Genomic Analysis of G2P[4] Group A Rotaviruses in Zambia Reveals Positive Selection in Amino Acid Site 7 of Viral Protein 3.

The G2P[4] genotype is among the rotavirus strains that circulate commonly in humans. Several countries have reported its immediate upsurge after the introduction of rotavirus vaccination, raising concern about sub-optimal vaccine effectiveness against this genotype in the long term. This study aimed to gain insight into the evolution of post-vaccine Zambian G2P[4] group A rotavirus (RVA) strains and their overall genetic make-up by analysis of sequence alignments at the amino acid (AA) level. Twenty-nine Zambian G2P[4] rotavirus strains were subjected to whole-genome sequencing using the Illumina MiSeq® platform. All the strains exhibited the typical DS-1-like genotype constellation, and the nucleotide sequences of the 11 genome segments showed high nucleotide similarities (>97%). Phylogenetic analyses together with representative global G2P[4] RVA showed that Zambian strains clustered into human lineages IV (for VP2, VP4, VP7, NSP1, and NSP5), V (for VP1, VP3, VP6, NSP2, and NSP3), and XXIII (for NSP4). The AA differences between the lineages where the study strains clustered and lineages of global reference strains were identified and analyzed. Selection pressure analysis revealed that AA site seven in the Viral Protein 3 (VP3) genome segment was under positive selection. This site occurs in the region of intrinsic disorder in the VP3 protein, and Zambian G2P[4] strains could potentially be utilizing this intrinsically disordered region to survive immune pressure. The Zambian G2P[4] strains from 2012 to 2016 comprised the G2P[4] strains that have been circulating globally since the early 2000s, highlighting the epidemiological fitness of these contemporary G2P[4] strains. Continuous whole-genome surveillance of G2P[4] strains remains imperative to understand their evolution during the post-vaccination period.

Whole Genome Analysis of Human Rotaviruses Reveals Single Gene Reassortant Rotavirus Strains in Zambia.

Rotarix® vaccine was implemented nationwide in Zambia in 2013. In this study, four unusual strains collected in the post-vaccine period were subjected to whole genome sequencing and analysis. The four strains possessed atypical genotype constellations, with at least one reassortant genome segment within the constellation. One of the strains (UFS-NGS-MRC-DPRU4749) was genetically and phylogenetically distinct in the VP4 and VP1 gene segments. Pairwise analyses demonstrated several amino acid disparities in the VP4 antigenic sites of this strain compared to that of Rotarix®. Although the impact of these amino acid disparities remains to be determined, this study adds to our understanding of the whole genomes of reassortant strains circulating in Zambia following Rotarix® vaccine introduction.

Molecular Characterisation of a Rare Reassortant Porcine-Like G5P[6] Rotavirus Strain Detected in an Unvaccinated Child in Kasama, Zambia.

A human-porcine reassortant strain, RVA/Human-wt/ZMB/UFS-NGS-MRC-DPRU4723/2014/G5P[6], was identified in a sample collected in 2014 from an unvaccinated 12 month old male hospitalised for gastroenteritis in Zambia. We sequenced and characterised the complete genome of this strain which presented the constellation: G5-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The genotype A8 is often observed in porcine strains. Phylogenetic analyses showed that VP6, VP7, NSP2, NSP4, and NSP5 genes were closely related to cognate gene sequences of porcine strains (e.g., RVA/Pig-wt/CHN/DZ-2/2013/G5P[X] for VP7) from the NCBI database, while VP1, VP3, VP4, and NSP3 were closely related to porcine-like human strains (e.g., RVA/Human-wt/CHN/E931/2008/G4P[6] for VP1, and VP3). On the other hand, the origin of the VP2 was not clear from our analyses, as it was not only close to both porcine (e.g., RVA/Pig-tc/CHN/SWU-1C/2018/G9P[13]) and porcine-like human strains (e.g., RVA/Human-wt/LKA/R1207/2009/G4P[6]) but also to three human strains (e.g., RVA/Human-wt/USA/1476/1974/G1P[8]). The VP7 gene was located in lineage II that comprised only porcine strains, which suggests the occurrence of independent porcine-to-human reassortment events. The study strain may have collectively been derived through interspecies transmission, or through reassortment event(s) involving strains of porcine and porcine-like human origin. The results of this study underline the importance of whole-genome characterisation of rotavirus strains and provide insights into interspecies transmissions from porcine to humans.

Risk Factors Associated With Increased Mortality From Intussusception in African Infants.

OBJECTIVES: Morbidity and mortality from intussusception, the leading cause of bowel obstruction in infants, is higher in Africa than in other regions of the world, but the reasons have not been well examined. We sought to identify risk and protective factors associated with death or intestinal resection following intussusception. METHODS: Infants with intussusception from 7 sub-Saharan African countries (Ethiopia, Ghana, Kenya, Malawi, Tanzania, Zambia, and Zimbabwe) were enrolled through active, hospital-based surveillance from February 2012 to December 2016. We examined demographic, clinical, and socioeconomic factors associated with death or intestinal resection following intussusception, using multivariable logistic regression. RESULTS: A total of 1017 infants <1 year of age with intussusception were enrolled. Overall, 13% of children (133/1017) died during the hospitalization, and 48% (467/966) required intestinal resection. In multivariable analyses, female sex [odds ratio (OR) 1.8, 95% confidence interval (CI) 1.2-3.3], longer duration of symptoms before presentation (OR 1.1; 95% CI 1.0-1.2), and undergoing intestinal resection (OR 3.4; 95% CI 1.9-6.1) were associated with death after intussusception. Diagnosis by ultrasound or enema (OR 0.4; 95% CI 0.3-0.7), and employment of a household member (OR 0.7; 95% CI 0.4-1.0) were protective against intestinal resection. CONCLUSIONS: Delays in hospital presentation and female sex were significantly associated with death, whereas higher socioeconomic status and availability of radiologic diagnosis reduced likelihood of undergoing resection. Efforts should be intensified to improve the awareness, diagnosis, and management of intussusception in sub-Saharan African countries to reduce morbidity and mortality from intussusception in these resource-limited settings.

Multicountry Analysis of Spectrum of Clinical Manifestations of Children <5 Years of Age Hospitalized with Diarrhea.

After introduction of rotavirus vaccine, other pathogens might become leading causes of hospitalizations for severe diarrhea among children <5 years of age. Our study in 33 hospitals in 7 countries found acute gastroenteritis accounted for most (84%) reported hospitalizations of children with diarrhea. Bloody and persistent diarrhea each accounted for <1%.

Declines in Pneumonia and Meningitis Hospitalizations in Children Under 5 Years of Age After Introduction of 10-Valent Pneumococcal Conjugate Vaccine in Zambia, 2010-2016.

BACKGROUND: Pneumococcus is a leading cause of pneumonia and meningitis. Zambia introduced a 10-valent pneumococcal conjugate vaccine (PCV10) in July 2013 using a 3-dose primary series at ages 6, 10, and 14 weeks with no booster. We evaluated the impact of PCV10 on meningitis and pneumonia hospitalizations. METHODS: Using hospitalization data from first-level care hospitals, available at the Ministry of Health, and from the largest pediatric referral hospital in Lusaka, we identified children aged <5 years who were hospitalized with pneumonia or meningitis from January 2010-December 2016. We used time-series analyses to measure the effect of PCV10 on monthly case counts by outcome and age group (<1 year, 1-4 years), accounting for seasonality. We defined the pre- and post-PCV10 periods as January 2010-June 2013 and July 2014-December 2016, respectively. RESULTS: At first-level care hospitals, pneumonia and meningitis hospitalizations among children aged <5 years accounted for 108 884 and 1742 admissions in the 42 months pre-PCV10, respectively, and 44 715 and 646 admissions in the 30 months post-PCV10, respectively. Pneumonia hospitalizations declined by 37.8% (95% confidence interval [CI] 21.4-50.3%) and 28.8% (95% CI 17.7-38.7%) among children aged <1 year and 1-4 years, respectively, while meningitis hospitalizations declined by 72.1% (95% CI 63.2-79.0%) and 61.6% (95% CI 50.4-70.8%), respectively, in these age groups. In contrast, at the referral hospital, pneumonia hospitalizations remained stable and a smaller but significant decline in meningitis was observed among children aged 1-4 years (39.3%, 95% CI 16.2-57.5%). CONCLUSIONS: PCV10 introduction was associated with declines in meningitis and pneumonia hospitalizations in Zambia, especially in first-level care hospitals.

Diversity of rotavirus strains circulating in children under five years of age who presented with acute gastroenteritis before and after rotavirus vaccine introduction, University Teaching Hospital, Lusaka, Zambia, 2008-2015.

BACKGROUND: Following the introduction of rotavirus vaccine into the routine immunization schedule, the burden of rotavirus disease has significantly reduced in Zambia. Although rotavirus vaccines appear to confer good cross-protection against both vaccine and non-vaccine strains, concerns about strain replacement following vaccine implementation remain. We describe the diversity of the circulating rotavirus strains before and after the Rotarix® vaccine was introduced in Lusaka from January 2012. METHODS: Under five children were enrolled through active surveillance at University Teaching Hospital using a standardized WHO case investigation form. Stool samples were collected from children who presented with ≥3 loose stool in 24 h and were admitted to the hospital for acute gastroenteritis as a primary illness. Samples were tested for group A rotavirus antigen enzyme-linked immunosorbent assay. Randomly selected rotavirus positive samples were analysed by reverse transcription polymerase chain reaction for G and P genotyping and and Nucleotide sequencing was used to confirm some mixed infections. RESULTS: A total of 4150 cases were enrolled and stool samples were collected from 4066 (98%) children between 2008 and 2011, before the vaccine was introduced. Rotavirus antigen was detected in 1561/4066 (38%). After vaccine introduction (2012 to 2015), 3168 cases were enrolled, 3092 (98%) samples were collected, and 977/3092 (32%) were positive for rotavirus. The most common G and P genotype combinations before vaccine introduction were G1P[8] (49%) in 2008; G12P[6] (24%) and G9P[8] (22%) in 2009; mixed rotavirus infections (32%) and G9P[8] (20%) in 2010, and G1P[6] (46%), G9P[6] (16%) and mixed infections (20%) in 2011. The predominant strains after vaccine introduction were G1P[8] (25%), G2P[4] (28%) and G2P[6] (23%) in 2012; G2P[4] (36%) and G2P[6] (44%) in 2013; G1P[8] (43%), G2P[4] (9%), and G2P[6] (24%) in 2014, while G2P[4] (54%) and G2P[6] (20%) continued to circulate in 2015. CONCLUSION: These continual changes in the predominant strains suggest natural secular variation in circulating rotavirus strains post-vaccine introduction. These findings highlight the need for ongoing surveillance to continue monitoring how vaccine use affects strain evolution over a longer period of time and assess any normal seasonal fluctuations of the rotavirus strains.

Review of Naturally Occurring Intussusception in Young Children in the WHO African Region prior to the Era of Rotavirus Vaccine Utilization in the Expanded Programme of Immunization.

Rotavirus vaccines, Rotarix and RotaTeq, are increasingly being introduced in national immunization programmes in Africa to prevent severe dehydrating acute gastroenteritis. A low-level risk of intussusception has been associated with rotavirus vaccines. We reviewed published data on intussusception in children <2 years of age in Africa. PubMed electronic database search was used to retrieve papers published on intussusception. The search was further refined to identify surveillance reports and case series conducted from 1980 to 2014, with at least 25 cases. The initial search identified 34 studies, and the refined search yielded 16. Intussusception occurred naturally in infants 2-4 months and peaked around 5-8 months of age. Delayed presentation was common and required surgical intervention in 87% (1008 of 1158) of cases with a high CFR, 10-33.7%. In African children, intussusception has been reported infrequently at a young age when the first dose of rotavirus vaccine is administered.

Safety and immunogenicity of a live attenuated pentavalent rotavirus vaccine in HIV-exposed infants with or without HIV infection in Africa.

OBJECTIVE: Although many HIV-infected (HIV+) and HIV-exposed but uninfected (HEU) infants have received live rotavirus vaccines since the WHO recommended universal administration of these vaccines to infants, there has been limited prospective information on their safety and immunogenicity in either group of infants. DESIGN/METHODS: We performed a randomized, double-blinded, placebo-controlled trial of the safety and immunogenicity of oral pentavalent rotavirus vaccine (RV5) administered to HIV+ and HEU infants in four African countries. Ninety-three percent of HIV+ infants were receiving antiretroviral therapy prior to vaccination. Participants were followed for safety. Immune responses were measured 14 days after three doses of RV5, including serum antirotavirus neutralizing and IgA antibodies, IgA antibody in stool, and antirotavirus memory B and T-cell FluoroSpot. Shedding of RV5 in stool was monitored. RESULTS: A total of 76 HIV+ and 126 HEU infants were enrolled from 2009 to 2013. No significant differences were found in adverse event rates, including grade 3 events, between RV5 and placebo recipients, for either HIV+ or HEU infants. The proportion of antirotavirus IgA responders (at least three-fold increase from baseline) after RV5 administration was 81% in both HIV+ and HEU infants, which was approximately 2.5-fold higher than in placebo recipients (P < 0.001). Neutralizing antibody responses to three of five serotypes were significantly higher after RV5 regardless of HIV status, and those of HIV+ infants were equal or greater than responses of HEU infants to all five serotypes. Only one HIV+ RV5 recipient had RV5 isolated from stool. CONCLUSION: RV5 was immunogenic in both HIV+ and HEU infants and no safety signals were observed.

Impact of Rotavirus Vaccination on Diarrheal Hospitalizations in Children Aged <5 Years in Lusaka, Zambia.

BACKGROUND: Monovalent rotavirus vaccine was introduced in the routine public health immunization program in Lusaka, Zambia, in January 2012 and was rolled out countrywide in November 2013. We examined the effect of rotavirus vaccination on hospitalization for all-cause acute gastroenteritis (AGE) and rotavirus-specific AGE at a large referral hospital in Lusaka. METHODS: Data were derived from ongoing hospital-based AGE surveillance from January 2009 to December 2014. Pre-rotavirus vaccine introduction (2009-2011) and post-rotavirus vaccine introduction (2013-2014) periods were compared for annual changes in hospitalizations for AGE and rotavirus; 2012 was excluded as a transition year. Hospital administrative discharge data were used to compare trends in all-cause diarrhea discharges and in-hospital diarrhea deaths captured by HIMS pre- and post-rotavirus vaccine introduction. RESULTS: Between January 2009 and December 2014, 5937 children <5 years of age presenting with AGE had their stools collected and tested for rotavirus by enzyme immunoassay. The rotavirus positivity rate declined from 40.1% (449/1121) in prevaccine years to 30.2% (250/828;P< .001) in 2013 and 24.7% (157/635;P< .001) in 2014. The greatest reduction was noted in infants, with the rotavirus positivity rate in this age group declining from 40.9% in prevaccine years to 34.0% (P= .009) in 2013 and 26.2% (P< .001) in 2014. Following rotavirus vaccine introduction, seasonal peaks of rotavirus and all-cause AGE were dwarfed. From HIMS data, compared to the prevaccine era, reductions of 18%-29% in all-cause diarrhea hospitalizations and 27%-33% in-hospital diarrhea deaths among children <1 year of age were observed in 2013 and 2014. CONCLUSIONS: We observed a significant reduction in AGE-associated in-hospital morbidity and mortality following rotavirus vaccine introduction. The greatest reduction was seen in infants <1 year who accounted for 84.4% of rotavirus hospitalizations prior to vaccine introduction.

Zinc absorption from biofortified maize meets the requirements of young rural Zambian children.

BACKGROUND: The zinc content of maize, a major global food staple, is generally insufficient alone to meet the requirements of young children. OBJECTIVES: The primary objective of this study was to determine whether substitution of biofortified maize (34 µg zinc/g grain) for control maize (21 µg zinc/g) was adequate to meet zinc physiologic requirements in young children for whom maize was the major food staple. A secondary objective was to compare total daily zinc absorption when maize flour was fortified with zinc oxide to a total concentration of 60 µg zinc/g. METHODS: Participants included 60 rural Zambian children with a mean age of 29 mo who were randomly assigned to receive 1 of 3 maize types (control, biofortified, or fortified) all of which were readily consumed (>100 g on 1 d). Total daily zinc intake (from maize and low-zinc relish) was determined from duplicate diet collections. Multiplication by fractional absorption of zinc, measured by a dual isotope ratio technique, determined the total daily zinc absorption on the day the test meals were given. RESULTS: The mean ± SD total daily zinc intake (milligrams per day) from the biofortified maize (5.0 ± 2.2) was higher (P < 0.0001) than for the control maize (2.3 ± 0.9). Intake of zinc from the fortified maize (6.3 ± 2.6) did not differ from the biofortified maize. Fractional absorption of zinc from control maize (0.28 ± 0.10) did not differ from the biofortified maize (0.22 ± 0.06). Total daily absorption of zinc (milligrams per day) from the biofortified maize (1.1 ± 0.5) was higher (P = 0.0001) than for the control maize (0.6 ± 0.2), but did not differ from the fortified maize (1.2 ± 0.4). CONCLUSIONS: These results indicate that feeding biofortified maize can meet zinc requirements and provide an effective dietary alternative to regular maize for this vulnerable population. This trial was registered at clinicaltrials.gov as NCT02208635.

Intussusception incidence rates in 9 Zambian hospitals, 2007-2011: prerotavirus vaccine introduction.

BACKGROUND: Intussusception, a rare adverse event associated with rotavirus vaccines in some settings, is a common cause of intestinal obstruction in infants and toddlers globally with a peak age of 4-6 months. This age group may overlap with the extended age of administering rotavirus vaccine. METHODS: A retrospective (January 2007 to June 2009) and prospective (July 2009 to June 2012) survey was conducted in 9 Zambian hospitals. Children between 0 and 24 months who were operated on for intestinal obstruction/intussusception were identified in theatre log books. In the latter part of the survey, patients were recruited prospectively. Demographic, clinical and surgical data from hospital files were collected for each patient. RESULTS: One-hundred and five children were identified to have undergone surgery for intussusceptions. Many were boys 57.6% (57/99). Of those with complete data, intussusception was common in infants 86.9% (86/99) and many children (68.0%) were between 3 and 8 months of age with a peak age of 5-6 months. Lusaka had the highest number of children with intussusception with an estimated annual incidence rate of 12/100,000 in children <2 years of age. The overall case fatality rate was very high 33.7% (31/92). CONCLUSION: Intussusception was common in infants with a peak age of 5-6 months, and of particular concern is the group of 2-4 months the age of rotavirus vaccination. The estimated incidence rate of 12/100,000 is an underestimate as many cases may not present for care. The high case fatality rate of 33.7% is due to both delayed presentation and diagnosis in hospital.

Burden and epidemiology of rotavirus diarrhea in selected African countries: preliminary results from the African Rotavirus Surveillance Network.

Severe rotavirus diarrhea in children <5 years of age is a major public health problem; however, limited regional and country specific data on rotavirus disease burden are available from sub-Saharan Africa. In June 2006, the World Health Organization Regional Office for Africa initiated rotavirus surveillance in selected African countries. With use of standardized methodology developed by the World Health Organization, children <5 years of age who were hospitalized with severe diarrhea were enrolled, and stool specimens were collected for detection of rotavirus strains with use of a commercial enzyme immunoassay. Rotavirus strains were further characterized for G and P types with use of a reverse-transcriptase polymerase chain reaction. From June 2006 through December 2008, rotavirus surveillance was established at 14 sites in 11 African countries. Of 5461 stool samples collected from children enrolled in 8 countries with 1 or 2 complete years of data, 2200 (40%) were positive for rotavirus. Ninety percent of all rotavirus hospitalizations occurred among children aged 3-12 months. Predominant types included G1P[8] (21%), G2P[4] (7%), and P [8] (29%); however, unusual types were also detected, including G8P[6] (5%), G8P[8] (1%), G12P[6] (1%), and G12P[6] (1%). A high percentage of mixed rotavirus infections was also detected. These preliminary results indicate that rotavirus is a major cause of severe diarrheal disease in African children.