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Journal of Clinical Microbiology, November 2004, p. 5403-5405, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5403-5405.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

An Unexpected Experimental Pitfall in the Molecular Diagnosis of Bacterial Endophthalmitis

Luana Ugahary,1 Wendy van de Sande,2 Jan C. van Meurs,1 and Alex van Belkum2*

The Rotterdam Eye Hospital,1 Department of Medical Microbiology & Infectious Diseases, Erasmus Medical Center, Rotterdam, The Netherlands2

Received 2 June 2004/ Returned for modification 5 July 2004/ Accepted 17 July 2004


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ABSTRACT
 
General primer-mediated ribosomal DNA amplification during endophthalmitis may improve the quality of diagnostic microbiology. However, extreme care needs to be taken not to introduce contaminating bacterial DNA during surgery procedures. The use of decontaminating iodine solutions can lead to such contamination due to the presence of DNA from Pseudomonas-like organisms.


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TEXT
 
Postoperative bacterial endophthalmitis is a rare complication of intraocular surgery or trauma. Bacterial toxins in particular are known to cause significant damage to retina and uvea, whereas the inflammatory process in itself also adds to the damage caused by the infection. Bacterial virulence is considered the main variable contributing to morbidity, and the absence of positive-testing cultures during endophthalmitis is a predictor for a positive outcome (1, 5). The present "gold standard" for the diagnosis of bacterial endophthalmitis is culture-based analysis of vitreous samples. Obtaining these samples, however, carries a risk for the patient and may lead to vitreous hemorrhage and retinal detachment. It would be a major improvement if vitreous samples could be replaced by samples derived from the anterior compartment of the eye. In general, however, these samples display a lower sensitivity in case of microbiological testing; consequently, a more sensitive laboratory tool for diagnosis is required (1). PCR could provide such a means, since this extremely sensitive method is capable of detecting limited numbers of DNA molecules (12). Several reports have described the application of PCR testing for the detection of bacterial endophthalmitis (9, 10, 11, 12, 13, 16). However, the general ribosomal DNA (rDNA) primed approach is highly sensitive to contamination. We applied bacterial rDNA amplification for diagnosing ocular infections and identified an unexpected source of bacterial DNA contamination.

From 1 September 2002 until 30 September 2002 we obtained undiluted vitreous or aqueous samples (n = 14) from several groups of patients requiring surgery at the Rotterdam Eye Hospital. This study has been approved by the institutional review board, and written informed consent was obtained from all patients.

Five patients presented with suspected bacterial endophthalmitis. Vitreal samples (from one of which an additional aqueous sample was obtained) from three patients were collected using a vitrectome after opening of the conjunctiva in the operation theatre, and a vitreous sample from two patients was obtained using a 27-gauge needle through the conjunctiva in the office. Eight control patients underwent vitrectomy for macular pucker (n = 3), macula hole (n = 1), or retinal detachment (n = 4). All control vitreous samples were obtained with the aid of a vitrectome after opening of the conjunctiva in the operation theatre.

All patients were treated twice with 0.3% Betadine iodine solution (Viatris, Diemen, The Netherlands) (10% stock) in a balanced salt solution (BSS; B. Braun, Meisingen, Germany) (0.9% NaCl) in the last 30 min before surgery. Immediately prior to surgery, bacterial decontamination of the eye was performed by generous application of a 1% dilution of Betadine iodine solution in BSS. The decontamination fluid was prepared fresh every day. Samples from the eye were collected during surgery, injected into sterile Eppendorf reaction vessels, and stored at –20°C.

Nucleic acids were extracted from the clinical specimens essentially according to the method described by Boom et al. (3). First, the samples were treated by lysozyme, lysostaphin, and proteinase K including 1% sodium dodecyl sulfate to destroy bacterial cell walls and bulk protein. DNA was stored in 10 mM Tris-HCl (pH 8.0) at –20°C. rDNA PCR amplification was performed using the primers EUB-L (5'-CTTTACGCCCATTTAATCCG-3') and EUB-R (5'-AGAGTTTGATCCTGGTTCAG-3') according to the method of Wilson et al. (18). The PCR fragments thus generated were analyzed directly by electrophoresis and restriction fragment length polymorphism (RFLP) using the restriction enzyme AluI (Boehringer-Mannheim, Mannheim, Germany) according to the manufacturer's instructions. DNA extraction, amplification, and RFLP analysis of fluids used for decontamination and sterilization before surgery were performed similarly to the negative process controls. Control samples included chlorhexidine, 0.9% NaCl solutions, 10% Betadin, 0.3% BSS, and 1% Betadin in BSS and tap water. In addition, the degree of DNA contamination of the Eppendorf tubes used for sample storage was assessed.

All clinical samples gave rise to successful DNA isolation, and the rDNA PCR results were positive in all instances. This was surprising, since not all samples were collected from patients suffering from obvious bacterial endophthalmitis, as patients operated on for noninfectious reasons were included as well. In addition, when the 550-bp-long PCR product was digested with the restriction enzyme AluI, strikingly similar RFLP patterns were produced (Fig. 1). Only in the cases of three samples (see lanes 11, 12, and 14 in Fig. 1) was a deviating pattern observed. Interestingly, two out of three of these samples were shown, by culture, to contain coagulase-negative staphylococci. To study this in more detail, one of the common PCR fragments was subjected to DNA sequencing. BLAST analysis of the sequence obtained revealed that the organisms involved was either identical to or at least very closely related to Pseudomonas sp. strain G2, Pseudomonas putida, and/or Pseudomonas gessardi. These are all species that have never before been identified as causal agents of bacterial endophthalmitis. The ribosomal DNA sequence of the PCR fragment was over 99% (514 of 519 nucleotides) identical to the sequence of Pseudomonas sp. strain G2. Upon further analysis it appeared that extracts from the fluids used to decontaminate the eye before surgery gave rise to identical PCR RFLP patterns. The BSS as well as the diluted iodine working solution scored strongly positive in the PCR. This ultimately pointed to the fluids used for eye decontamination as important sources of contaminating DNA. Nonsterilized tap water showed the same PCR signal, although it was far less intense (results not shown); the chlorhexidine solution results were negative, as were most of the nonsterilized Eppendorf tube results. It has to be emphasized that microbiological cultivation of the decontamination fluids did not result in growth of the microorganism (results not shown).



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  		FIG. 1. General ribosomal primer-mediated amplification of DNA extracted from perioperational antiseptic fluids versus vitreous and aqueous samples from patients suffering from endophthalmitis. Panel A shows the amplified products, which are approximately 550 bp in length. In panel B, 15 AluI restriction digests of the same amplicons are displayed together with two molecular size markers (100-bp ladder; Invitrogen, Breda, The Netherlands) (lanes M). On the right side of the panel, the 100-bp-long marker molecule is indicated by an arrow. The nature of the samples from which the DNA was extracted is indicated at the bottom of the picture. Lanes 1 and 2 represent the results for BSS and diluted iodine solutions. The other samples derived from patients as follows: aqueous (A) samples from cases of endophthalmitis (E) lanes 3 to 5, 7, 8, and 11 to 15); an aqueous sample from a case of retinal detachment (lane 6); and vitreous (V) samples from a case of retinal detachment lanes 9 and 10). The arrows show those fragments that are indicative of the presence of specific bacterial species beyond the DNA of the contaminating Pseudomonas spp. The samples represented in lanes 13 and 15 yielded coagulase-negative staphylococci upon microbiological cultivation.

The finding that antiseptics and surgical materials may be contaminated with bacteria is not new, with the first reports dating back as far as the mid-1970s. By that time it had already been demonstrated by bacterial culturing that viable bacteria can be present in benzalkonium antiseptic (6, 8), povidone iodine (2, 4, 14, 17), or even chlorhexidine, which was shown not to be a source of contamination here (17). The organisms most frequently observed included Burkholderia cepacia and Enterobacter spp. Also, distilled water to be used for injection or for the preparation of dilutions can be a source of infection (15). Modern sterilization technology obviously solved this latter problem. However, we here show that the sterilized products may still contain significant amounts of the DNA of the microorganisms that apparently thrived in the not-yet-sterilized formulation of the products. This issues a warning to those who would like to use PCR for the quality control of pharmaceutical materials: in the end, an undesired large fraction of all materials may not pass quality control due to the presence of (harmless?) microbial DNA (7).

This short report highlights the finding that the procedure for collection of samples during common surgical procedures may severely compromise molecular diagnosis of bacterial endophthalmitis. We observed that the contamination process was independent of the location where surgery was performed, the clinical syndrome that was treated, and the nature of the sample obtained. This led to the hypothesis that various decontamination fluids were the most likely sources of the DNA contamination rather than, for instance, transient contamination of the exterior eye. The level of contamination was high, which resulted in competition between contaminating templates and the templates derived from the genuine infectious agents, false-negative results being the ultimate consequence. Before embarking on large-scale diagnostic studies, one needs to verify not only that the laboratory reagents are free of contaminants but also that the materials used before and during the operation are checked for unsuspected DNA contamination.


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FOOTNOTES
 
* Corresponding author. Mailing address: Erasmus MC, Department of Medical Microbiology & Infectious Diseases, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Phone: 00-31-10-4635813. Fax: 00-31-10-4633875. E-mail: a.vanbelkum{at}erasmusmc.nl. Back


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Journal of Clinical Microbiology, November 2004, p. 5403-5405, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5403-5405.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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