YKL-40 and Lysosome-Associated Membrane Proteins as Potential Discriminative Biomarkers in Central Nervous System Infections

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting study that highlights the promising diagnostic role of YKL-40. The sample classification and inclusion criteria are well-defined. However, improvements are needed in organizing experimental data, statistical analysis, and patient information presentation in the Results section.

Title: "Proteins YKL-40, LAMP-1, and LAMP-2 in Central Nervous System Infections"

The title is ambiguous and does not effectively summarize the major findings of the study. Consider revising it to better reflect the key results and implications.

1. Introduction

"However, none of them focused on the gene and protein expression of the protein nor on its relationship with autophagy in CNS infections."

A correlation analysis should be performed to assess the relationship between YKL-40 and autophagy-associated proteins, as proposed by the authors in the Introduction.

2. Materials and Methods

The demographic and laboratory characteristics of patients with CNS infections and the control group should be provided in a structured format, such as Table 1, for clarity.

2.3.1. Microbiological Evaluation

This section provides a well-detailed microbiological examination of CSF samples included in the study. However, the corresponding results are not presented in the paper.

• A summary of the microbiological evaluation results for each patient should be included in the Results section or Supplementary Materials.
• It would also be informative to assess the diagnostic value of YKL-40 in specific viral or bacterial infections. For example, evaluating YKL-40 expression in Neisseria meningitidis CNS infections could provide additional insights.

Figures 1–3

• Gene/protein expression levels for each sample in both the control and patient groups should be displayed as individual data points within the columns to reflect the overall variability of test results.
• Each dataset should be separated into distinct figure panels (e.g., A, B, C, etc.) for clarity.

Figure 3

• The LAMP-2 relative mRNA expression graph is missing and should be included.
• Data on plasma and CSF levels of LAMP-1/LAMP-2 are absent and should be reported.

Comments on the Quality of English Language

A revision by native English speakers is needed.

Author Response

Answers to reviewer:

Title: "Proteins YKL-40, LAMP-1, and LAMP-2 in Central Nervous System Infections"

The title is ambiguous and does not effectively summarize the major findings of the study. Consider revising it to better reflect the key results and implications.

The title is revised. We propose:

YKL-40, LAMP-1, and LAMP-2 as potential discriminative biomarkers of central nervous system infections

 

Introduction

However, none of them focused on the gene and protein expression of the protein nor on its relationship with autophagy in CNS infections."

A correlation analysis should be performed to assess the relationship between YKL-40 and autophagy-associated proteins, as proposed by the authors in the Introduction.

 

An addition to the text is made (lines 275-277). Also, a new graph is presented in the combined Figure 3.

 

2. Materials and Methods

The demographic and laboratory characteristics of patients with CNS infections and the control group should be provided in a structured format, such as Table 1, for clarity.

We accept the reviewer’s suggestion and provide a Table, as follows:

Table 1. Demographic and laboratory characteristics of the studied patients

№	Age	M/F	Microbiological result	CSF WBC [x10^6/l]	CSF Protein [g/l]	CSF glucose [mmol/l]	Serum/ CSF glucose ratio	CSF appear-ance	Clinical course	Study group definition
1	55	M	no etiology	20.00	0.77	3.70	0.58	clear	mild	suspected viral
2	94	F	no etiology	528.00	0.66	3.20	0.60	Clear	mild	suspected viral
3	17	M	no etiology	2.00	1.79	7.80	0.62	Clear	mild	suspected viral
4	68	M	no etiology	11.00	0.84	4.70	0.70	Clear	mild	suspected viral
5	32	M	no etiology	3.00	1.40	2.70	0.56	Clear	mild	suspected viral
6	49	F	no etiology	176.00	1.10	2.20	0.32	Clear	mild	suspected viral
7	32	M	no etiology	1.00	0.64	2.00	0.51	Clear	mild	suspected viral
8	48	M	no etiology	126.00	1.30	4.00	0.72	Clear	mild	suspected viral
9	67	M	no etiology	2.00	1.10	4.00	0.77	Clear	mild	suspected viral
10	63	M	no etiology	7.00	0.68	5.50	0.57	Clear	mild	suspected viral
11	69	F	no etiology	3.00	0.32	9.40	1.20	Clear	mild	suspected viral
12	70	F	no etiology	6.00	0.82	6.10	0.91	Clear	mild	suspected viral
13	79	M	no etiology	18.00	0.58	4.20	0.79	Clear	mild	suspected viral
14	49	F	no etiology	6.00	0.45	4.00	0.65	Clear	mild	suspected viral
15	69	M	VZV	32.00	0.87	3.40	0.63	Clear	moderate	viral
16	62	M	HSV-1	288.00	1.77	2.90	0.51	Clear	moderate	viral
17	47	M	no etiology	1.00	0.83	5.20	0.85	Clear	mild	suspected viral
18	49	M	no etiology	21	0.3	6	0.77	Clear	mild	suspected viral
19	8	M	no etiology	2	0.21	3.7	0.79	Clear	mild	suspected viral
20	42	F	no etiology	12	0.37	3	0.67	Clear	mild	suspected viral
21	80	F	no etiology	9	0.46	6.9	0.68	Clear	mild	suspected viral
22	49	F	no etiology	0	0.26	4.1	0.61	Clear	mild	suspected viral
23	35	M	S. pneumoniae	1190.00	6.53	0.00	0	Turbid	severe	bacterial
24	25	M	no etiology	129.00	2.40	0.60	0.09	Turbid	severe	suspected bacterial
25*	43	M	no etiology	1.00	0.37	3.00	0.42	Turbid	moderate	suspected bacterial
26	22	M	N. meningitidis C	9386.00	7.07	0.00	0	Turbid	severe	bacterial
27	82	F	L. monocytogenes	64	5.43	2	0.29	Turbid	severe	bacterial
28	34	F	H. influenzae b	480	1.81	2.3	0.30	Turbid	severe	bacterial
29	70	M	S. pneumoniae	2218	2.2	0.2	0.03	Turbid	severe	bacterial

*HIV-positive patient, immunocompromised; Age presented in years; M-males; F-females; CSF-cerebrospinal fluid; WBC-white blood cells;

•  
• 3.1. Microbiological Evaluation

This section provides a well-detailed microbiological examination of CSF samples included in the study. However, the corresponding results are not presented in the paper.

We addressed the reviewer’s remark and included the following text in the manuscript’s Results section:

1. Microbiological results and groups of observation

Based on the microbiological evaluation of CSF specimens we managed to culture the following bacterial pathogens – S. pneumoniae (n=2), N. meningitidis serogroup C (n=1), L. monocytogenes (n=1), and H. influenzae type b. All the bacterial pathogens were culture-positive, confirmed by the mPCR ME Panel, and also identified by the MALDI-TOF MS. Microbial cultures showed no fungal growth on SDA agar plates in all the tested CSF samples. Among the CSF samples, we managed to detect only two viral pathogens by the mPCR ME Panel – VZV and HSV-1. Data are summarized in Table 1.

• A summary of the microbiological evaluation resultsfor each patient should be included in the Results section or Supplementary Materials.

We addressed the reviewer's remark and included that information in the new Table 1 (see above)

 

• It would also be informative to assess the diagnostic value of YKL-40 in specific viral or bacterial infections. For example, evaluating YKL-40 expressionin Neisseria meningitidis CNS infections could provide additional insights.

 

Single microbiologically confirmed clinical cases are insufficient to draw any statistically significant conclusion on pathogen-specific diagnostic values of tested parameters.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript of Kazakova et al., aim to study the inflammatory marker (YKL-40) and autophagy-related proteins 23 (LAMPs) in patients with central nervous system (CNS) infections viral or bacterial. The topic is interesting and recent references support the use of these molecules as biomarkers.

The following major points should be addressed:

1. page 3 line 86: "consistent with an acute CNS infection" . It is well known that an acute infection always produce inflammation. YKL -40 is reported to be associated with inflammation and YKL-40 serum levels are increased with age, in various cardiovascular, metabolic, and systemic inflammatory diseases. I suggest to clarify what type of viral and bacterial infections of the clinical samples are included. There is need to know exactly for what viral or bacterial infection are hospitalized. Its virus has a different kinetic of infection and different target tissues. As it is presented (page 4 lanes-134-139) is  a general description with no value except of the description. Viruses belongs to different families and the same for bacteria.

2. page 4 line 129: "A selective medium for fungi (Sabouraud-dextrose agar) was also inoculated" . This is a description in the materials/methods  but there is no other result or  comment about fungi in the manuscript.

3. page 5 lane 183: "potential viral". Please clarify what the authors mean by potential viral infection? How the know that is a virus?

4. page 6 lanes 208-211 : "We detected statistically elevated concentrations of both plasma and CSF YKL-40 in 208 patients compared to the control group (p=0.0005 and p<0.03 respectively). In addition, 209 we observed that YKL-40 levels in CSF of patients were significantly higher compared to 210 plasma levels (p=0.0021)". Please clarify about the state of infection. How many days post infection?

5. page 7 lanes 221-222: "Higher CSF YKL-40 levels compared to plasma levels in patients with viral infection were detected. There was no change in plasma or CSF levels in bacterial infections." As reported in recent papers, YKL-40 is a biomarker for inflammatory diseases, diagnosis and prediction and concentration increases with age and has variations in healthy population. So it will be important to know the stage and the type of the viral infection.

6. in table 2 : needs a line between patient numbers and concentration in viral infection similar to the presentation of  the bacterial one

7. page 9 lane 286: "Our results revealed that YKL-40 plasma levels could 286 be used as potential markers in CNS infections." Please clarify under what conditions.

8. page 11 lane 342: "YKL-40 levels in CSF and serum were highly ele-342 vated in different pathologies of the CNS, compared to those of the controls". It is known that in the brain, YKL-40 is upregulated in several neurological disorders such as stroke, lentiviral encephalitis, traumatic brain injury, amylotrophic lateral sclerosis, multiple sclerosis and Alzheimer’s disease (AD) Although YKL-40 was initially associated with the macrophage lineage evidence suggests that during neuroinflammatory processes, its expression is abundant in reactive astrocytes and residual in microglial cells. Please clarify if the clinical samples were between these categories.

9. page 11 lane 382: "Our results revealed higher CRP concentrations in patients indicating ongoing in-352 flammation but no correlation between YKL-40 and CRP was detected. "  As CRP is an acute marker do the authors suggest that the present results are more relevant for chronic infections?

Author Response

Answers to reviewer

The manuscript of Kazakova et al., aim to study the inflammatory marker (YKL-40) and autophagy-related proteins 23 (LAMPs) in patients with central nervous system (CNS) infections viral or bacterial. The topic is interesting and recent references support the use of these molecules as biomarkers.

The following major points should be addressed:

1. page 3 line 86: "consistent with an acute CNS infection" . It is well known that an acute infection always produce inflammation. YKL -40 is reported to be associated withinflammation and YKL-40 serum levels are increased with age, in various cardiovascular, metabolic, and systemic inflammatory diseases. I suggest to clarify what type of viral and bacterial infections of the clinical samples are included. There is need to know exactly for what viral or bacterial infection are hospitalized. Its virus has a different kinetic of infection and different target tissues. As it is presented (page 4 lanes-134-139) is  a general description with no value except of the description. Viruses belongs to different families and the same for bacteria.

We accept the reviewer’s suggestion and provided a Table, as follows:

Table 1. Demographic and laboratory characteristics of the studied patients

№	Age	M/F	Microbiological result	CSF WBC [x10^6/l]	CSF Protein [g/l]	CSF glucose [mmol/l]	Serum/ CSF glucose ratio	CSF appear-ance	Clinical course	Study group definition
1	55	M	no etiology	20.00	0.77	3.70	0.58	clear	mild	suspected viral
2	94	F	no etiology	528.00	0.66	3.20	0.60	Clear	mild	suspected viral
3	17	M	no etiology	2.00	1.79	7.80	0.62	Clear	mild	suspected viral
4	68	M	no etiology	11.00	0.84	4.70	0.70	Clear	mild	suspected viral
5	32	M	no etiology	3.00	1.40	2.70	0.56	Clear	mild	suspected viral
6	49	F	no etiology	176.00	1.10	2.20	0.32	Clear	mild	suspected viral
7	32	M	no etiology	1.00	0.64	2.00	0.51	Clear	mild	suspected viral
8	48	M	no etiology	126.00	1.30	4.00	0.72	Clear	mild	suspected viral
9	67	M	no etiology	2.00	1.10	4.00	0.77	Clear	mild	suspected viral
10	63	M	no etiology	7.00	0.68	5.50	0.57	Clear	mild	suspected viral
11	69	F	no etiology	3.00	0.32	9.40	1.20	Clear	mild	suspected viral
12	70	F	no etiology	6.00	0.82	6.10	0.91	Clear	mild	suspected viral
13	79	M	no etiology	18.00	0.58	4.20	0.79	Clear	mild	suspected viral
14	49	F	no etiology	6.00	0.45	4.00	0.65	Clear	mild	suspected viral
15	69	M	VZV	32.00	0.87	3.40	0.63	Clear	moderate	viral
16	62	M	HSV-1	288.00	1.77	2.90	0.51	Clear	moderate	viral
17	47	M	no etiology	1.00	0.83	5.20	0.85	Clear	mild	suspected viral
18	49	M	no etiology	21	0.3	6	0.77	Clear	mild	suspected viral
19	8	M	no etiology	2	0.21	3.7	0.79	Clear	mild	suspected viral
20	42	F	no etiology	12	0.37	3	0.67	Clear	mild	suspected viral
21	80	F	no etiology	9	0.46	6.9	0.68	Clear	mild	suspected viral
22	49	F	no etiology	0	0.26	4.1	0.61	Clear	mild	suspected viral
23	35	M	S. pneumoniae	1190.00	6.53	0.00	0	Turbid	severe	bacterial
24	25	M	no etiology	129.00	2.40	0.60	0.09	Turbid	severe	suspected bacterial
25*	43	M	no etiology	1.00	0.37	3.00	0.42	Turbid	moderate	suspected bacterial
26	22	M	N. meningitidis C	9386.00	7.07	0.00	0	Turbid	severe	bacterial
27	82	F	L. monocytogenes	64	5.43	2	0.29	Turbid	severe	bacterial
28	34	F	H. influenzae b	480	1.81	2.3	0.30	Turbid	severe	bacterial
29	70	M	S. pneumoniae	2218	2.2	0.2	0.03	Turbid	severe	bacterial

*HIV-positive patient, immunocompromised; Age presented in years; M-males; F-females; CSF-cerebrospinal fluid; WBC-white blood cells;

In addition, we addressed the reviewer’s remark by including the following text in the manuscript’s Results section:

 

1. Microbiological results and groups of observation

Based on the microbiological evaluation of CSF specimens we managed to culture the following bacterial pathogens – S. pneumoniae (n=2), N. meningitidis serogroup C (n=1), L. monocytogenes (n=1), and H. influenzae type b. All the bacterial pathogens were culture-positive, confirmed by the mPCR ME Panel, and also identified by the MALDI-TOF MS. Microbial cultures showed no fungal growth on SDA agar plates in all the tested CSF samples. Among the CSF samples, we managed to detect only two viral pathogens by the mPCR ME Panel – VZV and HSV-1. Data are summarized in Table 1.

2. page 4 line 129: "A selective medium for fungi (Sabouraud-dextrose agar) was also inoculated" .This is a description in the materials/methods  but there is no other result or  comment about fungi in the manuscript.

We addressed the reviewer’s remark and included the following text in the manuscript’s Results section:

1. Microbiological results and groups of observation

Based on the microbiological evaluation of CSF specimens we managed to culture the following bacterial pathogens – S. pneumoniae (n=2), N. meningitidis serogroup C (n=1), L. monocytogenes (n=1), and H. influenzae type b. All the bacterial pathogens were culture-positive, confirmed by the mPCR ME Panel, and also identified by the MALDI-TOF MS. Microbial cultures showed no fungal growth on SDA agar plates in all the tested CSF samples. Among the CSF samples, we managed to detect only two viral pathogens by the mPCR ME Panel – VZV and HSV-1. Data are summarized in Table 1.

3. page 5 lane 183:"potential viral". Please clarify what the authors mean by potential viral infection? How the know that is a virus?

The new Table 1 provides detailed data on how we defined the groups of observation. However, we included the following text in the Material and Methods section, 2.1. Study design and patients’ selection:

Patients were classified into two groups of observation based on the medical history, measured CSF abnormalities, CSF appearance, clinical course of the neuroinfection, and microbiological result (where present).

4. page 6 lanes 208-211 : "We detected statistically elevated concentrations of both plasma and CSF YKL-40 in 208 patients compared to the control group (p=0.0005 and p<0.03 respectively). In addition, 209 we observed that YKL-40 levels in CSF of patients were significantly higher compared to 210 plasma levels (p=0.0021)". Please clarify about the state of infection. How many days post infection?

To address the reviewer’s suggestion and to make the text clearer we included the following text in the Material and Methods section 2.3. Laboratory methods:

 All the clinical samples, including both blood and CSF, were collected within several hours after hospital admission in patients presenting with an acute CNS infection.

 

5. page 7 lanes 221-222: "Higher CSF YKL-40 levels compared to plasma levels in patients with viral infection were detected. There was no change in plasma or CSF levels in bacterial infections." As reported in recent papers, YKL-40 is a biomarker for inflammatory diseases, diagnosis and prediction and concentration increases with age and has variations in healthy population. So it will be important to know the stage and the type of the viral infection.

We included the patients’ age and the type of the viral infections in the new Table 1.

6. in table 2 : needs a line between patient numbers and concentration in viral infection similar to the presentation of  the bacterial one

Corrected.

7. page 9 lane 286:"Our results revealed that YKL-40 plasma levels could 286 be used as potential markers in CNS infections." Please clarify under what conditions.

A clarification is included in the main text (line 323-324)

To determine whether plasma YKL-40   could serve as a potential biomarker to distinguish patients from healthy controls, a ROC curve was constructed.

8. page 11 lane 342: "YKL-40 levels in CSF and serum were highly ele-342 vated in different pathologies of the CNS, compared to those of the controls". It is known that in the brain, YKL-40 is upregulated in several neurological disorders such as stroke, lentiviral encephalitis, traumatic brain injury, amylotrophic lateral sclerosis, multiple sclerosis and Alzheimer’s disease (AD) Although YKL-40 was initially associated with the macrophage lineage evidence suggests that during neuroinflammatory processes, its expression is abundant in reactive astrocytes and residual in microglial cells. Please clarify if the clinical samples were between these categories.

To address the reviewer’s suggestions, we included the following text in the Material and Methods section (line 107-109)

Patients with the following neurological disorders were not included in the current study – stroke, lentiviral encephalitis, traumatic brain injury, amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer’s disease.

9. page 11 lane 382: "Our results revealed higher CRP concentrations in patients indicating ongoing in-352 flammation but no correlation between YKL-40 and CRP was detected."  As CRP is an acute marker do the authors suggest that the present results are more relevant for chronic infections?

 

Based on the medical history, clinical and laboratory results, as well as on the defined selection criteria, the patients included in the current study presented only with acute infection of the CNS, not with chronic. These findings could be explained by the relatively small number of patients included in the study groups we defined.

To address the reviewer’s remark and to make the text clearer we changed the word ongoing to supporting.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The study is devoted to the behavior of proteins YKL-40, LAMP-1, and LAMP-2 in CNS inflammations of viral or bacterial etiology. The authors found differences in the levels of gene expression and production of the YKL-40 protein in healthy donors and patients with signs of CNS disease. The data on the ROC curves of YKL-40 plasma levels to discriminate between neuroinfections and healthy controls look quite convincing. However, the data on the LAMP-1 and LAMP-2 proteins are largely at the level of statistical error in themselves, and when compared with each other, they also do not reveal significant differences. Whether they can be used as a biomarker remains questionable. Perhaps the authors should slightly change the interpretation of the data obtained, emphasizing that the YKL-40 protein can indeed serve as such a marker, while LAMP-1 and LAMP-2 are questionable, but in this situation they are good as a control. This is a major point for reflection. There are also minor points that would be good to address before considering publishing the work in this journal.

Minor points:

Lines 112-114 -- How correct is it to compare results obtained on healthy donors with results obtained on cadavers, even if they were healthy before death? Please confirm the possibility of correct interpretation of the results by references to similar studies, if any.

Line 196 -- The text continues here. – Please delete the phrase.

The caption to Figure 2 is not sufficiently informative. Please expand.

Lines 219-225 -- The paragraph has some semantic ambiguity. It is difficult to follow the authors' thoughts. The results are not clearly presented. Please rewrite.

Lines 230-232 -- Why is there a positive correlation between LAMP-1 and LAMP-2, while the level of LAMP-2, according to the authors, does not change?

Please also provide the data for LAMP-2 in Figure 3, similar to LAMP-1, even if there are no special changes. This is necessary for clarity.

Author Response

Answers to reviewer:

 

The study is devoted to the behavior of proteins YKL-40, LAMP-1, and LAMP-2 in CNS inflammations of viral or bacterial etiology. The authors found differences in the levels of gene expression and production of the YKL-40 protein in healthy donors and patients with signs of CNS disease. The data on the ROC curves of YKL-40 plasma levels to discriminate between neuroinfections and healthy controls look quite convincing. However, the data on the LAMP-1 and LAMP-2 proteins are largely at the level of statistical error in themselves, and when compared with each other, they also do not reveal significant differences. Whether they can be used as a biomarker remains questionable. Perhaps the authors should slightly change the interpretation of the data obtained, emphasizing that the YKL-40 protein can indeed serve as such a marker, while LAMP-1 and LAMP-2 are questionable, but in this situation they are good as a control. This is a major point for reflection. There are also minor points that would be good to address before considering publishing the work in this journal.

We slightly changed the interpretation of results (line 398-401). The title is also modified.

The results from the ROC curves of YKL-40 plasma levels illustrate the potential of plasma YKl-40 to serve as a discriminative marker. In contrast, the data about  LAMPs in CNS infections are unconving and  indicate  the  biased and contradictory role of the glycoproteins.

Minor points:

Lines 112-114 -- How correct is it to compare results obtained on healthy donors with results obtained on cadavers, even if they were healthy before death? Please confirm the possibility of correct interpretation of the results by references to similar studies, if any.

We used the post-mortem CSF samples (healthy deceased individuals) only to compare with CSF from patients.

The use of  healthy deceased controls is common in different studies. A systemic review identified 134 reports using deceased controls. A basic reason to include deceased individuals is  the lack of appropriate controls from healthy living individuals and the counteracting bias associated with living controls. (https://doi.org/10.1093/aje/kwx052;  https://doi.org/10.3390/ jcm12093080)

 In our previous investigations we used cadavers as a control group to obtain CSF or normal brain tissue. We compared gene and protein expression of potential biomarkers in patients with glioblastoma and traumatic brain injury (https://archtrauma.kaums.ac.ir/article_156932_a7ce1812c8faea07f2d4ccfd2526665c.pdf ; https://doi.org/10.14712/fb2014060060261)

Line 196 -- The text continues here. – Please delete the phrase.

The phrase is removed from the main text.

The caption to Figure 2 is not sufficiently informative. Please expand.

The caption of Figure is expanded as follows:

Plasma and CSF levels of YKL-40 in controls and patients with CNS infections. Glycoprotein levels in both biological specimens were compared. In addition, a parallel group analysis was performed, between plasma and CSF samples of patients.

Lines 219-225 -- The paragraph has some semantic ambiguity. It is difficult to follow the authors' thoughts. The results are not clearly presented. Please rewrite.

The paragraph was rewritten.

Lines 230-232 -- Why is there a positive correlation between LAMP-1 and LAMP-2, while the level of LAMP-2, according to the authors, does not change?

There is a positive linear correlation between LAMP-2 and YKL-40 gene expression in the patients group. An addition to the text is made. Also, a new graph is presented in the combined Figure 3.

Please also provide the data for LAMP-2 in Figure 3, similar to LAMP-1, even if there are no special changes. This is necessary for clarity.

A new graph is added to the figure.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have taken into account all the reviewer's comments. Now the text of the manuscript and its illustrative part look better. There is only one small remark left. Please, in the captions to figures 2 and 3, decipher the letter designations of their components - what do a, b, c mean? After eliminating this inaccuracy, the article can be published.

Author Response

Thank for comments.

We included the required information. We clarified the letters.