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Expert Review of Anticancer Therapy 05/2013; 11(5):443-6. · 3.28 Impact Factor
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Jennifer L Marshall,
Adriana Flores-Langarica,
Robert A Kingsley,
Jessica R Hitchcock,
Ewan A Ross,
Constantino López-Macías,
Jeremy Lakey,
Laura B Martin,
Kai-Michael Toellner, Calman A Maclennan,
Ian C Maclennan,
Ian R Henderson,
Gordon Dougan,
Adam F Cunningham
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ABSTRACT: Vaccination with purified capsular polysaccharide Vi Ag from Salmonella typhi can protect against typhoid fever, although the mechanism for its efficacy is not clearly established. In this study, we have characterized the B cell response to this vaccine in wild-type and T cell-deficient mice. We show that immunization with typhoid vi polysaccharide vaccine rapidly induces proliferation in B1b peritoneal cells, but not in B1a cells or marginal zone B cells. This induction of B1b proliferation is concomitant with the detection of splenic Vi-specific Ab-secreting cells and protective Ab in Rag1-deficient B1b cell chimeras generated by adoptive transfer-induced specific Ab after Vi immunization. Furthermore, Ab derived from peritoneal B cells is sufficient to confer protection against Salmonella that express Vi Ag. Expression of Vi by Salmonella during infection did not inhibit the development of early Ab responses to non-Vi Ags. Despite this, the protection conferred by immunization of mice with porin proteins from Salmonella, which induce Ab-mediated protection, was reduced postinfection with Vi-expressing Salmonella, although protection was not totally abrogated. This work therefore suggests that, in mice, B1b cells contribute to the protection induced by Vi Ag, and targeting non-Vi Ags as subunit vaccines may offer an attractive strategy to augment current Vi-based vaccine strategies.
The Journal of Immunology 11/2012; · 5.79 Impact Factor
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Chinyere K Okoro,
Robert A Kingsley,
Thomas R Connor,
Simon R Harris,
Christopher M Parry,
Manar N Al-Mashhadani,
Samuel Kariuki,
Chisomo L Msefula,
Melita A Gordon,
Elizabeth de Pinna, [......],
Robert S Heyderman,
Stephen Obaro,
Pedro L Alonso,
Inacio Mandomando, Calman A Maclennan,
Milagritos D Tapia,
Myron M Levine,
Sharon M Tennant,
Julian Parkhill,
Gordon Dougan
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ABSTRACT: A highly invasive form of non-typhoidal Salmonella (iNTS) disease has recently been documented in many countries in sub-Saharan Africa. The most common Salmonella enterica serovar causing this disease is Typhimurium (Salmonella Typhimurium). We applied whole-genome sequence-based phylogenetic methods to define the population structure of sub-Saharan African invasive Salmonella Typhimurium isolates and compared these to global Salmonella Typhimurium populations. Notably, the vast majority of sub-Saharan invasive Salmonella Typhimurium isolates fell within two closely related, highly clustered phylogenetic lineages that we estimate emerged independently ∼52 and ∼35 years ago in close temporal association with the current HIV pandemic. Clonal replacement of isolates from lineage I by those from lineage II was potentially influenced by the use of chloramphenicol for the treatment of iNTS disease. Our analysis suggests that iNTS disease is in part an epidemic in sub-Saharan Africa caused by highly related Salmonella Typhimurium lineages that may have occupied new niches associated with a compromised human population and antibiotic treatment.
Nature Genetics 09/2012; · 35.53 Impact Factor
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Seung-Joo Lee,
Li Liang,
Silvia Juarez,
Minelva R Nanton,
Esther N Gondwe,
Chisomo L Msefula,
Matthew A Kayala,
Francesca Necchi,
Jennifer N Heath,
Peter Hart,
Renée M Tsolis,
Robert S Heyderman, Calman A MacLennan,
Philip L Felgner,
D Huw Davies,
Stephen J McSorley
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ABSTRACT: Despite the importance of Salmonella infections in human and animal health, the target antigens of Salmonella-specific immunity remain poorly defined. We have previously shown evidence for antibody-mediating protection against invasive Salmonellosis in mice and African children. To generate an overview of antibody targeting in systemic Salmonellosis, a Salmonella proteomic array containing over 2,700 proteins was constructed and probed with immune sera from Salmonella-infected mice and humans. Analysis of multiple inbred mouse strains identified 117 antigens recognized by systemic antibody responses in murine Salmonellosis. Importantly, many of these antigens were independently identified as target antigens using sera from Malawian children with Salmonella bacteremia, validating the study of the murine model. Furthermore, vaccination with SseB, the most prominent antigenic target in Malawian children, provided mice with significant protection against Salmonella infection. Together, these data uncover an overlapping immune signature of disseminated Salmonellosis in mice and humans and provide a foundation for the generation of a protective subunit vaccine.
Proceedings of the National Academy of Sciences 02/2012; 109(13):4998-5003. · 9.68 Impact Factor
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ABSTRACT: The complement cascade includes heat-labile proteins and care is required when handling serum in order to preserve its functional integrity. We have previously used a whole human serum bactericidal assay to show that antibody and an intact complement system are required in blood for killing of invasive isolates of Salmonella. The aim of the present study was to evaluate the conditions under which human serum can be stored and manipulated while maintaining complement integrity. Serum bactericidal activity against Salmonella was maintained for a minimum of 35 days when stored at 4°C, eight days at 22°C and 54 hours at 37°C. Up to three freeze-thaw cycles had no effect on the persistence of bactericidal activity and hemolytic complement assays confirmed no effect on complement function. Delay in the separation of serum for up to four days from clotted blood stored at 22°C did not affect bactericidal activity. Dilution of serum resulted in an increased rate of loss of bactericidal activity and so serum should be stored undiluted. These findings indicate that the current guidelines concerning manipulation and storage of human serum to preserve complement integrity and function leave a large margin for safety with regards to bactericidal activity against Salmonella. The study provides a scheme for determining the requirements for serum handling in relation to functional activity of complement in other systems.
PLoS ONE 01/2012; 7(11):e49147. · 4.09 Impact Factor
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ABSTRACT: Nontyphoidal Salmonella (NTS) serovars are a common cause of bacteraemia in young children and HIV-infected adults in Malawi and elsewhere in sub-Saharan Africa. These patient populations provide diverse host-immune environments that have the potential to drive bacterial adaptation and evolution. We therefore investigated the diversity of 27 multidrug resistant (MDR) Salmonella Typhimurium strains isolated over 6 years (2002-2008) from HIV-infected adults and children and HIV-uninfected children. Sequence reads from whole-genome sequencing of these isolates using the Illumina GA platform were mapped to the genome of the laboratory strain S. Typhimurium SL1344 excluding homoplastic regions that contained prophage and insertion elements. A phylogenetic tree generated from single nucleotide polymorphisms showed that all 27 strains clustered with the prototypical MDR strain D23580. There was no clustering of strains based on host HIV status or age, suggesting that these susceptible populations acquire S. Typhimurium from common sources or that isolates are transmitted freely between these populations. However, 7/14 of the most recent isolates (2006/2008) formed a distinct clade that branched off 22 SNPs away from the cluster containing earlier isolates. These data suggest that the MDR bacterial population is not static, but is undergoing microevolution which might result in further epidemiology change.
PLoS ONE 01/2012; 7(7):e42085. · 4.09 Impact Factor
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Dhaarini Raghunathan,
Timothy J Wells,
Faye C Morris,
Robert K Shaw,
Saeeda Bobat,
Sarah E Peters,
Gavin K Paterson,
Karina Tveen Jensen,
Denisse L Leyton,
Jessica M A Blair, [......],
Jessica R Hitchcock,
Claudia T P Moraes,
Roxane M F Piazza,
Duncan J Maskell,
Mark A Webber,
Robin C May, Calman A MacLennan,
Laura J Piddock,
Adam F Cunningham,
Ian R Henderson
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ABSTRACT: Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. We demonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection against Salmonella.
Infection and immunity 08/2011; 79(11):4342-52. · 4.21 Impact Factor
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ABSTRACT: Nontyphoidal strains of Salmonella are a major cause of fatal bacteremia in Africa. Developing a vaccine requires an improved understanding of the relevant mechanisms of protective immunity, and the mouse model of Salmonella infection is useful for studying immunity to Salmonella in vivo. It is important to appreciate the similarities and differences between immunity to Salmonella in mice and men. Ab is important for protection against nontyphoidal Salmonella in both species, and we have previously found an important role for Ab in cell-free complement-mediated bactericidal activity against Salmonella in Africans. It is unclear whether this modality of immunity is relevant in the mouse model. C57BL/6, BALB/c, and C3H mice immunized with heat-killed Salmonella Typhimurium strains D23580 (African invasive strain) and SL1344 and live-attenuated strain SL3261 produced a Salmonella-specific Ab response. Sera from these mice deposited reduced levels of C3 on Salmonella compared with human sera and were unable to kill both wild-type and galE(-) rough mutant of D23580, indicating absent cell-free killing via classical and alternative complement pathways. Supplementing immune mouse sera with human complement enabled killing of Salmonella, whereas addition of human anti-Salmonella Ab to immune mouse sera had no effect. These findings indicate that mouse serum cannot effect [corrected] cell-free complement-dependent killing of Salmonella, because of the reduced mouse complement ability to kill these bacteria compared with human complement. This difference in Ab-dependent immunity to Salmonella in mice and men must be considered when applying findings from the mouse model of Salmonella disease and vaccination response to man.
The Journal of Immunology 02/2011; 186(4):2365-71. · 5.79 Impact Factor
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Calman A MacLennan,
James J Gilchrist,
Melita A Gordon,
Adam F Cunningham,
Mark Cobbold,
Margaret Goodall,
Robert A Kingsley,
Joep J G van Oosterhout,
Chisomo L Msefula,
Wilson L Mandala, [......],
Esther N Gondwe,
Saeeda Bobat,
Constantino López-Macías,
Rainer Doffinger,
Ian R Henderson,
Eduard E Zijlstra,
Gordon Dougan,
Mark T Drayson, Ian C M MacLennan,
Malcolm E Molyneux
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ABSTRACT: Nontyphoidal Salmonellae are a major cause of life-threatening bacteremia among HIV-infected individuals. Although cell-mediated immunity controls intracellular infection, antibodies protect against Salmonella bacteremia. We report that high-titer antibodies specific for Salmonella lipopolysaccharide (LPS) are associated with a lack of Salmonella-killing in HIV-infected African adults. Killing was restored by genetically shortening LPS from the target Salmonella or removing LPS-specific antibodies from serum. Complement-mediated killing of Salmonella by healthy serum is shown to be induced specifically by antibodies against outer membrane proteins. This killing is lost when excess antibody against Salmonella LPS is added. Thus, our study indicates that impaired immunity against nontyphoidal Salmonella bacteremia in HIV infection results from excess inhibitory antibodies against Salmonella LPS, whereas serum killing of Salmonella is induced by antibodies against outer membrane proteins.
Science 04/2010; 328(5977):508-12. · 31.20 Impact Factor
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ABSTRACT: Bacteremia caused by nontyphoidal strains of Salmonella is endemic among African children. Case-fatality rates are high and antibiotic resistance increasing, but no vaccine is currently available. T cells are important for clearance of Salmonella infection within macrophages, but in Africa, invasive Salmonella disease usually manifests in the blood and affects children between 4 months and 2 y of age, when anti-Salmonella antibody is absent. We have previously found a role for complement-fixing bactericidal antibody in protecting these children. Here we show that opsonic activity of antibody and complement is required for oxidative burst and killing of Salmonella by blood cells in Africans. Induction of neutrophil oxidative burst correlated with anti-Salmonella IgG and IgM titers and C3 deposition on bacteria and was significantly lower in African children younger than 2 y compared with older children. Preopsonizing Salmonella with immune serum overcame this deficit, indicating a requirement for antibody and/or complement. Using different opsonization procedures, both antibody and complement were found to be necessary for optimal oxidative burst, phagocytosis and killing of nontyphoidal Salmonella by peripheral blood cells in Africans. Although most strains of African nontyphoidal Salmonella can be killed with antibody and complement alone, phagocytes in the presence of specific antibody and complement can kill strains resistant to killing by immune serum. These findings increase the likelihood that an antibody-inducing vaccine will protect against invasive nontyphoidal Salmonella disease in African children.
Proceedings of the National Academy of Sciences 02/2010; 107(7):3070-5. · 9.68 Impact Factor
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ABSTRACT: Nontyphoidal Salmonellae frequently cause life-threatening bacteremia in sub-Saharan Africa. Young children and HIV-infected adults are particularly susceptible. High case-fatality rates and increasing antibiotic resistance require new approaches to the management of this disease. Impaired cellular immunity caused by defects in the T helper 1 pathway lead to intracellular disease with Salmonella that can be countered by IFNγ administration. This report identifies the lymphocyte subsets that produce IFNγ early in Salmonella infection.
Intracellular cytokine staining was used to identify IFNγ production in blood lymphocyte subsets of ten healthy adults with antibodies to Salmonella (as evidence of immunity to Salmonella), in response to stimulation with live and heat-killed preparations of the D23580 invasive African isolate of Salmonella Typhimurium. The absolute number of IFNγ-producing cells in innate, innate-like and adaptive lymphocyte subpopulations was determined.
Early IFNγ production was found in the innate/innate-like lymphocyte subsets: γδ-T cells, NK cells and NK-like T cells. Significantly higher percentages of such cells produced IFNγ compared to adaptive αβ-T cells (Student's t test, P<0.001 and ≤0.02 for each innate subset compared, respectively, with CD4(+)- and CD8(+)-T cells). The absolute numbers of IFNγ-producing cells showed similar differences. The proportion of IFNγ-producing γδ-T cells, but not other lymphocytes, was significantly higher when stimulated with live compared with heat-killed bacteria (P<0.0001).
Our findings indicate an inherent capacity of innate/innate-like lymphocyte subsets to produce IFNγ early in the response to Salmonella infection. This may serve to control intracellular infection and reduce the threat of extracellular spread of disease with bacteremia which becomes life-threatening in the absence of protective antibody. These innate cells may also help mitigate against the effect on IFNγ production of depletion of Salmonella-specific CD4(+)-T lymphocytes in HIV infection.
PLoS ONE 01/2010; 5(10):e13667. · 4.09 Impact Factor
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ABSTRACT: CD4(+)T lymphocyte measurements are the most important indicator of mortality in HIV-infected individuals in resource-limited settings. There is currently a lack of comprehensive immunophenotyping data from African populations to guide the immunologic assessment of HIV infection.
To quantify variation in absolute and relative lymphocyte subsets with age in healthy Malawians.
Lymphocyte subsets in peripheral blood of 539 healthy HIV-uninfected Malawians stratified by age were enumerated by flow cytometry.
B and T-lymphocyte and T-lymphocyte subset absolute concentrations peaked in early childhood then decreased to adult levels, whereas lymphocyte subset proportions demonstrated much less variation with age. Adult lymphocyte subsets were similar to those in developed countries. In contrast, high B-lymphocyte and CD8(+)T-lymphocyte levels among children under 2 years, relative to those in developed countries, resulted in low CD4(+)T-lymphocyte percentages that varied little between 0 and 5 years (35% to 39%). The CD4(+)T-lymphocyte percentages in 35% of healthy children under 1 year and 18% of children age 1 to 3 years were below the World Health Organization threshold defining immunodeficiency in HIV-infected children in resource-limited settings. Thirteen percent of healthy children under 18 months old had a CD4:CD8T-lymphocyte ratio <1.0, which is commonly associated with HIV infection. All immunologic parameters except absolute natural killer lymphocyte concentration varied significantly with age, and percentage and overall absolute CD4(+)T-lymphocyte counts were higher in females than males.
Although lymphocyte subsets in Malawian adults are similar to those from developed countries, CD4(+)T-lymphocyte percentages in young children are comparatively low. These findings need to be considered when assessing the severity of HIV-related immunodeficiency in African children under 3 years.
The Journal of allergy and clinical immunology 11/2009; 125(1):203-8. · 9.17 Impact Factor
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Robert A Kingsley,
Chisomo L Msefula,
Nicholas R Thomson,
Samuel Kariuki,
Kathryn E Holt,
Melita A Gordon,
David Harris,
Louise Clarke,
Sally Whitehead,
Vartul Sangal,
Kevin Marsh,
Mark Achtman,
Malcolm E Molyneux,
Martin Cormican,
Julian Parkhill, Calman A MacLennan,
Robert S Heyderman,
Gordon Dougan
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ABSTRACT: Whereas most nontyphoidal Salmonella (NTS) are associated with gastroenteritis, there has been a dramatic increase in reports of NTS-associated invasive disease in sub-Saharan Africa. Salmonella enterica serovar Typhimurium isolates are responsible for a significant proportion of the reported invasive NTS in this region. Multilocus sequence analysis of invasive S. Typhimurium from Malawi and Kenya identified a dominant type, designated ST313, which currently is rarely reported outside of Africa. Whole-genome sequencing of a multiple drug resistant (MDR) ST313 NTS isolate, D23580, identified a distinct prophage repertoire and a composite genetic element encoding MDR genes located on a virulence-associated plasmid. Further, there was evidence of genome degradation, including pseudogene formation and chromosomal deletions, when compared with other S. Typhimurium genome sequences. Some of this genome degradation involved genes previously implicated in virulence of S. Typhimurium or genes for which the orthologs in S. Typhi are either pseudogenes or are absent. Genome analysis of other epidemic ST313 isolates from Malawi and Kenya provided evidence for microevolution and clonal replacement in the field.
Genome Research 11/2009; 19(12):2279-87. · 13.61 Impact Factor
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Calman A MacLennan,
Esther N Gondwe,
Chisomo L Msefula,
Robert A Kingsley,
Nicholas R Thomson,
Sarah A White,
Margaret Goodall,
Derek J Pickard,
Stephen M Graham,
Gordon Dougan,
C Anthony Hart,
Malcolm E Molyneux,
Mark T Drayson
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ABSTRACT: Nontyphoidal strains of Salmonella (NTS) are a common cause of bacteremia among African children. Cell-mediated immune responses control intracellular infection, but they do not protect against extracellular growth of NTS in the blood. We investigated whether antibody protects against NTS bacteremia in Malawian children, because we found this condition mainly occurs before 2 years of age, with relative sparing of infants younger than 4 months old. Sera from all healthy Malawian children tested aged more than 16 months contained anti-Salmonella antibody and successfully killed NTS. Killing was mediated by complement membrane attack complex and not augmented in the presence of blood leukocytes. Sera from most healthy children less than 16 months old lacked NTS-specific antibody, and sera lacking antibody did not kill NTS despite normal complement function. Addition of Salmonella-specific antibody, but not mannose-binding lectin, enabled NTS killing. All NTS strains tested had long-chain lipopolysaccharide and the rck gene, features that resist direct complement-mediated killing. Disruption of lipopolysaccharide biosynthesis enabled killing of NTS by serum lacking Salmonella-specific antibody. We conclude that Salmonella-specific antibody that overcomes the complement resistance of NTS develops by 2 years of life in Malawian children. This finding and the age-incidence of NTS bacteremia suggest that antibody protects against NTS bacteremia and support the development of vaccines against NTS that induce protective antibody.
Journal of Clinical Investigation 05/2008; 118(4):1553-62. · 15.39 Impact Factor
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ABSTRACT: Rapid expansion of antiretroviral therapy in Malawi has occurred in the relative absence of suitable pediatric CD4 counting facilities. We have recently validated in adults a simplified affordable flow cytometric CD4 counting method, the Blantyre count. There is a need for this technology to transfer to government laboratories run by local staff, and to be validated in children, where %CD4/lymphocyte values are required.
We assessed agreement of %CD4/lymphocyte values determined by the Blantyre count and Panleucogate methods on an EPICS XL-MCL flow cytometer on 113 venous blood samples from HIV-seropositive children in Blantyre, Malawi. All assays were performed by two Malawian laboratory technicians.
Overall bias between the two methods was -0.13% (95% CI -0.37 to 0.11) and limits of agreement were -2.69 to 2.43% (95% CI -3.11 to -2.27 and 2.01 to 2.85). Limits of agreement were within -3.00 and 3.00 for each laboratory technician. Coefficient of variation for the Blantyre count assay was 2.0% and samples showed good stability over 5 days.
The Blantyre count method can accurately determine %CD4/lymphocyte values in blood of HIV-seropositive children on an EPIC XL-MCL flow cytometer at a reagent cost of US $0.21 per test or less. The assay can be competently carried out by local laboratory technicians.
Cytometry Part B Clinical Cytometry 02/2008; 74 Suppl 1:S90-7. · 2.53 Impact Factor
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ABSTRACT: To assess the diagnostic accuracy and clinical utility of a simplified low cost method for measuring absolute and percentage CD4 counts with flow cytometry.
A CD4 counting method (Blantyre count) using a CD4 and CD45 antibody combination with reduced blood and reagent volumes. Diagnostic accuracy was assessed by measuring agreement of the index test with two other assays (TruCount and FACSCount). Clinical utility was investigated by comparing CD4 counts with the new assay with WHO clinical staging in patients with HIV.
Research laboratories and antiretroviral therapy clinic at a medical school and large government hospital in southern Malawi.
Assay comparisons were performed on consecutive blood samples sent for CD4 counting from 129 patients with HIV. Comparison of CD4 count with staging was conducted on 253 consecutive new patients attending the antiretroviral therapy clinic.
Limits of agreement with 95% confidence intervals between index test and reference standards.
The limits of agreement for Blantyre count and TruCount were excellent (cell count -48.9 to 27.0 x10(9)/l for absolute counts in the CD4 range <400x10(9)/l and -2.42% to 2.37% for CD4 percentage). The assay was affordable with reagent costs per test of $0.44 ( pound0.22, euro0.33) for both absolute count and CD4 percentage, and $0.11 for CD4 percentage alone. Of 193 patients with clinical stage I or II disease, who were ineligible for antiretroviral therapy by clinical staging criteria, 73 (38%) had CD4 counts <200x10(9)/l. By contrast, 12 (20%) of 60 patients with stage III or IV disease had CD4 counts >350x10(9)/l.
This simplified method of counting CD4 cells with flow cytometry has good agreement with established commercial assays, is affordable for routine clinical use in Africa, and could improve clinical decision making in patients with HIV.
BMJ (Clinical research ed.). 08/2007; 335(7612):190.
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ABSTRACT: The objective of this study was to investigate the utility of finger-prick blood samples for CD4 cell counting. We estimated agreement between CD4 cell counts in paired finger-prick and venous samples from 110 HIV-infected adults from Malawi. Bias was 6.6 cells/microl (limits of agreement -50.7 and 63.7 cells/microl) for absolute counts and 0.71% (limits of agreement -2.07 and 3.48%) for %CD4/lymphocyte suggesting that finger-prick blood samples can be used interchangeably with venous samples for CD4 cell counting.
AIDS 08/2007; 21(12):1643-5. · 6.24 Impact Factor
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Robert A Kingsley,
Chisomo L Msefula,
Nicholas R Thomson,
Samuel Kariuki,
Kathryn E Holt,
Melita A Gordon,
David Harris,
Louise Clarke,
Sally Whitehead,
Vartul Sangal,
Kevin Marsh,
Mark Achtman,
Malcom E. Molyneux,
Martin Cormican,
Julian Parkhill, Calman A Maclennan,
Robert S Heyderman,
Gordon Dougan
Genome Research, v.19, 2279-2287 (2009).
