In resource-poor countries, the major constraint in the use of DNA PCR for diagnosis of HIV-1 infection in infants under the age of 18 months is the cost of the equipment and the reagents. In addition, highly trained laboratory personnel and stringent quality assurance measures are needed to run this assay for routine diagnosis of HIV infection in infants.
In Zimbabwe, enzyme linked immunosorbent assay (ELISA) is routinely performed in both public and private laboratories for diagnosis of various infections including HIV-1. However, because of the transplacental transfer of maternal IgG antibodies, which may persist in infants for up to 18 months, ELISA is not suitable for diagnosis of HIV-1 infection in these infants. Therefore alternative methods are needed for this purpose. In addition, both public and private laboratories have flow cytometers or FACSCount machines for enumeration of T cell subset profile and automated haematological analysers for routine full blood counts with differential. With this equipment available in the country we evaluated the CD4/CD8 ratio as an alternative diagnostic test for infant HIV-1 infection.
In our investigation we used Epics XL Coulter flow cytometer equipped with System II software (Beckman Coulter, Miami Florida, USA) for the following reasons. With this instrument, CD4/CD8 ratios can be conveniently obtained. In addition, when used in double platform setting tandem with a haematological analyzer, the results also show absolute CD4+ cell counts and both %CD4 and %CD8 values among lymphocytes. The %CD4 value among lymphocytes is the recommended parameter for analyzing pediatric samples, as absolute counts for infants are age sensitive and variable. A simpler single platform system such as the FACScount (Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA), is not fully suited for pediatric work as it provides CD4+, CD8+, CD3+ T lymphocyte counts and CD4/CD8 ratio but %CD4 are not available. Of note, %CD4 expressed among CD3+ T lymphocytes, is a different parameter from the customary %CD4 expressed among lymphocytes. The %CD4 expressed among lymphocytes and not the %CD4 expressed among CD3+ T lymphocytes is recommended for decision making to initiate ARV therapy in children under the age of 18 months .
In Zimbabwe, CD4+ cell count, is less expensive than PCR and may provide additional information of value to the clinician with respect to prognosis, and the need for prophylaxis and treatment. Optimal flow cytometry for determination of T cell subset profile, offers the added advantage that CD4/CD8 ratio will determine the infection status of the infant while the % CD4 will provide information on decision-making for commencement of HAART.
Overall, the CD4/CD8 ratio had a ≥ 98% sensitivity for diagnosis of infant HIV-1 infection and a specificity ≥ 98%. Both sensitivity and specificity were 100% for infants in the 12–18 months age group. Interestingly, in parallel studies performed in 250 HIV-1 seropositive adults, 249 had a CD4/CD8 ratio of <1. The CD4/CD8 ratio of the one patient was 1 at enrollment and has remained so for over one year.
When interpreting our data, it is important to note that normal T cell subset values among African children differ from those of other populations [14–16]. A study in Guinea Bissau , reported that Guinean children under the age of 2 years had lower %CD4 and CD4/CD8 ratios and higher %CD8 when compared to their counterparts from developed countries. Interestingly, girls had higher CD4/CD8 ratios and lower %CD8 than boys. In our study, there were no statistically significant differences in absolute T cells, or percentages or CD4/CD8 ratio between boys and girls (Table 3 "refer to Additional file 1").
A very few studies in Africa have compared T cell subset profiles between HIV-1 infected and uninfected infants under the age of 2 years [14, 17]. Moodley and colleagues in South Africa reported that the most marked changes in lymphocyte subset of HIV-1 infected children aged between 3 and 15 months were a lower %CD4 and higher %CD8 relative to uninfected infants . In addition, CD4/CD8 ratio was a good predictor of poor clinical outcome at 3 months. The authors concluded that CD4/CD8 ratio and %CD4 among lymphocytes are reliable markers of HIV-1 infection in an African pediatric population. Furthermore a raised CD8+ cell count rather than a CD4+ cell count was a more specific prognostic marker of disease progression in HIV infected children.
Embree and colleagues, in Kenya, also reported that HIV-1 infected children had lower %CD4 and higher %CD8 by 3 months when compared to uninfected children . The authors concluded that %CD4 and %CD8 among lymphocytes could be useful as an adjunct in HIV-1 diagnosis.
The two African studies mentioned above, have documented the clinical value of %CD4, CD4/CD8 ratio and CD8 counts in HIV-1 infection in infants.
In summary, the CD4/CD8 ratio may be useful in identifying infected infants while %CD4 will identify infants who may benefit from cotrimoxazole prophylaxis and/or initiation of HAART, and for management of HIV-infected infants in developing countries in general. We thus propose use of flow cytometry, where available, as a point of care diagnostic tool for ill infants admitted to hospitals with clinical symptoms suggestive of HIV infection and/or AIDS.