Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco

Acute gastroenteritis is a very common disease in young children. It is characterized by massive inflammation of the gastrointestinal tract membranes, leading to frequent vomiting and/or diarrhea. Although regarded as a short-term intestinal disorder, acute gastroenteritis can cause severe dehydration, leading to further complications and hospitalization [1–3].

Enteric viruses are recognized as the most significant etiological agent of acute gastroenteritis, accounting for approximately 70% of episodes [2]. Four viral families are commonly associated with acute gastroenteritis: Reoviridae (group A rotaviruses), Caliciviridae (noroviruses), Adenoviridae (adenoviruses 40/41), and Astroviridae (astroviruses) [4–6]. Worldwide, rotavirus is considered the most common viral cause of severe acute gastroenteritis, causing 453,000 deaths and over 2 million hospitalizations among children under the age of 5 years [7].

Of the two major outer-shell proteins, VP7 defines the G genotype (G for Glycoprotein) and VP4 defines the P genotype (P for Protease-sensitive) and these are the main criteria upon which the dual classification system for the rotaviruses into P and G serotypes and genotypes [8]. Currently, 27 G genotypes (G1-G27) and 35 P genotypes (P[1]-P[35]) have been described for rotavirus [9]. To what extent each genotype defines an antigenically distinct VP7 or VP4 protein is not known.

Globally, the five most common G/P combinations among strains infecting humans are G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8], [10]. Remarkably, the G/P types of the uncommon strains show wide variation from one region to the next. For instance, a surveillance program directed by the World Health Organization noted that in 2010 the predominant uncommon strains were G12P[8] and G12P[6] viruses in Southeast Asia; G2P[6], G3P[6], and G1P[6] viruses in sub-Saharan Africa; G1P[4] and G2P[8] viruses in the Western Pacific; and G9P[4] viruses in the Americas [11]. The uncommon strains that can be spread throughout the world to become common global strain are difficult to predict. Indeed, the G9P[8] strains represent the only clear example of a previously rare G/P genotype combination that has become dominant within the landscape of globally circulating rotavirus [12–14].

Norovirus is considered the second most frequent cause of severe childhood gastroenteritis after rotavirus [15]. Its prevalence in children with acute gastroenteritis is in the range of 6–48% [16]. Although more than 30 genotypes, within genogroups GI, GII, and GIV, can infect humans [17], a single genotype, GII.4, has been associated with the vast majority of norovirus-related outbreaks and sporadic cases of gastroenteritis worldwide [18–20]. The GII.4 norovirus strains undergo continuous processes of genetic/antigenic diversification and periodically generate novel strains through the accumulation of punctuate mutations or recombinations, and novel GII.4 variants emerge every 2–3 years [21, 22].

Few data are available on the etiology of acute gastroenteritis among young children in Morocco. Only one study reports the detection of norovirus in shellfish in Morocco. Norovirus were detected in 30% of samples, with an equal representation of GI and GII strains, but were much more frequently found in cockles or clams than in oysters [23].

The report of a national survey performed in 1998, describing the causes of child deaths, revealed that a high rate of mortality among children aged less than 5 years was attributed to diarrheal diseases. The number of these deaths in 1 year is estimated to be 6000 [24]. In 2006, a national program for rotavirus surveillance was established by the Ministry of Health, in partnership with the World Health Organization, at four different sites. Data were collected during a 5-year survey and confirmed the rotaviral etiology of infective gastroenteritis among children aged less than 5 years in Morocco. Subsequently, isolated rotavirus strains have been investigated and their molecular epidemiology assessed [25]. These data supported the introduction of a rotavirus vaccine to the Moroccan National Immunization Program in 2010. The vaccine coverage rate was greater than 80% [26].

In countries where the rotaviral vaccine has been introduced, there has been a decline in morbidity related to rotavirus-associated gastroenteritis. However, a mathematical increase of the percentage involved in gastroenteritis cases related to norovirus has been reported. Norovirus has consequently been designated the predominant cause of gastroenteritis in hospitalized children [3, 27, 28].

To address the paucity of data regarding norovirus infections, we assessed the features of acute gastroenteritis related to rotavirus and norovirus in four regions of Morocco.

In this study, acute gastroenteritis with a rotaviral or noroviral etiology was investigated in children aged less than 5 years. This is the first study to investigate both viruses simultaneously in Morocco. Of the 335 children with acute gastroenteritis enrolled in the study, 26.6% (n = 89) tested positive for rotavirus and 16.1% (n = 54) for norovirus. Our results are consistent with several studies undertaken elsewhere, indicating that norovirus is the most common cause of acute gastroenteritis, after rotavirus [36–38].

After the introduction of a rotavirus vaccine in the United States, hospitalizations related to acute gastroenteritis were 45% lower than 2 years earlier [39]. A substantial decline in rotavirus-associated acute gastroenteritis was also observed. Therefore, norovirus is now considered the leading cause of severe gastroenteritis outbreaks in the United States [27, 40].

In Morocco, prior the implementation of the national immunization program with rotavirus vaccine, circulating isolates of rotavirus were genotyped in our facilities. From June 2006 to May 2009, 1388 stool samples were collected from children less than 5 years of age admitted for diarrhea in different regions of Morocco. Overall, 41.7% (579 of 1388) of the stools samples tested were positive for rotavirus. Genotyping of 548 (95%) samples demonstrated that G1P[8] (55%) was the most prevalent strain, followed by G9P[8] (11.3%), G2P[4] (9.1%), G4P[8] (0.9%), and G3P[8] (0.4%). Several other strains were identified including G1P[4] (0.2%), G1P[6] (0.9%), G2P[6] (4.3%), G2P[8] (0.2%), G3P[6] (0.4%), G3P[4] (0.2%), and G9P[6] (0.2%). A high prevalence of mixed infections was found (15% of all samples) of which G1G2P[8] (4%) and G1G3P[8] (3.6%) accounted for the majority. Considerable diversity of rotavirus genotypes was present among circulating strains in Morocco [25]. After introduction of rotavirus vaccination in Morocco, the most common genotype combination of rotavirus strains was G1[P8] (51.7%), followed by G2[P4] (10.1%), G2[P8] (4.5%), G9[P8] (3.4%), G4[P8] (3.4%) and G1[P6] (2.3%). A high prevalence of mixed strains (22.5% of the total) was found. The prevalence of rotavirus gastroenteritis decreased from 41.7% to 26.6%. The involvement of the vaccine introduction in the decreasing of the prevalence and the changing in molecular diversity of rotavirus strains in Morocco needs further study. The selective pressures coming from the vaccine may be subtle; many years are required before making a conclusion concerning this phenomenon [41].

Because the Moroccan childhood immunization program was implemented with a rotavirus vaccine in 2010, it is likely that norovirus-associated gastroenteritis will increase relative to rotavirus-associated gastroenteritis, as described by Patel et al. [15] and Payne et al. [27]. A prospective survey is required to determine the point at which the switch in this trend occurs.

Both viruses were detected in all age groups examined in this study. The peak detection rate occurred between 6 and 12 months of age for rotavirus, with a median age of 12 months, and between 12 and 24 months of age for norovirus, with a median age of 14 months, as was also shown in a French study [36]. These findings confirm that both viruses usually occur in early childhood, which may indicate that protective immunity is present after the age of 2 years [42], and also highlights the importance of implementing prevention strategies in the early years of life. The overall severity score was significantly higher for rotavirus gastroenteritis than for norovirus infections. Similar results have been reported in Spain, Poland, and Libya [3, 37, 43]. Those studies showed that rotavirus-infected children experienced longer periods of diarrhea and were more severely dehydrated than children infected with norovirus.

During this 1-year survey, cases of rotavirus- and norovirus-associated gastroenteritis were recorded throughout the year, except in October and February, when no case of rotavirus and norovirus was detected, respectively. We observed one peak for rotavirus in January and two peaks for norovirus, in June. The increased rates of noroviral infection in spring and summer have been documented previously in Libya and Spain [43, 44]. The summertime peak may be explained by the emergence of a new virus variant, as reported by Lopman et al. [45].

In Morocco, spring and summer seasons are commonly associated with increased consumption of seafood particularly oysters, which is known as a risk factor for acquiring norovirus infection [23, 46]. Rabat and Tanger which are coastal cities had the highest percentages of norovirus positivity, during the study period. The study needs to be longer so that a conclusion could be drawn.

Norovirus GII is predominantly responsible for gastroenteritis worldwide, as described in most studies [47–52], and our findings (77.8% GII and 22.2% GI) are consistent with these findings. A subset of norovirus-positive GII samples (n = 33) was sequenced and grouped into five different genotypes: GII.4 (n = 27) was the predominant genotype, followed by GII.3 (n = 2) and GII.13 (n = 2), GII.16 (n = 1), and GII.17 (n = 1). Several global studies have demonstrated the prevalence of the GII.4 genotype and our results are consistent with them [18, 19, 43, 53].

Unexpectedly, our results showed the presence of the GII.4 variant Sydney2012 in two samples, from the northern region of Morocco (GenBank accession numbers KJ162365 [Tanger/M462/2011/MOR] and KJ162362 [Tanger/M687/2011/MOR]). This variant was first identified in March 2012 in Australia [54] and in December 2012 in Denmark [55], one year after the sampling for our study. This supports the idea that patients with sporadic gastroenteritis are a reservoir for emerging epidemic noroviral strains [52].

Dual rotavirus-norovirus infections were detected in 2.7% of our study samples. Approximately the same rate was reported (2.1%) in a Polish study [3], but our rate is lower than that reported in France by Tran et al. [36]. As reported in the present study and the French study [36], there was no significant correlation between the severity of gastroenteritis and combined infections.

Our findings highlight the need to strengthen the surveillance of gastroenteritis and extend it to others regions in Morocco. This will allow us to monitor changes in the epidemiology of rotavirus- and norovirus-associated gastroenteritis and to identify dynamic shifts in the circulating strains.