Newborn Screening Program for Cystic Fibrosis in Cuba: Three Years' Experience

ABSTRACT In Cuba, newborn screening (NBS) for cystic fibrosis (CF) was introduced in January 2019. The results from the first three years of the CF NBS program are presented. An IRT/IRT protocol was followed using a cut-off value of 50 ng/mL. In this period 281,717 neonates were screened, 2,197 samples had increased IRT values, and a second sample was necessary (recall rate=0.78%). In 686 (0.24%) neonates, IRT was still elevated, and they were referred for clinical evaluation. Twenty-one children were confirmed by sweat test and molecular biology. Eighteen newborns presented variant F508del. A false negative case was reported. Demographic data of 32,764 neonates were collected. The average age of sampling was six days with results available at 11 days of life, but 1.7% of the samples were collected 20 days after birth. The mean IRT value was 12.7±11.7 ng/mL (ranging 0-283 ng/mL) with a calculated 98.5 percentile value of 42.4 ng/mL. On average, the samples were processed five days after collection and two days after they were received at the laboratory. Although CF NBS program in Cuba is just beginning, it can be predicted that CF will be one of the most frequent inherited-metabolic diseases in the Cuban population.

Pilot study for cystic fibrosis neonatal screening: the Cuban experience.

Background In Cuba, no screening program for cystic fibrosis (CF) has been implemented yet. The ultramicro enzyme-linked immunosorbent assay (UMELISA)® TIR NEONATAL has been developed for the measurement of immunoreactive trypsin (IRT) in dried blood spots on filter paper. The analytical performance of the kit was evaluated in the national network of laboratories. Methods Newborn dried blood samples (DBS) were evaluated in 16 laboratories. An IRT/IRT/DNA protocol was followed using a cut-off value of 50 ng/mL. The mean, median and percentiles of the distribution were calculated and a two-sample t-test with unequal variance was used for statistical analysis. Influence of perinatal factors on IRT levels was analyzed. Results From January to June 2018, 6470 newborns were studied, obtaining a mean IRT value of 12.09 ng/mL (ranging 0-358 ng/mL) and a median of 8.99 ng/mL. Fifty-two samples (0.78%) were above the cut-off level and 16 samples (0.24%) were elevated in the re-screening process. One of them was confirmed positive by molecular biology (phe508del/c.3120 + 1G > A), constituting the first newborn screened and diagnosed early in Cuba. Second DBS samples were collected on average at 14 days and processed in the laboratory at 16 days of birth. Significant differences were observed (p < 0.05) when evaluating the influence of gender, birth weight (BW) and gestational age (GA) on the IRT values. Lower IRT concentrations were found in samples processed after 10 days of collection. Conclusions The performance of UMELISA® TIR NEONATAL in the laboratories has been satisfactory; hence CF newborn screening (NBS) was extended throughout the country from January 2019.

Identification of Vimentin as a Potential Therapeutic Target against HIV Infection.

A combination of antiviral drugs known as antiretroviral therapy (ART) has shown effectiveness against the human immunodeficiency virus (HIV). ART has markedly decreased mortality and morbidity among HIV-infected patients, having even reduced HIV transmission. However, an important current disadvantage, resistance development, remains to be solved. Hope is focused on developing drugs against cellular targets. This strategy is expected to prevent the emergence of viral resistance. In this study, using a comparative proteomic approach in MT4 cells treated with an anti-HIV leukocyte extract, we identified vimentin, a molecule forming intermediate filaments in the cell, as a possible target against HIV infection. We demonstrated a strong reduction of an HIV-1 based lentivirus expressing the enhanced green fluorescent protein (eGFP) in vimentin knockdown cells, and a noteworthy decrease of HIV-1 capsid protein antigen (CAp24) in those cells using a multiround infectivity assay. Electron micrographs showed changes in the structure of intermediate filaments when MT4 cells were treated with an anti-HIV leukocyte extract. Changes in the structure of intermediate filaments were also observed in vimentin knockdown MT4 cells. A synthetic peptide derived from a cytoskeleton protein showed potent inhibitory activity on HIV-1 infection, and low cytotoxicity. Our data suggest that vimentin can be a suitable target to inhibit HIV-1.

HIV-1 Genetic Variability in Cuba and Implications for Transmission and Clinical Progression.

INTRODUCTION Serological and molecular HIV-1 studies in Cuba have shown very low prevalence of seropositivity, but an increasing genetic diversity attributable to introduction of many HIV-1 variants from different areas, exchange of such variants among HIV-positive people with several coinciding routes of infection and other epidemiologic risk factors in the seropositive population. The high HIV-1 genetic variability observed in Cuba has possible implications for transmission and clinical progression. OBJECTIVE Study genetic variability for the HIV-1 env, gag and pol structural genes in Cuba; determine the prevalence of B and non-B subtypes according to epidemiologic and behavioral variables and determine whether a relationship exists between genetic variability and transmissibility, and between genetic variability and clinical disease progression in people living with HIV/AIDS. METHODS Using two molecular assays (heteroduplex mobility assay and nucleic acid sequencing), structural genes were characterized in 590 people with HIV-1 (480 men and 110 women), accounting for 3.4% of seropositive individuals in Cuba as of December 31, 2013. Nonrandom sampling, proportional to HIV prevalence by province, was conducted. Relationships between molecular results and viral factors, host characteristics, and patients' clinical, epidemiologic and behavioral variables were studied for molecular epidemiology, transmission, and progression analyses. RESULTS Molecular analysis of the three HIV-1 structural genes classified 297 samples as subtype B (50.3%), 269 as non-B subtypes (45.6%) and 24 were not typeable. Subtype B prevailed overall and in men, mainly in those who have sex with men. Non-B subtypes were prevalent in women and heterosexual men, showing multiple circulating variants and recombinant forms. Sexual transmission was the predominant form of infection for all. B and non-B subtypes were encountered throughout Cuba. No association was found between subtypes and transmission or clinical progression, although the proportion of deaths was higher for subtype B. Among those who died during the study period, there were no differences between subtypes in the mean time from HIV or AIDS diagnosis to death. CONCLUSIONS Our results suggest that B and non-B HIV-1 subtypes found in Cuba do not differ in transmissibility and in clinical disease progression. KEYWORDS HIV-1, AIDS, molecular epidemiology, transmissibility, clinical progression, subtypes, circulating recombinant forms, pathogenesis, Cuba.

Phylogenetic analysis of human T cell lymphotropic virus type 1 isolated from Cuban individuals.

The presence of infection by human T cell lymphotropic virus type 1 (HTLV-1) in Cuba has been previously documented. However, genetic information on the strains that circulate in the Cuban people remains unknown. The present work constitutes the first study of phylogenetic relationship of HTLV-1 Cuban isolates. Twelve Cuban patients who were diagnosed with HTLV-1 infection and had different clinical manifestations were studied. The 3' LTR sequences were analyzed for the construction of a phylogenetic tree with reference sequences of HTLV-1 of different geographic origins. Phylogenetic analysis of the 3' LTR gene showed that all the Cuban samples clustered in the Transcontinental subgroup of the Cosmopolitan subtype. Phylogenetic analysis suggests multiple introductions of HTLV-1 in Cuba as well as a possible African origin of the samples. The results of the study will reinforce the program of epidemic surveillance of the infection in Cuba.

Transmitted HIV type 1 drug resistance in newly diagnosed Cuban patients.

Knowledge of the associated mutations to transmitted drug resistance (TDR) in strains of human immunodeficiency virus type 1 (HIV-1) constitutes a fundamental premise in epidemiological surveillance. In this present study, TDR from 200 Cuban patients who were diagnosed with HIV-1 between 2009 and 2011 was analyzed. By partial reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing of the HIV pol gene, an HIV subtype and transmitted resistance profile were determined. The prevalence of associated mutations to the TDR in the individuals studied was 21.5%. In the region of the reverse transcriptase, the most common mutations were K103N and M184V, while in the region of the protease they were L33F and M46L. The results of this study provide evidence of TDR in the Cuban seropositive population and suggest the necessity of making resistance assays before beginning antiretroviral therapy in HIV-1-infected patients in Cuba.

HIV­2 antibody detection after indeterminate or negative HIV­1 Western blot in Cuba, 2005-2008.

INTRODUCTION Differentiating between HIV-1 and HIV-2 infection is the first step to understanding HIV transmission, epidemiology and pathogenesis in geographical areas where both viruses circulate. In Cuba, positive results in mixed HIV-1/2 screening assays are confirmed by HIV-1 Western blot. Indeterminate results constitute the main limitation of this test and HIV-2 infection is among their possible causes; hence the importance of second-stage screening and confirmatory tests for HIV-2 infection. OBJECTIVE Investigate the contribution of HIV-2 antibodies to negative or indeterminate HIV-1 Western blot results in serum samples from 2005 through 2008 in Cuba. METHODS HIV-2 reactivity was studied using the ELISA DAVIH-VIH-2 diagnostic kit (Cuba) in 1723 serum samples with negative or indeterminate results for HIV-1 Western blot from January 2005 through December 2008. Duplicate sera reactive by ELISA were confirmed by HIV-2 Western blot, results interpreted according to WHO criteria. The epidemiological interview established by Cuba's National Program for Prevention and Control Sexually-Transmitted Diseases and HIV/AIDS was applied to HIV-2 Western blot-positive patients. RESULTS Among all sera studied, HIV-2 ELISA identified 12 reactive serum samples (0.70%) and 1711 non-reactive (99.30%). Western blot analysis of the 12 ELISA-reactive samples confirmed two positive samples (16.67%), 4 negative (33.33%) and 6 indeterminate (50%). Positive samples reacted against the p16, p26, gp36, p53, p56, p68 and gp105 proteins. All 12 ELISA-reactive samples belonged to the HIV-1 Western blot indeterminate group. The two HIV-2-positive samples showed well defined reactivity to gp160, p53, p55 and p34 of HIV-1. HIV-1 seroconversion was observed in all 10 remaining samples during serological followup. CONCLUSIONS Two new HIV-2 seropositive cases were diagnosed using DAVIH-VIH-2 and HIV-2 Western blot in indeterminate HIV-1 Western blot samples. Results support the recommendation that HIV-2 Western blot be included in the diagnostic algorithm for HIV-1/2 to followup negative or indeterminate HIV-1 Western blot results. KEYWORDS Diagnosis, laboratory techniques and procedures, antibodies, HIV-2, Western blot, enzyme-linked immunosorbent assay, algorithm, Cuba.

Purificacion de la Proteina de 24 KD natural (P24) del virus de inmunodeficiencia humana tipo-1 (VIH-1) utilizando inmunoafinidad / Purification of HIV-1 24 KD natural protein (p24)by immunoaffinity

En el seguimiento de las personas afectadas con el VIH-1 resulta de gran utilidad el empleo de sistemas para detectar la presencia de proteina de 24 KD y los niveles de anticuerpos a esta; para el desarrollo de estos sistemas es necesario contar con esta proteina purificada. El presente trabajo describe la purificacion de p24 a partir de lisado rival semipurificado sobre gradiente de sacarosa, empleando cromatografia de inmunoafinidad con anticuerpos monoclonales acoplados a Sefarosa 4B activada con bromuro de cianogeno. Para la caracterizacion del producto purificado se utilizo electroforesis en gel de poliacrilamida con tincion de plata e inmunoelectrotransferencia. Como resultado de este trabajo se obtuvo p24 util para ser empleada en el desarrollo de medios diagnosticos y anticuerpos monoclonales

Purificación de la Proteína de 24 KD natural (P24) del virus de inmunodeficiencia humana tipo-1 (VIH-1) utilizando inmunoafinidad / Purification of HIV-1 24 KD natural protein (p24)by immunoaffinity

En el seguimiento de las personas afectadas con el VIH-1 resulta de gran utilidad el empleo de sistemas para detectar la presencia de proteína de 24 KD y los niveles de anticuerpos a ésta; para el desarrollo de estos sistemas es necesario contar con esta proteína purificada. El presente trabajo describe la purificación de p24 a partir de lisado rival semipurificado sobre gradiente de sacarosa, empleando cromatografía de inmunoafinidad con anticuerpos monoclonales acoplados a Sefarosa 4B activada con bromuro de cianógeno. Para la caracterización del producto purificado se utilizó electroforesis en gel de poliacrilamida con tinción de plata e inmunoelectrotransferencia. Como resultado de este trabajo se obtuvo p24 útil para ser empleada en el desarrollo de medios diagnósticos y anticuerpos monoclonales(AU)