Multiplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assays for Diagnostic Testing of Severe Acute Respiratory Syndrome Coronavirus 2 and Seasonal Influenza Viruses: A Challenge of the Phase 3 Pandemic Setting.

BACKGROUND: Pandemic coronavirus disease 2019 (COVID-19) disease represents a challenge for healthcare structures. The molecular confirmation of samples from infected individuals is crucial and therefore guides public health decision making. Clusters and possibly increased diffuse transmission could occur in the context of the next influenza season. For this reason, a diagnostic test able to discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza viruses is urgently needed. METHODS: A multiplex real-time reverse-transcription polymerase chain reaction (PCR) assay was assessed using 1 laboratory protocol with different real-time PCR instruments. Overall, 1000 clinical samples (600 from samples SARS-CoV-2-infected patients, 200 samples from influenza-infected patients, and 200 negative samples) were analyzed. RESULTS: The assay developed was able to detect and discriminate each virus target and to intercept coinfections. The limit of quantification of each assay ranged between 5 and 10 genomic copy numbers, with a cutoff value of 37.7 and 37.8 for influenza and SARS-CoV-2 viruses, respectively. Only 2 influenza coinfections were detected in COVID-19 samples. CONCLUSIONS: This study suggests that multiplex assay is a rapid, valid, and accurate method for the detection of SARS-CoV-2 and influenza viruses in clinical samples. The test may be an important diagnostic tool for both diagnostic and surveillance purposes during the seasonal influenza activity period.

Plasmodium vivax exflagellated microgametes in human peripheral blood: a potential diagnostic dilemma.

Both malaria and relapsing fever Borrelia are infectious diseases characterized by fever, headache, myalgia, hepatosplenomegaly and tendency to relapse. Exflagellation of microgametocyte in malarial parasites is seen only in the definitive host, i.e., mosquitoes. Here we report an unusual case of a 23-year-old man who presented Plasmodium vivax infection with multiple exflagellated microgametes in the peripheral blood smear.

Tache Noire in a Patient with Acute Q Fever.

OBJECTIVE: To describe a rare case of acute Q fever with tache noire. CLINICAL PRESENTATION AND INTERVENTION: A 51-year-old man experienced acute Q fever showing tache noire, generally considered a pathognomonic sign of Mediterranean spotted fever (MSF) and MSF-like illness, but not a clinical feature of Q fever. The patient was treated with doxycycline 100 mg every 12 h. CONCLUSION: In the Mediterranean area, tache noire should be considered pathognomonic of MSF but it should not rule out Q fever. Clinical diagnosis should be supported by accurate laboratory diagnostic tests to guide proper management.

Respiratory diphtheria due to Corynebacterium ulcerans transmitted by a companion dog, Italy 2014.

A serious respiratory tract infection due to Corynebacterium ulcerans was observed in a 70-year-old woman. Clinical presentation included pseudomembranes in the upper respiratory tract and lung involvement. C. ulcerans was recovered from the nose of the patient's dog. Both dog's and patient's isolates belonged to Sequence Type 331.

Mediterranean spotted fever-like illness in Sardinia, Italy: a clinical and microbiological study.

INTRODUCTION: Rickettsioses represent a group of emerging infectious diseases in Europe. Climate changes and the anthropization of rural environment have favored vectors' biological cycle and geographic spread. In Sardinia, Mediterranean spotted fever (MSF) is endemic and represents an important public health problem. PURPOSE: We investigated the etiology and the clinical presentation of MSF-like illness in northern Sardinia by enrolling patients admitted to the Infectious Disease Unit of the University of Sassari. RESULTS: Diagnostic tests included ELISA, Indirect immunofluorescence (IFI), DNA isolation from blood and from eschar samples with real-time PCR and genotyping. Eighty-seven patients with a mean age of 53 ± 14 years, of whom 65 (75 %) males, were included in the study. The most common diagnosis was MSF (79 %), followed by Q fever (8 %), and anaplasmosis (2 %). A tache noire was found in 58 % of rickettioses and 28 % of Coxiella burnetii infections. MSF was confirmed in 47 % of the cases by IFI and 43 % by ELISA antibody tests. The isolation of rickettsial DNA from the eschar was positive in 10/13 (77 %) of the cases due to Rickettsia conorii. Using this method, we identified the first case of R. monacensis infection in Italy. CONCLUSIONS: In conclusion, antibody-based tests confirmed the diagnosis in less than 50 % of the cases, whereas DNA isolation confirmed the diagnosis in 77 % of tested cases and allowed the identification of a new pathogenic species in Italy. Therefore, DNA isolation should be implemented to better identify the etiology of MSF-like illnesses and help the clinician in the management of patients.

A fatal case of streptococcal toxic shock syndrome caused by Streptococcus suis carrying tet (40) and tet (O/W/32/O), Italy.

We report the first human fatal case of streptococcal toxic shock syndrome (STSS) caused by Streptococcus suis serotype 2 carrying the tetracycline efflux tet (40) gene and the tetracycline ribosomal protection tet (O/W/32/O) gene. The patient was splenectomized. The case was characterized by multi-organ dysfunction and disseminated intravascular coagulopathy, in accordance with the clinical parameters of STSS. More investigations are needed to improve the epidemiology and the pathogenesis of S. suis in human infection.

Chlamydophila pneumoniae phospholipase D (CpPLD) drives Th17 inflammation in human atherosclerosis.

Phospholipases are produced from bacterial pathogens causing very different diseases. One of the most intriguing aspects of phospholipases is their potential to interfere with cellular signaling cascades and to modulate the host-immune response. Here, we investigated the role of the innate and acquired immune responses elicited by Chlamydophila pneumoniae phospholipase D (CpPLD) in the pathogenesis of atherosclerosis. We evaluated the cytokine and chemokine production induced by CpPLD in healthy donors' monocytes and in vivo activated T cells specific for CpPLD that infiltrate atherosclerotic lesions of patients with C. pneumoniae antibodies. We also examined the helper function of CpPLD-specific T cells for monocyte matrix metalloproteinase (MMP)-9 and tissue factor (TF) production as well as the CpPLD-induced chemokine expression by human venular endothelial cells (HUVECs). We report here that CpPLD is a TLR4 agonist able to induce the expression of IL-23, IL-6, IL-1ß, TGF-ß, and CCL-20 in monocytes, as well as CXCL-9, CCL-20, CCL-4, CCL-2, ICAM-1, and VCAM-1 in HUVECs. Plaque-derived T cells produce IL-17 in response to CpPLD. Moreover, CpPLD-specific CD4(+) T lymphocytes display helper function for monocyte MMP-9 and TF production. CpPLD promotes Th17 cell migration through the induction of chemokine secretion and adhesion molecule expression on endothelial cells. These findings indicate that CpPLD is able to drive the expression of IL-23, IL-6, IL-1ß, TGF-ß, and CCL-20 by monocytes and to elicit a Th17 immune response that plays a key role in the genesis of atherosclerosis.

Enzymatic characterization of Chlamydophila pneumoniae phospholipase D.

Chlamydophila pneumoniae, an aetiological agent of respiratory infection, is also thought to play an immuno-pathogenetic role in atherosclerosis by contributing to inflammation and plaque instability. Phospholipase D (PLD) is an enzyme involved in lipid metabolism and may have a direct or indirect impact on virulence and the inflammatory response. Some aspects of the developmental cycle of C. pneumoniae suggest a direct implication of its PLD (CpPLD) in the pathogenesis, specifically by affecting the regulation of lipid metabolism and lipid exchange between C. pneumoniae and host cells. Our previous studies disclosed a specific anti-CpPLD antibody response in patients with acute coronary syndromes chronically infected with C. pneumoniae, and demonstrated that this antigen is a factor able to drive the inflammatory process in atherosclerosis. Due to the intriguing aspects of the CpPLD, the present study investigated CpPLD enzymatic activity of the protein and the two domains that include one HKD motif each polypeptide. Our results showed that CpPLD was able to synthesize the cardiolipin (CL) but unable to hydrolyze phospholipids. It was also observed that each single HKD motif has an independent CL synthetase activity. This enzymatic activity of CpPLD could be important in the inflammatory process within the atherothrombotic events.

Increasing Detection of Legionnaires' Disease in a Large Italian Hospital in the Period 2016-2023.

The pandemic marked the beginning of an era of dynamic and rapid changes in the diagnosis of respiratory infections. Herein we describe Legionnaires' disease trend in the years 2016-2023 in a large Italian hospital showing how improvements in diagnostic algorithms impact on its detection.

Virus investigation in ticks from migratory birds in Italy.

The role of migratory birds in circulation tick-borne viruses needs to be better defined. In order to assess the potential role of migratory birds in exotic virus spread, we conducted a study to identify ticks collected from migratory birds in the Central Region of Italy, and performed molecular investigation for Crimea-Congo Hemorrhagic Fever (CCHFV), West Nile fever (WNFV) and Usutu (USUV) in the vectors. A total of 137 competent ticks were collected with predominance of Hyalomma species. Although, negative results were obtained for all viruses considered, the high proportion of Hyalomma ticks highlights the potential risk for the dissemination of tick-borne viruses through infested migratory birds.

Nasopharyngeal carriage of Haemophilus influenzae among adults with co-morbidities.

After the widespread use of Haemophilus influenzae type b (Hib) vaccine, H. influenzae invasive disease is now commonly due to non-encapsulated (NTHi), affecting mostly the youngest and the elderly. The objective of this study was to investigate H. influenzae nasopharyngeal carriage rate in adults with co-morbidities and possible associated risk factors. METHODS: Patients aged >50 years with co-morbidities attending medical centres were examined. A nasopharyngeal swab was analysed for H. influenzae presence by cultural and molecular methods (RT-PCR). Univariable and multivariable analysis of risk factors for H. influenzae carriage were performed. Serotype of isolates was determined by PCR capsular genotyping. Minimum inhibitory concentration (MIC) was determined by MIC gradient test and ß-lactamase production was detected by the nitrocephin test. Genotyping was performed by Multilocus sequence typing (MLST). Phylogenetic relationships among carriage and invasive NTHi strains were assessed. RESULTS: Among 248 enrolled patients (median age: 73 years), the carriage rate was 5.6% and 10.5% by cultural method or RT-PCR, respectively. Colonization with H. influenzae was significantly associated with the presence of acute respiratory symptoms (adjusted OR = 12.16, 95% CI: 3.05-48.58, p < 0.001). All colonizing isolates were NTHi. Three isolates (3/14, 21.4%) were resistant to ampicillin and beta-lactamase positive. MLST revealed a high degree of genetic diversity, with 11 different STs from 14 isolates. Eight out of the 11 (72.7%) STs were shared among carriage and invasive isolates. CONCLUSIONS: Adults ≥50 years old with co-morbidities are occasionally colonized by H. influenzae, even if the presence of co-morbidities is not a risk factor for colonization. The presence of acute respiratory symptoms is the only factor associated with H. influenzae colonization. Colonizing H. influenzae are all NTHi. Colonizing H. influenzae often belong to the same STs of invasive disease isolates.

First evidence of blaNDM-1 and blaOXA-23 carbapenemase genes in human body lice infesting a second-hand T-shirt in a street market in Italy.

BACKGROUND: The spread of carbapenems resistance is a public health concern. The main group of carbapenemases encoding the ß-lactamases activity (bla genes) is the Metallo-ß-lactamases (MBLs). METHODS: The presence of carbapenemase blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, blaOXA-58-like, and blaNDM-1 genes was screened by real time PCR in 26 Pediculus humanus insects identified from second-hand clothes in a local market in Central Italy. Bacteria diversity was also characterized through shotgun metagenomic amplification for a deep sequencing of the host-associated bacterial microbiomes. RESULTS: The blaOXA-23 and blaNDM-1 carbapenemases genes were found and metagenomic analysis showed a great presence of Acinetobacter species. CONCLUSIONS: These results suggest a new potential transmission path for carbapenemase gene spread through bacteria ingested by insects infesting humans.

Laboratory management for SARS-CoV-2 detection: a user-friendly combination of the heat treatment approach and rt-Real-time PCR testing.

The RNA purification is the gold standard for the detection of SARS-CoV-2 in swab samples, but it is dependent on the availability of chemical reagents. In this study, we evaluated the heat treatment method without RNA extraction as a reliable option to nucleic acid purification.

Fourteen years of severe arthralgia in a man without gastrointestinal symptoms: atypical Whipple's disease.

We report here the case of Whipple's disease in a 60-year-old man with severe arthralgia and systemic disorders but without gastrointestinal manifestations. The patient had different clinical diagnoses over a period of 14 years. We identified Tropheryma whipplei by real-time PCR. Molecular typing was also performed by sequencing the 16S-23S rRNA intergenic spacer region and domain III of the 23S rRNA gene.

Report of the human body louse (Pediculus humanus) from clothes sold in a market in central Italy.

BACKGROUND: Pediculus humanus, the human body louse, is widespread where overcrowding and lack of hygiene are present, in areas of the world affected by poverty, war, famine and presence of refugees. It has recently been considered re-emerging among homeless populations in developed countries. In Italy, it was last reported in 1945. Pediculus humanus is a vector of highly relevant human pathogens. METHODS: In October 2018, a woman found small insects on a T-shirt bought second-hand in a local street market in a village 35 km south of Rome (central Italy). Insects were identified both morphologically and by molecular analysis. Moreover, they were analyzed molecularly for the presence of Rickettsia prowazekii, Borrelia recurrentis, Bartonella quintana, Coxiella burnetii and Yersinia pestis. RESULTS: Morphological and molecular analyses of the insects identified them as 26 lice (12 females, 10 males and 4 nymphs) of the species P. humanus. Many nits were found on the T-shirt seams. DNA of the investigated pathogens was not detected in any of the lice. CONCLUSIONS: The exceptionality of the described case lies both in the report of P. humanus from a country where it had not been reported since 1945, and in its finding from second-hand clothes for sale in a market, constituting a potential source of infection for people buying this type of goods. The question arises, how did adults and nits of P. humanus infest clothes for sale on a market stall in a country where it had not been reported for decades. Given that the body louse requires frequent blood meals to survive and develop, its arrival on clothes imported from abroad is highly improbable. Hence, it must be presumed that people infected with the human body louse are present in Italy. This report points out a serious regulatory problem regarding the management of second-hand clothes prior to sale and, more generally, of controls in street markets.

Primary Whipple disease of the Central Nervous System presenting with rhombencephalitis.

Primary Whipple disease of the Central Nervous System is a rare entity whose outcome might be fatal if not promptly diagnosed and treated. Few cases are reported in the literature with heterogeneous clinical and radiological presentations which often make the diagnosis extremely challenging. We report a case of primary Whipple disease of the Central Nervous System presenting with rhombencephalitis in a female patient in immunosuppressive treatment for rheumatoid arthritis. We describe the management of our patient and discuss the features of this rare clinical entity.