Assessment of the diagnostic performance of serological tests in areas where Leishmania infantum and Leishmania tarentolae occur in sympatry.

BACKGROUND: Visceral leishmaniosis caused by infection with the zoonotic protozoan Leishmania infantum is a life-threatening disease affecting dogs and humans. The sympatric occurrence of L. infantum and Leishmania tarentolae in an area of southern Italy endemic for canine leishmaniosis, where dogs are also exposed to the latter species, suggests the persistence of herpetophilic L. tarentolae in a non-permissive host, therefore raising questions about the performance of serological diagnostic tests routinely employed. METHODS: The diagnostic performance of serological tests such as the immunofluorescence antibody test (IFAT), two commercial immunoenzymatic assays (i.e. NovaTec VetLine Leishmania ELISA® and rK39 ICT®) and an in-house enzyme-linked immunosorbent assay (ELISA) was evaluated in healthy dogs seropositive to L. infantum, whereas the only IFAT available was used to detect antibodies to L. tarentolae. RESULTS: With the IFAT, out of a total of 104 dogs tested, 15 were seronegative for L. infantum of which three were L. tarentolae seropositive' and 89 were L. infantum seropositive. Of the latter 89 dogs, representing the highest proportion of seropositive animals (85.6%) detected by IFAT' 66 were also seropositive for L. tarentolae. Cohen's kappa (κ) agreement coefficient between the IFAT results and those of all the other tests was very low, and the IFAT results were significantly different from those of all the other serological tests as calculated by Cochran's Q-test. Analysis using the Bayesian latent class (Bayes-LCA) showed that the in-house ELISA and IFAT contributed the most towards identifying infected and non-infected dogs, respectively. The IFAT test showed low positive predictive value (59.5%), but high negative predictive value (100%). CONCLUSIONS: These results demonstrate that the IFAT for L. infantum, although highly sensitive, may not be considered a useful diagnostic test due to its low specificity. Therefore, an accurate serological tool with high specificity is mandatory for avoiding cross-reaction in epidemiological contexts where the two species of Leishmania occur in sympatry.

Seroprevalence of Leishmania infantum in outdoor workers from southern Italian endemic regions.

Visceral leishmaniasis is a zoonotic vector-borne disease caused by Leishmania infantum. The infection often remains asymptomatic, though clinical forms may occur in immunosuppressed individuals. Although data on leishmaniasis in humans are available worldwide, the exposure to L. infantum of workers conducting outdoor activities has been scantly investigated, and it is limited to military personnel operating in endemic regions. This study aimed to assess the seroprevalence of L. infantum in different groups of outdoor workers and the occupational risk factors. The cross-sectional study was performed on 229 workers including forestry guards, farmers, veterinarians, geologists and agronomists from three regions of southern Italy (i.e., Apulia, Basilicata and Campania). All serum samples were screened for L. infantum-specific IgG/IgM by using automated indirect chemiluminescent immunoassays. Overall, 5.7% (13/229) of workers was positive to anti-L. infantum antibodies, with the highest seroprevalence in veterinarians (13.6%). An increased occupational health surveillance for L. infantum infection in outdoor workers is essential to better understand the risk of exposure in specific jobs. Furthermore, guidelines and education along with a One Health collaboration among veterinarians, physicians, parasitologists and occupational health care professionals are crucial for the prevention of this disease.

Meropenem/vaborbactam activity in vitro: a new option for Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae treatment.

Aim: Infections by Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae represent a major challenge because of limited treatment strategies. New ß-lactam/ß-lactamase inhibitor associations may help to deal with this challenge. The aim of this study is to evaluate the in vitro susceptibility of KPC-producing K. pneumoniae for meropenem/vaborbactam in comparison with ceftazidime/avibactam against. Materials and methods: Twenty-eight strains isolated from blood cultures were evaluated. Testing for susceptibility to meropenem/vaborbactam and ceftazidime/avibactam was performed by E-test gradient strip. Results: All the clinical isolates were susceptible to meropenem/vaborbactam, while one strain was resistant to ceftazidime/avibactam with a MIC of 32 µg/ml. The median MIC of ceftazidime/avibactam evaluated after standardization was higher compared with that of meropenem/vaborbactam. Conclusion: Meropenem/vaborbactam could be an important turning point in the treatment of KPC-producing K. pneumoniae infections, especially considering the emergence of ceftazidime/avibactam resistance.

Lay abstract Carbapenem-resistant Klebsiella pneumoniae is responsible, in critically ill patients, for nosocomial infections that are difficult to treat due to limited therapeutic options. Today, new antibiotics are available for treating these infections. The aim of this study is to evaluate the antimicrobial activity of meropenem/vaborbactam versus ceftazidime/avibactam. The results demonstrate that meropenem/vaborbactam could be an important turning point in the treatment of carbapenem-resistant K. pneumoniae infections, considering the emergence of ceftazidime/avibactam resistance.

Epidemiology of Shiga Toxin-Producing Escherichia coli Infections in Southern Italy after Implementation of Symptom-Based Surveillance of Bloody Diarrhea in the Pediatric Population.

Shiga toxin-producing Escherichia coli (STEC) infections result in a significant public health impact because of the severity of the disease that, in young children especially, can lead to hemolytic-uremic syndrome (HUS). A rise in the number of HUS cases was observed in the Apulia region of Italy from 2013 to 2017, and so, in 2018, a symptom-based surveillance system for children with bloody diarrhea (BD) was initiated in order to detect and manage STEC infections. The objective of the study was to describe the epidemiology of STEC infections in children from June 2018 to August 2019. Children <15 years old with BD were hospitalized and tested for STEC. Real-time PCR for virulence genes (stx1, stx2, eae) and serogroup identification tests were performed on stool samples/rectal swabs of cases. STEC infection was detected in 87 (10.6%) BD cases. The median age of STEC cases was 2.7 years, and 60 (68.9%) were <4. Of these 87 cases, 12 (13.8%) came from households with diarrhea. The reporting rate was 14.2/100,000, with the highest incidence in cases from the province of Bari (24.2/100,000). Serogroups O26 and O111 were both detected in 22/87 (25.3%) cases. Co-infections occurred in 12.6% of cases (11/87). Twenty-nine STEC were positive for stx1, stx2, and eae. Five cases (5.7%) caused by O26 (n = 2), O111 (n = 2), and O45 (n = 1) developed into HUS. A risk-oriented approach based on the testing of children with BD during the summer may represent a potentially beneficial option to improve the sensitivity of STEC surveillance, not only in Italy but also in the context of Europe as a whole.

Emerging high-risk ST101 and ST307 carbapenem-resistant Klebsiella pneumoniae clones from bloodstream infections in Southern Italy.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CR-KP) is an urgent public health issue in Italy. This pattern of resistance is due mainly to dissemination of carbapenemase genes. Molecular characterization of carbapenem-resistant Klebsiella pneumoniae (CR-KP) strains was performed over a three-year period. In-depth analysis was performed on a subset of emerging CR-KP ST101 and ST307 clones. METHODS: A prospective study was performed on 691 patients with CR-KP bloodstream infections hospitalized in 19 hospitals located in three large provinces in Southern Italy. Carbapenemase genes were identified via genotyping methods. Multi-locus sequence typing (MLST) and Whole Genome Sequencing (WGS) were carried out on ST101 and ST307 isolates. RESULTS: Among the CR-KP isolates, blaKPC was found in 95.6%, blaVIM was found in 3.5%, blaNDM was found in 0.1% and blaOXA-48 was found in 0.1%. The blaKPC-3 variant was identified in all 104 characterized KPC-KP isolates. MLST of 231 representative isolates revealed ST512 in 45.5%, ST101 in 20.3% and ST307 in 18.2% of the isolates. cgMLST of ST307 and ST101 isolates revealed presence of more than one beta-lactam resistance gene. Amino acid substitution in the chromosomal colistin-resistance gene pmrB was found in two ST101 isolates. CONCLUSIONS: ST512 is widespread in Southern Italy, but ST101 and ST307 are emerging since they were found in a significant proportion of cases. Aggressive infection control measures and a continuous monitoring of these high-risk clones are necessary to avoid rapid spread of CR-KP, especially in hospital settings.

First detection of autochthonous extensively drug-resistant NDM-1 Pseudomonas aeruginosa ST235 from a patient with bloodstream infection in Italy, October 2019.

BACKGROUND: Pseudomonas aeruginosa (PA) is one of the most common and serious causes of healthcare-associated bacteremia. The emergence and dissemination of multidrug-resistant (MDR) and extensively drug-resistant (XDR) PA strains pose a major clinical concern. ST235-PA is a high-risk clone which shows a high capacity to acquire antibiotic resistance. Here we describe the first autochthonous New Delhi metallo-ß-lactamase (NDM)-producing Pseudomonas aeruginosa ST235 identified in Italy. CASE PRESENTATION: In October 2019, a patient residing in an elderly health care and rehabilitation facility, was hospitalized and died from sepsis caused by an XDR-PA. The strain belonged to the high-risk clone sequence type ST235. Whole genome sequencing (WGS) revealed the presence of genes encoding NDM-1 and multiple ß-lactamases, many clinically significant multidrug efflux pump complexes and also the virulence gene ExoU, which is associated with a high cytotoxic phenotype. CONCLUSIONS: Few strains of NDM-1-PA have been identified worldwide, all belonging to ST235. The combination of ST235 and ExoU is a predictor of highly unfavorable prognosis. The potential spread of these high-risk clones in healthcare settings is worrisome because treatment options are limited. Early identification of high-risk clones could help in outbreaks investigation and infections control.

High Public-Health Impact in an Influenza-B-Mismatch Season in Southern Italy, 2017-2018.

BACKGROUND: Yearly influenza epidemics have considerable effects on public health worldwide. The 2017-2018 influenza season in Italy was of greater severity than previous seasons. The aim of this study was to describe the 2017-2018 influenza season in Southern Italy and the molecular characteristics of the circulating viral strains. METHODS: The incidence of influenza-like illness (ILI) was analysed. Nasopharyngeal swabs collected from patients with ILI from week 46/2017 to week 17/2018 were tested to identify influenza A viruses (IAV) and influenza B viruses (IBV). Sequencing and phylogenetic analysis of haemagglutinin genes were also performed on 73 positive samples (35 IBV, 36 IAV H1, and 2 IAV H3 strains). RESULTS: During the 2017-2018 season, the peak incidence was 14.32 cases per 1,000 inhabitants. IBV strains were identified in 71.0% of cases. The 35 characterised IBV strains belonged to Yamagata lineage clade 3, the 36 A/H1N1pdm09 strains clustered with the genetic subgroup 6B.1, and the 2 A/H3N2 strains clustered with the genetic subgroup 3C.2a. Intensive-care unit (ICU) admission was required in 50 cases of acute respiratory distress syndrome (ARDS). Among the >64-year age group, 18 out of 26 ICU-ARDS cases (69.2%) were caused by IBV, and 14 of these (77.8%) were B/Yamagata lineage. CONCLUSIONS: The 2017-2018 influenza season was one of the most severe in a decade in Southern Italy. IBV mismatch between the trivalent vaccine and the circulating strains occurred. The high number of ICU-ARDS cases caused by B/Yamagata strains in the >64-year age group suggests that further data on the effectiveness of the available influenza vaccines are needed to determine the best way to protect the elderly against both IBV lineages.

First Description of Macrolide-Resistant Mycoplasma pneumoniae in Adults with Community-Acquired Pneumonia in Italy.

BACKGROUND: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP). This cross-sectional study aimed to determine the prevalence of macrolide-resistant M. pneumoniae strains in a convenience series of 234 adult hospitalised and nonhospitalised subjects with a diagnosis of CAP in January 2013 to April 2015 in South Italy. METHODS: Respiratory samples were subjected to real-time PCR. In M. pneumoniae-positive samples, domain V of 23S rRNA was sequenced to detect resistance-conferring point mutations. P1 major adhesion protein typing and multiple loci variable-number tandem repeat analysis (MLVA) were also performed. RESULTS: Of the 234 samples, 15 (6.4%) were positive for M. pneumoniae. Three of these had a macrolide-resistant genotype: two and one had A2063G and A2064G mutations, respectively. Fourteen of the 15 strains were subtyped: half had subtype 1 and half had subtype 2. Eight strains underwent MLVA profiling: one each had the J, A, and Z type. The remainder was unclassifiable. CONCLUSIONS: This novel discovery of macrolide-resistant M. pneumoniae strains in adults with CAP in Italy suggests that there may be increasing circulation of these strains in the population. To facilitate rapid optimization of the antibiotic strategy in Italy, macrolide resistance should be monitored by a surveillance system that is based on molecular methods.

Sentinel hospital-based surveillance for norovirus infection in children with gastroenteritis between 2015 and 2016 in Italy.

Noroviruses are one of the leading causes of gastro-enteric diseases worldwide in all age groups. Novel epidemic noroviruses with GII.P16 polymerase and GII.2 or GII.4 capsid type have emerged worldwide in late 2015 and in 2016. We performed a molecular epidemiological study of the noroviruses circulating in Italy to investigate the emergence of new norovirus strains. Sentinel hospital-based surveillance, in three different Italian regions, revealed increased prevalence of norovirus infection in children (<15 years) in 2016 (14.4% versus 9.8% in 2015) and the emergence of GII.P16 strains in late 2016, which accounted for 23.0% of norovirus infections. The majority of the strains with a GII.P16 polymerase showed a GII.2 capsid genotype (79.5%). Also, a marked circulation of strains with a GII.17 capsid (14.0%) was observed, chiefly in early 2016. The emergence and global spread of non-GII.4 noroviruses pose challenges for the development of vaccine strategies.

Seroprevalence of Dengue Virus, West Nile Virus, Chikungunya Virus, and Zika Virus in International Travelers Attending a Travel and Migration Center in 2015-2017, Southern Italy.

International travelers to areas endemic for vector-borne diseases (VBDs) may be at risk of contracting and spreading these diseases. The aim of this study was to evaluate the prevalence of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies that are specific for Dengue Virus (DV), West Nile Virus (WNV), Chikungunya Virus (CHIKV), or Zika Virus (ZV) in a cohort of international travelers. The study enrolled travelers who attended the Travel Medicine and Migration outpatient service of Local Health Unit of Bari, Italy, in March 2015-June 2017 for counseling and vaccine prophylaxis before travel. After receiving informed consent, post-travel blood samples were tested for IgM and IgG antibodies specific for DV, WNV, CHIKV, and ZV. Of the 207 travelers attending the vaccine service, 156 (75%) were enrolled. Of the 156 subjects, 23 (14.7%) had IgM and/or IgG antibodies specific for at least one VBD. Of these, 12 (52%) were asymptomatic. Nineteen (12.2% of the whole cohort), nine (5.8%), nine (5.8%), and two (1.3%) subjects had IgM and/or IgG antibodies specific for DV, WNV, CHIKV, and ZV, respectively. Ten subjects (6.4%) harbored antibodies that were specific for more than one VBD. A significant number of the international travelers were DV-positive. Our findings suggest that international travelers should undergo serological surveillance, particularly those who travel frequently and for long periods to areas that are endemic for hemorrhagic dengue. Due to a possible risk of introducing VBDs into nonendemic areas, increased awareness among physicians and travelers and appropriate laboratory detection are crucial. There are currently no licensed vaccines for these VBDs in Italy or other European countries; the main preventive measures are protection from mosquito bites and vector control.

Resurgence of Pertussis and Emergence of the Ptxp3 Toxin Promoter Allele in South Italy.

BACKGROUND: Despite universal immunization programs, pertussis remains a major public health concern. This study aimed to describe the pertussis epidemiology in the Puglia region in 2006-2015 and to identify recent polymorphisms in Bordetella pertussis virulence-associated genes. METHODS: The pertussis cases in 2006-2015 were identified from the National Hospital Discharge Database and the Information System of Infectious Diseases. Samples of pertussis cases in 2014-2016 that were confirmed by the Regional Reference Laboratory were subjected to ptxA, ptxP and prn gene sequencing and, in 10 cases, multiple-locus variable-number tandem repeat analysis. RESULTS: In Puglia in 2006-2015, the pertussis incidence rose from an average of 1.39/100,000 inhabitants in 2006-2013 to 2.56-2.54/100,000 in 2014-2015. In infants <1 year of age, the incidence rose from an average of 60.4/100,000 infants in 2006-2013 to 149.9/100,000 in 2015. Of the 661 cases recorded in 2006-2015, 80.3% required hospitalization; of these, 45.4% were <1 year of age. Of the 80 sequenced samples, the allelic profile ptxA1-ptxP3-prn2 was detected in 74. This variant was detected in both vaccinated and unvaccinated people. Six Bordetella pertussis samples were prn deficient. The multiple-locus variable-number tandem repeat analysis cases exhibited multiple-locus variable-number tandem repeat analysis-type 27. CONCLUSIONS: The pertussis incidence in Puglia has risen. The hypervirulent strain was also found in vaccinated people. This suggests bacterial adaptation to the vaccine and raises questions about acellular vaccine effectiveness. Prevention of infant pertussis cases is best achieved by immunizing the pregnant mother. Enhanced surveillance and systematic laboratory confirmation of pertussis should be improved in Italy.

Norovirus GII.17 as Major Epidemic Strain in Italy, 2015-16.

In winter 2015-16, norovirus GII.17 Kawasaki 2014 emerged as a cause of sporadic gastroenteritis in children in Italy. Median patient age was higher for those with GII.17 than GII.4 infection (55 vs. 24 months), suggesting limited cross-protection for older children.

Molecular characterization of two rare human G8P[14] rotavirus strains, detected in Italy in 2012.

Since 2007, the Italian Rotavirus Surveillance Program (RotaNet-Italy) has monitored the diversity and distribution of genotypes identified in children hospitalized with rotavirus acute gastroenteritis. We report the genomic characterization of two rare human G8P[14] rotavirus strains, identified in two children hospitalized with acute gastroenteritis in the southern Italian region of Apulia during rotavirus strain surveillance in 2012. Both strains showed a G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genomic constellation (DS-1-like genomic background). Phylogenetic analysis of each genome segment revealed a mixed configuration of genes of animal and zoonotic human origin, indicating that genetic reassortment events generated these unusual human strains. Eight out of 11 genes (VP1, VP2, VP3, VP6, VP7, NSP3, NSP4 and NSP5) of the Italian G8P[14] strains exhibited close identity with a Spanish sheep strain, whereas the remaining genes (VP4, NSP1 and NSP2) were more closely related to human strains. The amino acid sequences of the antigenic regions of outer capsid proteins VP4 and VP7 were compared with vaccine and field strains, showing high conservation between the amino acid sequences of Apulia G8P[14] strains and human and animal strains bearing G8 and/or P[14] proteins, and revealing many substitutions with respect to the RotaTeq™ and Rotarix™ vaccine strains. Conversely, the amino acid analysis of the four antigenic sites of VP6 revealed a high degree of conservation between the two Apulia strains and the human and animal strains analyzed. These results reinforce the potential role of interspecies transmission and reassortment in generating novel rotavirus strains that might not be fully contrasted by current vaccines.

Clonal Spread of a Unique Strain of Macrolide-Resistant Mycoplasma Pneumoniae Within a Single Family in Italy.

Macrolide-resistant Mycoplasma pneumoniae (MR-MP) is an increasing problem worldwide. This study describes the clonal spread of a unique strain of MR-MP within a single family. On January 23, 2015, nasopharyngeal swabs and sputum samples were collected from the index case (a 9-year-old girl) in southern Italy. The patient had pneumonia and was initially treated with clarithromycin. MR-MP infection was suspected due to prolonged symptoms despite appropriate antibiotic therapy. Two further cases of pneumonia occurred in relatives (a 7-year-old cousin and the 36-year-old mother of the index case); therefore, respiratory samples were also collected from other family members. Sequence analysis identified mutations associated with resistance to macrolides. Both P1 major adhesion protein typing and multiple loci variable-number tandem repeat analysis (MLVA) typing were performed to assess the relatedness of the strains. The index case, the cousin, the mother, and another 4 family members (twin siblings of the index case, a 3-year-old cousin, and the grandmother) were positive for MR-MP. All strains harbored the mutation A2063G, had the same P1 subtype (1), and were MLVA (7/4/5/7/2) type Z. In addition, the index case's aunt (31 years of age and the probable source of infection) harbored an M pneumoniae strain with the same molecular profile; however, this strain was susceptible to macrolides. This cluster of MR-MP infection/carriage caused by a clonal strain suggests a high transmission rate within this family and highlights the need for increased awareness among clinicians regarding the circulation of MR-MP. Novel strategies for the treatment and prevention of M pneumoniae infections are required.

Key Role of Sequencing to Trace Hepatitis A Viruses Circulating in Italy During a Large Multi-Country European Foodborne Outbreak in 2013.

BACKGROUND: Foodborne Hepatitis A Virus (HAV) outbreaks are being recognized as an emerging public health problem in industrialized countries. In 2013 three foodborne HAV outbreaks occurred in Europe and one in USA. During the largest of the three European outbreaks, most cases occurred in Italy (>1,200 cases as of March 31, 2014). A national Task Force was established at the beginning of the outbreak by the Ministry of Health. Mixed frozen berries were early demonstrated to be the source of infection by the identity of viral sequences in patients and in food. In the present study the molecular characterization of HAV isolates from 355 Italian cases is reported. METHODS: Molecular characterization was carried out by PCR/sequencing (VP1/2A region), comparison with reference strains and phylogenetic analysis. RESULTS: A unique strain was responsible for most characterized cases (235/355, 66.1%). Molecular data had a key role in tracing this outbreak, allowing 110 out of the 235 outbreak cases (46.8%) to be recognized in absence of any other link. The data also showed background circulation of further unrelated strains, both autochthonous and travel related, whose sequence comparison highlighted minor outbreaks and small clusters, most of them unrecognized on the basis of epidemiological data. Phylogenetic analysis showed most isolates from travel related cases clustering with reference strains originating from the same geographical area of travel. CONCLUSIONS: In conclusion, the study documents, in a real outbreak context, the crucial role of molecular analysis in investigating an old but re-emerging pathogen. Improving the molecular knowledge of HAV strains, both autochthonous and circulating in countries from which potentially contaminated foods are imported, will become increasingly important to control outbreaks by supporting trace back activities, aiming to identify the geographical source(s) of contaminated food, as well as public health interventions.