Abstract

Introduction: Rotaviruses are the leading cause of severe diarrhea in infants and young children worldwide. So far, 32 different G and 47 different P genotypes have been identified in group A. After the outbreak of the COVID-19 pandemic, various measures have been taken in our country. These are effective in reducing many infectious diseases such as acute gastroenteritis with COVID-19. In the current study, we investigate whether the COVID-19 pandemic measures cause a change in the rotavirus genotype distribution.

Materials and Methods: A total of 128 stool samples positive for rotavirus antigen, 64 pre-pandemic and 64 during the pandemic period, were taken into further study for genotyping. Rotavirus RNA was detected in 50 (78%) of the samples by Reverse Transcription-polymerase chain reaction in the pre-pandemic period and in 51 (80%) of the samples during the pandemic period.

Results: In the pre-pandemic period, the following results were found in the patients we studied: G9P[8] in 24 (48%) of them, G1P[8] in 14 (28%) G2P[8] in 5 (10%), G2P[4] in 3 (6%), G3P[8] in 2(4%), G4P[8] in 1 (2%) and G9P[4] in 1 (2%). During the pandemic period, the following results were found in the patients we studied: G9P[8] in 28 (54%) of them, G1P[8] in 12 (24%), G2P[8] in 6 (12%), G2P[4] in 2 (4%), G3P[8] in 1 (2%),G4P[8] in 1 (2%) and G9P[4] in 1 (2%).

Conclusion: As a result, in our study, G9P[8] was found to be the dominant genotype in both periods. It was found that there was no difference in rotavirus genotypes between the pre-pandemic and pandemic period.

Introduction

Rotaviruses can cause severe diarrhea, especially in infants and children under 5 years of age. In 2008, it was determined that rotavirus was responsible for 37% of deaths caused by diarrhea in children under 5 years of age. Among all deaths, deaths caused by rotavirus are 5%. It has been stated that more than 50% of deaths caused by rotavirus occur in developing countries ^[1]. Rotavirus infection is seen in 40% of children under the age of 5 who are hospitalized due to diarrhea ^[2].

Rotavirus is a virus with a segmented genome containing double-stranded RNA. It is a non-enveloped virus. It has a genome consisting of 11 segments encoding six structural (VP1, VP2, VP3, VP4, VP6 and VP7) and six non-structural proteins (NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6). Group specificity of rotaviruses is based on the VP6 protein (A-E). P serotyping of group A viruses is done according to VP4, an outer capsid protein. G serotyping of group A viruses is done according to the VP7 major outer capsid protein encoded by gene segment 8 or 9 ^[3].

So far, 32 different G and 47 different P genotypes have been identified in group A. Among these, six G genotype (G1, G2, G3, G4, G9 and G12) and three P genotype  are the frequently seen genotypes. G1P[8],G2P[4],G3P[8],G4P[8],G9P[8] and G12P[8] strains constitute 90% of group A rotaviruses seen worldwide. Group A rotavirus strain distribution may vary depending on geographical differences and vaccine application. The number of disease-causing strains in developing countries is higher than in developed countries ^[4].

With the Coronavirus Diseases-2019 (COVID-19) pandemic, various measures have been taken in our country, as well as all over the world. The main ones are maintaining social distance, wearing a mask, providing hand hygiene, closing schools and working from home. These measures have been proven to be effective in reducing many infectious diseases such as acute gastroenteritis along with COVID-19 ^{[5, 6]}. It has been shown that the number of cases of acute gastroenteritis caused by rotavirus has decreased due to school closures, activity restrictions, and decreased transmission due to patients' indifference to hospital admission during the pandemic ^{[5, 7]}.

It is essential to know the rotavirus genotypes in circulation in order to shed light on vaccine development studies in our country. For this purpose, it was aimed to investigate whether the COVID-19 pandemic measures had an effect on the rotavirus genotypes in circulation.

MATERIALS AND METHODS

Two periods were examined in the study. The period between March 11, 2019 and March 10, 2020 was considered pre-pandemic period. The period between March 11, 2020 and March 10, 2021 was considered the during the pandemic period. For this purpose, stool samples sent to our hospital's microbiology laboratory with suspicion of acute diarrhea and detected positive for rotavirus antigen by immunochromatographic card test were collected. Laboquick Rotavirus – Adenovirus Ag Combo Test (In Vitro Diagnostic Test, Izmir, Turkey) was used for the detection of rotavirus from stool samples. The rotavirus rapid test cassette contains a membrane band using red gold conjugated monoclonal antibodies against the VP6 antigen of Group A Rotavirus, which works with the immunochromatographic principle, and solid phase specific rotavirus antibodies. Since the rotavirus positivity rate was low during the pandemic period, all rotavirus antigen positive stools were included in the study. In the pre-pandemic period, rotavirus antigen positive stools were randomly selected to have an equal number of samples compared to the pandemic period.

RNA extraction was utilized the EZ1 virus Mini Kit (QiagenGmbH, Hilden, Germany) as described by Durmaz et al. ^[8]. VP7 and VP4 gene amplifications were performed using reverse transcription polymerase chain reaction (RT-PCR) according to the specific primers and conditions detailed in the previous study by Durmaz et al ^[8]. VP7 gene amplification employed the Superscript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA, USA). For VP4 gene amplification, cDNA synthesis was first performed with a cDNA synthesis kit (Thermo Scientific, Carlsbad, CA, USA). cDNA amplification was performed using VP4-F/VP4-R primers. G and P genotypes were determined using a semi-nested multiplex PCR method based on the obtained VP4 and VP7 gene amplicons, with specific primers as listed in Table 1.

For G typing, PCR utilized VP7-R primer along with specific forward primers for G1, G2,G3,G4,G8,G9,G10, and G12. P typing was conducted using specific reverse primer sets for P[4],P[6],P[8],P[9],P[10] and P[11] together with VP4-F primer. Amplified products were analyzed via 2% agarose gel electrophoresis, G and P genotypes were determined based on their expected sizes ^[8].

Statistical analysis was carried out using IBM SPSS Statistics version 20(IBM, USA), with the chi-square test employed for comparing categorical variables.

The sample size of the study was determined by power analysis. The sample size was determined as 100 for  the study to reach 95% test power at a 5% error level (G Power 3.1).

RESULTS:

In  the pre-pandemic period, a total of 2.955 stool samples were sent to our laboratory with a preliminary diagnosis of acute gastroenteritis. 178 of these were found positive for rotavirus antigen. The rotavirus positivity rate was determined as 6.7%. During the pandemic period, rotavirus antigen was detected in 64 of the 2.335 stool samples. The rotavirus positivity rate was found as 2.74%.  It was determined that the rotavirus positivity rate decreased significantly during the pandemic period compared to pre-pandemic period (p<0.001). All rotavirus antigen positive stool samples during the pandemic period were included in the further study for genotyping. In  the pre-pandemic period, the same number of stool samples as the pandemic period were randomly selected and included in the further study. A total of 128 stool samples positive for rotavirus antigen, 64  pre-pandemic period and 64 during the pandemic period, were taken into further study for genotyping. By RT-PCR, rotavirus RNA was detected in 50 (78%) of the samples in  the pre-pandemic period and in 51 (80%) of the samples during the pandemic period. Rotavirus RNA could not be detected in 27 (21%) samples. In the pre-pandemic period, the age range of rotavirus RNA positive patients was found to be 1-96 months. The duration of pandemic period patients was found to be 2-105 months. In the pre-pandemic period, 29 (58%) of the patients were male and 21 (42%) were female. During the pandemic period, 25 (49%) of the patients were male and 26 (51%) were female.

As a result of the genotyping performed on rotavirus RNA-positive stool samples in  the pre-pandemic period, 5 different G genotypes and 2 different P genotypes were detected. Among G genotypes, G9 is the most common (n: 25, 50%), followed by G1 (n: 14, 28%), G2 (n: 8, 16%), G3 (n: 2, 4%) and G4 (n: 4). 1,%2) genotypes. Among the P genotypes, P[8] (n:46, 92%) and P[4] (n:4,8%) genotypes were detected. 7 different combinations of G and P were identified. G9P[8] in 24 (48%) of the patients, G1P[8] in 14 (28%), G2P[8] in 5 (10%), G2P[4] in 3 (6%), G3P[8] in 2 (4%), G4P[8] in 1 (2%) and G9P[4] in 1 (2%)genotype was detected (Table 2).

During the pandemic period, 5 different G genotypes and 2 different P genotypes were detected. Among G genotypes, G9 is the most common (n: 29, 57%), followed by G1 (n: 12, 28%), G2 (n: 8, 16%), G3 (n: 1, 4%) and G4 (n: 4). 1,%2) genotypes. Among the P genotypes, P[8] (n:48, 92%) and P[4] (n:3,8%) genotypes were detected. 7 different combinations of G and P were identified. G9P[8] in 28 (54%) of the patients, G1P[8] in 12 (24%), G2P[8] in 6 (12%), G2P[4] in 2 (4%), G3P[8] in 1 (2%), G4P[8] in 1 (2%) and G9P[4] in 1 (2%) genotype  was detected (Table 2).

When  the pre-pandemic period and during the pandemic period were compared in terms of the frequency of genotypes, no significant difference was detected between the two periods (Table 2).

DISCUSSION:

Rotaviruses are one of the most important causes of acute diarrhea in young children, associated with high mortality and morbidity. It is thought to cause approximately 130.000 deaths and 258.000.000 episodes of diarrhea in children under the age of 5 in 2016 alone ^[9]. It was observed that the highest death rate was in Sub-Saharan Africa, Southeast Asia and South Asia.  In the last decade, the prevalence of rotavirus has decreased worldwide due to public health measures such as improved sanitation and the inclusion of rotavirus vaccine in the national vaccination programs of more than 112 countries. It is estimated that 28.000 rotavirus-related deaths were prevented worldwide in 2016 with the application of rotavirus vaccine ^[9]. It was reported that the incidence of rotavirus decreased from 36% to 13% with the inclusion of rotavirus vaccines in the national program in Italy ^[10]. All these results show that vaccination is extremely important in preventing rotavirus disease. For new vaccine studies or to determine the effectiveness of currently used vaccines, it is necessary to know the circulating rotavirus genotypes.

In countries where rotavirus vaccine is not administered, rotavirus disease still poses a threat to children under the age of 5 ^[9]. In our country, rotavirus vaccine has not yet been included in the national vaccination program. However, RotaTeq® (Merck & Co., West Point, PA, ABD) and Rotarix^TM (GlaxoSmithKline Biologicals, Rixensart, Belgium) vaccines for rotavirus are available in our country. While rotarix consists of a single human strain G1P[8], rotateq is a human-bovine reassortant vaccine consisting of G1P[5], G2P[5], G3P[5], G4P[5] and G6P[8] strains ^[9].

 The genotype variations of rotavirus may vary from year to year depending on vaccine application and geographical region ^[8]. In the study conducted by Bonuro et al. in Italy, G1P[8] was found to be the dominant genotype before vaccine application, and G2P[4] was found to be the dominant genotype after vaccine application ^[10]. Likewise, in the study conducted by Hungerford et al. in England, G1P[8] was reported as the dominant genotype before vaccination and G2P[4] after vaccination ^[11]. The results of these studies show us that the dominant genotypes circulating in the society can change with the application of vaccination.

Both vaccines for rotavirus are available in our country. However, it is not included in our country's national vaccination program. In the study conducted by Artıran et al. in 2012-2013, in which rotavirus genotypes were investigated in our country, G9P[8] (40%), G1P[8] (17%) and G3P[8] (9.6%) genotypes were found most frequently ^[12]. Bozdayi et al. in their study conducted in Ankara covering the years 2006-2011, they again found G9P[8] (28%) as the dominant genotype. This was followed by G1P[8] (16.3%) and G2P[8] (15.9%) ^[13]. In Durmaz et al.'s study covering 23 cities and 35 different centers between 2012 and 2014, the dominant genotype was found to be G9P[8] (40.5%). This was followed by G1P[8] (21.6%) and G2P[8] (9.3%), as in other studies ^[8]. However, Durmaz et al. in the study covering 20 different centers in 15 cities between 2014 and 2016, G1P[8] (24.6%) was found to be the dominant genotype. This was followed by G3P[8] (19.6%) and G9P[8] (12.2%) ^[14]. Tapissiz et al. In a meta-analysis study compiling rotavirus studies conducted in our country, they reported that G1P[8], which was the dominant genotype between 2001-2006, shifted to G9P[8] between 2018-2013 ^[15]. Gündeşlioğlu et al. in their study examining the years 2013-2016 in Adana, they found that the prevalence of G9P8 decreased from 40% to 8.1% and the prevalence of G1P8 increased from 21.3% to 48.6%. G3P8 isolates, which were not seen in the first two years, were detected at rates of 18.7% and 13.5% in 2015 and 2016, respectively ^[16]. In Caneriği and Şafak's studies covering the years 2018-2019, the most common genotypes were G1P[4] (44%), G2P[9] (20%), G9P[4] (20%), G2P[4] (8%) ^[17]. Bulut et al. in their study from the same region as our study covering the years 2012-2015, the most common genotypes were G9P[8] (28.9%), G1P[8] (26.5%) and G2P[4] (9.6%) ^[3]. In our study examining the years 2019-2020, the most common genotypes were G9P[8], G1P[8]. When we look at the studies conducted in our country, it has been seen that genotypes vary from year to year and depending on the region. In addition, since rotavirus vaccine is not included in the national vaccination program in our country, not all children are vaccinated, the dominant genotype will vary depending on the number of vaccinated children in the cohort in which the study was conducted. The vaccination information of the patients included in our study is unknown. Therefore, no comment could be made on the effect of the vaccine on genotypes.

With the COVID-19 pandemic, various measures have been taken around the world. The obligation to wear a mask, restriction of public activities, closure of schools and kindergartens, and online education are the main measures implemented in our country ^[18]. These measures taken have effected the incidence of other infectious agents as well as COVID-19. Various studies have shown that rotavirus infection rates, which are especially common in children under the age of 5, decreased significantly during the pandemic period ^{[5, 19]}. Farkas et al. reported that the number of rotavirus cases reported in 2020 was 18% less than in 2019 and 27% less than the average of the past 5 years in Australia ^[20]. In the 2020 annual report of EuroRotaNet, which compiled the rotavirus data of 12 European countries, it was reported that the number of samples received in the 2019/2020 season decreased compared to previous years ^[21]. In our country, Duman et al reported that the monthly median positivity rate of rotavirus decreased significantly during the pandemic period ^[22]. Also in our country, in the study conducted by Alici and Çam, it was reported that hospital admissions and the number of samples decreased significantly due to acute gastroenteritis during the pandemic period ^[7]. In our study, similar to previous studies, it was found that the rotavirus positivity rate decreased significantly with pandemic measures.

In our study comparing the Rotavirus genotypes of the pre-pandemic period and the pandemic period, the most common genotype was G9P[8] in both periods. This was followed by the G1P[8] strain in both periods. Pre-pandemic and pandemic periods were found to be similar in terms of rotavirus genotypes. Farkas et al., in their annual report published within the scope of the Australian rotavirus surveillance program, reported that the G3P[8] (27%) genotype was the most common in 2020. They stated that the most frequently detected genotype for 3 consecutive years was G3P[8] ^[20]. However, in the Australian rotavirus 2021 annual report, they reported that the G8P[8] genotype, which was 1% in 2020, was found to be 87.5% in 2021 ^[23]. In EuroRotaNet's 2020 annual report, which examined a 12-year period, it was reported that G1P[8] was the most common genotype in every season from the 2006/07 season to the 2014/15 season. For the first time in the 2015/16 season, another genotype other than G1P[8], G9P[8], was found to be the dominant genotype. During the 2019/20 pandemic period, G3P[8] was reported as the dominant genotype, while G1P[8] was found at a rate of 10%. It was also reported that there was no newly emerged rotavirus strain in the 2019/20 season ^[21]. This study is the first study conducted in our country to compare pre-pandemic and pandemic period rotavirus genotypes. However in our study, unlike the studies conducted abroad mentioned above, rotavirus genotypes during the pandemic period were found to be similar to the studies conducted before the pandemic. This suggested that pandemic measures had no effect on rotavirus genotypes in our country. The small sample size and the fact that it was a single-center study may have caused this result.

STUDY LIMITATIONS:

The limitations of the study are that it is likely to be a single center study and was conducted with a limited number of samples.

CONCLUSION:

As a result, in our study, G9P[8] was found to be the dominant genotype in both periods. It was found that there was no difference in rotavirus genotypes between the pre-pandemic and pandemic period. However, our study was a single center study and a limited number of patient samples could be studied. There is a need for multicenter studies across the country that include a larger number of patient samples.

DECLARATIONS:

DECLARATION OF COMPETING INTEREST:

No conflict of interest was present.