Lindsay M. Eyzaguirre

Protocol for Nearly Full-Length Sequencing of HIV-1 RNA from Plasma

Nearly full-length genome sequencing of HIV-1 using peripheral blood mononuclear cells (PBMC) DNA as a template for PCR is now a relatively routine laboratory procedure. However, this has not been the case when using virion RNA as the template and this has made full genome analysis of circulating viruses difficult. Therefore, a well-developed procedure for sequencing of full-length HIV-1 RNA directly from plasma was needed. Plasma from U.S. donors representing a range of viral loads (VL) was used to develop the assay. RNA was extracted from plasma and reverse-transcribed. Two or three overlapping regions were PCR amplified to cover the entire viral genome and sequenced for verification. The success of the procedure was sensitive to VL but was routinely successful for VL greater than 105 and the rate declined in proportion to the VL. While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads. This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies.

Buenos Aires cohort of men who have sex with men: prevalence, incidence, risk factors, and molecular genotyping of HIV type 1.

The prevalence, incidence, risk factors, and molecular genotyping of HIV-1 infection among men who have sex with men (MSM) were assessed through a prospective cohort study. The study was conducted in Buenos Aires from February 2003 to December 2004. Sociodemographic, sexual risk behavior data, and blood samples for HIV testing were collected at baseline and at 6 and 12 months. Cox regression analysis was applied to determine risk factors associated with HIV seroconversion. HIV-positive samples were analyzed by partial (pro/RT) and full-length genome sequencing. Of 811 HIV-negative participants evaluated at baseline, 327 volunteers that fulfilled the inclusion criteria were enrolled. Retention rates at 6 and 12 months were 97.2% and 91.5%, respectively. Twelve MSM seroconverted for HIV infection [incidence rate = 3.9 (95% CI = 2.0-6.7) per 100 person-years]. HIV seroconversion was associated with a greater number of different sexual contacts in the preceding 6 months (> or =10, hazard ratio = 3.3, 95% CI: 1.1-10.4). By partial pro/RT genotyping analysis, 83% HIV-positive samples were subtype B and 17% samples were BF recombinants, most of these being unique recombinant forms. This study describes for the first time the recruitment and follow-up of a cohort of MSM in Argentina. Retention rates and HIV incidence rate were high. These data should be considered as a promising potential population for HIV vaccine trials.

Healthcare-Associated Infection and Hospital Readmission

OBJECTIVE Hospital readmissions are a current target of initiatives to reduce healthcare costs. This study quantified the association between having a clinical culture positive for 1 of 3 prevalent hospital-associated organisms and time to hospital readmission. DESIGN Retrospective cohort study. PATIENTS AND SETTING Adults admitted to an academic, tertiary care referral center from January 1, 2001, through December 31, 2008. METHODS The primary exposure of interest was a clinical culture positive for methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), or Clostridium difficile obtained more than 48 hours after hospital admission during the index hospital stay. The primary outcome of interest was time to readmission to the index facility. Multivariable Cox proportional hazards models were used to model the adjusted association between positive clinical culture result and time to readmission and to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS Among 136,513 index admissions, the prevalence of hospital-associated positive clinical culture result for 1 of the 3 organisms of interest was 3%, and 35% of patients were readmitted to the index facility within 1 year after discharge. Patients with a positive clinical culture obtained more than 48 hours after hospital admission had an increased hazard of readmission (HR, 1.40; 95% CI, 1.33-1.46) after adjusting for age, sex, index admission length of stay, intensive care unit stay, Charlson comorbidity index, and year of hospital admission. CONCLUSIONS Patients with healthcare-associated infections may be at increased risk of hospital readmission. These findings may be used to impact health outcomes after discharge from the hospital and to encourage better infection prevention efforts.

HIV-1 recombinants with multiple parental strains in low-prevalence, remote regions of Cameroon: Evolutionary relics?

BackgroundThe HIV pandemic disseminated globally from Central West Africa, beginning in the second half of the twentieth century. To elucidate the virologic origins of the pandemic, a cross-sectional study was conducted of the genetic diversity of HIV-1 strains in villagers in 14 remote locations in Cameroon and in hospitalized and STI patients. DNA extracted from PBMC was PCR amplified from HIV(+) subjects. Partial pol amplicons (N = 164) and nearly full virus genomes (N = 78) were sequenced. Among the 3956 rural villagers studied, the prevalence of HIV infection was 4.9%; among the hospitalized and clinic patients, it was 8.6%.ResultsVirus genotypes fell into two distinctive groups. A majority of the genotyped strains (109/164) were the circulating recombinant form (CRF) known to be endemic in West Africa and Central West Africa, CRF02_AG. The second most common genetic form (9/164) was the recently described CRF22_01A1, and the rest were a collection of 4 different subtypes (A2, D, F2, G) and 6 different CRFs (-01, -11, -13, -18, -25, -37). Remarkably, 10.4% of HIV-1 genomes detected (17/164) were heretofore undescribed unique recombinant forms (URF) present in only a single person. Nearly full genome sequencing was completed for 78 of the viruses of interest. HIV genetic diversity was commonplace in rural villages: 12 villages each had at least one newly detected URF, and 9 villages had two or more.ConclusionsThese results show that while CRF02_AG dominated the HIV strains in the rural villages, the remainder of the viruses had tremendous genetic diversity. Between the trans-species transmission of SIVcpz and the dispersal of pandemic HIV-1, there was a time when we hypothesize that nascent HIV-1 was spreading, but only to a limited extent, recombining with other local HIV-1, creating a large variety of recombinants. When one of those recombinants began to spread widely (i.e. became epidemic), it was recognized as a subtype. We hypothesize that the viruses in these remote Cameroon villages may represent that pre-epidemic stage of viral evolution.

Role of HIV-1 matrix protein p17 variants in lymphoma pathogenesis.

Significance Non-Hodgkin lymphomas (NHLs) are associated with HIV-1 infection, but the HIV1 genome is not detectable in malignant B cells. Here we show that variants of the HIV-1 matrix protein p17 (vp17s) are detected in the NHL specimens of HIV+ patients. These vp17s are more frequently detected in HIV+ patients with NHL than in patients without NHL. These vp17s display a potent B-cell growth-promoting activity, which is exerted by activating the Akt signaling pathway. Results obtained by CD spectroscopy and thermal denaturation suggest that mutation-induced protein destabilization may lead to a conformational change potentially responsible for the viral protein to promote B-cell growth. Our results suggest that vp17s may have a role in sustaining lymphomagenesis, thus offering new opportunities to prevent and/or treat HIV-associated NHLs. Although in decline after successful anti-HIV therapy, B-cell lymphomas are still elevated in HIV-1-seropositive (HIV+) persons, and the mechanisms are obscure. The HIV-1 matrix protein p17 persists in germinal centers long after HIV-1 drug suppression, and some p17 variants (vp17s) activate Akt signaling and promote growth of transformed B cells. Here we show that vp17s derived from four of five non-Hodgkin lymphoma (NHL) tissues from HIV+ subjects display potent B-cell growth-promoting activity. They are characterized by amino acid insertions at position 117–118 (Ala–Ala) or 125–126 (Gly–Asn or Gly–Gln–Ala–Asn–Gln–Asn) among some other mutations throughout the sequence. Identical dominant vp17s are found in both tumor and plasma. Three of seven plasma samples from an independent set of NHL cases manifested multiple Ala insertions at position 117–118, and one with the Ala–Ala profile also promoted B-cell growth and activated Akt signaling. Ultradeep pyrosequencing showed that vp17s with C-terminal insertions are more frequently detected in plasma of HIV+ subjects with than without NHL. Insertion of Ala–Ala at position 117–118 into reference p17 (refp17) was sufficient to confer B-cell growth-promoting activity. In contrast, refp17 bearing the Gly–Asn insertion at position 125–126 did not, suggesting that mutations not restricted to the C terminus can also account for this activity. Biophysical analysis revealed that the Ala–Ala insertion mutant is destabilized compared with refp17, whereas the Gly–Asn form is stabilized. This finding provides an avenue for further exploration of structure function relationships and new treatment strategies in combating HIV-1–related NHL.

Elevated hypermutation levels in HIV-1 natural viral suppressors☆

Mutations in the HIV-1 proviral genomes delay the progression of the disease. We compared the mutation status in full-length proviral genomes of 23 HIV-infected patients with undetectable viral loads in the absence of therapy named natural viral suppressors (NVS) or Elite Controllers with 23 HIV-infected controls (10 patients on HAART treatment and 13 untreated patients). Provirus DNA was extracted from PBMC for amplification and sequencing to determine the mutation status. Nine (39 %) of the 23 NVS patients had defective proviral genomes, compared to 4 of the treated controls (40%, p = 0.96) and only one of the untreated controls (8%, p = 0.059). Most of the defective genomes resulted from Gto-A hypermutation. Among patients with hypermutation, the rate ratio for mutation was significantly higher for the NVS compared to treated controls (p = 0.043). Our data suggests that inactivation of the virus through the APOBEC3G system may contribute to the NVS phenotype.

Associations between Cognition, Gender and Monocyte Activation among HIV Infected Individuals in Nigeria

The potential role of gender in the occurrence of HIV-related neurocognitive impairment (NCI) and associations with markers of HIV-related immune activity has not been previously examined. In this study 149 antiretroviral-naïve seropositive subjects in Nigeria (SP, 92 women and 57 men) and 58 seronegative (SN, 38 women and 20 men) were administered neuropsychological testing that assessed 7 ability domains. From the neuropsychological test scores was calculated a global deficit score (GDS), a measure of overall NCI. Percentages of circulating monocytes and plasma HIV RNA, soluble CD163 and soluble CD14 levels were also assessed. HIV SP women were found to be younger, more educated and had higher CD4+ T cell counts and borderline higher viral load measures than SP men. On the neuropsychological testing, SP women were more impaired in speed of information processing and verbal fluency and had a higher mean GDS than SN women. Compared to SP men, SP women were also more impaired in speed of information processing and verbal fluency as well as on tests of learning and memory. Numbers of circulating monocytes and plasma sCD14 and sCD163 levels were significantly higher for all SP versus all SN individuals and were also higher for SP women and for SP men versus their SN counterparts. Among SP women, soluble CD14 levels were slightly higher than for SP men, and SP women had higher viral load measurements and were more likely to have detectable virus than SP men. Higher sCD14 levels among SP women correlated with more severe global impairment, and higher viral load measurements correlated with higher monocyte numbers and sCD14 and sCD14 levels, associations that were not observed for SP men. These studies suggest that the risk of developing NCI differ for HIV infected women and men in Nigeria and, for women, may be linked to effects from higher plasma levels of HIV driving activation of circulating monocytes.

HIV-1 epidemic in the Caribbean is dominated by subtype B.

Background The molecular epidemiology of HIV-1 in the Caribbean has been described using partial genome sequencing; subtype B is the most common subtype in multiple countries. To expand our knowledge of this, nearly full genome amplification, sequencing and analysis was conducted. Methodology/Principal Findings Virion RNA from sera collected in Haiti, Dominican Republic, Jamaica and Trinidad and Tobago were reverse transcribed, PCR amplified, sequenced and phylogenetically analyzed. Nearly full genomes were completed for 15 strains; partial pol was done for 67 strains. All but one of the 67 strains analyzed in pol were subtype B; the exception was a unique recombinant of subtypes B and C collected in the Dominican Republic. Of the nearly full genomes of 14 strains that were subtype B in pol, all were subtype B from one end of the genome to the other and not inter-subtype recombinants. Surprisingly, the Caribbean subtype B strains clustered significantly with each other and separate from subtype B from other parts of the pandemic. Conclusions The more complete analysis of HIV-1 from 4 Caribbean countries confirms previous research using partial genome analysis that the predominant subtype in circulation was subtype B. The Caribbean strains are phylogenetically distinct from other subtype B strains although the biological meaning of this finding is unclear.

Tenofovir-based regimens associated with less drug resistance in HIV-1-infected Nigerians failing first-line antiretroviral therapy

Background:In resource-limited settings, HIV-1 drug resistance testing to guide antiretroviral therapy (ART) selection is unavailable. We retrospectively conducted genotypic analysis on archived samples from Nigerian patients who received targeted viral load testing to confirm treatment failure and report their drug resistance mutation patterns. Methods:Stored plasma from 349 adult patients on non-nucleoside reverse transcriptase inhibitor (NNRTI) regimens was assayed for HIV-1 RNA viral load, and samples with more than 1000 copies/ml were sequenced in the pol gene. Analysis for resistance mutations utilized the IAS-US 2011 Drug Resistance Mutation list. Results:One hundred and seventy-five samples were genotyped; the majority of the subtypes were G (42.9%) and CRF02_AG (33.7%). Patients were on ART for a median of 27 months. 90% had the M184V/I mutation, 62% had at least one thymidine analog mutation, and 14% had the K65R mutation. 97% had an NNRTI resistance mutation and 47% had at least two etravirine-associated mutations. In multivariate analysis tenofovir-based regimens were less likely to have at least three nucleoside reverse transcriptase inhibitor (NRTI) mutations after adjusting for subtype, previous ART, CD4, and HIV viral load [P < 0.001, odds ratio (OR) 0.04]. 70% of patients on tenofovir-based regimens had at least two susceptible NRTIs to include in a second-line regimen compared with 40% on zidovudine-based regimens (P = 0.04, OR = 3.4). Conclusions:At recognition of treatment failure, patients on tenofovir-based first-line regimens had fewer NRTI drug-resistant mutations and more active NRTI drugs available for second-line regimens. These findings can inform strategies for ART regimen sequencing to optimize long-term HIV treatment outcomes in low-resource settings.

Neurocognitive impairment associated with predominantly early stage HIV infection in Abuja, Nigeria

Detailed neuropsychological testing was performed on 133 human immunodeficiency virus (HIV) seropositive (SP) and 77 HIV seronegative (SN) individuals, 86 % with early stage HIV infection in Nigeria, to determine the frequency of HIV-related neurocognitive impairment among the HIV-infected group. The tests were administered to assess the following seven ability domains: speed of information processing, attention/working memory, executive functioning, learning, memory, verbal fluency, and motor function motor. Demographically corrected individual test scores and scores for each domain or reflecting a global deficit (a global deficit score, or GDS) were compared for the SP and SN groups. SP participants were older, had fewer years of education, were more likely to be married, differed in ethnicity, and had higher depression scores than SN individuals. Within the seven ability domains, SP performed worse than SN with respect to speed of information processing, executive function, learning, memory, and verbal fluency and also on the global measure. SP were also more frequently impaired on tests of SIP, and there was a borderline increase in the frequency of global impairment. On the individual tests, SP performed worse than SN on four tests that assessed learning, verbal fluency, memory, and motor function (the Timed Gait). SP subjects, however, performed better than SN on the Finger-tapping test, also a motor task. Performance by SP subjects was not associated on the timed gait which showed a borderline statistically significant correlation with CD4 counts. However, there were significant correlations between viral load measurements and individual tests of speed of information processing, executive function, learning, and verbal fluency and with overall executive function and a borderline correlation with the GDS. Depression scores for SP were associated with impairment on only a single test of executive function. These results demonstrate the ability of these assessments to identify areas of impairment that may be specifically linked to a history of HIV infection among individuals in Nigeria. Confirmation of these findings awaits analyses using data from a larger number of control subjects.