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1.  α-2,3-Sialyltransferase Expression Level Impacts the Kinetics of Lipooligosaccharide Sialylation, Complement Resistance, and the Ability of Neisseria gonorrhoeae to Colonize the Murine Genital Tract 
mBio  2015;6(1):e02465-14.
ABSTRACT 
Neisseria meningitidis and Neisseria gonorrhoeae modify the terminal lacto-N-neotetraose moiety of their lipooligosaccharide (LOS) with sialic acid. N. gonorrhoeae LOS sialylation blocks killing by complement, which is mediated at least in part by enhanced binding of the complement inhibitor factor H (FH). The role of LOS sialylation in resistance of N. meningitidis to serum killing is less well defined. Sialylation in each species is catalyzed by the enzyme LOS α-2,3-sialyltransferase (Lst). Previous studies have shown increased Lst activity in N. gonorrhoeae compared to N. meningitidis due to an ~5-fold increase in lst transcription. Using isogenic N. gonorrhoeae strains engineered to express gonococcal lst from either the N. gonorrhoeae or N. meningitidis lst promoter, we show that decreased expression of lst (driven by the N. meningitidis promoter) reduced LOS sialylation as determined by less incorporation of tritium-labeled cytidine monophospho-N-acetylneuraminic acid (CMP-NANA; the donor molecule for sialic acid). Diminished LOS sialylation resulted in reduced rates of FH binding and increased pathway activation compared to N. gonorrhoeae promoter-driven lst expression. The N. meningitidis lst promoter generated sufficient Lst to sialylate N. gonorrhoeae LOS in vivo, and the level of sialylation after 24 h in the mouse genital tract was sufficient to mediate resistance to human serum ex vivo. Despite demonstrable LOS sialylation in vivo, gonococci harboring the N. meningitidis lst promoter were outcompeted by those with the N. gonorrhoeae lst promoter during coinfection of the vaginal tract of estradiol-treated mice. These data highlight the importance of high lst expression levels for gonococcal pathogenesis.
IMPORTANCE  Neisseria gonorrhoeae has become resistant to nearly every therapeutic antibiotic used and is listed as an “urgent threat” by the Centers for Disease Control and Prevention. Novel therapies are needed to combat drug-resistant N. gonorrhoeae. Gonococci express an α-2,3-sialyltransferase (Lst) that can scavenge sialic acid from the host and use it to modify lipooligosaccharide (LOS). Sialylation of gonococcal LOS converts serum-sensitive strains to serum resistance, decreases antibody binding, and combats killing by neutrophils and antimicrobial peptides. Mutant N. gonorrhoeae that lack Lst (cannot sialylate LOS) are attenuated in a mouse model. Lst expression levels differ among N. gonorrhoeae strains, and N. gonorrhoeae typically expresses more Lst than Neisseria meningitidis. Here we examined the significance of differential lst expression levels and determined that the level of LOS sialylation is critical to the ability of N. gonorrhoeae to combat the immune system and survive in an animal model. LOS sialylation may be an ideal target for novel therapies.
IMPORTANCE 
Neisseria gonorrhoeae has become resistant to nearly every therapeutic antibiotic used and is listed as an “urgent threat” by the Centers for Disease Control and Prevention. Novel therapies are needed to combat drug-resistant N. gonorrhoeae. Gonococci express an α-2,3-sialyltransferase (Lst) that can scavenge sialic acid from the host and use it to modify lipooligosaccharide (LOS). Sialylation of gonococcal LOS converts serum-sensitive strains to serum resistance, decreases antibody binding, and combats killing by neutrophils and antimicrobial peptides. Mutant N. gonorrhoeae that lack Lst (cannot sialylate LOS) are attenuated in a mouse model. Lst expression levels differ among N. gonorrhoeae strains, and N. gonorrhoeae typically expresses more Lst than Neisseria meningitidis. Here we examined the significance of differential lst expression levels and determined that the level of LOS sialylation is critical to the ability of N. gonorrhoeae to combat the immune system and survive in an animal model. LOS sialylation may be an ideal target for novel therapies.
doi:10.1128/mBio.02465-14
PMCID: PMC4324315  PMID: 25650401
2.  Phenotypic and genetic characterisation of bacterial sexually transmitted infections in Bissau, Guinea-Bissau, West Africa: a prospective cohort study 
BMJ Open  2012;2(2):e000636.
Background
Knowledge regarding characteristics and transmission of Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium and antibiotic resistance in N gonorrhoeae in Guinea-Bissau, West Africa, is entirely lacking.
Objectives
To characterise N gonorrhoeae, C trachomatis and M genitalium samples from Guinea-Bissau and to define bacterial populations, possible transmission chains and for N gonorrhoeae spread of antibiotic-resistant isolates.
Design
Prospective cohort study.
Setting
Two sexual health and family planning clinics, Bissau, Guinea-Bissau.
Participants
Positive samples from 711 women and 27 men.
Material and methods
Positive samples for N gonorrhoeae (n=31), C trachomatis (n=60) and M genitalium (n=30) were examined. The gonococcal isolates were characterised with antibiograms, serovar determination and N gonorrhoeae multiantigen sequence typing (NG-MAST). The C trachomatis ompA gene and the M genitalium mgpB gene were sequenced, and phylogenetic analyses were performed.
Results
For N gonorrhoeae, the levels of resistance (intermediate susceptibility) to ciprofloxacin, erythromycin, rifampicin, ampicillin, tetracycline, penicillin G and cefuroxime were 10% (0%), 6% (10%), 13% (10%), 68% (0%), 74% (0%), 68% (16%) and 0% (84%), respectively. All isolates were susceptible to cefixime, ceftriaxone, spectinomycin and azithromycin, and the minimum inhibitory concentrations of kanamycin (range: 8–32 mg/l) and gentamicin (range: 0.75–6 mg/l) were low (no resistance breakpoints exist for these antimicrobials). 19 NG-MAST sequence types (STs) (84% novel STs) were identified. Phylogenetic analysis of the C trachomatis ompA gene revealed genovar G as most prevalent (37%), followed by genovar D (19%). 23 mgpB STs were found among the M genitalium isolates, and 67% of isolates had unique STs.
Conclusions
The diversity among the sexually transmitted infection (STI) pathogens may be associated with suboptimal diagnostics, contact tracing, case reporting and epidemiological surveillance. In Guinea-Bissau, additional STI studies are vital to estimate the STI burden and form the basis for a national sexual health strategy for prevention, diagnosis and surveillance of STIs.
Article summary
Article focus
Knowledge regarding characteristics and transmission of Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium and antibiotic resistance in N gonorrhoeae in Guinea-Bissau, West Africa, is entirely lacking.
We aimed to phenotypically and genetically characterise N gonorrhoeae, and genetically characterise C trachomatis and M genitalium samples from women attending two sexual health clinics in Bissau, Guinea-Bissau and to define the bacterial populations, possible transmission chains and for N gonorrhoeae also to define the presence and spread of antibiotic-resistant isolates from women and additionally a group of symptomatic men.
Key messages
In Guinea-Bissau, N gonorrhoeae isolates displayed high level of resistance to traditional gonorrhoea antimicrobials but have remained susceptible to extended-spectrum cephalosporins, spectinomycin and azithromycin.
Genovar G, D and F were the most prevalent C trachomatis genovars, the usually most common genovar among heterosexuals, that is, genovar E, was rare.
The diversity among the bacterial STI pathogens may be associated with suboptimal diagnostics, contact tracing, case reporting and epidemiological surveillance. Additional studies are vital to estimate the STI burden and form the basis for a national sexual health strategy for prevention, diagnosis and surveillance.
Strengths and limitations of this study
This is the first time N gonorrhoeae, C trachomatis and M genitalium samples from Bissau, Guinea-Bissau, have been phenotypically and genetically characterised to define the bacterial populations, possible transmission chains and antibiotic resistance in N gonorrhoeae.
The study sample was relatively small, and in this setting, it is difficult to perform appropriate sample transportation and storage, as well as optimised diagnostics, which may have resulted in that true-positive samples were lost.
doi:10.1136/bmjopen-2011-000636
PMCID: PMC3329603  PMID: 22436137
3.  Byssinosis: a follow-up study of workers exposed to fine grade cotton dust 
Thorax  1972;27(4):459-462.
A follow-up study was made in a group of cotton workers processing fine grade cotton fibres. Respiratory symptoms and ventilatory capacity were first recorded in 1962 and later in 1971. In 1962, 20 men and 23 women were examined whereas in 1971 only 15 of the men and 13 of the women were still in the mill. In 1962 no case of byssinosis was recorded. The prevalence of chronic cough and chronic bronchitis was higher among men than women. In 1971 the incidence of byssinosis was 20% in men and 23% in women and the prevalence of all other respiratory symptoms had also increased.
In men there was a significant mean decrease of the forced expired volume in 0·75 sec (FEV0.75) over the shift both in 1962 (P<0·05) and in 1971 (P<0·01). In women the mean acute decrease of FEV0.75 over the shift in 1962 was not statistically significant (P>0·05), but in 1971 it was (P<0·05). The mean acute reductions of FEV0·75 were considerably higher in 1971 than in 1962. In byssinotics these changes were considerably higher than in non-byssinotics. The chronic effect on ventilatory capacity of exposure to fine cotton dust was not observed in this study.
PMCID: PMC469951  PMID: 5075616
4.  Performance of Three Nucleic Acid Amplification Tests for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Use of Self-Collected Vaginal Swabs Obtained via an Internet-Based Screening Program ▿  
Journal of Clinical Microbiology  2009;47(6):1663-1667.
Use of self-obtained vaginal specimens processed by nucleic acid amplification tests (NAATs) has significantly increased the utilization of nontraditional locations for Chlamydia trachomatis and Neisseria gonorrhoeae screening programs. One important emerging source of such venues includes home-based self-sampling kits available via the Internet. The objective of our study was to evaluate the performance of three commercially available NAATs (Becton-Dickinson ProbeTec SDA, Gen-Probe Aptima Combo2 TMA, and Roche Amplicor PCR) for detection of C. trachomatis and N. gonorrhoeae in vaginal samples obtained via an Internet-based screening program. From July 2004 to August 2005, 500 self-collected vaginal swabs were tested for C. trachomatis and N. gonorrhoeae by using all three NAATs. Another 500 samples were collected between August 2005 and November 2007 and tested by ProbeTec and Combo2; PCR testing was discontinued due to low specificity for N. gonorrhoeae. All tests were conducted according to the manufacturers' procedures; the “gold standard” for an infected C. trachomatis or N. gonorrhoeae patient was defined as ≥2 positive NAAT results. Of the first 500 swabs submitted, 46 were C. trachomatis infected (9.2%) and 5 were N. gonorrhoeae infected (1.0%), and 3 of these were coinfected (0.6%). All C. trachomatis and N. gonorrhoeae Combo2-positive/ProbeTec-negative samples were confirmed as true positives by an alternative NAAT. For C. trachomatis, ProbeTec, Combo2, and PCR had sensitivities of 82.6%, 100%, and 100%, with specificities of 100%, 100%, and 99.3%, respectively. For N. gonorrhoeae, ProbeTec, Combo2, and PCR had sensitivities of 80%, 100%, and 100%, with specificities of 100%, 100%, and 98.8%, respectively. Of the total 1,000 swabs submitted, 92 were C. trachomatis infected (9.2%) and 15 were N. gonorrhoeae infected (1.5%), and 7 of these were coinfected (0.7%). There were no ProbeTec-positive/Combo2-negative samples. For C. trachomatis, ProbeTec and Combo2 had sensitivities of 81.5% and 100%, with specificities of 100% and 100%, respectively. For N. gonorrhoeae, ProbeTec and Combo2 had sensitivities of 80% and 100%, with specificities of 100% and 100%, respectively. Overall, ProbeTec had 17 C. trachomatis false-negative results (1.7%) and 3 N. gonorrhoeae false-negative results (0.3%), while Combo2 had none. Our results were consistent with the sensitivities and specificities stated by the manufacturers. NAATs perform well for detection of chlamydia and gonorrhea with self-obtained vaginal swabs shipped in a dry state to a laboratory. For 1,000 self-collected vaginal swabs tested by NAATs, the sensitivities for C. trachomatis and N. gonorrhoeae for Combo2 were 100% and 100%, while they were 81.5% and 80%, respectively, for ProbeTec. For 500 PCR samples, the C. trachomatis sensitivity was 100% and the N. gonorrhoeae sensitivity was 100%, with specificities of 99.3% and 98.8%, respectively.
doi:10.1128/JCM.02387-08
PMCID: PMC2691063  PMID: 19386838
5.  A bacterial PriB with weak single-stranded DNA binding activity can stimulate the DNA unwinding activity of its cognate PriA helicase 
BMC Microbiology  2011;11:189.
Background
Bacterial DNA replication restart pathways facilitate reinitiation of DNA replication following disruptive encounters of a replisome with DNA damage, thereby allowing complete and faithful duplication of the genome. In Neisseria gonorrhoeae, the primosome proteins that catalyze DNA replication restart differ from the well-studied primosome proteins of E. coli with respect to the number of proteins involved and the affinities of their physical interactions: the PriA:PriB interaction is weak in E. coli, but strong in N. gonorrhoeae, and the PriB:DNA interaction is strong in E. coli, but weak in N. gonorrhoeae. In this study, we investigated the functional consequences of this affinity reversal.
Results
We report that N. gonorrhoeae PriA's DNA binding and unwinding activities are similar to those of E. coli PriA, and N. gonorrhoeae PriA's helicase activity is stimulated by its cognate PriB, as it is in E. coli. This finding is significant because N. gonorrhoeae PriB's single-stranded DNA binding activity is weak relative to that of E. coli PriB, and in E. coli, PriB's single-stranded DNA binding activity is important for PriB stimulation of PriA helicase. Furthermore, a N. gonorrhoeae PriB variant defective for binding single-stranded DNA can stimulate PriA's helicase activity, suggesting that DNA binding by PriB might not be important for PriB stimulation of PriA helicase in N. gonorrhoeae. We also demonstrate that N. gonorrhoeae PriB stimulates ATP hydrolysis catalyzed by its cognate PriA. This activity of PriB has not been observed in E. coli, and could be important for PriB stimulation of PriA helicase in N. gonorrhoeae.
Conclusions
The results of this study demonstrate that a bacterial PriB homolog with weak single-stranded DNA binding activity can stimulate the DNA unwinding activity of its cognate PriA helicase. While it remains unclear if N. gonorrhoeae PriB's weak DNA binding activity is required for PriB stimulation of PriA helicase, the ability of PriB to stimulate PriA-catalyzed ATP hydrolysis could play an important role. Thus, the weak interaction between N. gonorrhoeae PriB and DNA might be compensated for by the strong interaction between PriB and PriA, which could result in allosteric activation of PriA's ATPase activity.
doi:10.1186/1471-2180-11-189
PMCID: PMC3179954  PMID: 21861872
6.  Gonorrhoea 
Clinical Evidence  2011;2011:1604.
Introduction
In the UK, diagnosis rates for gonorrhoea in 2008 were 152/100,000 for men aged 20 to 24 years and 135/100,000 for women aged 16 to 19 years. Resistance to one or more antimicrobial agent is reported in more than one quarter of isolates. Co-infection with Chlamydia trachomatis is reported in 10% to 40% of people with gonorrhoea in the US and UK.
Methods and outcomes
We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of treatments for uncomplicated infections in men and non-pregnant women; and in pregnant women? What are the effects of treatments for disseminated gonococcal infection? What are the effects of dual treatment for gonorrhoea and chlamydia infection? We searched: Medline, Embase, The Cochrane Library, and other important databases up to March 2010 (Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA).
Results
We found 24 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions.
Conclusions
In this systematic review we present information relating to the effectiveness and safety of the following interventions: antibiotic regimens (dual treatment, multiple dose, single dose).
Key Points
Gonorrhoea is caused by infection with Neisseria gonorrhoeae. In men, uncomplicated urethritis is the most common manifestation, while in women less than half of cases produce symptoms (such as vaginal discharge and dyspareunia). Rates of diagnosed gonorrhoea infection in the UK fell by more than 30% between 2002 and 2009.In the UK in 2008, diagnosis rates for gonorrhoea were 152/100,000 for men aged 20 to 24 years, and 135/100,000 for women aged 16 to 19 years.Rates are highest in men aged 20 to 24 years and women aged 16 to 19 years.In the UK, some studies have shown that 28% of isolates are resistant to ciprofloxacin and 8% to penicillin. There is evidence of reduced susceptibility to cephalosporins.Co-infection with Chlamydia trachomatis is reported in 10% to 40% of people with gonorrhoea in the US and UK.
Single-dose antibiotic regimens have achieved cure rates of 95% and higher in men and non-pregnant women with urogenital or rectal gonorrhoea, although we don't know how different single-dose antibiotic regimens compare with each other. Single-dose antibiotics are also effective for curing gonorrhoea in pregnant women.
In people with disseminated gonococcal infection, there is consensus that multiple-dose regimens using cephalosporins or fluoroquinolones (when the infecting organism is known to be susceptible) are the most effective treatments, although evidence supporting this is somewhat sparse.
We found insufficient evidence to judge the best treatment for people with both gonorrhoea and chlamydia, although theory, expert opinion, and clinical experience suggest that a combination of antimicrobials active against both N gonorrhoeae and C trachomatis is effective.
PMCID: PMC3275146  PMID: 21401969
7.  Gonorrhoea 
Clinical Evidence  2007;2007:1604.
Introduction
In the UK, diagnoses rates for gonorrhoea in 2005 were 196/100,000 for 20-24 year old men, and 133/100,000 for 16-19 year old women. Co-infection with Chlamydia trachomatis is reported in 10-40% of people with gonorrhoea in the USA and UK.
Methods and outcomes
We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of treatments for uncomplicated infections in men and non-pregnant women; and in pregnant women? What are the effects of treatments for disseminated gonococcal infection? What are the effects of dual treatment for gonorrhoea and chlamydia infection? We searched: Medline, Embase, The Cochrane Library and other important databases up to July 2006 (BMJ Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA).
Results
We found 21 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions.
Conclusions
In this systematic review we present information relating to the effectiveness and safety of the following interventions: antibiotic regimens (dual treatment, multiple dose, single dose).
Key Points
Gonorrhoea is caused by infection with Neisseria gonorrhoeae. In men, uncomplicated urethritis is the most common manifestation while in women only about half of cases produce symptoms (such as vaginal discharge and dyspareunia). In the UK, diagnoses rates for gonorrhoea were 196/100 000 for 20-24 year old men and 133/100 000 for 16-19 year old women in 2005.Co-infection with Chlamydia trachomatis is reported in 10-40% of people with gonorrhoea in the USA and UK.
Single dose antibiotic regimens have achieved cure rates of 95% or higher in men and non-pregnant women with urogenital or rectal gonorrhoea. However, resistance to many widely available antibiotics (e.g. penicillins, tetracylines, fluoroquinolones) continues to spread, making it necessary to consider local N gonorrhoeae susceptibility patterns when choosing a treatment regimen. Single dose antibiotics are also effective for curing gonorrhoea in pregnant women.
In people with disseminated gonococcal infection, there is consensus that multidose regimens using injectable cephalosporins or fluoroquinolones (when the infecting organism is known to be susceptible) are the most effective treatments, although evidence supporting this is somewhat sparse.
We did not find any sufficient evidence to judge the best treatment for people with both gonorrhoea and chlamydia, although theory, expert opinion, and clinical experience suggest a combination of antimicrobials active against both N gonorrhoeae and C trachomatis are effective.
PMCID: PMC2943790  PMID: 19454057
8.  Decline in age at menarche among Spanish women born from 1925 to 1962 
BMC Public Health  2009;9:449.
Background
While the timing of reproductive events varies across populations, a downward trend in age at menarche has nevertheless been reported in most of the developed world over the past century. Given the impact of change in age at menarche on health conditions, this study sought to examine secular trends in age at menarche among women living in Navarre (Northern Spain) who participated in a population-based breast cancer screening programme.
Methods
The study was based on 110545 women born from 1925 to 1962. Trends were tested using a linear regression model, in which year of birth was entered continuously as the predictor and age at menarche (years) as the response variable, using size of town and region of birth as covariates.
Results
Among women born in Navarre between 1925 and 1962, age at menarche declined steadily from an average of 13.72 years in the 1925-1929 birth-cohorts to 12.83 years in the 1958-1962 birth-cohorts. Controlling for size of town or city of birth, age at menarche declined by an average of 0.132 years every 5 years over the period 1925-1962. This decline was greater in women born in rural versus urban settings. Trends were also different among regions of birth.
Conclusion
We report a population-based study showing a downward trend in age of onset of menarche among Spanish women born in the period 1925-1962, something that is more pronounced among women born in rural settings and varies geographically.
doi:10.1186/1471-2458-9-449
PMCID: PMC2796666  PMID: 19961593
9.  Evaluation of Self-Collected Glans and Rectal Swabs from Men Who Have Sex with Men for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Use of Nucleic Acid Amplification Tests▿  
Journal of Clinical Microbiology  2009;47(6):1657-1662.
Self-collected glans and rectal swab specimens from men who have sex with men (MSM) may be appropriate, convenient specimens for testing. We evaluated the use of self-collected swabs for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by a transcription-mediated amplification test (AC2; Aptima Combo 2; Gen-Probe Inc.) and a strand displacement amplification test (SDA; ProbeTec; Becton Dickinson Co.) in MSM seen at the city sexually transmitted disease clinic in San Francisco, CA. For the glans swab specimen, subjects enrolled early in the study rolled a Dacron swab across the meatus three times (method 1). A slightly more invasive procedure was performed later in the study: the subjects inserted the swab 1/4 in. into the urethra, rotated the swab, and then withdrew the swab (method 2). MSM self-collected a rectal swab specimen and also provided first-catch urine (FCU). Additional rectal swab samples were then obtained by the clinician. For the detection of C. trachomatis and N. gonorrhoeae, all swabs were evaluated by AC2 and SDA, FCU was tested by AC2, and the clinician-collected rectal swabs were cultured. A rectal true-positive (TP) result was defined as a culture-positive result for C. trachomatis or N. gonorrhoeae, two or more positive nucleic acid amplification test (NAAT) results, or a single NAAT-positive result confirmed by an alternate amplification method (the Aptima C. trachomatis or N. gonorrhoeae test). A glans TP result was defined as a positive result for FCU, positive results for both glans specimens (one tested by AC2 and one tested by SDA), or a positive result for a single glans specimen confirmed by an alternate amplification method. The prevalence rates of C. trachomatis and N. gonorrhoeae by testing of FCU were 6.8% (60/882 specimens) and 12.2% (108/882 specimens), respectively. Mixed results were obtained with the glans swab: N. gonorrhoeae detection by AC2 and SDA (method 1) had the best performance (sensitivities, >92%) with samples from a population with a higher prevalence of infection, but their performance for the detection of C. trachomatis was poor and varied by collection method (sensitivities, 56 to 68%). The prevalence rates of C. trachomatis and N. gonorrhoeae in the rectum were 7.3% (66/907 specimens) and 9.4% (83/882 specimens), respectively. The sensitivities of the tests with self-collected and clinician-collected rectal swab specimens were comparable (for C. trachomatis, 41% and 44%, respectively, by SDA and 82% and 71%, respectively, by AC2; for N. gonorrhoeae, 77% and 68%, respectively, by SDA and 84% and 78%, respectively, by AC2). AC2 and SDA were far superior to culture for the detection of C. trachomatis and N. gonorrhoeae in the rectum, with both tests detecting at least twice as many infections. While we found self-collected rectal swabs from MSM to be valid specimens for testing, the sensitivities of the tests with glans swab specimens were disappointing except for those from patients with symptomatic N. gonorrhoeae infections. Self-collected glans swab specimens may not be appropriate for the detection of C. trachomatis or for the detection of N. gonorrhoeae in low-risk or asymptomatic patients by AC2 and SDA, and we would not recommend their use on the basis of our results. Further studies are needed.
doi:10.1128/JCM.02269-08
PMCID: PMC2691064  PMID: 19369445
10.  Antimicrobial susceptibility/resistance and genetic characteristics of Neisseria gonorrhoeae isolates from Poland, 2010-2012 
Background
In Poland, gonorrhoea has been a mandatorily reported infection since 1948, however, the reported incidences are likely underestimated. No antimicrobial resistance (AMR) data for Neisseria gonorrhoeae has been internationally reported in nearly four decades, and data concerning genetic characteristics of N. gonorrhoeae are totally lacking. The aims of this study were to investigate the AMR to previously and currently recommended gonorrhoea treatment options, the main genetic resistance determinant (penA) for extended-spectrum cephalosporins (ESCs), and genotypic distribution of N. gonorrhoeae isolates in Poland in 2010-2012.
Methods
N. gonorrhoeae isolates cultured in 2010 (n = 28), 2011 (n = 92) and 2012 (n = 108) in Warsaw and Bialystok, Poland, were examined using antimicrobial susceptibility testing (Etest), pyrosequencing of penA and N. gonorrhoeae multi-antigen sequence typing (NG-MAST).
Results
The proportions of N. gonorrhoeae isolates showing resistance were as follows: ciprofloxacin 61%, tetracycline 43%, penicillin G 22%, and azithromycin 8.8%. No isolates resistant to ceftriaxone, cefixime or spectinomycin were found. However, the proportion of isolates with an ESC MIC = 0.125 mg/L, i.e. at the resistance breakpoint, increased significantly from none in 2010 to 9.3% and 19% in 2012 for ceftriaxone and cefixime, respectively. Furthermore, 3.1% of the isolates showed multidrug resistance, i.e., resistance to ciprofloxacin, penicillin G, azithromycin, and decreased susceptibility to cefixime (MIC = 0.125 mg/L). Seventy-six isolates (33%) possessed a penA mosaic allele and 14 isolates (6.1%) contained an A501V/T alteration in penicillin-binding protein 2. NG-MAST ST1407 (n = 58, 25% of isolates) was the most prevalent ST, which significantly increased from 2010 (n = 0) to 2012 (n = 46; 43%).
Conclusions
In Poland, the diversified gonococcal population displayed a high resistance to most antimicrobials internationally previously recommended for gonorrhoea treatment and decreasing susceptibility to the currently recommended ESCs. The decreasing susceptibility to ESCs was mostly due to the introduction of the internationally spread multidrug-resistant NG-MAST ST1407 in 2011. It is essential to promptly revise the gonorrhoea treatment guidelines, improve the gonorrhoea laboratory diagnostics, and implement quality assured surveillance of gonococcal AMR (ideally also treatment failures) in Poland.
doi:10.1186/1471-2334-14-65
PMCID: PMC3922028  PMID: 24502606
Neisseria gonorrhoeae; Gonorrhoea; Poland; Antimicrobial resistance (AMR); Extended-spectrum cephalosporins (ESCs); Ceftriaxone; Cefixime; penA; NG-MAST
11.  Incidence and antimicrobial susceptibility of Neisseria gonorrhoeae isolates from patients attending the national Neisseria gonorrhoeae reference laboratory of Hungary 
BMC Infectious Diseases  2014;14(1):433.
Background
The Hungarian national guidelines for the treatment of gonorrhoea were published in 2002 but are now widely considered to be outdated. Improved knowledge is needed with respect to the epidemiology and antimicrobial susceptibility of Neisseria gonorrhoeae strains currently circulating in Hungary not least for the construction of updated local recommendations for treating gonorrhoea. European guidelines are based mostly on western European data raising concerns locally that recommended treatments might not be optimised for the situation in Hungary. We report our recent study on the distribution of antibiotic resistance in various Hungarian (East European) Neisseria gonorrhoeae strains isolated from patients with gonorrhoea over the past four years.
Methods
Between January 2010 and December 2013, isolates of N. gonorrhoeae were obtained from sexually active individuals during medical examination at the STD Center of Semmelweis University in Budapest. The minimal inhibitory concentrations (MIC) of azithromycin, cefixime, ceftriaxone, ciprofloxacin, penicillin, tetracycline and spectinomycin were determined to establish the antimicrobial susceptibility of the strains currently circulating in patients that attend our clinic.
Results
Among the 9097 patients tested, 582 had an N. gonorrhoeae infection as detected by culture. The isolates were all sensitive to ceftriaxone and spectinomycin and 581/582 strains were sensitive to cefixime. In contrast, the number of detected strains with elevated azithromycin MIC did increase over the time period examined to approximately 16% in 2013. There was a high percentage of detected resistance to penicillin (77%), tetracycline (86%), and ciprofloxacin (66%) in the isolates examined in this study.
Conclusion
Current European guidelines recommend 2 g azithromycin in addition to 500 mg ceftriaxone as first choice therapy for gonorrhoea. For the purposes of revising the Hungarian national treatment guidelines, apparent increasing resistance to azithromycin during the last four years should be accounted for. It is also clear that penicillin, tetracycline and ciprofloxacin are inappropriate treatment measures at least locally. We also recommend that culture should form part of the diagnostic pathway of gonorrhoea, followed by antibiotic susceptibility testing with MIC determination. This will provide valuable continued monitoring of antibiotic resistance development in strains of Neisseria gonorrhoeae circulating in Hungary.
doi:10.1186/1471-2334-14-433
PMCID: PMC4155111  PMID: 25098185
Neisseria gonorrhoeae; Antimicrobial resistance; Incidence; MIC; Hungary
12.  Risk of coinfection with Chlamydia trachomatis and Neisseria gonorrhoeae in Nova Scotia 
BACKGROUND:
The frequency of Chlamydia trachomatis and Neisseria gonorrhoeae coinfection can vary depending on their individual incidence and prevalence rates.
OBJECTIVE:
To determine the frequency of C trachomatis and N gonorrhoeae coinfections by evaluating the results of testing in 2007 and 2008 to better inform testing and treatment decisions.
METHODS:
Specimens from the same patient submitted on the same day served as the basis for the present study. The age, sex and the source of the specimen were also linked to the accession number. Infection and coinfection rates were analyzed in both males and females.
RESULTS:
Concurrent testing was performed on 41,567 female specimens and 1827 male specimens, of which, 1495 female samples (3.6%) tested positive for C trachomatis infection and 88 (0.2%) tested positive for N gonorrhoeae infections. Only 31 females were coinfected; however, for those between 11 and 25 years of age, 25 of 61 females (40.1%) with N gonorrhoeae infection also tested positive for C trachomatis infection; conversely, 25 of 1248 females (2.0%) with C trachomatis infection also tested positive for N gonorrhoeae infection. For males, 213 (11.7%) tested positive for C trachomatis infection, and 59 (3.2%) tested positive for N gonorrhoeae infection. In 30 males with N gonorrhoeae between 11 and 25 years of age, and 149 males with C trachomatis, eight coinfections were observed (26.7% and 5.3%, respectively). Of those older than 25 years of age, only five of 905 men and six of 19,465 women were coinfected. None of the 10,935 women who were 30 years of age or older had coinfections.
CONCLUSION:
The N gonorrhoeae coinfection rate in males with C trachomatis may justify empirical antimicrobials; however, in females, the proportion of coinfected may not justify empirical treatment for N gonorrhoeae infection when the C trachomatis test is positive and N gonorrhoeae testing has not been performed.
PMCID: PMC2912101  PMID: 21629610
Chlamydia trachomatis; Coinfection; Neisseria gonorrhoeae
13.  Trends in the Supply and Distribution of Physicians in Saskatchewan: January 1962 to June 1964 
Using all the readily available sources of information, a study was designed to describe the changes in supply and distribution of physicians in Saskatchewan during the inception of the Province's controversial Medical Care Insurance Act. Baselines were obtained in January 1962, and comparisons made in June 1963 and June 1964. The physician:population ratio was 1:1037 in January 1962 and 1:980 in June 1964. The average yearly physician turnover rate of 10% was exceeded in 1960 and in 1961 and then levelled off. The levelling off continued from January 1962 to June 1963. But between June 1963 and June 1964 the total number of physicians was sufficient both to re-establish the rising trend of the previous decade and to offset some of the losses of 1960-1962.
PMCID: PMC1928160  PMID: 14275041
14.  Chlamydial and gonococcal reinfection among men: a systematic review of data to evaluate the need for retesting 
Sexually Transmitted Infections  2006;83(4):304-309.
This study aimed to systematically review and describe the evidence on chlamydia and gonorrhoea reinfection among men, and to evaluate the need for retesting recommendations in men. PubMed and STI conference abstract books from January 1995 to October 2006 were searched to identify studies on chlamydia and gonorrhoea reinfection among men using chlamydia and gonorrhoea nucleic acid amplification tests or gonorrhoea culture. Studies were categorised as using either active or passive follow‐up methods. The proportions of chlamydial and gonococcal reinfection among men were calculated for each study and summary medians were reported. Repeat chlamydia infection among men had a median reinfection probability of 11.3%. Repeat gonorrhoea infection among men had a median reinfection probability of 7.0%. Studies with active follow‐up had moderate rates of chlamydia and gonorrhoea reinfection among men, with respective medians of 10.9% and 7.0%. Studies with passive follow‐up had higher proportions of both chlamydia and gonorrhoea reinfections among men, with respective medians of 17.4% and 8.5%. Proportions of chlamydia and gonorrhoea reinfection among men were comparable with those among women. Reinfection among men was strongly associated with previous history of sexually transmitted diseases and younger age, and inconsistently associated with risky sexual behaviour. Substantial repeat chlamydia and gonorrhoea infection rates were found in men comparable with those in women. Retesting recommendations in men are appropriate, given the high rate of reinfection. To optimise retesting guidelines, further research to determine effective retesting methods and establish factors associated with reinfection among men is suggested.
doi:10.1136/sti.2006.024059
PMCID: PMC2598678  PMID: 17166889
15.  Comparative assessment of CDS, CLSI disc diffusion and Etest techniques for antimicrobial susceptibility testing of Neisseria gonorrhoeae: a 6-year study 
BMJ Open  2012;2(4):e000969.
Background
A variety of techniques are available for antimicrobial susceptibility testing of Neisseria gonorrhoeae.
Objective
The aim of this study was to find a cost-effective, reliable and easily applicable microbiological method to detect antimicrobial susceptibilities of N. gonorrhoeae in resource-poor countries.
Design
Prospective study.
Setting
Male and female STD clinic of Regional STD Teaching, Training and Research Centre, New Delhi, India.
Participants
N. gonorrhoeae isolates from all male and female patients presenting with acute gonococcal urethritis and cervical discharge.
Material and methods
A total of 295 consecutive N. gonorrhoeae isolates during 2005–2010 was used to compare the Clinical and Laboratory Standards Institute (CLSI) and CDS disc diffusion technique with Etest by performing antimicrobial susceptibility testing in parallel for penicillin, tetracycline, ceftriaxone, ciprofloxacin and spectinomycin. WHO reference strains were used as controls.
Results
CDS disc diffusion zones of inhibition showed that complete percentage agreement for penicillin, ciprofloxacin and tetracycline was high with their analogous Etest minimal inhibitory concentrations in comparison to CLSI disc diffusion technique, that is, 91.5%, 92.9% and 99.3% versus 87.5%, 88.5% and 74.9%, respectively. CDS results had less number of major and minor category discrepancies in comparison to CLSI and CDS method showed excellent correlation coefficient (r=1) with Etest for all five antimicrobial agents tested in comparison to CLSI (r=0.92). It was very poor (r=0.61) by CLSI method for tetracycline. The correlation coefficients between the two methods and the Etest were identical if tetracycline was removed from the CLSI analysis.
Conclusions
The CDS technique is an attractive alternative for N. gonorrhoeae susceptibility testing and is recommended for monitoring the antimicrobial susceptibility in less developed and resource-poor settings to facilitate enhanced antimicrobial resistance surveillance when the WHO Gonococcal Antimicrobial Surveillance Programme is undergoing expansion to meet the ongoing challenges of surveillance and control of gonococcal antimicrobial resistance.
Article summary
Article focus
A variety of techniques are available for antimicrobial susceptibility testing of N. gonorrhoeae.
The aim of this study is to find a cost-effective, reliable and easily applicable microbiological method to detect antimicrobial susceptibilities of N. gonorrhoeae in resource-poor countries.
Key messages
This study highlighted that the CDS disc diffusion technique yielded excellent category agreement with their analogous Etest minimal inhibitory concentrations in comparison to CLSI technique.
CDS offers the advantage for those laboratories that process small numbers of specimens.
CDS technique is cost-effective, reliable and easily applicable microbiological method to detect antimicrobial susceptibilities of N. gonorrhoeae in resource-poor countries.
This technique will facilitate enhanced antimicrobial resistance surveillance and direct and meaningful comparison of resistance data generated within different national and international laboratories.
Strengths and limitations of this study
This is the first study to determine the comparability of CDS and CLSI disc diffusion method with Etest minimal inhibitory concentrations and to accurately and reproducibly assess N. gonorrhoeae susceptibilities for penicillin, tetracycline, ceftriaxone, ciprofloxacin and spectinomycin, commonly used for susceptibility testing.
Susceptibility testing for cefixime, cefpodoxime, gentamicin and azithromycin was performed only by one method because of non-availability of interpretation criteria for these antimicrobials or limitation of resources. Therefore, comparison of above three techniques could not be carried out for these antibiotics.
doi:10.1136/bmjopen-2012-000969
PMCID: PMC3391364  PMID: 22761285
16.  Rectal gonorrhoea as an independent risk factor for HIV infection in a cohort of homosexual men. 
Genitourinary Medicine  1995;71(3):150-154.
OBJECTIVE--To determine whether certain sexually transmitted diseases are independent risk factors for HIV transmission in a cohort of homosexual men. METHODS--Eligible cases were identified as those who had seroconverted between November 1982 and November 1990. Two persistently HIV-seronegative control participants were randomly selected for each case from all participants who remained seronegative in November 1990. For cases, risk factor data were taken from an index visit which was defined as the first seropositive visit, while for controls these data were obtained from a matched visit which occurred within two months of the index visit for the corresponding case. Mantel-Haenszel methods and logistic regression were used to compare differences in risk factors for seroconversion between cases and controls. RESULTS--A total of 125 cases and 250 controls were eligible for this study. Cases were significantly more likely to have had reported any gonorrhoea (17% versus 6%; OR = 2.94; 95% CI: 1.51-5.73) or syphilis (7% versus 2%; OR = 3.78; 95% CI: 1.33-10.79) than controls during the seroconversion period. Multivariate logistic regression revealed rectal gonorrhoea to be independently associated with risk of seroconversion (odds ratio = 3.18; p = 0.044), whereas urethral gonorrhoea (p = 0.479) and pharyngeal gonorrhoea (p = 0.434) were not after inclusion of rectal gonorrhoea. In addition, the following variables were also shown to exert an independent effect on seroconversion: frequency of anal intercourse, use of illicit drugs, number of male sexual partners, and lack of a post-secondary education. CONCLUSIONS--In this observational study, rectal gonorrhoea was found to be associated with HIV seroconversion after adjustment for a number of HIV risk factors. We cannot rule out that rectal gonorrhoea was not directly associated with HIV infection but rather with other residual lifestyle factors not fully adjusted for in the analysis. However, the relationship with gonococcal involvement of a specific anatomic site lends support to a biological association between gonorrhoea and HIV infection, rather than to alternative non-biologic explanations. Our findings are consistent with previous studies reporting an association between HIV infection and non-ulcerative sexually transmitted diseases. Such a direct association might be explained by postulating that gonorrhoea results in inflamed rectal mucosa and compromised epithelial integrity, thereby predisposing an individual to subsequent HIV infection.
PMCID: PMC1195487  PMID: 7635489
17.  Neisseria gonorrhoeae Infection Protects Human Endocervical Epithelial Cells from Apoptosis via Expression of Host Antiapoptotic Proteins▿ † 
Infection and Immunity  2009;77(9):3602-3610.
Several microbial pathogens can modulate the host apoptotic response to infection, which may contribute to immune evasion. Various studies have reported that infection with the sexually transmitted disease pathogen Neisseria gonorrhoeae can either inhibit or induce apoptosis. N. gonorrhoeae infection initiates at the mucosal epithelium, and in women, cells from the ectocervix and endocervix are among the first host cells encountered by this pathogen. In this study, we defined the antiapoptotic effect of N. gonorrhoeae infection in human endocervical epithelial cells (End/E6E7 cells). We first established that N. gonorrhoeae strain FA1090B failed to induce cell death in End/E6E7 cells. Subsequently, we demonstrated that stimulation with N. gonorrhoeae protected these cells from staurosporine (STS)-induced apoptosis. Importantly, only End/E6E7 cells incubated with live bacteria and in direct association with N. gonorrhoeae were protected from STS-induced apoptosis, while heat-killed and antibiotic-killed bacteria failed to induce protection. Stimulation of End/E6E7 cells with live N. gonorrhoeae induced NF-κB activation and resulted in increased gene expression of the NF-κB-regulated antiapoptotic genes bfl-1, cIAP-2, and c-FLIP. Furthermore, cIAP-2 protein levels also increased in End/E6E7 cells incubated with gonococci. Collectively, our results indicate that the antiapoptotic effect of N. gonorrhoeae in human endocervical epithelial cells results from live infection via expression of host antiapoptotic proteins. Securing an intracellular niche through the inhibition of apoptosis may be an important mechanism utilized by N. gonorrhoeae for microbial survival and immune evasion in cervical epithelial cells.
doi:10.1128/IAI.01366-08
PMCID: PMC2738021  PMID: 19546192
18.  Performance of the Abbott RealTime CT/NG for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae▿  
Journal of Clinical Microbiology  2010;48(9):3236-3243.
A multicenter clinical study was conducted to evaluate the performance characteristics of the Abbott RealTime CT/NG assay, a multiplex real-time PCR assay, for simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. The specimens were collected from a total of 3,832 male and female subjects at 16 geographically diverse sites. Specimens included male and female urine samples, male urethral swabs, female endocervical swabs, and self-collected and clinician-collected vaginal swabs. Specimens were tested with the automated Abbott RealTime CT/NG assay, Aptima Combo 2 assay (Gen-Probe), ProbeTec ET CT/GC assay (Becton Dickinson), and culture for N. gonorrhoeae. The Aptima Combo 2 assay, the ProbeTec assay, and the N. gonorrhoeae culture were used as the reference assays. For each subject, a patient infected status (PIS) was determined based on the combined results from the reference assays. The overall prevalence in female subjects was 8.9% for C. trachomatis and 3.8% for N. gonorrhoeae. The overall male prevalence was 18.2% for C. trachomatis and 16.7% for N. gonorrhoeae. The overall sensitivity and specificity of the Abbott RealTime CT/NG assay were 92.4% and 99.2% for C. trachomatis and 96.9% and 99.7% for N. gonorrhoeae, respectively. In comparison, the sensitivity and specificity, respectively, for the Aptima Combo 2 assay were 94.5% and 99.0% for C. trachomatis and 96.1% and 99.5% for N. gonorrhoeae, and those for the ProbeTec ET assay were 90.3% and 99.5% for C. trachomatis and 92.0% and 97.3% for N. gonorrhoeae in this study. The Abbott RealTime CT/NG assay offers C. trachomatis and N. gonorrhoeae dual detection with high sensitivity and specificity. The automated assay provides a useful alternative nucleic acid amplification assay for clinical laboratories and clinicians.
doi:10.1128/JCM.01019-10
PMCID: PMC2937681  PMID: 20668135
19.  The role of acidification in the inhibition of Neisseria gonorrhoeae by vaginal lactobacilli during anaerobic growth 
Background
Vaginal lactobacilli protect the female genital tract by producing lactic acid, bacteriocins, hydrogen peroxide or a local immune response. In bacterial vaginosis, normal lactobacilli are replaced by an anaerobic flora and this may increase susceptibility to Neisseria gonorrhoeae, a facultative anaerobe. Bacterial interference between vaginal lactobacilli and N. gonorrhoeae has not been studied in liquid medium under anaerobic conditions. By co-cultivating N. gonorrhoeae in the presence of lactobacilli we sought to identify the relative contributions of acidification and hydrogen peroxide production to any growth inhibition of N. gonorrhoeae.
Methods
Three strains of N. gonorrhoeae distinguishable by auxotyping were grown in the presence of high concentrations (107-108 cfu/mL) of three vaginal lactobacilli (L. crispatus, L. gasseri and L. jensenii) in an anerobic liquid medium with and without 2-(N-morpholino)-ethanesulfonic (MES) buffer. Fusobacterium nucleatum was used as an indicator of anaerobiosis. Bacterial counts were performed at 15, 20 and 25 h; at 25 h pH and hydrogen peroxide concentrations were measured.
Results
Growth of F. nucleatum to >108 cfu/mL at 25 h confirmed anaerobiosis. All bacteria grew in the anaerobic liquid medium and the addition of MES buffer had negligible effect on growth. L. crispatus and L. gasseri produced significant acidification and a corresponding reduction in growth of N. gonorrhoeae. This inhibition was abrogated by the addition of MES. L. jensenii produced less acidification and did not inhibit N. gonorrhoeae. Hydrogen peroxide was not detected in any experiment.
Conclusions
During anaerobic growth, inhibition of N. gonorrhoeae by the vaginal lactobacilli tested was primarily due to acidification and abrogated by the presence of a buffer. There was no evidence of a specific mechanism of inhibition other than acid production under these conditions and, in particular, hydrogen peroxide was not produced. The acidification potential of vaginal lactobacilli under anaerobic conditions may be their most important characteristic conferring protection against N. gonorrhoeae infection.
doi:10.1186/1476-0711-10-8
PMCID: PMC3045876  PMID: 21329492
20.  Association of Neisseria gonorrhoeae OpaCEA with Dendritic Cells Suppresses Their Ability to Elicit an HIV-1-Specific T Cell Memory Response 
PLoS ONE  2013;8(2):e56705.
Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4+ T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific OpaCEA, but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, OpaCEA binding to CEACAM1 reduced the DCs’ capacity to stimulate an allogeneic T cell proliferative response. Moreover, OpaCEA-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with OpaCEA-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why infection of N. gonorrhoeae fails to trigger an effective specific immune response or develop immune memory, and may affect the potent synergy between gonorrhea and HIV-1 infection.
doi:10.1371/journal.pone.0056705
PMCID: PMC3570455  PMID: 23424672
21.  The Neisseria Lipooligosaccharide-Specific α-2,3-Sialyltransferase Is a Surface-Exposed Outer Membrane Protein  
Infection and Immunity  2002;70(7):3744-3751.
Neisseria gonorrhoeae and Neisseria meningitidis express an ∼43-kDa α-2,3-sialyltransferase (Lst) that sialylates the surface lipooligosaccharide (LOS) by using exogenous (in all N. gonorrhoeae strains and some N. meningitidis serogroups) or endogenous (in other N. meningitidis serogroups) sources of 5′-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA). Sialylation of LOS can protect N. gonorrhoeae and N. meningitidis from complement-mediated serum killing and from phagocytic killing by neutrophils. The precise subcellular location of Lst has not been determined. We confirm and extend previous studies by demonstrating that Lst is located in the outer membrane and is surface exposed in both N. gonorrhoeae and N. meningitidis. Western immunoblot analysis of subcellular fractions of N. gonorrhoeae strain F62 and N. meningitidis strain MC58⊄3 (an acapsulate serogroup B strain) performed with rabbit antiserum raised against recombinant Lst revealed an ∼43-kDa protein exclusively in outer membrane preparations of both pathogens. Inner membrane, periplasmic, cytoplasmic, and culture supernatant fractions were devoid of Lst, as determined by Western blot analysis. Consistent with this finding, outer membrane fractions of N. gonorrhoeae were significantly enriched for sialyltransferase enzymatic activity. A trace of enzymatic activity was detected in inner membrane fractions, which may have represented Lst in transit to the outer membrane or may have represented inner membrane contamination of outer membrane preparations. Subcellular preparations of an isogenic lst insertion knockout mutant of N. gonorrhoeae F62 (strain ST01) expressed neither a 43-kDa immunoreactive protein nor sialyltransferase activity. Anti-Lst rabbit antiserum bound to whole cells of N. meningitidis MC58⊄3 and wild-type N. gonorrhoeae F62 but not to the Lst mutant ST01, indicating the surface exposure of the enzyme. Although the anti-Lst antiserum avidly bound enzymatically active, recombinant Lst, it inhibited Lst (sialyltransferase) activity by only about 50% at the highest concentration of antibody used. On the contrary, anti-Lst antiserum did not inhibit sialylation of whole N. gonorrhoeae cells in the presence of exogenous CMP-NANA, suggesting that the antibody did not bind to or could not access the enzyme active site on the surface of viable Neisseria cells. Taken together, these results indicate that Lst is an outer membrane, surface-exposed glycosyltransferase. To our knowledge, this is the first demonstration of the localization of a bacterial glycosyltransferase to the outer membrane of gram-negative bacteria.
doi:10.1128/IAI.70.7.3744-3751.2002
PMCID: PMC128106  PMID: 12065517
22.  Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Enzyme Immunoassay, Culture, and Three Nucleic Acid Amplification Tests 
Journal of Clinical Microbiology  2001;39(5):1751-1756.
The purpose of this study was to evaluate and compare three commercially available nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis. Roche PCR and Becton Dickinson strand displacement amplification (SDA) were performed on 733 endocervical swab specimens from commercial sex workers. Abbott ligase chain reaction (LCR) was performed on a subset of 396 samples. Endocervical specimens from all women were also tested by culture for N. gonorrhoeae and by Syva MicroTrak enzyme immunoassay (EIA) for C. trachomatis. A positive N. gonorrhoeae result was defined as a positive result by culture or by two NAATs, and a positive C. trachomatis result was defined as a positive result by two tests. According to these definitions, the sensitivities and specificities for the subsample of 396 specimens of N. gonorrhoeae culture, PCR, SDA, and LCR were 69.8, 95.2, 88.9, and 88.9% and 100, 99.4, 100, and 99.1%, respectively; the sensitivities and specificities of C. trachomatis EIA, PCR, SDA, and LCR were 42.0, 98.0, 94.0, and 90.0% and 100, 98.0, 100, and 98.6%, respectively. The performance characteristics of N. gonorrhoeae culture, PCR, and SDA and C. trachomatis EIA, PCR, and SDA for all 733 specimens were defined without inclusion of LCR results and by discrepant analysis after resolution of discordant N. gonorrhoeae PCR results and of discordant C. trachomatis EIA and PCR results by LCR testing. The sensitivities of N. gonorrhoeae culture, PCR, and SDA before and after LCR resolution were 67.8, 95.7, and 93.9% and 65, 95.8, and 90.0%, respectively. The sensitivities of C. trachomatis EIA, PCR, and SDA decreased from 39.4, 100, and 100% to 38.7, 98.7, and 94.7%, respectively. All three NAATs proved to be superior to N. gonorrhoeae culture and to C. trachomatis EIA. The accuracies of the different NAATs were quite similar. SDA was the only amplification assay with 100% specificity for detection of both N. gonorrhoeae and C. trachomatis in endocervical specimens.
doi:10.1128/JCM.39.5.1751-1756.2001
PMCID: PMC88020  PMID: 11325985
23.  Relation of health literacy to gonorrhoea related care 
Sexually Transmitted Infections  2001;77(3):206-211.
Objective: To assess the relation between health literacy and receipt of a screening test for gonorrhoea in the past year.
Methods: Study design was multisite, cross sectional survey of subjects enrolled from clinics, from community based organisations, and by street intercept. Data were obtained using face to face interview. The dependent variable was self reported receipt of a test for gonorrhoea in the past year. Health literacy was measured by the Rapid Estimate of Adult Literacy in Medicine (REALM), recoded to represent 8th grade or lower reading or 9th grade and higher reading level. Statistical analyses were adjusted to account for selection bias in literacy assessment.
Results: 54% of the sample reported at least one gonorrhoea test in the previous year. 65% of the sample read at a 9th grade level or higher. REALM score was moderately correlated with the respondent's years of education. After adjustment for missing REALM data, past suspicion of gonorrhoea, self inspection for gonorrhoea, self efficacy for care seeking, REALM score of 9th grade reading level or higher, and younger age were independently associated with gonorrhoea testing in the previous year. For the average respondent, REALM reading grade level of 9th grade or higher is associated with a 10% increase in the probability of having a gonorrhoea test in the past year.
Conclusions: Low literacy appears to pose a barrier to care for sexually transmitted infections such as gonorrhoea.
Key Words: literacy; care seeking behaviour; gonorrhoea
doi:10.1136/sti.77.3.206
PMCID: PMC1744316  PMID: 11402232
24.  Susceptibility of N. gonorrhoeae to Antibiotics 
Gonorrhea has increased in incidence over the past five years in Manitoba as elsewhere. Cases which did not respond to routine penicillin treatment were noted to be occuring more often. In 1948, strains of N. gonorrhoeae isolated in Canada were all sensitive to 0.06 unit of penicillin per c.c. in vitro. Commencing in May 1962, strains of N. gonorrhoeae were isolated from 100 patients of each sex attending the clinic at the St. Boniface General Hospital. Eighteen per cent required concentrations of more than 0.05 unit of penicillin per ml. to inhibit growth; 31% were not inhibited by the 2 μg. disc of dihydrostreptomycin; but only one strain was found resistant to sulfisoxazole and none to oxytetracycline. Results of penicillin treatment were markedly less successful in the patients with strains that demonstrated reduced penicillin sensitivity. Use of streptomycin and a sulfonamide proved to be a satisfactory substitute. Intramuscular oxytetracycline was less successful despite the laboratory findings.
PMCID: PMC1921705  PMID: 14063939
25.  Global Analysis of Neutrophil Responses to Neisseria gonorrhoeae Reveals a Self-Propagating Inflammatory Program 
PLoS Pathogens  2014;10(9):e1004341.
An overwhelming neutrophil-driven response causes both acute symptoms and the lasting sequelae that result from infection with Neisseria gonorrhoeae. Neutrophils undergo an aggressive opsonin-independent response to N. gonorrhoeae, driven by the innate decoy receptor CEACAM3. CEACAM3 is exclusively expressed by human neutrophils, and drives a potent binding, phagocytic engulfment and oxidative killing of Opa-expressing bacteria. In this study, we sought to explore the contribution of neutrophils to the pathogenic inflammatory process that typifies gonorrhea. Genome-wide microarray and biochemical profiling of gonococcal-infected neutrophils revealed that CEACAM3 engagement triggers a Syk-, PKCδ- and Tak1-dependent signaling cascade that results in the activation of an NF-κB-dependent transcriptional response, with consequent production of pro-inflammatory cytokines. Using an in vivo model of N. gonorrhoeae infection, we show that human CEACAM-expressing neutrophils have heightened migration toward the site of the infection where they may be further activated upon Opa-dependent binding. Together, this study establishes that the role of CEACAM3 is not restricted to the direct opsonin-independent killing by neutrophils, since it also drives the vigorous inflammatory response that typifies gonorrhea. By carrying the potential to mobilize increasing numbers of neutrophils, CEACAM3 thereby represents the tipping point between protective and pathogenic outcomes of N. gonorrhoeae infection.
Author Summary
Gonorrhea is a sexually transmitted infection caused by the bacteria Neisseria gonorrhoeae. These bacteria have re-emerged as a public health priority due to its acquisition of resistance to multiple antibiotics, leading to fears of untreatable infection. The symptoms of gonorrhea include an intense inflammatory response that may lead to pus discharged from the infected genital tract and scarring of the reproductive tract caused by neutrophils recruited to the site of infection. Past studies have detailed molecular interactions that lead to neutrophil binding and engulfment of N. gonorrhoeae, yet it remains unclear why N. gonorrhoeae elicits such a pathogenic inflammatory response. In this study, we reveal that N. gonorrhoeae binding to the human innate decoy receptor, CEACAM3, elicits a potent intracellular signaling cascade that leads to neutrophil expression of cytokines that actively recruit other neutrophils to the infected tissues. As they encounter the gonococci, the next wave of neutrophils becomes similarly activated, leading to the progressive expansion in phagocytic cell numbers until they overwhelm the infected tissues. While this process promotes a rapid response to a troubling pathogen early during infection, the unrestrained recruitment of neutrophils and their toxic antimicrobial arsenal also lead to the pathogenic consequences associated with gonorrhea.
doi:10.1371/journal.ppat.1004341
PMCID: PMC4154863  PMID: 25188454

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