Adeno-associated virus 2 infection in children with non-A-E hepatitis.

An outbreak of acute hepatitis of unknown aetiology in children was reported in Scotland1 in April 2022 and has now been identified in 35 countries2. Several recent studies have suggested an association with human adenovirus with this outbreak, a virus not commonly associated with hepatitis. Here we report a detailed case-control investigation and find an association between adeno-associated virus 2 (AAV2) infection and host genetics in disease susceptibility. Using next-generation sequencing, PCR with reverse transcription, serology and in situ hybridization, we detected recent infection with AAV2 in plasma and liver samples in 26 out of 32 (81%) cases of hepatitis compared with 5 out of 74 (7%) of samples from unaffected individuals. Furthermore, AAV2 was detected within ballooned hepatocytes alongside a prominent T cell infiltrate in liver biopsy samples. In keeping with a CD4+ T-cell-mediated immune pathology, the human leukocyte antigen (HLA) class II HLA-DRB1*04:01 allele was identified in 25 out of 27 cases (93%) compared with a background frequency of 10 out of 64 (16%; P = 5.49 × 10-12). In summary, we report an outbreak of acute paediatric hepatitis associated with AAV2 infection (most likely acquired as a co-infection with human adenovirus that is usually required as a 'helper virus' to support AAV2 replication) and disease susceptibility related to HLA class II status.

Investigation into cases of hepatitis of unknown aetiology among young children, Scotland, 1 January 2022 to 12 April 2022.

On 31 March 2022, Public Health Scotland was alerted to five children aged 3-5 years admitted to hospital with severe hepatitis of unknown aetiology. Retrospective investigation identified eight additional cases aged 10 years and younger since 1 January 2022. Two pairs of cases have epidemiological links. Common viral hepatitis causes were excluded in those with available results. Five children were adenovirus PCR-positive. Other childhood viruses, including SARS-CoV-2, have been isolated. Investigations are ongoing, with new cases still presenting.

The experience of point-of-care testing for influenza in Scotland in 2017/18 and 2018/19 - no gain without pain.

BackgroundDuring the 2017/18 and 2018/19 influenza seasons, molecular amplification-based point-of-care tests (mPOCT) were introduced in Scotland to aid triaging respiratory patients for hospital admission, yet communication of results to national surveillance was unaccounted for.AimThis retrospective study aims to describe steps taken to capture mPOCT data and assess impact on influenza surveillance.MethodsQuestionnaires determined mPOCT usage in 2017/18 and 2018/19. Searches of the Electronic Communication of Surveillance in Scotland (ECOSS) database were performed and compared with information stored in laboratory information management systems. Effect of incomplete data on surveillance was determined by comparing routine against enhanced data and assessing changes in influenza activity levels determined by the moving epidemic method.ResultsThe number of areas employing mPOCT increased over the two seasons (6/14 in 2017/18 and 8/14 in 2018/19). Analysis of a small number of areas (n = 3) showed capture of positive mPOCT results in ECOSS improved between seasons and remained high (> 94%). However, capture of negative results was incomplete. Despite small discrepancies in weekly activity assessments, routine data were able to identify trend, start, peak and end of both influenza seasons.ConclusionThis study has shown an improvement in capture of data from influenza mPOCT and has highlighted issues that need to be addressed for results to be accurately captured in national surveillance. With the clear benefit to patient management we suggest careful consideration should be given to the connectivity aspects of the technology in order to ensure minimal impact on national surveillance.

Value of repeat testing using Cepheid GeneXpert CT/NG for indeterminate PCR results when diagnosing Chlamydia trachomatis and Neisseria gonorrhoeae.

Nucleic acid amplification tests (NAATs) are the most sensitive method for diagnosing chlamydia and gonorrhoea. We use the COBAS 4800 CT/NG combined assay (Roche Molecular Diagnostics, CA, USA), and whilst the majority of samples yield definitive results, a small proportion are reported as indeterminate. In these instances, it is usual practice to request repeat samples which delays diagnosis. This audit was twofold: first to establish the proportion of indeterminate results with current NAAT testing requiring re-sampling. Second, to determine whether a second NAAT such as Cepheid GeneXpert CT/NG assay (Cepheid, CA, USA) could be used on initial indeterminate samples to resolve indeterminate results, therefore reducing need for repeat sampling. During 2012, 144/21,931 (0.66%) samples were indeterminate for Neisseria gonorrhoeae, Chlamydia trachomatis or both, and a repeat sample was received in only 51.77% of patients with final results being delayed for more than 24 h. Over the next six months, there were 77/9472 (0.81%) indeterminate results. After an evaluation and introduction of the Cepheid assay, the number of indeterminate results fell to 9 (0.10%). Thus, use of the Cepheid assay significantly reduced indeterminate results, reduced reliance on a repeat sampling and significantly improved turnaround time, laboratory workflow and patient experience.

Lopinavir/ritonavir monotherapy after 24 weeks of second-line antiretroviral therapy in Africa: a randomized controlled trial (SARA).

BACKGROUND: Boosted protease inhibitor (bPI) monotherapy (bPImono) potentially has substantial cost, safety and operational benefits. It has never been evaluated as second-line antiretroviral therapy (ART) in Africa. METHODS: After 24 weeks of lopinavir/ritonavir-containing second-line therapy, DART participants were randomized to remain on combination therapy (CT), or change to bPImono maintenance (SARA trial; ISRCTN53817258). Joint primary end points were CD4(+) T-cell changes 24 weeks later and serious adverse events (SAEs); retrospectively assayed viral load (VL) was a secondary end point. Analyses were intention-to-treat. RESULTS: A total of 192 participants were randomized to CT (n=95) or bPImono (n=97) and followed for median 60 weeks (IQR 45-84). Participants received median 4.0 years (IQR 3.5-4.4) first-line ART. Median CD4(+) T-cell count at first-line failure was 86 cells/mm(3) (47-136), increasing to 245 cells/mm(3) (173-325) after 24-week induction when 77% had VL<50 copies/ml. Overall, 44 (23%) were receiving second-line therapy with bPI and nucleoside reverse transcriptase inhibitors (NRTI) only, and 148 (77%) with bPI plus non-NRTI (NNRTI) with or without NRTI. At 24 weeks after randomization to CT versus bPImono, mean CD4(+) T-cell increase was 42 (CT, n=85) versus 49 cells/mm(3) (bPImono, n=88; adjusted difference 13 [95% CI -15, 43], P=0.37; non-inferior compared with predetermined non-inferiority margin [-33]). Virological suppression was greater for CT versus bPImono (trend P=0.009): 77% (70/91) versus 60% (56/94) were <50 copies/ml, and 5% (5) versus 14% (13) were ≥1,000 copies/ml, respectively. A total of 0 (0%) versus 5 (5%) participants had major protease inhibitor mutations and 3 (3%) versus 0 (0%) new NNRTI/NRTI mutations were detected during follow-up. Two participants (1 CT and 1 bPImono) died >24 weeks after randomization, and 5 (2 CT and 3 bPImono) experienced SAEs (P=0.51). CONCLUSIONS: bPImono following a 24-week second-line induction was associated with similar CD4(+) T-cell response, but increased low-level viraemia, generally without protease inhibitor resistance. Longer-term trials are needed to provide definitive evidence about effectiveness in Africa.

Evolution of drug resistance during 48 weeks of zidovudine/lamivudine/tenofovir in the absence of real-time viral load monitoring.

OBJECTIVES: To describe the resistance mutations selected by a first-line regimen of zidovudine/lamivudine/tenofovir in the absence of real-time viral load monitoring. DESIGN: A substudy of 300 participants from the Development of Antiretroviral Therapy in Africa trial in Uganda and Zimbabwe, which compared managing antiretroviral therapy with and without laboratory monitoring. METHODS: Stored plasma samples from selected time points were assayed retrospectively for HIV-1 RNA. The pol gene in all baseline samples and those with HIV RNA >1000 copies per milliliter at weeks 24 and 48 were sequenced. RESULTS: The proportion with HIV RNA >1000 copies per milliliter increased from 15% at 24 weeks to 24% at 48 weeks. Eighteen of 31 (58%) genotyped samples at 24 weeks had ≥ 1 major nucleoside reverse transcriptase inhibitor-associated mutations compared with 41 of 47 (87%) at 48 weeks. Excluding 1 nonadherent patient, a mean of 2.0 (95% confidence interval: 1.3 to 2.8) thymidine analogue mutations (TAMs) developed between weeks 24 and 48 among 14 patients with HIV RNA >1000 copies per milliliter at both time points. K65R was detected in 8 of 63 (13%) patients and was negatively associated with number of TAMs (P = 0.01) but not viral subtype (P = 0.30). CONCLUSIONS: A high rate of acquisition of TAMs, but not of K65R, among patients with prolonged viraemia was observed. However, most patients were virologically suppressed at 48 weeks, and long-term clinical and immunological outcomes in the Development of Antiretroviral Therapy in Africa trial were favorable.

Novel subtype C human immunodeficiency virus type 1 envelopes cloned directly from plasma: coreceptor usage and neutralization phenotypes.

Human immunodeficiency virus type 1 (HIV-1) is classified into different phylogenetic subtypes, with subtype C representing more than half of the novel infections globally. However, there are relatively few subtype C envelopes available for study. We amplified 18 unique env genes from 13 patients who were infected with subtype C HIV-1 in six African countries and in Scotland to create replication-competent viruses. These envelopes are phylogenetically diverse across the subtype C spectrum, and have on average more N-linked glycosylation sites and slightly longer variable loops than previously described C envelopes. We found that CCR3 coreceptor usage is less prevalent in subtype C than in subtype B viruses, and these envelopes have varied sensitivity to neutralization. The subtype C chimeric viruses generated in this study will be useful for evaluating the breadth of neutralizing antibodies and other entry inhibitors.

Viral rebound and emergence of drug resistance in the absence of viral load testing: a randomized comparison between zidovudine-lamivudine plus Nevirapine and zidovudine-lamivudine plus Abacavir.

BACKGROUND: We investigated virological response and the emergence of resistance in the Nevirapine or Abacavir (NORA) substudy of the Development of Antiretroviral Treatment in Africa (DART) trial. METHODS: Six hundred symptomatic antiretroviral-naive human immunodeficiency virus (HIV)-infected adults (CD4 cell count, <200 cells/mm(3)) from 2 Ugandan centers were randomized to receive zidovudine-lamivudine plus abacavir or nevirapine. Virology was performed retrospectively on stored plasma samples at selected time points. In patients with HIV RNA levels >1000 copies/mL, the residual activity of therapy was calculated as the reduction in HIV RNA level, compared with baseline. RESULTS: Overall, HIV RNA levels were lower in the nevirapine group than in the abacavir group at 24 and 48 weeks (P < .001), although no differences were observed at weeks 4 and 12. Virological responses were similar in the 2 treatment groups for baseline HIV RNA level <100,000 copies/mL. The mean residual activity at week 48 was higher for abacavir in the presence of the typically observed resistance pattern of thymidine analogue mutations (TAMs) and M184V (1.47 log(10) copies/mL) than for nevirapine with M184V and nonnucleoside reverse-transcriptase inhibitor mutations, whether accompanied by TAMs (0.96 log(10) copies/mL) or not (1.18 log(10) copies/mL). CONCLUSIONS: There was more extensive genotypic resistance in both treatment groups than is generally seen in resource-rich settings. However, significant residual activity was observed among patients with virological failure, particularly those receiving zidovudine-lamivudine plus abacavir.

Host HLA B*allele-associated multi-clade Gag T-cell recognition correlates with slow HIV-1 disease progression in antiretroviral therapy-naïve Ugandans.

BACKGROUND: Some HIV infected individuals remain asymptomatic for protracted periods of time in the absence of antiretroviral therapy (ART). Virological control, CD4 T cell loss and HIV-specific responses are some of the key interrelated determinants of HIV-1 disease progression. In this study, possible interactions between viral load, CD4 T cell slopes, host genetics and HIV-specific IFN-gamma responses were evaluated in chronically HIV-1-infected adults. METHODOLOGY/PRINCIPAL FINDINGS: Multilevel regression modeling was used to stratify clade A or D HIV-infected individuals into disease progression groups based on CD4 T cell slopes. ELISpot assays were used to quantify the frequency and magnitude of HIV-induced IFN-gamma responses in 7 defined rapid progressors (RPs) and 14 defined slow progressors (SPs) at a single time point. HLA typing was performed using reference strand conformational analysis (RSCA). Although neither the breadth nor the magnitude of the proteome-wide HIV-specific IFN-gamma response correlated with viral load, slow disease progression was associated with over-representation of host immunogenetic protective HLA B* alleles (10 of 14 SPs compared to 0 of 7; p = 0.004, Fisher's Exact) especially B*57 and B*5801, multiclade Gag T-cell targeting (71%, 10 of 14 SPs compared to 14%, 1 of 7 RPs); p = 0.029, Fisher's Exact test and evident virological control (3.65 compared to 5.46 log10 copies/mL in SPs and RPs respectively); p<0.001, unpaired student's t-test CONCLUSIONS: These data are consistent with others that associated protection from HIV disease with inherent host HLA B allele-mediated ability to induce broader Gag T-cell targeting coupled with apparent virological control. These immunogenetic features of Gag-specific immune response which could influence disease progression may provide useful insight in future HIV vaccine design.

Molecular evidence for polyphyletic origin of human immunodeficiency virus type 1 subtype C in Bangladesh.

HIV-1 positive blood samples were collected between 1999 and 2005 from population groups most at risk of HIV infection in Bangladesh through the national surveillance, from clients of the Voluntary Counseling and Testing (VCT) Unit for HIV at ICDDR,B and a survey of HIV in patients with tuberculosis. Partial sequences of the gag gene were used for subtyping the HIV strains by nested PCR using selective primers. Of the 198 HIV strains tested, subtype C (41.4%) was the commonest strain identified. Phylogenetic analysis of Bangladeshi subtype C strains showed that they clustered in polyphyletic branches representing HIV strains from different parts of the world. Most of the strains from injecting drug users (IDU) clustered together and were similar to Indian strains. The VCT strains however were very heterogeneous and clustered with strains from India, Myanmar, Ethiopia and Zimbabwe. Data suggest that there have been few introductions into the IDU population where the epidemic is driven by indigenous transmission. On the other hand there have been many and regular introductions of subtype C viruses through migrant workers in the VCT group. Very little overlap was observed in the strains obtained from IDU and those from other population groups.

Tat mutations in an African cohort that do not prevent transactivation but change its immunogenic properties.

Humoral responses against extra-cellular HIV-1 Tat may be beneficial as Tat has been implicated in the viral pathogenesis associated with HIV-1 disease progression. We determined the levels of anti-Tat IgG in sera of HIV-1 seropositive individuals from the Rural Clinical Cohort in Uganda using nine different Tat proteins representative of the major subtypes presently accounting for 97% of infections worldwide. We observed the presence of anti-Tat IgG able to react against the various subtypes tested, although none cross-reacted against all nine variants. We show that 46.25% of seropositive patients were able to recognise at least one Tat variant with 1:1000 sera dilution. We also show that the C terminus of Tat is the most variable region and an important epitope that might explain the limitation of cross-recognition of Tat antibodies regarding Tat variants. This study shows in seropositive patients that Tat can tolerate mutations without modification of its primary function but with changes in its immunogenic properties. These findings should be considered when designing Tat-based HIV-1 vaccines.

Relation between chemokine receptor use, disease stage, and HIV-1 subtypes A and D: results from a rural Ugandan cohort.

OBJECTIVES: To determine whether there are differences in coreceptor use in subjects infected with HIV-1 envelope subtypes A and D that could explain the differences in progression rates between these subtypes in a rural Ugandan cohort. METHODS: HIV-1 was subtyped in env by V3 sequencing or heteroduplex mobility assay. Coreceptor use was determined by the ability of the isolates to replicate in U87 CD4 cells expressing different coreceptors. The Fisher exact test was used to examine the relation between coreceptor use and subtype, clinical stage, and V3 charge. The Kruskall-Wallis nonparametric test was used to examine the association between median CD4 cell counts, coreceptor use, and subtype. Logistic regression was used to examine predicted coreceptor use at different CD4 groupings. RESULTS: Isolates from 66 participants were analyzed. Thirty-one were infected with subtype A, and 35 were infected with subtype D. Although this work was based on a small sample size, we found statistically significant differences. The probability of having an X4 virus was higher in subtype D infections than in subtype A infections among those with a non-AIDS clinical status (Fisher exact test, P = 0.040). Logistic regression analysis, in which we predicted X4 use by subtype and stratified by CD4 group, confirmed these findings among those with a CD4 count >200 cells/microL (likelihood ratio test, P = 0.003). R5 viruses were associated with higher median CD4 cell counts than X4 or X4/R5 (Kruskall-Wallis test, P = 0.0045). A V3 charge of +5 and greater was highly associated with X4 virus (Fisher exact test, P = 0.006). CONCLUSIONS: These subtype differences in coreceptor use may partially explain the faster progression rates we have previously reported in individuals infected with subtype D compared with subtype A. Our observations may have implications for the future use of coreceptor inhibitors in this population.

Genotypic variation in the pol gene of HIV type 1 in an antiretroviral treatment-naive population in rural southwestern Uganda.

The majority of studies of HIV-1 drug resistance have involved subtype B viruses. Here we have characterized subtype distribution and determined the levels of polymorphism at protease (PR) and reverse transcriptase (RT) drug resistance positions, in antiretroviral treatment-naive HIV-positive Ugandan patients. We have also investigated codon usage variability at these positions and assessed intersubtype recombination within the pol gene. The study population consisted of 187 patients, from a cohort established by the UK Medical Research Council Programme on AIDS in Uganda in 1990. Results indicate that 28.3% of patients were infected with subtype A (n = 53), 64.2% subtype D (n = 120), 6.4% A/D recombinant (n = 12), and 1.1% subtype C (n = 2). Variation in amino acid usage at drug resistance-associated positions was minimal between the two main subtypes (A and D) in RT, but there was appreciable variation in PR. Codon usage, however, was considerably more variable between subtypes A and D in both PR and RT. Thus, while no natural high-level resistance to antiretroviral therapy was detected in this cohort, subtypes A and D may possess different genetic barriers to be overcome in order to achieve resistance. With the increasing introduction of antiretroviral therapy into Africa, such information will be vital in our understanding and evaluation of the development of drug resistance as it occurs, and how to interpret resistance data the type of which has rarely previously been seen. This analysis also significantly increases the number of Ugandan PR and RT sequences characterized to date.

The relationship between HIV type 1 disease progression and V3 serotype in a rural Ugandan cohort.

Antigenic properties of the V3 region are reflected by HIV-1 serotypes. These may represent biological properties of the virus. We serotyped HIV-1 in 142 serum samples from participants in a rural Uganda cohort who seroconverted between August 1991 and December 2001. Clinical progression was assessed using Cox proportional hazards and Kaplan-Meier methods. Of 112 (79%) samples successfully serotyped, 36% were serotype A, 17% serotype B, 18% serotype C, and 29% serotype D. Median follow-up time, age at enrollment, and first CD4 count were similar in each serotype group. Clinical progression was faster for serotype D than other serotypes to AIDS or death, death, and CD4 count <200 cells/mm(3) (all p < 0.05). HIV-1 V3 serotypes are associated with variations in the pathogenicity of HIV-1 and should be taken into account when studying the biological relevance of HIV-1 diversity.

Full-length HIV-1 Tat protein necessary for a vaccine.

AIDS vaccines now use a truncated version of 86 residues of the Tat protein related to the HIV-1 HXB2 strain predominant in Europe and North America. We compared antibodies raised in rabbits using a B subtype short Tat HXB2(86) and a full-length Tat HXB2(100). Serum against HXB2(86) recognizes only B and D subtypes while serum against HXB2(100) recognizes B, D, and C subtype variants. Conformational epitopes appear to be involved in the capacity of anti-Tat HXB2 sera to recognized non-homologous Tat variants. A linear B-epitope identified in sequence 71-81 in HXB2(86) disappears in HXB2(100), which has a new linear B-epitope identified at the C-terminus. Anti-HXB2(100) serum has a higher titer in neutralizing antibody against homologous and non-homologous variants compared to anti-HXB2(86) serum. We suggest that a Tat vaccine should contain a Tat variant with regular size, up to 99-101 residues now found in the field.

The glutamine-rich region of the HIV-1 Tat protein is involved in T-cell apoptosis.

Human immunodeficiency virus (HIV) infection and the progression to AIDS are characterized by the depletion of CD4(+) T-cells. HIV-1 infection leads to apoptosis of uninfected bystander cells and the direct killing of HIV-infected cells. This is mediated, in part, by the HIV-1 Tat protein, which is secreted by virally infected cells and taken up by uninfected cells. We chemically synthesized two 86-residue subtype D Tat proteins, Ug05RP and Ug11LTS, from two Ugandan patients who were clinically categorized as either rapid progressor or long-term survivor, with non-conservative mutations located essentially in the glutamine-rich region. Structural heterogeneities were revealed by CD, which translate into differing trans-activational and apoptotic effects. CD data analysis and molecular modeling indicated that the short alpha-helix observed in subtype D Tat proteins from rapid progressor patients such as Tat Mal and Tat Ug05RP is not present in Ug11LTS. We show that Tat Ug05RP is more efficient than Tat Ug11LTS in its trans-activational role and in inducing apoptosis in binding tubulin via the mitochondrial pathway. The glutamine-rich region of Tat appears to be involved in the Tat-mediated apoptosis of T-cells.

HIV type 1-specific inter- and intrasubtype cellular immune responses in HIV type 1-infected Ugandans.

Investigations concerning the extent and nature of subtype-specific and intersubtype immune responses in HIV-1-infected persons are necessary for the development of appropriate candidate vaccines. In the cross-sectional study described here, 26 HIV-1-positive Ugandan patients were tested for their ability to mount HIV antigen-specific cellular immune responses. Subjects were infected with either HIV-1 subtypes A, C, or D. Recombinant vaccinia virus (rVV)-based and peptide-based enzyme-linked immunospot (Elispot) assays were used to evaluate HIV-1-specific gamma-interferon (IFN-gamma) cellular responses. rVV expressing gag, pol, or env proteins derived from HIV-1 subtypes A, B, and D were evaluated for their ability to induce whole HIV-1-protein-specific IFN-gamma responses in 14 patients. A panel of previously identified HLA class I-restricted peptides based on representative sequences from HIV-1 subtypes A, B, C, and D and restricted through HLA-A2, -A29, -B42, -B53, and -B57 alleles were used to evaluate the presence of HIV-1-peptide-specific T cells in 19 patients. Using rVV, 27 of a possible 38 subtype-specific responses (71%) and 56 of a possible 110 intersubtype responses (51%) were observed. When appropriate peptides were used 18 of 39 (46.2%) subtype-specific and 13 of 39 (33.3%) intersubtype responses were observed. Peptide responses were higher quantitatively than those seen when rVV were used. In 7 patients, both rVV and specific peptides were evaluated; in 3 of 7 individuals, global responses were seen despite a lack of measurable HLA-restricted peptide-specific responses demonstrating the need to evaluate a broader range of HIV-specific immune responses.