Design and Medical Effects of a Vaginal Cleaning Device Generating Plasma-Activated Water with Antimicrobial Activity on Bacterial Vaginosis

Round 1

Reviewer 1 Report

Comments on manuscript entitled

«Design and medical effects of a vaginal cleaning device generating plasma-activated water with antimicrobial activity on bacterial vaginosis» by Yuan Hwang et al.

In the manuscript, the authors investigated the antibacterial effect of the water activated by low temperature plasma. The title is descriptive and accurately describes the paper. The results have been analyzed in the context of the perspectives of low temperature plasmas for clinical medicine. Thus, the paper subject and its content are appropriate for the Plasma journal. The study could also be of interest to practicing physicians.

However, the idea and the proposed approach are not original. Many plasma-generating devices for water treatment have been designed by now. There are a large number of studies concerning bacteria inactivation by plasma-treated water published in the last five years or even earlier. Therefore, the detailed comparison of the effectiveness of the applied plasma generator with the results obtained by other researchers should be performed (may be as Discussion section) to reveal the advantages of the proposed technique.

My other comments are as follows:

• In the Sections 2.1 and 2.2 the construction of the used plasma generator is given. However, it is not clear whether this set-up is originally designed by the authors or is a commercial device. The
• The main characteristics of the plasma generator and the produced plasma should be given (the frequency and the power of the discharge, the distance between the electrodes, etc.). What were the volume of the water to be treated and the treatment time? How the water flow or water mixing was organized during the procedure? Otherwise, it is very hard to estimate the accuracy of the obtained data (even clinical results).
• Figure 3: the units on the axis “Time after plasma treatment” are not indicated. Please, specify.
• Section 3.1: How were the measurements of hypochlorous acid concentration were carried out? Some brief comments should be included into the Section. It is worth including speculations why the acid concentration in the plasma-treated water rises sharply after long lag-period and does not depend on the duration of the treatment.
• Oxygen- and nitrogen-containing particles produced in water during its plasma treatment are also very active antibacterial agents. Some convincing arguments that the chlorine-containing particles in the plasma-treated water play the main role in the antibacterial effect should be given.
• It is well known that the changes of pH are induced in plasma-solution systems and the water pH strongly affects bacterial activity and growth. Therefore, the data concerning pH of the plasma-treated water used in clinical trial should be given (see also comment #5).
• Figure 4 is not clear enough and should be changed significantly to improve its understanding by readers. Please clarify the term “degree of bacterial contamination”. For example, what do terms “many, some, few, a few” in the legend mean? What is the quantitative difference between these groups? What are the units for the “degree of bacterial contamination” (Figure 4, B) and for the “Quantity of Gram(+) bacilli” (Figure 4, A)? Looking at Figure 4 B it seems that betadine provides a more significant and stable antibacterial effect than the plasma-treated water. Is it really so?
• The used plasma device was equipped with a laser light and UV-radiation was generated during clinical treatment manipulations. Did authors performed control clinical experiments to be sure that the antibacterial effect is due to the plasma-treated water and is not related to the UV-action? Please, comment.

Thus, the paper is not appropriate for publication in the present form and should be reconsidered after major revisions.

Author Response

Reviewer 1

In the manuscript, the authors investigated the antibacterial effect of the water activated by low temperature plasma. The title is descriptive and accurately describes the paper. The results have been analyzed in the context of the perspectives of low temperature plasmas for clinical medicine. Thus, the paper subject and its content are appropriate for the Plasma journal. The study could also be of interest to practicing physicians. However, the idea and the proposed approach are not original. Many plasma-generating devices for water treatment have been designed by now. There are a large number of studies concerning bacteria inactivation by plasma-treated water published in the last five years or even earlier. Therefore, the detailed comparison of the effectiveness of the applied plasma generator with the results obtained by other researchers should be performed (may be as Discussion section) to reveal the advantages of the proposed technique.

We really appreciate the reviewer’s constructive comments. Responses to the questions are listed below. The main text and supporting information have been revised according to the valuable comments of the referees, and all changes are highlighted in red.

As the reviewer pointed out, numerous studies have provided the antimicrobial activity of plasma-activated water in various fields. For a practical application to medical field, we thought that the design of the medical device considering a certain disease is important in order to apply the anti-bacterial activity of plasma-activated water for treatment for the disease. As mentioned in the manuscript, vaginitis is a common female disease caused by a vaginal infection due to an overgrowth of bacteria, and is the most common gynecological disease encountered by obstetricians, and in the United States, approximately 10 million patients visit obstetrics and gynecology clinics every year. Generally, some topical antiseptics and disinfectants, such as betadine, have been used to treat bacterial vaginosis, but these agents are generally difficult to apply deep in the vaginal tract due to the structural curvature of the vaginal tact. Thus, spraying antibacterial plasma-activated water to the vaginal tract would have a great advantage in delivering it in depth. Therefore, we designed a vaginal cleaning device with a spray nozzle that contains both a clean outlet and a suction outlet to spray and recover the plasma water generated using the tap water. As a result, clinical trial results support that plasma-activated water has an antiseptic effect on vaginal bacterial contamination similar to betadine. Even if it is similar to the effect of betaine or a little less, if plasma-activated water can replace the chemical betadine, it is believed that it has a great advantage in treating the patients with vaginitis.

 

My other comments are as follows:

1. In the Sections 2.1 and 2.2 the construction of the used plasma generator is given. However, it is not clear whether this set-up is originally designed by the authors or is a commercial device.

[Response] First, we are really sorry for missing a detailed information about plasma generator used in this study. We have designed the plasma generator ourselves for this study. A detailed description of the specifications was introduced in the following questions.

2. The main characteristics of the plasma generator and the produced plasma should be given (the frequency and the power of the discharge, the distance between the electrodes, etc.). What were the volume of the water to be treated and the treatment time? How the water flow or water mixing was organized during the procedure? Otherwise, it is very hard to estimate the accuracy of the obtained data (even clinical results).

[Response] As the reviewer pointed out, we have revised a detailed description on the raised issue as below.

The plasma generation modules include a pair of plasma electrodes and an insulating frame placed between the plasma electrodes, and a conductive screw applying different polarity power to each plasma electrode (Figure 1C). The plasma electrode is a disc-shaped grid electrode with a diameter of 77 mm and a thickness of 0.5 mm, and titanium is used as the electrode material to prevent corrosion due to plasma. In the disc-shaped grid electrodes, the diameter and pitch of the grid were 1.07 mm and 3.92 mm, respectively. According to the dimension of the grid, the percentage of open space was 50%. In addition, a platinum thin film with a thickness of 300 nm was deposited on the grid surface using electroless plating in order to function as a catalyst for increasing the amount of plasma generated. The plasma is generated between two electrodes of different polarities, and the distance between the two electrodes was kept constant by a 2 mm thick insulating frame.

3. Figure 3: the units on the axis “Time after plasma treatment” are not indicated. Please, specify.

[Response] In the revised Figure 3, we have added the unit on the x- axis as the reviewer pointed out.

4. Section 3.1: How were the measurements of hypochlorous acid concentration were carried out? Some brief comments should be included into the Section. It is worth including speculations why the acid concentration in the plasma-treated water rises sharply after long lag-period and does not depend on the duration of the treatment.

[Response] As the reviewer suggested, we additionally inserted a measurement method of hypochlorous acid in the revised manuscript. Briefly, the concentration of residual chlorine in water is determined by colorimetric method based on N,N-diethyl-p-phenylenediamine by using colorimeter instrument (Sinsche Technology, Q-CL501P) according to manufacturer’s instruction.

As shown in Figure 9 and 10 of the referenced study below, Jirásek and colleagues showed that atmospheric pressure plasma discharge during 20 min showed an increased pattern of hypochlorous acid’s concentration after long-lag period with decreasing pH value, and the lowing pH leads to the increase of oxidation–reduction potential. Thus, they suggest that the ORP increase can be attributed to the associated pH decrease and to the increase of the [HOCl]/[OCl−] ratio, since the HOCl has a higher standard reduction potential (1.482 V) than OCl− (0.81 V). To confirm this possibility in our system, we measured pH value over time after plasma discharge. Unexpectedly, there was no a remarkable change in pH value by plasma exposure (please refer to response 6 below). Therefore, in-depth study is further required on why active free chlorines have increased by the plasma treatment to a supplied tap water.

5. Oxygen- and nitrogen-containing particles produced in water during its plasma treatment are also very active antibacterial agents. Some convincing arguments that the chlorine-containing particles in the plasma-treated water play the main role in the antibacterial effect should be given.

[Response] In the revised manuscript, we have further provided possible roles of chlorine-containing chemicals in the antibacterial activity in the discussion as below.

Plasma-activated media or water has largely known to produce reactive oxygen and nitrogen species containing solutions, which is believed to exhibit the antimicrobial activity when exposed to infected bacteria [21]. In addition, it has known that chlorine-based active species are created by the plasma treatment. These free active chlorines such as hypochlorous acid and hypochlorite are commonly considered as the active ingredient of the deactivation of pathogens in drinking water, swimming pool water and wastewater. Indeed, numerous studies have been supporting the antimicrobial activity of chlorine-base active species including hypochlorous acid as a reactive chlorine species [22]. Because of highly reactive nature as a strong oxidant, these chlorine-base active species have known to interact with most cellular macromolecules, lipids, and nucleotides. For example, hypochlorous acid rapidly reacts with sulfur-containing residues (cysteine, methionine, or glutathione) and amines of the protein. In the present study, we found an increased residual free chlorine concentration after plasma discharge. To more clarify the antibacterial effect of our plasma-activated water, further studies are additionally warranted whether reactive oxygen and nitrogen species are created in plasma-activated water produced from our device.

21. Kaushik, N.K.; Ghimire, B.; Li, Y.; Adhikari, M.; Veerana, M.; Kaushik, N.; Jha, N.; Adhikari, B.; Lee, S.J.; Masur, K., et al. Biological and medical applications of plasma-activated media, water and solutions. Biol Chem 2018, 400, 39-62, doi:10.1515/hsz-2018-0226.
22. Gray, M.J.; Wholey, W.Y.; Jakob, U. Bacterial responses to reactive chlorine species. Annu Rev Microbiol 2013, 67, 141-160, doi:10.1146/annurev-micro-102912-142520.

 

6. It is well known that the changes of pH are induced in plasma-solution systems and the water pH strongly affects bacterial activity and growth. Therefore, the data concerning pH of the plasma-treated water used in clinical trial should be given (see also comment #5).

[Response] Same as the conditions for measuring free chlorines, we measured pH value after the plasma discharge at 10 (circle, blue), 20 (rectangular, green), and 30 (triangle, red) minutes. The initial pH value of supplied tap water was approximately 7.2, and plasma treatment induced a slight increase in pH to 7.4, but there was a remarkable difference in pH value in the water after the plasma discharge. Therefore, it is likely that antimicrobial activity of plasma-activated water in our system is not dependent on pH changes. To clarify antibacterial effect of our plasma-activated water, further studies are needed how to increase free chlorine concentrations by the plasma discharge in our system.

7. Figure 4 is not clear enough and should be changed significantly to improve its understanding by readers. Please clarify the term “degree of bacterial contamination”. For example, what do terms “many, some, few, a few” in the legend mean? What is the quantitative difference between these groups? What are the units for the “degree of bacterial contamination” (Figure 4, B) and for the “Quantity of Gram(+) bacilli” (Figure 4, A)? Looking at Figure 4 B it seems that betadine provides a more significant and stable antibacterial effect than the plasma-treated water. Is it really so?

[Response] First, we apologize for unclear descriptions of the Figure 4. In the revised manuscript, we have further clarified the terms/sentences and added detailed explanation on clinical treating conditions of plasma activated water to help the readers understand as below. In addition, betadine is a common antiseptic used for skin disinfection, and its effect on killing the bacteria has well known in the patients with bacterial vaginosis. However, some side effects such as skin irritation have reported. In the present study, we show that plasma-activated water has an antiseptic effect on vaginal bacterial contamination similar to betadine. Even if it is similar to the effect of betaine or a little less, if plasma-activated water can replace the chemical betadine, it is believed that it has a great advantage in treating the patients with vaginitis. 

Briefly, the gram-positive test and bacterial culture test were examined from vaginal secretion of clinical trial participants with vaginitis (44 patients) at the first visit (VISIT 1). After one week, they were randomly divided into the control group (22 patients) and experimental group (20 patients). Our vaginal cleaning device was utilized for vaginal cleaning in the experimental group, and a topical antiseptic, betadine, was applied in the control group. For using plasma-activated water, the plasma discharged for 10 min in the cleaning solution container with 3 liters of tap water. The plasma-activated water sprayed for 30~60 seconds (approximately 150~200 mL) to the patients of the experimental group through spraying nozzle, and subsequently exposed an ultraviolet light inside the vagina for 5 seconds through light-generating nozzle. Thereafter, the gram-positive test and bacterial culture tests were performed in the patients (VISIT 2). After 2 days, we examined the gram-positive test and bacterial culture test (VISIT 3) and additionally conducted the tests after 2 days (VISIT 4).

Consequently, a total of 20 patients in the experimental group and 22 patients in the control group were examined in this study. The gram-positive test and bacterial culture tests were performed 4 times in total for each patient, and the effects of cleaning were analyzed as the test results at VISIT 1 and VISIT 3. The results are shown in Table 1 (control group) and Table 2 (experimental group). As shown in Figure 4A, 6 out of 20 patients in the experimental group showed a quantitative decrease in gram (+) bacilli between VISIT 1 (4.0 ± 0.0) and VISIT 3 (2.6 ± 0.9), and 5 out of 22 in the control group showed a quantitative decrease in gram (+) bacilli between VISIT 1 (4.0 ± 0.0) and VISIT 3 (2.8 ± 0.4). The quantity of gram (+) bacilli is indicated by the number of times that gram-stained bacilli was visible in the field when the microscopic field moved at random 10 times. For example, 4+ means that the fields with gram-stained bacilli were 4 times in a total of 10 fields. In addition, we classified the degree of bacterial contamination into four stages (many, some, a few, and few) according to the degree of spreading bacterial colony in the bacterial culture test. If bacteria grow in three parts when the petri dish is quartered, they are classified as many, some in two parts, a few in one part, and few if they grow little. As a result, we found a decrease in the degree of bacterial contamination in both the control and experimental groups (Figure 4B).

8. The used plasma device was equipped with a laser light and UV-radiation was generated during clinical treatment manipulations. Did authors performed control clinical experiments to be sure that the antibacterial effect is due to the plasma-treated water and is not related to the UV-action? Please, comment.

[Response] As the reviewer pointed out, we are not sure whether plasma-activated water only influences on the antimicrobial activity in the clinical experiments. As well known, its prominent and reliable antibacterial effect has proved by numerous studies. So, we strongly believe that the clinical effect on vaginal bacterial contamination is largely attributed to the treatment of plasma-activated water. Despite the expected antimicrobial action of UV radiation, it is generally difficult to apply deep in the curved vaginal tract. In this regard, plasma-activated water has the great advantage of being able to handle deeply, so it is expected to have a clinically synergy effect when used with UV light for treating the bacterial vaginosis.

Reviewer 2 Report

The authors describe a novel device based on plasma-activated water and UV light to treat vaginosis. In particular they present results from clinical trials that are of high interest to the plasma medicine community. Overall, the manuscript is well-written but can benefit from some revisions. Firstly the figures do not have units. For example, figure 3 has labels 10,20,30 and an x-axis label of Time after plasma treatment with no units. Similarly, Figure 3 quantifies the `quantity of Gram(+) bacilli` and `the degree of bacteria` in uncertain units. Further discussion and precise explanation of the included figures could greatly improve the readability of the manuscript and make results easier to understand.

Moreover, the paper lacks some detail particularly in areas regarding the characteristics of the used plasma device. Some electrical and chemical characterization of the device, and if available some in-vitro studies on the particular conditions used to generate the plasma-activated water would be of great interest. A more detailed description of the particular treatment applied (how long was the plasma run, how was the plasma activation process optimized, etc.) would also be of benefit. Lastly, it would be good to have some insight on how much of the treatment effect can be attributed to plasma-activated water versus UV sterilization. 

Overall I congratulate the authors for their work, particularly for undertaking the arduous task of organizing and executing clinical trials with a plasma medical device. I encourage the authors to where possible apply some of the suggestions above and provide more detail to increase the impact of the manuscript for publication.

Author Response

Reviewer 2

The authors describe a novel device based on plasma-activated water and UV light to treat vaginosis. In particular they present results from clinical trials that are of high interest to the plasma medicine community. Overall, the manuscript is well-written but can benefit from some revisions.

We really appreciate the reviewer’s constructive comments. Responses to the questions are listed below. The main text and supporting information have been revised according to the valuable comments of the referees, and all changes are highlighted in red.

 

1. Firstly the figures do not have units. For example, figure 3 has labels 10,20,30 and an x-axis label of Time after plasma treatment with no units. Similarly, Figure 3 quantifies the `quantity of Gram(+) bacilli` and `the degree of bacteria` in uncertain units. Further discussion and precise explanation of the included figures could greatly improve the readability of the manuscript and make results easier to understand.

[Response] First, we apologize for missing a detailed information of Figure 3 and unclear descriptions of the Figure 4. As the reviewer point out, we have revised the raised points in the revised manuscript. The x-axis of Figure 3 was minute (min). Moreover, we have introduced a more detailed description on critical results in the revised manuscript as below.

Consequently, a total of 20 patients in the experimental group and 22 patients in the control group were examined in this study. The gram-positive test and bacterial culture tests were performed 4 times in total for each patient, and the effects of cleaning were analyzed as the test results at VISIT 1 and VISIT 3. The results are shown in Table 1 (control group) and Table 2 (experimental group). As shown in Figure 4A, 6 out of 20 patients in the experimental group showed a quantitative decrease in gram (+) bacilli between VISIT 1 (4.0 ± 0.0) and VISIT 3 (2.6 ± 0.9), and 5 out of 22 in the control group showed a quantitative decrease in gram (+) bacilli between VISIT 1 (4.0 ± 0.0) and VISIT 3 (2.8 ± 0.4). The quantity of gram (+) bacilli is indicated by the number of times that gram-stained bacilli was visible in the field when microscopic field moved at random 10 times. For example, 4+ means that the fields with gram-stained bacilli were 4 times in a total of 10 fields. In addition, we classified the degree of bacterial contamination into four stages (many, some, a few, and few) according to the degree for spreading bacterial colony in the bacterial culture test. If bacteria grow in three parts when the petri dish is quartered, they are classified as many, some in two parts, a few in one part, and few if they grow little. As a result, we found a decrease in the degree of bacterial contamination in both the control and experimental groups (Figure 4B).

 

2. Moreover, the paper lacks some detail particularly in areas regarding the characteristics of the used plasma device. Some electrical and chemical characterization of the device, and if available some in-vitro studies on the particular conditions used to generate the plasma-activated water would be of great interest. A more detailed description of the particular treatment applied (how long was the plasma run, how was the plasma activation process optimized, etc.) would also be of benefit.

[Response] In the revised manuscript, we have revised the raised points.

1) the characteristics of the used plasma device as below.

The plasma generation modules include a pair of plasma electrodes and an insulating frame placed between the plasma electrodes, and a conductive screw applying different polarity power to each plasma electrode (Figure 1C). The plasma electrode is a disc-shaped grid electrode with a diameter of 77 mm and a thickness of 0.5 mm, and titanium is used as the electrode material to prevent corrosion due to plasma. In the disc-shaped grid electrodes, the diameter and pitch of the grid were 1.07 mm and 3.92 mm, respectively. According to the dimension of the grid, the percentage of open space was 50%. In addition, a platinum thin film with a thickness of 300 nm was deposited on the grid surface using electroless plating in order to function as a catalyst for increasing the amount of plasma generated. The plasma is generated between two electrodes of different polarities, and the distance between the two electrodes was kept constant by a 2 mm thick insulating frame.

2) in vitro particular conditions used to generate the plasma-activated water as below.

For using plasma-activated water in vitro experiment, we treated the plasma during at 10, 20, and 30 minutes in the cleaning solution container with 3 liters of tap to optimize the plasma activation. According to above conditions, we measured the concentration of free chlorines in the water. The concentration of residual chlorine in water is determined by colorimetric method based on N,N-diethyl-p-phenylenediamine by using colorimeter instrument (Sinsche Technology, Q-CL501P) according to manufacturer’s instruction.

 

3. Lastly, it would be good to have some insight on how much of the treatment effect can be attributed to plasma-activated water versus UV sterilization.

[Response] As the reviewer pointed out, we are not sure whether plasma-activated water only influences on the antimicrobial activity in the clinical experiments. As well known, its prominent and reliable antibacterial effect has proved by numerous studies. So, we strongly believe that the clinical effect on vaginal bacterial contamination is largely attributed to the treatment of plasma-activated water. Despite the expected antimicrobial action of UV radiation, it is generally difficult to apply deep in the curved vaginal tract. In this regard, plasma-activated water has the great advantage of being able to handle deeply, so it is expected to have a clinically synergy effect when used with UV light for treating the bacterial vaginosis.

 

4. Overall I congratulate the authors for their work, particularly for undertaking the arduous task of organizing and executing clinical trials with a plasma medical device. I encourage the authors to where possible apply some of the suggestions above and provide more detail to increase the impact of the manuscript for publication.

[Response] We really appreciate the reviewer’s suggestion for the manuscript for publication.

 

 

 

Round 2

Reviewer 1 Report

The reviewer thanks the authors for their comprehensive answers and comments. The article can be accepted for publication in Plasma.