To our knowledge, this case represents the first demonstration that an EVD can provide an abiotic surface for biofilm formation, that a substantial biofilm can develop even in the lumen of the device and, importantly, that the presence of a biofilm infection can remain undetected by standard surveillance culture of CSF drawn via the EVD device. In this case, routine surveillance cultures performed daily were uniformly negative, and only the final broth culture on the explanted EVD was positive at 5 days. In fact, the organism isolated by broth culture (CoNS) was not consistent with the organisms identified by PCR within the catheter (S. aureus),
and it is not certain whether the CoNS was indeed present on the catheter or may represent an artifactual or contaminating finding. Given the direct micrographic and molecular evidence for the presence of S. aureus
within the catheter, this case highlights the inadequacy of the routine culture of sampled CSF as a reliable surveillance tool. There are multiple possible reasons for the culture insensitivity of biofilm bacteria. Although CSF withdrawn from the EVD was necessarily passing over the biofilm, sufficient bacteria may not have been detaching from the biofilm foci so as to reach the level of detectability by culture. It is also possible that the biofilm in this case was producing slow-growing ‘small colony variants’. In orthopedic infections, the presence of such staphylococcal small colony variants has been identified as a possible explanation for the high rate of false negatives associated with conventional culture techniques [22
It remains unclear whether the explanted VP shunt that necessitated the placement of this EVD was itself infected since it was not retained for confocal or molecular evaluation. It is possible that bacteria shed from the VP shunt colonized and propagated within the EVD; indeed, given that no other cause for the shunt failure was immediately apparent, this would seem to be the most parsimonious explanation, but direct evidence is lacking. The absence of culture positivity from the explanted VP shunt does not exclude a biofilm resident on the shunt, which would echo the culture negativity of the EVD despite a clearly demonstrable biofilm therein.
In this patient, fortuitously, a positive late culture finding at the time of EVD removal (even though it may have been artifactual) prompted treatment with intravenous nafcillin prior to placement of yet another VP shunt (during which time a replacement EVD was in place). This new VP shunt remained in place and functional for some 9 months. It appears, therefore, that the antibiosis administered may have been effective in inhibiting transmission of the biofilm bacteria to the new foreign body. Any biofilm that had formed on the (replacement) EVD may have been removed with the drain, and any bacteria shed in the CSF may have proven more susceptible to antibiotics in planktonic form.
Our findings in this case largely fulfill the Parsek-Singh criteria for the diagnosis of biofilm infection. Briefly, these include: (1) the presence of pathogenic bacteria associated with a surface, (2) the presence of bacteria aggregated in cell clusters, and (3) the infection being localized, with dissemination being a rare event [5
]. Given that the patient exhibited no meningeal signs while the infected EVD was in place, the infection can be considered to have remained localized to the EVD and clinically silent although the potential for acute exacerbation and dissemination was present. A fourth criterion, that the biofilm infection may be impossible to eradicate by antibiosis, is untested in this case since the foreign body hosting the biofilm (the infected EVD) was explanted, thereby physically removing the nidus of infection, while any planktonic bacteria shed by the biofilm would likely prove more susceptible to antibiosis. Both micrographic and molecular analyses in this instance showed that a clinical biofilm need not be uniform; thus, failure to detect bacteria from clinical specimens may be due to sampling error as well as other factors (e.g. simple physical dislodgment of an attached biofilm in the process of removing the device or tissue).
The direct documentation of infection within the catheter despite culture negativity has important clinical implications and especially highlights the need for more reliable diagnostic measures that can offer better clinical guidance. Confocal microscopy is unlikely to become a standard diagnostic in its present format: the preparation and examination of specimens are labor-intensive and can only be performed after a shunt or drain has been removed. However, the PCR-based detection of nucleic acids from pathogens in the CSF offers the ability to detect minute quantities of biofilm bacteria despite their low cultivability and deserves further evaluation. One report comparing PCR detection of bacterial DNA with cultural results in CSF from shunt/ventriculostomy patients with clinically suspected infection has demonstrated that PCR significantly increases the number of patients found to have evidence of bacteria in the CSF [23
Our data support the hypothesis by Stevens et al. [11
] that a major contributor to infection associated with EVD may be biofilm formation. Hayhurst et al. [24
] reported that, of 27 pediatric patients with EVD, 4 EVD were infected (by clinical signs and apparent response to treatment) even though all patients had sterile CSF throughout surveillance. Occult infection has also been associated with peritoneal catheters: Gorman et al. [25
] examined 32 peritoneal catheters from patients with a history of peritonitis by electron and confocal microscopy. Bacteria were cultured after removal by sonication and vortexing. Biofilms were found in 4 of 4 catheters removed for peritonitis, but were also observed in 17 of 21 catheters removed for renal transplant from patients with no diagnosis of clinical infection; 41% of the infected catheters had mixed biofilms of CoNS and S. aureus.
Biofilm formation in the intraluminal compartment might also explain the absence of early clinical signs of infection. In the early stages of biofilm formation, an intraluminal biofilm would be sequestered from the host, which might explain the limited inflammation associated with EVD-related infections [1
The recognition of a biofilm involvement has significance for the design of devices specifically designed to prevent shunt (or EVD) infection (and presumably biofilm formation). Several studies have examined whether antibiotic-impregnated shunts can reduce the rate of infection with somewhat encouraging results. Hayhurst et al. [24
] and Parker et al. [26
] reported that antibiotic-impregnated shunts reduced the incidence of CSF infection in pediatric patients. However, it was noted that when S. aureus
was cultured from patients with antibiotic-impregnated shunts, the strains were more likely to be oxacillin-resistant than from patients with conventional shunts[26
], illustrating the potential danger of selecting for antibiotic resistance by this strategy. Lastly, it has been noted that the simple placement of an EVD, whether culture-positive or -negative, is itself a risk factor for the infection even of a subsequent antibiotic-impregnated shunt [25
]. These results again suggest that a culture-negative EVD may still host a hard-to-detect biofilm infection which may transfer to a subsequent device, again reinforcing the need for better methods for the detection and combat of biofilm.