Lissette Pérez

CRF19_cpx is an Evolutionary fit HIV-1 Variant Strongly Associated With Rapid Progression to AIDS in Cuba

Background Clinicians reported an increasing trend of rapid progression (RP) (AIDS within 3 years of infection) in Cuba. Methods Recently infected patients were prospectively sampled, 52 RP at AIDS diagnosis (AIDS-RP) and 21 without AIDS in the same time frame (non-AIDS). 22 patients were sampled at AIDS diagnosis (chronic-AIDS) retrospectively assessed as > 3 years infected. Clinical, demographic, virological, epidemiological and immunological data were collected. Pol and env sequences were used for subtyping, transmission cluster analysis, and prediction of resistance, co-receptor use and evolutionary fitness. Host, immunological and viral predictors of RP were explored through data mining. Findings Subtyping revealed 26 subtype B strains, 6 C, 6 CRF18_cpx, 9 CRF19_cpx, 29 BG-recombinants and other subtypes/URFs. All patients infected with CRF19 belonged to the AIDS-RP group. Data mining identified CRF19, oral candidiasis and RANTES levels as the strongest predictors of AIDS-RP. CRF19 was more frequently predicted to use the CXCR4 co-receptor, had higher fitness scores in the protease region, and patients had higher viral load at diagnosis. Interpretation CRF19 is a recombinant of subtype D (C-part of Gag, PR, RT and nef), subtype A (N-part of Gag, Integrase, Env) and subtype G (Vif, Vpr, Vpu and C-part of Env). Since subtypes D and A have been associated with respectively faster and slower disease progression, our findings might indicate a fit PR driving high viral load, which in combination with co-infections may boost RANTES levels and thus CXCR4 use, potentially explaining the fast progression. We propose that CRF19 is evolutionary very fit and causing rapid progression to AIDS in many newly infected patients in Cuba.

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.

Diagnosis and Screening for Cytomegalovirus Infection in Pregnant Women in Cuba as Prognostic Markers of Congenital Infection in Newborns 2007-2008

Background: Human cytomegalovirus (HCMV) has established itself as the most significant cause of congenital infection in the developed world. The objective of this research was prenatal identification of pregnant women at risk for developing active infection due to HCMV as well as to diagnose congenitally infected newborns. Methods: A diagnostic algorithm based on specific immunoglobulin G (IgG), IgM, and, IgG avidity was used to screen serum from 1131 pregnant women enrolled prospectively from 3 municipalities from Havana City, Cuba during 2007–2008. Qualitative multiplex nested PCR and quantitative real time-based PCR testing for HCMV DNA were performed on urine and saliva specimens from women detected with active infection and from their newborns. Results: Most women were seropositive to HCMV (92.7%), with 2.38% (27 women) having active infection. Primary infection was detected in 20 pregnant women (1.77%) while 7 patients (0.62%) had active nonprimary infection. HCMV DNA was detected in specimens from 9 of the 27 pregnant women by both PCR methods. HCMV congenital infection was diagnosed in 12 (1.06%) of the 26 live children born from 25 mothers with active infection, for a vertical transmission rate of 46.2%. Two fetal deaths were reported from 2 women with active infection; furthermore 2 newborns were symptomatic at birth and 2 showed sequelae during the follow-up done until 6 months age. Conclusions: Mothers with active infection during the pregnancy and with HCMV excretion had significant risks, RR = 1.16 and RR = 1.35, respectively, to have congenitally infected children.

The effect of highly active antiretroviral therapy on outcome of central nervous system herpesviruses infection in Cuban human immunodeficiency virus-infected individuals

With the rapid progress in the development of highly active antiretroviral therapy (HAART), the observed patterns in human immunodeficiency virus (HIV) encephalitis has changed, allowing herpesvirus (HV) infection to be controlled. HAART was first administered to HIV patients in Cuba in 2001. Consequently with the aim of investigate the behavior of the HVs causing neurological disorders in this population in the post-HAART era, the authors perform a clinical evaluation by a multiplex nested polymerase chain reaction (PCR) assay for simultaneous detection of human HVs—herpes simplex virus (HSV), varicellazoster virus (VZV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and Epstein-Barr virus (EBV). The authors studied 241 samples of cerebrospinal fluid (CSF) received at the Sexually Transmitted Diseases Laboratory between 2001 and 2005 inclusive. Of the 241 CSF studied, 10.4% resulted positive for HV infections. Of these, 92% of patients were acquired immunodeficiency syndrome (AIDS) individuals at the C3 stage. CMV (44%), EBV (28%), and dual-HV (16%) infections were the most important agents identified. The principal clinical manifestations were fever, headache, vomiting, and focal abnormalities; the latter being associated with an increased risk of death. A statistically significant result was observed when central nervous system (CNS) disease evolution was compared between patients who were under HAART against those who were not, before they developed encephalitis. It was therefore concluded that it is more likely that HIV individuals receiving HAART have a better recovery of CNS infections than those who are not receiving it.

HCMV seroprevalence and associated risk factors in pregnant women, Havana City, 2007 to 2008.

To prenatally identify pregnant women at risk of developing congenital infection due to human cytomegalovirus (HCMV).

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.

Simultaneous quantification of human herpesvirus 8 DNA by real time PCR in different tissues of HIV infected cuban patients with Kaposi's sarcoma.

In Cuba, previous reports have shown an increase of epidemic KS, reaching a total of 120 cases by the end of 2007, despite the use of HAART. To evaluate and compare the role of human herpes virus 8 (HHV-8) viral loads in different compartments of AIDS-related Kaposis sarcoma (AIDS-KS) patients real-time polymerase chain reaction (RT-PCR) was used to determine the genome copy number of HHV-8 in plasma, saliva, tissue and peripheral blood mononuclear cells (PBMC) of 49 AIDS-KS patients. Overall, 98% of AIDS-KS patients harbored detectable HHV-8. HHV-8 could be detected in 91.6% of KS tissue lesions showing the highest viral load (median log = 3.14 copies/100 ng DNA) followed by saliva and PBMC which were positive in 78%, and 69.2%; respectively. In contrast, HHV-8 was detected in only 37% of plasma samples, which also showed lower viral loads. Men who had sex with men (MSM) were more likely to have three-times higher HHV-8 genome copies in KS lesions when compared with tissues from heterosexuals individuals (OR 3; 95% CI 1.1 to 12.5). These results emphasize the systemic nature of HHV-8-infection and demonstrate the possible role of saliva in HHV-8 transmission among MSM.

Prospective, comprehensive, and effective viral monitoring in Cuban children undergoing solid organ transplantation

PurposeIn Cuba, viral monitoring in the post-transplant period was not routinely performed. The aim of this research is to identify the most frequent viruses that affect transplanted Cuban children, by implementing a viral follow-up during the post-transplant period.MethodsThe study population included all Cuban pediatric patients who underwent solid organ transplantation (SOT) between November 2009 and December 2012. A total of 34 transplanted pediatric patients of kidney (n = 11) and liver (n = 23) were prospectively monitored during a 34-week period for viral DNAemia and DNAuria by simultaneous detection of cytomegalovirus (CMV), Epstein-Barr virus, herpes simplex virus type 1 and 2, varicella zoster virus, human herpesvirus 6, human adenovirus, and polyomaviruses (BKV and JCV) using quantitative real-time polymerase chain reaction (qRT-PCR).ResultsViral genome of at least one virus was detected in 21 of 34 recipients, 18 patients excreted virus in urine while 12 presented DNAemia. CMV (41.2%) and BKV (35.3%) were the most frequent viruses detected during the follow-up. CMV was the virus mainly associated with clinical symptoms and DNAemia. Its excretion in urine (with cut off value of 219 copies/mL) was associated with detection in plasma (p < 0.001); furthermore, CMV viruria was predictive of CMV viremia (OR:8.4, CI:2.4-29.1, p = 0.001). There was no association between high viral load and clinical complications, due to the prompt initiation of preemptive ganciclovir. Conclusion: This comprehensive viral monitoring program effectively prevents the development of critical viral disease, thus urge the implementation of qRT-PCR as routine for viral monitoring of transplanted Cuban organ recipients.

Distinct genotypic distribution of cytomegalovirus (CMV) envelope glycoprotein B (gB) in a Cuban cohort of patients with different CMV diseases

To investigate the association between human CMV glycoprotein B (gB) genotypes and CMV disease, we retrospectively analysed 73 biological samples from 56 Cuban patients with different CMV-related diseases using a multiplex nested PCR for detection of the reported 5 CMV gB genotypes. All 4 main genotypes 1 to 4 were found in the clinical samples while no genotype 5 was detected. Among the individuals analysed, genotype gB-2 was the most prevalent (38%) followed by gB-1 (30%) and mixed infections (16%) being mainly detected among immunosuppressed patients (7 out of 9), although there was no association between mixed infections and CMV rejection in transplant recipients. Genotype gB-4 was the least frequent (5 patients), which was almost exclusively detected in mixed infections (4 out of 5, p<0.0001). Genotype gB-1 was more frequently detected in AIDS patients (47%) although it was not statistically significant, while 68% of transplant patients showed mixed infections (p<0.05). This study represents the first report of human CMV gB genotypes in Cuban patients; however, the study is limited by the small number patients, thus making it difficult to draw firm conclusions about the distribution of CMV genotypes in Cuba. Nevertheless, this preliminary report has allowed us to identify that the main 4 CMV genotypes are present in the Cuban population, with genotypes 2 and 1 being the most frequent strains.

Kaposi's Sarcoma and Human Herpesvirus 8 in Cuba: evidence of subtype B expansion.

OBJECTIVE To evaluate the temporal distribution (1991-2009) and associated variation of KSHV subtypes in Cuba. METHOD Phylogenetic characterization based on the KSHV K1 gene was performed using 90 KSHV positive samples. RESULTS Molecular characterization confirmed the prevalence of a wide range of KSHV subtypes (A: n=48 [A5=12]; C: n=15; B: n=22; and E: n=5). In the current study, we observed a significant increase in HHV-8 subtype B after 2004 (p=0.0063). This Subtype B in Cuba was associated with: heterosexual behaviour (OR: 3.63, CI: 1,2-10,98; p=0.03), with the antecedent of acquiring HIV/KSHV in Africa (p=0.0003), with nodular stage of KS lesions (OR 4.2, CI: 1.1 to 15.7; p=0.04). CONCLUSION Our study is the first to report KSHV Subtype B expansion in Cuba, that might be reflective of a change in human behavioural pattern.