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1.  Persistence of HIV-1 Transmitted Drug Resistance Mutations 
The Journal of Infectious Diseases  2013;208(9):1459-1463.
There are few data on the persistence of individual human immunodeficiency virus type 1 (HIV-1) transmitted drug resistance (TDR) mutations in the absence of selective drug pressure. We studied 313 patients in whom TDR mutations were detected at their first resistance test and who had a subsequent test performed while ART-naive. The rate at which mutations became undetectable was estimated using exponential regression accounting for interval censoring. Most thymidine analogue mutations (TAMs) and T215 revertants (but not T215F/Y) were found to be highly stable, with NNRTI and PI mutations being relatively less persistent. Our estimates are important for informing HIV transmission models.
PMCID: PMC3789571  PMID: 23904291
persistence; transmitted; HIV-1; resistance; mutations
2.  Low frequency of genotypic resistance in HIV-1-infected patients failing an atazanavir-containing regimen: a clinical cohort study 
Dolling, David I. | Dunn, David T. | Sutherland, Katherine A. | Pillay, Deenan | Mbisa, Jean L. | Parry, Chris M. | Post, Frank A. | Sabin, Caroline A. | Cane, Patricia A. | Aitken, Celia | Asboe, David | Webster, Daniel | Cane, Patricia | Castro, Hannah | Dunn, David | Dolling, David | Chadwick, David | Churchill, Duncan | Clark, Duncan | Collins, Simon | Delpech, Valerie | Geretti, Anna Maria | Goldberg, David | Hale, Antony | Hué, Stéphane | Kaye, Steve | Kellam, Paul | Lazarus, Linda | Leigh-Brown, Andrew | Mackie, Nicola | Orkin, Chloe | Rice, Philip | Pillay, Deenan | Phillips, Andrew | Sabin, Caroline | Smit, Erasmus | Templeton, Kate | Tilston, Peter | Tong, William | Williams, Ian | Zhang, Hongyi | Zuckerman, Mark | Greatorex, Jane | Wildfire, Adrian | O'Shea, Siobhan | Mullen, Jane | Mbisa, Tamyo | Cox, Alison | Tandy, Richard | Hale, Tony | Fawcett, Tracy | Hopkins, Mark | Ashton, Lynn | Booth, Claire | Garcia-Diaz, Ana | Shepherd, Jill | Schmid, Matthias L. | Payne, Brendan | Hay, Phillip | Rice, Phillip | Paynter, Mary | Bibby, David | Kirk, Stuart | MacLean, Alasdair | Gunson, Rory | Coughlin, Kate | Fearnhill, Esther | Fradette, Lorraine | Porter, Kholoud | Ainsworth, Jonathan | Anderson, Jane | Babiker, Abdel | Fisher, Martin | Gazzard, Brian | Gilson, Richard | Gompels, Mark | Hill, Teresa | Johnson, Margaret | Kegg, Stephen | Leen, Clifford | Nelson, Mark | Palfreeman, Adrian | Post, Frank | Sachikonye, Memory | Schwenk, Achim | Walsh, John | Huntington, Susie | Jose, Sophie | Thornton, Alicia | Glabay, Adam | Orkin, C. | Garrett, N. | Lynch, J. | Hand, J. | de Souza, C. | Fisher, M. | Perry, N. | Tilbury, S. | Gazzard, B. | Nelson, M. | Waxman, M. | Asboe, D. | Mandalia, S. | Delpech, V. | Anderson, J. | Munshi, S. | Korat, H. | Welch, J. | Poulton, M. | MacDonald, C. | Gleisner, Z. | Campbell, L. | Gilson, R. | Brima, N. | Williams, I. | Schwenk, A. | Ainsworth, J. | Wood, C. | Miller, S. | Johnson, M. | Youle, M. | Lampe, F. | Smith, C. | Grabowska, H. | Chaloner, C. | Puradiredja, D. | Walsh, J. | Weber, J. | Ramzan, F. | Mackie, N. | Winston, A. | Leen, C. | Wilson, A. | Allan, S. | Palfreeman, A. | Moore, A. | Wakeman, K.
Journal of Antimicrobial Chemotherapy  2013;68(10):2339-2343.
To determine protease mutations that develop at viral failure for protease inhibitor (PI)-naive patients on a regimen containing the PI atazanavir.
Resistance tests on patients failing atazanavir, conducted as part of routine clinical care in a multicentre observational study, were randomly matched by subtype to resistance tests from PI-naive controls to account for natural polymorphisms. Mutations from the consensus B sequence across the protease region were analysed for association and defined using the IAS-USA 2011 classification list.
Four hundred and five of 2528 (16%) patients failed therapy containing atazanavir as a first PI over a median (IQR) follow-up of 1.76 (0.84–3.15) years and 322 resistance tests were available for analysis. Recognized major atazanavir mutations were found in six atazanavir-experienced patients (P < 0.001), including I50L and N88S. The minor mutations most strongly associated with atazanavir experience were M36I, M46I, F53L, A71V, V82T and I85V (P < 0.05). Multiple novel mutations, I15S, L19T, K43T, L63P/V, K70Q, V77I and L89I/T/V, were also associated with atazanavir experience.
Viral failure on atazanavir-containing regimens was not common and major resistance mutations were rare, suggesting that adherence may be a major contributor to viral failure. Novel mutations were described that have not been previously documented.
PMCID: PMC3772741  PMID: 23711895
HIV; drug resistance mutations; naive patients; protease inhibitors; virological failure
3.  HIV-1 virologic failure and acquired drug resistance among first-line antiretroviral experienced adults at a rural HIV clinic in coastal Kenya: a cross-sectional study 
An increasing number of people on antiretroviral therapy (ART) in sub-Saharan Africa has led to declines in HIV related morbidity and mortality. However, virologic failure (VF) and acquired drug resistance (ADR) may negatively affect these gains. This study describes the prevalence and correlates of HIV-1 VF and ADR among first-line ART experienced adults at a rural HIV clinic in Coastal Kenya.
HIV-infected adults on first-line ART for ≥6 months were cross-sectionally recruited between November 2008 and March 2011. The primary outcome was VF, defined as a one-off plasma viral load of ≥400 copies/ml. The secondary outcome was ADR, defined as the presence of resistance associated mutations. Logistic regression and Fishers exact test were used to describe correlates of VF and ADR respectively.
Of the 232 eligible participants on ART over a median duration of 13.9 months, 57 (24.6% [95% CI: 19.2 – 30.6]) had VF. Fifty-five viraemic samples were successfully amplified and sequenced. Of these, 29 (52.7% [95% CI: 38.8 – 66.3]) had at least one ADR, with 25 samples having dual-class resistance mutations. The most prevalent ADR mutations were the M184V (n = 24), K103N/S (n = 14) and Y181C/Y/I/V (n = 8). Twenty-six of the 55 successfully amplified viraemic samples (47.3%) did not have any detectable resistance mutation. Younger age (15–34 vs. ≥35 years: adjusted odd ratios [95% CI], p-value: 0.3 [0.1–0.6], p = 0.002) and unsatisfactory adherence (<95% vs. ≥95%: 3.0 [1.5–6.5], p = 0.003) were strong correlates of VF. Younger age, unsatisfactory adherence and high viral load were also strong correlates of ADR.
High levels of VF and ADR were observed in younger patients and those with unsatisfactory adherence. Youth-friendly ART initiatives and strengthened adherence support should be prioritized in this Coastal Kenyan setting. To prevent unnecessary/premature switches, targeted HIV drug resistance testing for patients with confirmed VF should be considered.
PMCID: PMC3922732  PMID: 24456757
HIV; Virologic failure; Acquired drug resistance; Correlates; Rural; Kenya
4.  Phylodynamic and Phylogeographic Patterns of the HIV Type 1 Subtype F1 Parenteral Epidemic in Romania 
In the late 1980s an HIV-1 epidemic emerged in Romania that was dominated by subtype F1. The main route of infection is believed to be parenteral transmission in children. We sequenced partial pol coding regions of 70 subtype F1 samples from children and adolescents from the PENTA-EPPICC network of which 67 were from Romania. Phylogenetic reconstruction using the sequences and other publically available global subtype F sequences showed that 79% of Romanian F1 sequences formed a statistically robust monophyletic cluster. The monophyletic cluster was epidemiologically linked to parenteral transmission in children. Coalescent-based analysis dated the origins of the parenteral epidemic to 1983 [1981–1987; 95% HPD]. The analysis also shows that the epidemic's effective population size has remained fairly constant since the early 1990s suggesting limited onward spread of the virus within the population. Furthermore, phylogeographic analysis suggests that the root location of the parenteral epidemic was Bucharest.
PMCID: PMC3423652  PMID: 22251065
5.  HIV Type 1 in a Rural Coastal Town in Kenya Shows Multiple Introductions with Many Subtypes and Much Recombination 
The extent of HIV-1 diversity was examined among patients attending a rural district hospital in a coastal area of Kenya. The pol gene was sequenced in samples from 153 patients. Subtypes were designated using the REGA, SCUEAL, and jpHMM programs. The most common subtype was A1, followed by C and D; A2 and G were also detected. However, a large proportion of the samples was found to be recombinants, which clustered within the pure subtype branches. Phylogeographic analysis of Kilifi sequences compared with those from other regions of Africa showed that while many sequences were closely related to sequences from Kenya, others were most closely related to known sequences from other parts of Africa, including West Africa. Overall, these data indicate that there have been multiple introductions of HIV-1 into this small rural town and surroundings with ongoing diversity being generated by recombination.
PMCID: PMC3275924  PMID: 21770741
6.  Low Prevalence of Transmitted HIV Type 1 Drug Resistance Among Antiretroviral-Naive Adults in a Rural HIV Clinic in Kenya 
Low levels of HIV-1 transmitted drug resistance (TDR) have previously been reported from many parts of sub-Saharan Africa (sSA). However, recent data, mostly from urban settings, suggest an increase in the prevalence of HIV-1 TDR. Our objective was to determine the prevalence of TDR mutations among HIV-1-infected, antiretroviral (ARV)-naive adults enrolling for care in a rural HIV clinic in Kenya. Two cross-sectional studies were carried out between July 2008 and June 2010. Plasma samples from ARV-naive adults (>15 years old) at the time of registering for care after HIV diagnosis and before starting ARVs were used. A portion of the pol subgenomic region of the virus containing the protease and part of the reverse transcriptase genes was amplified and sequenced. TDR mutations were identified and interpreted using the Stanford HIV drug resistance database and the WHO list for surveillance of drug resistance strains. Overall, samples from 182 ARV-naive adults [mean age (95% CI): 34.9 (33.3–36.4) years] were successfully amplified and sequenced. Two TDR mutations to nucleoside reverse transcriptase inhibitors [n=1 (T215D)] and protease inhibitors [n=1 (M46L)] were identified, giving an overall TDR prevalence of 1.1% (95% CI: 0.1–3.9). Despite reports of an increase in the prevalence of HIV-1 TDR in some urban settings in sSA, we report a prevalence of HIV-1 TDR of less than 5% at a rural HIV clinic in coastal Kenya. Continued broader surveillance is needed to monitor the extent of TDR in sSA.
PMCID: PMC3537300  PMID: 22900472
7.  Influenza Surveillance Among Children With Pneumonia Admitted to a District Hospital in Coastal Kenya, 2007–2010 
The Journal of Infectious Diseases  2012;206(Suppl 1):S61-S67.
Background. Influenza data gaps in sub-Saharan Africa include incidence, case fatality, seasonal patterns, and associations with prevalent disorders.
Methods. Nasopharyngeal samples from children aged <12 years who were admitted to Kilifi District Hospital during 2007–2010 with severe or very severe pneumonia and resided in the local demographic surveillance system were screened for influenza A, B, and C viruses by molecular methods. Outpatient children provided comparative data.
Results. Of 2002 admissions, influenza A virus infection was diagnosed in 3.5% (71), influenza B virus infection, in 0.9% (19); and influenza C virus infection, in 0.8% (11 of 1404 tested). Four patients with influenza died. Among outpatients, 13 of 331 (3.9%) with acute respiratory infection and 1 of 196 without acute respiratory infection were influenza positive. The annual incidence of severe or very severe pneumonia, of influenza (any type), and of influenza A, was 1321, 60, and 43 cases per 100 000 <5 years of age, respectively. Peak occurrence was in quarters 3–4 each year, and approximately 50% of cases involved infants: temporal association with bacteremia was absent. Hypoxia was more frequent among pneumonia cases involving influenza (odds ratio, 1.78; 95% confidence interval, 1.04–1.96). Influenza A virus subtypes were seasonal H3N2 (57%), seasonal H1N1 (12%), and 2009 pandemic H1N1 (7%).
Conclusions. The burden of influenza was small during 2007–2010 in this pediatric hospital in Kenya. Influenza A virus subtype H3N2 predominated, and 2009 pandemic influenza A virus subtype H1N1 had little impact.
PMCID: PMC3502370  PMID: 23169974
8.  Group- and Genotype-Specific Neutralizing Antibody Responses Against Respiratory Syncytial Virus in Infants and Young Children With Severe Pneumonia 
The Journal of Infectious Diseases  2012;207(3):489-492.
The effect of genetic variation on the neutralizing antibody response to respiratory syncytial virus (RSV) is poorly understood. In this study, acute- and convalescent-phase sera were evaluated against different RSV strains. The proportion of individuals with homologous seroconversion was greater than that among individuals with heterologous seroconversion among those infected with RSV group A (50% vs 12.5%; P = .0005) or RSV group B (40% vs 8%; P = .008). Seroconversion to BA genotype or non-BA genotype test viruses was similar among individuals infected with non-BA virus (35% vs 50%; P = .4) or BA virus (50% vs 65%; P = .4). The RSV neutralizing response is group specific. The BA-associated genetic change did not confer an ability to escape neutralizing responses to previous non-BA viruses.
PMCID: PMC3541697  PMID: 23175761
respiratory syncytial virus; neutralizing antibody; immunity
9.  Genetic Relatedness of Infecting and Reinfecting Respiratory Syncytial Virus Strains Identified in a Birth Cohort From Rural Kenya 
The Journal of Infectious Diseases  2012;206(10):1532-1541.
Background. Respiratory syncytial virus (RSV) reinfects individuals repeatedly. The extent to which this is a consequence of RSV antigenic diversity is unclear.
Methods. Six-hundred thirty-five children from rural Kenya were closely monitored for RSV infection from birth through 3 consecutive RSV epidemics. RSV infections were identified by immunofluorescence testing of nasal washing samples collected during acute respiratory illnesses, typed into group A and B, and sequenced in the attachment (G) protein. A positive sample separated from a previous positive by ≥14 days was defined as a reinfection a priori.
Results. Phylogenetic analysis was undertaken for 325 (80%) of 409 identified infections, including 53 (64%) of 83 reinfections. Heterologous group reinfections were observed in 28 episodes, and homologous group reinfections were observed in 25 episodes; 10 involved homologous genotypes, 5 showed no amino acid changes, and 3 were separated by 21–24 days and were potentially persistent infections. The temporal distribution of genotypes among reinfections did not differ from that of single infections.
Conclusions. The vast majority of infection and reinfection pairs differed by group, genotype, or G amino acid sequence (ie, comprised distinct viruses). The extent to which this is a consequence of immune memory of infection history or prevalent diversity remains unclear.
PMCID: PMC3475639  PMID: 22966119
10.  Improved Detection of Respiratory Viruses in Pediatric Outpatients with Acute Respiratory Illness by Real-Time PCR Using Nasopharyngeal Flocked Swabs▿ 
Journal of Clinical Microbiology  2011;49(9):3365-3367.
Detection of respiratory viruses by real-time multiplexed PCR (M-PCR) and of respiratory syncytial virus (RSV) by M-PCR and immunofluorescence (IF) was evaluated using specimens collected by nasopharyngeal flocked swabbing (NFS) and nasal washes (NW). In children with mild respiratory illness, NFS collection was superior to NW collection for detection of viruses by M-PCR (sensitivity, 89.6% versus 79.2%; P = 0.0043). NFS collection was noninferior to NW collection in the detection of RSV by IF.
PMCID: PMC3165583  PMID: 21775539
11.  Three Residues in HIV-1 Matrix Contribute to Protease Inhibitor Susceptibility and Replication Capacity▿  
Other than cleavage site mutations, there is little data on specific positions within Gag that impact on HIV protease inhibitor susceptibility. We have recently shown that non-cleavage site mutations in gag, particularly within matrix protein can restore replication capacity and further reduce protease inhibitor drug susceptibility when coexpressed with a drug-resistant (mutant) protease. The matrix protein of this patient-derived virus was studied in order to identify specific changes responsible for this phenotype. Three amino acid changes in matrix (R76K, Y79F, and T81A) had an impact on replication capacity as well as drug susceptibility. Introduction of these three changes into wild-type (WT) matrix resulted in an increase in the replication capacity of the protease mutant virus to a level similar to that achieved by all the changes within the mutant matrix and part of the capsid protein. Pairs of changes to wild-type matrix led to an increased replication capacity of the protease mutant (although less than with all three changes). Having only these three changes to matrix in a wild-type virus (with wild-type protease) resulted in a 5- to 7-fold change in protease inhibitor 50% effective concentration (EC50). Individual changes did not have as great an effect on replication capacity or drug susceptibility, demonstrating an interaction between these positions, also confirmed by sequence covariation analysis. Molecular modeling predicts that each of the three mutations would result in a loss of hydrogen bonds within α-helix-4 of matrix, leading to the hypothesis that more flexibility within this region or altered matrix structure would account for our findings.
PMCID: PMC3067102  PMID: 21149628
12.  The evolution of HIV-1 reverse transcriptase in route to acquisition of Q151M multi-drug resistance is complex and involves mutations in multiple domains 
Retrovirology  2011;8:31.
The Q151M multi-drug resistance (MDR) pathway in HIV-1 reverse transcriptase (RT) confers reduced susceptibility to all nucleoside reverse transcriptase inhibitors (NRTIs) excluding tenofovir (TDF). This pathway emerges after long term failure of therapy, and is increasingly observed in the resource poor world, where antiretroviral therapy is rarely accompanied by intensive virological monitoring. In this study we examined the genotypic, phenotypic and fitness correlates associated with the development of Q151M MDR in the absence of viral load monitoring.
Single-genome sequencing (SGS) of full-length RT was carried out on sequential samples from an HIV-infected individual enrolled in ART rollout. The emergence of Q151M MDR occurred in the order A62V, V75I, and finally Q151M on the same genome at 4, 17 and 37 months after initiation of therapy, respectively. This was accompanied by a parallel cumulative acquisition of mutations at 20 other codon positions; seven of which were located in the connection subdomain. We established that fourteen of these mutations are also observed in Q151M-containing sequences submitted to the Stanford University HIV database. Phenotypic drug susceptibility testing demonstrated that the Q151M-containing RT had reduced susceptibility to all NRTIs except for TDF. RT domain-swapping of patient and wild-type RTs showed that patient-derived connection subdomains were not associated with reduced NRTI susceptibility. However, the virus expressing patient-derived Q151M RT at 37 months demonstrated ~44% replicative capacity of that at 4 months. This was further reduced to ~22% when the Q151M-containing DNA pol domain was expressed with wild-type C-terminal domain, but was then fully compensated by coexpression of the coevolved connection subdomain.
We demonstrate a complex interplay between drug susceptibility and replicative fitness in the acquisition Q151M MDR with serious implications for second-line regimen options. The acquisition of the Q151M pathway occurred sequentially over a long period of failing NRTI therapy, and was associated with mutations in multiple RT domains.
PMCID: PMC3113953  PMID: 21569325
13.  Intrapatient Variation of the Respiratory Syncytial Virus Attachment Protein Gene▿  
Journal of Virology  2010;84(19):10425-10428.
Intrapatient variability of the attachment (G) protein gene of respiratory syncytial virus (RSV) was examined using both population and single-genome sequencing. Samples from three patients infected with a group B virus variant which has a 60-nucleotide duplication in the G protein gene were examined. These samples were chosen because occasional mixed sequence bases were observed. In a minority of RSV genomes from these patients considerable variability was found, including point mutations, insertions, and deletions. Of particular note, the deletion of the exact portion of the gene which had been duplicated in some isolates was observed in viral RNAs from two patients.
PMCID: PMC2937772  PMID: 20660195
14.  Patterns of resistance development with integrase inhibitors in HIV 
Raltegravir, the only integrase (IN) inhibitor approved for use in HIV therapy, has recently been licensed. Raltegravir inhibits HIV-1 replication by blocking the IN strand transfer reaction. More than 30 mutations have been associated with resistance to raltegravir and other IN strand transfer inhibitors (INSTIs). The majority of the mutations are located in the vicinity of the IN active site within the catalytic core domain which is also the binding pocket for INSTIs. High-level resistance to INSTIs primarily involves three independent mutations at residues Q148, N155, and Y143. The mutations significantly affect replication capacity of the virus and are often accompanied by other mutations that either improve replication fitness and/or increase resistance to the inhibitors. The pattern of development of INSTI resistance mutations has been extensively studied in vitro and in vivo. This has been augmented by cell-based phenotypic studies and investigation of the mechanisms of resistance using biochemical assays. The recent elucidation of the structure of the prototype foamy virus IN, which is closely related to HIV-1, in complex with INSTIs has greatly enhanced our understanding of the evolution and mechanisms of IN drug resistance.
PMCID: PMC3108751  PMID: 21694910
raltegravir; elvitegravir; integrase inhibitors; HIV; drug resistance
15.  Viral etiology of severe pneumonia among Kenyan young infants and children 
Pneumonia is the leading cause of childhood death in sub-Saharan Africa. Comparative estimates of the contribution of causative pathogens to the burden of disease are essential for targeted vaccine development.
To determine the viral etiology of severe pneumonia among infants and children at a rural Kenyan hospital using comprehensive and sensitive molecular diagnostic techniques.
Design, Setting & Participants
Prospective observational and case control study during 2007 in a rural Kenyan district hospital. We recruited children age 1 day to 12 years who were resident in a systematically enumerated catchment area: i) those admitted to Kilifi District Hospital meeting WHO clinical criteria for ‘severe pneumonia’ or ‘very severe pneumonia’; ii) those presenting with mild upper respiratory tract infection but not admitted and iii) well infants and children attending for immunization.
Main Outcome Measures
The presence of respiratory viruses and the odds ratio for admission with severe disease.
759/922 (83%) eligible admissions were sampled (median age 9 months). One or more respiratory viruses were detected in 425/759 (56%, 95% CI 52 to 60%). Respiratory syncytial virus (RSV) was detected in 260 (34%, 95% CI 31 to 38%) and other respiratory viruses in 219 (29%, 95% CI 26 to 32%), the commonest being human coronavirus 229E (n=51, 6.7%, 95% CI 5.0 to 8.7%), influenza type A (n=44, 5.8%, 95% CI 4.2 to 7.7%), parainfluenza type 3 (n=29, 3.8%, 95% CI 2.6 to 5.4%), adenovirus (n=29, 3.8%, 95% CI 2.6 to 5.4%) and human metapneumovirus (n=23, 3.0%, 95% CI 1.9 to 4.5%). Compared to well controls, detection of RSV was associated with severe disease (4% in controls, adjusted odds Ratio 6.11 [95% CI 1.65 to 22.6]) whilst collectively, other respiratory viruses were not (23% in controls, adjusted odds Ratio 1.27 [95% CI 0.64 to 2.52]).
In a sample of Kenyan infants and children admitted with severe pneumonia to a rural hospital, RSV was the predominant viral pathogen.
PMCID: PMC2968755  PMID: 20501927
16.  Duration of shedding of respiratory syncytial virus in a community study of Kenyan children 
Our understanding of the transmission dynamics of respiratory syncytial virus (RSV) infection will be better informed with improved data on the patterns of shedding in cases not limited only to hospital admissions.
In a household study, children testing RSV positive by direct immunofluorescent antibody test (DFA) were enrolled. Nasal washings were scheduled right away, then every three days until day 14, every 7 days until day 28 and every 2 weeks until a maximum of 16 weeks, or until the first DFA negative RSV specimen. The relationship between host factors, illness severity and viral shedding was investigated using Cox regression methods.
From 151 families a total of 193 children were enrolled with a median age of 21 months (range 1-164 months), 10% infants and 46% male. The rate of recovery from infection was 0.22/person/day (95% CI 0.19-0.25) equivalent to a mean duration of shedding of 4.5 days (95%CI 4.0-5.3), with a median duration of shedding of 4 days (IQR 2-6, range 1-14). Children with a history of RSV infection had a 40% increased rate of recovery i.e. shorter duration of viral shedding (hazard ratio 1.4, 95% CI 1.01-1.86). The rate of cessation of shedding did not differ significantly between males and females, by severity of infection or by age.
We provide evidence of a relationship between the duration of shedding and history of infection, which may have a bearing on the relative role of primary versus re-infections in RSV transmission in the community.
PMCID: PMC2822777  PMID: 20096106
17.  The Level and Duration of RSV-Specific Maternal IgG in Infants in Kilifi Kenya 
PLoS ONE  2009;4(12):e8088.
Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants. The rate of decay of RSV-specific maternal antibodies (RSV-matAb), the factors affecting cord blood levels, and the relationship between these levels and protection from infection are poorly defined.
A birth cohort (n = 635) in rural Kenya, was studied intensively to monitor infections and describe age-related serological characteristics. RSV specific IgG antibody (Ab) in serum was measured by the enzyme linked immunosorbent assay (ELISA) in cord blood, consecutive samples taken 3 monthly, and in paired acute and convalescent samples. A linear regression model was used to calculate the rate of RSV-matAb decline. The effect of risk factors on cord blood titres was investigated.
The half-life of matAb in the Kenyan cohort was calculated to be 79 days (95% confidence limits (CL): 76–81 days). Ninety seven percent of infants were born with RSV-matAb. Infants who subsequently experienced an infection in early life had significantly lower cord titres of anti-RSV Ab in comparison to infants who did not have any incident infection in the first 6 months (P = 0.011). RSV infections were shown to have no effect on the rate of decay of RSV-matAb.
Maternal-specific RSV Ab decline rapidly following birth. However, we provide evidence of protection against severe disease by RSV-matAb during the first 6–7 months. This suggests that boosting maternal-specific Ab by RSV vaccination may be a useful strategy to consider.
PMCID: PMC2779853  PMID: 19956576
Military recruits receiving training are vulnerable to acute respiratory disease and a significant proportion of illness is due to unidentified pathogens. While some countries use surveillance programs to monitor such illness, few data exist for recruits of the British Armed Forces.
Through active surveillance of approximately 1000 Royal Navy trainees during 2001, we sought to describe and determine the aetiology of acute respiratory illness.
Standard viral culture was used together with serology and a novel highly sensitive real-time PCR and molecular beacon probe assay for RSV detection.
Among 54 Royal Navy recruits with respiratory symptoms adenovirus was identified in 35%, influenza viruses in 19% and RSV in 14% of this group. All of the recruits were absent from training for almost a week, most of whom were confined to the sickbay.
This study is the first to document adenovirus and RSV as important causes of acute respiratory illness among Royal Navy trainees. The study findings demonstrate the clinical significance and challenges of diagnosing RSV infection in young adults.
PMCID: PMC2564797  PMID: 18846262
Adults; Human RSV; Military personnel; Respiratory tract infections
19.  Identifying Infections with Respiratory Syncytial Virus by Using Specific Immunoglobulin G (IgG) and IgA Enzyme-Linked Immunosorbent Assays with Oral-Fluid Samples▿  
Journal of Clinical Microbiology  2008;46(5):1659-1662.
Currently, respiratory syncytial virus (RSV) infection is identified in epidemiological studies by virus antigen or nucleic acid detection in combination with serology. Oral-fluid specimens may provide a noninvasive alternative to blood, and oral fluid is more suitable for sampling outside of the clinic setting. We evaluated an indirect enzyme-linked immunosorbent assay for the detection of RSV-specific immunoglobulin G (IgG) and IgA by using oral-fluid samples collected from individuals with RSV infections confirmed by an immunofluorescent antibody test. For five children sampled repeatedly from birth, antibody profiles in oral fluid quite consistently tracked those in paired sera, and RSV infections were detected by rising titers of antibodies of at least one Ig class. Specific IgG responses were generally more reliable than IgA responses, except in early infancy, where the reverse was sometimes true. For a further five young children from whom oral fluid was collected weekly following RSV infection, boosted antibody responses, frequently of a transient nature, lasting a few weeks, were observed; specific IgG responses were of longer duration and more pronounced than specific IgA responses. Our data show significant promise for the use of oral fluid alone in RSV infection surveillance. The observed rapid dynamics of the antibody responses are informative in defining study sampling intervals.
PMCID: PMC2395083  PMID: 18305129
20.  Factors associated with increased risk of progression to respiratory syncytial virus-associated pneumonia in young Kenyan children 
To identify factors associated with developing severe respiratory syncytial virus (RSV) pneumonia and their commonality with all-cause lower respiratory tract infection (LRTI), in order to isolate those risk factors specifically associated with RSV-LRTI and identify targets for control.
A birth cohort of rural Kenyan children was intensively monitored for acute respiratory infection (ARI) over three RSV epidemics. RSV was diagnosed by immunofluorescence of nasal washings collected at each ARI episode. Cox regression was used to determine the relative risk of disease for a range of co-factors.
A total of 469 children provided 937 years of follow-up, and experienced 857 all-cause LRTI, 362 RSV-ARI and 92 RSV-LRTI episodes. Factors associated with RSV-LRTI, but not RSV-ARI, were severe stunting (z-score ≤−2, RR 1.7 95%CI 1.1–2.8), crowding (increased number of children, RR 2.6, 1.0–6.5) and number of siblings under 6 years (RR 2.0, 1.2–3.4). Moderate and severe stunting (z-score ≤−1), crowding and a sibling aged over 5 years sleeping in the same room as the index child were associated with increased risk of all-cause LRTI, whereas higher educational level of the primary caretaker was associated with protection.
We identify factors related to host nutritional status (stunting) and contact intensity (crowding, siblings) which are distinguishable in their association with RSV severe disease in infant and young child. These factors are broadly in common with those associated with all-cause LRTI. The results support targeted strategies for prevention.
PMCID: PMC2635480  PMID: 18482199
respiratory syncytial virus; risk factors; disease; Kenya
21.  Molecular analysis of respiratory syncytial virus reinfections in infants from coastal Kenya 
Individuals are reinfected with respiratory syncytial virus (RSV) repeatedly. The nature of reinfections in relation to RSV genetic and antigenic diversity is ill defined and has implications to persistence and vaccine control.
We examined the molecular relatedness of RSV causing primary and repeat infections by phylogenetic analysis of the attachment (G) gene in 12 infants from a birth cohort in rural Kenya, using nasal washings collected over a 16 month period in 2002-03 spanning two successive epidemics.
Six infants were infected in both epidemics, 4 with RSV-A in the first epidemic followed by RSV-B in the second epidemic and 2 infected with RSV-A strains in both epidemics with no significant G gene sequence variability between samples. Two children showed infection and reinfection with different RSV-A strains within the same epidemic. Possible viral persistence was suspected in the remaining 4 infants, although reinfection with same variant cannot be excluded.
These are the first data specifically addressing strain-specific reinfections in infancy in relation to the primary infecting variant. The data strongly suggest that following primary infection some infants lose strain-specific immunity within 7-9 months (between epidemics) and group-specific immunity within 2-4 months (within an epidemic period).
PMCID: PMC2384051  PMID: 16323133
Respiratory syncytial virus; infants; reinfection
22.  Respiratory Syncytial Virus Infection and Disease in Infants and Young Children Studied from Birth in Kilifi District, Kenya 
Within the developing country setting data are few that characterise the disease burden due to RSV and which clearly define the age group to target vaccine intervention.
Children numbering 635, recruited 2002-03, were intensively monitored until each experienced three RSV epidemics. RSV was diagnosed by use of immunofluorescence on nasal washings collected on occurence of acute respiratory infection. Incidence estimates were adjusted for seasonality in RSV exposure.
From 1187 child years of observation (cyo) a total of 409 RSV episodes were identifed; 365 primary and 82 repeat. Adjusted incidence estimates (per 1000cyo) of lower respiratory tract infection (LRTI), severe LRTI and hospital admission were 90, 43, and 10, respectively, and corresponding estimates in infants were 104, 66 and 13. The proportion of all-cause LRTI, severe-LRTI and hospitalizations in the cohort due to RSV was 13%, 19% and 5%, respectively. 55-65% of RSV LRTI and severe-LRTI occured in children over 6 months old. The risk of RSV disease following primary symptomatic infection remained significant beyond the first year of life and a quarter of all re-infections were associated with LRTI.
RSV accounts for a substantial proportion of the total respiratory disease in this rural population: we estimate 85,000 infant cases of severe LRTI annually in Kenya. The majority of this morbidity occurs in late infancy and early childhood; ages at which the risk of disease following infection remains significant. Disease from re-infection is common. Our results inform the debate on vaccine target age group and effectiveness.
PMCID: PMC2358944  PMID: 18171213
respiratory syncytial virus; incidence; burden of disease; vaccination strategy; Kenya
23.  Discordances between Interpretation Algorithms for Genotypic Resistance to Protease and Reverse Transcriptase Inhibitors of Human Immunodeficiency Virus Are Subtype Dependent 
The major limitation of drug resistance genotyping for human immunodeficiency virus remains the interpretation of the results. We evaluated the concordance in predicting therapy response between four different interpretation algorithms (Rega 6.3, HIVDB-08/04, ANRS [07/04], and VGI 8.0). Sequences were gathered through a worldwide effort to establish a database of non-B subtype sequences, and demographic and clinical information about the patients was gathered. The most concordant results were found for nonnucleoside reverse transcriptase (RT) inhibitors (93%), followed by protease inhibitors (84%) and nucleoside RT inhibitor (NRTIs) (76%). For therapy-naive patients, for nelfinavir, especially for subtypes C and G, the discordances were driven mainly by the protease (PRO) mutational pattern 82I/V + 63P + 36I/V for subtype C and 82I + 63P + 36I + 20I for subtype G. Subtype F displayed more discordances for ritonavir in untreated patients due to the combined presence of PRO 20R and 10I/V. In therapy-experienced patients, subtype G displayed a lot of discordances for saquinavir and indinavir due to mutational patterns involving PRO 90 M and 82I. Subtype F had more discordance for nelfinavir attributable to the presence of PRO 88S and 82A + 54V. For the NRTIs lamivudine and emtricitabine, CRF01_AE had more discordances than subtype B due to the presence of RT mutational patterns 65R + 115 M and 118I + 215Y, respectively. Overall, the different algorithms agreed well on the level of resistance scored, but some of the discordances could be attributed to specific (subtype-dependent) combinations of mutations. It is not yet known whether therapy response is subtype dependent, but the advice given to clinicians based on a genotypic interpretation algorithm differs according to the subtype.
PMCID: PMC1366873  PMID: 16436728
24.  Impact of HIV-1 Subtype and Antiretroviral Therapy on Protease and Reverse Transcriptase Genotype: Results of a Global Collaboration 
PLoS Medicine  2005;2(4):e112.
The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate.
Methods and Findings
To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates.
Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.
Most research has focused on HIV-1 subtype B, although most infections worldwide are non-B. However, this paper suggests that the same drug resistance mutations occur across subtypes
PMCID: PMC1087220  PMID: 15839752
25.  Antigenic and Genetic Variability of Human Metapneumoviruses 
Emerging Infectious Diseases  2004;10(4):658-666.
Human metapneumovirus (HMPV) is a member of the subfamily Pneumovirinae within the family Paramyxoviridae. Other members of this subfamily, respiratory syncytial virus and avian pneumovirus, can be divided into subgroups based on genetic or antigenic differences or both. For HMPV, the existence of different genetic lineages has been described on the basis of variation in a limited set of available sequences. We address the antigenic relationship between genetic lineages in virus neutralization assays. In addition, we analyzed the genetic diversity of HMPV by phylogenetic analysis of sequences obtained for part of the fusion protein (n = 84) and the complete attachment protein open reading frames (n = 35). On the basis of sequence diversity between attachment protein genes and the differences in virus neutralization titers, two HMPV serotypes were defined. Each serotype could be divided into two genetic lineages, but these did not reflect major antigenic differences.
PMCID: PMC3323073  PMID: 15200856
Paramyxoviridae; Pneumovirinae; human metapneumovirus; genetic variability; antigenic variability; serotypes; fusion protein; attachment protein

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