Improving influenza surveillance in sub-Saharan Africa.

PROBLEM: Little is known about the burden of influenza in sub-Saharan Africa. Routine influenza surveillance is key to getting a better understanding of the impact of acute respiratory infections on sub-Saharan African populations. APPROACH: A project known as Strengthening Influenza Sentinel Surveillance in Africa (SISA) was launched in Angola, Cameroon, Ghana, Nigeria, Rwanda, Senegal, Sierra Leone and Zambia to help improve influenza sentinel surveillance, including both epidemiological and virological data collection, and to develop routine national, regional and international reporting mechanisms. These countries received technical support through remote supervision and onsite visits. Consultants worked closely with health ministries, the World Health Organization, national influenza laboratories and other stakeholders involved in influenza surveillance. LOCAL SETTING: Influenza surveillance systems in the target countries were in different stages of development when SISA was launched. Senegal, for instance, had conducted virological surveillance for years, whereas Sierra Leone had no surveillance activity at all. RELEVANT CHANGES: Working documents such as national surveillance protocols and procedures were developed or updated and training for sentinel site staff and data managers was organized. LESSONS LEARNT: Targeted support to countries can help them strengthen national influenza surveillance, but long-term sustainability can only be achieved with external funding and strong national government leadership.

High human cytomegalovirus loads and diverse linked variable genotypes in both HIV-1 infected and exposed, but uninfected, children in Africa.

Human cytomegalovirus, HCMV, was analysed using real-time quantitative PCR in symptomatic or asymptomatic pediatric cohorts from HIV-1 infected, exposed (HIV-1+ mothers), or uninfected groups in Zambia, an HIV-1/AIDS endemic region of Africa. HCMV infections were identified in 94% samples from HIV-1+ respiratory pediatric mortalities, 50% with high DNA loads of 10(3)-10(8) copies/10(6) cells. In comparison, HCMV viremia with high DNA loads, indicative of acute infections, were in 10% hospitalised febrile infants, with 50% HIV-1+. Whereas high sera loads were in 1% of asymptomatic infants, with 2% HIV-1+, and higher levels in both HIV-1 infected or exposed, but negative infants. All 8 linked-hypervariable glycoprotein gN-gO genotypes were shown, including identification of a new gN4d group with gO5 linkage (previously only Merlin reference strain), and samples with multiple infections. Overall, this shows global genotypes in Africa (unlike some herpesviruses) and acute pediatric HCMV infections in both HIV-1+ plus exposed, but uninfected infants, an emerging group.

Diverse genotypes of Kaposi's sarcoma associated herpesvirus (KSHV) identified in infant blood infections in African childhood-KS and HIV/AIDS endemic region.

Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) has been associated with several neoplasias, including childhood endemic Kaposi's sarcoma (KS). It is possible that strain genotypes could contribute to the differences in regional presentation (mainly sub-Saharan Africa), childhood infection, lack of male sex bias, distinct disseminated forms and rapid fatality observed for childhood endemic KS. Early studies, at the advent of the HIV/AIDS epidemic, identified only the K1-A5 genotype in childhood KS biopsies as well as blood of a few HIV positive and negative febrile infants in Zambia, a highly endemic region. This current enlarged study analyses blood infections of 200 hospitalized infants (6-34 months age) with symptoms of fever as well as upper respiratory tract infection, diarrhoea, rash or rhinitis. KSHV and HIV viraemia and were prevalent in this group, 22% and 39%, respectively. Multiple markers at both variable ends of the genome (K1, K12, and K14.1/K15) were examined, showing diverse previously adult-linked genotypes (K1 A2, A5, B, C3, D, with K12 B1 and B2 plus K14.1/K15 P or M) detected in both HIV positive and negative infants, demonstrating little restriction on KSHV genotypes for infant/childhood transmission in a childhood endemic KS endemic region. This supports the interpretation that the acquisition of childhood KSHV infections and subsequent development of KS are due to additional co-factors.

Antigenic and genetic analyses of influenza B viruses isolated in Lusaka, Zambia in 1999.

Previous studies of the hemagglutinin (HA) genes of various influenza B virus isolates demonstrated the existence of two antigenically distinct virus lineages represented by B/Victoria/2/87 and B/Yamagata/16/88, respectively. Here, we investigated the antigenic and genetic characteristics of influenza B viruses isolated from children living in Lusaka, Zambia between January and May 1999. Antigenic analysis with chicken antiviral sera showed that all the Zambian isolates had the HA protein belonging to B/Yamagata/16/88-related lineage. Furthermore, phylogenetic analyses of the eight RNA segments performed by using the total or partial nucleotide sequences of the two representative Zambian strains (B/Lusaka/270/99 and B/Lusaka/432/99) as well as the previously reported sequences suggested that the Zambian viruses are closely related to the recently circulating reassortants represented by B/Shiga/T30/98 and B/Yamanashi/166/98 which acquired the genes coding for three polymerase proteins (PB2, PB1, and PA), HA, nucleoprotein, and matrix protein from a B/Yamagata/16/88-like parent and the gene encoding nonstructural proteins (NS1 and NS2) from a B/Guandong/8/93-like parent.

Detecting undetected HIV-1 variants in African children using degenerate polymerase chain reaction and sequence analyses.

PIP: This paper addresses HIV detection and its variation using degenerate polymerase chain reaction (PCR) and sequence analysis among African children. A total of 6 genomes of HIV-1 from AIDS patients, representing geographically distinct regions of Zambia and the major circulating genotypes (A, B, D, and O) were examined. Sequence multiple alignment was used to determine matches of HIV-1 and its variation, but none was suitable; hence, alignments were re-examined to design new primers. Standard PCR conditions were used with a modified cycling protocol. The new primers were tested on blood DNA from 53 HIV-negative, 60 HIV-positive febrile infants, and 9 HIV-positive children with Kaposi's sarcoma (KS). HIV status was determined in an enzyme-linked immunosorbent assay. 6 of the HIV-negative infants, 43 of those who were seropositive, and all children with KS were positive for HIV-1 proviral DNA. One PCR-positive, HIV-seronegative infant and the 9 KS samples were examined further using automated DNA sequencing and showed no evidence for contamination. Multiple alignment and phylogeny analyses using the Clustal program showed most similarity (94%) to the published adult Zambian strains. Variations in PCR detection rate in the HIV-positive infants (72%) and the KS children (100%) were brought about by confounding factors such as maternal HIV-1 infection during pregnancy without seroconversion, impaired B-cell function, and diversity of HIV-1 genotype circulating in Zambia.^ieng

Sequence analyses of human herpesvirus-8 strains from both African human immunodeficiency virus-negative and -positive childhood endemic Kaposi's sarcoma show a close relationship with strains identified in febrile children and high variation in the K1 glycoprotein.

Human herpesvirus-8 (HHV-8) DNA sequences have been identified in all forms of Kaposi's sarcoma (KS), a cancer found primarily in adult AIDS patients. We have identified HHV-8 strains in a rare human immunodeficiency virus (HIV)-negative form of KS, which is endemic in children in parts of sub-Saharan Africa. This was shown in Zambia, where we also had identified HHV-8 sequences in blood from HIV-negative febrile children without KS. In order to investigate the relationship of these Zambian strains to each other and to those from other forms of KS, we compared them to strains we have characterized from European AIDS KS (Denmark) and all published sequences from all forms of KS. Four distinct genomic regions were examined by PCR and sequencing: ORF26, ORF75, gH and K1. The results showed a distinct grouping of strains from both sets of Zambian children in all genomic regions studied, but which was most pronounced in the K1 glycoprotein gene. This gene was highly variable, encoding up to 25% amino acid sequence variation. In contrast, the Zambian groups were closely related to each other, with only 2% variation. Similar results were found in comparisons to the K1 sequences from HIV-positive febrile infants or KS children. The data raise the possibility that in areas where rare childhood endemic KS occurs, geographical variation in HHV-8 may relate to differences in virulence or transmission.

Characterization of VP6 subgroup, VP7 and VP4 genotype of rotavirus strains in Lusaka, Zambia.

In a previous study, rotavirus infection was determined in young children at the University Teaching Hospital in Lusaka. In this study, selected rotavirus strains were characterized by monoclonal antibody assay to the VP6 subgroup antigen present and by hybridization analysis of the VP7 and VP4 genes carried by the virus. The majority of the strains were characterized as a VP6 subgroup II, VP7 serotype G1 strain with a long electropherotype and bearing the VP4 P8 genotype. A further four minor rotavirus strains with a long RNA electropherotype and subgroup II antigen were also observed to be circulating bearing G1 or G4 VP7 genes and the VP4 P8 genotype. Two electrophoretic strains with differing short RNA electropherotypes and subgroup I antigenicity were also present. These strains hybridized to the VP7 type G2 and VP4 P4 genotype probes.

Laboratory diagnosis of acute measles infections in hospitalized children in Zambia.

Laboratory diagnosis of measles infection is rarely performed in developing countries and tends to depend on clinical symptoms alone. We evaluated detection of immunoglobulin M (IgM) antibodies for confirmation of acute measles infection in Zambia. In 149 hospitalized children with clinical diagnosis of measles, IgM antibodies were detected in 88.6% (132/149). The IgM-positive rate increased with time after onset of skin rash and all samples were positive after 4 days. In addition to IgM antibody test, virus isolations from throat swabs using B95a cells were also performed. These were positive in only 20.9% (14/67), and both IgM and virus isolation in combination increased the positive rate to 92.5% (62/67). Vaccinated children had higher neutralizing (Nt) antibody responses and, among IgM-negative patients, all 4 vaccinated children had high Nt antibodies while all 10 unvaccinated children had negative or low Nt results. The IgM antibody test was proved to be a sensitive method for laboratory confirmation of measles virus infection in developing countries.

Epidemiology of influenza virus infections in children with acute respiratory infections in Zambia.

A viral aetiological and epidemiological study of acute respiratory infections (ARI) in children was carried out in Lusaka, Zambia between June 1993 and September 1995. A total of 3,760 throat swab specimens were collected for virus isolation from children under 5 years of age who had ARI and were attending three health centres in Lusaka. Between June and November 1993, 52 cases of the influenza A/H3N2 viruses were isolated. Between May and July 1994, 34 influenza B cases were isolated. In 1995, one A/H3N2 influenza virus was isolated in January and then the same type of influenza virus was isolated from 55 samples between June and August. The isolation rate of influenza virus was highest at 14.3% (20/139) in August 1993, at 15.1% (18/119) in June 1994 and at 25.4% (43/169) in July 1995. This is the first report of a consecutive study of influenza virus infections in Zambia and the results reveal that influenza virus infections are one of the most important pathogens of ARI in children in the cool, dry season (June-August) in this country.

Infection with AIDS-related herpesviruses in human immunodeficiency virus-negative infants and endemic childhood Kaposi's sarcoma in Africa.

Novel herpesviruses have been described recently. These include human herpesviruses 6, 7 and 8 (HHV-6, -7, -8). HHV-6 has at least two strain groups, variants A and B. The B strains are predominant in the West and can account for over 97% of infections in infants. In contrast, the A strains are rare and the few well-characterized isolates have been from adult African AIDS patients. It is not clear whether the HHV-6 variant A strains are AIDS-related and/or whether they can also be acquired as childhood infections and may reactivate later during adulthood. What contribution geographical variation plays has yet to be assessed. HHV-8 has been associated with AIDS-related epidemic Kaposi's sarcoma (KS), but has also been identified in endemic KS. In regions of Africa where KS is endemic, the onset of AIDS has led to increased prevalence of KS. In this report, we examine in Zambia, an AIDS epidemic and KS endemic region, infection with these novel herpesviruses during infancy. In blood samples from human immunodeficiency virus-negative infants with first febrile episode, both semi-quantitative PCR and sequence analyses were used to identify HHV-8 in 8% and HHV-6 in 30%, with 44% of these variant A; in childhood endemic KS biopsies HHV-8 was detected in 100% and HHV-6 in none. The high viral-DNA loads in the infant blood samples were consistent with viraemia. This is the first demonstration that HHV-6 variant A and HHV-8 may be acquired as common childhood infections.

Prevalence of hepatitis B antigens in human immunodeficiency virus type 1 seropositive and seronegative pregnant women in Zambia.

PIP: During April-October 1992 in Zambia, 1861 pregnant women attending prenatal clinics in 3 urban health centers in Lusaka and 5 district hospitals in various provinces were recruited to examine the association between HIV infection and hepatitis B virus (HBV) infection. 340 (18.3%) tested positive for HIV infection. HIV-positive pregnant women were more likely to test positive for the hepatitis B surface antigen (HBsAg) than were HIV-negative pregnant women, but the difference was not significantly different (7.1% vs. 5.4%; p = 0.23). On the other hand, among the HBsAg-positive pregnant women, HIV- infected women were more likely to test positive for hepatitis B e antigen (HBeAg) than were HIV-negative women (25% vs. 12.3%; p 0.05), suggesting more HBV replication in HIV-infected people. Only women younger than 30 tested positive for HBeAg. If HIV does indeed facilitate HBV replication and increases its rate of vertical transmission, the HBV epidemiological pattern in sub-Saharan African could change. Further studies focusing on the epidemiological impact of HIV on HBV infection in sub-Saharan Africa are needed.^ieng

An outbreak of influenza A/H3N2 in a Zambian school dormitory.

There was an outbreak of "a mysterious disease" at a Zambian school dormitory in September, 1993. Investigation with questionnaire and collection of throat swab specimens for virus isolation were carried out on 46 patients to identify the causative agent. In this outbreak, most of the patients showed similar symptoms such as fever, headache, sore throat, cough, etc. The disease had spread to all dormitories within a couple of days after the onset of the first cases. From these patients, 13 influenza viruses A/H3N2 were isolated on MDCK cell line. This was a first ever confirmed outbreak of influenza virus infection in Zambia.

Association of rotavirus and human immunodeficiency virus infection in children hospitalized with acute diarrhea, Lusaka, Zambia.

In Lusaka, Zambia, rotavirus (RV) and human immunodeficiency virus (HIV) infection commonly coexist; 132 (25%) of 537 consecutively studied infants < 5 years old hospitalized with diarrhea were positive for both viral infections. Infants with RV infection were younger than those who were RV-negative (P > .05), and infants with both viruses more frequently experienced dehydration (P < .05). HIV-infected children more often exhibited respiratory symptoms on admission to the study (P < .0001) and were more frequently underweight (P < .0001) than were HIV-negative children, independent of RV infection. The mortality rate was highest in HIV-positive infants (P < .05), and coinfection with RV did not increase the risk of fatality. This study demonstrates that while RV and HIV infections commonly coexist in one region of Africa, RV infection is no more common nor is the illness more severe in HIV-positive infants.