Plasma HIV burden in Malawian children co-infected with rotavirus

Fifty-eight HIV-infected children with acute rotavirus diarrhea were tested for plasma HIV RNA. There was no difference between acute and convalescent mean viral loads, and little change in the CD4 cell counts. Compared with the 16 children who died within 4 weeks, 31 survivors had slightly lower viral loads at presentation and significantly higher CD4 cell counts. Low CD4 cell counts, but not HIV-1-RNA concentrations, were predictive of death. Local, enteric rotavirus infection did not appear to affect blood HIV viral load or CD4 cell counts in this small group of children. Rotavirus is an important cause of severe gastroenteritis in infants and young children worldwide. In developing countries, rotavirus infections account for 10–20% of deaths associated with gastroenteritis [1]. HIV is also prevalent in many developing countries, especially those in sub-Saharan Africa. In Malawi, approximately 30% of pregnant women are infected with HIV and 25–40% of their infants will become infected [2]. It is thus common for infants and children to be co-infected with these pathogens. Concurrent infections with several pathogens have been associated with transient, but significant, increases in the blood plasma HIV load [3–7]. In other cases, the HIV load was significantly increased in the semen of men with urethritis [8] or bronchoalveolar lavage of individuals with Pneumocystis pneumonia [9], but not in concurrent blood specimens. The purpose of this study was to evaluate the difference in plasma HIV-1-RNA levels between acute and convalescent samples in a subset of children co-infected with rotavirus [10]. The parent study [10] enrolled 1186 children under 5 years of age with acute gastroenteritis. A total of 102 children were infected with both HIV and rotavirus. Of these, 44 were not sampled primarily because of lack of parental consent. Therefore, 58 HIV-infected children ranging in age from 1 to 19 months were tested for HIV-1 RNA. The Malawi National Health Sciences and Research Committee gave ethical approval for the study. Written, informed consent was obtained from the child's parents or guardians before enrolment. A 10–20% (w/v) fecal suspension in phosphate buffered saline was tested for rotavirus antigen by enzyme-linked immunosorbent assay (ELISA; Rotaclone, Meridian Diagnostics, Cincinnati, OH, USA). Children older than 15 months were initially screened for HIV-1/HIV-2 antibodies using a rapid method (Serocard HIV, Trinity Biotech, Dublin, Ireland) and then screened by ELISA (Ortho Diagnostic Systems Ltd., Amersham, UK). Specimens that were reactive in the Ortho ELISA were confirmed using the Recombigen HIV-1/HIV-2 Rapid Test Device (Cambridge Diagnostics Ireland Ltd., Galway, Ireland). For children less than 15 months of age, a diagnosis of HIV infection was determined by an HIV DNA polymerase chain reaction using DNA from dried blood spots [11]. CD4 and CD8 T cell subsets were determined by FACScan flow cytometry (Becton-Dickinson, San Jose, CA, USA). HIV RNA was measured using the NucliSens assay (Organon Teknika, Durham, NC, USA) according to the manufacturer's instructions. Previous studies had determined that clade C is the predominant HIV-1 subtype in Malawi [12] and that the NucliSens assay accurately detects clade C [13,14]. An exploratory data analysis strategy was used. All reported P values should be interpreted accordingly. Linear mixed models were used to investigate the relationship between viral load (or CD4 cell count) and visit and survival. Logistic regression was used to model the probability of survival as a function of viral load and CD4 cell count. Transformations to log10 HIV-1-RNA copies/ml and to square root CD4 cell counts were used in the statistical analyses. Fifty-eight HIV-infected children (28 females and 30 males) with acute rotavirus diarrhea were included. Their median age was 6.5 months (range 1–19 months). Thirty-one children were examined at follow-up 3–4 weeks later. Eleven children had unknown survival status: nine children refused follow-up before the final visit and two children were lost to follow-up. A total of 16 children died. There was no difference in median (mean ± SD) viral load between baseline and follow-up time-points for those who survived 3 weeks or more: 306 902 HIV-RNA copies/ml (5.5 ± 0.7 log10 copies/ml) versus 289 734 copies/ml (5.5 ± 0.9 log10 copies/ml) (Fig. 1a). The baseline viral load was slightly higher (P = 0.101) for those who died compared with those who survived: 767 361 HIV-RNA copies/ml (5.9 ± 1.0 log10 copies/ml) versus 306 902 HIV-RNA copies/ml (5.5 ± 0.7 log10 copies/ml) (Fig. 1a).Fig. 1.: (a) Baseline HIV-RNA levels in blood of 16 children who died and 36 (out of 37) who survived and follow-up HIV-RNA levels of 29 (out of 31) children who survived. (b) Baseline CD4 cell counts of 16 children who died and 25 (out of 36) children who survived and follow-up CD4 cell counts of 20 (out of 31) children who survived.Similarly, the median (mean ± SD) CD4 cell counts at baseline and follow-up were not substantially different for the children who survived 3 weeks or longer: 706 CD4 cells (729 ± 144 CD4 cells) versus 779 CD4 cells (729 ± 100 CD4 cells) (Fig. 1b). In contrast, those who died had substantially lower median baseline CD4 cell counts (P = 0.012) compared with those who survived: 357 CD4 cells (289 ± 49 CD4 cells) versus 706 CD4 cells (729 ± 144 CD4 cells;P = 0.007) (Fig. 1b). This study suggests that local enteric rotavirus infection did not affect the systemic blood viral load in this small group of dually infected children. Data from different studies regarding co-infections and changes in plasma viral load have yielded conflicting results. However, in some studies in which no changes were observed in the blood viral load, significant differences in the HIV viral burden were documented in the affected body compartment [8,9]. In addition, Fransje et al.[15] demonstrated an increased detectability of HIV RNA in the stool of individuals who had diarrhea (62%) compared with those without diarrhea (43%). It is possible that rotavirus infection increases HIV-RNA levels in stool, but not in the peripheral blood. There was no difference in the CD4 cell counts between the baseline and follow-up visits. Children who died within 4 weeks of acute rotavirus infection had lower CD4 cell counts at baseline, but did not demonstrate substantial differences in viral load when compared with children who survived. There are several important limitations of this study. We do not have control data from HIV-infected Malawian children who were not co-infected with rotavirus, and thus we cannot exclude an effect on viral load that persists for 3–4 weeks. Despite the large initial sample size of 1186 patients, only 102 were co-infected with both HIV and rotavirus and only 58 contributed to the study. It is partly for this reason that we have treated the investigation as an exploratory study. In summary, it does not appear that rotavirus infection had a systemic effect on the HIV-RNA burden in the co-infected children studied. However, short-term survival in these children may be dependent on the CD4 cell count. This finding, if substantiated, would warrant the development of an algorithm that could identify and target these co-infected children with low CD4 cell counts for additional treatment. The safety of rotavirus vaccination in HIV-infected infants needs to be determined. Acknowledgements The authors gratefully acknowledge the children who participated in this study and their parents and guardians as well as the technical assistance of Mr B.D.M. Thindwa. Charles Jeread Nigel A. Cunliffeefh Irving F. Hoffmanb Paul W. Stewartc Rakhi Kilaruc Robin L. Broadheade Malcolm E. Molyneuxgh C. Anthony Hartf Susan A. Fiscusa