Neisseria gonorrhoeae (N. gonorrhoeae) is one of the most common sexually transmitted pathogens. Its high morbidity and associated medical and socio-economic costs make it one of the major public health issues in the U.S. and in the world. Approximately 300,000 cases are reported to the CDC each year . Because many cases could be asymptomatic, a recent estimate indicates that there may be more than 820,000 infected individuals in the U.S . Globally, many areas are experiencing significant rise in reported cases of gonorrhea. According to the World Health Organization (WHO), in 2008 there were 106 million new cases of gonorrhea worldwide .
CDC currently recommends that for uncomplicated genital, rectal, and pharyngeal gonorrhea, a combination of two drugs should be used; specifically, ceftriaxone 250 mg is administered intramuscularly (IM) in a single dose, plus azithromycin 1 g orally in a single dose. When ceftriaxone is not available, cefixime 400 mg orally in a single dose is recommended to replace ceftriaxone in combination with 1 g oral azithromycin [4, 5]. Treatment guidelines also recommend individuals with pharyngeal infections return for medical consultation 1 week post therapy to ensure success of therapy.
It is not clear whether combining two antimicrobials with different mechanisms of action (e.g., ceftriaxone plus azithromycin) can delay the rise of isolates that are resistant to the extended spectrum cephalosporins (ESC) . A recent report from Canada showed a significant decline in decreased susceptibility to cephalosporins but at the same time increased azithromycin resistance .
Despite the use of dual drug therapy, both CDC and the WHO have warned that gonorrhea will soon become more difficult to treat. This is because gonococcus is well known for its ability to develop antimicrobial resistance against first-line therapies within a short duration of a drug’s introduction [8–11].
Until new drugs for treating gonorrhea are discovered, there is urgent need for public health professionals to closely monitor antimicrobial susceptibility of gonococci. Quick identification of isolates that potentially possess reduced susceptibility to cephalosporins is critical to controlling the spread of drug-resistant gonococcal organisms. The recent emergence of isolates with reduced antibiotic susceptibility across the country has further alerted physicians and epidemiologists to the urgent need to closely monitor such activities .
Three common tests are routinely used by laboratories to determine the antimicrobial susceptibility of N. gonorrhoeae. The agar dilution test is the gold standard and is used mostly by reference laboratories. We reported recently that the Etest® (bioMerieux, France) is suitable to serve as an alternative test . Both agar dilution and Etest methods report an MIC value which can be easily used by physicians or epidemiologists to determine treatment options. These two tests are appropriate for finding organisms with increased MIC values. While these tests are our first choice, they are not routinely used by all laboratories.
A third test, the disk diffusion test, is more commonly used by microbiological laboratories and hospitals world-wide to determine the antimicrobial susceptibility of many organisms against wide-spectrum antibiotics [9, 12–15]. The test is simple and widely accepted by many laboratories. Its use, however, is not without limitations. Disk diffusion test results are observed as a diameter of inhibition-zone. The diameter is relatively imprecise and often cannot convert to a MIC value, but rather provides a categorical classification of susceptible, intermediate, or resistant phenotypes . For the treatment of gonococci with cephalosporins, only susceptible and non-susceptible categories are accepted for classifications [13, 14, 16].
In the past 20 years, the number of laboratories using the disk diffusion method for N. gonorrhoeae antimicrobial susceptibility testing has declined. In 1989, 86% of public health laboratories surveyed used the disk diffusion method . In a study performed in New York in 2000, approximately 37.4% of laboratories used disk diffusion ; in a 2012 study , 47% of laboratories were utilizing disk diffusion tests for N. gonorrhoeae antimicrobial susceptibility. Thus the disk diffusion method remains the choice of many reference and regional laboratories and hospitals to survey gonococcal drug susceptibility. However, without sufficient clinically ESC resistant isolates, it is not clear whether the disk diffusion method can reliably detect future ESC resistance.
In this report, we compared the disk diffusion method with the current gold standard agar dilution and a potential alternative Etest method. We analyzed the suitability and reliability of disk diffusion to monitor susceptibility of N. gonorrhoeae isolates of the most commonly used cephalosporins (ceftriaxone and cefixime). In addition, we included 10 laboratory generated mutants with raised MIC level against these common ESCs to simulate non-susceptible isolates. Cefpodoxime was also included in this study because it was used in selecting laboratory generated mutants.