Macrolide resistance in Mycoplasma genitalium in Catalonia, Spain: a 1 year prospective study.

BACKGROUND: Mycoplasma genitalium is an emergent cause of sexually transmitted disease (STD). The first-line treatment is azithromycin, but macrolide resistance is increasing due to mutations in the 23S rRNA gene. OBJECTIVES: To determine the rates of M. genitalium infection and macrolide resistance in an area adjacent to Barcelona. METHODS: This 1 year prospective study was performed in a heterogenous population that included both low- and high-risk patients. M. genitalium was detected in all specimens sent to our institution for STD detection. Epidemiological and relevant clinical data were collected in the positive cases. Characterization of macrolide-associated resistance was performed by 23S rDNA sequencing. RESULTS: Of the 3540 patients included, 132 (3.7%) were positive for M. genitalium. Another sexually transmitted bacteria was detected in 20.4% of the M. genitalium cases, and Chlamydia trachomatis (11%) was the most frequently co-detected microorganism. Only 61.4% of patients received an adequate initial treatment against M. genitalium. The test of cure (TOC) was performed in 42% of patients, and therapeutic failure was detected in 10 cases. The rate of macrolide resistance was 12.6% and the most prevalent mutation was A2058G. There was an association between macrolide resistance and a previous history of M. genitalium detection (P < 0.001). CONCLUSIONS: Our results support the contribution of the previous use of macrolides in resistant strains. Given the difficulties in performing TOC in all patients, the inclusion of macrolide resistance in the detection test should be mandatory.

Contingent screening for Down syndrome completed in the first trimester: a multicenter study.

OBJECTIVE: To assess a new contingent screening strategy for Down syndrome completed in the first trimester. METHODS: First-trimester screening combining nuchal translucency thickness measurement and assessment of serum analytes (combined test) was offered to pregnant women who presented for prenatal care during the first trimester to nine health centers and community hospitals in the area served by the Catalan Public Health Service. If an intermediate risk (1/101-1/1000) for Down syndrome was identified, women were referred to the Hospital Clinic Barcelona for risk reassessment that included the use of secondary ultrasound markers (nasal bone, ductus venosus blood flow and tricuspid flow). Intermediate-risk women were divided into two subgroups for further analysis: high-intermediate risk (1/101-1/250) and low-intermediate risk (1/251-1/1000). We compared feasibility and efficacy of both combined and contingent screening strategies. RESULTS: The combined test, the first screening stage, was performed in 16 001 pregnant women, of whom 1617 (10.1%) were found to have an intermediate risk. Further division of this group showed that 1.8% (n = 289) of women were at high-intermediate risk and 8.3% (n = 1328) at low-intermediate risk. The contingent screening strategy significantly reduced the false-positive rate, from 3.0% to 1.3-1.8% (P < 0.001), without affecting the detection rate (which was 75-79% and 76%, with and without the contingent screening strategy, respectively). However, only 45% of intermediate-risk patients underwent the second screening step due to a preference among high-intermediate-risk (1/101-1/250) women for invasive testing and to low uptake among low-intermediate-risk (1/251-1/1000) women. CONCLUSIONS: The proposed first-trimester contingent strategy reduces the screen false-positive rate without impacting on the detection rate of Down syndrome. The low compliance observed in our study may prevent its use in certain populations.

Changes in Corneal Expression of MUC5AC after Autologous Serum Eyedrop Treatment in Patients with Limbal Stem Cell Deficiency.

Purpose: To evaluate the effect of autologous serum eyedrops treatment on corneal expression of the MUC5AC in patients with limbal deficiency. Methods: A prospective and comparative interventional case series study of 42 eyes of 21 patients was performed before and 8 weeks after treatment with autologous serum. All patients underwent a complete ophthalmic assessment of the tear film and ocular surface, corneal impression cytology (IC) and MUC5AC detection by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results: Forty-one of the 42 eyes studied were available for both conventional cytology and MUC5AC analysis prior to and after treatment. Differences between outcomes obtained by impression cytology and MUC5AC detection were found in 9 of 82 samples (11%). We found changes in the corneal expression of MUC5AC after treatment in 19 of 41 eyes (46.3%): 18 of them (94.7%) changed from positive to negative expression, and 1 eye (5.3%) changed from negative to positive MUC5AC expression after autologous serum eyedrops. These changes were related with the corneal involvement prior to treatment (15 of them (78.9%) occurred in patients with slight corneal involvement), and with the improvement in the degree of squamous metaplasia after treatment (P = .001 and P = .003, respectively). Conclusions: The treatment significantly improved tear stability, squamous metaplasia, and subjective patient perception. Autologous serum eyedrops treatment diminished the corneal expression of MUC5AC mainly in patients with slight corneal involvement before treatment.

[Protected penetrating keratoplasty: surgical technique and endothelial response]. / Queratoplastia penetrante protegida: técnica quirúrgica y respuesta del endotelio.

PURPOSE: To describe the technique and to evaluate the safety of protected penetrating keratoplasty (PPK). METHODS: A technique for penetrating keratoplasty is described. The postoperative endothelial cell counts of 17 eyes in which this operative technique was used were compared with those in 24 eyes in whom the standard operative technique for penetrating keratoplasty (PK) was used. The post-operative time periods were grouped as follows: 3-6 months, 7-12 months and >12 months. For statistical analysis, the Mann-Whitney U non-parametric test was employed. RESULTS: There was no case where tissue extrusion occurred during the procedure. The endothelial cell count was similar in both groups for the 3-6 month period (PK = 2,086, DS 566; PPK = 1,858, DS 671; p = 0.2702) and >12 months period (PK = 1,574, DS 745; PPK = 1,419, DS 810; p = 0.2882). There was a significant difference in the 7-12 month period (PK = 2,255, DS 831; PPK = 1,569, DS 623; p = 0.0397). CONCLUSIONS: The described technique of PPK may reduce the risk of per-operative complications. Damage to the endothelium is not increased compared with that seen following the standard PK procedure.

Keratoconus: an inflammatory disorder?

Keratoconus has been classically defined as a progressive, non-inflammatory condition, which produces a thinning and steepening of the cornea. Its pathophysiological mechanisms have been investigated for a long time. Both genetic and environmental factors have been associated with the disease. Recent studies have shown a significant role of proteolytic enzymes, cytokines, and free radicals; therefore, although keratoconus does not meet all the classic criteria for an inflammatory disease, the lack of inflammation has been questioned. The majority of studies in the tears of patients with keratoconus have found increased levels of interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α), and matrix metalloproteinase (MMP)-9. Eye rubbing, a proven risk factor for keratoconus, has been also shown recently to increase the tear levels of MMP-13, IL-6, and TNF-α. In the tear fluid of patients with ocular rosacea, IL-1α and MMP-9 have been reported to be significantly elevated, and cases of inferior corneal thinning, resembling keratoconus, have been reported. We performed a literature review of published biochemical changes in keratoconus that would support that this could be, at least in part, an inflammatory condition.

Tear proteome and protein network analyses reveal a novel pentamarker panel for tear film characterization in dry eye and meibomian gland dysfunction.

Dry eye and meibomian gland dysfunction are common ocular surface disorders. Discrimination of both conditions often may be difficult given the overlapping of signs and symptoms, and the lack of correlation with clinical parameters. A total of 144 individuals were included in this study. To search for proteome differences, tear proteins were collected by Merocel sponge and analyzed using 2D-PAGE. Comparative tear protein profile analysis indicated changes in the expression levels of fifteen proteins. Subsequent to MALDI-TOF/TOF protein identification, network analysis revealed expression/interaction connections with other proteins, thereby identifying additional putative markers. A screening validation assay demonstrated the discriminative power of six candidate biomarkers. A further validation study using multiplexed-like ELISA assays in tear samples collected with both sponge and capillary confirmed the high discriminatory power of five biomarkers: S100A6, annexin A1 (ANXA1), annexin A11 (ANXA11), cystatin-S (CST4), and phospholipase A2-activating protein (PLAA) with an area under ROC curve (AUC)≥ 97.9% (sensitivity ≥ 94.3%; specificity ≥ 97.6%) when comparing dry eye and control individuals. This panel also discriminated between dry eye, meibomian gland dysfunction and control individuals, with a global correct assignment (CA) of 73.2% between all groups. Correct assignment was not found to be significantly dependent on the tear collection method.

[Allergic mediators in tear from children with seasonal and perennial allergic conjunctivitis]. / Mediadores alérgicos en lágrimas de niños con conjuntivitis alérgica estacional y perenne.

PURPOSE: To evaluate the concentration of allergic mediators in tears of children with seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC) compared with controls. METHODS: Twenty children with allergic conjunctivitis (17 SAC, and 3 PAC) and sixteen healthy children were included in this study. Tear samples were collected using a Merocel sponge (Oasis, 0525), and immediately eluted by incubation in elution buffer and subsequent centrifugation at 20,000 rpm for 30 min at 4°C. Concentrations of histamine (HIS), tryptase (TPS), eosinophil chemotactic factor (ECF), major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), IgE and E-selectin were measured using enzyme linked immunosorbent assays (ELISA). Data were compared with the Mann-Whitney U test (P<.05), and multivariate analyses were also performed. RESULTS: Tear levels of TPS (P=.014), MBP (P=.032), ECP (P=.0041), IgE (P=.014) and EDN (P=.00077) showed significant differences in children with SAC and PAC compared to controls. CONCLUSION: The simultaneous analyses of allergic mediators in the tears of children with SAC and PAC showed a significant elevated concentration in EDN, ECP and MBP in allergic group and decreased levels in IgE and TPS. Statistical analyses showed a diagnostic accuracy of 94.4% using the eight molecules panel.

Changes in tear protein profile in keratoconus disease.

PURPOSE: To analyze tear protein profile variations in patients with keratoconus (KC) and to compare them with those of control subjects. SUBJECTS AND METHODS: Tears from 12 normal subjects and 12 patients with KC were analyzed by two-dimensional gel electrophoresis (2-DE) and liquid chromatography-mass spectrometry (LC-MS). Analysis of the 2-DE gels was performed using Progenesis SameSpots software (Nonlinear Dynamics). Proteins exhibiting high variation in expression levels (P-value <0.05) were identified using matrix-assisted laser desorption/ionization-TOF spectrometry. For LC-MS analysis, a label-free quantification approach was used. Tears were digested with trypsin, subjected to data-independent acquisition (MS(E)) analysis, and identified proteins were relatively quantified using ProteinLynx Global Server software (Waters). RESULTS: The 2-DE and LC-MS analyses revealed a significant decrease in the levels of members of the cystatin family and an increase in lipocalin-1 in KC patients. A 1.43-fold decrease was observed for cystatin-S by 2-DE, and 1.69- and 1.56-fold for cystatin-SN and cystatin-SA by LC-MS, respectively. The increase in lipocalin-1 was observed by both methods with fold changes of 1.26 in the 2-DE approach and 1.31 according to LC-MS. Significant protein upregulation was also observed for Ig-κ chain C and Ig J chain proteins by 2-DE. Levels of lipophilin-C, lipophilin-A, and phospholipase A2 were decreased in tears from KC patients according to LC-MS. Serum albumin was found to be increased in KC patients according to LC-MS. CONCLUSION: The results show differences in the tear protein profile of KC and control subjects. These changes are indicative of alterations in tear film stability and in interactions with the corneal surface in KC patients.