Renin-angiotensin system gene polymorphisms and endometrial cancer.

Endometrial cancer (EC) is the most common gynaecological malignancy and its incidence is increasing. Dysregulation of the endometrial renin-angiotensin system (RAS) could predispose to EC; therefore, we studied the prevalence of RAS single nucleotide polymorphisms (SNPs) in Australian women with EC. SNPs assessed were AGT M235T (rs699); AGTR1 A1166C (rs5186); ACE A240T and T93C (rs4291, rs4292) and ATP6AP2 (rs2968915). They were identified using TaqMan SNP Genotyping Assays. The C allele of the AGTR1 SNP (rs5186) was more prevalent in women with EC (odds ratio (OR) 1.7, 95% confidence interval (CI) (1.2-2.3), P=0.002). The CC genotype of this SNP is associated with upregulation of the angiotensin II type 1 receptor (AGTR1). The G allele of AGT rs699, which is associated with higher angiotensinogen (AGT) levels, was less prevalent in women with EC (OR 0.54, 95% CI (0.39-0.74), P<0.001) compared with controls. AGT and AGT formed by removal of angiotensin I (des(Ang I)AGT) are both anti-angiogenic. In women with EC who had had hormone replacement therapy (HRT), the prevalence of the AGTR1 SNP (rs5186) and the ACE SNPs (rs4291 and rs4292) was greater than in women who had no record of HRT; SNP rs4291 is associated with increased plasma ACE activity. These data suggest there is an interaction between genotype, oestrogen replacement therapy and EC. In conclusion, the prevalence of two SNPs that enhance RAS activity was different in women with EC compared with healthy controls. These genetic factors may interact with obesity and hyperoestrogenism, predisposing ageing, obese women to EC.

The prevalence of unique SNPs in the renin-angiotensin system highlights the need for pharmacogenetics in Indigenous Australians.

Genetic differences between ethnic populations affect susceptibility to disease and efficacy of drugs. This study examined and compared the prevalence of single nucleotide polymorphisms (SNPs) in genes of the renin-angiotensin system (RAS) in a desert community of Indigenous Australians and in non-Indigenous Australians. The polymorphisms were angiotensinogen, AGT G-217A (rs5049); AGT G+174A (rs4762); Angiotensin II type 1 receptor, AGTR1 A+1166C (rs5186); angiotensin converting enzyme, ACE A-240T (rs4291), ACE T-93C (rs4292); renin, REN T+1142C (rs5706). They were measured using allelic discrimination assays. The prevalence of REN T+1142C SNP was similar in the two populations; 99% were homozygous for the T allele. All other SNPs were differently distributed between the two populations (P < 0.0001). In non-Indigenous Australians, the A allele at position 204 of ACE rs4291 was prevalent (61.8%) whereas in the Indigenous Australians the A allele was less prevalent (28%). For rs4292, the C allele had a prevalence of 37.9% in non-Indigenous Australians but in Indigenous Australians the prevalence was only 1%. No Indigenous individuals were homozygous for the C allele of AGTR1 (rs5186). Thus the prevalence of RAS SNPs in this Indigenous Australian desert community was different from non-Indigenous Australians as was the prevalence of cytokine SNPs (as shown in a previous study). These differences may affect susceptibility to chronic renal and cardiovascular disease and may alter the efficacy of drugs used to inhibit the RAS. These studies highlight the need to study the pharmacogenetics of drug absorption, distribution, metabolism and excretion in Indigenous Australians for safe prescribing guidelines.

Effects of maternal inflammation and exposure to cigarette smoke on birth weight and delivery of preterm babies in a cohort of indigenous Australian women.

Sudden infant death syndrome (SIDS), neonatal deaths, and deaths from infection are higher among Indigenous Australians. This study aimed to determine the effects of inflammatory responses and exposure to cigarette smoke, two important factors associated with sudden death in infancy, on preterm birth, and birth weight in a cohort of Indigenous mothers. Indigenous Australian women (n = 131) were recruited as part of a longitudinal study while attending antenatal care clinics during pregnancy; blood samples were collected up to three times in pregnancy. Serum cotinine, indicating exposure to cigarette smoke, was detected in 50.4% of mothers. Compared with non-Indigenous women, the cohort had 10 times the prevalence of antibodies to Helicobacter pylori (33 vs. 3%). Levels of immunoglobulin G, antibodies to H. pylori, and C-reactive protein (CRP) were all inversely correlated with gestational age (P < 0.05). CRP levels were positively associated with maternal body mass index (BMI; ρ = 0.449, P = 0.001). The effects of cigarette smoke (cotinine) and inflammation (CRP) were assessed in relation to risk factors for SIDS: gestational age at delivery and birth weight. Serum cotinine levels were negatively associated with birth weight (ρ = -0.37, P < 0.001), this correlation held true for both male (ρ = -0.39, P = 0.002) and female (ρ = -0.30, P = 0.017) infants. Cotinine was negatively associated with gestational age at delivery (ρ = -0.199, P = 0.023). When assessed by fetal sex, this was significant only for males (ρ = -0.327, P = 0.011). CRP was negatively associated with gestational age at delivery for female infants (ρ = -0.46, P < 0.001). In contrast, maternal BMI was significantly correlated with birth weight. These data highlight the importance of putting programs in place to reduce cigarette smoke exposure in pregnancy and to treat women with chronic infections such as H. pylori to improve pregnancy outcomes and decrease risk factors for sudden death in infancy.

Effects of gender, cytokine gene polymorphisms and environmental factors on inflammatory responses.

Previous studies have indicated that cytokine gene polymorphisms of Indigenous Australians were predominantly associated with strong pro-inflammatory responses. We tested the hypothesis that cells of donors with genetic profiles of inflammatory cytokine single nucleotide polymorphisms (SNPs) similar to Indigenous Australians produce higher pro-inflammatory responses. PBMCs from 14 donors with genetic profiles for a high risk of strong pro-inflammatory responses and 14 with low-risk profiles were stimulated with endotoxin and effects of gender, IFN-γ, cigarette smoke extract (CSE) and testosterone on cytokine responses analysed. Cytokines were calculated from standard curves (Luminex 2.3 software). No significant differences were associated with SNP profile alone. Lower pro-inflammatory responses were observed for cells from males with low- or high-risk profiles. For cells from females with high-risk profiles, anti-inflammatory IL-10 responses were significantly reduced. There was no effect of testosterone levels on responses from males. For females, results from IFN-γ-treated cells showed positive correlations between testosterone levels and IL-1ß responses to endotoxin for both risk groups and TNF-α for the high-risk group. If interactions observed among CSE, IFN-γ, genetic background and testosterone reflect those in vivo, these might contribute to increased incidences of hospitalisations for infectious diseases among Indigenous women.

The male excess in sudden infant deaths.

The peak age at which sudden infant death syndrome (SIDS) occurs corresponds to the developmental period in which infants are dependent on their innate responses to infection. There is a growing body of evidence indicating that dysregulation of inflammatory responses might contribute to the physiological changes leading to these sudden deaths. This study examined the effects of three important risk factors for SIDS on inflammatory responses: cigarette smoke, virus infection and male sex. Cytokine responses of peripheral monocytic blood cells of healthy, non-smoking males and females to endotoxin were measured. Surrogates for virus infection or cigarette smoke were assessed using IFN-γ or water-soluble cigarette smoke extract (CSE). For most conditions, cells from males had lower pro-inflammatory cytokine responses than those of females. An opposite trend was observed for IL-10. Significantly lower levels of some cytokines were noted for cells from male donors exposed to CSE. In females, there were significant correlations between testosterone levels and levels of pro-inflammatory cytokines, but none for males. Testosterone levels in females correspond to those among male infants in the age range at greatest risk of SIDS. The effects of the testosterone surge in male infants need to be examined in relation to changes in cortisol levels that occur during the same period of infant development.

Induction of inflammatory responses from THP-1 cells by cell-free filtrates from clinical isolates of Alloiococcus otitidis.

In our model system using the THP-1 monocytic cell line, whole heat-killed cells of Alloiococcus otitidis elicited several pro-inflammatory cytokines identified in ear effusions of children with otitis media (OM). Levels of these cytokines were equivalent to or greater than those elicited by a standard Gram-positive otopathogen, Streptococcus pneumoniae. The current study examined the hypothesis that extracellular material produced by A. otitidis might also contribute to the inflammatory responses in OM. Cell-free culture filtrates of recent A. otitidis isolates (n = 39) were tested for induction of pro-inflammatory cytokines from THP-1 cells primed with IFN-γ. The highest responses were from IL-8 followed by IL-1ß, and the lowest from IL-6. Filtrates from nine isolates were treated with lysozyme or proteinase K to assess the nature of the extracellular stimulants. Peptidoglycan was not a major component eliciting the responses. There was no correlation between colony type or ß-haemolysin production. Proteinase K treatment indicated extracellular proteins might induce the inflammatory responses, particularly the 70-75 ku band. Further studies on the role of the extracellular proteins of A. otitidis and cytokine responses in pathogenesis of ear infections are needed.

In vitro inflammatory responses elicited by isolates of Alloiococcus otitidis obtained from children with otitis media with effusion.

Alloiococcus otitidis is usually detected in children with otitis media (OM) by PCR as it is not often detected by routine culture. Our improved method for its isolation obtained A. otitidis from nearly 50% of 78 children with OM with effusion. The role of A. otitidis in pathogenesis of OM is unclear. This study tested two hypothesis: (1) that fresh isolates of A. otitidis would elicit pro-inflammatory cytokines from THP-1 monocytic cells equivalent to those induced by Streptococcus pneumoniae; (2) priming THP-1 cells with interferon-gamma (IFN-γ) a surrogate for virus infection, would enhance pro-inflammatory responses. Recent clinical isolates of A. otitidis, S. pneumoniae (ATCC 49619) and a blood culture isolate of S. pneumoniae (SP2) were used in the assays. Cytokines were quantified by BioRad bead assay and Luminex 200. IFN-γ priming enhanced cytokine responses. S. pneumoniae ATCC 49619 induced lower responses than SP2 for IL-1ß, IL-6, TNF-α. A. otitidis LW 27 elicited higher IL-1ß and TNF-α responses than either pneumococcal isolate. Small green colony types of A. otitidis induced higher responses than large white colony types for IL-8 and IL-1ß. The hypothesis that A. otitidis elicits cytokines observed in middle ear effusions was supported; the need to use recent clinical isolates in studies of pathogenesis was highlighted.

Cytokine gene polymorphism among Indigenous Australians.

The health profile of Indigenous Australians is characterised by high rates of classic 'lifestyle' diseases. Potential roles of inflammation in pathophysiology of these diseases requires investigation. It is not clear if genetic regulation of inflammation in Indigenous Australians is similar to other populations. This study characterised frequencies of single nucleotide polymorphisms (SNPs) for eight cytokine genes for 100 individuals from a remote Indigenous Australian community and assessed novel genetic variants in four cytokine genes. We used a commercially-available allelic discrimination assay for SNP genotyping; re-sequencing was undertaken by standard Sanger sequencing methodologies for 26 samples. Frequencies of cytokine gene SNPs differed significantly from the Caucasian population (P < 0.001-0.044). Twenty-five novel variants were identified across four re-sequenced genes; frequencies ranged from <5% to 100%. Genotype frequencies observed in Indigenous Australians did not consistently resemble reported HapMap frequencies in Northern and Western European populations, Yoruba Nigerian or Han Chinese. Our findings indicate Indigenous Australians might have an inherited propensity for strong inflammatory responses. Preliminary evidence of novel genetic variants highlights the need to catalogue the extent of genetic variation in specific population groups. Improved understanding of differences in genetic variation between specific population groups could assist in assessment of risk for lifestyle diseases.

Comparison of cytokine gene polymorphisms among Greek patients with invasive meningococcal disease or viral meningitis.

High levels of pro-inflammatory cytokines are implicated in the severity of invasive meningococcal disease (IMD) and viral meningitis (VM). This study compared single-nucleotide polymorphisms (SNPs) in pro- and anti-inflammatory cytokine genes among patients with VM or IMD. Patient DNA samples were prepared by the National Meningitis Reference Laboratory in Athens: n=98 for IMD and n=53 for VM. The results for both patient groups were compared with data published for healthy Greek control data. Real-time PCR was used to assess the interleukin (IL) gene SNPs IL6 G-174C, IL1B C-511T, IL1RN T+2018C, IL10 G-1082A and IL8 A-251T and the tumour necrosis factor α (TNF-α) SNP TNFA G-308A. Differences were compared by Fisher's exact test. The genotype for high IL-6 responses was predominant among IMD (51%, P=0.0008) and VM (74.5%, P<0.0001) patients compared with the controls (31%). The genotype associated with high TNF-α responses was 5% among controls and lower for IMD (1.1%, P=0.0014) and VM (0%, P=0.052). There was no difference for IL-8 SNPs between controls and IMD (P=0.162), but the difference was significant for VM (P=0.0025). IL-6 (P=0.024) and IL-8 (P=0.00004) SNPs differed between IMD and VM. Reports on associations between IL-8 SNPs and cytokine responses differ. Because of its role in neutrophil attraction, differences in frequencies of the IL-8 SNP for IMD and VM require further investigation.

Isolation of Alloiococcus otitidis from Indigenous and non-Indigenous Australian children with chronic otitis media with effusion.

During the last decade Alloiococcus otitidis has been identified in specimens from patients with chronic otitis media with effusion. Whereas most of those studies employed molecular techniques, we used minor modifications of conventional microbiological methods to isolate and identify A. otitidis in samples obtained from 20/50 (40%) children referred for myringotomy. Alloiococcus otitidis was isolated from 10/22 (45%) Indigenous and 10/28 (36%) non-Indigenous children. This is the first report of isolation of A. otitidis from Australian children with chronic otitis media. All isolates were sensitive to penicillin, but 14/20 (70%) of the isolates were resistant or partially resistant to erythromycin as assessed by the E-test.

IL6 G-174C associated with sudden infant death syndrome in a Caucasian Australian cohort.

The aims of this study were to analyze IL6 G-174C in relation to high interleukin (IL)-6 concentrations found in some sudden infant death syndrome (SIDS) infants, and to assess the effects of IL6 G-174C, smoking status, and gender on IL-6 responses. SIDS infants, parents of SIDS infants, and populations with high (Aboriginal Australian), medium (Caucasian) or low (Bangladeshi) SIDS incidences were genotyped. Leukocytes were stimulated in vitro with endotoxin and IL-6 responses were assessed in relation to IL6 G-174C genotype, smoking status, and gender. The study findings showed that GG genotype, associated with high IL-6 responses, was predominant among Australian SIDS infants (58%) compared with control subjects (38%, p = 0.02), as well as Bangladeshis (94%) and Aboriginal Australians (88%) compared with Caucasians (42%, p < 0.01). GC smokers had higher median IL-6 responses (8.4 ng/ml(-1)) than GG (3.5 ng/ml(-1), p = 0.01) or CC smokers (2.4 ng/ml(-1), p < 0.01). GG nonsmokers had higher median IL-6 responses (4.9 ng/ml(-1)) than GG smokers (p < 0.05). Gender did not affect IL-6 responses. In conclusion, an association between IL6 G-174C and Australian SIDS infants was observed. IL6 G-174C alone cannot explain observed differences in the incidence of SIDS in the Bangladeshi and Aboriginal Australian populations. Further investigations are needed on interactions between smoking and gene polymorphisms in relation to proinflammatory responses implicated in SIDS.

Cytokine responses and sudden infant death syndrome: genetic, developmental, and environmental risk factors.

Despite the success of the campaigns to reduce the risk of sudden infant death syndrome (SIDS), it still remains the major cause of postneonatal mortality. The incidence of SIDS is higher among ethnic groups in which there are also high incidences of serious infectious diseases. The risk factors for SIDS parallel those for susceptibility to infection, and recent data have provided evidence to support the mathematical model of the common bacterial toxin hypothesis. One current hypothesis for the etiology of SIDS is that the deaths are a result of overwhelming proinflammatory responses to bacterial toxins; as in inflammatory responses to sepsis, cytokines, induced by bacterial toxins, cause physiological changes leading to death. The genetic, developmental, and environmental risk factors for SIDS are reviewed in relation to colonization by potentially harmful bacteria and the inflammatory responses induced in the nonimmune infant to microorganisms or their products.

Ethnicity, infection and sudden infant death syndrome.

Epidemiological studies found the incidence of SIDS among Indigenous groups such as Aboriginal Australians, New Zealand Maoris and Native Americans were significantly higher than those for non-Indigenous groups within the same countries. Among other groups such as Asian families in Britain, the incidence of SIDS has been lower than among groups of European origin. Cultural and childrearing practices as well as socio-economic factors have been proposed to explain the greater risk of SIDS among Indigenous peoples; however, there are no definitive data to account for the differences observed. We addressed the differences among ethnic groups in relation to susceptibility to infection because there is evidence from studies of populations of European origin that infectious agents, particularly toxigenic bacteria might trigger the events leading to SIDS. The risk factors for SIDS parallel those for susceptibility to infections in infants, particularly respiratory tract infections which are also major health problems among Indigenous groups. Many of the risk factors identified in epidemiological studies of SIDS could affect three stages in the infectious process: (1) frequency or density of colonisation by the toxigenic species implicated in SIDS; (2) induction of temperature-sensitive toxins; (3) modulation of the inflammatory responses to infection or toxins. In this review we compare genetic, developmental and environmental risk factors for SIDS in ethnic groups with different incidences of SIDS: low (Asians in Britain); moderate (European/Caucasian); high (Aboriginal Australian). Our findings indicate: (1) the major difference was high levels of exposure to cigarette smoke among infants in the high risk groups; (2) cigarette smoke significantly reduced the anti-inflammatory cytokine interleukin-10 responses which control pro-inflammatory responses implicated in SIDS; (3) the most significant effect of cigarette smoke on reduction of IL-10 responses was observed for donors with a single nucleotide polymorphism for the IL-10 gene that is predominant among both Asian and Aboriginal populations. If genetic makeup were a major factor for susceptibility to SIDS, the incidence of these deaths should be similar for both populations. They are, however, significantly different and most likely reflect differences in maternal smoking which could affect frequency and density of colonisation of infants by potentially pathogenic bacteria and induction and control of inflammatory responses.

Interleukin-10 and sudden infant death syndrome.

Uncontrolled pro-inflammatory responses to infections or bacterial toxins have been suggested to play a role in triggering the physiological events leading to sudden infant death syndrome (SIDS). We tested the hypothesis that these uncontrolled responses might be due to interactions between the gene polymorphisms inducing low levels of IL-10 and exposure to cigarette smoke. In vitro, the IL-10 (G-1082A) polymorphism was associated with low IL-10 levels and the -1082G allele was associated with high levels. The first objective was to assess the distribution of this polymorphism among SIDS infants, parents of SIDS infants and controls, and two ethnic groups: Aboriginal Australians who have a high incidence of SIDS; and Bangladeshis who in Britain have a low incidence of SIDS compared with Europeans. The second objective was to assess effects of human recombinant IL-10 on interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) responses of human leukocytes to staphylococcal toxins implicated in SIDS. The third objective was to assess IL-10 responses to endotoxin and toxic shock syndrome toxin (TSST) from leukocytes of smokers and non-smokers in relation to the IL-10 (G-1082A) polymorphism. There were major differences in the distributions of these polymorphisms between Europeans and Bangladeshis (p=0.00) and between Europeans and Aboriginal Australians (p=0.00); however, they were similar for the Bangladeshi and Aboriginal Australian subjects. There were no significant differences in the distribution of these polymorphisms among SIDS infants or parents of SIDS infants compared to control groups. IL-10 significantly reduced IL-6 and TNF-alpha responses to TSST and staphylococcal enterotoxins A and C. At 50 ng ml(-1), IL-10 significantly increased TNF-alpha but not IL-6 responses to TSST and enterotoxin A. Although IL-10 responses to endotoxin were lower from leukocytes of smokers who were homozygous for the G allele, the differences were not significant; however, significantly lower IL-10 responses were found for smokers who were homozygous for the A allele (p=0.01) and heterozygotes (p=0.04). The pooled data found smokers had significantly lower levels of IL-10 responses to TSST, but there were no significant differences for smokers compared with non-smokers for the three genotypes. The high incidence of SIDS and serious respiratory infections among Aboriginal Australian infants and the low incidence of these conditions among Bangladeshi infants might be explained in part by our findings of differences in IL-10 responses between smokers and non-smokers. The lowest levels of IL-10 responses were observed among smokers who were homozygous for the A allele which is most prevalent among the Aboriginal Australians (83%) and Bangladeshis (84%). The major difference between the risk factors for SIDS in these two groups is the level of exposure of infants to cigarette smoke associated with maternal smoking.

Interleukin 1-beta responses to bacterial toxins and sudden infant death syndrome.

We tested the hypothesis that significantly higher IL-1beta responses to toxic shock syndrome toxin (TSST) noted for parents of sudden infant death syndrome (SIDS) infants might be due in part to genetic factors such as the IL-1beta (C-511T) and IL-1RN (T+2018C) single nucleotide polymorphisms (SNP). The first objective was to assess the distribution of these polymorphisms among SIDS infants, parents of SIDS infants and controls, and two ethnic groups: Aboriginal Australians who have a high incidence of SIDS; and Bangladeshis who in Britain have a low incidence of SIDS compared with Europeans. The second objective was to assess IL-1beta responses to endotoxin and toxic shock syndrome toxin (TSST) from leukocytes of smokers and non-smokers in relation to these polymorphisms. There were major differences in the distributions of the IL-1beta (C-511T) SNP between Europeans and Bangladeshis (p=0.00) and between Europeans and Aboriginal Australians (p=0.00); however, they were similar for the Bangladeshi and Aboriginal Australian subjects. The allele frequency distribution of the IL-1RN (T+2018C) SNP for the Aboriginal Australians was statistically different from the European group (p=0.00), but it was not different from the Bangladeshi group (p=0.09). Compared with controls of European origin, there were no significant differences in the distribution of these polymorphisms among SIDS infants or parents of SIDS infants. For the IL-1beta (C-511T) SNP, the highest IL-1beta responses to endotoxin were obtained with leukocytes of non-smokers with the heterozygous CT genotype. Smokers had significantly lower levels of IL-1beta in response to endotoxin (p=0.01) and these differences were significant for donors with the wild type CC (p=0.00) and CT (p=0.03) genotypes. Similar patterns were observed for IL-1beta responses to TSST, but the differences were not significant. For the IL-1RN (T+2018C) SNP, the highest IL-1beta responses to endotoxin were obtained with leukocytes from non-smoker donors with the wildtype TT genotype and significantly lower responses were found with leukocytes from donors with the TC genotype (p=0.02). The responses of smokers were lower but the differences were significant only for donors with the TT genotype (p=0.00). Similar patterns were observed for IL-1beta responses to TSST, but the differences were not significant. IL-1beta responses to both endotoxin and TSST were increased for the small number of smokers with the TT genotype of the IL-1beta (C-511T) SNP. The TT genotype of the IL-1beta (C-511T) was found predominantly among Aboriginal Australian and Bangladeshi individuals but only a small proportion of Europeans. Smokers with the AA genotype of the IL-10 (G-1082A) SNP which is found predominantly among these two groups had significantly lower levels of IL-10 responses. If cigarette smoke enhances pro-inflammatory responses and reduces anti-inflammatory responses in individuals with these genotypes, this might partly explain the increased susceptibility of Aboriginal Australian infants to infections and SIDS.

Neisseria meningitidis, Neisseria lactamica and Moraxella catarrhalis share cross-reactive carbohydrate antigens.

Carriage of commensal bacteria species is associated with the development of natural immunity to meningococcal disease, with lipo-oligosaccharides (LOS) of meningococci being one of the main virulence factors associated with severity of meningococcal disease. Meningococcal reference strains and isolates from the commensal species Neisseria lactamica and Moraxella catarrhalis were assessed for the presence of cross-reactive glycoconjugate antigens. Binding of human blood group antibodies of the P and Ii system to meningococcal immunotype reference strains were in accordance with the presence of known LOS carbohydrate structures. Binding studies with meningococcal immunotyping antibodies and blood group phenotyping antibodies to N. lactamica strains from different European countries showed, that a greater number of isolates obtained from native Greek and Scottish adults and children bound anti-meningococcal L(3, 7, 9) immunotyping (P < 0.001), pK (P = 0.035) and paragloboside (P < 0.001) blood group typing antibodies compared to isolates obtained from children of Russian immigrants in Greece. A greater number of M. catarrhalis strains isolated from children in Scotland bound anti-L(3, 7, 9) antibodies (38.2%) compared to strains isolated from adults (22.2%) (P = 0.017). These findings provide evidence that blood group like glycoconjugate antigens found on the commensal species N. lactamica and M. catarrhalis might be involved in the development of natural immunity to meningococcal endotoxins during childhood, and might be exploited as anti-meningococcal vaccine candidates.

Carriage of Neisseria meningitidis and Neisseria lactamica in northern Greece.

In response to an increase in the number of cases of invasive meningococcal disease (IMD) in northern regions of Greece, a survey was carried out to determine if there was an increase in carriage of Neisseria meningitidis, particularly in areas where there have been increases in immigrant populations from neighbouring countries. The second objective was to determine if there was an increase in the serogroup C:2a:P1.5,2 a phenotype associated with recent outbreaks or changes in antibiotic sensitivities. As carriage of Neisseria lactamica is associated with development of natural immunity to IMD, the third objective was to determine the carriage rate of N. lactamica in this population. Among 3167 individuals tested, meningococci were isolated from 334 (10.5%). Compared with our previous studies, the proportion of meningococcal carriers was significantly increased among children in secondary education (11.3%) (chi2=9.67, P<0.005) and military recruits (37.4%) (chi2=21.11, P<0.000). Only 5/334 (1.5%) isolates expressed the phenotype associated with the increase in IMD in Greece. N. lactamica was isolated from 146/3167 (4.6%) participants. It was isolated from 71/987 (7.2%) children attending primary or nursery schools; however, the highest proportion of carriers (11.3%) was found in the boarding school for young Albanian men. In the 21-59-year age range, the majority of N. lactamica isolates (22/25, 88%) were from women, probably due to closer or more prolonged contact with children in the primary school age range. Smoking was significantly associated with isolation of meningococci from men but not from women. Penicillin-insensitive strains (25/334, 7.5%) were identified in all four regions examined; the majority (14/25, 56%) were obtained from military personnel. We conclude that there was a higher proportion of carriers in the population of northern Greece; however, the increase in carriage rate was not associated with the influx of immigrants from neighbouring countries, and there was not a higher incidence of the C:2a:P1.5,2 strain responsible for increased disease activity in Greece in either the immigrant or local populations.

Evaluation of non-culture diagnosis of invasive meningococcal disease by polymerase chain reaction (PCR).

Antibiotic treatment prior to transport or admission to hospital has reduced the proportion of cases of invasive meningococcal disease (IMD) from which Neisseria meningitidis can be isolated by standard microbiological techniques. Identification of meningococci by polymerase chain reaction (PCR) was assessed in relation to microbiological diagnosis for cases over a 4-year period between 1998 and 2001. A screening assay for the IS1106 gene was used to detect meningococcal DNA and five additional assays for siaD and orf-2 genes were performed to determine the serogroup. PCR results were compared with results of bacteriological culture, other laboratory test results and clinical data. The sensitivity of the PCR assay for culture-confirmed cases was 98.5%. The specificity of the assay was 96% based on test results for patients from whom other bacteria were isolated, children with viral meningitis and afebrile negative controls. The siaD B/C/W-135 and Y as well as the orf-2 gene for serogroup A PCR assays were able to determine the serogroup for 75.2% of cases that were positive by PCR screening assay. When isolates from patients with IMD were tested by both agglutination and PCR, the results agreed in all cases. PCR is a useful tool for diagnosis of IMD when Gram stain and culture tests are negative due to antibiotic treatment prior to collection of samples for microbiological analyses.

Comparison of Moraxella catarrhalis isolates from children and adults for growth on modified New York City medium and potential virulence factors.

Initial studies found that Moraxella catarrhalis isolates from adults that grew on modified New York City medium (MNYC(+)) that contained antibiotics selective for pathogenic neisseriae differed from strains that did not grow on this medium (MNYC(-)) in their potential virulence properties. It was predicted that higher usage of antibiotics to treat respiratory illness in children might result in higher proportions of MNYC(+) isolates if antibiotics were an important selective pressure for this phenotype. Two of 100 adult isolates (2 %) were MNYC(+), compared to 88 of 88 isolates (100 %) from children (P = 0.000). MNYC(+) strains were serum-resistant and bound in higher numbers to HEp-2 cells that were infected with respiratory syncytial virus (RSV). Endotoxin from an MNYC(+) isolate induced significantly higher pro-inflammatory response levels than endotoxin from an MNYC(-) strain. MNYC(-) adult isolates expressed haemagglutinins and bound in lower numbers to RSV-infected cells, but serum resistance was variable. All isolates from children were MNYC(+), serum-resistant and bound in greater numbers to RSV-infected cells. These results indicate that both RSV infection and antibiotic usage select for the MNYC(+) phenotype.