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Keywords:

  • HIV screening;
  • pregnant women;
  • assays;
  • prevalence;
  • WHO strategies;
  • Republic of Congo

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Different strategies can be applied for the screening of HIV infection, depending on the local seroprevalence. Within a WHO type III strategy, we compared the results of two different second-line methods for HIV screening of a population of pregnant women in the Republic of Congo. Sera from 3614 consecutive pregnant women were tested for HIV with Genescreen Plus Ag/Ab EIA assay; positive specimens were retested with two different second-line methods. (Determine HIV-1/2 rapid test and Vironostika HIV Ag/Ab specific EIA assay). Discordant samples were tested with HIV-1/2 Western Blot and, if necessary, HIV RNA molecular assay. Of the 3614 sera, 221 were positive with Genscreen. Among them, 21 and 10 tested negative with Vironostika and Determine, respectively. A 100% correspondence with 3rd line confirmation test results was found in Genscreen positive/Vironostika negative samples, whereas a 5.5% overestimation of HIV seroprevalence was observed when Determine, instead of Vironostika, was used as second-line test. The choice of appropriate assays in adequate sequence, within the correct WHO strategy, is pivotal to minimize the risk of overtreatment of HIV infection.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In recent years, several initiatives for patient screening have been implemented in developing countries with variable levels of HIV seroprevalence (Fassinou et al. 2004; Thior et al. 2006; Marazzi et al. 2007; Sakarovitch et al. 2007). This has been determined by constraints including the cost of the tests, the portability on the field, the organization of the local socio-sanitary system and the possible poor performance of the tests with respect to locally circulating HIV strains/clades. Recent evaluations of the tests used to detect incident HIV cases in West Africa, for example, showed that the performance of different assays for the early detection of HIV infection was only partially satisfactory (Sakarovitch et al. 2007).

Depending on the ultimate goals of the projects, which may range from prevention of mother-to-child transmission (MTCT) to treatment of symptomatic patients, different screening strategies to detect HIV infection are applied. The World Health Organization (WHO) recommends that any HIV-testing strategy has to be based on the three main criteria, including (i) the aim of the test (surveillance, blood screening or diagnosis in individual patients), (ii) the performance of the methods (sensitivity and specificity) and (iii) the HIV seroprevalence within the study population. According to these parameters, three strategies have been identified, which may include up to three sequential tests (WHO 2001).

As recently shown (Gray et al. 2007; Powers et al. 2007; Sakarovitch et al. 2007), clinical studies that require the identification of established infection for subsequent inclusion of patients in treatment protocols might fraught with false positive and false negative identification of screened subjects. In the case of false positive results, inappropriate diagnosis and treatment may be administered to healthy individuals.

In 2004, we started an interventional program for the prevention of MTCT of HIV in the city of Pointe Noire, Republic of Congo (‘Kento-Muana’ Project), in which all pregnant women attending three antenatal clinics in urban and peripheral areas were screened for HIV seropositivity. A WHO type III algorithm was applied, as indicated by local, pre-existing, seroprevalence data (CREDES 2003). In order to clarify possible discrepancies between laboratory and clinical data and to avoid potential unnecessary treatment of HIV false-positive women, we decided to compare different screening tests, with the aim of finding the best approach to minimize the risk of misdiagnosing HIV infection. The present work shows that the possibility of unnecessary treatment of falsely positive women and their offspring is possible, depending on the tests used within the recommended screening strategy.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The study is a prospective evaluation of the performance of two different diagnostic approaches within a type III WHO strategy in an area of HIV seroprevalence of 4–8% (WHO 2001; CREDES 2003). From September 2005 to December 2006, 3614 consecutive pregnant women were tested for HIV infection. Serum specimens (one for each patient) were coded and centrally analysed in the project’s dedicated laboratory. All specimens were tested with the same initial test (Genscreen Plus HIV Ag/Ab EIA; BIO-RAD, Marne la Coquette, France) and then the positive specimens (221) were re-tested with second-line methods, using both a rapid screening test (Determine HIV-1/2 rapid, immunochromatography, Abbott Laboratories, Tokio, Japan), as commonly used locally, and a highly specific EIA (Vironostika HIV Ag/Ab UniForm EIA by Biomerieux Laboratories, Boxtel, NL, USA). Discordant samples were then tested with HIV-1 Western Blot (New Lav Blot I; BIO-RAD, Marne la Coquette, France) (WB1), whose results were interpreted according to CDC criteria (Centers for Disease Control (1989)). Samples still indeterminate after WB1 testing were assayed for HIV-RNA by currently used commercial assays and HIV-2 WB (New Lav Blot II; BIO-RAD, Marne la Coquette, France) (WB2).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Of the 3614 sera, 221 (6.1%) were positive with Genscreen. Of them, 21 (9.5%) and 10 (4.5%) tested negative with Vironostika and Determine, respectively. Of the 21 Vironostika-negative samples, 10 were also negative with Determine and were confirmed as true negative by WB1 test, while the 11 Determine positive samples were actually negative (7) or indeterminate (4) with WB1. The four WB1 indeterminate samples tested negative by HIV-RNA and WB2 assays. All these samples were considered as true negative (i.e. false positive at Determine), as shown in the Fig. 1. Overall, a 5.5% overestimation of seroprevalence (from 5.8% to 5.5%) would have been observed, if Determine instead of Vironostika had been used as second-line test. On the basis of these results, the sensitivity specificity and positive predictive value (PPV) were 100%, 99.68% e 94.8%, respectively, when Determine was used as second-line test as opposed to 100% in all instances when Vironostika assay was performed. Analysis of total reagent costs, including WB1 in discordant sera, showed superimposable expenses of approximately 1.9 € for each sample (when computed on the 3614 sera: 6,794 € and 6,713 €, using Determine or Vironostika as second-line test, respectively). If we had included these women in our prophylactic project (including antiretroviral therapy during pregnancy, at delivery, and afterwards, and prophylaxis in the newborn, including formula feeding supply for 3 months), this would have led to an estimated cost increase of 682 € for every mother/child pair with a total of 7502 € for 11 pairs.

image

Figure 1.  The figure summarizes the algorithms used and the problems encountered in the HIV seropositivity screening process. The 221 Genscreen (G) positive samples were subsequently tested with Determine (D) and Vironostika (V). The 10 G-positive but D-negative samples were confirmed as true negative with Western-Blot. By contrast, among the 211 G-positive and D-positive samples, 11 were actually false-positive as shown by negative WB (seven cases) and HIV-RNA (four cases). These samples were all V-negative.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Several enzyme immunoassay (EIA) tests for the detection of HIV-1 and HIV-2 serum antibodies have been developed and are available for the diagnosis of HIV infection. In areas with HIV seroprevalence <10%, the algorithm of choice is represented by an initial screening with a sensitive assay, followed by the confirmation of positive specimens with a more specific assay. Final confirmation in discordant cases is usually obtained with a positive Western Blot. Differences, however, exist in the overall performance of distinct algorithms, depending on several factors, including not only HIV seroprevalence but also economic resources, availability of laboratory equipments and the composition of prevailing circulating HIV-1 strains. Our study compared a very specific test vs. a sensitive test in the second diagnostic step. Using an oversensitive test in the confirmation step is actually inappropriate, but this is what was being done in the Republic of Congo, according to a survey conducted in the few local laboratories. However, soon after starting the project, we realized that some patients, identified as HIV positive with the diagnostic approach that put in sequence Genscreen and Determine, were probably cases of false-positivity, at least based on clinical evaluations. Biomolecular studies confirmed the clinical impression, because 5.2% (11/211) of positive patients were actually not infected when tested with WB and/or biomolecular techniques. The same individuals would have been correctly diagnosed as HIV negative if, since the beginning, Vironostika were used as second-line test. Although the difference in specificity was low (99.68%vs. 100%), this diagnostic mistake would have led to the unnecessary treatment of mother and child, with obvious consequences in terms of toxicity and cost, without considering the psychological and social impact of misdiagnosing a dreadful infection like HIV. Both diagnostic approaches were in agreement with WHO guidelines, but the choice of each test was crucial in influencing the overall performance of the diagnostic approach. Indeed, false positive testing appeared to be related to the performance of each assay: a highly specific EIA assay as second confirmatory step allows to correctly identify these samples when compared with less specific ones. Previous experiences in low-income countries suggest the usefulness of rapid field testing (Brattegaard et al. 1993; Stetler et al. 1997; Nkengasong et al. 1999; Koblavi-Deme et al. 2001; Rouet et al. 2004). However, a recent study in Uganda evidences the risk of over diagnosis of HIV infection using an algorithm based on three rapid tests: Gray et al. (2007) show a significant increase in PPV when weak bands were not classified as positives. Moreover, it has to be noted that these strategies were applied to WHO II areas with >10% seroprevalence, while the present study was performed in an area with a 5% prevalence. Similar data reported from the Indian region are in agreement with our findings (Iqbal et al. 2005). It is possible that in regions with median/low seroprevalence as in the Republic of Congo, intervention efforts may favour the use of rapid field testing in order to increase the screening efficiency especially in rural areas (Giuliano et al. 2007). Our work shows that rapid testing could be maintained, but, as suggested by Rouet et al. (2004), a serial-testing strategy using at least one assay with high specificity has to be used to avoid unnecessary treatment of uninfected mother–child pairs.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The project was financially supported by Ente Nazionale Idrocarburi, S.A., the Italian oil company.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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