Arch Virol DOI 10.1007/s00705-015-2458-3
ORIGINAL ARTICLE
Detection of rotavirus and other enteropathogens in children hospitalized with acute gastroenteritis in Havana, Cuba Marı´a de los Angeles Ribas1 • Yahisel Tejero1 • Yanislet Cordero1 • Marı´a de los Angeles Leo´n2 • Misladys Rodriguez1 • Jorge Perez-Lastre1 Thelma Triana3 • Mabel Guerra4 • Lucı´a Ayllo´n5 • Gladys Escalante6 • Jorge Hadad7
•
Received: 6 February 2015 / Accepted: 15 May 2015 Ó Springer-Verlag Wien 2015
Abstract The aim of the study was to diagnose infections with rotavirus and other enteric pathogens in children under five years old with acute gastroenteritis and to identify the most common epidemiological and clinical characteristics of these pathogens. The study was conducted using 110 stool samples from the same number of children under five years old who were inpatients at three paediatric hospitals in Havana, Cuba, between October and December 2011. The samples were tested for rotavirus and other enteric pathogens using traditional and molecular microbiological methods. Pathogens were detected in 85 (77.3 %) of the children. Rotavirus was the most commonly found, appearing in 54.5 % of the children, followed by bacteria (29 %) and parasites (10.9 %). Other viral pathogens detected included adenovirus (6.4 %) and astrovirus (3.6 %). In rotavirus-positives cases, at least one other pathogen was detected, usually a bacterium (26.6 %). More than three episodes of watery diarrhea in 24 hours were observed in 78.3 % of the cases. Dehydration was found in 30 (50 %) rotavirus-positive children, of whom
& Marı´a de los Angeles Ribas [email protected] 1
Pedro Kourı´ Institute of Tropical Medicine, Autopista Novia del Mediodı´a Km 61/2, La Lisa, Havana, Cuba
2
Ministry of Health, 23 y O. Vedado, Habana, Cuba
3
Provincial Hygiene, Epidemiology and Microbiology Centre, Habana, Cuba
4
Juan Manuel Marquez Pediatric Hospital, Habana, Cuba
5
Willian Soler Pediatric Hospital, Habana, Cuba
6
Havana Centre Pediatric Hospital, Habana, Cuba
7
Pan-American Health Organization (PAHO/WHO), Habana, Cuba
seven (11.6 %) were transferred to an intensive care unit due to complications of metabolic acidosis. Rotavirus was most commonly observed among children under 12 months old (65 %). The highest incidence of infection occurred in children who were under the care of a relative at home (78.3 %), had not been breastfed (65 %), or had been breastfed for less than six months (28.3 %). The genotype combinations most frequently found were G9P8 (28.3 %) and G1P8 (10 %). This study demonstrates the presence of rotavirus and other enteric pathogens as causes of gastroenteritis in hospitalized infants and young children in Cuba.
Introduction Acute gastroenteritis (AGE) is caused by viruses, bacteria and parasites. Is widely distributed in countries with different developmental and income patterns and infects people in diverse age groups belonging to all socio-economic strata, with the rate of infection much higher in localities with poor access to basic health care facilities and medical attention [1, 2]. Rotavirus (RoV) is responsible of 5 % of all deaths among children worldwide. In Latin America and the Caribbean countries, it is estimated that there are 15,000 deaths, 75,000 hospitalizations, two million clinical visits, and ten million diarrhea cases each year [3]. RoV is a member of the family Reoviridae. The virion contains 11 segments of double-stranded RNA, and two outer-layer proteins, VP4 and VP7, define the P (protease VP4) and G (glycoprotein VP7) serotype, respectively. Many G/P combinations are possible, but G1P [8], G2P [4], G3P [8], G4P [8] and G9P [8] are by far the most commonly
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M. A. Ribas et al.
identified genotypes worldwide. The RoV antigenic serogroups A, B and C have been recognised as causing gastroenteritis in humans. Group A has become the leading cause of AGE in children under five years old [4, 5]. The clinical presentation of RoV includes a wide range of symptoms, from asymptomatic infection to severe dehydrating diarrhea. Previous studies of this virus have detected coinfections with other viruses, bacteria and parasites [6]. The virus is highly contagious and is transmitted by fecal oral spread, through close person-to-person contact and by fomites. In tropical countries, transmission occurs year round, whereas in countries with a temperate climate, annual winter epidemics occur [2]. In Cuba, gastroenteritis is still a major cause of morbidity in infants and young children and accounts for a large proportion of severe episodes leading to clinic or hospital visit. In the country, systematic studies of AGE in patients requiring hospitalization usually do not include testing for a broad panel of pathogens, especially viruses. However, several studies have suggested a role of RoV, with an infection rate of 60 % of all episodes of gastroenteritis among children younger than five years old [7, 8]. An RoV vaccine is not included in the enhanced program of immunization (EPI) in Cuba. Further studies of RoV disease will be crucial to support the implementation of this vaccine into the Cuban program to prevent the infection and reduce the burden of disease. The aim of this study was to identify infections with RoV and other enteropathogens in children less than five years old with AGE who were hospitalized in three paediatric hospitals in Havana, Cuba, in the period between October and December 2011 and to assess their clinical and epidemiological characteristics.
Materials and methods In this study, we analyzed 110 fecal samples from the same number of children under five years old with AGE admitted to Juan Manuel Marquez, Havana Centre, and William Soler, the three largest paediatric hospitals in Havana, Cuba, between October and December 2011. Samples were sent to the National Reference Laboratory of Viral Diarrhea at the Pedro Kourı´ Institute of Tropical Medicine for diagnosis of RoV and other viruses. Bacteria and parasites were studied in the microbiology laboratory of each paediatric hospital. AGE was defined as three or more liquid evacuations within 24 hours and up to 14 days of duration in children less than five years old (4 years 11 months and 29 days) hospitalized with diarrhea. Clinical and epidemiological data were gathered by interviewing the parents or guardians before discharge from
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the hospital. The information obtained included age, sex, place of residence, and symptoms such as fever, vomiting, diarrhea, duration and number of episodes of diarrhea and vomiting within 24 hours, as well as information on consumption of food, dehydration, antibiotic therapy before hospital admission, and other matters. All patients were evaluated by paediatricians. Stool samples were collected in sterile containers from all enrolled patients and stored at -20 °C after written consent was given by their parent or guardian at the time when the clinical and epidemiological questionnaire was filled out.
Laboratory methods Rotavirus detection All samples were screened for group A RoV by enzymelinked immunosorbent assay (ELISA; IDEIA TM Rotavirus, OXOID Ltd., United Kingdom) to detect the VP6 antigen according to the manufacturer’s instructions. Genotyping of rotavirus ELISA-positive samples were examined by reverse transcription polymerase chain reaction (RT-PCR) to determine G (VP7) and P (VP4) genotypes [9–11]. The viral dsRNA was extracted from the sample using a QIAamp Viral RNA Mini Kit (QIAGEN Sciences, Maryland, USA) following the manufacturer’s instructions. The RT-PCR was performed using a OneStep RT-PCR Kit (QIAGEN Sciences, Maryland, USA) following the manufacturer’s instructions. The reaction was performed in 50-ll reaction volumes containing the specific amplification primer set [9]. This mixture was treated at 50 °C for 30 min with an activation step at 95 °C for 15 min, followed by 35 cycles of amplification (94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s) and a final extension step at 72 °C for 10 min in a thermal cycler. Multiplex RT-PCR PCR typing was also performed from dsDNA that was obtained for the first amplification. In this case 1 ll of the amplicon served as template for the second typing amplification, using a pool of primers to determine G1, G2, G3, G4, G8, G9, G10 and P [4], P [6], P [8], P [9], P [10], P [11] genotypes [9–11]. The conditions for PCR have been described by Gouvea et al. and Wu et al. [9, 12]. PCR products were separated by electrophoresis in an agarose gel stained with 1.5 % ethidium bromide and visualized under ultraviolet light.
Rotavirus in hospitalized children
Adenovirus and astrovirus detection All stool specimens were screened for adenovirus and astrovirus antigens. A rapid test for adenovirus (RIDAQUICK, R-Biopharm AG, Germany) was performed according to the manufacturer’s instructions. Astrovirus was detected by RT-PCR as described by Yan et al. [13]. Detection of bacteria Detection and identification of enteric pathogens of bacterial origin (Escherichia coli, Shigella sp, Salmonella sp, Citrobacter sp, and others (Klebsiella, Proteus, Morganella) were done by conventional culture procedures [14]. Detection of parasites Parasites (Giardia lamblia, Entamoeba histolytica, Blastocystis hominis, Cryptosporidium and yeast were investigated using conventional microscopy and staining methods (1 % eosin and Lugol’s solution) [15]. Statistical Analysis Statistical analysis was performed using SPSS software version 17.0 (SPSS, Chicago, IL, USA). Differences in proportion were assessed by chi-square or Fisher’s exact test. The values of variables were counted and summarized in frequency tables. A p-value less than 0.05 was considered statistically significant. Ethical considerations The study was conducted in accordance with ethical principles expressed in the World Medical Association Declaration of Helsinki. Ethical clearance was obtained from the ethical committee at Pedro Kourı´ Institute of Tropical Medicine, and the Juan Manuel Marquez, Havana Centre, and William Soler paediatric hospitals. The activities of this study caused no risk to the patients. Parents and guardians of children participating in the study were informed of the purpose and design of the study, and an information form was attached that the parents could return if they did not want to let their children participate in the study (Reference No.CEI-IPK-3712).
Results A total of 110 stool samples from children under five years old with AGE hospitalized in three paediatric hospitals, Havana Centre (43, 39 %), Juan Manuel Marquez (32,
29 %), and William Soler (35, 31.8 %), in Havana, Cuba, were studied. The median age of the patients was 4.42 months (range, 0 to 60 months). The percent of girls (50.9 %; 56/110) was slightly higher in comparison to boys (49 %; 54/110). Of the patients, 88.1 % (97/110) were hospitalized for more than 72 hours (mean, 4.7 days; SD, 2.26; 95 % CI: 4.26-5.13). During the study period, no deaths were reported. Pathogens were detected in 85 (77.3 %) of the 110 children. RoV was the most important gastroenteritiscausing agent, appearing in 60 (54.5 %) of the children, followed by bacteria (32/110; 29 %) and parasites (12/110; 10.9 %). Other viral pathogens that were detected included adenovirus (7/110; 6.4 %) and astrovirus (4/110; 3.6 %). The demographic and clinical characteristics of the patients and the enteric pathogens identified in RoV-positive cases are shown in Table 1. Of the positive cases 51.6 % (31/60) occurred in girls and 48.3 % in boys (29/60). This difference was not statistically significant (p [ 0.05; OR, 1.069; 95 % CI, 0.5-2.26). In 43.3 % (26/60) of the cases, at least one more pathogen was found, in most cases, bacteria (16/60; 26.6 %). Among the bacteria, Escherichia coli (7/60; 11.6 %) was found most frequently, followed by Shigella sp (2/60; 3.3 %). Mixed infections with parasites were observed in 5 % (3/60) of the children. Cryptosporidium, Blastocystis hominis and Entamoeba histolytica were the pathogens identified. Coinfection with adenovirus and astrovirus was found in 4 (6.7 %) and 2 (3.3 %) cases, respectively. All participants had diarrhea, with a mean duration of 5.65 days. More than three episodes of diarrhea in 24 hours were observed in 86.6 % (52/60) of cases. In 90 % (54/60), the appearance of the stool was watery. Vomiting was the second most frequent symptom observed (39/60; 65 %). More than three episodes of vomiting in 24 hours occurred in 36.7 % (22/60) of the children. Fever and upper respiratory infection (cough and nasal secretion) were identified in 61.7 % (37/60) and 45 % (27/60) of cases, respectively. At hospital admission, dehydration was observed in 50 % (30/60) of the patients, and the difference between RoV-positive and RoV-negative patients was statistically significant (p \ 0.05). Seven (11.6 %) of these children were transferred to the intensive care unit (ICU) for more than 3 days due to complications of metabolic acidosis (3/ 60; 5 %). All RoV-positive children had a similar clinical profile to those infected with the other agents; however, in cases of parasite infection, fever and upper respiratory symptoms were not present. The age distribution of acute RoV gastroenteritis is shown in Figure 1. Rotaviral gastroenteritis was most commonly observed in children 0 to 6 (20/60; 33.3 %) and
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M. A. Ribas et al. Table 1 Demographic information, enteric pathogens and clinical characteristics identified in children under 5 years old positive for rotavirus Characteristics
No. of RoV*-infected children (n = 60) (%)
No. of RoV-uninfected children (n = 50) (%)
Odds ratio (95 % CI)
P-value (\0.05)
1.069 (0.5-2.26)
0.1
Demographics Girls
31 (51.6)
25 (50)
Boys
29 (48.3)
25 (50)
Enteric pathogens Bacteria
16 (26.6)
16 (32)
Escherichia coli
7 (11.6)
7 (14)
Shigella sp Salmonella sp
2 (3.3) 1 (1.7)
1 (2) 1 (2)
Others**
6 (10)
7 (14)
Parasites
3 (5)
8 (16)
Cryptosporidium
1 (1.7)
5 (10)
Blastocystis hominis
1 (1.7)
0
Entamoeba hystolitica
1 (1.7)
0
Viruses
6 (10)
5 (10)
Adenovirus
4 (6.7)
3 (6)
Astrovirus
2 (3.3)
2 (4)
Norovirus
0
0
0.77 (0.33-1.76)
0.67
0.27 (0.69-1.10)
0.1
0.96 (0.27-3.35)
0.1
1.16 (0.46-2.93)
0.81
2.0 (0.64-6.23)
0.2
Clinical characteristics Diarrhea
60 (100)
0
[3 (24 h)
47 (78.3)
37 (74)
\3 (24 h)
12 (20)
11 (22)
Appearance of diarrhea Watery Watery/mucoid
54 (90)
42 (84)
4 (6.7)
2 (4)
Watery/bloody
0
4 (8)
Watery/mucoid/bloody
2 (3.3)
1 (2)
39 (65)
29 (58)
[3 (24 h)
22 (36.7)
11 (22)
\3 (24 h)
10 (16.7)
10 (20)
Vomiting
28 (46.7)
29 (58)
Fever
Unknown
37 (61.7)
33 (66)
0.82 (0.37-1.81)
0.69
Upper respiratory infection
27 (45)
28 (56)
0.64 (0.3-1.36)
0.33
Dehydration
30 (50)
12 (24)
0.31 (0.13-0.71)
0.006
Metabolic acidosis
3 (5)
0
Admission to ICU***
7 (11.6)
0
* Rotavirus (RoV) ** Others (Citrobacter, Edwalsiella, Klebisella, Morganella, Enterobacter, Proteus) *** Intensive care unit (ICU)
7 to 12 months old (19/60; 31.6 %), decreasing to 26.6 % (16/60) in children 13 to 24 months of age and 8.3 % (5/ 60) in children 25 to 60 months of age (p [ 0.05). In children between 0 to 6 months old, Escherichia coli (25 %; 4/16), adenovirus, and astrovirus were identified. No parasites were detected in children younger than 6 months old; however, in those between 7 and 12 months, Cryptosporidium and Blastocystis hominis were identified.
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Entamoeba histolytica was detected in older children (1324 months old). Before hospital admission (Table 2), 65 % (39/60) of RoV-infected children received oral rehydration therapy (ORT), 33.3 % (20/60) antipyretics, 13.3 % antiemetics (8/ 60), and 8.3 % (5/60) antibiotic treatment. The main antibiotics employed were azithromycin, cefalexin and sulfamethoxazole. After admission, seven (11.7 %) of the
Rotavirus in hospitalized children
The uncommon RoV genotypes G2P8 (1/60; 1.7 %) and G9P4 (2/60; 3.3 %) were also found. Thirteen (21.6 %) samples were partially typed and eleven (18.3 %) remained untypeable.
patients with otitis media received antibiotic therapy (ceftriaxone). Possible risks factors for RoV positivity are shown in Table 3. The highest incidence of infection occurred in children who were under the care of a relative at home (78.3 %, 47/60), had not been breastfed (65 %, 39/60), or had been breastfed for less than 6 months (28.3 %; 17/60) (p [ 0.05). In order to identify the circulating strains of RoV, genotyping was performed (Fig. 2). G9 (22/60; 36.7 %) and G1 (11/60; 18.3 %) were the most prevalent circulating genotypes during the collection period. Mixed genotypes accounted for 8.3 % (5/60) of the samples (G1 ? G9, G9 ? G2). The predominant P genotypes were P8 (58.3 %; 35/60) and P4 (16.7 %; 10/60). The P and G type combinations most frequently found were G9P8, in 28.3 % (17/60) of the cases, followed by G1P8 (6/60; 10 %).
Discussion The present study highlights the contribution of RoV and other pathogens (viruses, bacteria and parasites) as an important cause of AGE in children under five years old hospitalized in three paediatric hospitals in Havana, Cuba. The study demonstrates the role of RoV as the main agent that causes AGE in children under five years old. This finding is consistent with previous national studies that show an RoV infection rate of 18 % to 60 % in the same age group [7, 8, 16]. Rotavirus was present in both genders, but a non-significant difference was found. Overseas studies have reported a slightly higher incidence in males than in females, and boys have been considered more susceptible to infection than girls, but this has been questioned [17–19]. The prevalence of enteric pathogens in the study (77.3 %) was lower than those reported previously by Kirkwood et al. (89 %) and Frieseman et al. (98 %), possibly due to a smaller number of samples [20, 21]. Adenovirus is considered the second most frequent causative agent of AGE in young children, and the peak of incidence occurs in summer [22–24]. The reason for the higher prevalence of RoV-positive cases and the difference in adenovirus detection rates may be associated with the lower sensitivity of the methodology employed in the diagnosis. Recently, the introduction of molecular methods
Fig. 1 Rotavirus positives cases in different age group
Table 2 Treatment of rotavirus-positive children before and after hospital admission
Treatment
No. of RoV-infected children (n = 60) (%)
No. of RoV-uninfected children(n = 50) (%)
Total (n = 110) (%)
ORT*
39 (65)
39 (78)
78 (70.9)
Antipyretics
20 (33.3)
15 (30)
35 (31.8)
Antiemetics
8 (13.3)
5 (10)
14 (12.7)
5 (8.3)
4 (8)
9 (8.2)
Before admission
Antibiotic before admisssion Azithromycin
1 (1.7)
0
1 (0.9)
Cefalexin
1 (1.7)
0
1 (0.9)
Sulfamethoxazole
1 (1.7)
0
1 (0.9)
Trifamox
1 (1.7)
0
1 (0.9)
Amoxicillin
0
1 (2)
1 (0.9)
Ceftriaxone
0
2 (4)
2 (1.8)
7 (11.7)
5 (0)
12 (10.9)
Antibiotic after admission Ceftriaxone * Oral rehidration
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M. A. Ribas et al. Table 3 Rotavirus positivity and possible risk factors Possible risk factors
No. of RoVinfected children (n = 60) (%)
No. of RoV uninfected children (n = 50) (%)
Consumption of food Exclusively breastfed
4 (6.6)
7 (14)
Partially breastfed
17 (28.3)
13 (26)
Not breastfed
39 (65)
30 (60)
17 (28.3)
16 (32)
Duration of breastfeeding \6 months [6 months Unknown
3 (5)
7 (14)
40 (66.6)
27 (54)
Child care center
9 (15)
12 (24)
Child caregiver (Not at home)
3 (5)
4 (8)
Child care
School attendance
1 (1.7)
1 (2)
Child care at home by relative
47 (78.3)
33 (66)
Fig. 2 Percentage of rotavirus G/P circulating genotypes
has led to a significant increase in the detection rate of these viruses in stool samples. To our knowledge, this is the first report of astrovirus as a cause of AGE in hospitalized infants in Cuba. As expected, few astrovirus cases (3.6 %) were detected, in agreement with some reports on this infection. Usually, this virus is not an important cause of diarrhea in children and causes less-severe symptoms than RoV infection [25–27]. Rotaviral and bacterial infections were more common in children between 0 to 6 months of age, with a high prevalence of Escherichia coli and a few Shigella cases. Parasites were detected in older children. The rate of bacterial infection in this study was 28.3 %, possibly because some bacteria were not tested for, and some of the children received antibiotic therapy before admission.
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The role of RoV and bacterial infection as important causes of infection requiring hospitalization has been discussed by different authors, and Escherichia coli and Shigella infections in young children are considered important health problems in developing countries. Nonetheless, in agreement with other studies conducted in hospitalized children with AGE, parasites were rarely found [28–30]. In a previous study performed in Cuba, Cryptosporidium was the parasite commonly detected in children under 12 months of age. However, Giardia lamblia and Blastocystis hominis were identified in children between 12 to 48 months old. Helminths were recognised in children older than five years old, principally in rural areas, due to bad hygienic, water and food conditions [31]. Dual infections involving RoV indicate that children have contact with a variety of enteric pathogens. In this study, RoV coinfection was infrequent in younger children and peaked in infants between 7 and 12 months old. These results are comparable with the percentages of mixed infections detected in earlier reports, which found a prevalence of 2 % to 19 % [32–34]. The reason to this prevalence is the role of maternal antibodies in the protection of infants and the immunity acquired through previous exposure [23, 29, 35]. There are limited data on mixed infection of RoV with other viruses in Cuba. In a previous study performed in children under five years old admitted to a paediatric hospital in Havana in 2011, using RT-PCR, sporadic cases of NoV (31.8 %) were observed and adenovirus was not detected (unpublished data). In our study, the 88 % of patients were hospitalized for more than 72 hours (mean: 4.7 days), and watery diarrhea and vomiting were the most commonly observed symptoms in patients infected with each agent. Fever and upper respiratory infection were not found in parasite infections. Consistent with previous studies, clinical presentation of patient with AGE is not generally indicative of a pathogen. It has been reported that symptoms, the length of hospitalization, and the number of diarrhea episodes are not significantly different for the different infectious agents [27, 33, 36]. In Cuba, dehydration is the most important cause of hospitalization of children with watery diarrhea and vomiting. Following the World Health Organization (WHO) guidelines, there are directed efforts towards the use of ORT, aimed at reducing mortality secondary to diarrhea. Treatment of children is focussed on rehydration therapy to maintain fluid and electrolyte balance [21]. Before admission, prescription of antibiotics was not as infrequent as was expected and did not depend on knowledge about resistance or their incorrect use. The most commonly used antibiotics were azithromycin, cefalexin,
Rotavirus in hospitalized children
trifamox and sulfamethoxazole. Improvements in detection of pathogens and education measures are critical with regard to the use of antibiotics [23]. Among the strategies for decreasing mor-bidity due to diarrhea, breastfeeding and hand washing are top priorities, but even with hand washing and cleaning with a disinfectant, it is very hard to prevent rotavirus infection. In the study, a high proportion of children who were not breastfed or were breastfed for less than six months had RoV infection. Children generally start to supplement with food in addition to breastfeeding before 6 months of age, which in low-income countries frequently leads to diarrhea and infection with viruses, parasites or bacteria [28]. In Cuba, children attend day care centre they reach one year of age, but some parents choose to have relatives care for their children, because they prefer to keep their children in a healthier home-like environment. Unexpectedly, a fairly high percentage (15 %) of RoV positivity was found in the group that attend day care centres. It has been reported that children in this group are likely to be infected with RoV or other enteric pathogens causing AGE, due to more close contact with other children in this environment [37]. The highest rate of RoV positivity (78.3 %) was found in children cared for by relatives at home. In this environment, it is common that children in more than one age group share the same room and the infectious disease risks increase. This demonstrates the need to keep parents and caregivers informed about rotavirus transmission and its symptoms and to implement initiatives to enhance public understanding and awareness of child health issues through health promotion, education and advocacy. The molecular epidemiology study showed a high proportion of the common genotypes G9P8 and G1P8, which is consistent with previous studied carried out in Cuba and in other countries in the American Region, where G9 has been an important emerging serotype [8, 38]. We also found a few cases of the unusual combination G2P8 and G9P4. These strains have undergone natural intergenogroup reassortment, as was reported in India and Germany [18, 39]. The G9P4 genotype was identified in Brazil in the 1990s, in Honduras in 2009 and in Mexico and Guatemala in 2010 [8, 40, 41]. In our study, it appears that this genotype was the result of the reassortment between G2P4 and G9P6, strains that were reported in Cuba previously [8]. Some samples (18 %) could not be genotyped, possibly due to genetic variation in the Cuban strains, which need to be sequenced to reduce the number of untyped strains. Unfortunately, the lab currently does not have the capacity to do sequence analysis. Some limitations of the study need to be addressed. The short time period of the investigation did not allow enough
samples to be collected to do seasonal studies. Correlations of pathogens with clinical symptoms and symptom severity also need to be investigated. Nevertheless, the investigation provides important data on the presence of enteric pathogens in children less than five years old who were hospitalized with diarrhea. The results of the present study emphasize the importance of RoV as a major cause of gastroenteritis in hospitalized Cuban children under five years old. The introduction of an RoV vaccine in the EPI should reduce the prevalence of infection and result in important changes in rotavirus epidemiology. Continuing RoV surveillance before and after vaccine introduction will provide information about the range of circulating strains in the country. Acknowledgments The authors would like to thank the hospital and laboratory staff at each of the paediatric hospitals and Pedro Kourı´ Institute of Tropical Medicine for their invaluable support of this study. Conflict of interest of interest.
The authors declare that they have no conflict
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M. A. Ribas et al. 10. Gentsch J, Glass R, Woods P, Gouvea V, Gorziglia M, Flores J, Das B, Bhan M (1992) Identification of group A Rotavirus gene 4 types by polymerase chain reaction. J Clin Microbiol 30(6):1365–1373 11. Iturriza Gomara M, Green J, Brown D, Desselberger U, Gray J (2000) Diversity within the VP4 gene of rotavirus P[8] strain: implications for reverse transcription—PCR genotyping. J Clin Microbiol 38(2):898–901 12. Wu H, Tanigushi K, Wakangi F, Ukae S, Chiba S, Ohseto M, Hasegawa A, Usagawa T, Usagawa S (1994) Survey on the distribution of the gene 4 alleles of human rotaviruses by polymerase chain. Epidemiol Infect 112:615–622 13. Yan H, Yagyu F, Okitsu S, Nishio O, Ushijima H (2003) Detection of norovirus (Gi, GII), sapovarus and astrovirus in fecal samples using reverse transcription single round multiplex PCR. J Virol Meth 114:37–44 14. Valde´s-Dapena MM (2001) Enterobacterias. In: Llops A, Valde´sDapena MM, Zuazo JL (eds) Microbiologı´a y Parasitologı´a Me´dicas, 1ra edicio´n. Ciencias Me´dicas, La Habana, pp 251–279 15. Pelayo L (2001) Parasitologı´a. In: LLops A, Valdes-Dapena MM, Zuazo JL (eds) Microbiologı´a y Parasitologı´a Me´dicas. La Habana. Ciencias Me´dicas, pp 3–21 16. De la Cruz F, Guzma´n M, Esquivel M, Grandio O, Va´zquez S, Bravo J (1990) Clinical and epidemiological aspects of acute diarrea caused by rotavirus in children. Cuba 1982-1984. Rev Cub Med Trop 42:178–187 17. Maldonado A, Franco M, Blanco A, Villalobos L, Martı´nez R, Hagel I, Gonzalez R, Bastardo J (2010) Caracterı´sticas clı´nicas y epidemiolo´gicas de la infeccio´n por rotavirus en nin˜os de Cumana´, Venezuela. Invset Clin 51(4):519–529 18. Saluja T, Sharma D, Gupta M, Kundu R, Kar S, Dutta A, Silveira M, Singh J, Kamth V, Chaudary A, Rao J, Ravi M, Murthy S, Badji S, Prasad R, Gujjula R, Rao R, Dhingra M (2014) A multicenter prospective hospital –based surveillance to estimate the burden of rotavirus gastroenteritis in children less than five years of age in India. Vaccine 32S:A13–A19 19. Junaid S, Umeh C, Olabode A, Banda J (2011) Incidence of rotavirus infection in children with gastroenteritis attending Jos University teaching hospital, Nigeria. Virol J 8(233):2–8 20. Kirkwood C, Bogdanovic-Sakran N, Branes G, Bispo R (2004) Rotavirus serotype G9[P8] and acute gastroenteritis outbreak in children, North Australia. Emerg Infect Dis 10(9):1593–1600 21. Friesema I, Boer R, Duizer E, Kortbeek L, Notermans D, Norbruis O, Bezemer D, van Heerbeek H, van Andel R, van Enk J, Fraaij P, Koopsman M, Kooistra-Smid A, van Duynhoven Y (2012) Etiology of acute gastroenteritis in children requiring hospitalization in the Netherlands. Eur J Clin Microbiol Infect Dis 31:405–415 22. Bon F, Fascia P, Dauvergne M, Tenenbaum D, Planson H, Petion A, Pothier P, Kohli E (1999) Prevalence of group A rotavirus, human calicivirus, astrovirus, and adenovirus type 40 and 41 infections among children with acute gastroenteritis in Dijon, France. J Clin Microbiol 37:3055–3058 23. Moyo S, Gro N, Kirsti V, Matee M, Kitundu J, Maselle S, Langeland N, Myrmel H (2007) Prevalence of enterophatogenic viruses and molecular characterization of group A rotavirus among children with diarrhea in Dar es Salaam Tanzania. BMC Public Health 7(359):1–6 24. Bicer S, ColD Ciler G, Giray T, Gurol Y, Yilmaz G, Vitrinel A, Ozelgun B (2014) A restropective analysis of acute gastroenteritis agents in children admitted to university hospital pediatric emergency unit. Jundishapur J Microbiol 7(4):1–7 25. Preeti C, Payne D, Szilagyi P, Edwards K, Staat M, Shirley H, Wikswo M, Nix W, Lu X, Parashar U, Vinje J (2013) Etiology of viral gastroenteritis in children \5 years of age in the United States, 2008–2009. JID 208:790–800
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26. Ham H, Oh S, Jang J, Jo S, Choi S, Pak S (2014) Prevalence of human astrovirus in patient with acute gastroenteritis. Ann Lab Med 34:145–147 27. So C, Kim D, Yu S, Cho J, Kim J (2013) Acute viral gastroenteritis in children hospitalized in Iksan, Korea during December 2010–June 2011. Korean J Pediatr 56(9):383–388 28. Nitiema L, Nordgren J, Ouermi D, Dianou D, Traore A, Svensson L, Simpore J (2011) Burden of rotavirus and other enterophatogens among children with diarrhea in Burkina Faso. Inter J Infect Dis 15:e646–e652 29. Nataro J, Mai V, Johnson J, Blackwelder W, Heimer R, Triller S, Edberg S, Braden C, Glenn M, Hirshon J (2006) Diarrheagenic Escherichia coli infection in Baltimore, Maryland and New Haven, Connecticut. Clin Infec Dis 43:402–407 30. Moyo S, Gro N, Matee M, Kitundu J, Myrmel H, Mylvaganam H, Maselle S, Langeland N (2011) Age specific aetiological agents of diarrhoea in hospitalized children aged less than five years in Dar es Salaam, Tanzania. BMC Pediatrics 11(19):2–6 31. Nun˜ez F, Gonzalez O, Bravo J, Escobedo A, Gonzalez I (2003) Para´sitos intestinales en nin˜os ingresados en el hospital universitario Pedia´trico del Cerro, La Habana, Cuba. Rev Cub Med Trop 55(1):19–26 32. De Oliveira C, Raboni S, Pereira L, Nogueira M, Renaud L, Monteiro S (2012) Viral acute gastroenteritis: clinical and epidemiological features of co infected infants. Braz J Infect Dis 16(3):267–272 33. Soli K, Maure T, Kas M, Brande G, Bebes S, Luang Suarkia D, Siba P, Morita A, Umezaki M, Greenhill A, Horwood P (2014) Detection of enteric viral and bacterial pathogens associated with paediatric diarrhoea in Goroka, Papua, New Guinea. Intern J Infect Dis 27:54–58 34. Ramani S, Kang G (2009) Viruses causing childhood diarrhea in the developing world. Curr Opin Infect Dis 22:477–482 35. Tran A, Talmud D, Lejeune B, Jovenin N, Renois F, Payan C, Leveque N, Andreoletti L (2010) Prevalence of rotavirus, adenovirus, norovirus and astrovirus infections and co infections among hospitalized children in Northen France. J Clin Microbiol 48(5):1943–1946 36. Hwang P, Kwak J, Lee T, Jeong S (2009) Clinical features of acute noroviral gastroenteritis in children: comparison with rotaviral gastroenteritis. Korean J Pediatr 52:453–457 37. Enserink R, Scholts R, Bruijning- Verhagen P, Duizer E, Vennema H, de Boer R, Kortbeek T, Roelfsema J, Smit H, KooistraSmid M, van Pelt W (2014) High detection rate of enteropathogens in asymptomatic children attending day care. Plos One 9(2):e89496 38. Rivera R, Forney K, Castro M, Rebolledo P, Mamani N, Patzi M, Halkyer P, Leon J, In˜iguez V (2013) Rotavirus genotype distribution during the pre vaccine period in Bolivia: 2007–2008. Inter J Infect Dis 17(9):e762–e767 39. Mas Marques A, Diedrich S, Huth C, Schreier E (2007) Group A rotavirus genotype in Germany during 2005/2006. Arch Virol 152:1743–1749 40. Quaye O, Mc Donald S, Esona M, Lyde F, Mijatovic S, Roy S, Castro D, Mencos Y, Chinchilla B, Gomez F, Garcia H, Rey G, de Oliveira L, Gentsch J, Bowen M (2013) Rotavirus G9P4 in 3 countries in Latin America, 2009–2010. Emerg Infect Dis 19(8):1332–1333 41. Rahman M, Matthijnssens J, Yang X, Delbeke T, Arijs I, Taniguchi K, Iturriza-Gomara M, Iftekharuddin N, Azim T, Van Ranst M (2007) Evolutionary history and global spread of the emerging G12 human rotaviruses. J Virol 81(5):2382–2390
ORIGINAL ARTICLE
Detection of rotavirus and other enteropathogens in children hospitalized with acute gastroenteritis in Havana, Cuba Marı´a de los Angeles Ribas1 • Yahisel Tejero1 • Yanislet Cordero1 • Marı´a de los Angeles Leo´n2 • Misladys Rodriguez1 • Jorge Perez-Lastre1 Thelma Triana3 • Mabel Guerra4 • Lucı´a Ayllo´n5 • Gladys Escalante6 • Jorge Hadad7
•
Received: 6 February 2015 / Accepted: 15 May 2015 Ó Springer-Verlag Wien 2015
Abstract The aim of the study was to diagnose infections with rotavirus and other enteric pathogens in children under five years old with acute gastroenteritis and to identify the most common epidemiological and clinical characteristics of these pathogens. The study was conducted using 110 stool samples from the same number of children under five years old who were inpatients at three paediatric hospitals in Havana, Cuba, between October and December 2011. The samples were tested for rotavirus and other enteric pathogens using traditional and molecular microbiological methods. Pathogens were detected in 85 (77.3 %) of the children. Rotavirus was the most commonly found, appearing in 54.5 % of the children, followed by bacteria (29 %) and parasites (10.9 %). Other viral pathogens detected included adenovirus (6.4 %) and astrovirus (3.6 %). In rotavirus-positives cases, at least one other pathogen was detected, usually a bacterium (26.6 %). More than three episodes of watery diarrhea in 24 hours were observed in 78.3 % of the cases. Dehydration was found in 30 (50 %) rotavirus-positive children, of whom
& Marı´a de los Angeles Ribas [email protected] 1
Pedro Kourı´ Institute of Tropical Medicine, Autopista Novia del Mediodı´a Km 61/2, La Lisa, Havana, Cuba
2
Ministry of Health, 23 y O. Vedado, Habana, Cuba
3
Provincial Hygiene, Epidemiology and Microbiology Centre, Habana, Cuba
4
Juan Manuel Marquez Pediatric Hospital, Habana, Cuba
5
Willian Soler Pediatric Hospital, Habana, Cuba
6
Havana Centre Pediatric Hospital, Habana, Cuba
7
Pan-American Health Organization (PAHO/WHO), Habana, Cuba
seven (11.6 %) were transferred to an intensive care unit due to complications of metabolic acidosis. Rotavirus was most commonly observed among children under 12 months old (65 %). The highest incidence of infection occurred in children who were under the care of a relative at home (78.3 %), had not been breastfed (65 %), or had been breastfed for less than six months (28.3 %). The genotype combinations most frequently found were G9P8 (28.3 %) and G1P8 (10 %). This study demonstrates the presence of rotavirus and other enteric pathogens as causes of gastroenteritis in hospitalized infants and young children in Cuba.
Introduction Acute gastroenteritis (AGE) is caused by viruses, bacteria and parasites. Is widely distributed in countries with different developmental and income patterns and infects people in diverse age groups belonging to all socio-economic strata, with the rate of infection much higher in localities with poor access to basic health care facilities and medical attention [1, 2]. Rotavirus (RoV) is responsible of 5 % of all deaths among children worldwide. In Latin America and the Caribbean countries, it is estimated that there are 15,000 deaths, 75,000 hospitalizations, two million clinical visits, and ten million diarrhea cases each year [3]. RoV is a member of the family Reoviridae. The virion contains 11 segments of double-stranded RNA, and two outer-layer proteins, VP4 and VP7, define the P (protease VP4) and G (glycoprotein VP7) serotype, respectively. Many G/P combinations are possible, but G1P [8], G2P [4], G3P [8], G4P [8] and G9P [8] are by far the most commonly
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identified genotypes worldwide. The RoV antigenic serogroups A, B and C have been recognised as causing gastroenteritis in humans. Group A has become the leading cause of AGE in children under five years old [4, 5]. The clinical presentation of RoV includes a wide range of symptoms, from asymptomatic infection to severe dehydrating diarrhea. Previous studies of this virus have detected coinfections with other viruses, bacteria and parasites [6]. The virus is highly contagious and is transmitted by fecal oral spread, through close person-to-person contact and by fomites. In tropical countries, transmission occurs year round, whereas in countries with a temperate climate, annual winter epidemics occur [2]. In Cuba, gastroenteritis is still a major cause of morbidity in infants and young children and accounts for a large proportion of severe episodes leading to clinic or hospital visit. In the country, systematic studies of AGE in patients requiring hospitalization usually do not include testing for a broad panel of pathogens, especially viruses. However, several studies have suggested a role of RoV, with an infection rate of 60 % of all episodes of gastroenteritis among children younger than five years old [7, 8]. An RoV vaccine is not included in the enhanced program of immunization (EPI) in Cuba. Further studies of RoV disease will be crucial to support the implementation of this vaccine into the Cuban program to prevent the infection and reduce the burden of disease. The aim of this study was to identify infections with RoV and other enteropathogens in children less than five years old with AGE who were hospitalized in three paediatric hospitals in Havana, Cuba, in the period between October and December 2011 and to assess their clinical and epidemiological characteristics.
Materials and methods In this study, we analyzed 110 fecal samples from the same number of children under five years old with AGE admitted to Juan Manuel Marquez, Havana Centre, and William Soler, the three largest paediatric hospitals in Havana, Cuba, between October and December 2011. Samples were sent to the National Reference Laboratory of Viral Diarrhea at the Pedro Kourı´ Institute of Tropical Medicine for diagnosis of RoV and other viruses. Bacteria and parasites were studied in the microbiology laboratory of each paediatric hospital. AGE was defined as three or more liquid evacuations within 24 hours and up to 14 days of duration in children less than five years old (4 years 11 months and 29 days) hospitalized with diarrhea. Clinical and epidemiological data were gathered by interviewing the parents or guardians before discharge from
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the hospital. The information obtained included age, sex, place of residence, and symptoms such as fever, vomiting, diarrhea, duration and number of episodes of diarrhea and vomiting within 24 hours, as well as information on consumption of food, dehydration, antibiotic therapy before hospital admission, and other matters. All patients were evaluated by paediatricians. Stool samples were collected in sterile containers from all enrolled patients and stored at -20 °C after written consent was given by their parent or guardian at the time when the clinical and epidemiological questionnaire was filled out.
Laboratory methods Rotavirus detection All samples were screened for group A RoV by enzymelinked immunosorbent assay (ELISA; IDEIA TM Rotavirus, OXOID Ltd., United Kingdom) to detect the VP6 antigen according to the manufacturer’s instructions. Genotyping of rotavirus ELISA-positive samples were examined by reverse transcription polymerase chain reaction (RT-PCR) to determine G (VP7) and P (VP4) genotypes [9–11]. The viral dsRNA was extracted from the sample using a QIAamp Viral RNA Mini Kit (QIAGEN Sciences, Maryland, USA) following the manufacturer’s instructions. The RT-PCR was performed using a OneStep RT-PCR Kit (QIAGEN Sciences, Maryland, USA) following the manufacturer’s instructions. The reaction was performed in 50-ll reaction volumes containing the specific amplification primer set [9]. This mixture was treated at 50 °C for 30 min with an activation step at 95 °C for 15 min, followed by 35 cycles of amplification (94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s) and a final extension step at 72 °C for 10 min in a thermal cycler. Multiplex RT-PCR PCR typing was also performed from dsDNA that was obtained for the first amplification. In this case 1 ll of the amplicon served as template for the second typing amplification, using a pool of primers to determine G1, G2, G3, G4, G8, G9, G10 and P [4], P [6], P [8], P [9], P [10], P [11] genotypes [9–11]. The conditions for PCR have been described by Gouvea et al. and Wu et al. [9, 12]. PCR products were separated by electrophoresis in an agarose gel stained with 1.5 % ethidium bromide and visualized under ultraviolet light.
Rotavirus in hospitalized children
Adenovirus and astrovirus detection All stool specimens were screened for adenovirus and astrovirus antigens. A rapid test for adenovirus (RIDAQUICK, R-Biopharm AG, Germany) was performed according to the manufacturer’s instructions. Astrovirus was detected by RT-PCR as described by Yan et al. [13]. Detection of bacteria Detection and identification of enteric pathogens of bacterial origin (Escherichia coli, Shigella sp, Salmonella sp, Citrobacter sp, and others (Klebsiella, Proteus, Morganella) were done by conventional culture procedures [14]. Detection of parasites Parasites (Giardia lamblia, Entamoeba histolytica, Blastocystis hominis, Cryptosporidium and yeast were investigated using conventional microscopy and staining methods (1 % eosin and Lugol’s solution) [15]. Statistical Analysis Statistical analysis was performed using SPSS software version 17.0 (SPSS, Chicago, IL, USA). Differences in proportion were assessed by chi-square or Fisher’s exact test. The values of variables were counted and summarized in frequency tables. A p-value less than 0.05 was considered statistically significant. Ethical considerations The study was conducted in accordance with ethical principles expressed in the World Medical Association Declaration of Helsinki. Ethical clearance was obtained from the ethical committee at Pedro Kourı´ Institute of Tropical Medicine, and the Juan Manuel Marquez, Havana Centre, and William Soler paediatric hospitals. The activities of this study caused no risk to the patients. Parents and guardians of children participating in the study were informed of the purpose and design of the study, and an information form was attached that the parents could return if they did not want to let their children participate in the study (Reference No.CEI-IPK-3712).
Results A total of 110 stool samples from children under five years old with AGE hospitalized in three paediatric hospitals, Havana Centre (43, 39 %), Juan Manuel Marquez (32,
29 %), and William Soler (35, 31.8 %), in Havana, Cuba, were studied. The median age of the patients was 4.42 months (range, 0 to 60 months). The percent of girls (50.9 %; 56/110) was slightly higher in comparison to boys (49 %; 54/110). Of the patients, 88.1 % (97/110) were hospitalized for more than 72 hours (mean, 4.7 days; SD, 2.26; 95 % CI: 4.26-5.13). During the study period, no deaths were reported. Pathogens were detected in 85 (77.3 %) of the 110 children. RoV was the most important gastroenteritiscausing agent, appearing in 60 (54.5 %) of the children, followed by bacteria (32/110; 29 %) and parasites (12/110; 10.9 %). Other viral pathogens that were detected included adenovirus (7/110; 6.4 %) and astrovirus (4/110; 3.6 %). The demographic and clinical characteristics of the patients and the enteric pathogens identified in RoV-positive cases are shown in Table 1. Of the positive cases 51.6 % (31/60) occurred in girls and 48.3 % in boys (29/60). This difference was not statistically significant (p [ 0.05; OR, 1.069; 95 % CI, 0.5-2.26). In 43.3 % (26/60) of the cases, at least one more pathogen was found, in most cases, bacteria (16/60; 26.6 %). Among the bacteria, Escherichia coli (7/60; 11.6 %) was found most frequently, followed by Shigella sp (2/60; 3.3 %). Mixed infections with parasites were observed in 5 % (3/60) of the children. Cryptosporidium, Blastocystis hominis and Entamoeba histolytica were the pathogens identified. Coinfection with adenovirus and astrovirus was found in 4 (6.7 %) and 2 (3.3 %) cases, respectively. All participants had diarrhea, with a mean duration of 5.65 days. More than three episodes of diarrhea in 24 hours were observed in 86.6 % (52/60) of cases. In 90 % (54/60), the appearance of the stool was watery. Vomiting was the second most frequent symptom observed (39/60; 65 %). More than three episodes of vomiting in 24 hours occurred in 36.7 % (22/60) of the children. Fever and upper respiratory infection (cough and nasal secretion) were identified in 61.7 % (37/60) and 45 % (27/60) of cases, respectively. At hospital admission, dehydration was observed in 50 % (30/60) of the patients, and the difference between RoV-positive and RoV-negative patients was statistically significant (p \ 0.05). Seven (11.6 %) of these children were transferred to the intensive care unit (ICU) for more than 3 days due to complications of metabolic acidosis (3/ 60; 5 %). All RoV-positive children had a similar clinical profile to those infected with the other agents; however, in cases of parasite infection, fever and upper respiratory symptoms were not present. The age distribution of acute RoV gastroenteritis is shown in Figure 1. Rotaviral gastroenteritis was most commonly observed in children 0 to 6 (20/60; 33.3 %) and
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M. A. Ribas et al. Table 1 Demographic information, enteric pathogens and clinical characteristics identified in children under 5 years old positive for rotavirus Characteristics
No. of RoV*-infected children (n = 60) (%)
No. of RoV-uninfected children (n = 50) (%)
Odds ratio (95 % CI)
P-value (\0.05)
1.069 (0.5-2.26)
0.1
Demographics Girls
31 (51.6)
25 (50)
Boys
29 (48.3)
25 (50)
Enteric pathogens Bacteria
16 (26.6)
16 (32)
Escherichia coli
7 (11.6)
7 (14)
Shigella sp Salmonella sp
2 (3.3) 1 (1.7)
1 (2) 1 (2)
Others**
6 (10)
7 (14)
Parasites
3 (5)
8 (16)
Cryptosporidium
1 (1.7)
5 (10)
Blastocystis hominis
1 (1.7)
0
Entamoeba hystolitica
1 (1.7)
0
Viruses
6 (10)
5 (10)
Adenovirus
4 (6.7)
3 (6)
Astrovirus
2 (3.3)
2 (4)
Norovirus
0
0
0.77 (0.33-1.76)
0.67
0.27 (0.69-1.10)
0.1
0.96 (0.27-3.35)
0.1
1.16 (0.46-2.93)
0.81
2.0 (0.64-6.23)
0.2
Clinical characteristics Diarrhea
60 (100)
0
[3 (24 h)
47 (78.3)
37 (74)
\3 (24 h)
12 (20)
11 (22)
Appearance of diarrhea Watery Watery/mucoid
54 (90)
42 (84)
4 (6.7)
2 (4)
Watery/bloody
0
4 (8)
Watery/mucoid/bloody
2 (3.3)
1 (2)
39 (65)
29 (58)
[3 (24 h)
22 (36.7)
11 (22)
\3 (24 h)
10 (16.7)
10 (20)
Vomiting
28 (46.7)
29 (58)
Fever
Unknown
37 (61.7)
33 (66)
0.82 (0.37-1.81)
0.69
Upper respiratory infection
27 (45)
28 (56)
0.64 (0.3-1.36)
0.33
Dehydration
30 (50)
12 (24)
0.31 (0.13-0.71)
0.006
Metabolic acidosis
3 (5)
0
Admission to ICU***
7 (11.6)
0
* Rotavirus (RoV) ** Others (Citrobacter, Edwalsiella, Klebisella, Morganella, Enterobacter, Proteus) *** Intensive care unit (ICU)
7 to 12 months old (19/60; 31.6 %), decreasing to 26.6 % (16/60) in children 13 to 24 months of age and 8.3 % (5/ 60) in children 25 to 60 months of age (p [ 0.05). In children between 0 to 6 months old, Escherichia coli (25 %; 4/16), adenovirus, and astrovirus were identified. No parasites were detected in children younger than 6 months old; however, in those between 7 and 12 months, Cryptosporidium and Blastocystis hominis were identified.
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Entamoeba histolytica was detected in older children (1324 months old). Before hospital admission (Table 2), 65 % (39/60) of RoV-infected children received oral rehydration therapy (ORT), 33.3 % (20/60) antipyretics, 13.3 % antiemetics (8/ 60), and 8.3 % (5/60) antibiotic treatment. The main antibiotics employed were azithromycin, cefalexin and sulfamethoxazole. After admission, seven (11.7 %) of the
Rotavirus in hospitalized children
The uncommon RoV genotypes G2P8 (1/60; 1.7 %) and G9P4 (2/60; 3.3 %) were also found. Thirteen (21.6 %) samples were partially typed and eleven (18.3 %) remained untypeable.
patients with otitis media received antibiotic therapy (ceftriaxone). Possible risks factors for RoV positivity are shown in Table 3. The highest incidence of infection occurred in children who were under the care of a relative at home (78.3 %, 47/60), had not been breastfed (65 %, 39/60), or had been breastfed for less than 6 months (28.3 %; 17/60) (p [ 0.05). In order to identify the circulating strains of RoV, genotyping was performed (Fig. 2). G9 (22/60; 36.7 %) and G1 (11/60; 18.3 %) were the most prevalent circulating genotypes during the collection period. Mixed genotypes accounted for 8.3 % (5/60) of the samples (G1 ? G9, G9 ? G2). The predominant P genotypes were P8 (58.3 %; 35/60) and P4 (16.7 %; 10/60). The P and G type combinations most frequently found were G9P8, in 28.3 % (17/60) of the cases, followed by G1P8 (6/60; 10 %).
Discussion The present study highlights the contribution of RoV and other pathogens (viruses, bacteria and parasites) as an important cause of AGE in children under five years old hospitalized in three paediatric hospitals in Havana, Cuba. The study demonstrates the role of RoV as the main agent that causes AGE in children under five years old. This finding is consistent with previous national studies that show an RoV infection rate of 18 % to 60 % in the same age group [7, 8, 16]. Rotavirus was present in both genders, but a non-significant difference was found. Overseas studies have reported a slightly higher incidence in males than in females, and boys have been considered more susceptible to infection than girls, but this has been questioned [17–19]. The prevalence of enteric pathogens in the study (77.3 %) was lower than those reported previously by Kirkwood et al. (89 %) and Frieseman et al. (98 %), possibly due to a smaller number of samples [20, 21]. Adenovirus is considered the second most frequent causative agent of AGE in young children, and the peak of incidence occurs in summer [22–24]. The reason for the higher prevalence of RoV-positive cases and the difference in adenovirus detection rates may be associated with the lower sensitivity of the methodology employed in the diagnosis. Recently, the introduction of molecular methods
Fig. 1 Rotavirus positives cases in different age group
Table 2 Treatment of rotavirus-positive children before and after hospital admission
Treatment
No. of RoV-infected children (n = 60) (%)
No. of RoV-uninfected children(n = 50) (%)
Total (n = 110) (%)
ORT*
39 (65)
39 (78)
78 (70.9)
Antipyretics
20 (33.3)
15 (30)
35 (31.8)
Antiemetics
8 (13.3)
5 (10)
14 (12.7)
5 (8.3)
4 (8)
9 (8.2)
Before admission
Antibiotic before admisssion Azithromycin
1 (1.7)
0
1 (0.9)
Cefalexin
1 (1.7)
0
1 (0.9)
Sulfamethoxazole
1 (1.7)
0
1 (0.9)
Trifamox
1 (1.7)
0
1 (0.9)
Amoxicillin
0
1 (2)
1 (0.9)
Ceftriaxone
0
2 (4)
2 (1.8)
7 (11.7)
5 (0)
12 (10.9)
Antibiotic after admission Ceftriaxone * Oral rehidration
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M. A. Ribas et al. Table 3 Rotavirus positivity and possible risk factors Possible risk factors
No. of RoVinfected children (n = 60) (%)
No. of RoV uninfected children (n = 50) (%)
Consumption of food Exclusively breastfed
4 (6.6)
7 (14)
Partially breastfed
17 (28.3)
13 (26)
Not breastfed
39 (65)
30 (60)
17 (28.3)
16 (32)
Duration of breastfeeding \6 months [6 months Unknown
3 (5)
7 (14)
40 (66.6)
27 (54)
Child care center
9 (15)
12 (24)
Child caregiver (Not at home)
3 (5)
4 (8)
Child care
School attendance
1 (1.7)
1 (2)
Child care at home by relative
47 (78.3)
33 (66)
Fig. 2 Percentage of rotavirus G/P circulating genotypes
has led to a significant increase in the detection rate of these viruses in stool samples. To our knowledge, this is the first report of astrovirus as a cause of AGE in hospitalized infants in Cuba. As expected, few astrovirus cases (3.6 %) were detected, in agreement with some reports on this infection. Usually, this virus is not an important cause of diarrhea in children and causes less-severe symptoms than RoV infection [25–27]. Rotaviral and bacterial infections were more common in children between 0 to 6 months of age, with a high prevalence of Escherichia coli and a few Shigella cases. Parasites were detected in older children. The rate of bacterial infection in this study was 28.3 %, possibly because some bacteria were not tested for, and some of the children received antibiotic therapy before admission.
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The role of RoV and bacterial infection as important causes of infection requiring hospitalization has been discussed by different authors, and Escherichia coli and Shigella infections in young children are considered important health problems in developing countries. Nonetheless, in agreement with other studies conducted in hospitalized children with AGE, parasites were rarely found [28–30]. In a previous study performed in Cuba, Cryptosporidium was the parasite commonly detected in children under 12 months of age. However, Giardia lamblia and Blastocystis hominis were identified in children between 12 to 48 months old. Helminths were recognised in children older than five years old, principally in rural areas, due to bad hygienic, water and food conditions [31]. Dual infections involving RoV indicate that children have contact with a variety of enteric pathogens. In this study, RoV coinfection was infrequent in younger children and peaked in infants between 7 and 12 months old. These results are comparable with the percentages of mixed infections detected in earlier reports, which found a prevalence of 2 % to 19 % [32–34]. The reason to this prevalence is the role of maternal antibodies in the protection of infants and the immunity acquired through previous exposure [23, 29, 35]. There are limited data on mixed infection of RoV with other viruses in Cuba. In a previous study performed in children under five years old admitted to a paediatric hospital in Havana in 2011, using RT-PCR, sporadic cases of NoV (31.8 %) were observed and adenovirus was not detected (unpublished data). In our study, the 88 % of patients were hospitalized for more than 72 hours (mean: 4.7 days), and watery diarrhea and vomiting were the most commonly observed symptoms in patients infected with each agent. Fever and upper respiratory infection were not found in parasite infections. Consistent with previous studies, clinical presentation of patient with AGE is not generally indicative of a pathogen. It has been reported that symptoms, the length of hospitalization, and the number of diarrhea episodes are not significantly different for the different infectious agents [27, 33, 36]. In Cuba, dehydration is the most important cause of hospitalization of children with watery diarrhea and vomiting. Following the World Health Organization (WHO) guidelines, there are directed efforts towards the use of ORT, aimed at reducing mortality secondary to diarrhea. Treatment of children is focussed on rehydration therapy to maintain fluid and electrolyte balance [21]. Before admission, prescription of antibiotics was not as infrequent as was expected and did not depend on knowledge about resistance or their incorrect use. The most commonly used antibiotics were azithromycin, cefalexin,
Rotavirus in hospitalized children
trifamox and sulfamethoxazole. Improvements in detection of pathogens and education measures are critical with regard to the use of antibiotics [23]. Among the strategies for decreasing mor-bidity due to diarrhea, breastfeeding and hand washing are top priorities, but even with hand washing and cleaning with a disinfectant, it is very hard to prevent rotavirus infection. In the study, a high proportion of children who were not breastfed or were breastfed for less than six months had RoV infection. Children generally start to supplement with food in addition to breastfeeding before 6 months of age, which in low-income countries frequently leads to diarrhea and infection with viruses, parasites or bacteria [28]. In Cuba, children attend day care centre they reach one year of age, but some parents choose to have relatives care for their children, because they prefer to keep their children in a healthier home-like environment. Unexpectedly, a fairly high percentage (15 %) of RoV positivity was found in the group that attend day care centres. It has been reported that children in this group are likely to be infected with RoV or other enteric pathogens causing AGE, due to more close contact with other children in this environment [37]. The highest rate of RoV positivity (78.3 %) was found in children cared for by relatives at home. In this environment, it is common that children in more than one age group share the same room and the infectious disease risks increase. This demonstrates the need to keep parents and caregivers informed about rotavirus transmission and its symptoms and to implement initiatives to enhance public understanding and awareness of child health issues through health promotion, education and advocacy. The molecular epidemiology study showed a high proportion of the common genotypes G9P8 and G1P8, which is consistent with previous studied carried out in Cuba and in other countries in the American Region, where G9 has been an important emerging serotype [8, 38]. We also found a few cases of the unusual combination G2P8 and G9P4. These strains have undergone natural intergenogroup reassortment, as was reported in India and Germany [18, 39]. The G9P4 genotype was identified in Brazil in the 1990s, in Honduras in 2009 and in Mexico and Guatemala in 2010 [8, 40, 41]. In our study, it appears that this genotype was the result of the reassortment between G2P4 and G9P6, strains that were reported in Cuba previously [8]. Some samples (18 %) could not be genotyped, possibly due to genetic variation in the Cuban strains, which need to be sequenced to reduce the number of untyped strains. Unfortunately, the lab currently does not have the capacity to do sequence analysis. Some limitations of the study need to be addressed. The short time period of the investigation did not allow enough
samples to be collected to do seasonal studies. Correlations of pathogens with clinical symptoms and symptom severity also need to be investigated. Nevertheless, the investigation provides important data on the presence of enteric pathogens in children less than five years old who were hospitalized with diarrhea. The results of the present study emphasize the importance of RoV as a major cause of gastroenteritis in hospitalized Cuban children under five years old. The introduction of an RoV vaccine in the EPI should reduce the prevalence of infection and result in important changes in rotavirus epidemiology. Continuing RoV surveillance before and after vaccine introduction will provide information about the range of circulating strains in the country. Acknowledgments The authors would like to thank the hospital and laboratory staff at each of the paediatric hospitals and Pedro Kourı´ Institute of Tropical Medicine for their invaluable support of this study. Conflict of interest of interest.
The authors declare that they have no conflict
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