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What screening method was evaluated in this study?

The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals.

All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL Advanced Biotechnologies Inc. .

What screening method was evaluated in this study?

The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals.

The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC.

What screening method was evaluated in this study?

The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification NINA devices that generate heat from the exothermic reaction of calcium oxide and water . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals.

For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300. The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC. To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system Bio-Rad Laboratories, Hercules, CA . Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain Invitrogen , which was subsequently visualized using the ChemiDoc XRS system. To compare temperature and amplification consistency, three NINA heaters were tested in parallel.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory 28uC; Table 2 .

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree. The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates 60% . For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler Table 2 .

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option. We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory. For increased applicability for use at the POC, several modifications can be made to the NINA heaters.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals. Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health PATH, Seattle, WA , as described . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide CaO; Sigma-Aldrich, St. Louis, MO and water.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices Table 4 .

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option.

What was used to measure the performance of the NINA heaters?

We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment.

All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes. In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid NINA heaters for amplification of HIV-1 using RT-LAMP.

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA Bio-Rad Laboratories, Redmond, WA , GS HIV-1 Western blot Bio-Rad Laboratories , Aptima HIV-1 RNA assay Gen-Probe, Inc., San Diego, CA , and Amplicor HIV-1 DNA assay Roche Diagnostics, Branchburg, NJ . Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus.

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals.

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL Advanced Biotechnologies Inc. .

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

An RNA linearity panel was obtained commercially PRD801; SeraCare Life Sciences, Mil- ford, MA and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit QIAGEN . Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD .

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences . All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA Bio-Rad Laboratories, Redmond, WA , GS HIV-1 Western blot Bio-Rad Laboratories , Aptima HIV-1 RNA assay Gen-Probe, Inc., San Diego, CA , and Amplicor HIV-1 DNA assay Roche Diagnostics, Branchburg, NJ .

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially PRD801; SeraCare Life Sciences, Mil- ford, MA and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM .

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay Gen-Probe, Inc., BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466 is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing.

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus . Rapid tests are currently a key component of HIV screening at the point-of-care POC , significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings. Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations.

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection .

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes. In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid NINA heaters for amplification of HIV-1 using RT-LAMP.

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

To date, the Aptima HIV-1 RNA assay Gen-Probe, Inc., BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466 is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing. To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time ,1 hour is desirable . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs .

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC. Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed .

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

LAMP has also proven to be less sensitive to biological inhibitors than PCR , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction.

What percentage of HIV-infected people go undetected in the United States?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

BACKGROUND: To date, the use of traditional nucleic acid amplification tests NAAT for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care POC settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification RT-LAMP , exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices.

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals.

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA Bio-Rad Laboratories, Redmond, WA , GS HIV-1 Western blot Bio-Rad Laboratories , Aptima HIV-1 RNA assay Gen-Probe, Inc., San Diego, CA , and Amplicor HIV-1 DNA assay Roche Diagnostics, Branchburg, NJ . Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus.

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples SeraCare Life Sciences were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL Advanced Biotechnologies Inc. .

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus . Rapid tests are currently a key component of HIV screening at the point-of-care POC , significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings. Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations.

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection .

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests NAAT are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay Gen-Probe, Inc., BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466 is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing.

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

Positivity of the clinical specimens was consistent between the thermalcycler and devices Table 4 . Amplification consistency was most evident with two of the patient samples patient #4 and #5 that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative data not shown .

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

An RNA linearity panel was obtained commercially PRD801; SeraCare Life Sciences, Mil- ford, MA and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit QIAGEN . Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD .

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially PRD801; SeraCare Life Sciences, Mil- ford, MA and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM .

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

All HIVnegative blood samples, included in each reaction, were negative data not shown . A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes. In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid NINA heaters for amplification of HIV-1 using RT-LAMP.

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices Table 4 .

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC. Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed .

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

To date, the Aptima HIV-1 RNA assay Gen-Probe, Inc., BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466 is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing. To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time ,1 hour is desirable . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs .

What percentage of patients do not return for followup after HIV testing?

Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus .

Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC and RNA extracted from HIV-2 NIH-Z purified virus Advanced Biotechnologies Inc., Columbia, MD . Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study , or obtained commercially SeraCare Life Sciences . All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA Bio-Rad Laboratories, Redmond, WA , GS HIV-1 Western blot Bio-Rad Laboratories , Aptima HIV-1 RNA assay Gen-Probe, Inc., San Diego, CA , and Amplicor HIV-1 DNA assay Roche Diagnostics, Branchburg, NJ .

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 . The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration MIC values i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism within the susceptible range was described previously . Thus, the emergence of MRSA and vancomycin-resistant S. aureus in the recent years as well makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria .

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA . For the MICs of vancomycin, see Table 1 . Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus VS-MSSA ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and quality control strain.

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL. In all cases, there were comparable MBC values for the clinical isolates of MRSA and the S. aureus reference strain. Bactericidal activity is defined as a ratio of MBC to MIC of ≤4 .

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay . MBC values of all tested compounds are recorded in Table 1 as well.

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin.

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 methicillin-susceptible as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study.

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected . All isolates were tested for the presence of the mecA gene encoding methicillin resistance . These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA .

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus VS-MSSA ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and quality control strain. The bacteria were stored at −80 ∘ C and were kept on blood agar plates Columbia agar base with 5% ovine blood between experiments. MBCs .

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds . The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC.

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log. CFU/mL reduction in bacterial count after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems .

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 methicillin-susceptible as the reference and quality control strain.

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC g/mL MBC g/mL 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 S -CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 S -CH CH 3 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 S -benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 R -CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 S -CH CH 3 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 S -CH CH 3 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br S -CH CH 3 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl S -CH CH 3 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl S -benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1 Time-kill assays were performed by the broth macrodilution method according to previously described methodology with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume.

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

Thus, the emergence of MRSA and vancomycin-resistant S. aureus in the recent years as well makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs . This fact should be considered during the development of effective and safe treatment options for MRSA infections.

What is the most common, clinically-relevant multiresistant pathogen in both healthcare and community acquired infections?

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC. Treatment failure exceeding 60% for S. aureus with vancomycin MIC of 4 g/mL resulted in the change of susceptibility breakpoint from 4 g/mL to 2 g/mL by the Clinical and Laboratory Standards Institute CLSI in 2006 as well as by the US Food and Drug Administration FDA in 2008 . It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered .

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered . It is of note that based on time-kill assays in the present study, all tested diamides particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL. The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower.

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

Thus, the emergence of MRSA and vancomycin-resistant S. aureus in the recent years as well makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs . This fact should be considered during the development of effective and safe treatment options for MRSA infections.

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

However, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load . Moreover, in the case of an immune system disorder e.g., immunosuppressive therapy, AIDS patients, etc. bactericidal agents are unequivocally indicated.

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 . The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration MIC values i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism within the susceptible range was described previously . Thus, the emergence of MRSA and vancomycin-resistant S. aureus in the recent years as well makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria .

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds . The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC.

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 methicillin-susceptible as the reference and quality control strain.

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC. Treatment failure exceeding 60% for S. aureus with vancomycin MIC of 4 g/mL resulted in the change of susceptibility breakpoint from 4 g/mL to 2 g/mL by the Clinical and Laboratory Standards Institute CLSI in 2006 as well as by the US Food and Drug Administration FDA in 2008 . It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs 2 g/mL , alternative therapy should be considered .

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems . Methicillin-resistant Staphylococcus aureus MRSA has become the most common clinically relevant multiresistant pathogen causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010 , followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010 .

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA. The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria.

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

A series of nine substituted 2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant Staphylococcus aureus MRSA and S. aureus ATCC 29213 as the reference and quality control strain. The minimum bactericidal concentration was determined by subculturing aliquots from MIC determination onto substance-free agar plates. The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N- 2S -3-methyl-1-oxo-1-{ 4- trifluoromethyl phenyl amino}butan-2-yl benzamide 1f , N-{ 2S -1- 4-bromophenyl amino -3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide 1g , and 4-chloro-N-{ 2S -1- 3,4-dichlorophenyl amino -3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide 1h was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points.

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents , the failure in the cell wall biosynthesis due to the inhibition of transglycosylases could be responsible for bactericidal activity of diamides against MRSA. Based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time. 1-oxobutan-2-yl}-2-hydroxybenzamide 1h were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain.

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected . All isolates were tested for the presence of the mecA gene encoding methicillin resistance . These three clinical isolates were classified as vancomycin-susceptible but with higher MIC of vancomycin equal to 2 g/mL VA2-MRSA within the susceptible range for MRSA 63718 methicillinresistant S. aureus VS-MRSA .

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

This fact should be considered during the development of effective and safe treatment options for MRSA infections. The history of clinical usage of salicylanilides 2-hydroxy-N-phenylbenzamides dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s . Nowadays, salicylanilides SALs are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic , antibacterial , antimycobacterial , antifungal , and antiviral , among others.

What is the treatment of choice for MRSA infections?

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log. CFU/mL reduction in bacterial count after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h. Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems .

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

Furthermore, salicylanilides have been recognised as inhibitors of some bacterial enzymes, such as sortase A from S. aureus , d-alanine-d-alanine ligase , or transglycosylases from S. aureus but not from M. tuberculosis . These enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids .

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus including MRSA cell wall biosynthesis . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane . Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents , the failure in the cell wall biosynthesis due to the inhibition of transglycosylases could be responsible for bactericidal activity of diamides against MRSA.

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC g/mL MBC g/mL 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 S -CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 S -CH CH 3 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 S -benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 R -CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 S -CH CH 3 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 S -CH CH 3 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br S -CH CH 3 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl S -CH CH 3 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl S -benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1 Time-kill assays were performed by the broth macrodilution method according to previously described methodology with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume.

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

Such new agents could be a solution to the resistance challenges. This study is a follow-up paper to a recently published article . The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported .

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

SALs have been found to inhibit the two-component regulatory systems TCS of bacteria . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis . Furthermore, salicylanilides have been recognised as inhibitors of some bacterial enzymes, such as sortase A from S. aureus , d-alanine-d-alanine ligase , or transglycosylases from S. aureus but not from M. tuberculosis .

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay . MBC values of all tested compounds are recorded in Table 1 as well.

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N- 2S -3-methyl-1-oxo-1-{ 4- trifluoromethyl phenyl amino}butan-2-yl benzamide 1f , N-{ 2S -1- 4-bromophenyl amino -3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide 1g , and 4-chloro-N-{ 2S -1- 3,4-dichlorophenyl amino -3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide 1h was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points. The most potent bactericidal agent was compound 1f exhibiting remarkable rapid concentration-dependent bactericidal effect even at 2x MIC at 4, 6, and 8 h with a reduction in bacterial count ranging from 3.08 to 3.75 log. CFU/mL and at 4x MIC at 4, 6, 8, and 24 h 5.30 log.

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin.

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

MBC values of all tested compounds are recorded in Table 1 as well. Based on the obtained results, all compounds assessed as active according to MIC values in our previous study 1f-i showed low or moderate MBC values against all four strains. The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL.

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

As mentioned above, SALs are known to exhibit a bacteriostatic effect , so it was very interesting to discover that diamides possess bactericidal activity. The amide bond -CONH- can cause interactions with a variety of enzymes ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against MRSA. The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus including MRSA cell wall biosynthesis .

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

MBCs . The MBCs i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium were determined by subculturing aliquots 20 L from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar MHA plates. The plates were incubated aerobically at 37 ∘ C for 24 h for colony count.

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time over 24 h , and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum.

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

There is a discrepancy between bactericidal results of MBC assay compared with time-kill kinetics. This difference could be caused by comparing microtiter MBC assay to macrobroth time-kill assay dilutions . Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents .

What enzyme is essential for the metabolism of fatty acids?

Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: i methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and ii isocitrate lyase, which is essential for the metabolism of fatty acids . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.

Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. The present study is the first evidence of bactericidal effect of SAL analogues. Against other strains, reliable bactericidal effect was maintained at 4x MIC at 24 h after incubation.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC g/mL MBC g/mL 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 S -CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 S -CH CH 3 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 S -benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 R -CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 S -CH CH 3 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 S -CH CH 3 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br S -CH CH 3 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl S -CH CH 3 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl S -benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1 Time-kill assays were performed by the broth macrodilution method according to previously described methodology with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

Regrowth at 24 h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. The reason for regrowth of the test organism at 24 h in the experiment is unknown. Most probably, selection of resistant mutants is responsible for this phenomenon ; degradation of the drug in the growth medium is not assumed, as regrowth was Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time over 24 h , and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 methicillin-susceptible as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis . ; and MRSA SA 3202 National Institute of Public Health, Prague, Czech Republic both of human origin. Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected .

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay . MBC values of all tested compounds are recorded in Table 1 as well.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

Briefly, flasks containing sterile fresh Mueller-Hinton broth MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots 20 L were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

Bactericidal activity i.e., ≥3 log 10 CFU/mL decrease is expressed in bold. For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718. Time was not the predictive factor influencing the antibacterial activity because log 10 differences in CFU/mL from the starting inoculum were the same for 4x MIC with the highest efficiency with a reduction in bacterial count of 5.30 log 10 CFU/mL or very similar for 2x MIC with a moderate regrowth after 24 h causing a loss of bactericidal activity over 24 h. The bactericidal effect was maintained even at 2x MIC at 4 h after incubation for this strain reduction of 3.08 log 10 CFU/mL .

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

The results were analysed by evaluating the numbers of strains that yielded Δ log 10 CFU/mL values of −1 corresponding to 90% killing , −2 99% killing , and −3 99.9% killing at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% ≥3 log 10 of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 methicillin-susceptible as the reference and quality control strain.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides, called diamides due to their skeleton for general structure see Table 1 , was described previously , and their antimycobacterial and antibacterial activities against various bacterial species were reported . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA. The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

The plates were incubated aerobically at 37 ∘ C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced ≥99.9% killing Table 1 : Chemical structures and in vitro MIC and MBC g/mL values of tested 5-and 4-chloro-2-hydroxy-N- 1-oxo-1- phenylamino alkan-2-yl benzamides bactericidal effect of individual compounds against particular strains marked in bold . after 24 h of incubation as compared to the colony count of the starting inoculum .