Norovirus infections in young children in Lusaka Province, Zambia: clinical characteristics and molecular epidemiology.

BACKGROUND: The burden, clinical features, and molecular epidemiology of norovirus infection in young children in southern Africa are not well defined. METHODS: Using data from a health facility-based surveillance study of children <5 years in Lusaka Province, Zambia presenting with diarrhea, we assessed the burden of norovirus infection. A convenience sample of 454 stool specimens was tested for norovirus using reverse-transcriptase polymerase chain reaction (RT-PCR). RT-PCR positive samples underwent additional nucleotide sequencing for genogroup and genotype identification. Clinical features and severity of diarrheal illnesses were compared between norovirus-positive and -negative subjects using Chi-squared and t-tests. RESULTS: Norovirus was detected in 52/454 (11.5%) specimens tested. Abdominal pain, fever, and vomiting were the most common presenting features in norovirus-associated illnesses. However, there were no significant differences in the clinical features of norovirus-positive compared to norovirus-negative illnesses. Of 43 isolates that were available for sequencing, 31 (72.1%) were genogroup II (GII) and 12 (27.9%) were genogroup I (GI). The distribution of genotypes was diverse. CONCLUSIONS: Noroviruses were detected in approximately 10% of young children with diarrhea in the Lusaka Province of Zambia, with GII representing the majority of infections. These findings support the role of norovirus in symptomatic diarrhea disease in Africa. Further studies are needed to confirm these observations and to evaluate prevention strategies.

A Preliminary Assessment of Rotavirus Vaccine Effectiveness in Zambia.

BACKGROUND: Diarrhea is the third leading cause of child death in Zambia. Up to one-third of diarrhea cases resulting in hospitalization and/or death are caused by vaccine-preventable rotavirus. In January 2012, Zambia initiated a pilot introduction of the Rotarix live, oral rotavirus vaccine in all public health facilities in Lusaka Province. METHODS: Between July 2012 and October 2013, we conducted a case-control study at 6 public sector sites to estimate rotavirus vaccine effectiveness (VE) in age-eligible children presenting with diarrhea. We computed the odds of having received at least 1 dose of Rotarix among children whose stool was positive for rotavirus antigen (cases) and children whose stool was negative (controls). We adjusted the resulting odds ratio (OR) for patient age, calendar month of presentation, and clinical site, and expressed VE as (1 - adjusted OR) × 100. RESULTS: A total of 91 rotavirus-positive cases and 298 rotavirus-negative controls who had under-5 card-confirmed vaccination status and were ≥6 months of age were included in the case-control analysis. Among rotavirus-positive children who were age-eligible to be vaccinated, 20% were hospitalized. Against rotavirus diarrhea of all severity, the adjusted 2-dose VE was 26% (95% confidence interval [CI], -30% to 58%) among children ≥6 months of age. VE against hospitalized children ≥6 months of age was 56% (95% CI, -34% to 86%). CONCLUSIONS: We observed a higher point estimate for VE against increased severity of illness compared with milder disease, but were not powered to detect a low level of VE against milder disease.

Causes of stillbirth, neonatal death and early childhood death in rural Zambia by verbal autopsy assessments.

OBJECTIVES: To describe specific causes of the high rates of stillbirth, neonatal death and early child childhood death in Zambia. METHODS: We conducted a household-based survey in rural Zambia. Socio-demographic and delivery characteristics were recorded, alongside a maternal HIV test. Verbal autopsy questionnaires were administered to elicit mortality-related information and independently reviewed by three experienced paediatricians who assigned a cause and contributing factor to death. For this secondary analysis, deaths were categorized into: stillbirths (foetal death ≥28 weeks of gestation), neonatal deaths (≤28 days) and early childhood deaths (>28 days to <2 years). RESULTS: Among 1679 households, information was collected on 148 deaths: 34% stillbirths, 26% neonatal and 40% early childhood deaths. Leading identifiable causes of stillbirth were intrauterine infection (26%) and birth asphyxia (18%). Of 32 neonatal deaths, 38 (84%) occurred within the first week of life, primarily because of infections (37%) and prematurity (34%). The majority of early childhood deaths were caused by suspected bacterial infections (82%). HIV prevalence was significantly higher in mothers who reported an early childhood death (44%) than mothers who did not (17%; P < 0.01). Factors significantly associated with mortality were lower socio-economic status (P < 0.01), inadequate water or sanitation facilities (P < 0.01), home delivery (P = 0.04) and absence of a trained delivery attendant (P < 0.01). CONCLUSION: We provide community-level data about the causes of death among children under 2 years of age. Infectious etiologies for mortality ranked highest. At a public health level, such information may have an important role in guiding prevention and treatment strategies to address perinatal and early childhood mortality.

Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single-genome amplification and sequencing.

Accurate identification of the transmitted virus and sequences evolving from it could be instrumental in elucidating the transmission of human immunodeficiency virus type 1 (HIV-1) and in developing vaccines, drugs, or microbicides to prevent infection. Here we describe an experimental approach to analyze HIV-1 env genes as intact genetic units amplified from plasma virion RNA by single-genome amplification (SGA), followed by direct sequencing of uncloned DNA amplicons. We show that this strategy precludes in vitro artifacts caused by Taq-induced nucleotide substitutions and template switching, provides an accurate representation of the env quasispecies in vivo, and has an overall error rate (including nucleotide misincorporation, insertion, and deletion) of less than 8 x 10(-5). Applying this method to the analysis of virus in plasma from 12 Zambian subjects from whom samples were obtained within 3 months of seroconversion, we show that transmitted or early founder viruses can be identified and that molecular pathways and rates of early env diversification can be defined. Specifically, we show that 8 of the 12 subjects were each infected by a single virus, while 4 others acquired more than one virus; that the rate of virus evolution in one subject during an 80-day period spanning seroconversion was 1.7 x 10(-5) substitutions per site per day; and that evidence of strong immunologic selection can be seen in Env and overlapping Rev sequences based on nonrandom accumulation of nonsynonymous mutations. We also compared the results of the SGA approach with those of more-conventional bulk PCR amplification methods performed on the same patient samples and found that the latter is associated with excessive rates of Taq-induced recombination, nucleotide misincorporation, template resampling, and cloning bias. These findings indicate that HIV-1 env genes, other viral genes, and even full-length viral genomes responsible for productive clinical infection can be identified by SGA analysis of plasma virus sampled at intervals typical in large-scale vaccine trials and that pathways of viral diversification and immune escape can be determined accurately.

Causes of morbidity among HIV-infected children on antiretroviral therapy in primary care facilities in Lusaka, Zambia.

OBJECTIVES: To describe the pattern of incident illness in children after initiation of antiretroviral therapy (ART) in a large public health sector programme in Lusaka, Zambia. METHODS: Systematic chart review to retrospectively extract data from medical records of children (i.e. <15 years) initiating ART in the Lusaka, Zambia public sector. Incident conditions were listed separately and then grouped according to broad categories. Predictors for incident diagnoses were determined using univariate and multivariable analysis. RESULTS: Between May 2004 and June 2006, 1705 HIV-infected children initiated ART. Of these, 1235 (72%) had their medical records reviewed. Median age at ART initiation was 77 months and 554 (45%) were females. Eight hundred and forty-one (68%) children had an incident condition during this period, with a median time of occurrence of 64 days from ART initiation. Twenty-eight incident conditions were documented. When categorized, the most common were mucocutaneous conditions [incidence rate (IR): 70.6 per 100 child-years, 95% CI: 64.5-77.2] and upper respiratory tract infection (IR: 70.1 per 100 child-years; 95% CI: 64.0-76.7). Children with severe immunosuppression (i.e. CD4 < 10%) were more likely to develop lower respiratory tract infection (16.3%vs. 10.2%; P = 0.003) and mucocutaneous conditions (43.9% vs. 35.3%; P = 0.005) than those with CD4 > or = 10%. CONCLUSION: There is a high incidence of new illness after ART initiation, emphasizing the importance of close monitoring during this period. Early initiation of ART and use of antimicrobial prophylaxis may also help to reduce the occurrence of such co-morbidities.

Six-month hemoglobin concentration and its association with subsequent mortality among adults on antiretroviral therapy in Lusaka, Zambia.

Little is known about changes in hemoglobin concentration early in the course of antiretroviral therapy and its subsequent relation to survival. We analyzed data for 40,410 HIV-infected adults on antiretroviral therapy in Lusaka, Zambia. Our main exposure of interest was 6-month hemoglobin, but we stratified our analysis by baseline hemoglobin to allow for potential effect modification. Patients with a 6-month hemoglobin <8.5 g/dL, regardless of baseline, had the highest hazard for death after 6 months (hazard ratio: 4.5; 95% confidence interval: 3.3 to 6.3). Future work should look to identify causes of anemia in settings such as ours and evaluate strategies for more timely diagnosis and treatment.

Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection.

Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4(+) T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12-20 mo, viruses exhibited concentrated mutations at 17-34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.

Ulceroglandular tularemia in a nonendemic area.

Two patients present with the abrupt onset of fever, malaise, anorexia, fatigue, progressive skin lesions and lymphadenitis. These patients represent two of the six cases of tularemia reported in Alabama over the last decade. The cases illustrate how mode of acquisition (direct versus vector-mediated) influences the clinical manifestations of ulceroglandular tularemia. In addition, a brief review of the epidemiology, differential diagnosis, clinical manifestations, and treatment of tularemia is provided.