Magnus Unemo

Composition of the Vaginal Microbiota in Women of Reproductive Age – Sensitive and Specific Molecular Diagnosis of Bacterial Vaginosis Is Possible?

Background and Objective Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis. Materials and Methods Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3–V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated. Results Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor. Conclusions Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.

Antimicrobial resistance in Neisseria gonorrhoeae: Global surveillance and a call for international collaborative action

In a Policy Forum, Teodora Wi and colleagues discuss the challenges of antimicrobial resistance in gonococci.

Molecular diagnostics for gonorrhoea: implications for antimicrobial resistance and the threat of untreatable gonorrhoea.

This Essay from Nicola Low and colleagues discusses the importance of the nucleic acid amplification tests for rapid detection of N. gonorrhoeae and its resistance determinants, as well as the importance of ensuring their rational use, as priorities for controlling both gonorrhoea and antimicrobial resistance. Please see later in the article for the Editors Summary

Multidrug-resistant gonorrhea: A research and development roadmap to discover new medicines

Emilie Alirol and colleagues discuss the development of new treatments for gonorrhea.

A Novel Factor H–Fc Chimeric Immunotherapeutic Molecule against Neisseria gonorrhoeae

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to almost every conventional antibiotic. There is an urgent need to develop novel therapies against gonorrhea. Many pathogens, including N. gonorrhoeae, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains 18–20 (FH18–20). We explored the use of fusing FH18–20 with IgG Fc (FH18–20/Fc) to create a novel anti-infective immunotherapeutic. FH18–20 also binds to select host glycosaminoglycans to limit unwanted complement activation on host cells. To identify mutation(s) in FH18–20 that eliminated complement activation on host cells, yet maintained binding to N. gonorrhoeae, we created four mutations in domains 19 or 20 described in atypical hemolytic uremic syndrome that prevented binding of mutated fH to human erythrocytes. One of the mutant proteins (D to G at position 1119 in domain 19; FHD1119G/Fc) facilitated complement-dependent killing of gonococci similar to unmodified FH18–20/Fc but, unlike FH18–20/Fc, did not lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15 sialylated clinical N. gonorrhoeae isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10 of 15 (67%) strains, and enhanced C3 deposition (≥10-fold above baseline levels) on each of the five isolates not directly killed by complement. FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistant strain by human polymorphonuclear neutrophils. FHD1119G/Fc administered intravaginally significantly reduced the duration and burden of gonococcal infection in the mouse vaginal colonization model. FHD1119G/Fc represents a novel immunotherapeutic against multidrug-resistant N. gonorrhoeae.

Test of cure for anogenital gonorrhoea using modern RNA-based and DNA-based nucleic acid amplification tests - a prospective cohort study

BACKGROUNDnThe use of nucleic acid amplification tests (NAATs) to diagnose Neisseria gonorrhoeae infections complicates the performance of a test of cure (TOC) to monitor treatment failure, if this is indicated. As evidence for the timing of TOC using modern NAATs is limited, we performed a prospective cohort study to assess time to clearance when using modern RNA- and DNA-based NAATs.nnnMETHODSnWe included patients with anogenital gonorrhoea visiting the Sexually Transmitted Infection Clinic Amsterdam from March through October 2014. After treatment with ceftriaxone mono- or dual therapy (with azithromycin or doxycycline), anal, vaginal, or urine samples were self-collected during 28 consecutive days, and analyzed using an RNA-based NAAT (Aptima Combo 2) and a DNA-based NAAT (Cobas 4800). Clearance was defined as 3 consecutive negative results, and blips as isolated positive results following clearance.nnnRESULTSnWe included 77 patients; 5 self-cleared gonorrhoea before treatment and 10 were lost to follow-up. Clearance rate of the remaining 62 patients was 100%. Median time to clearance was 2 days, with a range of 1-7 days for RNA-based NAAT and 1-15 days for DNA-based NAAT. The risk of finding a blip after clearance was 0.8% and 1.5%, respectively. One patient had a reinfection.nnnCONCLUSIONSnIf indicated, we recommend that TOC be performed for anogenital gonorrhoea at least 7 or 14 days after administering therapy, when using modern RNA- or DNA-based NAATs, respectively. When interpreting TOC results for possible treatment failure, both the occurrence of blips and a possible reinfection need to be taken into account.

2014 European guideline on the management of syphilis: giving evidence priority.

neurospyhilis displays today. In addition, viable T. pallidum or T. pallidum DNA are detected in CSF following standard BPG treatment in several cases. As a consequence, we should not resign ourselves to accept suboptimal results as those CDC/JUSTI guidelines provide, but we should look forward to defining an optimal therapeutic regimen, possibly by considering other antibiotics. We have recently adopted an enhanced treatment protocol for early/late syphilis, regardless of HIV infection, employing ceftriaxone and doxycycline after the recommended BPG treeatment. This regimen provides CSF treponemicidal levels and is suitable for administration in the outpatient setting. As regards the follow-up, the treatment response is currently assessed according to the decrease over time of the antibody titres. NTT titres are usually considered the gold standard. Regrettably, however, NTT titres do not decrease in all patients after treatment and might become non-reactive even without therapy or persist for a long time after therapy, even lifetime, and are, therefore, unreliable for the follow-up. We believe that TT should be added, especially in late latent syphilis (LLS), in which NTT are often negative. In our experience, among 37 LLS patients, only three were VDRL-positive and TT titres always decreased after therapy. Incidentally, we suggest that gastrointestinal involvement should be considered in secondary syphilis and the definition of early latent syphilis should be revised: seroconversion or fourfold or greater increase in titre of a NTT is indicative of early latent syphilis.

The Sequencing Bead Array (SBA), a Next-Generation Digital Suspension Array

Here we describe the novel Sequencing Bead Array (SBA), a complete assay for molecular diagnostics and typing applications. SBA is a digital suspension array using Next-Generation Sequencing (NGS), to replace conventional optical readout platforms. The technology allows for reducing the number of instruments required in a laboratory setting, where the same NGS instrument could be employed from whole-genome and targeted sequencing to SBA broad-range biomarker detection and genotyping. As proof-of-concept, a model assay was designed that could distinguish ten Human Papillomavirus (HPV) genotypes associated with cervical cancer progression. SBA was used to genotype 20 cervical tumor samples and, when compared with amplicon pyrosequencing, was able to detect two additional co-infections due to increased sensitivity. We also introduce in-house software Sphix, enabling easy accessibility and interpretation of results. The technology offers a multi-parallel, rapid, robust, and scalable system that is readily adaptable for a multitude of microarray diagnostic and typing applications, e.g. genetic signatures, single nucleotide polymorphisms (SNPs), structural variations, and immunoassays. SBA has the potential to dramatically change the way we perform probe-based applications, and allow for a smooth transition towards the technology offered by genomic sequencing.

Rapid accurate point-of-care tests combining diagnostics and antimicrobial resistance prediction for Neisseria gonorrhoeae and Mycoplasma genitalium

In addition to inadequate access to early diagnosis and treatment with antimicrobial agents for patients and sexual contacts, management and control of STIs is significantly challenged by emergence and spread of antimicrobial resistance (AMR), particularly for STIs such as Neisseria gonorrhoeae and Mycoplasma genitalium. This is further compounded by use of nucleic acid amplification techniques for diagnosis, resulting in reduced phenotypic AMR testing for N. gonorrhoeae and absence or suboptimal AMR surveillance for guiding treatment of both STIs in many settings. Rapid accurate point-of-care (POC) tests for diagnosis of all STIs would be valuable but to significantly impact treatment precision and management of N. gonorrhoeae and M. genitalium infections, combinations of rapid POC diagnostic and AMR testing (POC-AMR) will likely be required. This strategy would combat STI burden and AMR emergence and spread by enabling diagnosis and individualised treatment at the first healthcare visit, potentially reducing selection pressure on recommended antimicrobials, reducing transmission of resistant strains and providing means for AMR surveillance. Microfluidic and nanotechnology platforms under development for rapid detection of STIs provide a basis to also develop molecular rapid POC-AMR prediction. A number of prototypic devices are in the pipeline but none as yet approved for routine use. However, particularly for N. gonorrhoeae, more knowledge is required to assess which antimicrobials lend themselves to a genotypic POC-AMR approach, in relation to genotypic–phenotypic associations and potential impact clinically and epidemiologically. Key for successful deployment will include also understanding cost-effectiveness, cost-consequences and acceptability for key stakeholders.

O21.3 Fitness Studies on Neisseria Gonorrhoeae Harboring Mosaic penA Alleles from Ceftriaxone-Resistant Isolates Predict the Spread of Resistance to Extended-Spectrum Cephalosporins

Background Approximately 106 million cases of gonorrhoea occur worldwide each year. Gonorrhea significantly affects reproductive health and increases transmission of HIV. Antibiotic treatment is a critical control measure; however, this strategy is threatened by the rapid evolution of resistance in Neisseria gonorrhoeae(Gc). Gc susceptibility to ceftriaxone, the last remaining option for antibiotic monotherapy, has decreased globally over the last decade. Recently Gc has been elevated to “superbug” status due to the emergence of ceftriaxone-resistant (CROR) strains. Dual antibiotic therapy is now recommended in the USA and Europe. Ceftriaxone resistance in Gc is conferred primarily by mosaic penA alleles that encode an altered penicillin-binding protein 2 with up to 70 amino acid substitutions. Whether acquisition of these mosaic alleles is accompanied by a fitness cost is unknown. Methods and Results Here we examined the impact of mosaic penA alleles from two well-characterised CROR clinical isolates, H041 (MIC = 2–4 µg/ml) and F89 (MIC = 1–2 µg/ml), on Gcfitness in vitro and in vivo. The wild-type penA allele of laboratory strain FA19 (CROS) was replaced by penA41or penA89 to create mutants FA19 penA41 and FA19 penA89, respectively. Acquisition of the mosaic alleles increased ceftriaxone resistance ≥ 500-fold. Both mutants grew significantly slower than FA19 in liquid culture. When cultured competitively with the parent strain, FA19 penA41 and FA19 penA89 demonstrated a fitness defect, as measured by competitive index. Mutants were attenuated relative to the parent strain during competitive murine infection. However, only CROR bacteria were recovered at later time points from 3 of 7 mice co-inoculated with FA19 penA41andFA19, suggesting selection of compensatory mutations in vivo. Conclusions Acquisition ofmosaic alleles significantly reduced fitness of Gc, but compensatory mutations can be selected in vivo that alleviate fitness defects while maintaining resistance. Our studies may be useful in predicting the national and international spread of CROR Gc.