Hemolytic Phenotype Research Articles

The Grndad Registry: Contemporary Natural History Data and an Analysis of Real-World Patterns of Use and Limitations of Disease Modifying Therapy in Adults with SCD

The Grndad Registry: Contemporary Natural History Data and an Analysis of Real-World Patterns of Use and Limitations of Disease Modifying Therapy in Adults with SCD

Traditional African remedies induce hemolysis in a glucose-6-phopshate dehydrogenase deficient zebrafish model

Traditional remedies are widely used throughout Africa in routine care for infants. However, such remedies could have detrimental effects. Acute bilirubin encephalopathy (ABE) and kernicterus spectrum disorder (KSD) are common newborn health conditions in the developing world, contributing to substantial neonatal mortality and morbidity. They frequently occur in children with glucose-6-phopshate dehydrogenase (G6PD) deficiency. Using our established zebrafish model of G6PD deficiency, we tested the effects of three traditional compounds used in the care of the newborn umbilical cord: eucalyptus oil, methylated spirits, and Yoruba herbal tea. We found that eucalyptus oil induced a 13.4% increase in a hemolytic phenotype versus control, while methylated spirits showed a 39.7% increase in affected phenotype. Yoruba herbal tea exposure showed no effect. While methylated spirits are already a known pro-oxidant, these data indicate that eucalyptus oil may also be a hemolytic trigger in those with G6PD deficiency. Discovering which agents may contribute to the pathophysiology of G6PD deficiency is critical to eliminate ABE and KSD, especially in countries with a high prevalence of G6PD deficiency. The next step in elucidating the role of these agents is to determine the clinical correlation between the use of these agents and ABE/KSD.

Virulence factors and antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis in Brazil.

This study aimed to evaluate virulence factors and genetic markers of antimicrobial resistance in 400 Staphylococcus aureus strains isolated from bovine mastitis in four Brazilian states, as well as to assess the association between these characteristics and field information. Virulence factors and drug resistance genes were identified by PCR screening. Biofilm-forming and hemolytic phenotype were detected using Congo red Tryptic Soy Broth and defibrinated sheep blood agar, respectively. Of all isolates, 83.5% were biofilm-forming and 98.5% strains exhibited biofilm gene icaAD, and a significant association between phenotype and genotype for biofilm was observed (P = 0.0005). Hemolysin genes were observed in 82.85% (hla+hlb+), 16.5% (hla+) and 0.75% (hlb+) isolates, whereas the hemolytic phenotype exhibited was complete and incomplete hemolysis in 64.25%, complete in 28.25%, incomplete in 4.75%, and negative in 2.75% of the strains. Virulence factors genes luk, seb, sec, sed, and tst were observed in 3.5%, 0.5%, 1%, 0.25%, and 0.74% isolates, respectively. The gene blaZ was detected in 82.03% of penicillin-resistant isolates, whereas tetK and aac(6')-Ie-aph(2')-Ia were observed in 33.87% and 45.15% of the tetracycline and aminoglycosides-resistant isolates, respectively. Fluoroquinolone resistance gene mepA was detected for the first time in S. aureus from bovine mastitis. Resistance genes tetM (3.22%), tetL (1.61%), ermA (14.29%), ermB (14.29%), ermC (33.3%), ermT (9.52%), ermY (4.76%), msrA (9.52%), and mphC (9.52%) were also detected among resistant isolates. No association between virulence factors or antimicrobial-resistant genes and year of isolation, geographic origin, or antimicrobial resistance profile was observed. Our results showed that S. aureus strains isolated from bovine mastitis in the four Brazilian states sampled are mainly biofilm-forming and hemolytic, whereas virulence genes associated with enterotoxins, luk and tst, were less frequently observed. Moreover, a wide variety of resistance genes that confer resistance to almost all classes of antimicrobial agents approved for use in animals and humans were found. Overall, the data point to a great pathogenic potential of S. aureus associated with bovine mastitis and to the non-negligible risks to public health of staphylococcal infections from animal origin.

Prevalence of Antimicrobial Resistance and Hemolytic Phenotypes in Culturable Arctic Bacteria.

Many Arctic biomes, which are populated with abundant and diverse microbial life, are under threat: climate change and warming temperatures have raised concerns about diversity loss and possible emergence of pathogenic microorganisms. At present, there is little information on the occurrence of Arctic virulence-associated phenotypes. In this study we worked with 118 strains of bacteria (from 10 sampling sites in the Arctic region, located in Greenland and the Svalbard Archipelago) isolated using R2A medium. These strains belong to 4 phyla and represent 36 different bacterial genera. Phenotypic resistance to 8 clinically important antimicrobials (ampicillin, chloramphenicol, ciprofloxacin, cefotaxime, erythromycin, imipenem, kanamycin, and tetracycline) and thermotolerance range were determined. In addition, a screening of all isolates on blood agar media and erythrocytes suspension of bovine and sheep erythrocytes for virulence-linked hemolytic activity was performed. Although antimicrobial resistance profiles varied among the isolates, they were consistent within bacterial families and genera. Interestingly, a high number of isolates (83/104) were resistant to the tested concentration of imipenem (4 mg/L). In addition, one third of the isolates showed hemolytic activity on blood agar, however, in only 5% of the isolates hemolytic activity was also observed in the cell extracts when added to erythrocyte suspensions for 60 min. The observed microbial phenotypes contribute to our understanding of the presence of virulence-associated factors in the Arctic environments, while highlighting the potential risks associated with changes in the polar areas in the light of climate change.

2605. Mixed Subpopulation of Hemolytic and Non-Hemolytic Phenotype in Clinical Staphylococcus aureus Blood Isolates

BackgroundAgr is a key regulator that controls expression of secreted exoproteins and surface protein in Staphylococcus aureus. It has been reported that mixed status of two different phenotypes including agr functional and nonfunctional subpopulations can coexist in vitro and in vivo. However, data on the natural course and clinical implication of the mixed agr status is limited. We thus investigated the frequency and characteristics of the mixed agr in clinical settings.MethodsWe evaluated isogenic paired MRSA isolates collected from patients with persistent S. aureus bacteremia (SAB) between October 2010 and April 2016, and then prospectively performed surveillance for the presence of mixed agr function in MRSA isolates from patients with SAB between May 2016 and December 2017. The mixed agr status was evaluated by single colony evaluation on sheep blood agar plate containing RN4220 supernatant (β-hemolysin) (Figure 1). Cross-streaking with RN4220 and RNAIII measurement were performed to confirm the agr functionality of each of hemolytic and non-hemolytic colonies, separately. The expression levels of RNAIII, hla, and saeS/saeR were measured by real-time reverse transcription polymerase chain reaction.ResultsA total of 161 first blood isolates were collected during study period, and 6 isolates (4%) displayed mixed phenotype by single colony test. The mixed hemolytic pattern was observed in 5 out of 52 ST72 isolates (10%) and 1 out of 82 ST5 isolates (1%) (Figure 1). No difference was found in the genotypes between hemolytic and non-hemolytic colonies from each isolate. Of the 6 isolates, three lost mixed hemolytic features in the follow-up blood cultures (Table 1). One ST72 and one ST5 isolate showed agr mixed pattern determined by different RNAIII levels, but remaining four ST72 isolates had mixed hemolytic pattern due to different expression of hla correlated with saeS/saeR expression (Figure 2).ConclusionThe mixture of agr function status among the clinical blood isolates of MRSA was rarely observed and isolates displaying heterogeneous hemolytic phenotype were largely due to differential expression of α-hemolysin. Further investigation is needed to unveil the clinical significance of mixture of different hemolytic phenotypes. Disclosures All authors: No reported disclosures.

Identification of Brachyspira species by cpn60 universal target sequencing is superior to NADH oxidase gene sequencing

The pig colon is the habitat of diverse Brachyspira species, of which only a few are of clinical importance. Methods for identification have shifted from phenotypic to molecular testing over the last two decades. Following the emergence of B. hampsonii it became evident that relying on species-specific PCRs carries the risk of overlooking important new species. Consequently, sequencing was proposed as an unbiased alternative for identification of isolates. So far, the main target for identification across species has been the NADH oxidase gene (nox). However, multiple copies of this gene in the genome and potential lateral gene transfer reduce confidence when using this gene. This study compared identification and phylogentic relationship inferred from nox sequencing to that inferred from sequencing of the cpn60 universal target using a collection of 168 isolates from different Brachyspira species. The majority of isolates had an identical identification with both methods. There were a few outliers in the trees with uncertain assignment to a species by BLAST analysis. A few major discrepancies pertained to the pathogenic species B. hampsonii (2), B. pilosicoli (1) and B. suanatina (1). Weakly haemolytic variants of B. hyodysenteriae were assigned to the correct species by both methods. Some of the isolates identified as B. hampsonii also had a weakly haemolytic phenotype.

Cutibacterium acnes persists despite topical clindamycin and benzoyl peroxide

Cutibacterium (formerly Propionibacterium) acnes persists in the dermis despite standard skin antiseptic agents, prompting some surgeons to use topical antimicrobials such as benzoyl peroxide and clindamycin prior to shoulder arthroplasty surgery. However, the efficacy of these topical agents has not been established. The upper backs of 12 volunteers were randomized into 4 treatment quadrants: topical benzoyl peroxide, topical clindamycin, combination topical benzoyl peroxide and clindamycin, and a negative control. The corresponding topical agents were applied to each site twice daily for 3 days. A 3-mm dermal punch biopsy specimen was obtained from each site and cultured for 14 days to assess for C acnes growth. Positive cultures were assessed for the hemolytic phenotype. The McNemar test was used to compare the proportion of positive cultures in each group. C acnes grew in 4 of 12control sites (33.3%), 1 of 12benzoyl peroxide sites (8.3%), 2 of 12clindamycin sites (16.7%), and 2 of 12combination benzoyl peroxide-clindamycin sites (16.7%). The C acnes hemolytic phenotype was present in 2 of 12control specimens (16.7%) compared with 0 (0.0%) in the benzoyl peroxide group, 2 of 12 (16.7%) in the clindamycin group, and 2 of 12 (16.7%) in the combination benzoyl peroxide-clindamycin group. There were no statistically significant differences between treatment arms. The topical application of benzoyl peroxide and clindamycin did not eradicate C acnes in all subjects. The clinical implications of these findings are yet to be determined.

Nonhemolysis of epidemic El Tor biotype strains of Vibrio cholerae is related to multiple functional deficiencies of hemolysin A

BackgroundHemolysis of bacteria is an important phenotype used for typing and characterizing strains with specific biomarkers and even a virulence factor in bacterial pathogenesis. In Vibrio cholerae, hemolysin HlyA is responsible for hemolysis of sheep red blood cells, and this hemolytic phenotype is used as a biotyping indicator and considered one of the virulence factors. At the beginning of the seventh cholera pandemic, the El Tor biotype strains of serogroup O1 were distinguished by hemolysis from the sixth pandemic O1 classical biotype strains, whereas during the following epidemics, nonhemolytic El Tor strains appeared, suggesting phenotypic and genetic variations in these strains. This study aimed to investigate the possible mechanisms involved in nonhemolysis of El Tor strains.ResultsFive sequence types of hlyA genes were found in the studied O1 El Tor strains isolated during the seventh pandemic. A 4-base deletion in hlyA caused the HlyA protein mutation and non-hemolytic phenotype. Some strains carry wildtype hlyA genes but are still non-hemolytic, and greatly reduced hlyA transcription and blocked secretion of hemolysin were observed in hemolysis tests of the subcellular components and transcription/expression analysis of hlyA.ConclusionsMechanisms responsible for nonhemolysis of the epidemic O1 El Tor strains are complex and not only confined to gene mutation but also deficiencies of transcription and extracellular transport of HlyA. Mutations in gene regulation and protein secretion systems of HlyA in the nonhemolytic V. cholerae strains should be areas of concern in future studies.

Association of pvl gene with incomplete hemolytic phenotype in clinical Staphylococcus aureus.

Purpose: This experiment aimed to evaluate the correlation between the hemolytic phenotype of Staphylococcus aureus and pvl gene in terms of characteristics of antibiotic resistance.Materials and methods: Two-hundred and eleven strains of hospital-acquired S. aureus and their bacterial susceptibility to 20 antibiotics were determined by MicroScan WalkAway96. All strains were cultured on Columbia sheep blood agar plates for 24 hours and then underwent ten passages for investigation of their hemolytic phenotypes. S. aureus produced incomplete β-hemolytic phenotype, termed as S. aureus strains with incomplete hemolytic phenotype (SIHP). The pvl gene was identified by PCR amplification followed by DNA sequencing. Statistical analyses of the data were performed using SPSS version 16.0 software.Results: Fifty-two (24.64%) strains were confirmed to maintain the incomplete hemolytic phenotype of S. aureus (SIHP). Meanwhile, 15 (7.11%) of 211 strains were found to carry the pvl gene, and eight of the 15 strains were SIHP. Compared with S. aureus strains with complete hemolytic phenotype (SCHP), SIHP showed higher susceptibility to seven of the 20 antibiotics (oxacillin, ciprofloxacin, gentamicin, ceftriaxone, cefoxitin, levofloxacin, and moxifloxacin) (P<0.05). The pvl-positive bacteria had a higher rate of resistance to four antibiotics (rifampin, ciprofloxacin, levofloxacin, and moxifloxacin) in comparison with the pvl-negative strains (P<0.05).Conclusion: SIHP had a high frequency of pvl gene. The pvl-positive isolates showed less resistance to rifampin, ciprofloxacin, levofloxacin, and moxifloxacin. Additionally, the majority of SIHP isolates (61.54%) were methicillin-resistant S. aureus. SIHP strains had significantly higher antibiotic resistance to cefoxitin when compared with SCHP, while SCHP strains had a high rate of antibiotic resistance to ciprofloxacin, gentamicin, ceftriaxone, levofloxacin, and moxifloxacin. The results may help to provide medical advice for selection of antibiotics for patients with SIHP-associated infections.

Weakly haemolytic variants of Brachyspira hyodysenteriae newly emerged in Europe belong to a distinct subclade with unique genetic properties

Brachyspira (B.) hyodysenteriae is widespread globally, and can cause mucohaemorrhagic colitis (swine dysentery, SD) with severe economic impact in infected herds. Typical strains of B. hyodysenteriae are strongly haemolytic on blood agar, and the haemolytic activity is believed to contribute to virulence in vivo. However, recently there have been reports of atypical weakly haemolytic isolates of B. hyodysenteriae (whBh). In this study, 34 European whBh and 82 strongly haemolytic isolates were subjected to comparative genomic analysis. A phylogenetic tree constructed using core single nucleotide polymorphisms showed that the whBh formed a distinct sub-clade. All eight genes previously associated with haemolysis in B. hyodysenteriae were present in the whBh. No consistent patterns of amino acid substitutions for all whBh were found in these genes. In contrast, a genome region containing six coding sequences (CDSs) had consistent nucleotide sequence differences between strongly and whBh isolates. Two CDSs were predicted to encode ABC transporter proteins, and a TolC family protein, which may have a role in the export of haemolysins from B. hyodysenteriae. Another difference in this region was the presence of three CDSs in whBh that are pseudogenes in strongly haemolytic isolates. One of the intact CDSs from whBh encoded a predicted PadR-like transcriptional repressor that may play a role in repression of haemolysis functions. In summary, a sub-clade of whBh isolates has emerged in Europe, and several genomic differences, that potentially explain the weakly haemolytic phenotype, were identified. These markers may provide targets for discriminatory molecular tests needed in SD surveillance.

Hemolysis Is a Diagnostic Adjuvant for Propionibacterium acnes Orthopaedic Shoulder Infections

The purpose of this study was to further evaluate the pathogenicity of hemolytic and nonhemolytic phenotypes of Propionibacterium acnes (P acnes) isolates from shoulders of orthopaedic patients. Thirty-one patient records were reviewed, which had a positive P acnes shoulder culture from joint aspiration fluid and/or intraoperative tissues for demographics, clinical course, culture, and laboratory data. Patients were categorized as definite infection, probable infection, or probable contaminant. Antibiotic resistance patterns and hemolysis characteristics were subsequently analyzed. Hemolysis demonstrated 100% specificity with a positive predictive value of 100% and 80% sensitivity with a negative predictive value of 73% for determining definite and probable infections. Hundred percent of the patients in the hemolytic group and only 27% of patients in the nonhemolytic group were classified as infected. Presenting inflammatory markers were markedly higher in the hemolytic group. Clindamycin resistance was found in 31% of the hemolytic strains, whereas no antibiotic resistance was observed in the nonhemolytic group. Hemolytic strains of P acnes exhibit enhanced pathogenicity to their host by eliciting a more prominent systemic inflammatory response, increased antibiotic resistance, and a more challenging clinical course. Hemolysis may serve as a specific marker for assisting in diagnosing true infection with P acnes. Level III retrospective comparative study.

Phenotypic and genotypic characterization of enterotoxigenic Escherichia coli isolated from diarrheic calves in Argentina.

Enterotoxigenic Escherichia coli (ETEC) is the most common and global cause of neonatal calf diarrhea, but there is a little information regarding calf ETEC strains in Argentina. In this study, five ETEC isolates from diarrheic dairy calves (2–10 d old) from Buenos Aires and Cordoba, Argentina were characterized on the basis of virulence gene (VG) pattern, O:H serotyping, hemolytic phenotype, phylogenetic group affiliation, antimicrobial (AM) resistance profile, and presence of integron class 1 and 2. The five isolates were examined by polymerase chain reaction (PCR) for the presence of 18 bovine VGs and showed the following genotypes: F5+/F41+/sta+ (D242), F5+/sta+ (D158), F5+/sta+ (D157), F5+ (D151-9), and F5+/iucD+ (D151-5). These VGs confer pathogenic potential and most of them are associated with the ETEC pathotype. The five isolates showed a non-hemolytic phenotype, belonged to five different serotypes: O101:H−, O141:H−, O60:H−, ONT:H10, and ONT:H−, and were assigned to the phylogenetic group A by the quadruplex Clermont PCR method. The AM resistance of the three isolates D242, D157, and D151-5 was determined by agar disk diffusion method for 24 AMs and they exhibited a multi-resistance phenotype (resistance to four different AM classes: Cephalosporins, Penicillins, Macrolides, and Ansamycins). In addition, class 1 integrons were found in the isolate D151-5 containing the dfrA17-aadA5 gene cassette and in the bovine ETEC reference strain FV10191 containing the dfrA1-aadA1 gene cassette. The present study revealed for the first time the occurrence of multi-resistant ETEC associated with neonatal diarrhea in dairy calves in Argentina. This finding may be used for diagnostic and therapeutic purposes.

Modeling Human Paroxysmal Nocturnal Hemoglobinuria Via CRISPR/Cas9 HSPC Gene Editing in Non-Human Primate

Recent advances in CRISPR/Cas9 technology allowing precise genome editing at a site of interest have enabled relevant human disease modeling and the development of corrective gene therapies for various genetic disorders. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematological disorder linked to acquired somatic loss-of-function mutations disrupting the X-linked PIG-A gene in hematopoietic stem and progenitor cells (HSPC), characterized by the clinical triad of intravascular hemolysis, thrombosis and bone marrow failure, as well as clonal expansion of HSPC defective in glycosylphophatidylinositol(GPI)-linked proteins, due to loss of PIG-A enzymatic activity. There have been many attempts to model PNH utilizing conditional knockout strategies in mice, but these do not recapitulate the hemolytic phenotype or clonal dominance. Whether clonal expansion is intrinsic to PIG-A deficient HSPC or extrinsic, resulting from relative protection of PIG-A deficient HSPC and progeny cells from immune attack, is unclear. To explore the pathophysiologic mechanisms of PNH HSPC clonal expansion, we generated a relevant non-human primate model for PNH, utilizing autologous transplantation of HSPC edited with CRISPR/Cas9 at the PIG-A locus.The most efficient guide RNAs (gRNAs) targeting several sites within PIG-A exon 2 locus on the X chromosome, where most of mutations occur in patients with PNH, were selected based on efficiency of editing as screened in FRhK-4 cell lines and mobilized macaque CD34+ HSPC. Compared to editing of the control AAVS1 locus and other genes of interest, editing of all PIG-A locus sites was relatively inefficient. Autologous HSPC were transduced with ribonucleotide protein (RNP) complexes of Cas9 protein and selected gRNAs targeting either PIG-A or the AAVS1 “safe harbor” site, as an internal control, followed by transplantation into one female (ZI35) and one male (ZL19) macaque, respectively. Both animals engrafted promptly and clones acquiring the PNH loss-of-function phenotype, defined by loss of binding of FLAER, a fluorescent compound that binds to all GPI anchors, and loss of expression of lineage-specific GPI-linked proteins (CD24 for granulocytes and CD14 for monocytes) were stably maintained at levels of 0.2-0.4% for up to 19 months post-transplantation, with no evidence for intrinsic clonal expansion of PIG-A edited cells (Fig. 1A). Upon targeted deep sequencing, 0.4-1% insertions and deletions (INDELs) induced by CRISPR/Cas9 were detected, and the predominant INDEL type was identified as a single base deletion at the +1 or -1 positions of the target site consistently in granulocytes and other mature hematopoietic lineage cells from peripheral blood (PB), suggesting that initial mutations occurred in long-lasting HSPCs rather than short-term progenitor cells. Furthermore, as the PIG-A gene is located on X chromosome, we sought to investigate the difference in genome editing efficiency depending on the number of X alleles or activation state. Note that the expected INDEL frequency in completely PIG-A deficient sorted FLAERneg cells would be 100%, however, large deletions or rearrangements are not detected by standard deep sequencing methodologies. Interestingly, the mutation frequencies in total granulocytes and more importantly in sorted FLAER negative PNH cells were always much higher in male (ZL19) macaque cells than in female (ZI35) macaque cells (Fig. 1B and C). Consistently, gene editing PIG-A allele efficiency with CRISPR/Cas9 was also higher in human male B-lymphoblastoid cell lines (LCL) compared to female cells, 23.2% versus 16.6% (n=5), respectively. The finding that edited allele frequency was consistently lower in sorted FLAERneg female than male cells suggests that editing of the active X allele may be favored, potentially due to poor accessibility of inactive loci to editing machinery.In conclusion, we have successfully established a rhesus macaque model for PNH utilizing autologous transplantation of CRISPR/Cas9 edited HSPC. To date, we found no evidence for intrinsic expansion of PIG-A deficiency HSPC and hematopoietic progeny. This modeling approach could be utilized for further investigation of extrinsic or intrinsic factor responsible for clonal expansion. Furthermore, our findings provide a better insight into the relationship between CRISPR/Cas9 editing efficiency and active versus inactive X-linked genes. [Display omitted] DisclosuresDunbar:National Institute of Health: Research Funding.

Risk Factors Predisposing Development of Priapism in Sickle Cell Disease Patients

Background and Purpose: Penile erection and detumescence are complex physiologic processes, which require delicate neurohormonal and cardiovascular response. Priapism is defined as a persistent and painful erection lasting longer than four hours without sexual stimulation. Stuttering priapism is characterized by a self-limited, recurrent, and intermittent erection, frequently occurring in patients with sickle cell disease(SCD). The aim of this study was to identify the clinical and laboratory features in SCD patients with priapism in Oman.Methodology: In this retrospective case-control study, medical records of all patients with SCD who developed priapism were retrieved from the hospital information system and compared in a 1: 1 ratio with age and gender matched SCD patients who did not ever had priapism. Addition information extracted include hematological and laboratory parameters, treatment, and complications. Data obtained were analyzed using IBM SPSS version 23. The study was conducted following approval from the hospital medical research and ethics committee.Results & Discussion: Amongst the forty-one SCD patients evaluated, in the 21 patients with priapism (mean age 24.7 yrs), there was a significantly higher WBC, platelet, retic counts, LDH and serum total bilirubin as compared to controls (p<0.05, student's t test). However, there was no significant differences in the incidence of vaso-occlusive crisis, acute chest syndrome, pulmonary hypertension or cerebrovascular accident (p >0.05, Chi Square test). Patients with priapism were managed with exchange transfusion (100%), aspiration (52%) and shunting (5%). Interestingly, none of these patients showed impotence, although 10% manifested with infertility.Conclusions: Priapism in this SCD cohort is a disease of the young, and seen predominantly in the hemolytic phenotype of the disease. Patients with priapism showed high WBC's, Platelets, Bilirubin, LDH and retics with a lower hemoglobin as compared to the controls. Favorable outcome is dependent on early intervention and almost half of these patients needed surgical intervention. DisclosuresNo relevant conflicts of interest to declare.

Coagulation Profile of Sickle Cell Patients with Leg Ulcers

BACKGROUNDLeg ulcers are a serious complication of Sickle Cell Disease (SCD), often linked to a hemolytic phenotype that includes priapism, pulmonary hypertension (PH) and renal disease. In the SCD pathogenesis, hemolysis and ischemia-reperfusion injuries lead to white blood cell (WBC), platelet and endothelial cell activation, pro-inflammatory cytokine production and subsequent activation of coagulation that tips the balance towards a prothrombotic and inflammatory state. Decreased levels of the naturally occurring anticoagulants (NOACs): protein C (PC), protein S (PS), antithrombin III (ATIII), have been observed in patients with SCD in steady state, with some reports stating no differences between steady state and crisis. This occurs due to decreased production, increased consumption, or a combination of both. However, the definite mechanisms have not yet been elucidated. Factor V Leiden (FVL) and prothrombin G20210A mutations are rare in African Americans and in patients with SCD. We speculated that patients with leg ulcers would have a more pronounced pro-thrombotic phenotype than patients who never developed them.METHODS127 adults with and without SCD leg ulcers were evaluated at steady state in the prospective cross-sectional INSIGHTS study - Insights into Microbiome and Environmental Contributions to Sickle Cell Disease and Leg Ulcers (ClinicalTrials.gov Identifier NCT02156102). At the time of enrollment, a clinical examination and a comprehensive past medical history were completed. Routine blood work including a full coagulation profile and analysis of blood clotting genetic mutations (thrombophilia genetic tests) were obtained. Laboratory results were compared between groups with and without leg ulcers using Chi-square or Fisher exact tests. Multivariable logistic regression analysis was conducted to test whether deep venous thrombosis (DVT) and pulmonary embolism (PE), clinical surrogates of a prothrombotic phenotype, were associated with the decreased NOACs (PC, PS), or leg ulcer.RESULTSThe study cohort consisted of 109 adults with Hb SS, 10 with Hb SC, 5 with Hb S beta thalassemia zero, and 3 with Hb S beta thalassemia plus. Of these, there were 35 subjects with leg ulcers. NOACs were found to be decreased in the sub-group of subjects with leg ulcers. Among those with leg ulcers, 49% had functional PC levels that were below the normal range, compared to 27% of those without leg ulcers (p=0.022) 74% had functional PS levels below the normal range, compared to 51% of study patients without leg ulcers (p=0.018); 23% had AT III levels below the normal range, compared to 17% of SCD patients without leg ulcers (p=0.482); and 29% tested positive for lupus anticoagulant (LA), compared to 25% of SCD patients without leg ulcers (p=0.682). Among the leg ulcer cohort, none of the subjects had the prothrombin gene mutation, while 3 subjects without leg ulcers were heterozygous for it. None of the subjects in our cohort had FVL mutation. The multivariable analysis showed that none of PC, PS, or leg ulcer was significantly associated with either DVT or PE.CONCLUSIONSIn SCD patients with leg ulcers, PC and PS levels are significantly lower than in SCD patients without leg ulcers. Additionally, in SCD patients with leg ulcers, there is a trend towards decreased levels of AT III and increased LA positivity. FVL and prothrombin gene mutations were rare in our cohort, consistent with the general population. The above findings did not correlate clinically with a history of DVT/PE in the same population.Our results of decreased NOACs confirm previous reports from our group that demonstrated similar findings. Of greater significance, however, is our finding that sickle cell patients with leg ulcers have further decreased levels of the NOACs compared to their counterparts without leg ulcers. This finding lends credence to the hypothesis that sickle cell patients with leg ulcers have a procoagulant phenotype (Am J Hematol. 2011 Aug; 86: 705-8, Am J Hematol. 2014 Jan; 89: 1-6). Whether this translates to an increased risk of thromboembolic events remains uncertain. From our findings, it may not be the case for DVTs and PEs. However, further studies evaluating other clinical manifestations such as priapism, PH, stroke, avascular necrosis, and retinopathy, are still needed. Future directions include further correlation with the above-mentioned manifestations to provide a more comprehensive picture. DisclosuresMinniti:Bluebird Bio: Other: Adjudicating Committee; Global Blood Therapeutics: Research Funding; Bayer: Research Funding; Teutona: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Mutation in cyl operon alters hemolytic phenotypes of Streptococcus agalactiae

Streptococcus agalactiae infects numerous fish species, causing considerable economic losses during fish cultivation. This study compared the phenotypic differences among S. agalactiae hemolytic variant isolates and investigated the genetic composition of their hemolysin genes. Hemolysin is encoded by the cyl operon and mainly regulated by covS/R, which also regulates encapsulation. In total, 45 S. agalactiae clinical isolates were collected from cultured fishes in Taiwan. Three different hemolytic phenotypes—α, β, and γ—were identified. Of the 45 isolates, 39 were β hemolytic, 3 were α hemolytic, and 3 were γ hemolytic. The γ-hemolytic isolates demonstrated significantly thicker encapsulation and slower growth rates than did the α- and β-hemolytic isolates. However, no isolate had mutations in the regulatory gene covS/R. A 1252-bp insertion sequence (IS) in the cyl operon of α-hemolytic isolates, located at cylF region, was found. This IS interrupted cylF through insertion at 23 bp downstream of starting codon, causing incomplete mRNA transcription. The β-hemolytic isolates showed no mutation in the cyl operon. By contrast, the γ-hemolytic isolates had lost the entire cyl operon; it had been replaced by a 14-kb genomic island containing genes for DNA recombinase and septum formation proteins. In summary, the differences in hemolysin genes between α- and β-hemolytic isolates were due to the IS in the cylF region, whereas in the γ-hemolytic isolates, the entire cyl operon was deleted and replaced. These findings explain different hemolysin expressions of the clinical S. agalactiae isolates taken from fish ponds in Taiwan. ImportanceStreptococcus agalactiae infects both warm- and cold-blooded animals and causes major aquatic cultivation loss. Pathogenic isolates from the outbreak of fish ponds were examined their cyl operon gene. α-Hemolytic isolate with mutant cyl operon was observed for the first time in aquaculture animals and was compared to intact or entire cyl operon deletion of β- and γ-hemolytic isolates. Hemolysis expression levels of Streptococcus agalactiae are explained.

Comparative Genomics of the First and Complete Genome of "Actinobacillus porcitonsillarum" Supports the Novel Species Hypothesis.

“Actinobacillus porcitonsillarum” is considered a nonpathogenic member of the Pasteurellaceae family, which phenotypically resembles the pathogen Actinobacillus pleuropneumoniae. Previous studies suggested that “A. porcitonsillarum” may represent a new species closely related to Actinobacillus minor, yet no full genome has been sequenced so far. We implemented the Oxford Nanopore and Illumina sequencing technologies to obtain the highly accurate and complete genome sequence of the “A. porcitonsillarum” strain 9953L55. After validating our de novo assembly strategy by comparing the A. pleuropneumoniae S4074T genome sequence obtained by Oxford Nanopore Technology combined with Illumina reads with a PacBio-sequenced S4074T genome from the NCBI database, we performed comparative analyses of the 9953L55 genome with the A. minor type strain NM305T, A. minor strain 202, and A. pleuropneumoniae S4074T. The 2,263,191 bp circular genome of 9953L55 consisted of 2168 and 2033 predicted genes and proteins, respectively. The lipopolysaccharide cluster resembled the genetic organization of A. pleuropneumoniae serotypes 1, 9, and 11, possibly explaining the positive reactions observed previously in serotyping tests. In contrast to NM305T, we confirmed the presence of a complete apxIICABD operon in 9953L55 and 202 accounting for their hemolytic phenotype and Christie-Atkins-Munch-Petersen (CAMP) reaction positivity. Orthologous gene cluster analysis provided insight into the differential ability of strains of the A. minor/“porcitonsillarum” complex and A. pleuropneumoniae to ferment lactose, raffinose, trehalose, and mannitol. The four strains showed distinct and shared transposable elements, CRISPR/Cas systems, and integrated prophages. Genome comparisons based on average nucleotide identity and in silico DNA-DNA hybridization confirmed the close relationship among strains belonging to the A. minor/“porcitonsillarum” complex compared to other Actinobacillus spp., but also suggested that 9953L55 and 202 belong to the same novel species closely related to A. minor, namely, “A. porcitonsillarum.” Recognition of the taxon as a separate species would improve diagnostics and control strategies of pig pleuropneumonia.

Total oxidant status of children with sickle cell anaemia: Correlation with rate of pain episodes and haematological indices

BackgroundTotal oxidative status (TOS) measures cumulative oxidant levels. In sickle cell anaemia (SCA), oxidative stress plays a significant role in the pathophysiology of vasoocclusive and haemolytic phenotypes. In this study, we evaluated serum levels of TOS in children with SCA and matched healthy controls, and determined its relationship with the rates of vasoocclusive crisis (VOC) and haematological profile. MethodOne hundred and sixty children, comprising of 80 with SCA aged 1–15 years and 80 age- and sex-matched haemoglobin (Hb) AA controls were studied. Serum TOS was determined by Human TOS Enzyme Linked Immunosorbent Assay. ResultThe mean serum TOS of subjects with SCA (13.27 ± 4.62 U/mL) was significantly higher than 9.69 ± 2.74 U/mL for the matched controls, p < 0.001. Among the subjects, TOS had significant positive correlation with rates of VOC (r = 0.24, p = 0.036) and leucocyte counts (r = 0.25, p = 0.027), and significant inverse correlation with haematocrit (r = −0.46, p < 0.001). There was no significant correlation with other haematological indices. ConclusionChildren with SCA had higher TOS than matched controls. Markers of oxidative stress had significant correlation with rates of pain episodes and haematocrit levels. Efforts should be made to minimize altered oxidative milieu in SCA.

Clinical and genetic predictors of atypical hemolytic uremic syndrome phenotype and outcome

Atypical hemolytic uremic syndrome (aHUS) is a rare, genetic, life-threatening disease. The Global aHUS Registry collects real-world data on the natural history of the disease. Here we characterize end-stage renal disease (ESRD)-free survival, the rate of thrombotic microangiopathy, organ involvement and the genetic background of 851 patients in the registry, prior to eculizumab treatment. A sex-specific difference was apparent according to age at initial disease onset as the ratio of males to females was 1.3:1 for childhood presentation and 1:2 for adult presentation. Complement Factor I and Membrane Cofactor Protein mutations were more common in patients with initial presentation as adults and children, respectively. Initial presentation in childhood significantly predicted ESRD risk (adjusted hazard ratio 0.55 [95% confidence interval 0.41-0.73], whereas sex, race, family history of aHUS, and time from initial presentation to diagnosis, did not. Patients with a Complement Factor H mutation had reduced ESRD-free survival, whereas Membrane Cofactor Protein mutation was associated with longer ESRD-free survival. Additionally extrarenal organ manifestations occur in 19%-38% of patients within six months of initial disease presentation (dependent on organ). Thus, our real-world results provide novel insights regarding phenotypic variables and genotypes on the clinical manifestation and progression of aHUS.

Bone marrow transplantation corrects haemolytic anaemia in a novel ENU mutagenesis mouse model of TPI deficiency.

ABSTRACTIn this study, we performed a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice to identify novel genes or alleles that regulate erythropoiesis. Here, we describe a recessive mouse strain, called RBC19, harbouring a point mutation within the housekeeping gene, Tpi1, which encodes the glycolysis enzyme, triosephosphate isomerase (TPI). A serine in place of a phenylalanine at amino acid 57 severely diminishes enzyme activity in red blood cells and other tissues, resulting in a macrocytic haemolytic phenotype in homozygous mice, which closely resembles human TPI deficiency. A rescue study was performed using bone marrow transplantation of wild-type donor cells, which restored all haematological parameters and increased red blood cell enzyme function to wild-type levels after 7 weeks. This is the first study performed in a mammalian model of TPI deficiency, demonstrating that the haematological phenotype can be rescued.