Centrifugation improves the detection of HIV-1 p24 antigen in plasma from children born to mothers infected with HIV-1

https://doi.org/10.1016/j.jviromet.2009.01.010Get rights and content

Abstract

Detection of HIV proteins and/or nucleic acids is necessary for the diagnosis of perinatal HIV infection. Despite its low sensitivity, detection of p24 antigen in plasma is a simple and economic method for the diagnosis of HIV in exposed children. The aim of this study was to improve the sensitivity of detection of p24 using centrifugation of plasma.

Forty-seven selected stored samples from 37 children (23 infected, 14 uninfected, median age of 137 days) were examined. Plasma samples (volume 0.3–1.5 ml) were defrosted, centrifuged at 23,500 × g at 4 °C for 60 min and determination of p24 was carried out in the resuspended pellet (0.12 ml).

In 32 plasma samples from infected children, p24 was found originally in 6 (18.7%) and resulted positive in 24 (75%) pellets. When only one sample per child was considered, sensitivity was significantly higher in pellets, 3/23 uncentrifuged plasma samples and 15/23 pellets (McNemar Test, p < 0.001). Specificity was 100%. The absorbance/cut-off ratio was always higher in the pellets from positive children (p = 0.028). Plasma samples with volumes of 1 ml or more achieved a higher sensitivity (91.7% vs. 36.4%, p = 0.009).

Centrifugation of plasma samples prior to determination of p24 in pediatric patients resulted in a significant increase in sensitivity.

Introduction

The human immunodeficiency virus (HIV) belongs to the family Retroviridae, genus Lentivirus. Vertical transmission of HIV, the major source of pediatric infection, has diminished dramatically in developed countries as a result of antiretroviral prophylaxis (Volmink et al., 2007). In Argentina, pediatric diagnosis requires at least two separate samples to determine the infection status (Ministerio de Salud de la Nación, 2004). A child is considered HIV positive if PCR and/or detection of p24 antigen are positive in both samples (CDC, 1994).

Inexpensive, easy-to-perform, technologically simpler assays for diagnosis are always needed in resource-limited settings in developing countries (Fiscus et al., 2006, Aledort et al., 2006).

An alternative approach to the qualitative PCR can be the determination of p24 antigen in plasma. Since the limitation of this test is related to low sensitivity, several methods that improve successfully detection are in use: sensitivity obtained by acid or heat treatment was 59% (Paul et al., 1997), 81% (Miles et al., 1993) and 96.7% (Zijenah et al., 2005), by heat treatment and signal amplification it was 91.7% (Fiscus et al., 2007) and a combination of these treatments resulted in a sensitivity of 97% (Tehe et al., 2006), 100% (Nadal et al., 1999), or 91.3% (Nouhin and Nguyen, 2006).

A previous study aimed at concentrating antigen in plasma from adult patients (Schüpbach, 2003). In these assays, most of the antigen was still found in the soluble phase, therefore, it was considered that it would not be part of virus particles. Several mechanisms can account for the predominance of soluble p24 antigen over p24 associated with virus particles once the immune response has started (Schüpbach, 2003). Additionally, other investigations showed that antibody-mediated complement lysis may also explain the presence of soluble p24 antigen (Huber et al., 2006). However, virolysis may be less effective in pediatric patients due to an immature immune system and that fewer maternal antibodies are present. This hypothesis led us to try to improve the detection of p24 antigen using pellets obtained by centrifugation of plasma samples from the pediatric population where more p24 antigen may be present in intact virions.

Section snippets

Materials and methods

This study was evaluated and approved by the local independent Institutional Review Board from the School of Medicine, University of Buenos Aires, Argentina.

Detection of p24 antigen in plasma from children

When determination of p24 antigen was performed in 32 uncentrifuged plasma samples from infected pediatric patients, this antigen was detected in 6 (18.7%) of them.

After centrifugation of plasma samples, p24 antigen was detected in 24/32 (75%) plasma pellets, including the 6 p24 positive uncentrifuged plasma samples. When one single sample from each child was considered, sensitivity was significantly higher in plasma pellets since p24 was positive originally in 3/23 uncentrifuged plasma samples

Discussion

Several modifications are applied currently to the basic determination of p24 antigen by ELISA in order to increase the sensitivity of the technique which result in a more accurate diagnosis of pediatric HIV (Fiscus et al., 2007, Miles et al., 1993, Nadal et al., 1999, Nouhin and Nguyen, 2006, Paul et al., 1997, Tehe et al., 2006, Zijenah et al., 2005). Another modification to the basic technique is suggested now as an alternative or in addition to those already in use. The results obtained

Acknowledgements

Special thanks to Biochemist Moira Vignoles for the technical assistance and to Carolina Berini (BS) and Mr. Sergio Mazzini for the revision of the manuscript.

References (17)

  • A. Tehe et al.

    Quantification of HIV-1 p24 by a highly improved ELISA: an alternative to HIV-1 RNA based treatment monitoring in patients from Abidjan. Côte d’Ivoire

    J. Clin. Virol.

    (2006)
  • J.E. Aledort et al.

    Reducing the burden of HIV/AIDS in infants: the contribution of improved diagnostics

    Nature

    (2006)
  • P.C. Aulicino et al.

    HIV-1 genetic diversity in Argentina and early diagnosis of perinatal infection

    Medicina (B Aires)

    (2006)
  • A. Ceballos et al.

    Efficacy of strategies to reduce mother-to-child HIV-1 transmission in Argentina, 1993–2000

    J. Acquir. Immune Defic. Syndr.

    (2002)
  • Centers for Disease Control and Prevention, 1994. Revised classification system for HIV infection in children less than...
  • S.A. Fiscus et al.

    HIV-1 viral load assays for resource-limited settings

    PLoS Med.

    (2006)
  • S.A. Fiscus et al.

    Ultrasensitive p24 antigen assay for diagnosis of perinatal human immunodeficiency virus type 1 infection

    J. Clin. Microbiol.

    (2007)
  • Huber, M., Fischer, M., Misselwitz, B., Manrique, A., Kuster, H., Niederöst, B., Weber, R., von Wyl, V., Günthard,...
There are more references available in the full text version of this article.
View full text