Jane Mullen

Maternal viral load, CD4 cell count and vertical transmission of HIV‐1

HIV load and CD4 cell numbers were measured among 95 HIV infected women during pregnancy in order to determine their value as prognostic markers for transmission of virus from mother to infant. Among the 94 live births, 13 children were infected with HIV, 69 were uninfected and 12 were of unknown infection status. HIV RNA levels, as measured by nucleic acid sequence based amplification, were significantly higher (P < 0.001) in women who transmitted virus than among those who did not transmit and maternal viral load was a stronger predictor of transmission than CD4 cell number. The predicted rate of transmission relative to maternal HIV RNA was 2% at 1,000 copies, 11% at 10,000 copies and 40% at 100,000 copies/ml. Little variation in viral load occurred during pregnancy and there was an association between viral load and prematurity, the mean gestation at delivery decreasing by 1.3 weeks for every 10‐fold increase in maternal HIV RNA (P = 0.007). This study demonstrates that a high level of maternal HIV RNA is a risk factor for transmission of virus to the infant and maternal viral load is of more value as a prognostic marker for transmission risk than CD4 cell number. High viral load is also associated with premature delivery. Maternal viral load is therefore a useful marker on which to base management decisions during pregnancy. J. Med. Virol. 54:113–117, 1998.

The natural history and clinical significance of intermittent viraemia in patients with initial viral suppression to < 400 copies/ml.

ObjectivesTo determine the prevalence and prognostic significance of intermittent viraemia (IV) in patients who attained an undetectable viral load (VL) < 400 copies/ml within 6 months on highly active antiretroviral therapy (HAART). MethodsRetrospective analysis of viral load rebound ⩾ 400 copies/ml and CD4 cell counts rise for 765 patients followed for ⩾ 12 months following initial VL undetectability, comparing the 226 (29.5%) who maintained an undetectable VL for > 1 year from initiation of HAART and 122 (15.9%) who had one or more episodes of IV. Genotypic resistance was evaluated at the time of the first episode of IV ⩾ 2000 copies/ml. ResultsPatients with IV had a threefold higher rate of sustained virological rebound [hazards ratio (HR), 3.15; 95% confidence interval (CI), 1.72–5.77;P < 0.001). For patients with and without IV, the Kaplan–Meier estimates at 24 and 36 months after initiation of HAART were 19.3% (95% CI, 8.9–21.5) versus 7.7% (95% CI, 4.5–13.0) and 31.6% (95% CI, 21.8–44.2) versus 12.9% (95% CI, 7.5–21.5), respectively (P < 0.001). The median CD4 cell count rise at 18 and 24 months was significantly lower in those with IV than in those without: 138 [interquartile range (IQR), 58–221] versus 224 × 106 cells/l (IQR, 119–357) (P = 0.0001) and 200 (IQR, 89–294) versus 260 × 106 cells/l (IQR, 125–384) (P = 0.003), respectively. In a subgroup of 16 patients, genotypic resistance mutations were found in the reverse transcriptase gene for five (31%) and in the protease gene in one. A probable contributing factor/event was identified for most patients with IV, such as poor adherence (42.6%), intercurrent infection (26.2%) or drug interaction (6.8%). ConclusionsPatients with IV > 400 copies/ml are three times more likely to experience sustained viral rebound and to have an impaired CD4 cell rise relative to those who maintain undetectable VL. This supports the adoption of a more pro-active approach to treatment intensification and the need for caution with structured treatment interruptions.

Evidence for horizontal and not vertical transmission of human herpesvirus 8 in children born to human immunodeficiency virus-infected mothers.

A survey of antibody responses to human herpesvirus 8 (HHV-8) was undertaken to examine the mode of transmission of this virus to children born to mothers with HIV. Methods. Serum samples from a cohort of 92 mother-infant pairs and a cross-sectional cohort of 100 children (median age, 4 years) were tested. In the cohort of mother-infant pairs, 14 infants were HIV-infected, 72 were not and the HIV status was unknown for 6. In the cohort of children 70 were HIV-infected and 30 were vertically exposed but uninfected. Serologic responses to two HHV-8 antigens, latency-associated nuclear antigen and the structural antigen encoded by open reading frame 65 were detected by immunofluorescent antibody test and enzyme-linked immunoassay. Results were confirmed by Western blot. Results. All HHV-8-seropositive mothers were African (17 of 92, 18.5%). Six of their infants were HHV-8-seronegative and 11 had at least 1 HHV-8-seropositive sample. One of the 11 infants tested only at birth had a lower antibody titer than the mother; the remaining 10 infants had decreasing titers up to 7 months of age and 6 became seronegative. No infants born to HHV-8-seronegative mothers had antibodies to the virus. The seroprevalence to HHV-8 was 6% in the cohort of children. All had African mothers and their median age was greater than that of the cohort (8.4 vs. 4.0 years). Five were coinfected with HIV. Conclusions. HHV-8 was not vertically transmitted by any of the HIV-coinfected mothers. Acquisition of antibody to HHV-8 occurred in older children, implying a horizontal route of transmission.

Assisted conception in HIV discordant couples: evaluation of semen processing techniques in reducing HIV viral load

Artificial insemination with motile spermatozoa prepared from HIV-infected men using standard procedures has been employed with many HIV-discordant couples. We have demonstrated that processing semen from HIV positive men can reduce HIV levels, measured as HIV1 RNA copies/ml using nucleic acid based sequence amplification (NASBA), to undetectable levels (less than 400 copies/ml) but not in all samples. We believe that all processed samples should be tested prior to insemination.

K65R and Y181C are less prevalent in HAART-experienced HIV-1 subtype A patients.

The vast majority of HIV-1 infections globally are caused by subtype A or C, although little is known about their drug resistance profiles. We found that HAART-experienced patients infected with subtype A had a lower prevalence of K65R and Y181C than those with subtypes B or C, despite similar exposure to antiretroviral agents that select for these mutations. If confirmed, this information may be important in the planning of antiretroviral regimens in patients infected with HIV-1 subtype A.

Significance of placental damage in vertical transmission of human immunodeficiency virus

The significance of physical breaches of the trophoblastic layer of the placenta in transmission of HIV from mother to infant was evaluated in 17 HIV‐infected pregnant women. Samples of peripheral blood were obtained from the women during pregnancy and at delivery, at which time a small piece of placental tissue was obtained from a random site and immediately placed into fixative. Blood samples were obtained from infants at or shortly after birth and thereafter at approximately 3‐month intervals, until the age of 18 months, in order to determine their HIV infection status. HIV RNA and p24 antigen were quantified in maternal plasma and CD4 cells enumerated. Paediatric diagnosis was conducted using polymerase chain reaction, virus isolation, detection of p24 antigen, and measurement of class‐specific antibodies. Placental damage was quantified and evaluated using transmission electron microscopy. Maternal viral load was low, with a mean RNA copy number of 8,237 per millilitre of plasma (range 230–37,233 copies/ml). Only two women were p24‐antigenaemic, and CD4 numbers ranged from 0.09 to 2.8 × 109/l. There was evidence of breaks in the trophoblastic surface to the depth of the basement membrane in all 17 placentas, and perivillous fibrinoid deposits were also observed to a varying degree in all samples. However, none of the 13 infants available for follow‐up had evidence of infection with HIV. Superficial damage to the trophoblastic surface of the placenta, with exposure of the basement membrane and potential exposure of CD4‐expressing cells, does not appear to be a significant factor in the transmission of HIV from mother to infant during pregnancy.

Problems in the interpretation of HIV-1 viral load assays using commercial reagents.

During routine monitoring of human immunodeficiency virus (HIV) viral load, two problems arose. First, a number of patients, the majority being African, were found to have low viral loads by the Chiron branched‐chain DNA assay in conjunction with low CD4+ cell numbers. In order to determine whether this was due to failure of the branched‐chain DNA assay to detect non‐B subtypes of HIV, selected samples were subtyped and HIV RNA quantified by branched‐chain DNA, NASBA, and the Roche Monitor RT‐PCR assay. Twenty‐eight (97%) of 29 Africans were infected with a non‐B subtype of HIV and 15 (93.7%) of 16 non‐Africans with subtype B. Twenty‐three samples had a low viral load by branched‐chain DNA, which was confirmed by the NASBA and RT‐PCR assays. All three assays detected B and non‐B subtypes with similar efficiency; NASBA failed to detect HIV RNA in a small number of non‐B samples. Discrepancies between viral load and CD4+ cell numbers did not appear therefore to be related to subtype. Second, while quantification of HIV RNA was being conducted using version 2 of the branched‐chain DNA assay (lower detection limit 500 HIV RNA copies/ml) the manufacturers had developed a more sensitive assay and a comparative evaluation was therefore conducted. In approximately 30% of samples the viral load was up to 10 times higher with the more sensitive assay. These experiences emphasise the importance of close collaboration between the clinic and the laboratory. J. Med. Virol. 61:187–194, 2000.

Genotyping of B and Non-B Subtypes of Human Immunodeficiency Virus Type 1

ABSTRACT Current HIV-1 genotyping assays were developed using subtype B viruses prevalent in Western countries. It is not clear whether these assays are appropriate for use among African patients, who are likely to be infected with non-B subtypes. We evaluated the Bayer TRUGENE HIV-1 genotyping (TG) assay using prospectively collected samples from HIV-1-infected individuals who acquired infection in either sub-Saharan Africa or the West (Europe, North America, and Australia). Plasma samples from 208 individuals with an HIV-1 viral load of >1,000 copies/ml were tested using version 1 primers supplied with the TG assay. If these failed, an alternative primer set version 1.5 was used. Of the 208 individuals, the likely origin of infection was Africa (n = 104), Western (n = 87) and “Others” (i.e., all other geographic locations or origin not certain; n = 17). Among the three groups, the version 1 primers were successful in 85 (82%), 77 (89%), and 13 (76%) individuals, respectively (P = 0.1). Of the remaining 32 samples, 30 were successfully amplified by using the version 1.5 primers. HIV-1 subtypes deduced from the reverse transcriptase sequences correlated with the likely origin of infection: Africa (28A, 3B, 33C, 13D, 6G, 4J, 2K, 5CRF01_AE, and 10CRF02_AG), Western (86B and 1K), and Others (1A and 16B). The success of the version 1 primers correlated with viral load (P < 0.014) and not with HIV-1 subtypes. A protocol based on version 1 primers, followed by 1.5 primers, was successful in sequencing 99% of the samples in this cohort.

Early hepatitis B virological rebound on entecavir through selection of lamivudine-associated mutations

G109A in the reverse transcriptase region, and I13V, L33I, L63P and V77I in the protease region. The last HAART regimen before transplantation, discontinued pre-operatively, was lamivudine 150 mg twice a day, tenofovir 300 mg once daily and fosampre-navir 1400 mg twice a day. Immunosuppression was initially achieved with cyclosporin (300 mg twice a day) and steroids, and then maintained with cyclosporin only. On the third post-operative day, HAART was resumed with emtricitabine 200 mg, tenofovir 300 mg once daily and a subcutaneous (sc) injection of enfuvirtide 90 mg twice a day. At 3 months after transplantation, the CD4þ T cell count and HIV RNA level were 263 cells/mm 3 (20%) and ,50 copies/mL, respectively, while HCV RNA was 4.09 log 10 copies/mL (Cobas Ampliprep/Cobas TaqMan HCV Test Roche Diagnostics). After 9 months, because of a further increase in HCV RNA levels (6.09 log 10 copies/mL) and of macro-and micronodular cirrhosis revealed by transjugular liver biopsy, the patient started anti-HCV treatment with pegylated interferon (PEG-IFN) alfa 2b (180 mg sc once a week) and riba-virin (1200 mg twice a day). Cyclosporin was administered twice daily and its dosage was adjusted individually to match target trough levels of 75– 125 mg/L. Four weeks later, because he had ongoing injection site reactions and complained of injection fatigue due to enfuvirtide and PEG-IFN, the patient switched from enfuvirtide to raltegravir (400 mg twice a day). Four weeks later, HIV RNA was still ,50 copies/mL, while the CD4þ T cell count had increased from 162 (18%) to 336 (23%). Self-reported therapeutic adherence was apparently optimal. No other potentially interacting drugs were administered. Cyclosporin levels were monitored at regular intervals during outpatient visits (Figure 1). Moreover, at week 4 and week 8 raltegravir concentrations were also measured by a validated HPLC method 7 before the morning dose and 3 h later and showed drug levels to be 60 and 5165 ng/mL at week 4, and 119 and 3386 ng/mL at week 8, respectively. No specific toxicity related to antiretroviral and immunosuppressive therapies was recorded. No administration of factor VIII for treatment of haemophilia A was requested. After 18 months of follow-up post-transplantation, the patient is alive and HIV RNA levels are undetectable. In our patient, a raltegravir-based regimen was chosen in order to avoid the potential deleterious effect of PIs on cyclos-porin. A new NNRTI-based regimen was excluded because etra-virine is a substrate for, and inhibitor …

Minority M184V variants in women exposed to 3TC/FTC-containing lopinavir-ritonavir (LPVr) regimens in pregnancy

7‐11 November 2010, Tenth International Congress on Drug Therapy in HIV Infection, Glasgow, UK