Correction: Cuban history of CRF19 recombinant subtype of HIV-1.

[This corrects the article DOI: 10.1371/journal.ppat.1009786.].

Cuban history of CRF19 recombinant subtype of HIV-1.

CRF19 is a recombinant form of HIV-1 subtypes D, A1 and G, which was first sampled in Cuba in 1999, but was already present there in 1980s. CRF19 was reported almost uniquely in Cuba, where it accounts for ∼25% of new HIV-positive patients and causes rapid progression to AIDS (∼3 years). We analyzed a large data set comprising ∼350 pol and env sequences sampled in Cuba over the last 15 years and ∼350 from Los Alamos database. This data set contained both CRF19 (∼315), and A1, D and G sequences. We performed and combined analyses for the three A1, G and D regions, using fast maximum likelihood approaches, including: (1) phylogeny reconstruction, (2) spatio-temporal analysis of the virus spread, and ancestral character reconstruction for (3) transmission mode and (4) drug resistance mutations (DRMs). We verified these results with a Bayesian approach. This allowed us to acquire new insights on the CRF19 origin and transmission patterns. We showed that CRF19 recombined between 1966 and 1977, most likely in Cuban community stationed in Congo region. We further investigated CRF19 spread on the Cuban province level, and discovered that the epidemic started in 1970s, most probably in Villa Clara, that it was at first carried by heterosexual transmissions, and then quickly spread in the 1980s within the "men having sex with men" (MSM) community, with multiple transmissions back to heterosexuals. The analysis of the transmission patterns of common DRMs found very few resistance transmission clusters. Our results show a very early introduction of CRF19 in Cuba, which could explain its local epidemiological success. Ignited by a major founder event, the epidemic then followed a similar pattern as other subtypes and CRFs in Cuba. The reason for the short time to AIDS remains to be understood and requires specific surveillance, in Cuba and elsewhere.

SARS-CoV-2: preliminary study of infected human nasopharyngeal tissue by high resolution microscopy.

BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.

Molecular epidemiology and phylogenetic analysis of human papillomavirus infection in women with cervical lesions and cancer from the coastal region of Ecuador.

The aim of the present study was to gather information regarding the molecular epidemiology of Human papillomavirus (HPV) and related risk factors in a group of women with low- and high-grade cervical lesions and cancer from the coastal region of Ecuador. In addition, we studied the evolution of HPV variants from the most prevalent types and provided a temporal framework for their emergence, which may help to trace the source of dissemination within the region. We analyzed 166 samples, including 57 CIN1, 95 CIN2/3 and 14 cancer cases. HPV detection and typing was done by PCR-sequencing (MY09/MY11). HPV variants and estimation of the time to most recent common ancestor (tMRCA) was assessed through phylogeny and coalescence analysis. HPV DNA was found in 54.4% of CIN1, 74.7% of CIN2/3 and 78.6% of cancer samples. HPV16 (38.9%) and HPV58 (19.5%) were the most prevalent types. Risk factors for the development of cervical lesions/cancer were the following: three or more pregnancies (OR=4.3), HPV infection (OR=3.7 for high-risk types; OR=3.5 for HPV16), among others. With regard to HPV evolution, HPV16 isolates belonged to lineages A (69%) and D (31%) whereas HPV58 isolates belonged only to lineage A. The period of emergence of HPV16 was in association with human populations (tMRCA=91052 years for HPV16A and 27000 years for HPV16D), whereas HPV58A preceded Homo sapiens evolution (322257 years). This study provides novel data on HPV epidemiology and evolution in Ecuador, which will be fundamental in the vaccine era.

Etiology of Genital Ulcer Disease in Male Patients Attending a Sexually Transmitted Diseases Clinic: First Assessment in Cuba.

BACKGROUND: Sexually transmitted diseases (STDs) and in particular genital ulcer disease (GUD) have a major impact on morbidity and mortality in developing countries. The World Health Organization recommends the use of syndromic guidelines for the treatment of sexually transmitted infections (STIs) in resource-constrained countries. Surveillance of autochthonous etiologies provides epidemiological information contributing to the prevention and treatment of STIs. We investigated the etiology and factors associated with GUD among male patients attending a STD clinic in Havana, Cuba. METHODS: Swabs from genital ulcers of 113 male patients, collected from May 2012 to June 2015, were analyzed using PCR for herpes simplex virus types 1 and 2, Treponema pallidum, Haemophilus ducreyi, and Chlamydia trachomatis. We also investigated the clinical and epidemiological characteristics associated with the presence of these pathogens in GUD. RESULTS: At least one of the pathogens was detected in 70% of patients. The occurrence of the pathogens was herpes simplex virus type 2 (HSV-2) (51.3%), T. pallidum (29.2%), and C. trachomatis (1.8%). Co-infections occurred as follows: T. pallidum-HSV-2 (10.6%), C. trachomatis-HSV-2 (0.9%) and C. trachomatis-T. pallidum (0.9%). Herpes simplex virus type 1 and H. ducreyi were not detected. Ages 15 to 40 years, HIV-positive serostatus, and no condom use were significant risk factors for the presence of HSV-2 in genital ulcers. CONCLUSIONS: Our preliminary results highlight the predominance of HSV-2 and T. pallidum as the leading GUD etiologies in the study population and identified risk factors associated with HSV-2. This information should help to inform guidelines for better management of GUD in Havana, Cuba.

Molecular evidence of high-risk human papillomavirus infection in colorectal tumours from Cuban patients.

The association between colorectal cancer and human papillomavirus (HPV) infection is still unproven. The aim of this study was to investigate the presence of high-risk HPV (HR-HPV) DNA in colorectal tissues from Cuban patients. A total of 63 colorectal formalin-fixed paraffin-embedded tissues were studied (24 adenocarcinoma, 18 adenoma, and 21 colorectal tissues classified as benign colitis). DNA from colorectal samples was analysed by quantitative real-time polymerase chain reaction to detect the most clinically relevant high HR-HPV types (HPV-16, -18, -31, -33, -45, -52, and -58). Associations between histologic findings and other risk factors were also analysed. Overall, HPV DNA was detected in 23.8% (15/63) of the samples studied. Viral infections were detected in 41.7% of adenocarcinoma (10/24) and 27.7% of adenoma cases (5/18). HPV DNA was not found in any of the negative cases. An association between histological diagnosis of adenocarcinoma and HPV infection was observed (odd ratio = 4.85, 95% confidence interval = 1.40-16.80, p = 0.009). The only genotypes identified were HPV 16 and 33. Viral loads were higher in adenocarcinoma, and these cases were associated with HPV 16. This study provides molecular evidence of HR-HPV infection in colorectal adenocarcinoma tissues from Cuban patients.

Coxsackievirus A6 and enterovirus 71 causing hand, foot and mouth disease in Cuba, 2011-2013.

Hand, foot and mouth disease (HFMD) is usually caused by coxsackievirus A16 or enterovirus 71 (EV71). Between 2011 and 2013, HFMD cases were reported from different Cuban provinces. A total of 42 clinical specimens were obtained from 23 patients. Detection, identification and phylogenetic analysis of enterovirus-associated HFMD were carried out by virus isolation, specific enterovirus PCR and partial VP1 sequences. HEV was detected in 11 HFMD cases. Emerging genetic variants of coxsackievirus A6 and EV71 were identified as the causative agents of the Cuban HFMD cases.

Molecular epidemiology of human papillomavirus infections in cervical samples from cuban women older than 30 years.

OBJECTIVE: This study aimed to provide information about the molecular epidemiology of human papillomavirus (HPV) in a group of Cuban women. MATERIALS AND METHODS: DNA from cervical samples was analyzed using a quantitative real-time polymerase chain reaction (PCR), which detects 6 of the clinically most relevant high-risk HPV types. Furthermore, end point PCR and sequencing were performed. Three hundred twenty-two women (211 with positive and 111 with negative cytologic results) aged between 30 and 69 years were enrolled. Risk factors associated with HPV infections and premalignant lesions were also investigated. RESULTS: HPV DNA was detected in 76.1% (245/322) of the studied population, and 34 different genotypes were found. There was an association between HPV infection and low educational level, history of oral contraceptives, menopausal stage, as well as cigarette and/or alcohol consumption. Besides, in a multivariate analysis, previous positive Pap test result and positive colposcopy finding were both predictor variables for HPV infections and for premalignant lesions. Human papillomavirus infection was found in 94.3% of women (199/211) with positive cytologic result and in 41.4% (46/111) of those with negative results, being more likely that the first group was infected with any HPV (odds ratio = 23.43; 95% CI = 11.70-46.92; p = .000). The most common genotypes were HPV types 16, 18, 31, 58, 33, and 45. All the cases with HPV positive findings had at least 1 high-risk HPV genotype. CONCLUSIONS: This is the first report of the molecular epidemiology of HPV in Cuban women, based on results from a DNA sequence and quantitative PCR. Most individuals were infected with high-risk HPV types. These findings support the inclusion of HPV vaccine in Cuba.

Epigenetics DNA methylation in the core ataxin-2 gene promoter: novel physiological and pathological implications.

Pathogenic CAG (cytosine-adenine-guanine) expansions beyond certain thresholds in the ataxin-2 (ATXN2) gene cause spinocerebellar ataxia type 2 (SCA2) and were shown to contribute to Parkinson disease, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Regulation of ATXN2 gene expression and the function of the protein product are not known. SCA2 exhibits an inverse correlation between the size of the CAG repeat and the age at disease onset. However, a wide range of age at onset are typically observed, with CAG repeat number alone explaining only partly this variability. In this study, we explored the hypothesis that ATXN2 levels could be controlled by DNA methylation and that the derangement of this control may lead to escalation of disease severity and influencing the age at onset. We found that CpG methylation in human ATXN2 gene promoter is associated with pathogenic CAG expansions in SCA2 patients. Different levels of methylation in a SCA2 pedigree without an intergenerational CAG repeat instability caused the disease anticipation in a SCA2 family. DNA methylation also influenced the disease onset in SCA2 homozygotes and SCA3 patients. In conclusion, our study points to a novel regulatory mechanism of ATXN2 expression involving an epigenetic event resulting in differential disease course in SCA2 patients.

Assessment of infection with polyomaviruses BKV, JCV and SV40 in different groups of Cuban individuals.

We investigated the frequency of BKV, JCV and SV40 reactivation in three groups of Cuban patients by multiplex nested PCR assay of 40 paraffin-embedded colorectal neoplasm tissues, 113 urine samples, and 125 plasma samples from 27 transplant recipients, and cerebrospinal fluid (CSF) from 67 HIV-1-infected individuals with central nervous system (CNS) disorders. None of these polyomaviruses were detected in colorectal neoplasms. JCV DNA was detected in 2 of 67 patients (2.9%) with CNS disorders, but neither BKV nor SV40 was identified. BKV was found in urine from 38.5% and 28.6% of adult and pediatric transplant recipients, respectively. In adult renal transplant recipients, excretion of BKV in urine was significantly associated with episodes of acute rejection (p=0.012) and with excretion of HCMV in urine (p= 0.008). In Cuba, the polyomaviruses studied here could not be related to colorectal neoplasms, and JCV was rarely detected in CSFs of HIV-1-infected individuals, whilst BKV reactivation was found to occur frequently in organ transplant recipients.

SARS-CoV-2: theoretical analysis of the proposed algorithms to the enhancement and segmentation of high-resolution microscopy images-Part II.

Today is a reality that the novel coronavirus SARS-Cov-2 has become a global pandemic. For this reason, the study of real microscopic images of this coronavirus is of great importance, as it allows us to carry out a more precise research on it. However, as we pointed out in a former paper as reported by Roberto Rodríguez (SARS-CoV-2: Enhancement and Segmentation of High-Resolution Microscopy Images. Part I", Sent to Signal, Image and Video Processing Video Processing, Springer, New York, 2020), many times these microscopic images present some blurring problems, which are always susceptible to be improved. The aim of this work is to carry out a theoretical analysis of the proposed algorithms to enhancement and segmentation of these microscopic images, which is important for the design and development of future algorithms before new epidemics.

Emergence and evolution of SARS-CoV-2 genetic variants during the Cuban epidemic.

The SARS CoV-2 D614G variant circulated in Cuba in 2020. New viral variants were detected after the opening of the border in November 2020. We show the results of the genomic surveillance in Cuba from December 28, 2020, to September 28, 2021 and their relationship to the epidemiological situation in the country. A total of 1,406 nasopharyngeal exudates from COVID-19 patients were processed for RNA extraction and the 1836 bp fragment of the spike gene was amplified and sequenced. The mutations present were determined using the GISAID database. Prevalence ratios were estimated by fitting Poisson univariate and multivariate regression models to investigate associations between SARS-CoV-2 variant group (VOC, non-VOC) and disease outcome. Seventeen genetic variants were detected including VOC Alpha, Beta, Gamma and Delta, one variant of interest (VOI) (Lambda) and two previous VOI (A.2.5.1 and Zeta/P.2). Beta (34.77%), Delta (24.89%) and D614G (19%) variants were the most frequently detected. By June, Delta increased in frequency, displacing Beta. Disease severity increased significantly with age and VOC (PR =1.98, IC 95%: 1.33-3.05, p <0.05). Genomic surveillance allowed us to identify the upsurge of novel variants. Coinciding with the higher epidemic period, multiple variants were co-circulating. Although we cannot rule out that failure in the transmission containment measures occurred, the increase in the number of cases associated with the circulation of several variants, particularly the Beta and Delta variants is highly suggestive. A greater association of Beta variant with clinical severity and Delta variant with a greater transmissibility was observed.

SARS-CoV-2: enhancement and segmentation of high-resolution microscopy images-Part I.

Possibly, and due to poor eating habits and unhealthy lifestyle, many viruses are transmitted to human people. Such is the case, of the novel coronavirus SARS-Cov-2, which has expanded of exponential way, practically, to whole world population. For this reason, the enhancement of real microscopic images of this coronavirus is of great importance. Of this way, one can highlight the S-spikes and visualizing those areas that show a high density, which are related to active zones of viral germination and major spread of the virus. The SARS-Cov-2 images were captured from nasopharyngeal samples of Cuban symptomatic individuals (RT-PCR positives for SARS-CoV-2) and processed via scanning electron microscopy. However, many times these microscopic images present some blurring problems, and the S-spikes do not look well defined. Therefore, the aim of this work is to propose new computational methods to carry out enhancement and segmentation of SARS-Cov-2 high-resolution microscopic images. The proposed strategy obtained very satisfactory results, and we validated its performance, together with specialist physicians, on a set of 1005 images. Due to the importance of the obtained results, this first work will be addressed to the application of the proposed algorithm. A second paper will deeply analyze the theory related to these algorithms.

Kaposi's sarcoma-associated herpesvirus load in asymptomatic contacts of Cuban epidemic KS patients.

To evaluate the pathogenic mechanisms and transmission routes involved in KSHV infection in 22 Cuban individuals who maintained close contact with epidemic KS patients, real-time PCR was used to quantify KSHV-DNA in clinical samples of plasma, saliva and peripheral blood mononuclear cells (PBMC). KSHV-DNA was detected in 72.7% (16/22) of the contacts. The highest levels of KSHV load were detected in saliva, followed by PBMC (average log copies/100 ng DNA = 1.28 and 1.12), while significantly lower levels were detected in plasma (average log copies/ml = 0.37). Two of three intra-domiciliary and two serodiscordant sexual contacts of AIDS-KS patients were infected with KSHV. The rate of KSHV-DNA detection in saliva and PBMC samples in men who have sex with men (MSM) was significantly higher than in heterosexuals (HT) (p = 0.014). MSM were more likely to harbor KSHV-DNA in saliva when compared with HT individuals (OR 4.33; 95% CI 1.117-16.8). These results emphasize that, in Cuba, KSHV horizontal transmission through saliva may occur, although homosexual behavior may predispose an individual to KSHV acquisition. Even in the absence of disease, KSHV could cause an asymptomatic systemic infection in individuals who maintain close contact with AIDS-KS patients.

First report on fatal myocarditis associated with adenovirus infection in Cuba.

Myocarditis is caused frequently by viral infections of the myocardium. In the past, enteroviruses (EV) were considered the most common cause of myocarditis in all age groups. Other viruses that cause myocarditis are adenovirus and influenza viruses. Parvovirus B19 infection is associated sometimes with myocarditis. Members of the Herpesviridae family, cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) have been associated occasionally with myocarditis. During an atypical outbreak of acute febrile syndrome, eight children, with ages from 5 months to 15 years, died in cardiogenic shock due to myocarditis in July-August 2005, in the city of Havana, Cuba. Nested polymerase chain reaction (nPCR) and nested reverse transcription-PCR (nRT-PCR) were carried out on fresh heart muscle and lung tissue to analyze the genomic sequences of adenovirus, CMV, HHV-6, herpes simplex virus, Epstein-Barr virus (EBV), varizella zoster virus, influenza virus A, B, C, respiratory syncytial virus (RSV) A and B, parainfluenza viruses, rhinoviruses, coronavirus, flaviruses and enteroviruses. Evidence was for the presence of the adenovirus genome in 6 (75%) of the children. Phylogenetic analyses of a conserved hexon gene fragment in four cases showed serotype 5 as the causal agent. No others viruses were detected. Histological examination was undertaken to detect myocardial inflammation. After exclusion of other possible causes of death, the results indicated that viral myocarditis was the cause of death in patients with adenovirus infection.

A myocarditis outbreak with fatal cases associated with adenovirus subgenera C among children from Havana City in 2005.

BACKGROUND: Among multiple causes of acute myocarditis, viral infection, especially that due to enteroviruses and adenoviruses, is the leading cause. In the summer 2005 an outbreak of a febrile syndrome accompanied by acute cardiac decompensation occurred in infants and young children in Havana City. Eleven patients had a rapid evolution of disease and there were 8 fatalities from cardiac failure secondary to myocarditis. OBJECTIVE: The aim of the present study was to determine the etiological agent responsible for this outbreak. STUDY DESIGN: Children admitted to the pediatric hospitals of Havana City from July 3 to August 2 with this clinical presentation were studied. Forty samples of necropsy tissue, cerebrospinal fluid, stools and serum were tested by molecular methods for 14 respiratory viruses, 6 herpesviruses and generic enteroviruses and flavirus and alfaviruses. Viral isolation was performed in A-549 cells. Isolated viruses were typed by sequence analysis. RESULTS: Adenovirus genome was detected in 6 of the 8 fatal cases-the lungs in 5 (63%) and the myocardium in 3 (37%). In two fatal cases, viral genome was detected in both lung and myocardium. Adenovirus was isolated in five fatal cases. In all three non-fatal cases, adenovirus genome was detected and adenovirus was isolated into two. Sequence analysis showed that adenovirus type 5 was the only isolate from fatal cases and adenovirus 1 the only isolate in non-fatal cases. No other viruses were found by PCR or isolation techniques. CONCLUSION: Adenovirus was the etiologic agent implicated in this myocarditis outbreak and adenovirus type 5 was associated with fatal outcome.

Nearly identical strains of human herpesvirus 8 in couples discordant for Kaposi's sarcoma.

Transmission of human herpesvirus 8 (HHV-8) may occur through various routes including breastfeeding and sexual intercourse. We attempted to detect HHV-8 infection in nine HIV-positive couples discordant for Kaposi's sarcoma who maintained a monogamous sexual relationship for at least one year. By quantitative real-time polymerase chain reaction and HHV-8 genotyping we provide strong evidence for the sexual transmission of HHV-8 in this unique cohort.

Limitations to contact tracing and phylogenetic analysis in establishing HIV type 1 transmission networks in Cuba.

Sequence analysis can be used to evaluate transmission networks. We have used retrospective samples to examine two HIV-1 transmission networks established by contact tracing. Regions of the HIV-1 region representing segments of gag and env were amplified by RT-PCR from frozen plasma samples and the sequence of each PCR product was determined. Within one of the networks (composed of 38 subjects) we found only a subset of the tested sequence clusters was consistent with the reported epidemiological linkage. Of 15 presumed transmission events where sequence data were available, 9 could be rejected either by subtype mismatch or by phylogenetic tests. In the other network (composed of 89 subjects) we were able to assess sequences for 26 presumed transmission events, 18 of which were rejected based on subtype discordance. Long lags in time between the time of transmission and the time of sequence sampling (ranging from 2 to 18 years) may limit the sensitivity for the detection of sequence linkage. Also, superinfection and incomplete epidemiological information are other factors that will limit the concordance of phylogenetic reconstruction and reported epidemiological linkage.

The Cuba-United States Thaw: Building Bridges Through Science and Global Health.

AbstractBeginning in 2014, there has been significant progress in normalization of relations between Cuba and the United States. Herein, we discuss the history and recent progress in scientific collaboration between the two countries as well as the continued challenges. Science and global health diplomacy can be key tools in reestablishing a trusting and productive relationship of mutual and global benefit, bringing about better and healthier lives for people in both Cuba and the United States.