Prevalence and mechanisms of erythromycin and clindamycin resistance in clinical isolates of beta-haemolytic streptococci of Lancefield groups A, B, C and G in Seville, Spain.

Susceptibility to erythromycin and clindamycin was determined in 860 consecutive clinical isolates of beta-haemolytic streptococci belonging to groups A (GAS, n = 134), B (GBS, n = 689), C (GCS, n = 19) and G (GGS, n = 18). Erythromycin resistance was 26.1% in GAS, 15.7% in GBS, 5.3% in GCS and 33.3% in GGS. The highest rate of clindamycin resistance (33.3%) was in GGS, followed by GBS (15.8%), GCS (15.8%) and GAS (5.2%). The M phenotype was predominant in GAS (80%), the constitutive MLS(B) phenotype was predominant in GBS (75%), and all GGS isolates showed the inducible MLS(B) phenotype. The uncommon erythromycin-susceptible and clindamycin-resistant phenotype was found in four GBS and two GCS isolates.     

Prevalence and mechanisms of erythromycin and clindamycin resistance in clinical isolates of β-haemolytic streptococci of Lancefield groups A, B, C and G in Seville, Spain

Susceptibility to erythromycin and clindamycin was determined in 860 consecutive clinical isolates of β-haemolytic streptococci belonging to groups A (GAS, n  =   134), B (GBS, n  =   689), C (GCS, n  =   19) and G (GGS, n  =   18). Erythromycin resistance was 26.1% in GAS, 15.7% in GBS, 5.3% in GCS and 33.3% in GGS. The highest rate of clindamycin resistance (33.3%) was in GGS, followed by GBS (15.8%), GCS (15.8%) and GAS (5.2%). The M phenotype was predominant in GAS (80%), the constitutive MLS_B phenotype was predominant in GBS (75%), and all GGS isolates showed the inducible MLS_B phenotype. The uncommon erythromycin-susceptible and clindamycin-resistant phenotype was found in four GBS and two GCS isolates.     

Clinical presentations and epidemiology of β-haemolytic streptococcal bacteraemia: a population-based study

In this population-based study, all 314 episodes of β-haemolytic streptococcal bacteraemia in adult patients in the Pirkanmaa area, Finland, during the 10-year period 1995–2004 were retrospectively reviewed. Altogether, 92 cases of bacteraemia caused by Lancefield group A β-haemolytic streptococci (GAS), 76 caused by group B β-haemolytic streptococci (GBS), 18 caused by group C β-haemolytic streptococci (GCS) and 128 caused by group G β-haemolytic streptococci (GGS) were identified. The most important finding was that the incidence of GGS increased during the study period. Disruption of the cutaneous barrier was a very common predisposing factor in GAS and GGS bacteraemias. Skin infections were the presenting clinical manifestations in two-thirds of GAS and GGS bacteraemias.     

Distribution of emm types and subtypes among noninvasive group A, C and G streptococcal isolates in western Norway

Characterization of the reservoir of beta-hemolytic streptococci in a community may shed light on the pathogenesis of severe infections caused by these bacteria. We used emm sequence typing to characterize group A streptococci (GAS), group C streptococci (GCS) and group G streptococci (GGS) in community isolates associated with noninvasive disease in western Norway. A total of 165 isolates during a 13-month period were examined. Skin and throat isolates accounted for 123 and 16, respectively, and the remaining 26 isolates were from other non-sterile sites. We identified 18 previously validated emm types and one novel subtype, emm11.7, among the 101 GAS isolates. The two predominant types, emm28 and 12, were found in 40.6% of the GAS isolates. Compared to other recent studies of noninvasive GAS infections from elsewhere in the world, we found a higher frequency of emm82 (5.9%) and emm87 (12.9%) and a lower frequency of emm1 (4.0%) and emm3 (4.0%). We found a different distribution of GAS emm types compared to a previous study from western Norway. Among the 64 isolates of GCS and GGS, 15 previously described emm types and four novel subtypes, stC1400.5, stCK401.3, stG6.3 and stG652.3, were found, stG6, stG643 and stG485 were the most prevalent types and accounted for 59.4% of the GCS and GGS isolates. The high proportion of skin isolates in the present study may indicate the existence of GAS, GCS and GGS strains with predominantly skin and soft tissue tropism in our community.     

Molecular epidemiological investigation of an outbreak of invasive β-haemolytic streptococcal infection in western Norway

During a decade-long, high endemic situation with severe group A streptococcal disease in western Norway, a cluster of 16 patients with invasive streptococcal disease was hospitalized during a period of 11 weeks. A study including clinical characteristics and molecular epidemiology of the outbreak was initiated. Relevant clinical information was obtained from the medical records of the patients. Nine of the 16 patients had soft tissue infection, and seven of these had streptococcal toxic shock syndrome (STSS). Mortality, both overall and among those with STSS, was 25%. Streptococcal isolates from these patients were characterized by serogrouping and emm sequence typing. The emm amplicons were further characterized by sequence analysis and restriction fragment length polymorphism ( emm RFLP) analysis. The streptococci were identified as group A streptococcus (GAS) in 11 patients and group G streptococcus (GGS) in four patients. The patients with GGS infection were older than the patients with GAS infection, and all patients infected with GGS had predisposing comorbidities. Isolates from 13 patients were available for emm gene analysis and found to belong to nine different emm types. Similar differentiation was obtained with emm RFLP in GAS. Hence, the outbreak was polyclonal. Results suggestive of horizontal gene transfer and recombination between the emm genes of GAS, group C streptococcus and GGS were found in the isolates from seven patients. Such genetic recombination events suggest a possible role in pathogenesis.     

Directional Gene Movement from Human-Pathogenic to Commensal-Like Streptococci

Group A streptococci (GAS) are highly pathogenic for humans, and their closest genetic relatives, group C and G streptococci (GCS and GGS, respectively), are generally regarded as commensals, although they can be found in association with human disease. As part of an effort to better understand the evolution of virulence, the phylogenetic relationships between GAS, GCS, and GGS were examined. The nucleotide sequence was determined for an internal portion of seven housekeeping (neutral) loci among >200 isolates of GAS and 34 isolates of GCS or GGS obtained from human subjects. Genotypic analysis failed to show support for the separation of GCS and GGS into two distinct populations. Unlike GAS, there was poor concordance between emm type and genetic relatedness among GCS and GGS. All housekeeping genes within GAS displayed relatively low levels of sequence diversity. In contrast, individual GCS and GGS strains had mosaic genomes, containing alleles at some loci that were similar or identical to GAS alleles, whereas the alleles at other loci were about 10 to 30% diverged. The data provide evidence for a history of recent interspecies transfer of neutral genes that exhibits a strong net directionality from GAS donors to GCS and GGS recipients. A model for the evolution of GAS and of GCS and GGS is described.     

Invasive group A,C and G streptococcal disease in western Norway: virulence gene profiles,clinical features and outcomes

Invasive group A streptococcal (iGAS) disease is endemic in Norway, but data on invasive group C and group G streptococcal (iGCS/GGS) disease are lacking. We investigated the characteristics of iGAS and iGCS/GGS infections in western Norway from March 2006 to February 2009. Clinical information was retrospectively obtained from medical records. GAS and GCS/GGS isolates were emm typed and screened for the presence of 11 superantigen (SAg) genes and the gene encoding streptococcal phospholipase A₂ (SlaA). GCS/GGS isolates were also subjected to PCR with primers targeting speG^dys. Sixty iGAS and 50 iGCS/GGS cases were identified, corresponding to mean annual incidence rates of 5.0 per 100 000 and 4.1 per 100 000 inhabitants, respectively. Skin and soft tissue infections were the most frequent clinical manifestations of both iGAS and iGCS/GGS disease, and 14 iGAS patients (23%) developed necrotizing fasciitis. The 30-day case fatality rates of iGAS and iGCS/GGS disease were 10% and 2%, respectively. emm1, emm3 and emm28 accounted for 53% of the GAS isolates, and these types were associated with severe clinical outcome. SAg gene and SlaA profiles were conserved within most of the GAS emm types, although five profiles were obtained within isolates of emm28. stG643 was the most prevalent GCS/GGS emm type, and speG^dys was identified in 73% of the GCS/GGS isolates. Neither GAS SAg genes nor SlaA were detected in GCS/GGS. Our findings indicate a considerable burden of both iGAS and iGCS/GGS disease and a high frequency of necrotizing fasciitis caused by GAS in our community.     

emm gene diversity, superantigen gene profiles and presence of SlaA among clinical isolates of group A, C and G streptococci from western Norway

In order to investigate molecular characteristics of beta-hemolytic streptococcal isolates from western Norway, we analysed the entire emm gene sequences, obtained superantigen gene profiles and determined the prevalence of the gene encoding streptococcal phospholipase A2 (SlaA) of 165 non-invasive and 34 contemporary invasive group A, C and G streptococci (GAS, GCS and GGS). Among the 25 GAS and 26 GCS/GGS emm subtypes identified, only emm3.1 was significantly associated with invasive disease. M protein size variation within GAS and GCS/GGS emm types was frequently identified. Two non-invasive and one invasive GGS possessed emm genes that translated to truncated M proteins as a result of frameshift mutations. Results suggestive of recombinations between emm or emm-like gene segments were found in isolates of emm4 and stG485 types. One non-invasive GGS possessed speC, speG, speH, speI and smeZ, and another non-invasive GGS harboured SlaA. speA and SlaA were over-represented among invasive GAS, probably because they were associated with emm3. speG ^dys was identified in 83% of invasive and 63% of non-invasive GCS/GGS and correlated with certain emm subtypes. Our results indicate the invasive potential of isolates belonging to emm3, and show substantial emm gene diversity and possible lateral gene transfers in our streptococcal population.     

Distribution of <Emphasis Type="Italic">emm</Emphasis> types in invasive and non-invasive group A and G streptococci

Our study describes the emm type distributions of invasive and non-invasive group A streptococci (GAS) and group G streptococci (GGS) strains in one of the biggest Health Districts in Finland. A total of 571 GAS or GGS were recovered from patients with invasive or non-invasive infections during a 1-year period in 2008–2009 in Pirkanmaa Health District in Finland. We describe here the emm type distributions of GAS and GGS collected from throat (n = 246), pus (n = 217), deep tissue (n = 56) and blood (n = 52). The most common emm types among GAS were emm77, emm1, emm28, emm89 and emm12. Among GGS, the most common emm types were stG480, stG643, stG6, stC6979 and stG485. Some emm types were found to associate with certain infection focus. In GAS, emm77 associated with pus isolates, whereas emm1 and emm12 were more frequent among throat isolates. In GGS, stG480 was more commonly found from throat isolates.     

Inverse relation between disease severity and expression of the streptococcal cysteine protease, SpeB, among clonal M1T1 isolates recovered from invasive group A streptococcal infection cases

The streptococcal cysteine protease (SpeB) is one of the major virulence factors produced by group A streptococci (GAS). In this study we investigated if differences exist in SpeB production by clonally related M1T1 clinical isolates derived from patients with invasive infections. Twenty-nine of these isolates were from nonsevere cases and 48 were from severe cases, including streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) cases. The expression and amount of the 28-kDa SpeB protein produced were determined by quantitative Western blotting, and protease activity was measured by a fluorescent enzymatic assay. A high degree of variation in SpeB expression was seen among the isolates, and this variation seemed to correlate with the severity and/or clinical manifestation of the invasive infection. The mean amount of 28-kDa SpeB protein and cysteine protease activity produced by isolates from nonsevere cases was significantly higher than that from STSS cases (P = 0.001). This difference was partly due to the fact that 41% of STSS isolates produced little or no SpeB compared to only 14% of isolates recovered in nonsevere cases. Moreover, the cysteine protease activity among those isolates that expressed SpeB was significantly lower for STSS isolates than for isolates from nonsevere cases (P = 0.001). Increased SpeB production was also inversely correlated with intact M protein expression, and inhibition of cysteine protease activity blocked the cleavage of the surface M protein. Together, the data support the existence of both an "on-off" and a posttranslational regulatory mechanism(s) controlling SpeB production, and they suggest that isolates with the speB gene in the "off" state are more likely to spare the surface M protein and to be isolated from cases of severe rather than nonsevere invasive infection. These findings may have important implications for the role of SpeB in host-pathogen interactions via regulation of the expression of GAS virulence genes and the severity of invasive disease.     

Characterization of group C and G streptococcal strains that cause streptococcal toxic shock syndrome

Twelve strains (the largest number ever reported) of group C and G(1) streptococci (GCS and GGS, respectively) that caused streptococcal toxic shock syndrome (STSS) were collected and characterized. Eleven strains were identified as Streptococcus dysgalactiae subsp. equisimilis, and one strain was identified as Streptococcus equi subsp. zooepidemicus. We found that it was the first reported case of STSS caused by S. equi subsp. zooepidemicus. Cluster analysis according to the 16S rRNA gene (rDNA) sequences revealed that the S. dysgalactiae strains belonged to clusters I and II, both of which were closely related. The emm types and the restriction patterns of chromosomal DNA measured by pulsed-field gel electrophoresis were highly variable in these strains except BL2719 and N1434. The 16S rDNA sequences and other characteristics of these two strains were indistinguishable, suggesting the clonal dissemination of this particular S. dysgalactiae strain in Japan. As the involvement of superantigens in the pathogenesis of group A streptococcus-related STSS has been suggested, we tried to detect known streptococcal superantigens in GCS and GGS strains. However, only the spegg gene was detected in seven S. dysgalactiae strains, with none of the other superantigen genes being detected in any of the strains. However, the sagA gene was detected in all of the strains except Tokyo1291. In the present study no apparent factor(s) responsible for the pathogenesis of STSS was identified, although close genetic relationships of GCS and GGS strains involved in this disease were suggested.     

Clonal relationships between invasive and noninvasive Lancefield group C and G streptococci and emm-specific differences in invasiveness

Lancefield group G and group C streptococci (GGS and GCS, respectively) are pathogens responsible for a number of life-threatening infections. A collection of 116 recent (1998 to 2004) invasive (n = 28) and noninvasive (n = 88) GGS and GCS clinical isolates from Portugal were characterized. All isolates were identified as Streptococcus dysgalactiae subsp. equisimilis and characterized by emm typing and DNA macrorestriction profiling using pulsed-field gel electrophoresis (PFGE). emm typing revealed the presence of 22 distinct types, including 3 novel types. PFGE identified 14 clones with more than two isolates, but over half of the isolates were concentrated in 3 large clones. Individual clones and emm types showed a low level of association, since the majority of the clones included more than one emm type and the same emm type was found among diverse genetic backgrounds. Two emm types, stg2078 and stg10, were significantly more frequent among invasive isolates, and another two, stg6792 and stg166b, were present only in noninvasive isolates, suggesting a correlation between emm type and invasive disease potential.     

Clinical aspects of invasive infections with Streptococcus dysgalactiae ssp. equisimilis in Japan: differences with respect to Streptococcus pyogenes and Streptococcus agalactiae infections

Streptococcus dysgalactiae ssp. equisimilis (SDSE) is increasingly being identified as a pathogen responsible for invasive and non-invasive infections. We compared the clinical features of invasive SDSE infections with those of invasive infections caused by Streptococcus pyogenes (group A streptococcus (GAS)) and Streptococcus agalactiae (group B streptococcus (GBS)). Active surveillance for invasive SDSE, GAS and GBS was maintained over 1 year at 142 medical institutions throughout Japan. Clinical information was collected together with isolates, which were characterized microbiologically. Two hundred and thirty-one invasive SDSE infections were identified, 97 other patients had infections with GAS, and 151 had infections with GBS. The median age of the SDSE patients was 75 years; 51% were male and 79% had underlying diseases. Forty-two SDSE patients (19%) presented to the emergency department. Among the 150 patients (65%) for whom follow-up was completed, 19 (13%) died and eight (5%) had post-infective sequelae (poor outcome). Insufficient white blood cell responses (<5000 cells/µL) and thrombocytopenia on admission each suggested significantly higher risk of poor outcome (ORs 3.6 and 4.5, respectively). Of 229 isolates, 55 (24%) showed an stG6792 emm type, which was significantly associated with poor outcome (OR 2.4). Clinical manifestations of invasive SDSE infections were distinct from those of invasive GBS infections. Primary-care doctors should consider invasive SDSE infections when treating elderly patients.     

Nationwide laboratory-based surveillance of invasive beta-haemolytic streptococci in Denmark from 2005 to 2011

The aim of this work was to describe national surveillance of invasive beta-haemolytic streptococci (BHS) in Denmark and to report overall trends and major findings by groups and types of BHS causing laboratory-confirmed disease from 2005 to 2011. A total of 3063 BHS isolates were received from 2872 patients. Based on confirmed cases the overall annual incidence increased from 6.2 to 8.9 per 100 000 persons between 2005 and 2011. In 2011 the incidences of group A, B, C and G streptococci were 3.1, 2.3, 0.9 and 2.6 per 100 000 persons, respectively. An increase was observed for all groups of BHS, but in particular for group G in men above 65 years of age. Among group A streptococci (GAS), five T-types (1, 28,12, 3,13,B3264 and B3264) represented 71% and five emm-types (1, 28, 3, 89 and 12) 76% of all isolates. Among group B streptococci (GBS) four types (III, Ia, V, Ib) represented 79% of the isolates. Potential coverage for future vaccines against GAS and GBS disease was 76% compared with the 26-valent GAS vaccine and 89% based on GBS serotypes Ia, Ib, II, III and V. The number of reported cases of invasive BHS disease increased in Denmark from 2005 to 2011. Nationwide laboratory-based surveillance of BHS is required to monitor epidemiological changes, explore potential outbreaks and determine potential vaccine coverage.     

Mechanism to cause streptococcal toxic-shock syndrome

Group A streptococci(GAS) are responsible for a number of infectious diseases in humans. The development of a variety of antibiotic drugs decreased the number of bacterial infections dramatically. Streptococcal toxic shock-like syndrome(TSLS), which is now also called streptococcal toxic shock syndrome(STSS) has, however, become an important disease because it causes severe life-threatening clinical symptoms. Several reports have described STSS during pregnancy. Although a number of GAS virulence factors have been examined, the critical causative agents for STSS has not yet been identified. The clinical symptoms and pathological examinations of STSS suggest that STSS is caused by multiple factors. We need to study the pathogenic mechanism of STSS from both the bacterial and host side. Our study indicated that the lethal activity and anti-phagocytic activity examined in mice and SLO may be a major pathogenic factor in STSS.     

Characterization of sil in Invasive Group A and G Streptococci: Antibodies against Bacterial Pheromone Peptide SilCR Result in Severe Infection

Group G beta-hemolytic streptococcus (GGS) strains cause severe invasive infections, mostly in patients with comorbidities. GGS is known to possess virulence factors similar to those of its more virulent counterpart group A streptococcus (GAS). A streptococcal invasion locus, sil, was identified in GAS. sil encodes a competence-stimulating peptide named SilCR that activates bacterial quorum sensing and has the ability to attenuate virulence in GAS infections. We found that sil is present in most GGS strains (82%) but in only 25% of GAS strains, with a similar gene arrangement. GGS strains that contained sil expressed the SilCR peptide and secreted it into the growth medium. In a modified murine model of GGS soft tissue infection, GGS grown in the presence of SilCR caused a milder disease than GGS grown in the absence of SilCR. To further study the role of the peptide in bacterial virulence attenuation, we vaccinated mice with SilCR to produce specific anti-SilCR antibodies. Vaccinated mice developed a significantly more severe illness than nonvaccinated mice. Our results indicate that the sil locus is much more prevalent among the less virulent GGS strains than among GAS strains. GGS strains express and secrete SilCR, which has a role in attenuation of virulence in a murine model. We show that the SilCR peptide can protect mice from infection caused by GGS. Furthermore, vaccinated mice that produce specific anti-SilCR antibodies develop a significantly more severe infection. To our knowledge, this is a novel report demonstrating that specific antibodies against a bacterial component cause more severe infection by those bacteria.     

Identification and characterization of an antigen I/II family protein produced by group A Streptococcus

Group A Streptococcus (GAS) is a gram-positive human bacterial pathogen that causes infections ranging in severity from pharyngitis to life-threatening invasive disease, such as necrotizing fasciitis. Serotype M28 strains are consistently isolated from invasive infections, particularly puerperal sepsis, a severe infection that occurs during or after childbirth. We recently sequenced the genome of a serotype M28 GAS strain and discovered a novel 37.4-kb foreign genetic element designated region of difference 2 (RD2). RD2 is similar in gene content and organization to genomic islands found in group B streptococci (GBS), the major cause of neonatal infections. RD2 encodes seven proteins with conventional gram-positive secretion signal sequences, six of which have not been characterized. Herein, we report that one of these six proteins (M28_Spy1325; Spy1325) is a member of the antigen I/II family of cell surface-anchored molecules produced by oral streptococci. PCR and DNA sequence analysis found that Spy1325 is very well conserved in GAS strains of distinct M protein serotypes. As assessed by real-time TaqMan quantitative PCR, the Spy1325 gene was expressed in vitro, and Spy1325 protein was present in culture supernatants and on the GAS cell surface. Western immunoblotting and enzyme-linked immunosorbent assays indicated that Spy1325 was produced by GAS in infected mice and humans. Importantly, the immunization of mice with recombinant Spy1325 fragments conferred protection against GAS-mediated mortality. Similar to other antigen I/II proteins, recombinant Spy1325 bound purified human salivary agglutinin glycoprotein. Spy1325 may represent a shared virulence factor among GAS, GBS, and oral streptococci.     

Antibiotic Susceptibilities of Group C and Group G Streptococci Isolated from Patients with Invasive Infections: Evidence of Vancomycin Tolerance among Group G Serotypes

A retrospective review of medical records for 32 patients with invasive group C streptococcus (GCS) or group G streptococcus (GGS) infections was performed. MICs and minimum bactericidal concentrations (MBCs) of penicillin, erythromycin, and vancomycin for all isolates were obtained. Tolerance of vancomycin, defined as an MBC 32 or more times higher than the MIC, was exhibited by 18 GGS isolates (54%). The identification of tolerance in clinical isolates of GGS and GCS may have clinical implications in treating these seriously ill patients.     

Epidemiology of invasive Streptococcus pyogenes infections in France in 2007

Invasive group A streptococcal (GAS) infections cause significant morbidity and mortality. A national survey was initiated to assess the burden of invasive GAS infections in France, describe their clinical characteristics, and assess the molecular characteristics of GAS strains responsible for these infections. The survey was conducted in 194 hospitals, accounting for 51% of acute care hospital admissions in France. Clinical data, predisposing factors, and demographic data were obtained, and all GAS isolates were emm sequence typed. We identified 664 cases of invasive GAS infections, with an annual incidence of 3.1 per 100,000 population. The case-fatality ratio was 14% and rose to 43% in the case of streptococcal toxic shock syndrome. Bacteremia without identified focus (22%) and skin/soft tissue infections (30%) were the most frequent clinical presentations. Necrotizing fasciitis was frequent in adults (18%) and uncommon in children (3%). The 3 predominant emm types were emm1, emm89, and emm28, accounting for 33%, 16%, and 10% of GAS isolates, respectively. The emm1 type was associated with fatal outcomes and was more frequent in children than in adults. Six clusters of cases were identified, with each cluster involving 2 invasive cases due to GAS strains which shared identical GAS emm sequence types. Four clusters of cases involved eight postpartum infections, one family cluster involved a mother and child, and one cluster involved two patients in a nursing home. Invasive GAS infection is one of the most severe bacterial diseases in France, particularly in persons aged ≥ 50 years or when associated with toxic shock syndrome.     

Long-term surveillance of invasive group A streptococcal disease in The Netherlands, 1994–2003

A nationwide laboratory-based surveillance study of invasive group A streptococcal (GAS) infections was conducted in The Netherlands from May 1994 until December 2003 (average population during this period was 15 729 704). Microbiologically invasive isolates were obtained from 1504 patients, with most (70%) isolates cultured from blood. There was a clear seasonal pattern in invasive streptococcal infections, with an estimated annual incidence that peaked in 1996 (4.0 cases/100 000 individuals/year) and was at its lowest in 1999 (2.0 cases/100 000 individuals/year). Twenty-eight different M-types were identified, of which the most frequent were M1 (339/1504, 23%), M3 (187/1504, 12%), M89 (174/1504, 12%), M28 (164/1504, 11%), M12 (109/1504, 7%) and M6 (55/1504, 4%). There was a high degree of variation in the relative annual contributions of the predominant M-types, but variations in M1 and M3 combined correlated with overall changes in the annual incidence. The contribution of the patient group aged > or = 56 years to all cases of invasive GAS disease increased during the study period, whereas that of the group aged 0-20 years decreased. A peak in the incidence of invasive GAS disease among the patient group aged 30-34 years did not vary during the study period, indicating that the high incidence of invasive GAS disease in this age group was age-specific rather than cohort-related.