Pontine A5 region modulation of the cardiorespiratory response evoked from the midbrain dorsolateral periaqueductal grey.

Connections between the midbrain dorsolateral periaqueductal grey (dlPAG) and the pontine A5 region have been shown. The stimulation of both regions evokes similar cardiovascular responses: tachycardia and hypertension. Accordingly, we have studied the interactions between dlPAG and A5 region in spontaneously breathing anesthetized rats. dlPAG was electrically stimulated (20-30 µA 1-ms pulses were given for 5 s at 100 Hz). Changes in the evoked cardiorespiratoy response were analysed before and after ipsilateral microinjections of muscimol (GABAergic agonist, 50 nl, 0.25 nmol, 5 s) within the A5 region. Electrical stimulation of the dlPAG produces, in the rat, a response characterized by tachypnoea (p < 0.001), hypertension (p < 0.001) and tachycardia (p < 0.001). The increase in respiratory rate was due to a decrease in expiratory time (p < 0.01). Pharmacological inhibition of the A5 region with muscimol produced a marked reduction of the tachycardia (p < 0.001) and the tachypnoea (p < 0.01) evoked from the dlPAG. Finally, to assess functional interactions between A5 and dlPAG, extracellular activity of putative A5 neurones were recorded during dlPAG electrical stimulation. Forty A5 cells were recorded, 16 of which were affected by dlPAG stimulation (40%). 4 cells showed activation, 5 cells excitation and 7 cells decreased spontaneous activity to dlPAG stimulation (p < 0.001). These results confirm a link between the A5 region and dlPAG. The potential role of these connections in the modulation of dlPAG evoked cardiorespiratory responses and their possible clinical implications are discussed.

High frequency of antiviral drug resistance and non-b subtypes in HIV-1 patients failing antiviral therapy in Cuba.

INTRODUCTION: Emergence of HIV-1 drug resistance may limit the sustained benefits of antiretroviral therapy (ART) in settings with limited laboratory monitoring and drug options. The objective is to implement the surveillance of drug resistance and subtypes in HIV-1 patients failing ART in Cuba. METHODS: This study compiled clinical and genotypic drug resistance data 588 ART-experienced HIV-1 patients attending a clinical center in Havana in 2009-2013. Drug resistance testing was performed as part of routine clinical care. Drug resistance mutations and levels were determined using Rega version 8.0.2. RESULTS: Eighty-three percent received solely ART containing at least three drugs. Patients from 2009 to 2010 were longer treated (median: 4.9 vs 2.7 years) and exposed to more ART regimens (median: 4 vs 2 regimens) compared to patients from 2011-2013. Nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside RTI (NNRTI) and PI mutations were present in 83.5, 77.4 and 52.0%. Full-class resistance (FCR) to NRTI, NNRTI, PI and multidrug resistance (MDR) were detected in 25.0, 33.7, 11.4 and 6.3%. FCR to NRTI, NNRTI, PI and MDR were present in 12.8, 28.7, 0 and 0% after first-line failure (164 patients) and in 23.1, 34.6, 3.8 and 3.1% after second-line failure (130 patients). Subtype B (32.5%), BG recombinants (19.6%) and CRF19_cpx (16.2%) were the most prevalent genetic forms. Subtype distribution did not change significantly between 2009-2010 and 2011-2013, except for BG recombinants that increased from 12.2 to 21.3% (p=0.002). CONCLUSIONS: Our study found a high prevalence of drug resistance and supports the need for appropriate laboratory monitoring in clinical practice and access to drug options in case of virological failure.

Antiretroviral drug resistance in HIV-1 therapy-naive patients in Cuba.

In Cuba, antiretroviral therapy rollout started in 2001 and antiretroviral therapy coverage has reached almost 40% since then. The objectives of this study were therefore to analyze subtype distribution, and level and patterns of drug resistance in therapy-naive HIV-1 patients. Four hundred and one plasma samples were collected from HIV-1 therapy-naive patients in 2003 and in 2007-2011. HIV-1 drug resistance genotyping was performed in the pol gene and drug resistance was interpreted according to the WHO surveillance drug-resistance mutations list, version 2009. Potential impact on first-line therapy response was estimated using genotypic drug resistance interpretation systems HIVdb version 6.2.0 and Rega version 8.0.2. Phylogenetic analysis was performed using Neighbor-Joining. The majority of patients were male (84.5%), men who have sex with men (78.1%) and from Havana City (73.6%). Subtype B was the most prevalent subtype (39.3%), followed by CRF20-23-24_BG (19.5%), CRF19_cpx (18.0%) and CRF18_cpx (10.3%). Overall, 29 patients (7.2%) had evidence of drug resistance, with 4.0% (CI 1.6%-4.8%) in 2003 versus 12.5% (CI 7.2%-14.5%) in 2007-2011. A significant increase in drug resistance was observed in recently HIV-1 diagnosed patients, i.e. 14.8% (CI 8.0%-17.0%) in 2007-2011 versus 3.8% (CI 0.9%-4.7%) in 2003 (OR 3.9, CI 1.5-17.0, p=0.02). The majority of drug resistance was restricted to a single drug class (75.8%), with 55.2% patients displaying nucleoside reverse transcriptase inhibitor (NRTI), 10.3% non-NRTI (NNRTI) and 10.3% protease inhibitor (PI) resistance mutations. Respectively, 20.7% and 3.4% patients carried viruses containing drug resistance mutations against NRTI+NNRTI and NRTI+NNRTI+PI. The first cases of resistance towards other drug classes than NRTI were only detected from 2008 onwards. The most frequent resistance mutations were T215Y/rev (44.8%), M41L (31.0%), M184V (17.2%) and K103N (13.8%). The median genotypic susceptibility score for the commonly prescribed first-line therapies was 2.5. This analysis emphasizes the need to perform additional surveillance studies to accurately assess the level of transmitted drug resistance in Cuba, as the extent of drug resistance might jeopardize effectiveness of first-line regimens prescribed in Cuba and might necessitate the implementation of baseline drug resistance testing.

SIDATRAT: informatics to improve HIV/AIDS care.

AIDS is a major cause of death in the Caribbean, a region with a high prevalence of HIV. However, prevalence in Cuba's population aged 15 to 49 years, despite a slight increase in recent years, is considered extremely low (0.1%). At the close of 2010, 5692 Cuban patients were receiving antiretroviral therapy. SIDATRAT, an informatics system, was developed at the Pedro Kourí Tropical Medicine Institute in Havana to ensure proper monitoring and followup of drug administration. Functioning on a web platform utilizing an Apache server, PHP and MySQL, it records patients' general information, CD4 counts, viral load and data from other laboratory tests, as well as endoscopic and imaging studies. It also compiles information on their AIDS classification, opportunistic infections, HIV subtype and resistance studies, followup consultations, drug regimen, adverse reactions to medications, changes in drug combinations, and survival; and tracks total number of individuals under treatment. SIDATRAT follows the client-server philosophy and enables access by authorized users throughout Cuba via the health informatics network. SIDATRAT has been found effective in supporting quality care for persons living with HIV/AIDS and universal access to antiretroviral therapy, compiling most of the information needed for decisionmaking on patient health and therapies. SIDATRAT has been offered to the UNDP office in Havana for sharing with other developing countries that may wish to adapt or implement it.

High frequency of antiviral drug resistance and non-B subtypes in HIV-1 patients failing antiviral therapy in Cuba.

BACKGROUND: Emergence of HIV-1 drug resistance may limit the sustained benefits of antiretroviral therapy (ART) in settings with limited laboratory monitoring and drug options. OBJECTIVES: Surveillance of drug resistance and subtypes in HIV-1 patients failing ART in Cuba. STUDY DESIGN: This study compiled data of ART-experienced HIV-1 patients attending a clinical center in Havana in 2003 and 2009-2011. The first period included results of a cross-sectional study, whereas in the second period genotyping was performed as part of routine care. Drug resistance mutations and levels were determined using HIVdb version 6.0.9. RESULTS: Seventy-six percent received solely ART containing at least 3 drugs, of which 79.1% ever receiving unboosted protease inhibitors (PI). Patients from 2009 to 2011 were longer treated and exposed to more ART regimens. Subtype B (39%) and CRF19_cpx (18%) were the most prevalent genetic forms. Subtype distribution did not change significantly between both periods, except for BG recombinants that increased from 6% to 14%. Nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside RTI (NNRTI) and PI mutations were present in 69.5%, 54.8% and 44.4%. Full-class resistance (FCR) to NRTI, NNRTI, PI and multidrug resistance (MDR) were detected in 31.8%, 37.9%, 18.5% and 15.4%. FCR to NRTI, NNRTI, PI and MDR were present in 9.8%, 14.1%, 0%, 0% after first-line failure and in 19.8%, 20.8%, 2.9% and 2.9% after second-line failure. CONCLUSIONS: Our study found a high prevalence of drug resistance and supports the need for appropriate laboratory monitoring in clinical practice and access to drug options in case of virological failure.

Antiretroviral therapy adherence in persons with HIV/AIDS in Cuba.

INTRODUCTION Cuba has an HIV prevalence of 0.1% in the population aged 15 to 49 years, very low despite increased incidence in recent years. In 2001, domestically-produced generic antiretroviral therapy was introduced and there has been complete coverage since 2003. In 2006, 1986 people with HIV/AIDS were receiving ART; by 2009, that figure reached 5034. Adherence to antiretroviral therapy is fundamental: nonadherence leads to treatment failure, development of resistance, progression to AIDS, and death. OBJECTIVE Measure levels of treatment adherence and its predictive factors in persons with HIV/AIDS receiving antiretroviral therapy in 2006 in Cuba. METHODS A cross-sectional study was carried out in 2006 of Cuban HIV-positive individuals receiving antiretroviral therapy. A sample size of 876 was calculated using two-stage sampling (first by strata, and then by simple random sampling in each stratum). An anonymous structured questionnaire was administered to participants. Reporting of doses taken on each of the three days and in the week preceding the survey was recoded into five categories. Participants were considered highly adherent if they reported taking ≥95.0% of their medication as prescribed. Reasons for nonadherence were described and logistic regression modeling used to develop hypotheses on associations between high adherence and its predictive factors. RESULTS Interviews were obtained with 847 individuals, 70.6% of whom self reported high adherence. There were no significant differences between highly adherent and less adherent patients with regard to sex, place of residence, treatment setting, time of diagnosis, or length of treatment. Variables associated with high adherence were communication with the specialist physician, change in treatment, memory, self-efficacy, as well as commitment to and opinions about treatment. CONCLUSIONS In Cuba, where treatment is free of charge to patients, adherence is good. Treatment adherence might be improved by achieving a closer doctor-patient relationship; taking measures to motivate patients and promote self-efficacy and commitment to treatment; publicizing treatment outcomes; and providing assistance to patients to help them remember their medication schedule. Further studies are required to determine current adherence levels; and longitudinal research to determine adherence over time.

Raising HIV/AIDS awareness through Cuba's Memorias project.

As long as there is no cure for AIDS, the only effective means of containing its spread is prevention, primarily through public education. Cuba's AIDS Prevention Group is a community-based organization whose main purpose is to support the National HIV/AIDS Program's prevention and education efforts. The Group's Memorias Project uses the creation and display of memorial quilts to put a human face on AIDS statistics and stimulate public reflection on issues related to the disease and society.

Role of the parabrachial complex in the cardiorespiratory response evoked from hypothalamic defense area stimulation in the anesthetized rat.

To analyze the role of parabrachial complex (PBc) in the modulation of cardiorespiratory response evoked from the hypothalamic defense area (HDA), cardiorespiratory changes were analyzed in spontaneously breathing anesthetised rats in response to electrical stimulation of the HDA (1 ms pulses, 30-50 microA, 100 Hz for 5 s) before and after the microinjection of muscimol (50 nl, 0.25 nmol, 5 s) within the PBc. HDA stimulation evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (p<0.001) due to a decrease in expiratory time (p<0.01). The respiratory response was accompanied by a pressor (p<0.001) and a tachycardic (p<0.001) response. Muscimol microinjection within the lateral parabrachial region (lPB) abolished the respiratory response to HDA stimulation (p<0.01) and decreased the pressor response (p<0.05). Muscimol within the medial parabrachial region and Kölliker-Fuse (mPB-KF) decreased the magnitude of the pressor (p<0.01) and tachycardic (p<0.05) responses to HDA stimulation. The respiratory response persisted unchanged. Finally, extracellular recording of putative neurons from these regions were obtained during HDA stimulation to confirm functional interaction between HDA and parabrachial regions. 105 pontine cells were recorded during HDA stimulation, 57 from the lPB and 48 from the mPB-KF. In mPB-KF 34/48 (71%) and in lPB 38/57 (67%) cells were influenced from HDA. The results indicate that neurons from different regions of the PBc have an important function in mediating the cardiorespiratory response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.

Genotypic resistance to antiretroviral drugs in patients infected with several HIV type 1 genetic forms in Cuba.

The main objective of this study is to evaluate the prevalence of resistance-associated mutations to reverse transcriptase (RT) and protease (PR) inhibitors (I) 2 years after the introduction of antiretroviral treatment in Cuba, analyzing the mutations corresponding to different HIV-1 genetic forms circulating in Cuba. A total of 425 plasma samples were collected in 2003, corresponding to 175 (41.2%) subtype B and 250 (58.8%) non-B genetic forms, including 56 (22.4 %) non-B subtypes, 112 (44.8%) circulating recombinant forms (CRFs), and 82 (32.8%) unique RFs (URFs). Of these, 175 (41.2%) patients were under highly active antiretroviral therapy (HAART) and 250 (58.8%) were treatment-naive. The presence of RT and PR resistance-associated mutations was established by sequencing. Levels of resistance were evaluated according to the Stanford Database program (http://hivdb.stanford.edu). The prevalence of resistance to RTI was 52.2% among RTI-treated patients, 51.5% for subtype B, and 53.2% for non-B genetic forms, including CRF18_cpx, CRF19_cpx, subtype C, and BG URF. In treatment-naive patients it was 6.4% in subtype B and 4.2% in non-B subtypes and RFs. The prevalence of resistance to PRI was 30% among PRI-treated patients, 28% in subtype B and 31% in non-B genetic forms, and 3.2% among treatment-naive subjects, mostly BG recombinants. In conclusion, significant differences in the prevalence of resistance to RTI and PRI were not detected among the most frequent genetic forms from treated patients, suggesting that the genetic diversity of HIV-1 in Cuba does not play a main role in the development of resistance to antiretroviral drugs. The presence of transmitted resistance mutations supports the study of resistance at baseline of treatment.

Identification of 3 phylogenetically related HIV-1 BG intersubtype circulating recombinant forms in Cuba.

BG intersubtype recombinants represented 11.6% of HIV-1 isolates in a recent survey in Cuba based on pol sequences, most of them forming a single clade further subdivided into 3 subclades. Here, we analyze 8 near full-length genomes and 1 gag-pol sequence from epidemiologically unlinked Cuban BG recombinants from these 3 subclades (3 from each). Near full-length sequences were also obtained from 3 subtype G and 2 subtype B Cuban viruses. Phylogenetic relationships were estimated via maximum likelihood, and mosaic structures of the recombinants were inferred with the bootscanning, MaxChi, Genconv, and GARD methods. For the near full-length genomes, all recombinants formed a strongly supported clade further subdivided into the same subclades previously defined in pol. Mosaic structures were identical within each subclade and different among subclades, although 5 breakpoints were coincident among all recombinants. Individual phylogenetic trees for nonrecombinant fragments (concatenated B and G subtype segments) indicated a common ancestry for the parental viruses and their relationships to local subtype B and G strains. These results allow us to identify 3 new BG intersubtype circulating recombinant forms in Cuba derived from a common recombinant ancestor, which originated from B and G subtype parental strains circulating in Cuba.

HIV Type 1 molecular epidemiology in cuba: high genetic diversity, frequent mosaicism, and recent expansion of BG intersubtype recombinant forms.

Highly diverse HIV-1 genetic forms are circulating in Cuba, including subtypes B and G and two recombinant forms of African origin (CRF18_cpx and CRF19_cpx). Here we phylogenetically analyze pol sequences from a large collection of recent samples from Cuba, corresponding to 425 individuals from all Cuban provinces, which represents approximately 12% of prevalent infections in the country. RNA from plasma was used to amplify a pol segment by reverse transcription-polymerase chain reaction; phylogenetic analyses were performed with neighbour-joining trees and bootscanning. The distribution of genetic forms was subtype B, 41.2%; CRF19_cpx, 18.4%; BG recombinants, 11.6%; CRF18_cpx, 7.1%; subtype C, 6.1%; subtype G, 3.8%; B/CRF18 recombinants, 2.6%; subtype H, 2.1%; B/CRF19 recombinants, 1.7%; and others, 5.4%. Seventy-five (17.6%) viruses were recombinant between genetic forms circulating in Cuba. In logistic regression analyses, adjusting by gender and region, subtype B was more prevalent (OR 5.0, 95% CI 2.0-12.3) and subtype G less prevalent (OR 0.1, 95% CI 0.0-0.5) among men who have sex with men (MSM) than among heterosexuals. Within the main genetic forms of Cuba there were phylogenetic subclusters, several of which correlated with risk exposure or region. BG recombinants formed three phylogenetically related subclusters, corresponding to three different mosaic structures; most of these recombinants were from MSM from Havana City, among whom they have expanded recently, reaching 31% HIV-1 infections diagnosed in 2003. This study confirms the high HIV-1 diversity and frequent recombination in Cuba and reveals the recent expansion of diverse related BG recombinant forms in this country.