Future and Emerging Treatments for Microbial Infections

Microbiologic diagnosis, particularly the use of molecular techniques.

New antimicrobials, and pharmacokinetic–pharmaco dynamic principles to achieve greater therapeutic efficacy.

Emerging antibiotic resistance, and what can be done to minimize the impact.

MICROBIOLOGIC DIAGNOSIS

The microbiologic diagnosis of postoperative or posttraumatic endophthalmitis remains problematic. Cultures from aqueous aspirates may be negative in up to 70% of cases, while vitreous cultures may be sterile in 30–40% of cases (1–3). In 1995, the Endophthalmitis Vitrectomy Study Group (EVS) examined the microbiology of postoperative endophthalmitis in one of the largest multicenter prospective studies of its kind (1). The overall culture positive rate was 69% among 420 patients with endophthalmitis within 6 weeks of cataract extraction or secondary intraocular lens implantation. Thus, the microbiologic diagnosis could not be established in fully one-third of these patients. Among those with positive cultures, gram-positive organisms were isolated from 94% of the patients, while gram-negative bacteria were isolated from 6% (Table 1). The most common isolates were coagulasenegative staphylococci (70%), followed by Staphylococcus aureus (10%), Streptococcus species (9%), Enterococcus species (2%), and miscellaneous gram-positive bacilli such as Propionibacterium acnes and Bacillus species (3%). In this study, a positive Gram stain from the aqueous or vitreous fluid was highly predictive of a positive culture from the eye, but a negative Gram stain had little predictive value for culture results (4). A more recent single-centre prospective study involving 206 vitreous samples from 206 patients yielded largely similar results (5). In this study, 46% of the specimens were sterile (Table 1). Among those with positive cultures, gram-positive bacteria (64%) were again more common than gram-negative bacteria (29%), which were recovered at a

Table 1 Microbiology of Postoperative or Posttraumatic Endophthalmitis

Postoperative cases included those following cataract extraction and lens implantation.

Note that the total is greater than 100% in each column because of polymicrobial infections.

higher rate than in previous reports. This study also identified Actinomycetes-related organisms (4%) and filamentous fungi (19%), mainly Aspergillus species. These results are contrasted to posttraumatic endophthalmitis in which a higher rate of Streptococcus species and gram-positive bacilli and a lower rate of coagulase-negative staphylococci were recovered (6) (Table 1).

Multiple studies have demonstrated that the usual source of organisms in postoperative endophthalmitis is endogenous from the lid or conjunctiva (7). Less common exogenous sources include airborne contaminants or contaminated intraocular solutions, lenses, or surgical instruments. The reasons for the low yield of positive cultures in postoperative endophthalmitis remain poorly explained. Cultures may be negative because of a small inoculum, sequestration of

bacteria in a biofilm environment on solid surfaces, the fastidious nature of some microorganisms, and the use of antibiotics prior to sampling (8). Nevertheless, the presence of microorganisms in many culture-negative cases has been documented by transmission or scanning microscopy and by other means (9,10). Thus, improving the method for detection of microorganisms in postoperative endophthalmitis is an important task, and molecular techniques such as polymerase chain reaction (PCR) appear to hold considerable promise.

One of the earliest reports of the successful application of PCR for the detection of microorganisms in delayed postoperative endophthalmitis, particularly Propionebacterium acnes, was by Hykin et al. (11). These authors used nested PCR with universal eubacterial primers complimentary to regions of 16S rDNA-conserved sequences to detect bacterial DNA in vitreous samples obtained from vitrectomy. Twenty-three samples from patients with delayed postoperative endophthalmitis and 29 samples from patients who underwent vitrectomy for reasons unrelated to infection were studied. Seventeen (74%) specimens from patients with endophthalmitis and four (14%) from uninfected individuals gave positive results. Positive results with the P. acnes primers were obtained from 8 (35%) of 23 ‘‘endophthalmitis’’ specimens and none of 29 ‘‘normal’’ samples. Subsequent to this, three separate studies have confirmed the promising results with PCR (12–14) (Table 2). There is excellent correlation between positive PCR and positive cultures. The addition of PCR to culture improved the diagnostic yield of culture alone by 29–76%. False positive rates have been low ( 5%). In addition, by using primers specific to gram-positive vs. gram-negative bacteria, the PCR method demonstrated an excellent capacity in discriminating grampositive from gram-negative organisms (15).

All the PCR studies reported above utilized universal eubacterial primers directed at conserved sequences of the 16S rDNA gene. Research in my own laboratory has investigated the use of broad-range degenerate primers that amplify the 60-kDa heat shock protein (hsp60) gene, which is highly conserved and universally present in all microorganisms (16,17). The universal hsp60 primers amplify a 600-bp

Table 2 Molecular Detection of Intraocular Microorganisms by PCR

product that contains a hypervariable region in which the DNA sequence is unique for a given bacterial genus or species (16,17) (Fig. 1). Furthermore, PCR amplification followed by direct sequencing, dot blot hybridization, or restriction enzyme digestion has provided a convenient method for species-specific identification of both gram-positive and gram-negative microorganisms. Furthermore, the hsp60 method was found to be more discriminative than 16SrDNA sequences for species identification (17–19). An important advantage of this methodology is that multiple pathogens can be detected with a single set of universal hsp60 primers in a single clinical specimen. We have demonstrated proof of concept of this approach in the detection and identification of putative respiratory pathogens in the respiratory tract of patients with community-acquired pneumonia, and the etiologic diagnosis of enteric pathogens in infectious diarrhea (18). Its utility in the diagnosis of ocular infections remains to be determined by prospective study.

There are a number of advantages to molecular detection of intraocular organisms by PCR. The technique is extremely sensitive and can detect DNA from a single infectious agent. Results can be obtained within hours rather than days or weeks by conventional culture methods. It is particularly

Figure 1 A schematic diagram depicting the 600-bp DNA product of the hsp60 gene amplified with our proprietary universal hsp60 degenerate primers. The primers are complimentary to highly conserved DNA sequences and amplify a hypervariable region, which contains sequences that are unique to each bacterial species or genus. The amplified product can be used as a DNA probe for diagnosis by hybridization, and can be analyzed by direct sequencing or restriction fragment length polymorphism.

suited for the detection of fastidious or non-cultivable microorganisms, and in cases where culture results may have been affected by the prior administration of antibiotics (8). Despite these advantages, PCR also has potential drawbacks. Because of the exquisite sensitivity of the test, scrupulous techniques are required to avoid contamination of the samples during collection and processing. Ocular specimens also contain substances that may inhibit the PCR reaction. This technical problem can be minimized by diluting the clinical samples or by the extraction of DNA prior to PCR (8). It should be noted that PCR does not directly test the ability of the microorganism to actively replicate, nor will it differentiate between an infection from contamination by extraneous or commensal organisms. Thus, clinical assessment of the patient remains critical for the evaluation of infection or inflammation, and PCR is unlikely to completely replace diagnostic cultures, which also provide information on antibiotic

susceptibility or resistance. Finally, a significant number of newly determined microbial DNA sequences cannot be accurately identified due to the inadequacy of available GenBank databases.

NEW ANTIMICROBIALS AND

PHARMACOLOGIC PRINCIPLES OF

ANTIMICROBIAL THERAPY

The EVS study conducted, in 1995 was the first randomized clinical trial that evaluated the role of pars plana vitrectomy and systemic antibiotics in the management of postoperative endophthalmitis (20). The study concluded that vitrectomy was necessary only for patients who presented with the worst vision (light perception only), and that systemic antibiotics were not helpful when used in addition to intravitreal antibiotics. However, both the choice of systemic antibiotics used in the study (amikacin plus ceftazidime) and the conclusion about their lack of efficacy have been questioned (21). Since amikacin plus ceftazidime have little activity against coagu- lase-negative staphylococci, the most common organisms associated with postoperative endophthalmitis, it is not surprising that the addition of these antibiotics made no difference in outcome. Even if vancomycin was chosen instead of amikacin and ceftazidime against coagulase-negative staphylococci, its poor penetration into the vitreous would still likely produce similar results. In a study reported by Ferencz et al. (22), 14 patients with acute postoperative endophthalmitis were treated with 1 g intravenous vancomycin prior to vitrectomy and collection of vitreous samples. Intravitreal vancomycin and ceftazidime were then administered intraoperatively. Vitreous samples were cultured and their vancomycin concentrations were assayed. The intravitreal vancomycin levels following intravenous administration were below the minimal inhibitory concentrations (MIC) of the causative organisms in 44% of patients with positive cultures. Vitreous cidal activity against the causative organism was achieved in only 10% of these cases. Following a 1 mg intravitreal injection,

vancomycin levels and vitreous cidal activity were all therapeutic in a smaller subset of the patients. Thus, newer agents with improved antimicrobial spectrum and pharmacologic properties are needed for the optimal management of these serious infections.

Some of the newer antimicrobials with potential for treating intraocular infections are listed in Table 3. These agents either have improved antimicrobial spectrum or bioavailability for organisms common in postoperative or posttraumatic endophthalmitis. Many are still under investigation and their efficacy in the treatment of endophthalmitis remains to be determined by prospective, randomized clinical trials. Among the newer antibacterial agents, the second or third generation fluoroquinolones (e.g., levofloxacin, moxifloxacin, and gatifloxacin) deserve special mention, as these agents exhibit improved activity against gram-positive and anaerobic pathogens, including penicillin-resistant pneumococci in contrast to ciprofloxacin (2,23,24). Furthermore, they can penetrate the eye even with oral administration (25,26). As a group, the

Table 3 New Antimicrobials with Potential Application in Endophthalmitis

fluoroquinolones have excellent activity against gram-nega- tive bacteria and thus may replace aminoglycosides or third generation cephalosporins in the treatment of bacterial endophthalmitis. However, intravenous or intravitreal administration may still be required since the concentrations achieved following oral administration may not be adequate (27). Whereas ofloxacin at intravitreal concentrations of 500 mg or higher may be toxic to the retina (28), ciprofloxacin and levofloxacin appear to be better tolerated (29,30).

The other newer antibacterials, listed in Table 2, all have improved activity against gram-positive bacteria. These include the ketolides (e.g., telithromycin) (31), which have improved activity against erythromycin resistant grampositive cocci, and four other classes of antimicrobials with improved activity against methicillin-resistant staphylococci, coagulase-negative staphylococci, and vancomycin-resistant enterococci. These include the oxazolidinones (e.g., linezolid) (32), streptogramins (e.g., quinupristin=dalfoprisin or synercid) (33), lipopeptides (e.g., daptomycin) (34) and glycopeptides (e.g., teicoplanin) (35,36).

New antifungals include the azoles (e.g., voriconazole) (37) and echinocandins (e.g., caspofungin) (38), both with enhanced activity including Aspergillus and Candida species, including the fluconazole-resistant Candida species. A detailed discussion of these agents is beyond the scope of this review.

Perhaps more important is an understanding of some emerging pharmacologic principles that may improve therapeutic efficacy of antimicrobial therapy by optimizing the appropriate dosing regimens. Appropriate dosing requires knowledge of both the pharmacokinetic and pharmacodynamic (PK=PD) properties of different antimicrobial agents (39). Pharmacokinetic parameters determine what drug concentrations can be achieved in serum and tissue; pharmacodynamic parameters determine what antimicrobial effect can be achieved (Fig. 2). It is the time course of antimicrobial activity that determines which pharmacokinetic properties of a drug are the most important determinants of clinical efficacy (40). The critical question to ask is what kind

Figure 2 Overview of pharmacokinetics and pharmacodynamics in antimicrobial chemotherapy.

of killing pattern the drug has and whether the effect is shortlived or prolonged. Does it have concentration-dependent killing where higher levels will result in better killing? Or is it more time-dependent where higher concentrations will not necessarily kill the organism better but maintaining the concentration above the MIC of the organism for a longer period will? The specific PK=PD parameters that have been found to correlate best with outcome include the ratio of peak-to-mini- mum inhibitory concentration (peak=MIC ratio), the ratio of trough-to-minimum inhibitory concentration (trough=MIC ratio), the ratio of the 24-hr area under the curve to MIC (AUC24=MIC ratio), and the duration of time when serum levels exceed the MIC, expressed as the percentage of the dosing interval (time above MIC) (41). The value of each of these PK=PD parameters in predicting a favorable outcome depends on the class of antibiotics.

Most antimicrobials fall into one of three categories based on their pattern of antimicrobial activity (Fig. 3). Agents such as aminoglycosides, fluoroquinolones, metronidazole, ketolides, and amphotericin B demonstrate concen tration-dependent killing and persistent antimicrobial effects (Fig. 3A) (40). The goal of therapy with these agents is to