of microbial growth on culture. The difference between the rate of microbial growths between the two groups (P = 0.485; odds ratio = 0.777; 95%CI: 0.141-2.815) was statistically insignificant (Table 2). Most common organism detected was Staphylococcus epidermidis (4.50%) followed by Staphylococcus aureus (2.70%) (Table 2).

Culture analysis of other ocular tissues in group I and II

In group I, microbial growth was seen in 12 (28.57%) conjunctival swabs, with Staphylococcus epidermidis (11.90%) and Staphylococcus aureus (7.14%) being the commonest identified organisms. Whereas in group II, 37 (16.66%) conjunctival swabs revealed positive microbial growth with Staphylococcus epidermidis (6.30%) and Staphylococcus aureus (3.60%) constituting the commonest organisms isolated (Table 2). No correlation was observed between the incidence of microbial growth in conjunctival culture between the two groups (P = 0.058; odds ratio = 1.99; 95%CI: 0.938-4.263) (Table 1). With reference to scleral culture, Group I showed microbial growth in four (9.52%) with Staphylococcus epidermidis being the most common isolated organism (Tables 1 and 2). Whereas in group II, 24 (10.81%) ocular tissue 2 revealed positive microbial growth with Staphylococcus epidermidis (5.40%) being the most common organism, however a nonsignificant correlation (P = 0.530; odds ratio = 0.868; 95%CI: 0.207-2.739) (Table 1).

Concerning aqueous and vitreous tissue samples, group I showed microbial growth in two (4.76%) and one (2.38%) sample respectively with Hemophilus influenzae as the only organism detected from all three samples (Table 2). Culture positive sample of vitreous, was from the same donor in which one aqueous sample tested positive. While among group II, microbial growth was obtained in 2.25% aqueous and 1.35% vitreous samples. Overall, results remained insignificant (P = 0.308; odds ratio = 2.162; 95%CI: 0.199-13.78, P = 0.502, odds ratio = 1.776, 95%CI: 0.033-22.74) (Table 1).

Correlation of post-mortem blood culture with tissue samples in study groups

In compliance with study objectives to analyze the two groups with regards to positive post-mortem blood culture and to further asses the possible risk of direct transmission of infection, ocular tissue from 16 post-mortem blood culture positive donors in group I, were compared with 210 post-mortem blood culture negative donors from group II. However, the analysis for any of the tissue samples from both the groups were not reflective of any statistically significant correlation (P > 0.05) (Table 3).

Table 3 Microbiological analysis of ocular tissues harvested from blood culture positive septicemic group and blood culture negative non-septicemic donors.

Group	Conjunctival swab			Corneal tissue			Scleral tissue			Aqueous tap			Vitreous tap
	+	-	P value	+	-	P value	+	-	P value	+	-	P value	+	-	P value
Septicemic (n = 16)	3	13	0.564	2	14	0.477	1	15	0.450	2	14	0.080	1	15	0.256
Non-septicemic (n = 210)	37	173		20	190		24	186		5	205		3	207
Odds ratio	1.079			1.355			0.518			5.768			4.543
95% confidence interval	0.188-4.207			0.140-6.603			0.012-3.672			0.506-39.39			0.082-60.7

+: Positive culture, -: Negative culture.

While analyzing different ocular tissue from referenced sixteen post-mortem blood culture positive group I donors, three conjunctival swabs, two ocular tissue 1, one ocular tissue 2, two ocular tissue 3 and one ocular tissue 4 sample showed positive growth on culture. However, only one ocular tissue 1, one ocular tissue 2 and one conjunctival swab revealed the true positive microbial growth (Klebsiella pneumoniae). Numerical codes from 1 to 4 were allotted to ocular tissue, where 1, 2, 3 and 4 denote corneal, scleral, aqueous and vitreous samples respectively. ocular tissue 1, ocular tissue 2 and conjunctival swab were from the same eye of a single donor. However, for the same donor, none of the ocular tissue from the other eye showed any microbial growth. Rest of the ocular tissues which showed microbial growth did not reveal similar microorganism as in post-mortem blood culture. Staphylococcus species followed by Hemophilus remained the most common organisms obtained from these tissues.

Correlation between DPT and culture positivity rate in study groups

DPT of all corneal tissues were analyzed and compared with the results of microbial growth. In group I for all the three corneal tissues which tested culture positive the DPT was > 12 hours showing a statistically significant correlation (P = 0.010) (Table 4). While among group II donors, seven and thirteen corneal tissues showed microbial growth respectively at ≤ 12 hours and > 12 hour of DPT. The results being statistically significant (P = 0.0003) implicating an increase in microbial growth with increasing duration of DPT (Table 4).

Table 4 Correlation of death to preservation time with microbial growth in study groups.

Group	Culture results	Death to preservation time		P value
		≤ 12 hours	12 hours
Septicemic donors (n = 42)	Positive	0	3	0.0104
	Negative	32	7
Non septicemic donors (n = 222)	Positive	7	13	0.0003
	Negative	153	49

DISCUSSION

Infectious keratitis and endophthalmitis are rare but devastating complications after corneal transplantation surgery[25,26]. Infected donor corneal tissue is considered as one of the major risk factors for corneal transplantation, justifiably rendering septicemia as a contraindication for corneal donation. Standards of Eye Banking and International guidelines advocate septicemia as one of the contraindications for utilization of corneal tissue for transplantation[7]. When compared to positive bacterial rim cultures, positive donor corneal rim fungal cultures at the time of keratoplasty may be more predictive of infectious keratitis and endophthalmitis[12]. The possibility of a higher risk of graft infection or endophthalmitis for grafts retrieved from septicemic donors has been investigated in the past, but results have proven equivocal[8,12-14]. Confirmatory evidence regarding the direct spread of blood specific infection to donor ocular tissue is still deficient, hence there is considerable ambiguity in ascertaining whether the infection in donated ocular tissues, is a result of septicemia or due to local contamination[27].

Term ‘septicemia’ as per Food and Drug Administration comprises of 10 clinical parameters, which need to be assessed from the treatment summary of the deceased at the time of corneal tissue retrieval[7,28]. Blood culture analysis at death is one of the parameters that can be considered as a definitive sign, which was observed to be independently associated with active septicemia[23]. In our study, we evaluated all donors in study groups for post-mortem blood culture. All the donors included in septicemic group did not test positive on post-mortem blood culture analysis, the positivity rate being meager 38.10%, which can be attributed to intravenous antibiotics in hospitalized patients and further correlated with half-life of antibiotics, duration, dos, sensitivity etc. Moreover, around 6% of blood cultures taken from non-septicemic group donors also showed microbial growth despite being categorized as non-septicemic based on death summary/medical records. Results seem to lack a correlation between clinical signs of sepsis and predict active septicemia. Similar results (34% vs 25%) were described by Mathur et al[15] in a cross-sectional study on seventeen donors.

The incidence of donor corneo-scleral rim contamination ranges from approximately 12% to 48%, depending on the methodology used for obtaining cultures[29-31], while the incidence of corneal and scleral tissue positivity in septicemic donors varies from 0%-28% irrespective of the blood culture results[15,18]. In our study, among the septicemic donor eyes, positive cultures were obtained in 28.57% conjunctival, 7.14% corneal and 9.52% scleral samples. Similar results were obtained in group II with 16.66% conjunctival, 9.01% corneal and 10.81% scleral sample positivity. The conjunctival tissue had the highest microbial culture positivity among the ocular tissues, and this is likely attributable to the presence of conjunctival commensal flora, increased environmental exposure of the ocular surface, and the absence of povidone-iodine application during the procedure. The rest of the ocular tissues did not show positivity similar to blood culture, which reflects that sterility of the ocular tissues is not affected by septicemia. On further sub-group analysis of post-mortem blood culture positive septicemic donors with the post-mortem blood culture negative non-septicemic donors, the proportion of positive tissue cultures obtained in the septicemic group was 18.75% in conjunctival, 12.5% in corneal and 6.25% in scleral samples. There was only one sample that tested positive for the same microbial growth in conjunctival, corneal and scleral culture, and a similar pathogen as that of the blood culture. However, the culture results from the other eye of the same donor revealed no microbial growth. The rest of the samples that tested positive on culture either reflected different pathogens as compared to blood culture or different pathogens in different tissue samples of the same eye. The microbiological spectrum of pathogens from these culture-positive tissues mainly consisted of the commensal pathogens, including Staphylococcus epidermidis, Staphylococcus aureus, coagulase-negative Staphylococcus species, Streptococcus and Klebsiella, which commonly constitute the ocular flora[32-34]. No definitive and significant correlation was observed between the donors of both groups.

There remains a possibility of direct spread of infection from blood to the ocular tissues through aqueous or limbal circulation. Considering the aforementioned probability, aqueous humour cultures should reflect the presence of the same organism with positive microbial growth on culture analysis. Keates et al[35] and Olson et al[36] in their study on donors’ eyes observed a significant contamination rate of aqueous. The authors suggested that septicemia could contaminate ocular tissues, perhaps via the aqueous or limbal circulations. However, there are other evidence in the literature that supports that anterior chamber sterility does not seem to be compromised by chronic debilitating disease or terminal bacteremia in patients with active septicemia. In a study on the cadaveric group, consisting mainly of elderly patients with documented high terminal bacteremia, Clark et al[16] observed that the aqueous is capable of maintaining its sterility, by virtue of an intact blood aqueous barrier. In another study by Mathur et al[15], 17% of aqueous tap cultures tested positive in a group of septicemic donors, but all with negative blood culture. While Nagaraja et al[18] observed a high polymerase chain reaction positivity rate (27%) of aqueous and vitreous, but none with a similar organism as those causing septicemia.

We observed microbial growth in just 4.76% of aqueous samples and 2.38% of vitreous samples, with none of the identified organisms corresponding to the sepsis-related pathogens in the donors. Additionally, the rate of microbial growth was almost similar between both septicemic and non-septicemic donors. The microbiological spectrum of pathogens obtained from aqueous and vitreous in both groups is reflective of commensal organisms commonly constituting the ocular flora. These observations indicate possible microbial contamination of the donor tissue, the most attributable reason being the suboptimal retrieval technique of donor tissue and not from the active septicemia of the deceased. Similarity in microbiologic profile was not evident in these two groups. Blood culture positivity was seen in 38.10% of group 1 and 5.40% of group 2. While microbial growth was seen in 7.14% and 9.01% of corneal tissue in groups I and II, respectively. Hence, it can be inferred that contamination of tissues can occur independently of systemic infection. Therefore, stringent aseptic precautions during postmortem tissue retrieval are strongly recommended.

In a study to identify significant factors influencing the suitability and main reasons for discarding organ-cultured corneas, Kramp et al[27] observed septicemia as a cause of death, which resulted in significantly higher discard rates due to increased contamination of the culture medium. Our study shows that the presence of septicemia does not have any definitive correlation with ocular tissue infection, nor does it have any consistent relationship between pathogens present in the blood and the ocular tissues. Study outcomes are similar to previous observations put forth by Spelsberg et al[17] and Mathur et al[15], who reported no such definitive correlation.

According to approved eye bank practices, death-to-retrieval time and DPT < 6 hours are preferred for retrieval and therapeutic utilization[7,37]. However, no clear correlation has been established between graft infection and DPT. In a study to assess DPT and its impact on utilization of donor corneas, Patel et al[38] did not observe any significant association of duration of DPT with graft infection. However, a significantly higher rate of pre-utilization culture positivity was observed with prolonged DPT[38]. In our study, we considered the effect of DPT on the culture positivity in both donor groups to evaluate any possible correlation, between the duration of DPT and rate of microbial growth. Study results showed a higher risk of donor tissue positivity in relation to DPT among both the groups. These findings further support the fact that, among both septicemic and non-septicemic donors, a significant proportion of tissues exhibited microbial growth and increased DPT can be an attributable factor for increased contamination of ocular tissue. Despite following all aseptic measures for corneal graft harvesting, the incidence of corneal graft contamination has been reported to be as high as 20%, driven by bacterial contamination of donor rims[11]. Furthermore, in the cornea preservation time study, even the utilization of culture-positive donor tissue was not found to be associated with an increased risk of post-keratoplasty infectious keratitis and endophthalmitis[39].

National Program for Control of Blindness, as well as other studies in the recent past, have reported that despite a consistent and significant increase in the number of donor eyes collected, the backlog of patients awaiting corneal transplantation remains inadequately addressed, owing partly to decreased utilization rate[40]. Failure to retrieve corneas from septicemic donors contributes considerably to donor cornea wastage. This concern mandates a justifiable need to reassess and reformulate currently prescribed contraindications.

Considering the profile of unutilized septicemic donor tissues in the present study, 17 donor tissues did not show microbial growth on blood culture and ocular tissue cultures. Out of such 17 donor corneas, 13 corneas were of ‘optical’ grade. Similarly, 11 donor tissues in the septicemic group tested positive on blood culture but did not show microbial growth in any of the ocular tissues. Out of such 11 donor corneas, 8 corneas were of ‘optical’ grade. In a favourable scenario of considering these tissues for transplantation, it would have increased the utilization rate for transplantation significantly with the addition of 21 optical-grade corneal tissues.

The lack of correlation between organisms isolated from septicemic blood cultures and corneal cultures suggests that systemic infection does not necessarily lead to corneal contamination. Hence, acceptance of corneas from septicemic donors with negative corneal and rim cultures can safely expand the donor pool and increase the availability of good-quality tissue, as many such donors provide clinically suitable corneas. Accordingly, corneas from septicemic donors should not be discarded solely on the basis of positive blood cultures. Current contraindications warrant re-evaluation to optimize tissue availability without compromising safety.

This study has certain limitations, mainly pertaining to the fact that the probability of an organism being attributed as being responsible for infection, if isolated from the blood as well as ocular tissue, remains an area of uncertainty, particularly for commensals of the skin. There remains a probability, even though small, for inadvertent creep in the samples. Additionally, it is not definitive that true positives will only be those organisms that exhibit identical isolation from blood and ocular tissues, since the culture yields are dependent on variables such as the organism load in the sample and its viability. These are also the inherent limitations of the currently available methods. Moreover, we have not considered the pre-mortem blood culture results in statistical analysis because of the following reasons: Unavailability of pre-mortem culture results in every case to maintain homogeneity among donor data, time frame of pre-mortem blood culture positivity duration, and effects of subsequent antibiotic therapy during hospitalization of the deceased.

CONCLUSION

The study showed that microbial growth was detected in only a small proportion of corneal and aqueous samples 7.14% and 4.76%, respectively, and none of the isolates corresponded to the pathogens responsible for donor sepsis. Furthermore, the rate of microbial growth was identical between tissues from septicemic and non-septicemic donors. Based on statistically significant evidence, septicemia does not appear to compromise the sterility of ocular tissues. The study demonstrates that ocular tissues from septicemic donors have equally low infection rates compared to those from non-septicemic donors, reflecting their suitability for transplantation.

These findings support the need to reevaluate current eye banking guidelines that categorize septicemia as a contraindication for tissue transplantation. Furthermore, the study highlights the importance of enhancing ocular surface sterilization and adhering to strict aseptic protocols to minimize contamination of transplantable ocular tissues.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the assistance of Dr. Abhijit P Pakhare, MD, Community and Family Medicine and Statistics Unit, for reviewing this manuscript and helping on statistical analysis, and Dr Shashank Purwar, MD, (All India Institute of Medical Sciences, Bhopal) microbiology for microbiological analysis of tissue samples.