M protein gene type distribution among group A streptococcal clinical isolates recovered in Mexico City,Mexico, from 1991 to 2000, and Durango,Mexico, from 1998 to 1999: overlap with type distribution within the United States

To examine the type distribution of pathogenic group A streptococcal (GAS) strains in Mexico, we determined the emm types of 423 GAS isolates collected from ill patients residing in Mexico (Durango or Mexico City). These included 282 throat isolates and 107 isolates from normally sterile sites. Of the other isolates, 38 were recovered from other miscellaneous infections. A total of 31 different emm types were found, revealing a broad overlap between commonly occurring emm types in Mexico and the United States. The information obtained in this study is consistent with the possibility that multivalent, M type-specific vaccines prepared for GAS strain distribution within the United States could theoretically protect against the majority of GAS strains causing disease in the two cities surveyed in Mexico.     

Synthesis, staining properties, and biocompatibility of a new cyanine dye for ILM peeling

Background

The aim of this work is to investigate the biocompatibility and staining properties of DSS: 3,3′-Di-(4-sulfobutyl)-1,1,1′,1′-tetramethyl-di-1H-benz[e]indocarbocyanine (DSS).

Methods

Dye concentrations of 0.5, 0.25, and 0.1% were evaluated (290 and 295?mOsm). Toxicity was assessed using a colorimetric test measuring the inhibition of ARPE 19 cell, human primary RPE cell, and human Müller cell proliferation. Exposure time was 30, 60, 120, and 300?s. Indocyanine green (ICG) (0.5, 0.25, and 0.1%) served as a control. Cells were also illuminated with plain white light (750?mW/cm²) for 10?min to assess phototoxic effects. Besides staining of porcine and human lens capsule, internal limiting membrane (ILM)-staining was assessed by applying 0.25 and 0.5% DSS over the macula in two human post-mortem eyes.

Results

DSS of 0.25 and 0.1% showed no toxic effect on primary RPE cells and MIO-M1cells, and 0.5, 0.25, and 0.1% for ARPE-19 cells. In MIO-M1cells, 0.5% dye showed a significant reduction of mitochondrial dehydrogenase activity only following an exposure time of 300?s. Following illumination, ICG showed a significantly more pronounced effect on cell viability in primary RPE cells and MIO-M1cells compared to DSS. The absorption maximum is found at 591?nm; the even more bathochromic fluorescence proceeds with a common Stokes’ shift where maxima at 620 and 660?nm with a quantum yield of 32% were found. The fluorescence is sufficiently hypsochromic and the fluorescence quantum yield high enough for an easy visual detection. The contrast and staining properties at the ILM were excellent and allowed for a controlled removal of the ILM during surgery. No penetration into deeper retinal layers was noted.

Conclusions

Our results indicate that this new cyanine dye DSS may represent an alternative for ILM staining due to its matched absorption concerning visibility and fluorescence qualities as well as its good biocompatibility.     

Myriophyllum aquaticum versus Lemna minor: sensitivity and recovery potential after exposure to atrazine

The relative sensitivity and recovery potential of two aquatic macrophyte species, Lemna minor and Myriophyllum aquaticum, exposed to atrazine (concentration ranges 80-1,280 μg/L and 40-640 μg/L, respectively) were evaluated using slightly adapted standard protocol for Lemna spp.: relative growth rates (RGR) and yield of both plants were measured in 3-d-long intervals during the exposure and recovery phase. Myriophyllum aquaticum was also exposed to atrazine-spiked sediment (0.1-3.7 μg/g) in a water-free system. The results of M. aquaticum sediment contact tests showed that root- and shoot-based growth parameters are equally sensitive endpoints. In the water (sediment-free) test system, L. minor recovered after short (3 d) and longer exposure (7 d) to all atrazine concentrations after only a 5- to 6-d-long recovery phase. The recovery of M. aquaticum after short exposure was slower and less efficient: after 12 d of recovery phase the final biomass of plants exposed to 380 and 640 μg/L was below the initial values. The last interval RGR provides a good indication of plant recovery potential regardless of species growth strategy. If compared to L. minor, the difference in growth rate, sensitivity, lag phase, recovery potential from water-column substances, and also suitability for studies investigating the effect of sediment-bound pollutants advocates the use of M. aquaticum as an additional macrophyte species in risk assessment.     

Genetically diverse group A streptococci from children in far-western Nepal share high genetic relatedness with isolates from other countries

The genetic diversity of group A streptococci (GAS) throughout much of the world has not been adequately explored. To assess genetic variation among GAS in western Nepal, 120 noninvasive GAS, collected from eight different villages, were genetically characterized using emm typing, sof sequencing, and multilocus sequence typing (MLST). A high level of genetic diversity was observed among these isolates, with 51 genotypes based upon 51 multilocus sequence types (STs), 45 emm sequence types, and 28 sof sequence types. On the basis of shared ST-emm and sof-emm associations, 40 of the 51 genotypes were identical or highly related to genotypes characterized from locations outside of Nepal, even though most of the emm sequence and clonal types are rare among GAS within the United States. When analyzing all known STs highly related to Nepal STs, only one example of similar STs shared between a sof PCR-positive strain and a sof PCR-negative strain was observed. Since previous data indicate free exchange of MLST loci between sof-positive and sof-negative strains, there is possibly selection against the expansion of subclones resulting from horizontal transfers of sof or emm genes between sof-positive and sof-negative strains. All 45 emm types encountered in Nepal have also been documented from other countries. These data, together with data encompassing the past decade of emm type surveillance, support the possibility that most existing GAS emm types have been discovered. Similarly, since most (40/51) strain types were highly related to strains found elsewhere, it is likely that a major fraction of the existing GAS clonal complexes have been discovered.     

Can follow-up study questions about correct and consistent condom use reduce respondent over-reporting among groups at high risk? An analysis of datasets from six countries

Background

This investigation describes features of patients undergoing in vitro fertilisation (IVF) and embryo transfer (ET) where both gametes were obtained from anonymous donors.

Methods

Gamete unsuitability or loss was confirmed in both members of seven otherwise healthy couples presenting for reproductive endocrinology consultation over a 12-month interval in Ireland. IVF was undertaken with fresh oocytes provided by anonymous donors in Ukraine; frozen sperm (anonymous donor) was obtained from a licensed tissue establishment. For recipients, saline-enhanced sonography was used to assess intrauterine contour with endometrial preparation via transdermal estrogen.

Results

Among commissioning couples, mean±SD female and male age was 41.9 ± 3.7 and 44.6 ± 3.5 yrs, respectively. During this period, female age for non dual anonymous gamete donation IVF patients was 37.9 ± 3 yrs (p < 0.001). Infertility duration was ≥3 yrs for couples enrolling in dual gamete donation, and each had ≥2 prior failed fertility treatments using native oocytes. All seven recipient couples proceeded to embryo transfer, although one patient had two transfers. Clinical pregnancy was achieved for 5/7 (71.4%) patients. Non-transferred cryopreserved embryos were available for all seven couples.

Conclusions

Mean age of females undergoing dual anonymous donor gamete donation with IVF is significantly higher than the background IVF patient population. Even when neither partner is able to contribute any gametes for IVF, the clinical pregnancy rate per transfer can be satisfactory if both anonymous egg and sperm donation are used concurrently. Our report emphasises the role of pre-treatment counselling in dual anonymous gamete donation, and presents a coordinated screening and treatment approach in IVF where this option may be contemplated.     

Genetic Relationships Deduced from emm and Multilocus Sequence Typing of Invasive Streptococcus dysgalactiae subsp. equisimilis and S. canis Recovered from Isolates Collected in the United States

Beta-hemolytic group C and G streptococci cause a considerable invasive disease burden and sometimes cause disease outbreaks. Little is known about the critical epidemiologic parameter of genetic relatedness between isolates. We determined the emm types of 334 Streptococcus dysgalactiae subsp. equisimilis isolates, and attempted emm typing of 5 Streptococcus canis isolates from a recent population-based surveillance for invasive isolates. Thirty-four emm types were observed, including one from S. canis. We formulated multilocus sequence typing (MLST) primers with six of the seven loci corresponding to the Streptococcus pyogenes MLST scheme. We performed MLST with 65 of the 334 surveillance isolates (61 S. dysgalactiae subsp. equisimilis isolates, 4 S. canis isolates) to represent each emm type identified, including 2 to 3 isolates for each of the 25 redundantly represented emm types. Forty-one MLST sequence types (STs) were observed. Isolates within 16 redundantly represented S. dysgalactiae subsp. equisimilis emm types shared identical or nearly identical STs, demonstrating concordance between the emm type and genetic relatedness. However, seven STs were each represented by two to four different emm types, and 7 of the 10 S. dysgalactiae subsp. equisimilis eBURST groups represented up to six different emm types. Thus, S. dysgalactiae subsp. equisimilis isolates were similar to S. pyogenes isolates, in that strains of the same emm type were often highly related, but they differed from S. pyogenes, in that S. dysgalactiae subsp. equisimilis strains with identical or closely similar STs often exhibited multiple unrelated emm types. The phylogenetic relationships between S. dysgalactiae subsp. equisimilis and S. pyogenes alleles revealed a history of interspecies recombination, with either species often serving as genetic donors. The four S. canis isolates shared highly homologous alleles but were unrelated clones without evidence of past recombination with S. dysgalactiae subsp. equisimilis or S. pyogenes.Streptococcus pyogenes, Streptococcus dysgalactiae subsp. equisimilis, and Streptococcus canis are beta-hemolytic pyogenic species that are highly genetically related on the basis of 16S rRNA sequence comparisons, with S. canis being the closest relative to S. pyogenes (39, 16). In recent years, S. dysgalactiae subsp. equisimilis has increasingly been reported as a cause of invasive disease (11, 24, 36), yet the epidemiology and population genetics of this species is poorly understood. S. canis is a commensal and opportunistic pathogen of dogs and other animals (13, 16). S. canis rarely causes disease in humans (7, 39); however, its incidence in human disease is unknown.As with S. pyogenes, S. dysgalactiae subsp. equisimilis isolates are almost always emm typeable (8, 11, 24, 39), with over 50 known emm types (emm types are downloadable from ftp://ftp.cdc.gov/pub/infectious_diseases/biotech/tsemm/). During 1998, we documented the occurrence of emm type stG1389 from an invasive S. canis infection in a dog (unpublished data), and we also recently noticed the same sequence in the GenBank database (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EU195120","term_id":"162424912","term_text":"EU195120"}}EU195120). To our knowledge, this is the only emm type documented from S. canis. Several other virulence determinants are known to be shared between S. dysgalactiae subsp. equisimilis and S. pyogenes (9, 10, 12, 19, 25). S. pyogenes exotoxin genes have been detected within S. dysgalactiae subsp. equisimilis and S. canis, and lysogenic transfers of prophages carrying superantigen genes between S. pyogenes and S. dysgalactiae subsp. equisimilis have been documented (20, 22, 31, 33, 38).M-protein gene (emm) typing has been useful as a simple genetic tool for identifying and resolving Streptococcus pyogenes strains for epidemiologic studies (18, 27, 29, 35). This is consistent with the observation that within given regions, group A streptococcal (GAS) emm types are often restricted to only one or two defined clonal complexes, and often, these common clones are predominant in diverse locations (15) (available at www.mlst.net). In direct contrast, a previous report described a poor concordance between the emm type and genetic relatedness in S. dysgalactiae subsp. equisimilis (23). That report additionally showed evidence of the lateral movement of housekeeping genes between the two species, with the majority of gene transfer events involving S. pyogenes donors and S. dysgalactiae subsp. equisimilis recipients.We recently carried out population-based surveillance for invasive disease caused by beta-hemolytic streptococci of groups other than A and B, and the results from 2 years of surveillance were reported (7). The majority of case isolates (about 80%) were S. dysgalactiae subsp. equisimilis. The only other emm-typeable species identified by active surveillance was S. canis, of which only one of five S. canis isolates was emm typeable. We found that the burden of invasive disease caused by S. dysgalactiae subsp. equisimilis is comparable to that caused by invasive GAS (29) and affects similar adult populations (7). In the work presented here, we describe newly discovered relationships between multilocus sequence types and emm types among an expanded collection of invasive S. dysgalactiae subsp. equisimilis and S. canis isolates recovered in the United States from 2002 to 2005.     

Array of M protein gene subtypes in 1064 recent invasive group A streptococcus isolates recovered from the active bacterial core surveillance

Using sequence analysis to detect variation within the hypervariable M protein N terminus, we found 41 emm types encompassing 81 subtypes, among 1064 consecutive invasive group A streptococcus isolates from a recent multistate, population-based surveillance. Seventeen of the 30 emm types represented by multiple isolates displayed multiple subtypes. Most subtypes differed from reference strain emm sequences as a result of single base substitutions or other alterations likely to be stably inherited. The Centers for Disease Control and Prevention database (available at: http://www.cdc.gov/ncidod/biotech/strep/strepblast.htm) currently contains 225 distinct emm types encompassing 450 subtypes. Although this subtyping scheme increases specificity, limited variation within individual types favors introduction of M protein type-specific vaccines.     

Cyanine dyes as optical contrast agents for ophthalmological surgery

Cyanine dyes were prepared as optical contrast media for supporting the surgery of the lamina limitans interna (LLI) of the retina and other structures of the human eye. Their absorption spectra were adapted both to the spectral sensitivity of the human eye and to standard illumination. The contrast could be further amplified by the application of the strong fluorescence of the dyes used. The binding of the dyes to various surfaces was studied. No toxic effects could be detected for the applied dyes.