Dissertation committee:
Ελένη Γιαμαρέλλου, Ομ. Καθητήτρια, Ιατρική Σχολή, ΕΚΠΑ
Γεώργιος Δαΐκος. Ομ. Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Λεωνίδας Τζουβελέκης, Αν. Καθηγητής,Ιατρική Σχολή, ΕΚΠΑ
Αναστασία Αντωνιάδου, Αν. Καθηγήτρια,Ιατρική Σχολή, ΕΚΠΑ
Αντώνιος Παπαδόπουλος, Αν. Καθηγητής,Ιατρική Σχολή, ΕΚΠΑ
Μιχαήλ Σαμάρκος, Αν. Καθηγητής,Ιατρική Σχολή, ΕΚΠΑ
Μήνα Ψυχογυιού, Επ. Καθηγήτρια,Ιατρική Σχολή, ΕΚΠΑ
Summary:
Carbapenem resistant K. pneumoniae admittedly represents a contemporary public health threat. This form of resistance is primarily caused by class A, B or D carbapenemases. These enzymes are highly transmissible via mobile genetic elements such as plamids, and therefore manifest a high potential to cause outbreaks, particularly across health care facilities. Their global prevalence has consequently reached alarming rates. In Greece, carbapenem resistant K. pneumoniae prevalence has exceeded 50%, according to recent data. Unfortunately, resistance does not only affect carbapenems and subsequently virtually all b-lactams, but because of additional resistance mechanisms that are co-carried by these isolates, it expands to many other antibiotic classes. Very few antibiotics remain variably active, such as colistin, tigecycline, gentamycin and fosfomycin and even these seem to suffer a decreasing level of effectiveness, as resistance is reported at a growing rate. The recently implemented combinations of ceftazidime/avibactam and meropenem/vaborbactam seem to constitute promising options, but clear in vivo evidence is lacking and early resistance reports are of concern, while they are inactive against class B carbapenemases.
Misfortunes do not end here. Infections caused by carbapenem resistant K. pneumoniae isolates primarily affect vulnerable patients severely ill, either chronically or acutely, or else debilitated with multiple comorbidities. Risk factors for initial colonization and subsequent infection from CRKP have been well established by multiple researchers and are all associated with compromised states of health, namely admission in Intensive Care Units, need of mechanical ventilation, use of central catheters or other invasive procedures, diabetes mellitus, malignancy, chemotherapy, prior multiple antibiotic use, poor functional status and extended length of hospital stay. Given their critical condition, treatment should be prompt and highly effective. Instead, it is compromised by the limited therapeutic options. Hence, very high mortality ensues, resulting primarily from bacteraemias due to said isolates.
Despite the ongoing battle of clinicians against such infections, there has been no consensus as to which is the indicated treatment strategy. Even though high quality evidence, in the form of randomized control trials, is lacking, there is data derived from several large scale retrospective studies which clearly supports combination therapy for these infections, claiming an association with lower mortality, particularly for patients at a high risk of death. Combination therapy seems to be the preferred approach by most experts, in the hope of achieving a result more favorable than the one expected by monotherapy.
Combined antimicrobials might act additively, indifferently or even antagonistically to one another. The best scenario is for them to act synergistically, generating an effect greater than the sum of their individual activities. In the era of limited or even absent therapeutic options, a synergistic antimicrobial combination might prove important for the clinician. Given the frequency at which combination therapy is employed for the treatment of resistant infections, as well as the expectations clinicians have placed upon it, a reliable and accurate method able to predict the efficacy of a specific combination would be invaluable. Although there is no definite gold standard synergy testing method, the two most common ones through literature are the Time Kill and the Checkerboard methods, neither of which is available for routine use in the clinical microbiology laboratory, due to their laborious nature. A method as reliable, but also easily performed in any laboratory, would be expedient, in that it would permit routine combination testing against strains isolated from patients.
In this context, this study assessed dual combinations of some of the last resort drugs against K. pneumoniae isolates producing carbapenemases of various stypes, namely combinations of tigecycline with either colistin, gentamicin, fosfomycin or meropenem. The Time Kill and Checkerboard nethods were employed, along with three different Etest methodologies, in order to determine whether one of the latter - much easier and simpler to perform - might prove suitable to replace the former. To that end, the Time Kill was used as a reference method, being considered by many to provide the most useful information from a clinical point of view, while it remains the sole standarised method for laboratory synergy testing.
According to the reference method, synergy was found for 11% of all combinations tested. The most synergistic one was that of tigecycline and colistin at a rate of 25%, followed by the one with gentamicin at 10%, meropenem at 8% and fosfomycin at 4.5%. These findings are in line with published research data on the activity of tigecycline in combination with other agents against carbapenem resistant K. pneumoniae, which show that when combined with colistin, tigecycline is significantly more effective than either agent alone in Time-Kill assays. This combination has also demonstrated a synergistic effect with the checkerboard method, which in this study reached 45%. There also exist sporadic synergy reports with fosfomycin and gentamicin, as also noted in the present study. In the latter, the checkerboard method produced in total a higher synergy rate of 26.5%, while the Etest methods rates ranged from 2 to 47%. The “strictest” result was generated by the Cross Formation Etest method.
The ultimate goal of the present study was to determine the degree of concordance between the reference and possible alternative methods. Despite the considerable rates of observed agreement between TKA and the rest of the methods tested, which ranged from 50% to 85%, analysis by Cohen’s kappa statistics revealed an almost nonexistent statistical correlation between them. There was one solitary exception, that of perfect statistical agreement between the reference and the cross formation Etest method for the combination of tigecycline with fosfomycin. Exploring correlations further than the Time Kill, there was only a fair agreement between two of the Etest methods with the checkerboard method.
Looking at published studies comparing synergy testing methods against a variety of resistant gram-negative microorganisms, one can find rates of concordance which vary greatly, from 0% to 100%, which in fact represents the highest possible variability. This is not surprising if a number of parameters adhering to the nature of each method is to be considered. Most importantly, the methods involved bear different endpoints. Thus, the Time Kill assay measures the rate of bacterial killing, whereas the checkerboard and Etest methods, the inhibition of bacterial growth. Furthermore, the Time Kill evaluates killing over time, while the rest determine results at a fixed time point. It also uses specific agent concentrations, unchanged throughout the assay, whereas the rest assess optimal inhibition concentrations of the agents tested. The present study might indeed reflect these differences between the Τime Κill and Checkerboard/Etest methods, as it yielded poor agreement between the former and the latter, but detected a fair one between the two out of three Etest methods and the checkerboard.
Further insight into the comparability of synergy testing methods reveals additional confounding factors, such as reproducibility issues, particularly for the checkerboard technique. On another note, each researcher may or may not evaluate parameters such as synergy at time points other than the 24 hours for the time kill assay, or the expression of additive, alongside synergistic, actions. Finally, there is an infinite variety of combinations of different drug concentrations acting on strains of different qualities, such as resistanse or sensitivity to the former, and with individual resistance mechanisms.
Such considerations imply that there is a need for optimization and standardization of in vitro combination studies. Still, and in light of the absence of a true gold standard for synergy, it would be difficult to discern which of these methods provides the most reliable and accurate results. The optimal way to find out would be to correlate their findings with clinical outcomes, so as to obtain clinical evidence to support their validity. Such validation, in the form of ramdomised controlled trials, is almost nonexistent throughout current literature. It seems that outcome-based studies are urgently needed so as to throw light on the matter of antimicrobial synergy testing.
