Molecular evolution of coxsackievirus A24v in Cuba over 23-years, 1986-2009.

Coxsackievirus A24 variant (CVA24v) is a major causative agent of acute hemorrhagic conjunctivitis outbreaks worldwide, yet the evolutionary and transmission dynamics of the virus remain unclear. To address this, we analyzed and compared the 3C and partial VP1 gene regions of CVA24v isolates obtained from five outbreaks in Cuba between 1986 and 2009 and strains isolated worldwide. Here we show that Cuban strains were homologous to those isolated in Africa, the Americas and Asia during the same time period. Two genotypes of CVA24v (GIII and GIV) were repeatedly introduced into Cuba and they arose about two years before the epidemic was detected. The two genotypes co-evolved with a population size that is stable over time. However, nucleotide substitution rates peaked during pandemics with 4.39 × 10-3 and 5.80 × 10-3 substitutions per site per year for the 3C and VP1 region, respectively. The phylogeographic analysis identified 25 and 19 viral transmission routes based on 3C and VP1 regions, respectively. Pandemic viruses usually originated in Asia, and both China and Brazil were the major hub for the global dispersal of the virus. Together, these data provide novel insight into the epidemiological dynamics of this virus and possibly other pandemic viruses.

Immunogenicity of Intramuscular Fractional Dose of Inactivated Poliovirus Vaccine.

BACKGROUNDS: Intradermal (id) fractional inactivated poliovirus vaccine ([fIPV] one fifth of normal IPV dose) is safe and immunogenic; however, id administration is perceived as difficult. We compared fIPV immunogenicity administered id or intramuscularly (im). METHODS: This noninferiority trial was conducted among polio vaccine-naive Cuban infants who received 2 IPV doses at 4 and 8 months of age. Infants were randomized into 4 arms: (A) fIPV, 0.1 mL im; (B) fIPV, 0.2 mL im; (C) fIPV, 0.1mL id; and (D) IPV, 0.5 mL im. Blood collected before and after vaccinations was tested for poliovirus-neutralizing antibodies. RESULTS: A total of 196 of 214 (91.6%) enrolled children completed study. Seroconversion after 2 IPV doses in each arm were as follows: (A) 97.3% (90.6-99.7), 98.7% (92.7-99.9), and 90.5% (81.5-96.1) for serotypes 1, 2, and 3, respectively; (B) 97.2% (90.3-99.7), 100%, 95.8% (88.3-99.1) for serotypes 1, 2, and 3, respectively; (C) 89.3% (71.8-97.7), 92.9% (76.5-99.1), 82.1% (63.1-93.9) for serotypes 1, 2, and 3, respectively; and (D) 100%, 100%, 100% for serotypes 1, 2, and 3, respectively. Seroconversion with fIPV im was noninferior to fIPV id for all serotypes. CONCLUSIONS: We demonstrated noninferiority of fIPV im compared with id when administered at 4 and 8 months of age. Further investigations in an earlier infant schedule should be pursued to explore fIPV im as option for dose-sparing strategy in countries reluctant to use fIPV id due to programmatic difficulties of id administration.

Boosting Immune Responses Following Fractional-Dose Inactivated Poliovirus Vaccine: A Randomized, Controlled Trial.

BACKGROUND: Fractional-dose administration of inactivated poliovirus vaccine (fIPV) could increase IPV affordability and stretch limited supplies. We assessed immune responses following fIPV administered intradermally, compared with full-dose IPV administered intramuscularly, among adults with a history of oral poliovirus vaccine (OPV) receipt. METHODS: We conducted a randomized, controlled noninferiority trial in Cuba. fIPV or IPV were administered on days 0 and 28; serum was collected on days 0, 7, 28, and 56 for analysis by a neutralization assay. The primary end point was seroconversion or a ≥4-fold rise in antibody titer. The noninferiority limit was 10%. The secondary end point was safety, assessed by the number and intensity of adverse reactions. RESULTS: A total of 503 of 534 enrolled participants (94.2%) completed all study requirements. Twenty-eight days after the first dose, 94.8%, 98.0%, and 98.0% of fIPV recipients had an immune response to poliovirus types 1, 2, and 3, respectively, compared with 98.1% (P = .06), 98.0% (P = 1.00), and 99.2% (P = .45) in the IPV arm. Noninferiority was achieved on days 7, 28, and 56 for all serotypes. No serious adverse events were reported. CONCLUSION: fIPV induced similar boosting immune responses, compared with full-dose IPV. This suggests that fIPV would be an effective strategy to boost population immunity in an outbreak situation. CLINICAL TRIALS REGISTRATION: ACTRN12615000305527.

Needle-free jet injector intradermal delivery of fractional dose inactivated poliovirus vaccine: Association between injection quality and immunogenicity.

INTRODUCTION: The World Health Organization recommends that as part of the polio end-game strategy a dose of inactivated poliovirus vaccine (IPV) be introduced by the end of 2015 in all countries currently using only oral poliovirus vaccine (OPV). Administration of fractional dose (1/5 of full dose) IPV (fIPV) by intradermal (ID) injection may reduce costs, but its conventional administration is with Bacillus Calmette-Guerin (BCG) needle and syringe (NS), which is time consuming and technically challenging. We compared injection quality achieved with BCG NS and three needle-free jet injectors and assessed ergonomic features of the injectors. METHODS: Children between 12 and 20 months of age who had previously received OPV were enrolled in the Camaguey, Cuba study. Subjects received a single fIPV dose administered intradermally with BCG NS or one of three needle-free injector devices: Bioject Biojector 2000® (B2000), Bioject ID Pen® (ID Pen), or PharmaJet Tropis® (Tropis). We measured bleb diameter and vaccine loss as indicators of ID injection quality, with desirable injection quality defined as bleb diameter ≥5mm and vaccine loss <10%. We surveyed vaccinators to evaluate ergonomic features of the injectors. We further assessed the injection quality indicators as predictors of immune response, measured by increase in poliovirus neutralizing antibodies in blood between day 0 (pre-IPV) and 21 (post-vaccination). RESULTS: Delivery by BCG NS and Tropis resulted in the highest proportion of subjects with desirable injection quality; health workers ranked Biojector2000 and Tropis highest for ergonomic features. We observed that vaccine loss and desirable injection quality were associated with an immune response for poliovirus type 2 (P=0.02, P=0.01, respectively). CONCLUSIONS: Our study demonstrated the feasibility of fIPV delivery using needle-free injector devices with high acceptability among health workers. We did not observe the indicators of injection quality to be uniformly associated with immune response.

Decay of Sabin inactivated poliovirus vaccine (IPV)-boosted poliovirus antibodies.

INTRODUCTION: We conducted a follow-on study to a phase I randomized, controlled trial conducted in Cuba, 2012, to assess the persistence of poliovirus antibodies at 21-22 months following booster dose of Sabin-IPV compared to Salk-IPV in adults who had received multiple doses of oral poliovirus vaccine (OPV) during childhood. METHODS: In 2012, 60 healthy adult males aged 19-23 were randomized to receive one booster dose, of either Sabin-inactivated poliovirus vaccine (Sabin-IPV), adjuvanted Sabin-IPV (aSabin-IPV), or conventional Salk-IPV. In the original study, blood was collected at days 0 (before) and 28 (after vaccination), respectively. In this study, an additional blood sample was collected 21-22 months after vaccination, and tested for neutralizing antibodies to Sabin poliovirus types 1, 2 and 3. RESULTS: We collected sera from 59/60 (98.3%) subjects; 59/59 (100%) remained seropositive to all poliovirus types, 21-22 months after vaccination. The decay curves were very similar among the study groups. Between day 28 and 21-22 months, there was a reduction of ⩾87.4% in median antibody levels for all poliovirus types in all study groups, with no significant differences between the study groups. CONCLUSION: The decay of poliovirus antibodies over a 21-22-month period was similar regardless of the type of booster vaccine used, suggesting the scientific data of Salk IPV long-term persistence and decay may be broadly applicable to Sabin IPV.

Immune responses after fractional doses of inactivated poliovirus vaccine using newly developed intradermal jet injectors: a randomized controlled trial in Cuba.

INTRODUCTION: The World Health Organization recommends that, as part of the new polio endgame, a dose of inactivated poliovirus vaccine (IPV) be introduced by the end of 2015 in all countries using only oral poliovirus vaccine (OPV). Administration of fractional dose (1/5th of full dose) IPV (fIPV) intradermally may reduce costs, but its administration is cumbersome with BCG needle and syringe. We evaluated performance of two newly developed intradermal-only jet injectors and compared the immune response induced by fIPV with that induced by full-dose IPV. METHODS: Children between 12 and 20 months of age, who had previously received two doses of OPV, were enrolled in Camaguey, Cuba. Subjects received a single dose of IPV (either full-dose IPV intramuscularly with needle and syringe or fIPV intradermally administered with one of two new injectors or with BCG needle or a conventional needle-free injector). Serum was tested for presence of poliovirus neutralizing antibodies on day 0 (pre-IPV) and on days 3, 7 and 21 (post-vaccination). RESULTS: Complete data were available from 74.2% (728/981) subjects. Baseline median antibody titers were 713, 284, and 113 for poliovirus types 1, 2, and 3, respectively. Seroprevalence at study end were similar across the intervention groups (≥ 94.8%). The immune response induced with one new injector was similar to BCG needle and to the conventional injector; and superior to the other new injector. fIPV induced significantly lower boosting response compared to full-dose IPV. No safety concerns were identified. INTERPRETATION: One of the two new injectors demonstrated its ability to streamline intradermal fIPV administration, however, further investigations are needed to assess the potential contribution of fIPV in the polio endgame plan.

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.

Priming after a fractional dose of inactivated poliovirus vaccine.

BACKGROUND: To reduce the costs of maintaining a poliovirus immunization base in low-income areas, we assessed the extent of priming immune responses after the administration of inactivated poliovirus vaccine (IPV). METHODS: We compared the immunogenicity and reactogenicity of a fractional dose of IPV (one fifth of a full dose) administered intradermally with a full dose administered intramuscularly in Cuban infants at the ages of 4 and 8 months. Blood was collected from infants at the ages of 4 months, 8 months, 8 months 7 days, and 8 months 30 days to assess single-dose seroconversion, single-dose priming of immune responses, and two-dose seroconversion. Specimens were tested with a neutralization assay. RESULTS: A total of 320 infants underwent randomization, and 310 infants (96.9%) fulfilled the study requirements. In the group receiving the first fractional dose of IPV, seroconversion to poliovirus types 1, 2, and 3 occurred in 16.6%, 47.1%, and 14.7% of participants, respectively, as compared with 46.6%, 62.8%, and 32.0% in the group receiving the first full dose of IPV (P<0.008 for all comparisons). A priming immune response to poliovirus types 1, 2, and 3 occurred in 90.8%, 94.0%, and 89.6% of participants, respectively, in the group receiving the fractional dose as compared with 97.6%, 98.3%, and 98.1% in the group receiving the full dose (P=0.01 for the comparison with type 3). After the administration of the second dose of IPV in the group receiving fractional doses, cumulative two-dose seroconversion to poliovirus types 1, 2, and 3 occurred in 93.6%, 98.1%, and 93.0% of participants, respectively, as compared with 100.0%, 100.0%, and 99.4% in the group receiving the full dose (P<0.006 for the comparisons of types 1 and 3). The group receiving intradermal injections had the greatest number of adverse events, most of which were minor in intensity and none of which had serious consequences. CONCLUSIONS: This evaluation shows that vaccinating infants with a single fractional dose of IPV can induce priming and seroconversion in more than 90% of immunized infants. (Funded by the World Health Organization and the Pan American Health Organization; Australian New Zealand Clinical Trials Registry number, ACTRN12610001046099.).

Type 1 diabetes associated and tissue transglutaminase autoantibodies in patients without type 1 diabetes and coeliac disease with confirmed viral infections.

Coeliac disease and type 1 diabetes are autoimmune diseases that may share the same initiating environmental factors. In this study, the occurrence of type 1 diabetes associated autoantibodies (GADA and IA-2A) and tissue transglutaminase autoantibodies (TGA) was determined in patients with confirmed viral infections and no signs of type 1 diabetes or coeliac disease. Serum samples from 82 Cuban patients tested positive for PCR and IgG specific to enterovirus (HEV, serotype echovirus 16, 20 samples), Epstein-Barr virus (EBV, 20 samples), cytomegalovirus (CMV, 21 samples), and hepatitis C virus (HCV, 21 samples); and sera from 164 controls negative serologically to EBV, CMV, HCV, and echovirus 16 were enrolled in the study. All subjects were screened for GADA, IA-2A, and TGA. The prevalence of TGA in patients infected with HEV, EBV, CMV, or HCV was 55% (11/20), 25% (5/20), 9.5% (2/21), and 9.5% (2/21), respectively. GADA and IA-2A were found in 15% (3/20) and 25% (5/20) of patients infected with HEV. None of the patients infected by EBV, CMV, and HCV had GADA or IA-2A. All children infected with HEV who were positive for type 1 diabetes-associated autoantibodies were also TGA-positive. None of the sera from uninfected subjects were positive for GADA, IA-2A or TGA. In conclusion, TGA can develop during infection with HEV, EBV, CMV, or HCV, while the emergence of islet cell related autoantibodies is restricted to HEV infections. The findings suggest that HEV may be a shared environmental factor for the development of islet and gut-related autoimmunity.

Isolation of Coxsackievirus A24 variant from patients with hemorrhagic conjunctivitis in Cuba, 2008-2009.

BACKGROUND: An outbreak of acute hemorrhagic conjunctivitis occurred in Cuba in 2008 and 2009. OBJECTIVE: To determinate the etiological agent associated with the Cuban outbreaks of acute hemorrhagic conjunctivitis during 2008 and 2009. STUDY DESIGN: Conjunctival swabs and/or faecal samples from 382 patients with clinical diagnosis suggestive of acute hemorrhagic conjunctivitis were subject to viral culture in HEp-2 human laryngeal epidermoid carcinoma cells. Positive samples were identified by a specific Coxsackievirus A24 variant PCR and the 3C protease region of 16 isolates was sequenced for phylogenetic analysis. RESULTS: Enterovirus cytopathic effect was observed in 138 cases (36%). A higher percent of CA24v was recovered from faecal samples, 19 out of 45 cases (42.2%), than from conjunctival swabs, 127 out of 355 samples (35.8%). All isolates were identified as Coxsackievirus A24 variant. Phylogenetic analysis revealed that 2008 and 2009 Cuban outbreaks were caused by the same virus strains and that isolates were closely related to those from Taiwan (2006-2007), China (2007-2008) and Singapore (2005) with a bootstrap value of 71%. CONCLUSIONS: Outbreaks of acute hemorrhagic conjunctivitis occurred in Cuba in 2008 and 2009 were caused by Coxsackievirus A24 variant. The faecal-oral route is another mode of transmission of CA24v in the acute hemorrhagic conjunctivitis outbreaks. Phylogenetic analysis of Cuban CA24v strains involved in an acute hemorrhagic conjunctivitis outbreak in 2008 and 2009 confirms a new introduction of the CA24 variant into the Americas from South-east Asia.