Human and Pathogen Factors Associated with Chlamydia trachomatis-Related Infertility in Women.

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen worldwide. Infection can result in serious reproductive pathologies, including pelvic inflammatory disease, ectopic pregnancy, and infertility, in women. However, the processes that result in these reproductive pathologies have not been well defined. Here we review the evidence for the human disease burden of these chlamydial reproductive pathologies. We then review human-based evidence that links Chlamydia with reproductive pathologies in women. We present data supporting the idea that host, immunological, epidemiological, and pathogen factors may all contribute to the development of infertility. Specifically, we review the existing evidence that host and pathogen genotypes, host hormone status, age of sexual debut, sexual behavior, coinfections, and repeat infections are all likely to be contributory factors in development of infertility. Pathogen factors such as infectious burden, treatment failure, and tissue tropisms or ascension capacity are also potential contributory factors. We present four possible processes of pathology development and how these processes are supported by the published data. We highlight the limitations of the evidence and propose future studies that could improve our understanding of how chlamydial infertility in women occurs and possible future interventions to reduce this disease burden.

Effects of vitamin D2 or D3 supplementation on glycaemic control and cardiometabolic risk among people at risk of type 2 diabetes: results of a randomized double-blind placebo-controlled trial.

AIMS: To investigate the effect of short-term vitamin D supplementation on cardiometabolic outcomes among individuals with an elevated risk of diabetes. METHODS: In a double-blind placebo-controlled randomized trial, 340 adults who had an elevated risk of type 2 diabetes (non-diabetic hyperglycaemia or positive diabetes risk score) were randomized to either placebo, 100,000 IU vitamin D2 (ergocalciferol) or 100,000 IU vitamin D3 (cholecalciferol), orally administered monthly for 4 months. The primary outcome was change in glycated haemoglobin (HbA1c) between baseline and 4 months, adjusted for baseline. Secondary outcomes included: blood pressure; lipid levels; apolipoprotein levels; C-reactive protein levels; pulse wave velocity (PWV); anthropometric measures; and safety of the supplementation. RESULTS: The mean [standard deviation (s.d.)] 25-hydroxyvitamin D [25(OH)D]2 concentration increased from 5.2 (4.1) to 53.9 (18.5) nmol/l in the D2 group, and the mean (s.d.) 25(OH)D3 concentration increased from 45.8 (22.6) to 83.8 (22.7) nmol/l in the D3 group. There was no effect of vitamin D supplementation on HbA1c: D2 versus placebo: -0.05% [95% confidence interval (CI) -0.11, 0.02] or -0.51 mmol/mol (95% CI -1.16, 0.14; p = 0.13); D3 versus placebo: 0.02% (95% CI -0.04, 0.08) or 0.19 mmol/mol (95% CI -0.46, 0.83; p = 0.57). There were no clinically meaningful effects on secondary outcomes, except PWV [D2 versus placebo: -0.68 m/s (95% CI -1.31, -0.05); D3 versus placebo -0.73 m/s (95% CI -1.42, -0.03)]. No important safety issues were identified. CONCLUSIONS: Short-term supplementation with vitamin D2 or D3 had no effect on HbA1c. The modest reduction in PWV with both D2 and D3 relative to placebo suggests that vitamin D supplementation has a beneficial effect on arterial stiffness.

Sero-epidemiological assessment of Chlamydia trachomatis infection and sub-fertility in Samoan women.

BACKGROUND: In our recent village-based cross-sectional study, the prevalence of nucleic acid amplification technique (NAAT) diagnosed Chlamydia trachomatis (CT) in sexually active Samoan women was very high (36 %), and test positivity was associated with sub-fertility. We conducted a serological and epidemiological analysis in these participants to identify if serological data can provide further insight into the potential contribution of CT to sub-fertility in this population. METHODS: Serological prediction of CT associated sub-fertility was conducted using a series of commercial tests. The correlation between fertility or sub-fertility, behavioral factors, and serologically predicted CT associated sub-fertility was determined. RESULTS: A positive antibody reaction against the Chlamydia Major Outer Membrane Protein (MOMP) was significantly associated with sub-fertility, with 50 % of infertile women being positive. Serum IgG and IgA antibodies against MOMP correlated with current infection measured by urine NAAT, suggesting longer term infections are common in this population. Chlamydia pneumoniae antibodies were frequently detected in this population (84 %), and unexpectedly, were significantly associated with sub-fertility. CONCLUSIONS: The high prevalence of chlamydial infection and of positive chlamydial sub-fertility results suggests that CT is an important and frequent contributory factor to sub-fertility in this population.

Changes in vitamin D and matrix metalloproteinase-9 in submariners during a submerged patrol.

OBJECTIVES: To quantify changes in vitamin D and matrix metalloproteinase-9 (MMP9) in submariners over a single long patrol and compare the data to a group of non-deploying servicemen from their base port. METHODS: A prospective time-series analysis was performed. Blood samples were taken from 49 submariners deploying on patrol and 43 shore-side controls from the base port (naval officers from base or non-deploying submariners), following a winter ashore at latitude 56° north. Samples were drawn immediately before the submarine sailed, in January, and again in the final week of patrol 85 days later. Paired pre-patrol and late samples from each individual were assayed together and changes in vitamin D and MMP9 were assessed. RESULTS: Mean pre-patrol vitamin D concentrations were 58 and 49 nmol/L for the controls and submariners, respectively. Mean vitamin D concentrations increased in controls as expected (mean increase 12.6 nmol/L), but not in the submariners (mean decrease 1.6 nmol/L). MMP9 levels were significantly higher in submariners pre-patrol, and increased significantly during the patrol. There was a significant inverse correlation between MMP9 and vitamin D levels (r=-0.41, p=0.01). CONCLUSIONS: This is the first study to quantify vitamin D and MMP levels in submariners. Circulating vitamin D concentrations on board were insufficient to prevent a rise in MMP. This has potential for adverse health effects and requires further study.

The relationship of serum anti-Mullerian hormone with polycystic ovarian morphology and polycystic ovary syndrome: a prospective cohort study.

STUDY QUESTION: What is the relationship of serum anti-Mullerian hormone (AMH) with polycystic ovarian morphology (PCOM) and polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Serum AMH concentrations are capable of differentiating between normal ovaries, PCOM and PCOS. WHAT IS KNOWN ALREADY: Serum AMH levels are high in PCOS reflecting the number of small antral follicles and an intrinsic defect of individual granulosa cells. STUDY DESIGN, SIZE, DURATION: Data were collected prospectively and analysed from three groups of women: those with PCOS according to Rotterdam criteria, those with PCOM but no symptoms and those with normal ovaries. PARTICIPANTS/MATERIAL, SETTING, METHODS: Women with PCOS (n = 90), with PCOM (n = 35) and with normal ovaries (controls, n = 90), matched for age and body mass index, were all being treated for infertility at Homerton University Hospital, a tertiary referral centre. MAIN RESULTS AND THE ROLE OF CHANCE: Using adequate numbers and statistical methods for demographically similar groups, there were significant differences in the mean serum AMH concentrations between women with PCOS [77.6 pmol/l (95% CI 64.8-90.3)], those with PCOM [52.2 pmol/l (95% CI 40.1-64.2)] and controls [23.6 pmol/l (95% CI 20.5-26.7)] (P < 0.001). The combination of AMH >48 pmol/l and LH > 6 IU/l diagnosed 82.6% of women with PCOS. The mean serum FSH was lower in both PCOS and PCOM compared with controls, whereas LH was higher in PCOS compared with PCOM and controls and correlated positively with AMH (r = 0.321, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: Further research is needed to determine the relationship of AMH, PCOS and PCOM. The study was restricted to women who sought out treatment for infertility. WIDER IMPLICATIONS OF THE FINDINGS: The study suggests that the severity of symptoms of PCOS is positively related to the number of small follicles and that AMH may play an important part in the pathophysiology of anovulation. STUDY FUNDING/COMPETING INTEREST: None.

Second European multi-disciplinary conference of national strategies for Chlamydia trach. and human papillomavirus NSCP conf. in Berlin, 2013 enhanced detection, management and surveillance of sexually transmitted infections in Europe are essential!

There is a need for updated guidance on detection, management and surveillance of sexually transmitted infections (STIs). Chlamydia, gonorrhoea and syphilisreporting needs to be mandatory in more European countries to aid collection of data. More widespread Chlamydia screening is needed in many countries as this is the only way to reduce complications. The role of Human Papillomavirus (HPV) screening in a situation where the prevalence of HPV infection has dropped significantly was also discussed in the context of the high cost of screening, the need for a relatively complex infrastructure, particularly in developing countries, and falling vaccination costs. An integrated HPV vaccination and screening policy could be the most appropriate with vaccination at 9-13 years as recommended by WHO and a single HPV screen at 35-39 years, possibly repeated thereafter every 10 years. Female and male HPV vaccination programmes could lead to near elimination of genital warts in both females and males. Surveillance of STIsshould be intensified where needed; additional or better quality data should be collected including reasons for testing, denominator data to estimate positivity rates, diagnostic methods, concurrent STIs, sexual orientation and country of acquisition; more analytical rather than descriptive epidemiology is needed.

Epidemiology of Chlamydia psittaci infections in pregnant Thoroughbred mares and foals.

Late-term foal loss due to the traditional avian pathogen Chlamydia psittaci recently emerged as a threat to the Australian Thoroughbred industry. A longitudinal study of 14 stud farms was undertaken to better understand C. psittaci infection in pregnant mares and their foals by evaluating C. psittaci prevalence, equine herpesvirus-1 (EHV-1) co-infection, avian reservoirs, and potential risk factors. Mucosal swabs taken from 228 healthy pregnant mares and their foals were tested for C. psittaci and EHV-1 using species-specific qPCR assays. No foal loss was recorded due to either pathogen, and no mare tested positive to either C. psittaci or EHV-1. However, healthy newborn foals tested positive to both pathogens, at low levels, with 13.2% (n = 30/228) and 14.5% (n = 33/228) prevalence for C. psittaci and EHV-1, respectively. Co-infection occurred in 1.3% (n = 3/228) of foals. In avian environmental faecal samples collected from the same studs, C. psittaci was detected at 5.3% (n = 5/94). Multiple logistic regression modelling found that foals born in winter were more likely to be infected with C. psittaci (adjusted odds ratio = 15.83; P < 0.001; Confidence Interval 5.12-48.49). Being a maiden mare, absence of prophylactic vaginal suture, interventions in the last trimester and residing on a farm with prior history of C. psittaci abortion posed no higher risk to infection in the newborn. Analysis of all reported C. psittaci abortion cases (Hunter Valley, 2016-2019) revealed a dominant C. psittaci sequence type (denoted ST24) and a significant correlation with frost events (Spearmans' rho = 0.44; P = 0.002).

Evidence that human Chlamydia pneumoniae was zoonotically acquired.

Zoonotic infections are a growing threat to global health. Chlamydia pneumoniae is a major human pathogen that is widespread in human populations, causing acute respiratory disease, and has been associated with chronic disease. C. pneumoniae was first identified solely in human populations; however, its host range now includes other mammals, marsupials, amphibians, and reptiles. Australian koalas (Phascolarctos cinereus) are widely infected with two species of Chlamydia, C. pecorum and C. pneumoniae. Transmission of C. pneumoniae between animals and humans has not been reported; however, two other chlamydial species, C. psittaci and C. abortus, are known zoonotic pathogens. We have sequenced the 1,241,024-bp chromosome and a 7.5-kb cryptic chlamydial plasmid of the koala strain of C. pneumoniae (LPCoLN) using the whole-genome shotgun method. Comparative genomic analysis, including pseudogene and single-nucleotide polymorphism (SNP) distribution, and phylogenetic analysis of conserved genes and SNPs against the human isolates of C. pneumoniae show that the LPCoLN isolate is basal to human isolates. Thus, we propose based on compelling genomic and phylogenetic evidence that humans were originally infected zoonotically by an animal isolate(s) of C. pneumoniae which adapted to humans primarily through the processes of gene decay and plasmid loss, to the point where the animal reservoir is no longer required for transmission.

Influence of a single oral dose of vitamin D(2) on serum 25-hydroxyvitamin D concentrations in tuberculosis patients.

SETTING: Newham Chest Clinic, London, UK. OBJECTIVE: To determine the safety and efficacy of the administration of bolus-dose vitamin D(2) in elevating serum 25-hydroxyvitamin D (25[OH]D) concentrations in tuberculosis (TB) patients. DESIGN: A multi-ethnic cohort of TB patients was randomised to receive a single oral dose of 2.5 mg vitamin D(2) (n = 11) or placebo (n = 14). Serum 25(OH)D and corrected calcium concentrations were determined at baseline and 1 week and 8 weeks post-dose, and compared to those of a multi-ethnic cohort of 56 healthy adults receiving an identical dose of vitamin D(2). RESULTS: Hypovitaminosis D (serum 25[OH]D < 75 nmol/l) was present in all patients at baseline. A single oral dose of 2.5 mg vitamin D2 corrected hypovitaminosis D in all patients in the intervention arm of the study at 1 week post-dose, and induced a 109.5 nmol/l mean increase in their serum 25(OH)D concentration. Hypovitaminosis D recurred in 10/11 patients at 8 weeks post-dose. No patient receiving vitamin D(2) experienced hypercalcaemia. Patients receiving 2.5 mg vitamin D(2) experienced a greater mean increase in serum 25(OH)D at 1 week post-dose than healthy adults receiving 2.5 mg vitamin D(2). CONCLUSION: A single oral dose of 2.5 mg vitamin D(2) corrects hypovitaminosis D at 1 week but not at 8 weeks post-dose in TB patients.

Antibody response against koala retrovirus (KoRV) in koalas harboring KoRV-A in the presence or absence of KoRV-B.

Koala retrovirus (KoRV) is in the process of endogenization into the koala (Phascolarctos cinereus) genome and is currently spreading through the Australian koala population. Understanding how the koala's immune system responds to KoRV infection is critical for developing an efficacious vaccine to protect koalas. To this end, we analyzed the antibody response of 235 wild koalas, sampled longitudinally over a four-year period, that harbored KoRV-A, and with or without KoRV-B. We found that the majority of the sampled koalas were able to make anti-KoRV antibodies, and that there was a linear increase in anti-KoRV IgG levels in koalas up to approximately seven years of age and then a gradual decrease thereafter. Koalas infected with both KoRV-A and KoRV-B were found to have slightly higher anti-KoRV IgG titers than koalas with KoRV-A alone and there was an inverse relationship between anti-KoRV IgG levels and circulating KoRV viral load. Finally, we identified distinct epitopes on the KoRV envelope protein that were recognized by antibodies. Together, these findings provide insight into the koala's immune response to KoRV and may be useful in the development of a therapeutic KoRV vaccine.

Induction of neutralizing antibody response against koala retrovirus (KoRV) and reduction in viral load in koalas following vaccination with recombinant KoRV envelope protein.

Koala retrovirus (KoRV) infects the majority of Australia's koalas (Phascolarctos cinereus) and has been linked to several life-threatening diseases such as lymphoma and leukemia, as well as Chlamydia and thus poses a threat to the continued survival of this species. While quarantine and antiretroviral drug treatment are possible control measures, they are impractical, leaving vaccination as the only realistic option. In this study, we examined the effect of a recombinant envelope protein-based anti-KoRV vaccine in two groups of South Australian koalas: KoRV infected or KoRV free. We report a successful vaccination response in the koalas with no vaccine-associated side effects. The vaccine induced a significant humoral immune response as well as the production of neutralizing antibodies in both groups of koalas. We also identified B-cell epitopes that were differentially recognized in KoRV-infected versus KoRV-free koalas following vaccination. Importantly, we also showed that vaccination had a therapeutic effect on koalas infected exogenously with KoRV by reducing their circulating viral load. Together, this study highlights the possibility of successfully developing a vaccine against KoRV infection in koalas.

Anti-P. gingivalis response correlates with atherosclerosis.

Significant associations between atherosclerosis and both Porphyromonas gingivalis, a major periodontopathogen, and the respiratory pathogen, Chlamydia pneumoniae, have been shown. Many individuals with evidence of atherosclerosis demonstrate seropositivity to these pathogens. The aim of the present study was to examine the atherogenic effect of repeated immunizations with either or both of these agents, and to determine if molecular mimicry of bacterial heat-shock protein (HSP), termed GroEL, and host (h) HSP60 was involved. Atherogenesis was examined in apolipoprotein-E-deficient (-/-) mice following intraperitoneal immunizations with P. gingivalis, C. pneumoniae, P. gingivalis, and C. pneumoniae or vehicle. Lesion area in the proximal aorta and levels of serum antibodies to P. gingivalis, C. pneumoniae, and GroEL were measured. The increased pathogen burden of P. gingivalis, but not of C. pneumoniae, enhanced atherosclerosis. hHSP60 was detected in lesions, and in P. gingivalis-immunized mice, lesion development was correlated with anti-GroEL antibody levels, supporting the involvement of molecular mimicry between GroEL and hHSP60.

Development of a quantitative gene expression assay for Chlamydia trachomatis identified temporal expression of sigma factors.

Chlamydia trachomatis is an important human pathogen which possesses a unique bi-phasic developmental cycle. We used lightcycler methodology to quantitatively measure gene transcript levels in C. trachomatis strain L2. By measuring 16S rRNA transcript levels, we determined C. trachomatis L2 to have a generation time of approximately 3 h and an inclusion burst size of 200-300 particles. The three chlamydial sigma factor genes rpoD (sigma66), rpsD (sigma28) and rpoN (sigma54) exhibited different patterns of temporal expression. rpoD was central to early chlamydial development, whereas rpsD and rpoN were temporally expressed, coinciding with elementary body (EB) to reticulate body (RB) conversion and RB to EB conversion, respectively.

cDNA clone encoding a high molecular weight antigen of Babesia bovis.

An expression library was constructed by inserting cDNA copied from mRNA of the blood stages of Babesia bovis isolate KA into bacteriophage lambda gt11-amp3. An antigen-positive cDNA clone detected by screening the library with antibodies from cattle vaccinated with the KA isolate was shown to encode part of a high-molecular weight polypeptide antigen of B. bovis. This molecule was a dominant immunogen and was found by immunofluorescence to be within the parasite in infected erythrocytes.

DNA polymorphisms and subpopulations in Babesia bovis.

Independent isolates of Babesia bovis differ by only a limited number of polypeptides, some of which may be important as host protective antigens. Avirulent derivatives of these parasites also differ from their virulent counterparts in only a few polypeptides. To identify genes encoding such polypeptides we have isolated cDNA clones corresponding to poly(A)+ RNAs that are expressed only in certain isolates. For this purpose a cDNA clone library was constructed from poly(A)+ RNA of the K-avirulent isolate (KA). These clones were screened by colony hybridization using [32P]cDNA complementary to poly(A)+ RNA from KA and from virulent isolates, in order to identify clones that selectively hybridize to one cDNA probe. Hybridization of DNA from three clones, designated pK4, pK5 and pK6 to poly(A)+ RNA from various isolates revealed different and complex patterns. The gene represented by clone pK5 appeared to be transcribed predominantly in avirulent parasites. Analysis of genomic DNA by the Southern procedure enabled each isolate to be distinguished and suggested that most isolates are comprised of a heterogeneous mixture of subpopulations. Analysis of genomic DNA from parasites obtained after passage of KA through the tick vector (Boophilus microplus) suggested that a subpopulation was being selected that more closely resembled KV than KA.

Development of ligase-assisted spacer addition for the measurement of microsatellites.

Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation detection. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capabilities as a genotyping method.

Reduced levels of gamma-interferon secretion in response to chlamydial 60 kDa heat shock protein amongst women with pelvic inflammatory disease and a history of repeated Chlamydia trachomatis infections.

Peripheral lymphocytes in uninfected fertile controls, women with various histories of Chlamydia trachomatis infection, pelvic inflammatory disease (PID) and infertility not due to C. trachomatis infection (endometriosis) were cultured overnight with PHA mitogen and the 60 kDa chlamydial heat shock protein. Plasma samples were then assayed for levels of gamma-interferon and IL-10 using a commercial ELISA system. Women with PID and those with a history of multiple C. trachomatis infections showed reduced gamma-interferon production in response to cHSP60, not seen in women infected only once and those with infertility due to other causes (endometriosis). Secretion of IL-10 in response to cHSP60 did not vary significantly across the various patient groups, though all patients showed elevated levels of total IL-10 compared with uninfected controls.

Chlamydia trachomatis infection: incidence, health costs and prospects for vaccine development.

Chlamydia trachomatis infection is now the most common sexually transmitted disease worldwide. World Health Organisation figures estimated that 89 million new cases of genital Chlamydia infections occurred in 1995, highlighting the worldwide prevalence of infections and the economic burden on healthcare delivery. A number of methods have been developed for detection of chlamydial infection, which vary in sensitivity and specificity. No single method has yet gained general acceptance and in many countries Chlamydia infections are not reported, suggesting that the above figures may be an underestimate of the problem. As yet there is no consensus as to what constitutes a protective immune response against genital Chlamydia infection. Studies in animal models have shown that cell-mediated immunity, both Th1-driven macrophage activation and cytotoxic T cell responses, as well as antibody can mediate protection at different stages of the chlamydial life cycle. A successful vaccine would probably need to elicit both a systemic cell-mediated immune response to limit/resolve established infections and a mucosal IgA response to reduce bacterial shedding and the resulting spread of infection to partners of infected individuals. The immune response to Chlamydia, either through natural infection or following immunisation, also has the potential to enhance inflammation and to act as a driving force for constant mutation in the variable regions of the major outer membrane protein. As a result a constant prevalence of infection is maintained even in an immune population through the emergence of new allelic variants. Immune responses against antigens such as the 60 kDa heat shock protein can exacerbate inflammation through molecular mimicry and must not be elicited as a result of vaccination. Thus there are many challenges for the development of a successful vaccine which must elicit immunity against multiple serovars while at the same time minimising damaging pro-inflammatory immune responses.

Failure of metronidazole in a patient with hepatic amebic abscess.

The therapeutic problems of multiple amebic liver abscesses in a patient who failed to respond to metronidazole are described, and the importance of an early change to an alternative therapy is emphasized.