Complementing Braden scale for pressure ulcer risk with clinical and demographic-related factors in a large cohort of hospitalized Italian patients.

AIMS: To determine the prevalence, risk, and determinants of pressure ulcer risk in a large cohort of hospitalized patients. DESIGN: A prospective cross-sectional study with data collection in January 2023. METHODS: Registered nurses collected data from 798 patients admitted to 27 health care units of an Italian hospital. The pressure ulcer risk was assessed using the Braden scale. The presence of comorbidities was collected from clinical reports. Obesity was assessed according to international indicators (Body Mass Index). The receiver operating characteristic (ROC) curve was used to estimate the sensitivity and specificity of different Braden scores for identifying participants with pressure ulcers. RESULTS: The prevalence of pressure ulcers was 9.5%, and 57.4% of the sample were at risk of developing pressure ulcers. The area under the ROC curve was 0.88. The best sensitivity and specificity were found for a Braden cutoff score of 15.5 (sensibility = 0.76; specificity = 0.85). The determinants of lower Braden scores were older age (p < 0.001), comorbidities (p < 0.001), wounds of other nature (p = 0.001), urinary incontinence (p < 0.001), fecal incontinence (p < 0.001), and urinary catheterization (p < 0.001). CONCLUSION: Several demographic factors and specific clinical indicators have been identified as determinants of the risk of developing pressure ulcers, which are easily ascertainable by healthcare providers; thus, they may routinely complement the Braden Scale in the assessment of pressure ulcer risk in order to reinforce and accelerate clinical judgment.

Immunogenicity and protective efficacy of tuberculosis subunit vaccines expressing PPE44 (Rv2770c).

In this study we have evaluated the vaccine potential of a Mycobacterium tuberculosis antigen of the PPE protein family, namely PPE44 (Rv2770c). PPE44-specific immune responses could be detected in mice acutely, chronically and latently infected with M. tuberculosis. Vaccination of mice with a plasmid DNA vaccine coding for PPE44 or recombinant PPE44 protein formulated in adjuvant generated strong cellular and humoral immune responses; immunodominant T cell epitopes were identified. Most importantly, vaccination of mice with both subunit vaccines followed by an intratracheal challenge with M. tuberculosis resulted in a protective efficacy comparable to the one afforded by BCG. Taken together these results indicate that PPE44 of M. tuberculosis is a protective antigen that could be included in novel subunit TB vaccines and that warrants further analysis.

Variation of the expression of Mycobacterium tuberculosis ppe44 gene among clinical isolates.

PPE44 is a member of the Mycobacterium tuberculosis PPE proteins, a polymorphic family of 69 glycine-rich proteins that predictively represent a source of antigenic variation. The genetic diversity of gene ppe44 among clinical isolates has been studied. No genomic polymorphism of ppe44 was found by a PCR-restriction fragment length polymorphism assay using three restriction enzymes. Nucleotide sequencing of gene ppe44 of a number of isolates, selected to represent the major phylogenetic lineages of M. tuberculosis, showed no nucleotide substitution, with the exception of isolates of the Beijing genotype. These findings indicate that gene ppe44 is basically conserved among M. tuberculosis strains. The expression of gene ppe44 was then determined at the transcriptional level by a real-time reverse transcriptase PCR assay. Extremely high quantitative variations in ppe44 expression were found among the isolates; ppe44 expression of the Beijing strains was significantly higher than the non-Beijing strains. To test whether differential expression of gene ppe44 has the potential to provide a dynamic antigen display, antibodies to PPE44 were titered in the sera of M. tuberculosis-infected subjects. Variation of antibody response to PPE44 was found with regard to both antibody titers and the proportion of responding subjects. These results indicate that the differential expression of genes ppe could influence the host's immune responsiveness, thus having implications in the immunopathogenesis of tuberculosis.

Three-year longitudinal study of genotypes of Mycobacterium tuberculosis isolates in Tuscany, Italy.

The genetic diversity of 829 strains of Mycobacterium tuberculosis isolated during a 3-year period in Tuscany, Italy, a country with a low prevalence of tuberculosis, from 480 Italian-born and 349 foreign-born patients was determined by spoligotyping. The predominant spoligotype families were T (30.2% of isolates), Haarlem (19.9%), and the Latino-American and Mediterranean family (LAM) (11.2%); the remaining isolates were distributed among the Beijing (6.5%), S (4.2%), East Africa-India (EAI) (3.0%), Bovis (2.3%), Central Asia (CAS) (2.1%), Africanum (1.3%), and X (1.2%) families or were undefined (2.7%) or orphan (14.1%) isolates. Isolates of the families T, Haarlem, Bovis, and X were distributed among Italian- and foreign-born patients almost proportionally to the patients' numbers. Isolates of the LAM family were prevalent in foreign-born people (13.5%, versus 9.6% in Italian-born patients). Isolates of the S family were found almost exclusively in Italian-born patients, while strains of families EAI and CAS were isolated almost exclusively from foreign-born patients; Africanum isolates were all from African-born patients. The isolates of the Beijing family showed a trend to a steady increase during the survey. The prevalence of Beijing strains was 11.7% among foreign-born people and 2.7% among Italian-born patients. The Beijing strains were typed by the standardized IS6110 restriction fragment length polymorphism assay, which yielded a total of 38 distinct IS6110 patterns; 21 isolates (39.6%) occurred in six distinct clusters; of these, three contained two isolates and the other three contained four, five and six isolates, thus demonstrating that Beijing strains caused several tuberculosis outbreaks in the region. These findings indicate that transmission of Beijing strains between immigrants and the autochthonous population has occurred frequently and suggests an ongoing active transmission of the Beijing genotype in the region.

Molecular analysis of clinical isolates of Mycobacterium bovis recovered from humans in Italy.

In order to achieve a better knowledge of Mycobacterium bovis epidemiology in Italy, 42 clinical isolates from humans were genotyped. Predominant molecular patterns were found in one cluster of 15 isolates sharing spoligotype (ST482), variable-number tandem repeat (VNTR), and IS6110-based restriction fragment length polymorphism (one 1.9-kb band) profiles and in two clusters of 6 and 3 Mycobacterium bovis BCG isolates differing by one VNTR character. The remaining 18 isolates yielded unique profiles. Our results confirm the potential utility of spoligotyping and VNTR typing as a major typing system of M. bovis isolates.

Mutations in mutT genes of Mycobacterium tuberculosis isolates of Beijing genotype.

Missense alterations in genes mutT4 and mutT2, which encode DNA repair enzymes, were sequenced from 30 clinical isolates of Mycobacterium tuberculosis of Beijing genotype, mostly from patients with primary tuberculosis, to evaluate their contribution to anti-mycobacterial drug resistance. The mutation Arg to Gly at codon position 48 (CGG to GGG) of mutT4 was found in 21 isolates; of these, 16 isolates also harboured the mutation Gly to Arg at position 58 (GGA to CGA) of mutT2. No statistically significant association was found between mutT4 and mutT2 mutations, and drug resistance. Furthermore, no mutations in mutT4 or mutT2 were found in any of 24 isolates resistant to multiple drugs, nor in 28 anti-mycobacterial drug-susceptible isolates of different genotypes. These data confirm that the polymorphism of mutT genes is characteristic and unique to the Beijing phylogenetic lineage. The mutator phenotype does not appear to increase prevalence of drug resistance, but further studies are required to investigate the mutation rates of Beijing isolates in response to drug exposure.

Genetic diversity, determined on the basis of katG463 and gyrA95 polymorphisms, Spoligotyping, and IS6110 typing, of Mycobacterium tuberculosis complex isolates from Italy.

Mycobacterium tuberculosis complex isolates (n = 248) collected during a 1-year period in Tuscany, Italy, were genotyped for the katG463 and gyrA95 polymorphisms and by standard spacer oligonucleotide typing (spoligotyping) and IS6110 restriction fragment length polymorphism (RFLP) assays. Most of the isolates (n = 212; 85.5%) belonged to genotypic groups 2 and 3, which included most isolates from Italian-born patients. The remaining isolates were genotypic group 1 organisms, which were prevalent among foreign-born patients (29 of 36; 80.6%). Spoligotype analysis detected 116 unique patterns and 34 clusters including 166 isolates. The combination of spoligotyping and IS6110 RFLP analyses yielded 28 distinct clusters including 65 identical isolates (26.2%)--22 clusters with 2 isolates, 4 clusters with 3 isolates, 1 cluster with 4 isolates, and 1 cluster with 5 isolates--thus proving a low transmission rate in the community. Predominant spoligotypes representing 50% of clustered isolates were found in six clusters that included widespread type ST53 (clade T1) with 29 isolates (11.7% of total isolates); types ST50 and ST47 (Haarlem family) with 18 isolates (7.3%) and 8 isolates (3.2%), respectively; type ST42 (Latino-American and Mediterranean clade) with 13 isolates (5.2%); new type ST1737 (named "Tuscany") with 8 isolates (3.2%); and type ST1 (W-Beijing family) with 7 isolates (2.8%). Other spoligotype families, such as the Mycobacterium africanum, East African-Indian (EAI2/Manila), and central Asia 1 (CAS1/Delhi) families (all including organisms of genotypic group 1) and the Cameroun family (genotypic group 2), were detected especially among immigrant patients. The occurrence of genotypes originally found in distant geographic areas with a high prevalence of tuberculosis may represent a hallmark for changes in the dynamics of transmission of tuberculosis in the region in the near future.

Immunogenicity of mycobacterial PPE44 (Rv2770c) in Mycobacterium bovis BCG-infected mice.

The Pro-Pro-Glu (PPE) protein family of Mycobacterium tuberculosis includes 69 glycine-rich proteins with a conserved N-terminal domain. Their role in tuberculosis is unknown, but it has been speculated that they may have an important immunological significance. In this investigation, the immunogenicity of the ppe44 (Rv2770c) gene product in BALB/c mice infected subcutaneously or intravenously with Mycobacterium bovis bacille Calmette-Guerin (BCG) was evaluated. Mice infected subcutaneously developed high titres of anti-PPE44 IgG1 antibodies, while PPE44-specific IgG2a antibodies were absent at all times tested. PPE44-primed cells from draining lymph nodes and spleen produced low levels of IFN-gamma, and a moderate degree of delayed-type hypersensitivity was observed following PPE44 intracutaneous challenge. In mice infected intravenously, the anti-PPE44 IgG1 antibody response was markedly higher compared with the subcutaneous infection; anti-PPE44 IgG2a antibodies at titres approximately 0.5-2.0 log(10) lower than IgG1 were detected. Interferon (IFN)-gamma production in PPE44-stimulated spleen-cell cultures was transient. These results indicate that PPE44 represents a novel mycobacterial antigen expressed during subcutaneous and intravenous infection by M. bovis BCG in BALB/c mice. Both infection models seem to polarize the immune response to PPE44 towards a Th2 phenotype, as testified by the IgG1 isotype being predominant over IgG2a and by the low IFN-gamma and delayed-type hypersensitivity responses.

Detection of Mycobacterium tuberculosis genotypic groups by a duplex real-time PCR targeting the katG and gyrA genes.

A duplex real-time PCR assay was developed for the assignment of Mycobacterium tuberculosis isolates to the three genotypic groups based on the katG463/gyrA95 polymorphism. The assay was as sensitive and specific as nucleotide sequencing and proved also able to detect unambiguously the isolate genotype in clinical specimens.

Most human isolates of Mycobacterium avium Mav-A and Mav-B are strong producers of hemolysin, a putative virulence factor.

Hemolysin was quantified in 58 isolates of Mycobacterium avium from human, animal, and environmental sources. Human Mav-A and Mav-B isolates were the strongest producers; in contrast, animal and environmental Mav-A isolates and human, animal, and environmental Mav-C organisms were low-level producers. Hemolysin production was not restricted to isolates causing invasive infections.

A real-time PCR assay for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates.

A real-time PCR genotypic assay was developed for the detection of isoniazid (INH) resistance in Mycobacterium tuberculosis. The assay detects mutations C(-15)T and, possibly, G(-24)T in the regulatory region of the inhA gene and proved as sensitive and specific as nucleotide sequencing in all the clinical isolates tested. Our assays mapped the mutations efficiently in 10 out of 35 resistant isolates, thereby covering 29% of all resistant strains.

Involvement of the fadD33 gene in the growth of Mycobacterium tuberculosis in the liver of BALB/c mice.

The potential pathogenic role of Mycobacterium tuberculosis H37Rv fadD33, a gene encoding an acyl-CoA synthase that is underexpressed in the attenuated strain H37Ra, was investigated. In a first approach, fadD33 was cloned and expressed in strain H37Ra to restore gene expression and fadD33-complemented bacteria were used to investigate whether fadD33 might confer any growth advantage to M. tuberculosis H37Ra in an infection model of BALB/c mice. No differences were found in the growth rates of M. tuberculosis H37Rv, H37Ra and fadD33-complemented H37Ra in the lungs and spleen. In contrast, in the liver, where the attenuated strain H37Ra showed impaired growth compared to the virulent strain H37Rv, complementation of the attenuated strain H37Ra with fadD33 restored bacterial replication. In a further approach, the fadD33 gene of strain H37Rv was disrupted by allelic exchange mutagenesis and the virulence of the mutant strain was tested by mouse infection. It was found that disruption of fadD33 decreased M. tuberculosis H37Rv growth in the liver, but not in the lungs or spleen, and complementation of the fadD33-disrupted mutant with fadD33 restored bacterial replication in the liver, but did not affect replication in the lungs and spleen. These findings suggest that fadD33 plays a role in M. tuberculosis virulence by supporting bacterial growth in the liver.