I don't know about you but scientific papers are offered in a format that is not the easiest thing to understand. I went through the Faro, et al, analysis just published in the medical journal and picked out what I thought were highlights for non-medical readers in order to enhance my own understanding of the importance of what was reported. I deleted some background material and added a few bracketed [N-Assay] insertions to help my own focus. If you look at the resulting outline of a scientific report now available to the world's medical professionals then it should leap off the "electronic" page that this N-Assay is a real breakthrough in medical care and testing. It is not only the speed advantage (which is very significant) but the ability to short cut the antibiotic selection and effectiveness of treatment processes for multiple infectious diseases in addition to the GBS. The improved treatment protocols, reduced use of prophylactic general antibiotics that otherwise result fairly quickly in reduced sensitivity by bacteria are extremely significant and now "proven" advantages of the N-Assay over traditional or current methods, including PCR that was considered state-of-the art.
I want to express my great thanks to John Faro, his co-researchers and NanoLogix management for achieving this breakthrough--and note that we all should be proud to be part of it not only for financial benefits but for the advancement of medical treatment that will help many people.
NNLX N Assay Paper Abstract
Background. Elucidation of a pathogen’s antimicrobial susceptibility requires subculture after the organism is first isolated. This takes several days, requiring patients to be treated with broad-spectrum antibiotics. This approach contributes to the development of bacterial resistance.
Findings. Group B streptococcus (GBS), Enterococcus faecalis, and Neisseria gonorrhoeae were each detected at 105 bacteria/mL [by the N-Assay] following a 20-minute incubation period.
Susceptibility to select antibiotics was discernable [by N-Assay] following a 6-hour incubation period (GBS and Enterococcus).
Sensitivity was increased to 10−2 bacteria/mL for GBS, 10−1 bacteria/mL for E. faecalis, and 101 bacteria/mL for N. gonorrhoeae following 18–24-hour culture. [N-Assay]
Conclusion. This novel assay [N-Assay technology] allows for the highly sensitive and specific identification of a pathogen and simultaneous determination of its antimicrobial susceptibility in a reduced time.
1. Introduction
When confronted with a patient battling an infectious disease, elucidation of the offending pathogen requires great effort. Time is critical: broad-spectrum antibiotics are initiated after cultures are collected, as identification of the offending microbe requires 24–48 hours.
To increase the sensitivity of culture, an enrichment step may be added [1]. The workup is not completed once a pathogen is identified: antimicrobial susceptibility must next be ascertained following additional subculture in the presence of select antibiotics for another 24–48 hours [2].
Several techniques have been developed to aid in reducing the time required to identify a pathogen. These techniques include the use of chromogenic agar/broth and nucleic acid amplification technology (NAAT) [3, 4].
While both of these techniques have been employed in screening for antenatal GBS colonization, the Centers for Disease Control and Prevention still recommend that culture be performed [5].
This is an absolute requirement for patients allergic to penicillin, as NAAT is not capable of providing antimicrobial susceptibility profiles.
Additionally, the resources and highly specialized training required to perform NAAT are not universally available [6].
Recently, the Infectious Diseases Society of America released a paper detailing the current approach towards identifying clinical pathogens. Described is a process characterized as having inadequate sensitivity and significant time delays, thereby contributing to the development of greater and greater antimicrobial resistance [7].
These concerns are all too-well illustrated in our approach towards treatment for GBS prophylaxis, in which increasing resistance to penicillin has been observed, and resistance to clindamycin is on the rise [8, 9].
Additionally, there is concern that this practice will contribute to E. coli resistance observed in premature neonates [10].
Already, we have seen the implications of antimicrobial resistance with Enterococcus, and hospital acquired infections with this pathogen are estimated to add $27,000.00 per infection [11, 12].
N. gonorrhoeae resistance has been increasing steadily, and multidrug resistance has recently been confirmed [13].
These three disparate organisms demonstrate a range in the microbial response to our directed approach to both prophylaxis and treatment.
Through a modification of a recently reported test, we have developed a method for the simultaneous identification of a pathogen and determination of its antibiotic susceptibility [14]. [The N-Assay technology]
We have modified this test so that GBS, E. faecalis, and N. gonorrhoeae may be detected at dilute concentrations after 6-hour incubation.
Additionally, we show that inducible resistance of GBS against clindamycin may be determined.
Finally, we show that following an overnight incubation, test organisms may be detected at concentrations rivaling those published for PCR.
As this test allows one to simultaneously identify a pathogen and determine its antimicrobial susceptibility, this novel technique provides a change in the clinician’s approach to managing infectious diseases. [Better than PCR and PCR doesn’t distinguish between live and dead bacteria cells]
Group B streptococcus clinical isolates 12386 and 01.12.76 were shown by disk diffusion to be susceptible or resistant to clindamycin, respectively.
Enterococcus faecalis ATCC 29212 was confirmed to be susceptible to vancomycin by disk diffusion, and ATCC strain 51299 was confirmed to be resistant.
Neisseria gonorrhoeae ATCC strain 31426 was shown to be resistant to penicillin. Strain 1279, a clinical isolate, was shown to be susceptible to penicillin. E. coli, S. aureus, Candida albicans, and Beta streptococcus groups A, C, F, and G were all clinical isolates.
3.2. Limit of Detection
Bacteria were next diluted in Fastidious Broth after first preparing a 0.5 McFarland. The ability to detect the test organism at greater dilutions increased when the incubation times were lengthened. GBS was detected down to 102 bacteria/mL after 2-hour incubation, 101 bacteria/mL after 6 hours, and 10−2 bacteria/mL after incubating overnight (Figure 2(a)).
N. gonorrhoeae was diluted out serially in Fastidious Broth, first starting with a 0.5 McFarland. After overnight incubation, the assay was run. N. gonorrhoeae was detected strongly at high concentrations, as well as at very low concentrations, 102 and 101 bacteria/mL (Figure 2(c)). The test was not capable of detecting bacteria less than 101 bacteria/mL following 24-hour incubation.
3.3. Determination of Antimicrobial Susceptibility
The development of microbial resistance to antibiotics has plagued contemporary clinicians. Following the introduction of each new antibiotic, resistant isolates have been detected, with resistance of Staphylococcus to Methicillin noted 2 yrs after the first use of this antibiotic in 1960, resistance of Enterococcus to vancomycin was noted 16 yrs after its introduction in 1972, and resistance of Staphylococcus to Linezolid was noted just 1 yr after its first use in 1996 [16].
The development of microbial resistance to an antibiotic has become the norm, and anticipated resistance has been confirmed again as recently as 2011 with the multidrug resistant N. gonorrhoeae strain reported in Japan [17].
That antimicrobial resistance has developed and continues to do so is not surprising. What is concerning is that the approach to managing patients with suspected infection/colonization has remained static, and in fact this approach continues to be reestablished, with the recent example of intrapartum GBS prophylaxis as recommended by the CDC in 2010 [5].
The increased prevalence of vancomycin resistant Enterococcus in hospitalized patients is yet another example. As Enterococci are to a large degree resistant to cephalosporins, use of this class of antibiotic for surgical prophylaxis allows for overgrowth of Enterococcus at sites previously colonized by cephalosporin-sensitive organisms and may contribute to the increased number of hospital associated surgical site infections [18].
Perhaps nowhere is this battle between pathogen and microbicide more apparent than with N. gonorrhoeae. From the first use of sulfonamides in the 1940s, this bacterium has countered the development of resistant strains [19]. After showing that this pathogen is capable of developing resistance to any antibiotic directed against it, including sulfonamides, penicillin, tetracycline, spectinomycin, quinolones, macrolides, and now cephalosporins, it is apparent that novel approaches are needed beyond the two-drug counterattack recommended just last year [20].
We recently developed an immunoassay [N-Assay] in which membranes coated with antibody against GBS are exposed to bacterial suspensions, and after incubating for a series of time-points, bound GBS is detected [14]. This assay was tested on patients’ vaginal-rectal specimens and was found to have a high degree of sensitivity and specificity, with the added benefit of being capable of detecting nonbeta hemolytic streptococcal strains of GBS [21].
In order to decrease the interobserver variation seen with dot-blot assays, we converted the test to an ELISA format. In this study, we have substituted the anti-GBS antibodies for those directed against either N. gonorrhoeae or Enterococcus. We show that these polyclonal antibodies provide a great deal of sensitivity in a nonselective broth, with little interference from other commonly isolated cocolonizers Figure 1 and Supplemental Figure 1.
Following a six-hour incubation period, the limit of detection increased to 101 bacteria per mL for GBS (Figure 2). Furthermore, we show that antibiotic susceptibility may be determined (Supplemental Figure 2). From this, one may envision a panel in which a series of wells are set up in which multiple clinically relevant antibiotics are tested, providing the clinician with a targeted approach to treatment in a greatly reduced timeframe (Figure 3).
A central facet of this assay involves cellular viability: the concentration of bacteria present in the well is related specifically to the incubation times utilized. By incubating overnight, the sensitivity of the test was increased significantly as live bacteria continued to grow and divide, with GBS being detected at 10−2 bacteria per mL, Enterococcus detected at 10−1 bacteria per mL, and N. gonorrhoeae detected at 101 bacteria per mL (Figure 2). This allows for a sensitivity greater than published results for several PCR-based methods of detection following an enrichment step, with the unique advantage being that this test is capable of detecting viable cells [22–25].
This assay [the N-Assay] provides the unique approach of targeting the growth of a specific clinically relevant pathogen through the use of a capture antibody. An immediate binding assay demonstrates whether a pathogen is present or not. By then culturing the organism in the presence or absence of selected antibiotics, one may show that cells are viable and susceptible to specific antimicrobials, thereby allowing the clinician to then make an informed decision more rapidly.
I want to express my great thanks to John Faro, his co-researchers and NanoLogix management for achieving this breakthrough--and note that we all should be proud to be part of it not only for financial benefits but for the advancement of medical treatment that will help many people.
NNLX N Assay Paper Abstract
Background. Elucidation of a pathogen’s antimicrobial susceptibility requires subculture after the organism is first isolated. This takes several days, requiring patients to be treated with broad-spectrum antibiotics. This approach contributes to the development of bacterial resistance.
Findings. Group B streptococcus (GBS), Enterococcus faecalis, and Neisseria gonorrhoeae were each detected at 105 bacteria/mL [by the N-Assay] following a 20-minute incubation period.
Susceptibility to select antibiotics was discernable [by N-Assay] following a 6-hour incubation period (GBS and Enterococcus).
Sensitivity was increased to 10−2 bacteria/mL for GBS, 10−1 bacteria/mL for E. faecalis, and 101 bacteria/mL for N. gonorrhoeae following 18–24-hour culture. [N-Assay]
Conclusion. This novel assay [N-Assay technology] allows for the highly sensitive and specific identification of a pathogen and simultaneous determination of its antimicrobial susceptibility in a reduced time.
1. Introduction
When confronted with a patient battling an infectious disease, elucidation of the offending pathogen requires great effort. Time is critical: broad-spectrum antibiotics are initiated after cultures are collected, as identification of the offending microbe requires 24–48 hours.
To increase the sensitivity of culture, an enrichment step may be added [1]. The workup is not completed once a pathogen is identified: antimicrobial susceptibility must next be ascertained following additional subculture in the presence of select antibiotics for another 24–48 hours [2].
Several techniques have been developed to aid in reducing the time required to identify a pathogen. These techniques include the use of chromogenic agar/broth and nucleic acid amplification technology (NAAT) [3, 4].
While both of these techniques have been employed in screening for antenatal GBS colonization, the Centers for Disease Control and Prevention still recommend that culture be performed [5].
This is an absolute requirement for patients allergic to penicillin, as NAAT is not capable of providing antimicrobial susceptibility profiles.
Additionally, the resources and highly specialized training required to perform NAAT are not universally available [6].
Recently, the Infectious Diseases Society of America released a paper detailing the current approach towards identifying clinical pathogens. Described is a process characterized as having inadequate sensitivity and significant time delays, thereby contributing to the development of greater and greater antimicrobial resistance [7].
These concerns are all too-well illustrated in our approach towards treatment for GBS prophylaxis, in which increasing resistance to penicillin has been observed, and resistance to clindamycin is on the rise [8, 9].
Additionally, there is concern that this practice will contribute to E. coli resistance observed in premature neonates [10].
Already, we have seen the implications of antimicrobial resistance with Enterococcus, and hospital acquired infections with this pathogen are estimated to add $27,000.00 per infection [11, 12].
N. gonorrhoeae resistance has been increasing steadily, and multidrug resistance has recently been confirmed [13].
These three disparate organisms demonstrate a range in the microbial response to our directed approach to both prophylaxis and treatment.
Through a modification of a recently reported test, we have developed a method for the simultaneous identification of a pathogen and determination of its antibiotic susceptibility [14]. [The N-Assay technology]
We have modified this test so that GBS, E. faecalis, and N. gonorrhoeae may be detected at dilute concentrations after 6-hour incubation.
Additionally, we show that inducible resistance of GBS against clindamycin may be determined.
Finally, we show that following an overnight incubation, test organisms may be detected at concentrations rivaling those published for PCR.
As this test allows one to simultaneously identify a pathogen and determine its antimicrobial susceptibility, this novel technique provides a change in the clinician’s approach to managing infectious diseases. [Better than PCR and PCR doesn’t distinguish between live and dead bacteria cells]
Group B streptococcus clinical isolates 12386 and 01.12.76 were shown by disk diffusion to be susceptible or resistant to clindamycin, respectively.
Enterococcus faecalis ATCC 29212 was confirmed to be susceptible to vancomycin by disk diffusion, and ATCC strain 51299 was confirmed to be resistant.
Neisseria gonorrhoeae ATCC strain 31426 was shown to be resistant to penicillin. Strain 1279, a clinical isolate, was shown to be susceptible to penicillin. E. coli, S. aureus, Candida albicans, and Beta streptococcus groups A, C, F, and G were all clinical isolates.
3.2. Limit of Detection
Bacteria were next diluted in Fastidious Broth after first preparing a 0.5 McFarland. The ability to detect the test organism at greater dilutions increased when the incubation times were lengthened. GBS was detected down to 102 bacteria/mL after 2-hour incubation, 101 bacteria/mL after 6 hours, and 10−2 bacteria/mL after incubating overnight (Figure 2(a)).
N. gonorrhoeae was diluted out serially in Fastidious Broth, first starting with a 0.5 McFarland. After overnight incubation, the assay was run. N. gonorrhoeae was detected strongly at high concentrations, as well as at very low concentrations, 102 and 101 bacteria/mL (Figure 2(c)). The test was not capable of detecting bacteria less than 101 bacteria/mL following 24-hour incubation.
3.3. Determination of Antimicrobial Susceptibility
The development of microbial resistance to antibiotics has plagued contemporary clinicians. Following the introduction of each new antibiotic, resistant isolates have been detected, with resistance of Staphylococcus to Methicillin noted 2 yrs after the first use of this antibiotic in 1960, resistance of Enterococcus to vancomycin was noted 16 yrs after its introduction in 1972, and resistance of Staphylococcus to Linezolid was noted just 1 yr after its first use in 1996 [16].
The development of microbial resistance to an antibiotic has become the norm, and anticipated resistance has been confirmed again as recently as 2011 with the multidrug resistant N. gonorrhoeae strain reported in Japan [17].
That antimicrobial resistance has developed and continues to do so is not surprising. What is concerning is that the approach to managing patients with suspected infection/colonization has remained static, and in fact this approach continues to be reestablished, with the recent example of intrapartum GBS prophylaxis as recommended by the CDC in 2010 [5].
The increased prevalence of vancomycin resistant Enterococcus in hospitalized patients is yet another example. As Enterococci are to a large degree resistant to cephalosporins, use of this class of antibiotic for surgical prophylaxis allows for overgrowth of Enterococcus at sites previously colonized by cephalosporin-sensitive organisms and may contribute to the increased number of hospital associated surgical site infections [18].
Perhaps nowhere is this battle between pathogen and microbicide more apparent than with N. gonorrhoeae. From the first use of sulfonamides in the 1940s, this bacterium has countered the development of resistant strains [19]. After showing that this pathogen is capable of developing resistance to any antibiotic directed against it, including sulfonamides, penicillin, tetracycline, spectinomycin, quinolones, macrolides, and now cephalosporins, it is apparent that novel approaches are needed beyond the two-drug counterattack recommended just last year [20].
We recently developed an immunoassay [N-Assay] in which membranes coated with antibody against GBS are exposed to bacterial suspensions, and after incubating for a series of time-points, bound GBS is detected [14]. This assay was tested on patients’ vaginal-rectal specimens and was found to have a high degree of sensitivity and specificity, with the added benefit of being capable of detecting nonbeta hemolytic streptococcal strains of GBS [21].
In order to decrease the interobserver variation seen with dot-blot assays, we converted the test to an ELISA format. In this study, we have substituted the anti-GBS antibodies for those directed against either N. gonorrhoeae or Enterococcus. We show that these polyclonal antibodies provide a great deal of sensitivity in a nonselective broth, with little interference from other commonly isolated cocolonizers Figure 1 and Supplemental Figure 1.
Following a six-hour incubation period, the limit of detection increased to 101 bacteria per mL for GBS (Figure 2). Furthermore, we show that antibiotic susceptibility may be determined (Supplemental Figure 2). From this, one may envision a panel in which a series of wells are set up in which multiple clinically relevant antibiotics are tested, providing the clinician with a targeted approach to treatment in a greatly reduced timeframe (Figure 3).
A central facet of this assay involves cellular viability: the concentration of bacteria present in the well is related specifically to the incubation times utilized. By incubating overnight, the sensitivity of the test was increased significantly as live bacteria continued to grow and divide, with GBS being detected at 10−2 bacteria per mL, Enterococcus detected at 10−1 bacteria per mL, and N. gonorrhoeae detected at 101 bacteria per mL (Figure 2). This allows for a sensitivity greater than published results for several PCR-based methods of detection following an enrichment step, with the unique advantage being that this test is capable of detecting viable cells [22–25].
This assay [the N-Assay] provides the unique approach of targeting the growth of a specific clinically relevant pathogen through the use of a capture antibody. An immediate binding assay demonstrates whether a pathogen is present or not. By then culturing the organism in the presence or absence of selected antibiotics, one may show that cells are viable and susceptible to specific antimicrobials, thereby allowing the clinician to then make an informed decision more rapidly.
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