A real-time PCR for specific detection of the Legionella pneumophila serogroup 1 ST1 complex.

OBJECTIVE: Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 1 is globally widespread in the environment and accounts for a significant proportion of Legionella infections, including nosocomial Legionnaires' disease (LD). This study aimed to design a sensitive and specific detection method for Lp ST1 that will underpin epidemiological investigations and risk assessment. METHODS: A total of 628 Lp genomes (126 ST1s) were analyzed by comparative genomics. Interrogation of more than 900 accessory genes revealed seven candidate targets for specific ST1 detection and specific primers and hydrolysis probes were designed and evaluated. The analytical sensitivity and specificity of the seven primer and probe sets were evaluated on serially diluted DNA extracted from the reference strain CIP107629 and via qPCR applied on 200 characterized isolates. The diagnostic performance of the assay was evaluated on 142 culture-proven clinical samples from LD cases and a real-life investigation of a case cluster. RESULTS: Of seven qPCR assays that underwent analytical validation, one PCR target (lpp1868) showed higher sensitivity and specificity for ST1 and ST1-like strains. The diagnostic performance of the assay using respiratory samples corresponded to a sensitivity of 95% (19/20) (95% CI (75.1-99.9)) and specificity of 100% (122/122) (95% CI (97-100)). The ST1 PCR assay could link two out of three culture-negative hospitalized LD cases to ST1 during a real-time investigation. CONCLUSION: Using whole genome sequencing (WGS) data, we developed and validated a sensitive and specific qPCR assay for the detection of Lp1 belonging to the ST1 clonal complex by amplification of the lpp1868 gene. The ST1 qPCR is expected to deliver an added value for Lp control and prevention, in conjunction with other recently developed molecular assays.

Quadrivalent versus trivalent influenza vaccine: clinical outcomes in two influenza seasons, historical cohort study.

OBJECTIVES: The quadrivalent influenza vaccine (QIV) contains two influenza B antigens (one of each B lineage), while the trivalent vaccine (TIV) contains solely one. As a result, a mismatch between the circulating B lineage and the lineage in the TIV occurs frequently. We aimed to compare the frequency of clinically significant outcomes in a large cohort of vaccinees receiving either TIV or QIV. METHODS: Historical cohort study of all inactivated influenza vaccinees (aged 3 years and older) in a Health Maintenance Organization insuring 1.2 million individuals, over two influenza seasons in which both vaccines were provided non-selectively. Primary outcome was hospital admissions during the influenza season. Multivariate analysis was performed using logistic regression to adjust for relevant covariates. RESULTS: Our cohort included 150 518 and 168 296 vaccinees in the first (S1) and second season (S2), respectively. The two influenza seasons were characterized by high Influenza B activity. Of those vaccinated with QIV, 2074 of 49 726 (4.2%) and 6563 of 121 741 (5.4%) were hospitalized compared with 7378 of 100 792 (7.3%) and 3372 of 46 555 (7.2%) of those vaccinated with TIV (S1 and S2, respectively). After multivariate analysis adjusting for several covariates (gender, age, socioeconomic status, chronic morbidity, timing of vaccination), compared with TIV recipients, QIV vaccinees had lower odds for hospitalization (OR = 0.92, 95% CI 0.87-0.98 and OR = 0.89, 95% CI 0.85-0.93) or emergency department visit (OR = 0.91, 95% CI 0.87-0.95 and OR = 0.84, 95% CI 0.81-0.87) in S1 and S2, respectively (p < 0.001). Lower odds of mortality and influenza-like illness were also observed in S2 (OR = 0.61, 95% CI 0.50-0.75 and OR = 0.92, 95% CI 0.90-0.95, respectively). CONCLUSIONS: In seasons with relatively high influenza B activity, QIV appeared more protective than TIV in Israel.

Surveillance of listeriosis in the Tel Aviv District, Israel, 2010-2015.

This study analyses the epidemiologic, clinical and molecular findings of all culture-confirmed cases of listeriosis notified from 2010 to 2015 in the Tel Aviv District, which is known to have high rates of listeriosis. All clinical isolates of Listeria monocytogenes were subtyped using two-enzyme pulsed-field gel electrophoresis. During the studied period, 102 cases of listeriosis were notified, including 23 pregnancy-associated cases (23%). Among 79 non-pregnancy-associated cases, 18 had neuro-invasive disease (21%). There were 26 deaths associated with the disease. Using molecular identification, we found a number of clusters of identical bacterial clones, which pointed to possible sources of infection. The high rates of morbidity and mortality resulting from listeriosis, as well as the diverse ways of infection demonstrated in this study, accentuate the need to boost public health actions, in order to raise awareness and better control high-risk contamination routes.

Real-time genomic investigation underlying the public health response to a Shiga toxin-producing Escherichia coli O26:H11 outbreak in a nursery.

Shiga toxin-producing Escherichia coli (STEC) is a significant cause of gastrointestinal infection and the haemolytic-uremic syndrome (HUS). STEC outbreaks are commonly associated with food but animal contact is increasingly being implicated in its transmission. We report an outbreak of STEC affecting young infants at a nursery in a rural community (three HUS cases, one definite case, one probable case, three possible cases and five carriers, based on the combination of clinical, epidemiological and laboratory data) identified using culture-based and molecular techniques. The investigation identified repeated animal contact (animal farming and petting) as a likely source of STEC introduction followed by horizontal transmission. Whole genome sequencing (WGS) was used for real-time investigation of the incident and revealed a unique strain of STEC O26:H11 carrying stx2a and intimin. Following a public health intervention, no additional cases have occurred. This is the first STEC outbreak reported from Israel. WGS proved as a useful tool for rapid laboratory characterization and typing of the outbreak strain and informed the public health response at an early stage of this unusual outbreak.

A bioinformatics tool for ensuring the backwards compatibility of Legionella pneumophila typing in the genomic era.

OBJECTIVES: Whole genome sequencing (WGS) has revolutionized the subtyping of Legionella pneumophila but calling the traditional sequence-based type from genomic data is hampered by multiple copies of the mompS locus. We propose a novel bioinformatics solution for rectifying that limitation, ensuring the feasibility of WGS for cluster investigation. METHODS: We designed a novel approach based on the alignment of raw reads with a reference sequence. With WGS, reads originating from either of the two mompS copies cannot be differentiated. Therefore, when non-identical copies were present, we applied a read-filtering strategy based on read alignment to a reference sequence via unique 'anchors'. If minimal read coverage was achieved after filtration (≥3X), a consensus sequence was built based on mapped reads followed by calling the sequence-based typing allele. The entire procedure was implemented using a Perl script. RESULTS: The method was validated using a diverse sample of 265 L. pneumophila genomes, consisting of 59 different sequence types (STs) and 23 mompS variants; 57 of the 265 (22%) had non-identical mompS copies. In 237 of the 265 samples (89.4%), mompS calling was successful and no erroneous calling occurred. A 98.1% success was recorded among 109 samples meeting quality requirements. The method was superior to alternative approaches. CONCLUSIONS: As WGS becomes more accessible, technical difficulties in routine clinical and surveillance work will arise. The case of mompS in L. pneumophila serves as an example for such limitations that necessitate the development of novel computational solutions that meet end-user demands.

Molecular epidemiology of Campylobacter jejuni infection in Israel-a nationwide study.

The incidence of Campylobacter infection in Israel, particularly among children <2 years of age, has risen over the last decade and became one of the highest among industrialized countries. This study explored the molecular epidemiology of Campylobacter jejuni in Israel over a decade (2003-2012) using multilocus sequence typing (MLST) combined with demographic metadata. Representative clinical isolates (438) from a large national repository together with selected veterinary isolates (74) were subject to MLST. The distribution of age groups, ethnicity and clinical source across various genotypes was evaluated using Poisson modelling. The 512 studied isolates were assigned 126 distinct sequence types (STs) (18.8% novel STs) grouped into 21 clonal complexes (CCs). Most human, poultry and bovine STs clustered together in the leading CCs. Three dominant STs (ST21, ST6608, ST4766) were detected only since 2006. Patients infected with the leading CCs were similarly distributed along densely populated areas. The frequency of blood isolates was higher in patients infected with CC353 (relative rate (RR)=2.0, 95% CI 1.03-3.9, adjusted p value (adj.p) 0.047) and CC42 (RR=4.4, 95% CI 1.7-11.6, adj.p 0.018) and lower with CC257 (RR=0.3, 95% CI 0.1-0.9, adj. p 0.047). The distribution of age groups and ethnicity also varied across the leading CCs. In conclusion, C. jejuni isolates in a national sample appeared highly diverse with a high proportion of new STs. Phylogenic analysis was compatible with poultry and cattle as possible food sources of clinical infection. Demographic characteristics of the infected patients coupled with strain invasiveness across different genotypes revealed a complex epidemiology of C. jejuni transmission in Israel.

Molecular characterization and antibiotic resistance of group G streptococci in Israel: comparison of invasive, non-invasive and carriage isolates.

Beta-hemolytic group G streptococci (GGS) are increasingly recognized as a source of substantial morbidity, causing mild to severe sporadic infections as well as outbreaks. The purpose of this study was to determine the genetic diversity and antibiotic resistance of GGS in Israel in order to aid in prevention and control. A total of 325 GGS isolates were collected in Israel between 2007 and 2011 from three determined settings: (1) carriage (n = 60), an observational longitudinal carriage study in the IF, (2) non-invasive (n = 166), clinical sporadic and epidemic non-invasive cases in the IDF, and (3) invasive (n = 99) cases of bacteremia collected during this period in Israel from a similar age group, at the national Streptococcal Reference Center. All isolates were characterized genetically and by their antibiotic-resistance profile. emm typing revealed 35 distinct types and subtypes among 228 S. dysgalactiae subsp. equisimilis (SDSE) isolates, with high genetic diversity. An additional 97 GGS were identified as Streptococcus anginosus (SAG). The proportion of SDSE was higher in the invasive (100 %) and non-invasive (63.8 %) isolates compared to the carriage ones (38.3 %). Clindamycin, erythromycin, azithromycin and tetracycline resistance was detected in 6.6 %, 8.6 %, 9.7 % and 37.6 % of isolates, respectively. Overall, the most resistant isolates were in the invasive group and the fewest were in the SAG group. Considerable genetic diversity and common antibiotic resistance were revealed among GGS strains which differed according to the epidemiologic settings. Further clinical, epidemiological and basic research of GGS as a pathogen is warranted.

A cluster of invasive meningococcal disease revealed by the characterization of a novel serogroup B meningococcal clone.

The incidence of invasive infections due to Neisseria meningitidis in Israel is about 1/100 000 population annually. Three cases of meningococcal meningitis were reported in employees at a single plant; the first case appeared in March 2013 and the second and third cases appeared in December, almost 9 months later. N. meningitidis serogroup B was isolated from cerebrospinal fluid samples. Multilocus sequence typing assigned the three meningococcal isolates to ST10418, a new sequence type and a member of the ST32 clonal complex. The clonality was confirmed by performance of pulsed-field gel electrophoresis. Post-exposure antibiotic prophylaxis was administered to close contacts of the first case. Upon the diagnosis of the additional two cases, post-exposure prophylaxis was administered to all the plant employees. This report demonstrates the importance of combining public health measures and advanced laboratory studies to confirm clonality and to prevent further disease spread in a closed setting.

Design and application of a core genome multilocus sequence typing scheme for investigation of Legionnaires' disease incidents.

Sequence-based typing (SBT) for Legionella pneumophila (Lp) has dramatically improved Legionnaires' disease (LD) cluster investigation. Microbial whole genome sequencing (WGS) is a promising modality for investigation but sequence analysis methods are neither standardised, nor agreed. We sought to develop a WGS-based typing scheme for Lp using de novo assembly and a genome-wide gene-by-gene approach (core genome multilocus sequence typing, cgMLST). We analysed 17 publicly available Lp genomes covering the whole species variation to define a core genome (1,521 gene targets) which was validated using 21 additional published genomes. The genomes of 12 Lp strains implicated in three independent cases of paediatric humidifier-associated LD were subject to cgMLST together with three 'outgroup' strains. cgMLST was able to resolve clustered strains and clearly identify related and unrelated strains. Thus, a cgMLST scheme was readily achievable and provided high-resolution analysis of Lp strains. cgMLST appears to have satisfactory discriminatory power for LD cluster analysis and is advantageous over mapping followed by single nucleotide polymorphism (SNP) calling as it is portable and easier to standardise. cgMLST thus has the potential for becoming a gold standard tool for LD investigation. Humidifiers pose an ongoing risk as vehicles for LD and should be considered in cluster investigation and control efforts.

The association of serotype and pulsed-field gel electrophoresis genotype in isolates of Streptococcus pneumoniae isolated in Israel.

The relationship between Streptococcus pneumoniae isolates causing invasive infections in children admitted to a single center in central Israel was examined by pulsed-field gel electrophoresis (PFGE) and serotyping. Although there was a close correlation between serotype and PFGE clone, the genetic diversity varied by serotype, with some genotypes comprising multiple serotypes. Additionally, clones C and D were associated with higher penicillin minimum inhibitory concentrations. Serotyping alone may be insufficient for epidemiological mapping of pneumococcal isolates in the era of pneumococcal conjugate polysaccharide vaccines.

Diabetes prevention program in a Mediterranean environment: individual or group therapy? An effectiveness evaluation.

INTRODUCTION: Diabetes as a multifactorial disorder requires prevention measures based upon the modification of several risk factors simultaneously; otherwise, there is insufficient potential for prevention. Following the success of the American Diabetes Prevention Program (DPP), we implemented an intervention program in a large Israeli healthcare organization with an emphasize on Mediterranean Diet (MedDiet) and physical activity. The objective was to evaluate the effectiveness of two types of intervention, individual and group therapies, in reducing risk factors and in preventing or delaying the development of type 2 diabetes. METHODS: Out of 180 primary care physicians, 85 who agreed to participate, were randomly assigned, between the years 2005 and 2006, into two groups: those who would refer pre-diabetes adult patients for individual therapy and those who would refer for group therapy. The two groups of patients consisted of 111 and 112 in each group. The intervention lasted for 6 months and discussed: the benefits of MedDiet, planning nutritional behavior and mindful eating, and the importance of physical activity. All patients were invited to participate in walking groups. Follow up lasted for 24 months and logistic, mixed models, and Cox regressions were employed. RESULTS: No statistically significant differences were detected between the two intervention groups in age; gender and clinical measurements at recruitment. Thirty nine percent of both groups developed diabetes (entered the DR by 2012), including 38.7% from the individual therapy and 39.3% from the group therapy (P=0.933). The mean time from 2005 until entry to the Diabetes Registry (DR) was 2.9 and 2.5 years for the individual and group therapy respectively (P=0.542). CONCLUSION: Both interventions were equally effective in achieving the desired outcomes and time until entry to the DR. For large health organizations with a high number of pre-diabetes patients and scarce resources, group therapy, where 12 people are reached out by one team member are preferable and more costly effective, than a one on one therapy.

Molecular epidemiology of Legionnaires' disease in Israel.

National surveillance of Legionnaires' disease (LD) is important to inform control measures and facilitate international networking for timely reporting. This study is the first to describe the molecular epidemiology of LD in Israel. Case notifications for 2006-2011, collated through mandatory reporting, were identified and demographic, clinical and laboratory data were extracted. Unrelated clinical and environmental Legionella pneumophila strains were characterized using standard procedures, Dresden panel of monoclonal antibodies and the ESCMID Study Group for Legionella Infections (ESGLI) Sequence-Based Typing scheme. In all, 294 cases were reported (crude incidence 0.67 cases/100 000; age-standardized incidence 1/100 000). LD epidemiological trends and features largely resembled those of the EU, except for a larger proportion of nosocomial cases. Of 28 clinical and 23 environmental strains analysed, 71.4% and 21.7% were serogroup (sg) 1 and the most common immunological subgroup was OLDA/Oxford (64%). Of the clinical strains, OLDA/Oxford, ST1 was the most common (43%) followed by Allentown/France, ST40 (14%). The unusual sg 3 ST338 was found in 17.4% of environmental strains. Novel STs were detected amongst 23.5% of strains. These findings warrant further molecular investigation. Molecular epidemiology data generated from neighbouring countries newly adopting the ESGLI typing scheme for L. pneumophila contribute to understanding of regional strain diversity.

Déjà vu: Ralstonia mannitolilytica infection associated with a humidifying respiratory therapy device, Israel, June to July 2011.

Following a bloodstream infection in June 2011 with Ralstonia mannitolilytica in a premature infant treated with a humidifying respiratory therapy device, an investigation was initiated at the Hadassah Medical Centres in Jerusalem. The device delivers a warmed and humidified mixture of air and oxygen to patients by nasal cannula. The investigation revealed colonisation with R. mannitolilytica of two of 15 patients and contamination of components of five of six devices deployed in the premature units of the Hadassah hospitals. Ten isolates from the investigation were highly related and indistinguishable from isolates described in an outbreak in 2005 in the United States (US). Measures successful in containing the US outbreak were not included in user instructions provided to our hospitals by the distributor of the device.

Comparative seroepidemiology of diphtheria in six European countries and Israel.

Serological surveys for diphtheria were conducted in six European countries including Czech Republic, Hungary, Ireland, Latvia, Luxembourg, Slovakia and one country outside Europe, Israel. For each country, a nationally representative population sample was collected across the entire age range and was tested for antibodies to diphtheria toxin. Although each national laboratory used its preferred assay, the results were all standardized to those of the in vitro neutralization test and expressed in international units (IU) which allowed comparative analyses to be performed. The results showed that increasing age is related to a gradual increase in seronegative subjects (<0·01 IU/ml of diphtheria antitoxin antibodies). This may reflect waning immunity following childhood vaccination without repeated booster vaccinations in adults. Differences in seronegativity were also found according to gender. In subjects aged 1-19 years, geometric mean titres of antitoxin are clearly related to the different vaccination schedules used in the participating countries. Although clinical disease remains rare, the susceptibility to diphtheria observed in these serosurveys highlights the importance of strengthened surveillance.

Primary care intervention programme to improve early detection of hearing loss in the elderly.

OBJECTIVE: To improve audiology screening in general practice, using an intervention programme aiming to empower older adults and their general practitioners. METHODS: We conducted a quasi-experimental community study comparing 206 patients and two control groups (the first being 101 people registered with the same general practitioner, and the second 87 people registered with another general practitioner). Outcome measures were: rates of hearing tests in the six months before interview, and screening recommendation by the general practitioner. RESULTS: Amongst patients, there was a significant increase in numbers undergoing a hearing test, from 19 per cent before the intervention to 49 per cent two years later, while in the two control groups there was little change. Twenty-two per cent of patients and 19 per cent of the first control group reported that their physicians suggested undergoing a hearing test; the second control group subjects (whose general practitioners received no specific educational intervention) showed no change. CONCLUSION: The two crucial factors for improving hearing screening uptake in the elderly are general practitioner education and patient empowerment.

Streptococcus pyogenes emm and T types within a decade, 1996-2005: implications for epidemiology and future vaccines.

Streptococcus pyogenes group A (GAS) is a primary human pathogen. We performed genetic emm sequence and serological T-antigen typing of 819 mostly invasive GAS isolates recovered in Israel during 1996-2005. Of the 72 emm types found, the six most prevalent types (1, 81, 89, 14, 28, 5) comprised 30.2% of all isolates, and emm-type changes were observed over the years. The predicted coverage of the 26-valent S. pyogenes vaccine formulated for usage in the USA was predicted to be only approximately 60%. On the basis of different emm-T antigen type associations, some Israeli strains are probably different clonal types than those found in USA. About 2% of GAS had emm types that were originally associated with S. dysgalactiae subsp. equisimilis emm genes. Therefore, routine emm typing allows meaningful GAS strain surveillance, and provides data relevant to better vaccine coverage.

School closure may be effective in reducing transmission of respiratory viruses in the community.

Proposed measures to contain pandemic influenza include school closure, although the effectiveness of this has not been investigated. We examined the effect of a nationwide elementary school strike in Israel in 2000 on the incidence of influenza-like illness. In this historical observational study of 1.7 million members of a preferred provider organization, we analysed diagnoses from primary-care visits during the winter months in 1998-2002. We calculated the weekly ratio of influenza-like diagnoses to non-respiratory diagnoses, and fitted regression models for school-aged children, children's household members, and all other individuals aged >12 years. For each population the steepest drop in the ratio of influenza-like diagnoses to non-respiratory diagnoses occurred in the strike year 2 weeks after the start of the strike. The changes in the weekly ratio of influenza-like diagnoses to non-respiratory diagnoses were statistically significant (P=0.0074) for school children for the strike year compared to other years. A smaller decrease was also seen for the adults with no school-aged children in 1999 (P=0.037). The Chanukah holiday had a negative impact on the ratio for school-aged children in 1998, 1999 and 2001 (P=0.008, 0.006 and 0.045, respectively) and was statistically significant for both adult groups in 1999 and for adults with no school-aged children in 2001. School closure should be considered part of the containment strategy in an influenza pandemic.

Invasive meningococcal disease in children in Jerusalem.

Neisseria meningitidis is an important cause of childhood meningitis and septicaemia. Between 1999 and 2005, 133 invasive meningococcal disease (IMD) cases occurred in Jerusalem, 112 (84.2%) of them in children aged 0-14 years. The annual incidence rate in Jerusalem was higher than the national average (2.45+/-0.6 vs. 1.13+/-0.16/100 000 population, P=0.002). Most of the children (82.1%) were from low socio-economic Arab and Jewish ultra-orthodox communities; mortality was higher among Arab than Jewish children (1.3 vs. 0.22/100 000 person-years, P=0.004). A cluster of 10 children with severe meningococcal sepsis (three fatalities) emerged in the winter of 2003-2004. Compared to the other 102 cases in 1999-2005 both meningococcaemia (100% vs. 51%, P=0.003) and mortality (30% vs. 6.9%, P=0.014) rates were higher. Serogroup B comprised 77.6% of the bacterial isolates. Pulsed-field gel electrophoresis showed considerable variability among cluster isolates, but significant resemblance in Arab cases throughout 1999-2005. The increased susceptibility of specific sub-populations to IMD necessitates further evaluation.

Extended-spectrum beta-lactamase-producing Shigella strains in Israel, 2000-2004.

Routine susceptibility testing of 5,616 Shigella isolates at the National Shigella Reference Centre in Israel over a 5-year period (2000-2004) revealed resistance to ceftriaxone in one strain of Shigella boydii 2 and in two strains each of Shigella flexneri 2a, S. flexneri 6, and Shigella sonnei. All seven isolates were confirmed as producers of extended-spectrum beta-lactamase (ESBL) by the combination disk method, the Vitek 1 system, and a modification of the double-disk synergy test, which is based on the inhibitory properties of clavulanic acid, tazobactam, and sulbactam. Tazobactam had the strongest effect in all seven strains. Molecular characterization of the ESBLs identified CTX-M-type enzymes, consisting of the CTX-M-9 group (n = 3), CTX-M-3 (n = 2), CTX-M-39 (n = 1), and CTX-M-2 group (n = 1). Three of the strains also carried bla-(OXA) genes and a bla-(TEM) gene. Although the prevalence of ESBLs in this study was low, further research is needed on the spread and transfer of resistance genes, both in hospitals and in the community.

Prevalence and correlates of diphtheria toxoid antibodies in children and adults in Israel.

A seroepidemiological study was performed to evaluate immunity to diphtheria and to determine the correlates of diphtheria toxoid antibody levels among children and adults in Israel. In total, 3,185 sera from an age-stratified sample of children and adults, obtained in 2000-2001, were tested for diphtheria toxoid antibodies by an in-house double-antigen ELISA. A level of or=0.1 IU/mL (full protection or seropositivity). Seronegativity increased significantly in subjects aged >50 years, reaching levels of 9.7%, 12.6% and 18.9% in the groups aged 50-54, 55-59 and >60 years, respectively (p 0.001), with rates of basic immunity following a similar pattern. Subjects born abroad had higher seronegativity rates than those born in Israel (7.7% vs. 4.9%; p 0.019). No difference in diphtheria toxoid antibody levels was found according to other demographical variables, such as gender, Jewish or Arab ethnicity, urban or rural settlements, and the subjects' place of residence. The level of immunity to diphtheria among children and adults in Israel was satisfactory, with the exception of individuals aged >50 years. The risk of diphtheria outbreaks is low, but sporadic cases may occur among individuals lacking basic immunity against the disease.