Trends in invasive bacterial diseases during the first 2 years of the COVID-19 pandemic: analyses of prospective surveillance data from 30 countries and territories in the IRIS Consortium.

BACKGROUND: The Invasive Respiratory Infection Surveillance (IRIS) Consortium was established to assess the impact of the COVID-19 pandemic on invasive diseases caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Streptococcus agalactiae. We aimed to analyse the incidence and distribution of these diseases during the first 2 years of the COVID-19 pandemic compared to the 2 years preceding the pandemic. METHODS: For this prospective analysis, laboratories in 30 countries and territories representing five continents submitted surveillance data from Jan 1, 2018, to Jan 2, 2022, to private projects within databases in PubMLST. The impact of COVID-19 containment measures on the overall number of cases was analysed, and changes in disease distributions by patient age and serotype or group were examined. Interrupted time-series analyses were done to quantify the impact of pandemic response measures and their relaxation on disease rates, and autoregressive integrated moving average models were used to estimate effect sizes and forecast counterfactual trends by hemisphere. FINDINGS: Overall, 116 841 cases were analysed: 76 481 in 2018-19, before the pandemic, and 40 360 in 2020-21, during the pandemic. During the pandemic there was a significant reduction in the risk of disease caused by S pneumoniae (risk ratio 0·47; 95% CI 0·40-0·55), H influenzae (0·51; 0·40-0·66) and N meningitidis (0·26; 0·21-0·31), while no significant changes were observed for S agalactiae (1·02; 0·75-1·40), which is not transmitted via the respiratory route. No major changes in the distribution of cases were observed when stratified by patient age or serotype or group. An estimated 36 289 (95% prediction interval 17 145-55 434) cases of invasive bacterial disease were averted during the first 2 years of the pandemic among IRIS-participating countries and territories. INTERPRETATION: COVID-19 containment measures were associated with a sustained decrease in the incidence of invasive disease caused by S pneumoniae, H influenzae, and N meningitidis during the first 2 years of the pandemic, but cases began to increase in some countries towards the end of 2021 as pandemic restrictions were lifted. These IRIS data provide a better understanding of microbial transmission, will inform vaccine development and implementation, and can contribute to health-care service planning and provision of policies. FUNDING: Wellcome Trust, NIHR Oxford Biomedical Research Centre, Spanish Ministry of Science and Innovation, Korea Disease Control and Prevention Agency, Torsten Söderberg Foundation, Stockholm County Council, Swedish Research Council, German Federal Ministry of Health, Robert Koch Institute, Pfizer, Merck, and the Greek National Public Health Organization.

Infant Escherichia coli urinary tract infection: is it associated with meningitis?

OBJECTIVES: Determine the prevalence of coexisting bacterial meningitis (BM) and sterile cerebrospinal fluid (CSF) with raised white cell count relative to age ('pleocytosis') in the presence of Escherichia coli urinary tract infection (UTI), with the addition of CSF E. coli PCR analysis. DESIGN: Single-centre, retrospective cohort study. SETTING: Tertiary paediatric hospital. PARTICIPANTS: Children aged 8 days to 2 years, with a pure growth of E. coli from urine and a CSF sample taken within 48 hours of a positive urine culture between 1 January 2014 and 30 April 2019. MAIN OUTCOME MEASURE: Prevalence of coexisting E. coli BM with UTI, defined as a pure growth E. coli from urine and a CSF culture with pure growth E. coli and/or positive E. coli PCR. RESULTS: 1903 patients had an E. coli UTI, of which 314 (16%) had a CSF sample taken within 48 hours. No cases of coexisting E. coli BM were identified. There were 71 (23%) cases of pleocytosis, 57 (80%) of these had PCR analysis, all of which were E. coli PCR not detected. Patients aged 1-6 months accounted for 72% of all lumbar punctures (LPs). CONCLUSION: The risk of E. coli UTI and coexisting E. coli BM is low. There is potential to reduce the number of routine LPs in infants with a diagnosis of E. coli UTI with the greatest impact in children up to 6 months of age. CSF E. coli PCR can help further reduce post-test probability of BM in the setting of pleocytosis.

Risk factors for carriage of meningococcus in third-level students in Ireland: an unsupervised machine learning approach.

The aim of this study was to examine the risk factors for pharyngeal carriage of meningococci in third-level students using an unsupervised machine learning approach. Data were gathered as part of meningococcal prevalence studies conducted by the Irish Meningitis and Sepsis Reference Laboratory (IMSRL). Pharyngeal swab cultures for meningococcal carriage were taken from each student once they had completed a single-page anonymous questionnaire addressing basic demographics, social behaviors, living arrangements, vaccination, and antibiotic history. Data were analyzed using multiple correspondence analysis through a machine learning approach.In total, 16,285 students who had a pharyngeal throat swab taken returned a fully completed questionnaire. Overall, meningococcal carriage rate was 20.6%, and the carriage of MenW was 1.9% (n = 323). Young Irish adults aged under 20 years and immunized with the meningococcal C vaccine had a higher MenW colonization rate (n = 171/1260, 13.5%) compared with non-Irish adults aged 20 years or older without the MenC vaccine (n = 5/81, 6%, chi-square = 3.6, p = .05). Unsupervised machine learning provides a useful technique to explore meningococcal carriage risk factors. The issue is very complex, and asked risk factors only explain a small proportion of the carriage. This technique could be used on other conditions to explore reasons for carriage.

Changes in the incidence of invasive disease due to Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis during the COVID-19 pandemic in 26 countries and territories in the Invasive Respiratory Infection Surveillance Initiative: a prospective analysis of surveillance data.

BACKGROUND: Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis, which are typically transmitted via respiratory droplets, are leading causes of invasive diseases, including bacteraemic pneumonia and meningitis, and of secondary infections subsequent to post-viral respiratory disease. The aim of this study was to investigate the incidence of invasive disease due to these pathogens during the early months of the COVID-19 pandemic. METHODS: In this prospective analysis of surveillance data, laboratories in 26 countries and territories across six continents submitted data on cases of invasive disease due to S pneumoniae, H influenzae, and N meningitidis from Jan 1, 2018, to May, 31, 2020, as part of the Invasive Respiratory Infection Surveillance (IRIS) Initiative. Numbers of weekly cases in 2020 were compared with corresponding data for 2018 and 2019. Data for invasive disease due to Streptococcus agalactiae, a non-respiratory pathogen, were collected from nine laboratories for comparison. The stringency of COVID-19 containment measures was quantified using the Oxford COVID-19 Government Response Tracker. Changes in population movements were assessed using Google COVID-19 Community Mobility Reports. Interrupted time-series modelling quantified changes in the incidence of invasive disease due to S pneumoniae, H influenzae, and N meningitidis in 2020 relative to when containment measures were imposed. FINDINGS: 27 laboratories from 26 countries and territories submitted data to the IRIS Initiative for S pneumoniae (62 837 total cases), 24 laboratories from 24 countries submitted data for H influenzae (7796 total cases), and 21 laboratories from 21 countries submitted data for N meningitidis (5877 total cases). All countries and territories had experienced a significant and sustained reduction in invasive diseases due to S pneumoniae, H influenzae, and N meningitidis in early 2020 (Jan 1 to May 31, 2020), coinciding with the introduction of COVID-19 containment measures in each country. By contrast, no significant changes in the incidence of invasive S agalactiae infections were observed. Similar trends were observed across most countries and territories despite differing stringency in COVID-19 control policies. The incidence of reported S pneumoniae infections decreased by 68% at 4 weeks (incidence rate ratio 0·32 [95% CI 0·27-0·37]) and 82% at 8 weeks (0·18 [0·14-0·23]) following the week in which significant changes in population movements were recorded. INTERPRETATION: The introduction of COVID-19 containment policies and public information campaigns likely reduced transmission of S pneumoniae, H influenzae, and N meningitidis, leading to a significant reduction in life-threatening invasive diseases in many countries worldwide. FUNDING: Wellcome Trust (UK), Robert Koch Institute (Germany), Federal Ministry of Health (Germany), Pfizer, Merck, Health Protection Surveillance Centre (Ireland), SpID-Net project (Ireland), European Centre for Disease Prevention and Control (European Union), Horizon 2020 (European Commission), Ministry of Health (Poland), National Programme of Antibiotic Protection (Poland), Ministry of Science and Higher Education (Poland), Agencia de Salut Pública de Catalunya (Spain), Sant Joan de Deu Foundation (Spain), Knut and Alice Wallenberg Foundation (Sweden), Swedish Research Council (Sweden), Region Stockholm (Sweden), Federal Office of Public Health of Switzerland (Switzerland), and French Public Health Agency (France).

Antibiotic susceptibility and molecular analysis of invasive Neisseria meningitidis recovered in the Republic of Ireland, 1996 to 2016.

This study examined the antimicrobial susceptibility of invasive meningococcal disease (IMD)-associated Neisseria meningitidis recovered in the Republic of Ireland between 1996 and 2016. In total, 1359 isolates representing over one-third of all laboratory-confirmed cases of IMD diagnosed each epidemiological year (EY; July 1-June 30) were analysed. All isolates were susceptible to ciprofloxacin, rifampicin and cefotaxime and 74% and 87% were susceptible to sulphonamide and penicillin, respectively. The proportion of isolates exhibiting reduced susceptibility to penicillin increased significantly during the study with no evidence of major clonal expansion or horizontal spread of a specific penA allele. Greater diversity observed among recently recovered meningococci and specifically among isolates exhibiting reduced penicillin susceptibility contributed to the overall increase in penA allele diversity throughout. The emergence and dissemination of strains with phenotypic and genotypic reduced susceptibility to penicillin increase the need for continued surveillance of antimicrobial susceptibility of meningococci in the Republic of Ireland especially in view of the recommendation of penicillin G as empiric treatment of choice for pre-hospital management.

Epidemiology of Haemophilus influenzae in the Republic of Ireland, 2010-2018.

The purpose of this study was to investigate H. influenzae epidemiology in the Republic of Ireland. We performed serotyping, multi-locus sequence typing (MLST) and susceptibility testing on H. influenzae isolates received by the Irish Meningitis and Sepsis Reference Laboratory from 2010 to 2018. Three hundred sixty-seven invasive and 41 non-invasive infection (NII) isolates were received. Invasive isolates were mostly recovered from paediatric (21%) and elderly (42%) populations. Invasive disease was more prevalent in females of childbearing age (72%) compared with males the same age (28%). Non-typeable H. influenzae (NTHi) predominated among invasive (83%) and NII (95%). Invasive Hib disease isolates were infrequent (4%, n = 15). Among invasive disease, Hif was the commonest encapsulated serotype (10%, n = 37), and the only encapsulated serotype detected in NII (5%, 2/41). The first PCR-confirmed serotypes d and a in Ireland were characterised among invasive disease in 2017 and 2018, respectively. MLST revealed a diverse NTHi population, while encapsulated serotypes were clonal. Sequence type (ST) 103 (n = 14) occurred exclusively in invasive NTHi disease. Ampicillin resistance (AmpR) was 18% among invasive isolates and 22% in NII. ß-Lactamase production was the main source of ampicillin resistance in invasive and NII isolates. We detected ß-lactamase negative ampicillin resistance (BLNAR) among invasive isolates. We report an NTHi fluoroquinolone-resistant clone: ST1524 among invasive (n = 2) and NII isolates (n = 2). The Hib vaccine has positively impacted on Hib disease in Ireland, given the low frequency of Hib. The dominance of NTHi, emergence of serotypes a and d and BLNAR suggest a changing H. influenzae epidemiology in Ireland.

Diversity of meningococci associated with invasive meningococcal disease in the Republic of Ireland over a 19 year period, 1996-2015.

This study examined the capsular phenotype and genotype of invasive meningococcal disease (IMD)-associated Neisseria meningitidis recovered in the Republic of Ireland (RoI) between 1996 and 2015. This time period encompasses both pre- (when IMD was hyperendemic in the RoI) and post- meningococcal serogroup C conjugate (MCC) vaccine introduction. In total, 1327 isolates representing over one-third of all laboratory-confirmed cases of IMD diagnosed each epidemiological year (EY), were characterised. Serogroups B (menB) and C (menC) predominated throughout, although their relative abundance changed; with an initial increase in the proportion of menC in the late 1990s followed by their dramatic reduction post-MCC vaccine implementation and a concomitant dominance of menB, despite an overall decline in IMD incidence. While the increase in menC was associated with expansion of specific clonal-complexes (cc), cc11 and cc8; the dominance of menB was not. There was considerable variation in menB-associated cc with declines in cc41/44 and cc32, and increases in cc269 and cc461, contributing to a significant increase in the clonal diversity of menB isolates over the study. This increase in diversity was also displayed among the serosubtyping data, with significant declines in proportions of menB isolates expressing p1.4 and p1.15 antigens. These data highlight the changing diversity of IMD-associated meningococci since 1996 in the RoI and emphasise the need for on-going surveillance particularly in view of the recent introduction of a menB vaccine.

cgMLST characterisation of invasive Neisseria meningitidis serogroup C and W strains associated with increasing disease incidence in the Republic of Ireland.

INTRODUCTION AND AIMS: Since 2013 MenC and MenW disease incidence and associated mortality rates have increased in the Republic of Ireland. From 2002/2003 to 2012/2013, the average annual MenC incidence was 0.08/100,000, which increased to 0.34/100,000 during 2013/2014 to 2017/18, peaking in 2016/17 (0.72/100,000) with an associated case fatality rate (CFR) of 14.7%. MenW disease incidence has increased each year from 0.02/100,000 in 2013/2014, to 0.29/100,000 in 2017/18, with an associated CFR of 28.6%. We aimed to characterise and relate recent MenC isolates to the previously prevalent MenC:cc11 ET-15 clones, and also characterise and relate recent MenW isolates to the novel 'Hajj' clones. METHODS: Using WGS we characterised invasive (n = 74, 1997/98 to 2016/17) and carried (n = 16, 2016/17) MenC isolates, and invasive (n = 18, 2010/11 to 2016/17) and carried (n = 15, 2016/17) MenW isolates. Genomes were assembled using VelvethOptimiser and stored on the PubMLST Neisseria Bacterial Isolate Genome Sequence Database. Isolates were compared using the cgMLST approach. RESULTS: Most MenC and MenW isolates identified were cc11. A single MenC:cc11 sub-lineage contained the majority (68%, n = 19/28) of recent MenC:cc11 disease isolates and all carried MenC:cc11 isolates, which were interspersed and distinct from the historically significant ET-15 clones. MenW:cc11 study isolates clustered among international examples of both the original UK 2009 MenW:cc11, and novel 2013 MenW:cc11clones. CONCLUSIONS: We have shown that the majority of recent MenC disease incidence was caused by strain types distinct from the MenC:cc11 ET-15 clone of the late 1990s, which still circulate but have caused only sporadic disease in recent years. We have identified that the same aggressive MenW clone now established in several other European countries, is endemic in the RoI and responsible for the recent MenW incidence increases. This data informed the National immunisation Advisory Committee, who are currently deliberating a vaccine policy change to protect teenagers.

The role of real-time PCR testing in the investigation of paediatric patients with community-onset osteomyelitis and septic arthritis.

BACKGROUND: Culture yield in osteomyelitis and septic arthritis is low, emphasising the role for molecular techniques. AIMS: The purpose of this study was to review the laboratory investigation of childhood osteomyelitis and septic arthritis. METHODS: A retrospective review was undertaken in an acute tertiary referral paediatric hospital from January 2010 to December 2016. Cases were only included if they had a positive culture or bacterial PCR result from a bone/joint specimen or blood culture, or had radiographic evidence of osteomyelitis. RESULTS: Seventy-eight patients met the case definition; 52 (66%) were male. The median age was 4.8 years. Blood cultures were positive in 16 of 56 cases (29%), with 11 deemed clinically significant (Staphylococcus aureus = 8, group A Streptococcus = 3). Thirty-seven of 78 (47%) bone/joint samples were positive by culture with S. aureus (n = 16), coagulase-negative Staphylococcus (n = 9) and group A Streptococcus (n = 4), being the most common organisms. Sixteen culture-negative samples were sent for bacterial PCR, and four were positive (Kingella kingae = 2, Streptococcus pneumoniae = 1, group A Streptococcus = 1). CONCLUSIONS: Sequential culture and PCR testing can improve the detection rate of causative organisms in paediatric bone and joint infections, particularly for fastidious microorganisms such as K. kingae. PCR testing can be reserved for cases where culture is negative after 48 h. These results have been used to develop a standardised diagnostic test panel for bone and joint infections at our institution.

Potential Coverage of the 4CMenB Vaccine against Invasive Serogroup B Neisseria meningitidis Isolated from 2009 to 2013 in the Republic of Ireland.

Neisseria meningitidis is a common cause of bacterial meningitis in children and young adults worldwide. The 4CMenB vaccine (Bexsero), developed to combat meningococcal serogroup B (MenB) disease, contains subcapsular antigens that may induce immunity against strains of N. meningitidis, regardless of serogroup. Owing to differential levels of expression and peptide diversity in vaccine antigens across meningococcal strains, the meningococcal antigen typing system (MATS) was developed to estimate the potential MenB strain coverage of 4CMenB. Prior to introducing the 4CMenB vaccine into routine use, we sought to estimate the potential 4CMenB coverage against invasive MenB strains isolated in the Republic of Ireland (RoI) over four consecutive epidemiological years. MATS was applied to a panel of 105 invasive MenB strains isolated during July 2009 to June 2013. Sequence data characterizing the multilocus sequence typing (MLST) alleles and the major 4CMenB target peptides were extracted from isolate genome sequence data, hosted in the Bacterial Isolate Sequencing database (BIGSdb). MATS data indicated that 4CMenB may induce protective immunity against 69.5% (95% confidence interval [CI95%], 64.8% to 84.8%) of circulating MenB strains. Estimated coverage was highest against the most prevalent disease-causing lineage, cc41/44, where the most frequently observed sequence types, ST-154 and ST-41 (21% of isolates, collectively), were typically covered by three antigens. No significant temporal trends were observed. Overall, these data provide a baseline of strain coverage prior to the introduction of 4CMenB and indicate that a decrease in invasive meningococcal disease (IMD) is predicted following the introduction of 4CMenB into the routine infant immunization schedule in the RoI.IMPORTANCE The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA) that measures both the levels of expression and the immune reactivity of the three recombinant 4CMenB antigens. Together with PorA variable-region sequence data, this system provides an estimation of how susceptible MenB isolates are to killing by 4CMenB vaccine-induced antibodies. Assays based on subcapsular antigen phenotype analyses, such as MATS, are important in situations where conventional vaccine coverage estimations are not possible. Subcapsular antigens are typically highly diverse across strains, and vaccine coverage estimations would require unfeasibly large efficacy trials and screening of an exhaustive strain panel for antibody functional activity. Here, MATS was applied to all invasive meningococcal serogroup B (MenB) strains isolated over four consecutive epidemiological years (n = 105) and predicted reasonably high 4CMenB vaccine coverage in the Republic of Ireland.

Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis.

Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.

Resolution of a Protracted Serogroup B Meningococcal Outbreak with Whole-Genome Sequencing Shows Interspecies Genetic Transfer.

A carriage study was undertaken (n = 112) to ascertain the prevalence of Neisseria spp. following the eighth case of invasive meningococcal disease in young children (5 to 46 months) and members of a large extended indigenous ethnic minority Traveller family (n = 123), typically associated with high-occupancy living conditions. Nested multilocus sequence typing (MLST) was employed for case specimen extracts. Isolates were genome sequenced and then were assembled de novo and deposited into the Bacterial Isolate Genome Sequencing Database (BIGSdb). This facilitated an expanded MLST approach utilizing large numbers of loci for isolate characterization and discrimination. A rare sequence type, ST-6697, predominated in disease specimens and isolates that were carried (n = 8/14), persisting for at least 44 months, likely driven by the high population density of houses (n = 67/112) and trailers (n = 45/112). Carriage for Neisseria meningitidis (P < 0.05) and Neisseria lactamica (P < 0.002) (2-sided Fisher's exact test) was more likely in the smaller, more densely populated trailers. Meningococcal carriage was highest in 24- to 39-year-olds (45%, n = 9/20). Evidence of horizontal gene transfer (HGT) was observed in four individuals cocolonized by Neisseria lactamica and Neisseria meningitidis One HGT event resulted in the acquisition of 26 consecutive N. lactamica alleles. This study demonstrates how housing density can drive meningococcal transmission and carriage, which likely facilitated the persistence of ST-6697 and prolonged the outbreak. Whole-genome MLST effectively distinguished between highly similar outbreak strain isolates, including those isolated from person-to-person transmission, and also highlighted how a few HGT events can distort the true phylogenetic relationship between highly similar clonal isolates.

Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF.

Target gene sequencing to characterize the penicillin G susceptibility of Neisseria meningitidis.

Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, Pen(I)) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3' half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the Pen(I) phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

Consecutive use of two multiplex PCR-based assays for simultaneous identification and determination of capsular status of nine common Neisseria meningitidis serogroups associated with invasive disease.

We developed two Neisseria meningitidis multiplex PCR assays to be used consecutively that allow determination of the serogroup and capsular status of serogroup A, B, C, 29E, W135, X, and Y cnl-3/cnl-1-like-containing N. meningitidis isolates by direct analysis of the amplicon size. These assays offer a rapid and simple method of serogrouping N. meningitidis.

Functional analysis of the phospholipase C gene CaPLC1 and two unusual phospholipase C genes, CaPLC2 and CaPLC3, of Candida albicans.

Phospholipases C are known to be important regulators of cellular processes but may also act as virulence factors of pathogenic microbes. At least three genes in the genome of the human-pathogenic fungus Candida albicans encode phospholipases with conserved phospholipase C (Plc) motifs. None of the deduced protein sequences contain N-terminal signal peptides, suggesting that these phospholipases are not secreted. In contrast to its orthologue in Sacharomyces cerevisiae, CaPLC1 seems to be an essential gene. However, a conditional mutant with reduced transcript levels of CaPLC1 had phenotypes similar to Plc1p-deficient mutants in S. cerevisiae, including reduced growth on media causing increased osmotic stress, on media with a non-glucose carbon source, or at elevated or lower temperatures, suggesting that CaPlc1p, like the Plc1p counterpart in S. cerevisiae, may be involved in multiple cellular processes. Furthermore, phenotypic screening of the heterozygous DeltaCaplc1/CaPLC1 mutant showed additional defects in hyphal formation. The loss of CaPLC1 cannot be compensated by two additional PLC genes of C. albicans (CaPLC2 and CaPLC3) encoding two almost identical phospholipases C with no counterpart in S. cerevisiae but containing structural elements found in bacterial phospholipases C. Although the promoter sequences of CaPLC2 and CaPLC3 differed dramatically, the transcriptional pattern of both genes was similar. In contrast to CaPLC1, CaPLC2 and CaPLC3 are not essential. Although Caplc2/3 mutants had reduced abilities to produce hyphae on solid media, these mutants were as virulent as the wild-type in a model of systemic infection. These data suggest that C. albicans contains two different classes of phospholipases C which are involved in cellular processes but which have no specific functions in pathogenicity.

PCR-based assay for detection of Neisseria meningitidis capsular serogroups 29E, X, and Z.

PCR-based assays for the identification of Neisseria meningitidis serogroups 29E, X, and Z by detection of specific regions of the ctrA gene are described. The specificities of these assays were confirmed using serogroups A, B, C, 29E, H, W135, X, Y, and Z and nongroupable meningococcal isolates.

PCR and restriction endonuclease assay for detection of a novel mutation associated with sulfonamide resistance in Neisseria meningitidis.

We identified a previously undocumented mutation in the dihydropteroate synthase (folP) gene associated with Neisseria meningitidis sulfonamide resistance. A PCR-based assay to detect this mutation, which is 100% predictive of sulfonamide resistance, was developed.

Multilocus restriction typing: a tool for Neisseria meningitidis strain discrimination.

Invasive disease-associated strains of Neisseria meningitidis were analysed by multilocus restriction typing (MLRT), which involves the restriction fragment-length polymorphism analysis of PCR products generated from the seven loci of housekeeping genes used in MLST. Several different restriction patterns (alleles) were observed for each of the seven loci examined. Greater allelic variation was observed with the fumC and pgm loci than with the abcZ and adk loci, suggesting that the latter were more conserved. The alleles at each of the seven loci were combined to give an allelic profile or restriction type (RT). A good correlation between RT and serogroup, serotype and serosubtype was observed, as all C 2ap1.2,5 strains were contained in a single RT, as were all but one strain of B 4p1.4. In this study, MLRT proved to be an efficient, effective and relatively inexpensive method for N. meningitidis strain characterization.

Genetic characterization of a phospholipase C gene from Candida albicans: presence of homologous sequences in Candida species other than Candida albicans.

Phospholipase C (PLC) enzymes are essential in regulating several important cellular functions in eukaryotes, including yeasts. In this study, PCR was used to identify a gene encoding PLC activity in Candida albicans, using oligonucleotide primers complementary to sequences encoding highly conserved amino acid regions within the X domains of previously characterized eukaryotic phospholipase C genes. The nucleotide sequence of the C. albicans gene, CAPLC1 (2997 bp), was determined from a recombinant clone containing C. albicans 132A genomic DNA; it encoded a polypeptide of 1099 amino acids with a predicted molecular mass of 124.6 kDa. The deduced amino acid sequence of this polypeptide (CAPLC1) exhibited many of the features common to previously characterized PLCs, including specific X and Y catalytic domains. The CAPLC1 protein also exhibited several unique features, including a novel stretch of 18-19 amino acid residues within the X domain and an unusually long N-terminus which did not contain a recognizable EF-hand Ca(2+)-binding domain. An overall amino acid homology of more than 27% with PLCs previously characterized from Saccharomyces cerevisiae and Schizosaccharomyces pombe suggested that the CAPLC1 protein is a delta-form of phosphoinositide-specific PLC (PI-PLC). PLC activity was detected in cell-free extracts of both yeast and hyphal forms of C. albicans 132A following 7 h and 24 h growth using the PLC-specific substrate p-nitrophenylphosphorylcholine (p-NPPC). In addition, CAPLC1 mRNA was detected by reverse transcriptase PCR in both yeast and hyphal forms of C. albicans 132A at the same time intervals. Expression of CAPLC1 activity was also detected in extracts of Escherichia coli DH5 alpha harbouring plasmids which contained portions of the CAPLC1 gene lacking sequences encoding part of the N-terminus. Southern hybridization and PCR analyses revealed that all C. albicans and Candida dubliniensis isolates examined possessed sequences homologous to CAPLC1. Sequences related to CAPLC1 were detected in some but not all isolates of Candida tropicalis, Candida glabrata and Candida parapsilosis tested, but not in the isolates of Candida krusei, Candida kefyr, Candida guillermondii and Candida lusitaniae examined. This paper reports the first description of the cloning and sequencing of a PLC gene from a pathogenic yeast species.