The Prevalence and Serological Association of Hepatitis D Virus Genotypes in Taiwan

Round 1

Reviewer 1 Report

The article entitled” The Clinical Significance of Hepatitis D Virus Genotypes in HBV/HDV Superinfection” is self-explanatory. The study indicates the motive of the investigation. Hepatitis Delta Virus (HDV) is not so common and thus there remains strong interest to develop insights about its epidemiology, pathogenesis, and management strategy. In the study, 26 patients with chronic hepatitis B (CHB) have been enrolled who were also infected with the HDV. The ultimately findings include reduced HDV load in HDV genotype IV. Also, there has been a comparison of platelet levels between cirrhotic and non-cirrhotic patients. Overall, the novelty of the study is low and there remains considerable jargons.

Comments;

1. The title of the article is “The Clinical Significance of Hepatitis D Virus Genotypes in 2 HBV/HDV Superinfection”. In the Abstract, it has been mentioned that “The prevalence of HDV genotypes in Taiwan is less discussed. Therefore, we used the cohort included 26 chronic HBV patients who received nucleos(t)ides (NUCs) 28 between January 2002 to July 2018 to determine HDV genotypes and genotype specific serological 29 association in chronic HBV carriers”. I am confused as to what was the purpose of the study:
2. Analysis of Clinical Significance of HDV genotype or
3. Checking the prevalence of HDV genotypes.

These two types of motive of study is not supportive. Rather, it makes the designing weak and irreverent.

2. Please highlight the clinical significance of HDV genotype as you have mentioned in Title.
3. The data about platelet count in cirrhotic and non-cirrhotic patients are mostly not ne4ccessary. These have been published by several investigators several hundred times. What is the novelty of the study?
4. The entire article is filled with data of laboratory markers. Where can I get clinical significance of HDV genotype?

Author Response

Comments;

1. The title of the article is “The Clinical Significance of Hepatitis D Virus Genotypes in 2 HBV/HDV Superinfection”. In the Abstract, it has been mentioned that “The prevalence of HDV genotypes in Taiwan is less discussed. Therefore, we used the cohort included 26 chronic HBV patients who received nucleos(t)ides (NUCs) 28 between January 2002 to July 2018 to determine HDV genotypes and genotype specific serological 29 association in chronic HBV carriers”. I am confused as to what was the purpose of the study:
2. Analysis of Clinical Significance of HDV genotype or
3. Checking the prevalence of HDV genotypes.

These two types of motive of study is not supportive. Rather, it makes the designing weak and irreverent.

In the present study, we first aimed to analyze the Hepatitis D Virus Genotype prevalence and serological association in the general Taiwanese population. The study showing the three genotypes evident in Taiwan were HDV-Ⅰ (4/24, 16.7%), HDV-Ⅱ (6/24, 25.0%) and HDV-Ⅳ (14/24, 58.3%). This further confirms that HDV-IV is the predominant HDV genotype in Taiwan. We highlight this in lines 203-208 by yellow label.

We next analyzed the serological associations and found that low levels of creatinine and HDV-RNA were observed in the HDV-Ⅳ group compared to the non-HDV-Ⅳ group. According to the reviewer comments, we have revised the abstract and made this clearer in lines 36-41 by yellow label. Indeed, the paper title has been revised to “The prevalence and serological association of Hepatitis D Virus Genotypes in Taiwan.”

 

1. Please highlight the clinical significance of HDV genotype as you have mentioned in Title.

According to the reviewer comments, we have revised the title to “Hepatitis D Virus Genotypes prevalence and serological association in Taiwan.” Indeed, we revised the abstract to describe the clinical significance in our study by “The serologic association study found low levels of creatinine (p = 0.047) and HDV-RNA (p = 0.009) were observed in the HDV-IV group compared to the non-HDV-IV group but no significant difference in AST, ALT, Bilirubin and other laboratory test factors were found.” Please see lines 36-39, page 1 by yellow label.

 

1. The data about platelet count in cirrhotic and non-cirrhotic patients are mostly not necessary. These have been published by several investigators several hundred times. What is the novelty of the study?

The data in cirrhotic and non-cirrhotic patients in Table 1 are baseline characteristic factors for 24 HBV/HDV superinfection patients in total. We defined CHB as being HBsAg-positive for at least six months or more and chronic hepatitis delta as viral infection for a period equal to or greater than six months. A small fraction of the patients presented with LC and HCC. Liver enzymes AST, alanine aminotransferase (ALT), platelets (PLT), bilirubin and other biomarkers, HBsAg, HBeAg, anti-hepatitis Be (anti-HBe) and anti-HDV were collected as clinical parameters for each patient, as shown in Table 1. Not only do the platelet counts and WBC numbers reveal significant association with liver cirrhosis as being reproducible, but they illustrate the basic factors in the HBV/HDV superinfection cohort as well.

 

1. The entire article is filled with data of laboratory markers. Where can I get clinical significance of HDV genotype?

We revised the abstract to describe the clinical significance in our study by “The serologic association study found low levels of creatinine (p = 0.047) and HDV-RNA (p = 0.009) were observed in the HDV-IV group compared to the non-HDV-IV group, but no significant difference in AST, ALT, Bilirubin and other laboratory test factors were found.” Please see lines 36-41, page 1 by yellow label.

Reviewer 2 Report

My main recommendation is not to include so much statistical data in the text because it is very difficult to track.
To authors,
This study may give updated data on new epidemiological evidence of HDV infection for researchers of this infectious disease. However, the authors should address to revise it in order to make it easy to understand. I left my comments and suggestions as follows. 
In the presented abstract, I do not find a logical representation of the data. The aim of the authors is to determine HDV genotypes in chronic HBV carriers [91], but I don’t see any data about HDV genotyping in the abstract.  
I would like to recommend the authors rewrite the abstract. The abstract is difficult to understand, especially statistics. First, you say that [28] used the cohort that included 26 chronic HBV patients. 
After that, you gave [30] HDV-positive genotypes in 19/24 (79.2%) males and 5/24(20.8%) females in chronic HBV patients were identified.
Where does the numbеr 24 come from?
In the present form, many parts of the manuscript are not understandable. Please, consider the academic proofreading service or at least the help of an English native speaker. 
[71-73] please, provide sufficient data on the HDV genotypes distribution and include more references.

Please, provide more data about HBV genotype distribution, chronification of HBV, and correlation between HBV genotypes and HDV distribution!

Please, provide more data about HDV genotypes and their influence on liver disease outcomes.

[94-112] This paragraph is filled with too many statistics that are difficult to understand. Please, reorganized it. This also applies to all tables. 

[119-65]: This paragraph is confusing. Please, better organize the information and rephrase it in a clearer way.

[310]: Please, describe the primer pairs used for HDV genotyping in MM!

Author Response

My main recommendation is not to include so much statistical data in the text because it is very difficult to track.
To authors,
This study may give updated data on new epidemiological evidence of HDV infection for researchers of this infectious disease. However, the authors should address to revise it in order to make it easy to understand. I left my comments and suggestions as follows. 
In the presented abstract, I do not find a logical representation of the data. The aim of the authors is to determine HDV genotypes in chronic HBV carriers [91], but I don’t see any data about HDV genotyping in the abstract.  
I would like to recommend the authors rewrite the abstract. The abstract is difficult to understand, especially statistics. First, you say that [28] used the cohort that included 26 chronic HBV patients. 
After that, you gave [30] HDV-positive genotypes in 19/24 (79.2%) males and 5/24(20.8%) females in chronic HBV patients were identified.
Where does the numbеr 24 come from?

From the 26 patients who were seropositive for HDV RNA, we further excluded 2 patients who had missing data during follow-up. We identified the 24 patients for HDV genotyping, 18 (75%) were male and 6 (25%) were female. The error has been revised in the abstract and in lines 30-33 by yellow label.

In the present form, many parts of the manuscript are not understandable. Please, consider the academic proofreading service or at least the help of an English native speaker. 

We have revised our manuscript according to the Reviewer’s suggestions and the revised manuscript has been edited by a professional editor.

[71-73] please, provide sufficient data on the HDV genotypes distribution and include more references.

The description and references have been provided in the Discussion section in lines 196-202, page 7 by yellow label as per the following:
“HDV-genotyping distribution observed in our study coincided with other notable studies in the Asian region [24]. Currently, eight HDV genotypes have been elucidated to date [21]. HDV-Ⅰ, Ⅱ, and Ⅳ were reported as prevalent in a few Asian countries such as: Mongolia (56.5%) [25], Pakistan (≥ 60%) [26], India (37%) [25], Taiwan (15%) [25], and Northern Vietnam (15.4%) [24]. This notwithstanding, a low prevalence of HDV has been observed in other Asian countries, notably Korea (0.32%) [27], Indonesia (< 0.5%) [28] and the Philippines (1.6%) [21].”

Please, provide more data about HBV genotype distribution, chronification of HBV, and correlation between HBV genotypes and HDV distribution!

In the present study, we first aimed to analyze the Hepatitis D Virus Genotypes prevalence and serological association in the general Taiwanese population. The study illustrated that the three genotypes evident in Taiwan were HDV-Ⅰ (4/24, 16.7%), HDV-Ⅱ (6/24, 25.0%) and HDV-Ⅳ (14/24, 58.3%). This further confirms that HDV-IV is the predominant HDV genotype in Taiwan. We highlight this in lines 203-208, page 7 by yellow label.

Next, the serologic association was analyzed and it was found that low levels of creatinine and HDV-RNA were observed in the HDV-Ⅳ group compared to the non-HDV-Ⅳ group in the HBV/HDV superinfection cohort to emphasize the HDV genotypes in this study. The HBV genotypes and how the different HDV genotypes hijack HBV mechanisms for replication will be explored in future in-depth research.  

Please, provide more data about HDV genotypes and their influence on liver disease outcomes.

There are limited studies that focus on HDV viral loads and liver disease outcomes linked to genotypes. We have provided some related articles in the discussion in lines 232-236, page 7-8 by yellow label as per the following:

“HBV/HDV co-infection demonstrates lower platelet counts compared to HBV mono-infection, regardless of liver disease stage, however the functional mechanism is still unknown [33, 34], where by alterings immune-related genes and immune response resulting in lower integrity of immune response or white blood cells [35]. HDV is associ-ated with increased risk of liver cirrhosis and advanced liver disease [34].”

[94-112] This paragraph is filled with too many statistics that are difficult to understand. Please, reorganized it. This also applies to all tables. 

We have rewritten this section in the Table 1 results. Please see lines 99-111, page 3 by yellow label for the following description:

“24 patients for HDV genotyping were identified; 18 (75%) were male, 6 (25%) were female, and the prevalence of HBV/HDV was indicated as being higher in men than in women. The mean age of the patients was 52.42 ± 11.86 (mean ± SD) years; mean levels of ALT were 211.13 ± 321.96 IU/L, AST 226.29 ± 316.23 IU/L, and bilirubin was 3.28 ± 4.64 mg/dL. The mean HDV RNA level was 2.15 ± 1.67 log10 copies/mL. Two patients were positive for HBeAg and five patients were positive for anti-HCV, while mean HBsAg and HBV DNA levels were 1,376.7 ± 1,616.95 IU/mL and 3.58 ± 2.31 Log10 IU/L, respectively. Furthermore, between cirrhotic and non-cirrhotic patients, the cirrhotic patients had significantly lower platelet (93.09 x 103 /μL vs 211.77 x 103 /μL; p = 0.003) and low white blood cell (WBC) counts compared to the non-cirrhotic patients WBC (5,003 cells/mL vs 7,841 cells/mL; p = 0.024) (Table 1). The baseline factors associated with patients with or without liver cirrho-sis in HBV/HDV co-infected patients showed no significant change in HBsAg and HBV DNA levels.”

[119-165]: This paragraph is confusing. Please, better organize the information and rephrase it in a clearer way.

We have rewritten this section as recommended. Please see lines 117-144, page 3-4 for the following description:

2.2. 2.2. Factors associated with HDV genotypes in HBV/HDV superinfection patients.

Specific-HDV primers were used to target highly conserved regions of the L-HDAg [21]. The first round PCR product band was 323 bp and second round PCR product was 234 bp by primer-specific targeting for L-HDAg (Supplementary Figures 1A-1D). Sample sequences retrieved from direct sequencing were aligned. The conservative sites showed unique diversity among the sample study cohort (Supplementary Table 1 and Supple-mentary Figure 2). HDV-genotyping and phylogenetic analyses revealed that the 24 pa-tients exhibited HDV-genotypes Ⅰ, Ⅱ and Ⅳ, which are associated with the general Taiwanese population (Figure 1). Of the three genotypes observed, HDV-Ⅳ (14/24, 58.3%) was predominant followed by HDV-Ⅱ (6/24, 25.0%) and HDV-Ⅰ (4/24, 16.7%) (Supple-mentary Table 2). Our findings demonstrate a higher prevalence of HDV genotype - IV in the general Taiwanese cohort.

HDV viremia was significantly lower in HDV-Ⅳgenotype patients compared to non-HDV-Ⅳ (HDV I, II) patients (1.34 log10 copies/mL vs 3.30 log10 copies/mL; p = 0.009) (Table 2). Logistics regression analysis revealed that HDV-Ⅳ was significantly inversely proportional to HDV RNA (OR/95% CI: 0.370/0.164-0.830; p = 0.017) (Table 2) , revealing an inverse relationship compared to non-HDV-IV patients; furthermore, this group had significantly lower creatinine levels (0.96 ± 0.50 mg/dL) compare to the HDV-I (2.69 ± 3.88 mg/dL) and HDV-Ⅱ (3.91 ± 3.64 mg/dL) groups (Table 3). HDV RNA exhibited significantly lower viral loads in HDV-IV (1.34 ± 0.99 log10 copies/mL) compared to HDV-I (3.19 ± 2.22 log10 copies/mL) and HDV-II (3.34 ± 1.72 log10 copies/mL) groups (Table 3). These indicate that the creatinine and HDV RNA associated with HDV genotypes in HBV/HDV superinfection patients.

[310]: Please, describe the primer pairs used for HDV genotyping in MM!

We have added primer sequences and reference in the HDV Genotyping section of the Materials and Methods in line 302-308, page 9 by yellow label as per the following description:

“The primer pairs HDV04-F (5’-GGATGCCCAGGTCGGACCG-3’) and HDV05-R (5’- AAGAAGAGRAGCCGGCCCGY’) were used in the first round PCR and primer pairs HDV06-F (5’-ATGCCATGCCGACCCGAAGA-3’) and HDV07-R (5’- GGG-GAGCGCCCGGDGGCGG -3’) were used in the second round PCR (Supplementary Table 1.). The PCR conditions were held at pre-heat temperature of 94⁰C / 5 mins, denatured at 94 ⁰C / 30 sec, annealing at 54 ⁰C / 45 sec, extension at 72⁰C / 45 sec, and an additional ex-tension at 72 ⁰C / 7 mins for both rounds [21].”

Round 2

Reviewer 1 Report

No specific comments. Improved considerably in revised version.

 

Author Response

We have done the second professional English editing in this file to improve the language quality in the second revision file.  

Reviewer 2 Report

The manuscript should be reconsidered for publication in Pathogens, following major revisions.

The revised manuscript does not cover the requirements for the clear presentation of the results. In the abstract the the presentation of the results is chaotic. Please, better organize the information.

A careful revision of English language is strictly necessary to correct grammatical errors and improve the level of standard English.  The manuscript should be fully revised for the use of punctuation. Please, consider the academic proofreading service.

[96] From the 26 patients seropositive for HDV RNA, two patients with missing data during follow-up were excluded. In the total cohort as shown in Table 1. Please, provide this information into MM, not into Results.  [96-99] rewrite the stance!

[100-110]: In my opinion, it is not necessary to report results of the statistic in the text, but only in Table 1

[132-142]: In my opinion, it is not necessary to report results of the statistic in the text, but only in Table 2

Author Response

We have sent the file to professional academic English editing to improve the language quality in the second revision file.  

[96] From the 26 patients seropositive for HDV RNA, two patients with missing data during follow-up were excluded. In the total cohort as shown in Table 1. Please, provide this information into MM, not into Results.  [96-99] rewrite the stance!

 We have added the missing data description in the material and methods. Please refer to lines 257-258.

[100-110]: In my opinion, it is not necessary to report results of the statistic in the text, but only in Table 1

According to the reviewer's opinion, we only provide the description for the significant p-value and number to clarify the finding on the cohort study table 1. as the following sentence as the lines 93-102 of the yellow lable.

    The total cohort as shown in Table 1, the prevalence of HBV/HDV is higher in men than in women. The mean age of the patients was 52 years old; the mean levels of ALT, AST, and bilirubin were 211 IU/L, 226 IU/L, and 3.28 mg/dL, respectively. The mean HDV RNA level was 2.15 log₁₀ copies/mL. Two patients were positive for HBeAg, and five patients were positive for anti-HCV, while mean HBsAg and HBV DNA levels were 1,376.7 IU/mL and 3.58 Log₁₀ IU/L, respectively. Furthermore, cirrhotic patients had significantly lower platelet (p = 0.003) and white blood cell (WBC) count (p = 0.024) than non-cirrhotic patients (Table 1). The baseline factors associated with patients with or without liver cirrhosis in HBV/HDV co-infected patients showed no significant change in HBsAg and HBV DNA levels.

[132-142]: In my opinion, it is not necessary to report results of the statistic in the text, but only in Table 2

According to the reviewer's opinion, we only provide the description for the significant p-value and number to clarify the finding on the cohort study table 2. as the following sentence as the lines 126-134 of the blue label.

HDV viremia was significantly lower in HDV-Ⅳ genotype patients than non-HDV-Ⅳ (HDV I, II) patients (1.34 log₁₀ copies/mL vs 3.30 log₁₀ copies/mL; p = 0.009) (Table 2). A logistics regression analysis revealed that HDV-Ⅳ was significantly inversely proportional to HDV RNA (p = 0.017) (Table 2); furthermore, creatinine levels were significantly lower in this group (0.96 mg/dL) than in HDV-I (2.69 mg/dL) and HDV-Ⅱ (3.91 mg/dL) groups (Table 3). HDV RNA exhibited significantly lower viral loads in HDV-IV (1.34 log₁₀ copies/mL) compared with those in HDV-I (3.19 log₁₀ copies/mL) and HDV-II (3.34 log₁₀ copies/mL) groups (Table 3). These indicate that creatinine and HDV RNA are associated with HDV genotypes in HBV/HDV superinfection patients.

 

Round 3

Reviewer 2 Report

I accepted the paper in present form.