Respiratory Tract Infections and Laboratory Diagnostic Methods: A Review with A Focus on Syndromic Panel-Based Assays.

Respiratory tract infections (RTIs) are the focus of developments in public health, given their widespread distribution and the high morbidity and mortality rates reported worldwide. The clinical spectrum ranges from asymptomatic or mild infection to severe or fatal disease. Rapidity is required in diagnostics to provide adequate and prompt management of patients. The current algorithm for the laboratory diagnosis of RTIs relies on multiple approaches including gold-standard conventional methods, among which the traditional culture is the most used, and innovative ones such as molecular methods, mostly used to detect viruses and atypical bacteria. The implementation of molecular methods with syndromic panels has the potential to be a powerful decision-making tool for patient management despite requiring appropriate use of the test in different patient populations. Their use radically reduces time-to-results and increases the detection of clinically relevant pathogens compared to conventional methods. Moreover, if implemented wisely and interpreted cautiously, syndromic panels can improve antimicrobial use and patient outcomes, and optimize laboratory workflow. In this review, a narrative overview of the main etiological, clinical, and epidemiological features of RTI is reported, focusing on the laboratory diagnosis and the potentialities of syndromic panels.

Characterization of Clostridioides difficile Strains from an Outbreak Using MALDI-TOF Mass Spectrometry.

The epidemiology of Clostridioides difficile infection (CDI) has changed over the last two decades, due to the emergence of C. difficile strains with clinical relevance and responsible for nosocomial outbreaks with severe outcomes. This study reports an outbreak occurred in a Long-term Care Unit from February to March 2022 and tracked by using a Matrix-Assisted Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) typing approach (T-MALDI); subsequently, a characterization of the toxigenic and antimicrobial susceptibility profiles of the C. difficile isolates was performed. A total of 143 faecal samples belonging to 112 patients was evaluated and C. difficile DNA was detected in 51 samples (46 patients). Twenty-nine C. difficile isolates were obtained, and three different clusters were revealed by T-MALDI. The most representative cluster accounted 22 strains and was considered to be epidemic, in agreement with PCR-Ribotyping. Such epidemic strains were susceptible to vancomycin (MIC ≤ 0.5 mg/mL) and metronidazole (MIC ≤ 1 mg/mL), but not to moxifloxacin (MIC > 32 mg/mL). Moreover, they produced only the Toxin A and, additionally, the binary toxin. To our knowledge, this is the first reported outbreak referable to a tcdA+/tcdB-/cdt+ genotypic profile. In light of these results, T-MALDI is a valid and rapid approach for discovering and tracking outbreaks.

Prevalence of Intestinal Parasitoses in a Non-Endemic Setting during a 10-Year Period (2011-2020): A Focus on Dientamoeba fragilis.

Dientamoeba fragilis is a cosmopolitan and neglected protozoan. Although little is known concerning its pathogenicity and its true prevalence worldwide, its role as enteric pathogen is emerging, as the occurrence of dientamoebiasis has increased also in industrialised countries. This study investigated the occurrence and prevalence of intestinal parasites, focusing on D. fragilis in a 10-year period (2011-2020) in a single tertiary-care hospital located in Northern Italy. A statistical evaluation of the correlation between dientamoebiasis and specific signs other than gastrointestinal-related ones was performed. The laboratory diagnosis was performed on 16,275 cases of suspected intestinal parasitoses. Intestinal parasites were detected in 3254 cases, 606 of which were associated to D. fragilis, which represented 18.6% (606/3254) of all the intestinal parasitoses with a 3.7% (606/16,275) prevalence and an increasing trend during the last five years (2011-2015: 2.8% vs. 2016-2020: 4.8%). D. fragilis was commonly detected in foreigners, especially those from developing countries, as well as in children; prevalence was equal in males and females. With regard to the clinical aspect, the only putative sign statistically related to dientamoebiasis was anal pruritus. Despite the controversial epidemiological knowledges on dientamoebiasis, the prevalence of D. fragilis found in this study highlights the need to consider this parasite in any differential diagnosis of gastrointestinal disease.

Rapid Identification of Escherichia coli Colistin-Resistant Strains by MALDI-TOF Mass Spectrometry.

Colistin resistance is one of the major threats for global public health, requiring reliable and rapid susceptibility testing methods. The aim of this study was the evaluation of a MALDI-TOF mass spectrometry (MS) peak-based assay to distinguish colistin resistant (colR) from susceptible (colS) Escherichia coli strains. To this end, a classifying algorithm model (CAM) was developed, testing three different algorithms: Genetic Algorithm (GA), Supervised Neural Network (SNN) and Quick Classifier (QC). Among them, the SNN- and GA-based CAMs showed the best performances: recognition capability (RC) of 100% each one, and cross validation (CV) of 97.62% and 100%, respectively. Even if both algorithms shared similar RC and CV values, the SNN-based CAM was the best performing one, correctly identifying 67/71 (94.4%) of the E. coli strains collected: in point of fact, it correctly identified the greatest number of colS strains (42/43; 97.7%), despite its lower ability in identifying the colR strains (15/18; 83.3%). In conclusion, although broth microdilution remains the gold standard method for testing colistin susceptibility, the CAM represents a useful tool to rapidly screen colR and colS strains in clinical practice.

Malaria Diagnosis in Non-Endemic Settings: The European Experience in the Last 22 Years.

Accurate, prompt, and reliable tools for the diagnosis of malaria are crucial for tracking the successes or drawbacks of control and elimination efforts, and for future programs aimed at global malaria eradication. Although microscopy remains the gold standard method, the number of imported malaria cases and the risk of reappearance of autochthonous cases stimulated several laboratories located in European countries to evaluate methods and algorithms suited to non-endemic settings, where skilled microscopists are not always available. In this review, an overview of the field evaluation and a comparison of the methods used for the diagnosis of malaria by European laboratories is reported, showing that the development of numerous innovations is continuous. In particular, the combination of rapid diagnostic tests and molecular assays with microscopy represents a reliable system for the early diagnosis of malaria in non-endemic settings.

Rapid Classification of Clostridioides difficile Strains Using MALDI-TOF MS Peak-Based Assay in Comparison with PCR-Ribotyping.

Typing methods are needed for epidemiological tracking of new emerging and hypervirulent strains because of the growing incidence, severity and mortality of Clostridioides difficile infections (CDI). The aim of this study was the evaluation of a typing Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS (T-MALDI)) method for the rapid classification of the circulating C. difficile strains in comparison with polymerase chain reaction (PCR)-ribotyping results. Among 95 C. difficile strains, 10 ribotypes (PR1-PR10) were identified by PCR-ribotyping. In particular, 93.7% of the isolates (89/95) were grouped in five ribotypes (PR1-PR5). For T-MALDI, two classifying algorithm models (CAM) were tested: the first CAM involved all 10 ribotypes whereas the second one only the PR1-PR5 ribotypes. Better performance was obtained using the second CAM: recognition capability of 100%, cross-validation of 96.6% and agreement of 98.4% (60 correctly typed strains, limited to PR1-PR5 classification, out of 61 examined strains) with PCR-ribotyping results. T-MALDI seems to represent an alternative to PCR-ribotyping in terms of reproducibility, set up time and costs, as well as a useful tool in epidemiological investigation for the detection of C. difficile clusters (either among CAM included ribotypes or out-of-CAM ribotypes) involved in outbreaks.

Detecting clinical activity in systemic lupus erythematosus with an archaeal poly(ADP-ribose) polymerase-like thermozyme: a pivotal study.

The clinical usefulness of an immunotest was evaluated by using purified poly(adenosine diphosphate (ADP)-ribose) polymerase from Sulfolobus solfataricus (PARPSso) as an antigen to detect the presence of abnormal anti-PARP antibodies in the sera of patients with systemic lupus erythematosus (SLE) at different clinical stages. Sera from 44 patients with SLE, subgrouped on the basis of disease activity (16 with inactive disease, 28 with active disease) were analysed with a new immunotest to detect anti-PARP antibodies, and with an immunofluorescent (IIF) assay for antinuclear antibodies (ANA) detection. ANA detection by IIF revealed that sera of healthy subjects were negative, whereas sera from patients with SLE were positive in all cases (13 positive at 1:80, 15 at 1:160, 15 at 1:320, 1 at 1:640, v/v). Anti-PARP activity was higher in ANA-positive patients than in controls (p = 0.005). Within the group of SLE sera, disease and anti-PARP activity was increased more significantly in patients with active than in those with inactive disease (p < 0.001 and p = 0.001, respectively). Correlation between anti-PARP and disease activity in SLE patients was statistically significant (p < 0.001). PARPSso seems to be suitable for detecting anti-PARP antibodies and could play a role as a serological marker of disease activity in patients with SLE.

The ADP-ribosylation of Sulfolobus solfataricus Sso7 modulates protein/DNA interactions in vitro.

The 7 kDa Sso7 is a basic protein particularly abundant in Sulfolobus solfataricus and is involved in DNA assembly. This protein undergoes in vitro ADP-ribosylation by an endogenous poly(ADP-ribose) polymerase-like enzyme. The circular dichroism spectrum of purified ADP-ribosylated Sso7 shows that this modification stabilizes the prevalent protein beta-conformation, as suggested by shifting of negative ellipticity minimum to 220 nm. Moreover, a short ADP-ribose chain (up to 6-mers) bound to Sso7 is able to reduce drastically the thermoprotective and DNA condensing ability of the protein, suggesting a possible regulatory role of ADP-ribosylation in sulfolobal DNA organization.

NEMO-binding domain peptide inhibits proliferation of human melanoma cells.

Melanoma is the most aggressive form of skin cancer, it originates from melanocytes and its incidence has increased in the last decade. Recent advances in the understanding of the underlying biology of the progression of melanoma have identified key signalling pathways that are important in promoting melanoma tumourigenesis, thus providing dynamic targets for therapy. One such important target identified in melanoma tumour progression is the Nuclear Factor-kappaB (NF-kappaB) pathway. In vitro studies have shown that NF-kappaB binding is constitutively elevated in human melanoma cultures compared to normal melanocytes. It has been found that a short cell-permeable peptide spanning the IKK-beta NBD, named NBD peptide, disrupted the association of NEMO with IKKs in vitro and blocked TNFalpha-induced NF-kappaB activation in vivo. In the present study we investigated the effect of the NBD peptide on NF-kappaB activity and survival of A375 human melanoma cells. We found that NBD peptide is able to inhibit the proliferation of A375 cells, which present constitutively elevated NF-kappaB levels. Inhibition of cell proliferation by NBD peptide was associated with direct inhibition of constitutive NF-kappaB DNA-binding activity and induction of apoptosis by activation of caspase-3 as confirmed by the cleavage and consequently inactivation of poly (ADP ribose) polymerase (PARP-1) known as the best marker of this process.

Poly(ADP-ribosyl)ation of proteins and germ cell development in hyperthyroid rat testes.

The effect of increased serum levels of thyroid hormone (triiodothyronine, T3) on young rat testis spermatogenesis was studied by analysing molecular and morphological parameters. Hyperthyroidism was induced by either T3-treatment or 2- and 10-day cold exposure. The poly(ADP-ribosyl)ation of proteins catalysed by poly(ADP-ribose) polymerase, which is particularly active at specific stages of rat spermatogenesis, was analysed as molecular index of DNA damage and cell stress. Poly(ADP-ribose) polymerase activity rose after both T3-treatment and 2- and 10-day cold exposure, with a trend of 10-day cold-exposed rats towards control values. In all hyperthyroid rats poly(ADP-ribose) turnover, as a contribution of both poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase), was enhanced with respect to euthyroid animals. Poly(ADP-ribosyl)ation of proteins occurred with long and branched polymers suggesting an increased involvement of the modification system in DNA repair. Morphological changes of germ tissue were observed in hyperthyroid rats, mainly a high reduction of mature cells in the seminiferous tubule, and evidence of germ cell apoptosis was obtained by TUNEL method. In control animals germ cell apoptosis was within physiological levels. Conversely, in hyperthyroid rats a dramatic increase in the number of TUNEL-positive cells (some spermatogonia and numerous primary spermatocytes) was found, even though the increase was lower in 10-day than in 2-day cold-exposed animals.

The ADP-ribosylating thermozyme from Sulfolobus solfataricus is a DING protein.

The partial amino acid sequence of the sulfolobal thermoprotein biochemically characterized as poly(ADP-ribose)polymerase-like enzyme overlaps those of DING proteins. This group of proteins, widely occurring in animals, plants and eubacteria, shows a characteristic and highly conserved N-terminus, DINGGGATL. The sequence of the N-terminal region and of the analyzed tryptic peptides of the sulfolobal thermozyme shows a high similarity with most of the DING proteins from databases. This is the first example of a DING protein from a sulfolobal source.

The (ADP-ribosyl)ation reaction in thermophilic bacteria.

Species of Alicyclobacillus, Bacillus and Thermus genera were selected in order to study the possible presence of the (ADP-ribosyl)ation system. These bacteria are thermophilic, aerobic, and were isolated from different geothermal sources. Both activity and expression of (ADP-ribosyl)ating proteins were tested in cells at different growth phases, and evidence of an active system was obtained in all analyzed microorganisms, with comparable enzymatic levels. Immunochemical analyses with polyclonal antibodies against both eukaryotic anti-(ADP-ribose) transferase and anti-poly(ADP-ribose) polymerase revealed, for all tested organisms, an immunosignal localized in the range of molecular masses between 43-53 kD. Several proteins of various molecular masses were found as ADP-ribose acceptors. Reaction product analyses showed mono(ADP-ribose) to be the only synthesized compound.

Yeast (ADPribosyl)ation: revisiting a controversial question.

The controversy about the occurrence of an (ADPribosyl)ating activity in yeast is still standing up. Here we discuss this topic on the basis of results obtained with classic experiments proposed over years as basis to characterize an (ADPribosyl)ation system in any organism. Independent results obtained in two different laboratories were in line with each other and went towards the occurrence of an active (ADPribosyl)ating system in Saccharomyces cerevisiae. In fact data collected from nuclear preparations of cultured cells matched those from baker's yeast and lyophilized yeast cells. Yeast (ADPribosyl)ating enzyme is a protein of 80-90 kDa, as determined by electrophoresis on polyacrylamide gel in sodium dodecyl sulphate, followed by immunoblotting with antibodies against anti-poly(ADPribose) polymerase catalytic site. It synthesizes products, that, after digestion with phosphodiesterase, co-migrates mainly with phosphoribosyl adenosine monophosphate after thin layer chromatography on silica gel plate.

Active poly(ADPribose) metabolism in DNAase- and salt-resistant rat testis chromatin with high transcriptional activity/competence.

A chromatin fraction, named pP fraction, was prepared from rat testis nuclei, which had been digested with nuclease in order to separate soluble and insoluble chromatin. This fraction resembled nuclear matrix as it was highly resistant to DNAase digestion, had a high content of proteins compared to the low DNA percentage, and a noticeable transcriptional activity. Moreover, poly(ADPribosyl)ation system (i.e., poly(ADPR)polymerase, poly(ADPribose), and acceptor proteins) was still present at high levels. In order to study whether it might be identified as the protein support surrounding chromatin loops, this pP fraction was further analyzed after 3 M NaCl extraction. The 3 M NaCl extract and the highly insoluble pellet, named Nuclear Matrix Pellet, were characterized as it regards DNA, newly synthesized RNA and proteins. Furthermore, poly(ADPribose) metabolism was analyzed by measuring both poly(ADPribose) polymerase and poly(ADPribose) glycohydrolase activities, poly(ADPribose) distribution and by identifying protein acceptors. The final pellet had features of nuclear matrix containing less than 10% DNA and high percentage of proteins; 28% of newly synthesized RNA was still associated with this fraction. Long and branched polyADPribose were found in the nuclear matrix-like pellet, although ADPribose acceptors (mainly H1 and core histones) appeared to be modified mostly with short ADPribose oligomers. Longest and branched polymers were retained on the top of protein gel, likely bound to automodified poly(ADPribose) polymerase.

Histone H1-like protein and a testis-specific variant in the reproductive tracts of Octopus vulgaris.

In this study, we have identified a 28-kDa protein resembling the linker H1 in the testis and prostate of the reproductive system of Octopus vulgaris. This protein, OvH1, was partially purified by reverse phase high-pressure liquid chromatography (HPLC) of the perchloric acid extract from testis nuclei. It showed electrophoretic mobility, CD spectrum and amino acid composition highly comparable with those of the mammalian histone. Moreover, it was microheterogeneous, as resulted from prostate and testis HPLC and mass spectrometry analyses. Such analysis showed that in testis there are two H1 subfractions, which do not appear in the prostate. Amino acid composition of the major testis specific variant (OvH1t) showed high similarity with rat testis specific H1t. The histone-like nature of OvH1 was confirmed by its ability to bind DNA as tested both by circular dichroism and protection of the nucleic acid toward deoxyribonuclease I activity. The circular dichroism spectra of Octopus DNA in the absence and presence of increasing amounts of the protein showed a dose-dependent effect, leading to a progressive compactness of the polynucleotide. OvH1/DNA complexes were also resistant to nuclease digestion. The presence of H1 in the testis and prostate of the reproductive system of Octopus is discussed in light of the fact that there is a similarity between its behavior and that of vertebrates.

Interaction of the ADP-ribosylating enzyme from the hyperthermophilic archaeon S. solfataricus with DNA and ss-oligo deoxy ribonucleotides.

The DNA-binding ability of the poly-ADPribose polymerase-like enzyme from the extremely thermophilic archaeon Sulfolobus solfataricus was determined in the presence of genomic DNA or single stranded oligodeoxyribonucleotides. The thermozyme protected homologous DNA against thermal denaturation by lowering the amount of melted DNA and increasing melting temperature. The archaeal protein induced structural changes of the nucleic acid by modifying the dichroic spectra towards a shape typical of condensing DNA. However, enzyme activity was slightly increased by DNA. Competition assays demonstrated that the protein interacted also with heterologous DNA. In order to characterize further the DNA binding properties of the archaeal enzyme, various ss-oligodeoxyribonucleotides of different base composition, lengths (12-mer to 24-mer) and structure (linear and circular) were used for fluorescence titration measurements. Intrinsic fluorescence of the archaeal protein due to tryptophan (excitation at 295 nm) was measured in the presence of each oligomer at 60 degrees C. Changes of tryptophan fluorescence were induced by all compounds in the same range of base number per enzyme molecule, but independently from the structural features of oligonucleotides, although the protein exhibited a slight preference for those adenine-rich and circular. The binding affinities were comparable for all oligomers, with intrinsic association constants of the same order of magnitude (K=10(6) M(-1)) in 0.01 M Na-phosphate buffer, pH 8.0, and accounted for a "non-specific" binding protein. Circular dichroism analysis showed that at 60 degrees C the native protein was better organized in a secondary structure than at 20 degrees C. Upon addition of oligonucleotides, enzyme structure was further stabilized and changed towards a beta-conformation. This effect was more marked with the circular oligomer. The analysed oligodeoxyribonucleotides slightly enhanced enzyme activity with the maximal increase of 50% as compared to the control. No activation was observed with the circular oligomer.

High stability binding of poly(ADPribose) polymerase-like thermozyme from S. solfataricus with circular DNA.

The poly(ADPribose) polymerase-like thermozyme from the hyperthermophilic archaeon S. solfataricus was found to bind DNA with high affinity and non-specifically. Binding was independent of base composition and length of the nucleic acid, and the protein showed a slight preference for the circular structure. By using pCMV-Neo-Bam plasmid as experimental model, the behaviour of the thermozyme upon binding with either circular or linear plasmid was analyzed. pCMV-Neo-Bam has a single HindIII site that allows to obtain the linear structure after digestion with the restriction enzyme. Intrinsic tryptophan-dependent fluorescence of poly(ADPribose) polymerase-like thermozyme noticeably changed upon addition of either circular or linear plasmid, showing the same binding affinity (K=2 x 10(9) M-1). However, experiments of protection against temperature and DNase I gave evidence that the thermozyme formed more stable complexes with the circular structure than with the linear pCMV-Neo-Bam. Increasing temperature at various DNA/protein ratios had a double effect to reduce the amount of circular DNA undergoing denaturation and to split the melting point towards higher temperatures. Nil or irrelevant effect was observed with the linear form. Similarly, DNase acted preferentially on the linear plasmid/protein complexes, producing an extensive digestion even at high protein/DNA ratios, whereas the circular plasmid was protected by the thermozyme in a dose-dependent manner. The complexes formed by archaeal poly(ADPribose) polymerase (PARPss) with the circular plasmid were visualized by bandshift experiments both with ethidium bromide staining and by labelling the circular plasmid with 32P. The stability of complexes was tested as a function of enzyme concentration and in the presence of a cold competitor and of 0.1% SDS. From the performed experiments, a number of 3-10 base pairs bound per molecule of enzyme was calculated, indicating a high frequency of binding. The presence of circular DNA was also able to increase by 80% the poly(ADPribose)polymerase-like activity, as compared to 25% activation induced by the linear pCMV-Neo-Bam.