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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.  The Reversal of Fortunes: Trends in County Mortality and Cross-County Mortality Disparities in the United States  
PLoS Medicine  2008;5(4):e66.
Background
Counties are the smallest unit for which mortality data are routinely available, allowing consistent and comparable long-term analysis of trends in health disparities. Average life expectancy has steadily increased in the United States but there is limited information on long-term mortality trends in the US counties This study aimed to investigate trends in county mortality and cross-county mortality disparities, including the contributions of specific diseases to county level mortality trends.
Methods and Findings
We used mortality statistics (from the National Center for Health Statistics [NCHS]) and population (from the US Census) to estimate sex-specific life expectancy for US counties for every year between 1961 and 1999. Data for analyses in subsequent years were not provided to us by the NCHS. We calculated different metrics of cross-county mortality disparity, and also grouped counties on the basis of whether their mortality changed favorably or unfavorably relative to the national average. We estimated the probability of death from specific diseases for counties with above- or below-average mortality performance. We simulated the effect of cross-county migration on each county's life expectancy using a time-based simulation model. Between 1961 and 1999, the standard deviation (SD) of life expectancy across US counties was at its lowest in 1983, at 1.9 and 1.4 y for men and women, respectively. Cross-county life expectancy SD increased to 2.3 and 1.7 y in 1999. Between 1961 and 1983 no counties had a statistically significant increase in mortality; the major cause of mortality decline for both sexes was reduction in cardiovascular mortality. From 1983 to 1999, life expectancy declined significantly in 11 counties for men (by 1.3 y) and in 180 counties for women (by 1.3 y); another 48 (men) and 783 (women) counties had nonsignificant life expectancy decline. Life expectancy decline in both sexes was caused by increased mortality from lung cancer, chronic obstructive pulmonary disease (COPD), diabetes, and a range of other noncommunicable diseases, which were no longer compensated for by the decline in cardiovascular mortality. Higher HIV/AIDS and homicide deaths also contributed substantially to life expectancy decline for men, but not for women. Alternative specifications of the effects of migration showed that the rise in cross-county life expectancy SD was unlikely to be caused by migration.
Conclusions
There was a steady increase in mortality inequality across the US counties between 1983 and 1999, resulting from stagnation or increase in mortality among the worst-off segment of the population. Female mortality increased in a large number of counties, primarily because of chronic diseases related to smoking, overweight and obesity, and high blood pressure.
Majid Ezzati and colleagues analyze US county-level mortality data for 1961 to 1999, and find a steady increase in mortality inequality across counties between 1983 and 1999.
Editors' Summary
Background.
It has long been recognized that the number of years that distinct groups of people in the United States would be expected to live based on their current mortality patterns (“life expectancy”) varies enormously. For example, white Americans tend to live longer than black Americans, the poor tend to have shorter life expectancies than the wealthy, and women tend to outlive men. Where one lives might also be a factor that determines his or her life expectancy, because of social conditions and health programs in different parts of the country.
Why Was the Study Done?
While life expectancies have generally been rising across the United States over time, there is little information, especially over the long term, on the differences in life expectancies across different counties. The researchers therefore set out to examine whether there were different life expectancies across different US counties over the last four decades. The researchers chose to look at counties—the smallest geographic units for which data on death rates are collected in the US—because it allowed them to make comparisons between small subgroups of people that share the same administrative structure.
What Did the Researchers Do and Find?
The researchers looked at differences in death rates between all counties in US states plus the District of Columbia over four decades, from 1961 to 1999. They obtained the data on number of deaths from the National Center for Health Statistics, and they obtained data on the number of people living in each county from the US Census. The NCHS did not provide death data after 2001. They broke the death rates down by sex and by disease to assess trends over time for women and men, and for different causes of death.
Over these four decades, the researchers found that the overall US life expectancy increased from 67 to 74 years of age for men and from 74 to 80 years for women. Between 1961 and 1983 the death rate fell in both men and women, largely due to reductions in deaths from cardiovascular disease (heart disease and stroke). During this same period, 1961–1983, the differences in death rates among/across different counties fell. However, beginning in the early 1980s the differences in death rates among/across different counties began to increase. The worst-off counties no longer experienced a fall in death rates, and in a substantial number of counties, mortality actually increased, especially for women, a shift that the researchers call “the reversal of fortunes.” This stagnation in the worst-off counties was primarily caused by a slowdown or halt in the reduction of deaths from cardiovascular disease coupled with a moderate rise in a number of other diseases, such as lung cancer, chronic lung disease, and diabetes, in both men and women, and a rise in HIV/AIDS and homicide in men. The researchers' key finding, therefore, was that the differences in life expectancy across different counties initially narrowed and then widened.
What Do these Findings Mean?
The findings suggest that beginning in the early 1980s and continuing through 1999 those who were already disadvantaged did not benefit from the gains in life expectancy experienced by the advantaged, and some became even worse off. The study emphasizes how important it is to monitor health inequalities between different groups, in order to ensure that everyone—and not just the well-off—can experience gains in life expectancy. Although the “reversal of fortune” that the researchers found applied to only a minority of the population, the authors argue that their study results are troubling because an oft-stated aim of the US health system is the improvement of the health of “all people, and especially those at greater risk of health disparities” (see, for example http://www.cdc.gov/osi/goals/SIHPGPostcard.pdf).
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050066.
A study by Nancy Krieger and colleagues, published in PLoS Medicine in February 2008, documented a similar “fall and rise” in health inequities. Krieger and colleagues reported that the difference in health between rich and poor and between different racial/ethnic groups, as measured by rates of dying young and of infant deaths, shrank in the US from 1966 to 1980 then widened from 1980 to 2002
Murray and colleagues, in a 2006 PLoS Medicine article, calculated US mortality rates according to “race-county” units and divided into the “eight Americas,” and found disparities in life expectancy across them
The US Centers for Disease Control has an Office of Minority Health and Health Disparities. The office “aims to accelerate CDC's health impact in the US population and to eliminate health disparities for vulnerable populations as defined by race/ethnicity, socioeconomic status, geography, gender, age, disability status, risk status related to sex and gender, and among other populations identified to be at-risk for health disparities”
Wikipedia has a chapter on health disparities (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
In 2001 the US Agency for Healthcare Research and Quality sponsored a workshop on “strategies to reduce health disparities”
doi:10.1371/journal.pmed.0050066
PMCID: PMC2323303  PMID: 18433290
3.  G1961E mutant allele in the Stargardt disease gene ABCA4 causes bull’s eye maculopathy 
Experimental eye research  2009;89(1):16-24.
The aim of this study was to characterize the pathological and functional consequences of the G1961E mutant allele in the Stargardt disease gene ABCA4. Data from 15 patients were retrospectively reviewed and all the patients had at least one G1961E mutation. Comprehensive ophthalmic examination, full-field and pattern electroretinograms, and fundus autofluorescence (FAF) imaging were performed on all patients. Microperimetry, spectral-domain optical coherence tomography (OCT), and fluorescein angiography were performed in selected cases. Genetic screening was performed using the ABCR400 micro-array that currently detects 496 disctinct ABCA4 variants. All patients had normal full-field scotopic and photopic electroretinograms (ERGs) and abnormal pattern electroretinograms (PERGs) performed on both eyes, and all the fundi had bull’s eye maculopathy without retinal flecks on FAF. On OCT, one patient had disorganization of photoreceptor outer segment, two had outer nuclear layer (ONL) thinning likely due to photoreceptor atrophy proximal to the foveal center, and three had additional retinal pigment epithelium (RPE) atrophy. On microperimetry, six patients had eccentric superior fixation and amongst this group, five had an absolute scotoma in the foveal area. DNA analysis revealed that three patients were homozygous G1961E/G1961E and the rest were compound heterozygotes for G1961E and other ABCA4 mutations. The G1961E allele in either homozygosity or heterozygosity is associated with anatomical and functional pathologies limited to the parafoveal region and a trend to delayed onset of symptoms, relative to other manifestations of ABCA4 mutations. Our observations support the hypothesis that the G1961E allele contributes to localized macular changes rather than generalized retinal dysfunction, and is a cause of bull’s eye maculopathy in either the homozygosity or heterozygosity state. In addition, genetic testing provides precise diagnosis of the underlying maculopathy, and current non-invasive imaging techniques could be used to detect photoreceptor damage at the earliest clinical onset of the disease.
doi:10.1016/j.exer.2009.02.001
PMCID: PMC2742677  PMID: 19217903
Stargardt; mutation; maculopathy; retinal degeneration; dystrophy
4.  Trends and Projected Estimates of GHG Emissions from Indian Livestock in Comparisons with GHG Emissions from World and Developing Countries 
This study presents trends and projected estimates of methane and nitrous oxide emissions from livestock of India vis-à-vis world and developing countries over the period 1961 to 2010 estimated based on IPCC guidelines. World enteric methane emission (EME) increased by 54.3% (61.5 to 94.9 ×109 kg annually) from the year 1961 to 2010, and the highest annual growth rate (AGR) was noted for goat (2.0%), followed by buffalo (1.57%) and swine (1.53%). Global EME is projected to increase to 120×109 kg by 2050. The percentage increase in EME by Indian livestock was greater than world livestock (70.6% vs 54.3%) between the years 1961 to 2010, and AGR was highest for goat (1.91%), followed by buffalo (1.55%), swine (1.28%), sheep (1.25%) and cattle (0.70%). In India, total EME was projected to grow by 18.8×109 kg in 2050. Global methane emission from manure (MEM) increased from 6.81 ×109 kg in 1961 to 11.4×109 kg in 2010 (an increase of 67.6%), and is projected to grow to 15×109 kg by 2050. In India, the annual MEM increased from 0.52×109 kg to 1.1×109 kg (with an AGR of 1.57%) in this period, which could increase to 1.54×109 kg in 2050. Nitrous oxide emission from manure in India could be 21.4×106 kg in 2050 from 15.3×106 kg in 2010. The AGR of global GHG emissions changed a small extent (only 0.11%) from developed countries, but increased drastically (1.23%) for developing countries between the periods of 1961 to 2010. Major contributions to world GHG came from cattle (79.3%), swine (9.57%) and sheep (7.40%), and for developing countries from cattle (68.3%), buffalo (13.7%) and goat (5.4%). The increase of GHG emissions by Indian livestock was less (74% vs 82% over the period of 1961 to 2010) than the developing countries. With this trend, world GHG emissions could reach 3,520×109 kg CO2-eq by 2050 due to animal population growth driven by increased demands for meat and dairy products in the world.
doi:10.5713/ajas.2013.13342
PMCID: PMC4093536  PMID: 25049993
Greenhouse Gas; Methane; Nitrous Oxide; Livestock; India; Developing Countries
5.  Recent History of Ischaemic Heart Disease and Duodenal Ulcer in Doctors 
British Medical Journal  1968;3(5620):701-704.
Data are presented on the incidence of ischaemic (coronary) heart disease and duodenal ulcer among the several thousand male medical practitioners aged 35–64 holding immediate sickness benefit policies with the Medical Sickness Annuity and Life Assurance Society Limited. Three periods are considered: 1947–50, 1957–60, and 1961–5.
The incidence of first clinical episodes of ischaemic heart disease in the doctors altered little between 1947–50 and 1957–60 but increased in 1961–5. Comparison of the late 1940s with the early 1960s shows a 60% rise of incidence at ages 45–54 but little change at other ages. Cases first presenting as “sudden” death increased between 1947–50 and 1961–5 by 111% at 45–54, and again changed little at 55–64. In two other occupational groups that have been studied—bus conductors and insurance salesmen—the increase of incidence was greater than for the doctors at 45–54 and it occurred also over 55 years of age. The increase from 1947–50 to 1961–5 in mortality during all episodes of ischaemic heart disease was the same in the doctors as in the male population of England and Wales at 45–54, but at 55–64 it was less.
The results in the doctors are not due to alterations over the period in length of sickness absence, or underwriting policy, or of the nomenclature used on the certificates.
Well-documented changes in the smoking habits of doctors may be partly responsible for what appears to have been a relatively favourable experience of ischaemic heart disease from 1947–50 to 1961–5, especially at ages 55–64.
Incidence of duodenal ulcer at ages 35–64 declined steadily in this population of doctors from 1947–50 to 1961–5. The decline is very likely to be real.
PMCID: PMC1989645  PMID: 5673959
6.  Pharmacokinetics of A40926 in rats after single intravenous and subcutaneous doses. 
A40926 is a new glycopeptide antibiotic with unique activity against Neisseria gonorrhoeae and high and prolonged levels in mouse blood (B. P. Goldstein, E. Selva, L. Gastaldo, M. Berti, R. Pallanza, F. Ripamonti, P. Ferrari, M. Denaro, V. Arioli, and G. Cassani, Antimicrob. Agents Chemother., 31:1961-1966, 1987). We studied the pharmacokinetics of A40926 in rats after single intravenous and subcutaneous 10-mg/kg (body weight) doses. Concentrations in plasma and urine were determined by microbiological assay. After intravenous administration, high concentrations of A40926, ranging from 132 mg/liter at 3 min to 0.7 mg/liter at 96 h, were found in plasma. Concentrations declined with a three-exponential decay correlated with a prolonged, biphasic distribution and a slow elimination (terminal half-life, 61.22 h). After completion of the distribution, the compound was widely distributed to the extravascular space. The rate-limiting step in the elimination of A40926 from the body appears to be the slow return from the deep compartment into the central one. A40926 was rapidly absorbed from the injection site after subcutaneous administration, and its availability was close to 90%. The percentage of the dose excreted in urine in 120 h was 35.9%.
PMCID: PMC172143  PMID: 3364946
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  On the utility of the lognormal model for analysis of breast cancer survival in Sweden 1961-1973. 
British Journal of Cancer  1985;52(6):875-883.
Parametric models have been suggested as an alternative to conventional life table techniques for interpretation of observed survival patterns in cancer. This paper extends earlier work on breast cancer by studying the fit of Boag's lognormal model to the survival of 8,170 breast cancer cases reported to the Swedish Cancer Registry during 1961-1963. The model was also used to analyse the upward survival trend for breast cancer cases in Sweden during 1961-1973. The model fitted the 1961-1963 data well for the entire case material and for patients aged less than 70 years. It was therefore used to help explain whether the upward survival trend was due to long term cures or merely to protracted survival with cancer. The estimated cured proportion among patients aged less than 70 years rose from 33% +/- 2% (s.e.) during 1961-1963 to 40% +/- 3% for cases 1971-1973 (P less than 0.05). The median survival of uncured cases, was found to be similar during both periods, 4.5 and 4.6 years respectively. The model did not fit data for patients aged greater than 70 years. It is possibly too simplistic, and perhaps does not accurately describe the forces of mortality or their interactions in old patients. Another disadvantage is that large case materials are necessary in order to obtain estimates with reasonably small standard errors.
PMCID: PMC1977276  PMID: 4074639
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

Results 1-25 (878388)