Viability-PCR for the selective detection of Lactobacillus acidophilus and Bifidobacterium bifidum in live bacteria-containing products.

To exert their beneficial effects, microorganisms used in live bacteria-containing products must be viable and present in certain amounts. In this study, we developed a viability assay based on quantitative PCR coupled with propidium monoazide for the identification and enumeration of viable Lactobacillus acidophilus and Bifidobacterium bifidum. In order to optimize the protocol, the thermal inactivation conditions for the two target microorganisms and the PMA concentration inhibiting DNA amplification from the dead cells while allowing it from the live cells were first determined. The viability-PCR protocol was then applied to analyze a commercial product containing the two microorganisms. The quantities of both microorganisms determined using viability-PCR in the tested product were significantly higher than those obtained using the standard plate count, suggesting the presence of bacteria in a viable but non-culturable physiological state. Moreover, lower amounts of the two microorganisms were detected using viability-PCR compared to those achieved using quantitative PCR, possibly because of the presence of dead cells in the samples. Our results suggest that the viability-PCR method proposed here is a suitable alternative for rapid and accurate quantification and assessment of the viability of L. acidophilus and B. bifidum and could be easily adopted in the quality control screening of live bacteria-containing products.

Phleboviruses detection in Phlebotomus perniciosus from a human leishmaniasis focus in South-West Madrid region, Spain.

BACKGROUND: Phlebotomus-borne (PhB-) viruses are distributed in large areas of the Old World and are widespread throughout the Mediterranean basin, where recent investigations have indicated that virus diversity is higher than initially suspected. Some of these viruses are causes of meningitis, encephalitis and febrile illnesses. In order to monitor the viral presence and the infection rate of PhB-viruses in a recently identified and well characterized human zoonotic leishmaniasis focus in southwestern Madrid, Spain, a sand fly collection was carried out. METHODS: Sand fly insects were collected in four stations using CDC light traps during 2012-2013 summer seasons. Screening for Phlebovirus presence both via isolation on Vero cells and via polymerase chain reaction (PCR), using degenerated primers targeting a portion of the L segment, was performed. The serological identity and phylogenetic relationships on the three genomic segments of the viral isolates were carried out. RESULTS: Six viral isolates belonging to different serological complexes of the genus Phlebovirus were obtained from fifty pools on a total of 963 P. perniciosus (202 females). Phylogenetic analysis and serological assays allowed the identification of two isolates of Toscana virus (TOSV) B genotype, three isolates strongly related to Italian Arbia virus (ARBV), and one isolate of a novel putative Phlebovirus related to the recently characterized Arrabida virus in South Portugal, tentatively named Arrabida-like virus. Positive male sand fly pools suggested that transovarial or venereal transmission could occur under natural conditions. CONCLUSIONS: Our findings highlighted the presence of different Phlebovirus species in the South-West area of the Madrid Autonomous Community where an outbreak of cutaneous and visceral human leishmaniasis has been recently described. The evidence of viral species never identified before in Spain, as ARBV and Arrabida-like virus, and TOSV B genotype focus stability was demonstrated. Environmental aspects such as climate change, growing urbanization, socio-economic development could have contributed to the genesis of this wide ecological niche of PhB-viruses and Leishmania spp. The potential role of vertebrates as reservoir for the phleboviruses identified and the possibility of Phleboviruses-Leishmania co-infection in the same sand fly should be assessed. Furthermore the PhB-viruses impact on human health should be implemented.

A unique HAV strain circulated in patients with acute HAV infection with different risk exposures in Tuscany, Italy.

BACKGROUND: Acute Hepatitis A Virus (HAV) is reported to be an emergent problem in several developed countries. OBJECTIVES AND STUDY DESIGN: The aim of this study was to analyse the HAV strains circulating among individuals with acute HAV infection, apparently transmitted by different routes, in several districts of Tuscany in central Italy, during the year 2008. RESULTS: An outbreak of acute HAV infection occurred from May to August 2008 in Arezzo; 32 individuals were admitted to the hospital, in 25 of them at least a linkage with an infected food handler and/or household contacts was reported and in 3 homosexuality was a possible risk factor. In Florence, from January 2008 to August 2008, 41 individuals mainly homosexual men were admitted to two hospitals with the diagnosis of acute HAV. The phylogenetic analysis of VP1/2A region of HAV was used to characterize these HAV isolates. All viral sequences were assigned to genotype IA. All clustered in the same branch (bootstrap 82%) of phylogenetic tree, thus indicating the same circulating isolate. Apart of one isolate from France and one from Germany which were similar with the "Tuscany" strain reported here, high heterogeneity with the other European HAV strains reported in the GenBank in the last years, was observed. CONCLUSION: The detection of a unique HAV isolate circulating in different Tuscany districts, suggests sequential transmission of HAV infection in this geographical area through possible links among acute hepatitis cases. The application of safe food handling practices and vaccination of homosexual men may contribute to the prevention of HAV infection.

Identification of low HBV-DNA levels by nucleic acid amplification test (NAT) in blood donors.

OBJECTIVE: To evaluate the presence of HBV-DNA in 22,765 consecutive blood donors, who donated blood in the period from January 2006 to August 2007 at a transfusion centre in Lazio, a region in central Italy with low HBV endemicity. METHODS: Each donation was individually tested using immunoenzymatic assays and nucleic acid amplification technologies (NAT). Samples that were reactive to generic NAT, Procleix Ultrio Assay were tested for HBV-DNA, HCV-RNA and HIV1-RNA by Discriminatory Procleix Ultrio NAT Assay. In samples that were reactive to generic NAT and negative for HBsAg, HCV-RNA and HIV1-RNA, HBV-DNA was further tested using Cobas TaqMan and an in-house nested PCR following an ultracentrifugation step. Sequence analysis confirmed HBV-DNA positivity. RESULTS: Generic NAT identified 31 (0.13%) reactive sera. HBV-DNA discriminatory NAT identified 15 positive sera; HBsAg was positive in 12 sera. Of the 5 generic NAT-reactive/discriminatory NAT-negative/HBsAg-negative sera and of the 3 HBsAg-negative/HBV-DNA discriminatory NAT-positive sera, 7 were positive to Cobas TaqMan or the in-house PCR after ultracentrifugation. The overall HBV-DNA positivity was 0.083% [19 of 22,765 donors: 12 HBsAg-positive (HBV-DNA range 10(2)-10(4) IU/mL), 7 HBsAg-negative/anti-HBc positive (HBV-DNA< 6 IU/mL)]. CONCLUSIONS: For blood transfusion safety, the significance of the finding of very low HBV-DNA levels should be further investigated. Our data indicate that in areas with a low HBV endemicity, single NAT assays may not always identify blood donations with very low HBV-DNA levels.

HCV genetic variability: from quasispecies evolution to genotype classification.

HCV is a ssRNA virus belonging to the Flaviviruses and is found worldwide worldwide in humans. Following primary infection, persistent infection develops in more than 85% of cases, which in up to 30% of cases, may progress to liver disease, cirrhosis and hepatocellular carcinoma. The virus presents a high degree of genetic variability owing to the combination of a lack of proofreading by the RNA-dependent RNA polymerase and a high level of viral replication. This genetic variability allows the classification of genotypes, subtypes, isolates and quasispecies to which epidemiological and pathogenetic significance may be associated. The features and biological implications of HCV variability and of quasispecies dynamics in infection transmission, mechanisms of chronicity and resistance to antiviral therapy are discussed.

Phylogenetic reconstruction of HCV genotype 1b dissemination in a small city centre: the Camporeale model.

Several seroepidemiological population-based surveys carried out in Italy have shown a high prevalence of hepatitis C virus (HCV) infection. Camporeale (CP), a small Sicilian town with a 10.4% prevalence of HCV mostly genotype 1b, probably represents a specific context, since intravenous drug addiction, and sexual promiscuity are almost absent. In order to reconstruct the pattern of introduction and diffusion of HCV in this ecological niche, the NS5 genomic region of 72 HCV genotype 1 isolates (39 from CP and 33 collected throughout Sicily) was amplified and sequenced. Sequences were aligned and analyzed by BioEdit, PAUP and BEAST, and their molecular evolution compared. Thirty-eight HCV genotype 1b isolates from CP were associated in a monophyletic "transmission cluster." By applying Monte Carlo Markov simulation, it was calculated that HCV was introduced between the end of the 1940s and the beginning of the 1950s. The phylogenetic distance between the CP cluster and other Sicilian isolates confirmed its uniqueness and the local diffusion from a common ancestor. The data obtained from classic phylogenetic analysis, combined with the application of the Bayesian analysis to the study of the coalescence of phylogenetic trees, have shown that, in CP, few HCV native strains have been transmitted in a limited length of time probably through iatrogenic routes, and then have not spread further.

Tat protein vaccination of cynomolgus macaques influences SHIV-89.6P cy243 epitope variability.

In a previous study we showed that vaccination with the native Tat protein controlled virus replication in five out of seven monkeys against challenge with the simian human immunodeficiency virus (SHIV)-89.6P cy243 and that this protection correlated with T helper (Th)-1 response and cytotoxic T lymphocyte (CTL) activity. To address the evolution of the SHIV-89.6P cy243 both in control and vaccinated infected monkeys, the sequence of the human immunodeficiency virus (HIV)-1 Tat protein and the C2-V3 Env region of the proviral-DNA-derived clones were analyzed in both control and vaccinated but unprotected animals. We also performed analysis of the T cell epitope using a predictive epitope model taking into consideration the phylogeny of the variants. Our results suggest that even though the viral evolution observed in both groups of monkeys was directed toward variations in the major histocompatibility complex (MHC)-I epitopes, in the control animals it was associated with mutational escape of such epitopes. On the contrary, it is possible that viral evolution in the vaccinated monkeys was linked to mutations that arose to keep high the viral fitness. In the vaccinated animals the reduction of epitope variability, obtained prompting the immune system by vaccination and inducing a specific immunological response against virus, was able to reduce the emergence of escape mutants. Thus the intervention of host's selective forces in driving CTL escape mutants and in modulating viral fitness appeared to be different in the two groups of monkeys. We concluded that in the vaccinated unprotected animals, vaccination with the Tat protein induced a broad antiviral response, as demonstrated by the reduced ability to develop escape mutants, which is known to help in the control of viral replication.

Acute hepatitis with severe cholestasis and prolonged clinical course due to hepatitis A virus Ia and Ib coinfection.

BACKGROUND: Acute viral hepatitis due to hepatitis A virus is a self-limited illness that infrequently has a severe clinical course. METHODS: We analyzed the virological characteristics of acute hepatitis A in a patient with a severe clinical presentation (peak total and conjugated bilirubin levels, 65.5 mg/dL and 40.1 mg/dL, respectively) and a course of disease that lasted 7 months. RESULTS: Hepatitis A virus sequencing revealed coinfection with 2 subgenotypes of hepatitis A virus (Ia and Ib) as etiological factors of the illness. CONCLUSIONS: Hepatitis A virus Ia and Ib coinfection may have accounted for the prolonged and severe course of illness.

An outbreak of hepatitis A virus infection with a high case-fatality rate among injecting drug users.

BACKGROUND/AIMS: In 2002, the first reported outbreak of hepatitis A virus (HAV) infection involving mostly intravenous drug users (IDU) occurred in Italy. We attempted a thorough evaluation of the outbreak, including epidemiological, clinical and virological analyses. METHODS: We conducted an epidemiological investigation, including a case-control study, to identify the source and the modes of HAV transmission. Hepatitis B and C (HCV) viruses and human immunodeficiency virus (HIV) coinfections were clinically analysed. Sequence analysis of the VP1/2A junction of the HAV isolates was also performed. RESULTS: Of the 47 symptomatic cases, 35 were IDUs. The only associated risk factor was contact (not related to injecting practices) with a jaundiced person (odds ratio: 5.8; 95% confidence interval: 1.3-29.9). Of the cases, 58% were anti-HCV positive and 4.7% anti-HIV positive. Three individuals died of acute liver failure: 2 were HCV-coinfected alcohol abusers, with underlying liver cirrhosis; 1 was HCV/HIV-coinfected. HAV-RNA was found in 15 of the 24 tested patients: genotype IB (8 cases) and IIIA (7 cases) were detected. CONCLUSIONS: HAV was probably transmitted through the fecal-oral route, although parenteral transmission cannot be excluded. The high fatality rate was probably due to severe underlying liver damage. The occurrence of this outbreak highlights the need for routine HAV vaccination for IDUs.