The surveillance was conducted at 3 sentinel hospitals located in the western province of Kinshasa (Centre Hospitalier Pédiatrique de Kalembelembe, CHPK; Centre Hospitalier de Kingasani, CHK) and the southern province of Katanga (Hôpital Général Sendwe, HGS) in the town of Lubumbashi (Fig. 1). These 3 hospitals are representative provincial hospitals, caring for most of the pediatric patients in the area. The surveillance of rotavirus started in DRC in 2009; August in CHPK, November in CHK and December in HGS.

From August 2009 to June 2012, stool specimens were collected within 48 hours of hospital admission, from children <5 years of age with a diagnosis of gastroenteritis. The WHO case definition of gastroenteritis was the occurrence of at least 3 looser than normal or watery stools in a 24-hour period and/or 2 or more episodes of vomiting unexplained by other reasons.¹¹

Samples collected from each sentinel site were first screened for group A rotavirus antigen by enzyme immunoassay (EIA), as described below and aliquots stored at −20°C before transportation to the Institut National de Recherche Biomedicale (INRB) where EIA results were confirmed by repeat EIA. Before 2011, the genotyping assays were performed using multiplex reverse transcription polymerase chain reaction (RT-PCR) at the rotavirus Regional Reference Laboratory in South Africa, Medical Research Council (MRC)/ Diarrhoeal Pathogens Research Unit, University of Limpopo, Medunsa Campus. Since 2011, the genotyping assays have been performed by the INRB laboratory, with the support of the SURVAC Project, using a multiplex RT-PCR technique. Samples subjected to genotyping were subsequently confirmed at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, for quality control.

A 10% suspension of specimens was prepared and analyzed using a sandwich EIA for detection of group A rotavirus in fecal specimens (ProSpecT Rotavirus Microplate Assay (Oxoid, Ltd., Basingstoke, Hampshire, United Kingdom) according to manufacturer’s instructions.

Viral RNA from rotavirus-positive samples was extracted from 140 μL of 10% stool suspension using a QIAamp viral RNA Mini kit (Qiagen, Inc., Valencia, CA) according to manufacturer’s instructions and extracted RNAs were stored at −80°C.

The RNA extracts first were subjected to RT-PCR. Two gene regions, VP7 (896 bp) and VP4 (876 bp), were reverse transcribed and amplified with primers 9Con1-L/VP7-R and Con3/Con2, respectively.^12,13 Reverse transcription of double strand RNA (dsRNA) was carried out with the OneStep RT-PCR Kit (Qiagen, Inc.). After 5 min denaturation at 97°C, the RNA was mixed with kit reagents and incubated at 42°C for 30 minutes to obtain complementary DNA (cDNA), immediately followed by the PCR reaction. These first round RT-PCR products then were used in a semi-nested, multiplexed PCR to identify G-types (G1, G2, G3, G4, G9 and G12) and P-types (P[4], P[6], P[8]).^12,13 All PCR products were identified by electrophoresis in 2% agarose gels containing 10% Gel Red (Biotium, Hayward, CA) and visualized under ultraviolet illumination.

Between August 2009 and June 2012, a total of 1614 children <5 years of age with diarrhea were enrolled at the 3 sentinel sites in DRC. They were distributed as follows: Kalembelembe Hospital 828/1614 (51%), Kingasani Hospital 628/1614 (39%) and Sendwe Hospital 159/1614 (10%). During the study period, rotavirus infection occurred year round but was more common from March through November, with a peak from April to August (Fig. 2). This corresponds to the dry season in these parts of the country.

During the study period, 1614 stool samples were tested for group A rotavirus antigen by EIA and rotavirus was identified in 990 (61%). The age distribution of rotavirus cases showed that 22% of rotavirus infections occurred during the first 6 months of life and peaked in the 6–11 month age group (54.3%; Table 1). Among children with rotavirus infection, 3 quarters of the patients (76.3%) were hospitalized before 12 months of age. Only 2% cases were observed in the 24–59 month age group.

Genotyping analyses were carried out to determine the type and distribution of rotavirus strains in the 3 sentinel sites. Of the 990 rotavirus-positive stool samples, 330 samples (33%) were characterized for both VP7 and VP4 types using multiplex RT-PCR. Among the G-types examined, 315 (95.5%) could be G-typed. The prevalence of G2, G1 and G8 strains was found to be 37%, 27% and 9%, respectively (Table 2). Among the P-types, 314 (95.2%) could be P-typed, the most common strains were P[6] (52%) followed by P[8] (22%) and P[4] (17%). Of all the strains that could be both G- and P-typed (299 strains, 90.6%), the most prevalent G-P combination was G2P[6] (18%) followed by G2P[4] (14%), G1P[8] (13%) and G1P[6] (11%). The uncommon G6P[6] strain was found and represented 5% of typed strains. Among the strains characterized in this study, 16 (5%) and 15 (5%) were P and G nontypeable, respectively (Table 2).

Temporal distribution of genotype combinations showed that G1P[8] and G1P[6] strains circulated during the entire study period, while G8P[6] and G12P[6] were not found in 2012 (Fig. 3). Mixed infection, defined as a sample containing multiple G and/or P-types, occurred in 44 cases (13.3%). Within these mixed infections, P[6] was involved in 25 cases (56%), of which 10 cases (40%) were P[6] in combination with G8 and G12 (Table 3). The age distribution for the “mixed infection” cases was proportional to the number of cases per age group and thus mixed genotype infections were detected at the same rate in all age groups.