Impact of a Pneumococcal Conjugate Vaccination Program on Carriage among Children in Norway

In July 2006, the seven-valent pneumococcal conjugate vaccine (PCV7) was introduced in Norway with a reduced (2 doses + 1 boost) dose schedule. Post-PCV7 shifts in pneumococcal reservoirs were assessed by two point prevalence studies of nasopharyngeal colonization among children in day care centers, before (2006) and after (2008) widespread use of PCV7. Nasopharyngeal swabs were obtained from 1,213 children, 611 in 2006 and 602 in 2008. A total of 1,102 pneumococcal isolates were recovered. Serotyping, multilocus sequence typing, and antimicrobial drug susceptibility testing were performed on all isolates. Although carriage of PCV7 serotypes decreased among both vaccinated and unvaccinated children, the overall prevalence of pneumococcal carriage remained high (80.4%) after vaccine introduction. The pneumococcal populations were diverse, and in the shift toward non-PCV7 serotypes, expansion of a limited number of established clonal complexes was observed. While non-antimicrobial-susceptible clones persisted among PCV7 serotypes, antimicrobial resistance did not increase among non-PCV7 serotypes. Direct and indirect protection of PCV7 against nasopharyngeal colonization was inferred from an overall decrease in carriage of PCV7 serotypes. No preference was found for nonsusceptible clones among the replacing non-PCV7 serotypes.Streptococcus pneumoniae is a leading cause of otitis media, sinusitis, pneumonia, and meningitis worldwide (35). S. pneumoniae colonizes the nasopharynx and is considered a part of the normal flora in early childhood (1). Following the implementation of childhood vaccination with the seven-valent conjugated pneumococcal vaccine (PCV7), reports from several locations have described declines in carriage of the seven serotypes included in the vaccine, i.e., serotypes 4, 6B, 9V, 14, 18C, 19F and 23F (4, 6, 13, 19, 24). Due to reduced transmission of PCV7 serotypes, the incidence of invasive pneumococcal disease (IPD) declines also among the unvaccinated, which is an indirect effect of conjugate pneumococcal vaccination. However, the effect of PCV7 may in part be eroded over time as non-PCV7 serotypes emerge as a more frequent cause of IPD (11). In the United States, non-antimicrobial-susceptible clones seem to have an advantage among the emerging and expanding non-PCV7 serotypes, both in asymptomatic colonization and IPD (10, 20). This is primarily demonstrated by increasing incidence rates of drug-resistant clones of serotype 19A (23).PCV7 was introduced into the Norwegian childhood vaccination program in a dose schedule of two doses and one boost (2 + 1 dose schedule) in July 2006 and has been offered free of charge to all children born in 2006 and since. A high level of effectiveness of the vaccination program among children was demonstrated quickly after vaccine introduction, and the effect included a decline in IPD caused by erythromycin-resistant S. pneumoniae (34).As part of the Norwegian surveillance of PCV7 introduction, a cross-sectional study of nasopharyngeal carriage of Streptococcus pneumoniae among children attending day care centers (DCC) was performed in the early autumn of 2006. Data from this study, with exception of data regarding 38 vaccinated participants, have been reported previously (33). To assess the impact of the 2 + 1 dose schedule PCV7 vaccination program on carriage of S. pneumoniae, a follow-up was performed in 2008. Serotyping, antimicrobial susceptibility testing, and genotyping of the isolates from 2008 were performed, and the results were compared to those from analyses of the previous collection. Shifts in clonal compositions of the pneumococcal populations were analyzed and are reported here.Limited outpatient use of antimicrobial agents is recommended in Norway, and the levels of nonsusceptibility to antimicrobials among S. pneumoniae isolates from both local infections and IPD are low (25). Hence, emphasis has been put on post-PCV7 changes in nonsusceptibility to antimicrobials and nonsusceptible clones in a setting with limited antimicrobial use and resistance.     

Immunization with a Combination of Three Pneumococcal Proteins Confers Additive and Broad Protection against Streptococcus pneumoniae Infections in Mice

Pneumococcal polysaccharide-based vaccines are effective in preventing pneumococcus infection; however, some drawbacks preclude their widespread use in developing and undeveloped countries. Here, we evaluated the protective effects of ATP-dependent caseinolytic protease (ClpP), pneumolysin mutant (ΔA146 Ply), putative lipoate-protein ligase (Lpl), or combinations thereof against pneumococcal infections in mice. Vaccinated mice were intraperitoneally and/or intranasally challenged with different pneumococcal strains. In intraperitoneal challenge models with pneumococcal strain D39 (serotype 2), the most striking protection was obtained with the combination of the three antigens. Similarly, with the intranasal challenge models, (i) additive clearance of bacteria in lungs was observed for the combination of the three antigens and (ii) a combination vaccine conferred complete protection against intranasal infections of three of the four most common pneumococcal strains (serotypes 14, 19F, and 23F) and 80% protection for pneumococcal strain 6B. Even so, immunity to this combination could confer protection against pneumococcal infection with a mixture of four serotypes. Our results showed that the combination vaccine was as effective as the currently used vaccines (PCV7 and PPV23). These results indicate that system immunization with the combination of pneumococcal antigens could provide an additive and broad protection against Streptococcus pneumoniae in pneumonia and sepsis infection models.Streptococcus pneumoniae (pneumococcus) commonly colonizes the upper respiratory tract asymptomatically and was estimated, in 2005, to kill 1.6 million people every year, most of whom were children aged <5 years in developing and undeveloped countries (36). As far as we know, 91 capsular polysaccharide serotypes have been identified in S. pneumoniae (33); among these, serotypes 23F, 19F, 14, and 6B are the four most epidemic strains worldwide (2, 5, 15, 17, 25, 26, 29). Moreover, and of recent concern, the widespread use of antibiotics, leading to the development of antibiotic resistance or multidrug resistance against S. pneumoniae, is increasing (9, 12, 26).Heptavalent protein-polysaccharide conjugate vaccine (PCV7) and 23-valent pneumococcal polysaccharide vaccine (PPV23) are the two vaccines currently being used against S. pneumoniae. Both of these vaccines are polysaccharide-based formulations and effective in preventing invasive pneumococcal infections; however, some drawbacks, such as high cost, the limited polysaccharides covered, poor immunogenicity in the very young and the very old, and serotype replacement (22, 24, 26, 36), limit their wider use.Alternatively, in an attempt to overcome the disadvantages of polysaccharide-based vaccines, a number of studies have been focusing on the screening and evaluation of protein-based vaccine candidates. Pneumococcal protein vaccine candidates, such as nontoxic pneumolysin derivates, pneumococcal surface proteins (PspA and PspC), pneumococcal surface adhesin (PsaA), and ATP-dependent caseinolytic proteases (ClpP), have been studied and shown to provide protection against S. pneumoniae. In addition, another surface protein, putative lipoate-protein ligase (Lpl), has been suggested to be a vaccine candidate, which could effectively elicit a high IgG titer and reduce the blood bacterial load (30). These vaccine candidates are shared by all S. pneumoniae. Of note, it is generally recognized that pneumolysin localized in the cytoplast in a soluble monomer, and its release was dependent on or independent of autolysin (3, 4, 18, 19). A recent study, which showed that pneumolysin was also partially localized on the cell wall (34), strengthened its utility as a vaccine candidate. Additive protections were obtained with combinations of these protein candidates. However, previous evaluations have been based only on intraperitoneal challenge models of pneumococcal disease and were not, to our knowledge, been performed in pneumonia models (7, 10, 13, 20, 23, 30, 37).In the present study, putative lipoate-protein ligase (Lpl), ClpP, and Ply toxoid were expressed, purified, and confirmed to express on all of the pneumococcal strains used here. A focal pneumococcal pneumonia model, mimicking the natural pneumococcal infection, was used to evaluate pneumococci on lung colonization. We also set up models of invasive diseases, which were used to evaluate their systemic protective effects against pneumococcal infections. System vaccination with the combination of three antigens was sufficient to provide complete protection against pneumococcal serotypes 14, 19F, and 23F. In addition, this vaccination regimen conferred protection against the intranasal infection of a mixture of serotypes 14, 6B, 19F, and 23F. We now report the details of the protective effects elicited by ΔA146 Ply, ClpP, Lpl, and combinations thereof against pneumococcal infections.     

Sortase A Confers Protection against Streptococcus pneumoniae in Mice

Streptococcus pneumoniae sortase A (SrtA) is a transpeptidase that is highly conserved among pneumococcal strains, whose involvement in adhesion/colonization has been reported. We found that intraperitoneal immunization with recombinant SrtA conferred to mice protection against S. pneumoniae intraperitoneal challenge and that the passive transfer of immune serum before intraperitoneal challenge was also protective. Moreover, by using the intranasal challenge model, we observed a significant reduction of bacteremia when mice were intraperitoneally immunized with SrtA, while a moderate decrease of lung infection was achieved by intranasal immunization, even though no influence on nasopharynx colonization was seen. Taken together, our results suggest that SrtA is a good candidate for inclusion in a multicomponent, protein-based, pneumococcal vaccine.Streptococcus pneumoniae colonizes the nasopharynx of humans and represents a leading cause of severe diseases, such as otitis media, pneumonia, and meningitis. S. pneumoniae is one of the major causes of bacterial pneumonia in developing countries (19). It is estimated that each year, nearly 1 million children worldwide die because of pneumococcal diseases (10). Besides children, groups at high risk of pneumococcal infection are immunocompromised subjects and the elderly, for whom a high case fatality rate is also observed. The last decades have seen an increase in investigations of protein antigens, and several protein candidates have been proposed for a vaccine for S. pneumoniae (2) to overcome the problems inherent to the currently available polysaccharide-based vaccines. In fact, the 23-valent polysaccharide pneumococcal vaccine is not effective in children under 2 years of age, whose immune systems are unable to mount a T-independent response to polysaccharides. On the other hand, the 7-valent polysaccharide conjugate vaccine, although efficacious, induces serotype replacement (5, 20). Moreover, while more than 90 S. pneumoniae serotypes are presently known, both polysaccharide pneumococcal vaccines and polysaccharide conjugate vaccines are effective only against the serotypes included in the vaccine. Efforts to identify new S. pneumoniae factors that play a role in colonization and pathogenesis may contribute to the indication of possible targets of either new therapeutic agents or vaccines.Sortase A (SrtA) is a membrane-anchored transpeptidase expressed by gram-positive bacteria (12). The role of SrtA in the processing of sorting signals at the LPXTG motif to anchor surface proteins to the cell wall envelope was first described for Staphylococcus aureus (21), in which an isogenic SrtA mutation resulted in a strongly reduced ability to infect animals (13, 23). SrtA has been shown to participate in the colonization and/or pathogenesis of several Streptococcus species (1, 6, 8, 22, 24).S. pneumoniae SrtA has been described as playing a role in adhesion to human pharyngeal cells in vitro (7), in nasopharyngeal colonization in chinchilla (3), and in pneumonia, bacteremia, and nasopharyngeal colonization in murine models (15). Although SrtA seems to be dispensable in pilus biogenesis, its possible role in repressing pilus islet expression has been very recently proposed (9). SrtA has been found to be widely expressed among S. pneumoniae isolates and highly conserved, with a DNA identity of 99 to 100% (15). Although all of these findings suggest that pneumococcal SrtA might be useful as a protein vaccine, to the best of our knowledge no data have been provided so far on the protective efficacy afforded by SrtA immunization in animal models. Thus, we investigated the protective role of SrtA in murine models of S. pneumoniae infection.     

Psychiatric heredity and posttraumatic stress disorder: survey study of war veterans

Aim

To explore the prevalence of psychiatric heredity (family history of psychiatric illness, alcohol dependence disorder, and suicidality) and its association with the diagnosis of stress-related disorders in Croatian war veterans established during psychiatric examination.

Methods

The study included 415 war veterans who were psychiatrically assessed and diagnosed by the same psychiatrist during an expert examination conducted for the purposes of compensation seeking. Data were collected by a structured diagnostic procedure.

Results

There was no significant correlation between psychiatric heredity of psychiatric illness, alcohol dependence, or suicidality and diagnosis of posttraumatic stress disorder (PTSD) or PTSD with psychiatric comorbidity. Diagnoses of psychosis or psychosis with comorbidity significantly correlated with psychiatric heredity (φ = 0.111; P = 0.023). There was a statistically significant correlation between maternal psychiatric illness and the patients’ diagnoses of partial PTSD or partial PTSD with comorbidity (φ = 0.104; P = 0.035) and psychosis or psychosis with comorbidity (φ = 0.113; P = 0.022); paternal psychiatric illness and the patients’ diagnoses of psychosis or psychosis with comorbidity (φ = 0.130; P = 0.008), alcohol dependence or alcohol dependence with comorbidity (φ = 0.166; P = 0.001); psychiatric illness in the primary family with the patients’ psychosis or psychosis with comorbidity (φ = 0.115; P = 0.019); alcohol dependence in the primary family with the patients’ personality disorder or personality disorder with comorbidity (φ = 0.099; P = 0.044); and suicidality in the primary family and a diagnosis of personality disorder or personality disorder with comorbidity (φ = 0.128; P = 0.009).

Conclusion

The study confirmed that parental and familial positive history of psychiatric disorders puts the individual at higher risk for developing psychiatric illness or alcohol or drug dependence disorder. Psychiatric heredity might not be necessary for the individual who was exposed to severe combat-related events to develop symptoms of PTSD.There are several risk factors associated with the development of posttraumatic stress disorder (PTSD), such as factors related to cognitive and biological systems and genetic and familial risk (1), environmental and demographic factors (2), and personality and psychiatric anamnesis (3).They are usually grouped into three categories: factors that preceded the exposure to trauma or pre-trauma factors; factors associated with trauma exposure itself; and post-trauma factors that are associated with the recovery environment (2,4).There are many studies which support the hypothesis that pre-trauma factors, such as ongoing life stress, psychiatric history, female sex (3), childhood abuse, low economic status, lack of education, low intelligence, lack of social support (5), belonging to racial and ethnic minority, previous traumatic events, psychiatric heredity, and a history of perceived life threat, influence the development of stress related disorders (6). Many findings suggest that ongoing life stress or prior trauma history sensitizes a person to a new stressor (2,7-9). The same is true for the lack of social support, particularly the loss of support from significant others (2,9-11), as well as from friends and community (12-14). If the community does not have an elaborated plan for providing socioeconomic support to the victims, then the low socioeconomic status can also be an important predictor of a psychological outcome such as PTSD (2,10,15). Unemployment was recognized as a risk factor for developing PTSD in a survey of 374 trauma survivors (16). It is known that PTSD commonly occurs in patients with a previous psychiatric history of mental disorders, such as affective disorders, other anxiety disorders, somatization, substance abuse, or dissociative disorders (17-21). Epidemiological studies showed that pre-existing psychiatric problems are one of the three factors that can predict the development of PTSD (2,22). Pre-existing anxiety disorders, somatoform disorders, and depressive disorders can significantly increase the risk of PTSD (23). Women have a higher vulnerability for PTSD than men if they experienced sexually motivated violence or had pre-existing anxiety disorders (23,24). A number of studies have examined the effects of gender differences on the predisposition for developing PTSD, with the explanation that women generally have higher rates of depression and anxiety disorders (3,25,26). War-zone stressors were described as more important for PTSD in men, whereas post-trauma resilience-recovery factors as more important for women (27).Lower levels of education and poorer cognitive abilities also appear to be risk factors (25). Golier et al (25) reported that low levels of education and low IQ were associated with poorer recall on words memorization tasks. In addition, this study found that the PTSD group with lower Wechsler Adult Intelligence Scale-Revised (WAIS-R) scores had fewer years of education (25). Nevertheless, some experts provided evidence for poorer cognitive ability in PTSD patients as a result or consequence rather than the cause of stress-related symptoms (28-31). Studies of war veterans showed that belonging to racial and ethnic minority could influence higher rates of developing PTSD even after the adjustment for combat exposure (32,33). Many findings suggest that early trauma in childhood, such as physical or sexual abuse or even neglect, can be associated with adult psychopathology and lead to the development of PTSD (2,5,26,34,35). Surveys on animal models confirm the findings of lifelong influences of early experience on stress hormone reactivity (36).Along with the reports on the effects of childhood adversity as a risk factor for the later development of PTSD, there is also evidence for the influence of previous exposure to trauma related events on PTSD (9,26,28). Breslau et al (36) reported that previous trauma experience substantially increased the risk for chronic PTSD.Perceived life threats and coping strategies carry a high risk for developing PTSD (9,26). For instance, Ozer et al (9) reported that dissociation during trauma exposure has high predictive value for later development of PTSD. Along with that, the way in which people process and interpret perceived threats has a great impact on the development or maintenance of PTSD (37,38).Brewin et al (2) reported that individual and family psychiatric history had more uniform predictive effects than other risk factors. Still, this kind of influence has not been examined yet.Keeping in mind the lack of investigation of parental psychiatric heredity on the development of stress-related disorders, the aim of our study was to explore the prevalence and correlation between the heredity of psychiatric illness, alcohol dependence, suicidality, and the established diagnosis of stress-related disorders in Croatian 1991-1995 war veterans.     

Urine fibroblast growth factor 23 levels in hypertensive children and adolescents

AimTo determine the correlation of urinary fibroblast growth factor 23 (FGF23) excretion with blood pressure and calcium-phosphorus metabolism.MethodsThe study included 42 hypertensive (17 girls) and 46 healthy children and adolescents (17 girls) aged 6-18 years admitted to the Department of Pediatrics and Nephrology, Medical University of Białystok between January 2013 and December 2013. FGF23 in urine was measured using Human Intact FGF-23 ELISA Kit.ResultsHypertensive participants had significantly higher urine FGF23/creatinine values than the reference group (8.65 vs 5.59 RU/mg creatinine, P = 0.007). Urine FGF23/creatinine positively correlated with systolic blood pressure in all participants. In hypertensive patients, urine FGF23/creatinine positively correlated with serum calcium and negatively with serum 25(OH)D, urinary calcium, phosphorus, and magnesium.ConclusionThis study found that FGF23 may play an important role in the pathogenesis of hypertension in children and adolescents, but our results should be confirmed by further studies.Hypertension is a chronic medical condition and a major risk factor for cardiovascular disease, heart failure, and chronic kidney disease (CKD). Hypertension was found to be associated with several factors, among them calcium-phosphorus imbalance, lack of vitamin D, and serum parathyroid hormone (PTH) (1-5). However, far too little attention has been paid to phosphates and hormonal mechanisms responsible for their regulation, especially since the consumption of phosphorus has considerably increased in recent years. Some studies have shown that serum phosphorus increases BP (11,12). However, recent studies have found that high phosphorus intake reduces BP, when the diet is rich in calcium (6-8), while other have shown that BP was reduced by low phosphorus and high calcium diet (9,10).Phosphate concentration is primarily regulated by PTH and fibroblast growth factor 23 (FGF 23) – phosphatonin, produced by osteoblasts/osteocytes in the bone, which, similarly to PTH, stimulates phosphaturia. FGF23 decreases renal calcitriol production and inhibits PTH secretion. Its main function is to maintain phosphate homeostasis by increasing urinary phosphate excretion and decreasing serum 1,25(OH)₂D (13,14) In patients with CKD, it positively correlated with PTH secretion (15,16). The increase in FGF23 in those patients led to an early development of secondary hypertension by suppression of 1,25(OH)₂D production (17), and low phosphate intake of phosphorus binders caused 35% decrease in plasma FGF23 level (18). However in healthy individuals no changes in FGF23 levels were observed after both phosphate deprivation and loading (19,20).FGF 23 is also involved in renal sodium handling (21) and, what is even more interesting, it suppresses the expression of angiotensin-converting enzyme-2 (ACE2) in CKD-mice and thereby activates renin-angiotensin-aldosterone system (RAAS) (22). FGF23 can also influence the RAAS indirectly through vitamin D (23), which probably reduces renin gene expression and secretory activity of the juxtaglomerular apparatus, the main place of production of renin (24).The investigation of the effect of FGF23 on hypertension is not confined to in vitro models. Hypertensive people were found to have significantly higher plasma FGF23 level than normotensive people (25). FGF23 was shown to have an association with markers of inflammation in individuals with CKD stages 2-4 (26), and with impaired endothelium-dependent vasodilatation in healthy individuals and early CKD patients (27). This effect of FGF23 might also result indirectly from a decrease in 1,25 (OH)₂D (28). FGF23 also correlated with asymmetrical dimethylarginin (ADMA), which is an endogenous inhibitor of NO synthase and a biomarker of endothelial dysfunction (29).So far, however, the relevance of FGF23 in primary arterial hypertension has been under-investigated. What is more, available data focus on adult hypertensive patients and possible relation of phosphorus intake and increased FGF23 concentration to elevated BP (25). There is a paucity of similar data in children and adolescents. The aims of this research were to determine whether urinary excretion of FGF23 in hypertensive children and adolescents was higher than in healthy controls and whether its urinary level correlated with serum calcium, phosphorus, vitamin D, and PTH concentrations. Reference group data were obtained from the OLAF study, which established the reference blood pressure range for Polish children and adolescents. A strong correlation between serum and urine FGF23 was previously confirmed (r = 0.92, P < 0.001) (30).     

Can laboratory evaluation differentiate between coronavirus disease-2019, influenza,and respiratory syncytial virus infections? A retrospective cohort study

AimTo identify clinical and laboratory parameters that can assist in the differential diagnosis of coronavirus disease 2019 (COVID-19), influenza, and respiratory syncytial virus (RSV) infections.MethodsIn this retrospective cohort study, we obtained basic demographics and laboratory data from all 685 hospitalized patients confirmed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus, or RSV from 2018 to 2020. A multiple logistic regression was employed to investigate the relationship between COVID-19 and laboratory parameters.ResultsSARS-CoV-2 patients were significantly younger than RSV (P = 0.001) and influenza virus (P = 0.022) patients. SARS-CoV-2 patients also displayed a significant male predominance over influenza virus patients (P = 0.047). They also had significantly lower white blood cell count (median 6.3 × 10⁶ cells/μ) compared with influenza virus (P < 0.001) and RSV (P = 0.001) patients. Differences were also observed in other laboratory values but were insignificant in a multivariate analysis.ConclusionsMale sex, younger age, and low white blood cell count can assist in the diagnosis of COVID-19 over other viral infections. However, the differences between the groups were not substantial enough and would probably not suffice to distinguish between the viral illnesses in the emergency department.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus causing coronavirus disease 2019 (COVID-19). First identified in the Chinese province of Hubei in late 2019, COVID-19 was declared a global pandemic by the World Health Organization in March 2020 (1).As of July 2021, there were more than 180 million confirmed COVID-19 cases and more than four million patients who died due to the disease complications (2). Moreover, the disease caused a substantial economic and social burden (3), and affected health care quality (4-7).The diagnosis of COVID-19 is currently determined primarily by molecular methods and antigen tests (8,9). Radiographic diagnosis is possible as well (10,11). This practice often consumes valuable time and expensive equipment (12). There is a growing need to accelerate the diagnostic process by enabling point-of care diagnosis in various ambulatory settings, while keeping it accurate to ensure the necessary precautionary measures (13).The clinical presentation of SARS-CoV-2 infection resembles that of other respiratory viruses, with predominant symptoms of fever, cough, fatigue, and dyspnea (14-17). Hematological abnormalities, including leukopenia, lymphopenia, and thrombocytopenia, are common among COVID-19 patients, as well as elevated levels of C-reactive protein (CRP), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and ferritin (14,15,18-21). Some of these inflammatory markers correlated with disease severity and mortality (22,23).The influenza season of 2021 in the Northern hemisphere was relatively weak in contrast with predictions. Low to zero rates of influenza were detected in several countries. This was attributed to social distancing, masks wearing, and a reduced number of air travelers (24). Despite a growing number of vaccinated individuals (25), the emergence of new SARS-CoV-2 variants suggest that COVID-19 is here to stay. Seasonal viruses such as influenza virus and respiratory syncytial virus (RSV) could rebound in the following winter, with the loosening of restrictions.Differentiating between COVID-19 and other respiratory viral illnesses on clinical grounds alone can be very challenging. These viral infections share similarities in the transmission route and symptoms (26-28). Several small studies attempted to delineate the differences in the clinical presentation of SARS-CoV-2 and influenza infections (29-31). In this study, we aimed to identify demographic and laboratory parameters that can assist in the early differentiation between SARS-CoV-2, influenza, and RSV infections in the emergency department.     

Prediction of eye color in the Slovenian population using the IrisPlex SNPs

Aim

To evaluate the accuracy of eye color prediction based on six IrisPlex single nucleotide polymorphisms (SNP) in a Slovenian population sample.

Methods

Six IrisPlex predictor SNPs (HERC2 – rs12913832, OCA2 – rs1800407, SLC45A2 – rs16891982 and TYR – rs1393350, SLC24A4 – rs12896399, and IRF4 – rs12203592) of 105 individuals were analyzed using single base extension approach and SNaPshot chemistry. The IrisPlex multinomial regression prediction model was used to infer eye color probabilities. The accuracy of the IrisPlex was assessed through the calculation of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and the area under the receiver characteristic operating curves (AUC).

Results

Blue eye color was observed in 44.7%, brown in 29.6%, and intermediate in 25.7% participants. Prediction accuracy expressed by the AUC was 0.966 for blue, 0.913 for brown, and 0.796 for intermediate eye color. Sensitivity was 93.6% for blue, 58.1% for brown, and 0% for intermediate eye color. Specificity was 93.1% for blue, 89.2% for brown, and 100% for intermediate eye color. PPV was 91.7% for blue and 69.2% for brown color. NPV was 94.7% for blue and 83.5% for brown eye color. These values indicate prediction accuracy comparable to that established in other studies.

Conclusion

Blue and brown eye color can be reliably predicted from DNA samples using only six polymorphisms, while intermediate eye color defies prediction, indicating that more research is needed to genetically predict the whole variation of eye color in humans.Prediction of human visible characteristics by genotyping informative polymorphisms in DNA opens up a new perspective in the forensic field. Multiple genes including HERC2, OCA2, MC1R, SLC24A5, SLC45A2, TYR, TYRP1, ASIP, SLC24A4, TPCN2, KITLG, and IRF4 have been associated with eye, hair, and skin color in European populations and they have been used in studies dealing with eye color prediction (1-14). Variation of iris color depends on the content of eumelanine, a brown light-absorbing biopolymer, which is present in higher concentrations in brown-eyed individuals (15,16). Although eye color is evidently a continuous variable, it has been often classified into three categories – blue, brown, and intermediate (4,14). Eye color variability is particularly striking in European populations, constituting a highly differentiating trait of potential use in forensic investigations (7,14,17). Recent studies have shown that a significant fraction of human iris color variation can be explained by polymorphisms within a single region in the human genome, comprising the evolutionary conserved HERC2 gene and the neighboring OCA2 gene located on the chromosome 15. It is assumed that the level of expression of the known pigmentation gene – OCA2 – is controlled by polymorphism rs12913832 on HERC2 locus (18,19). The remaining genes that have been shown to contribute to eye color variation are SLC24A4, SLC45A2, TYR, and IRF4 (4,20,21). However, their impact on eye color prediction is lower and it seems to vary between populations (8,14,22,23). Since such differences may potentially affect accuracy of prediction in various populations, we further addressed this issue and analyzed a population sample of individuals with defined eye color from Slovenia.Several prediction models have already been proposed to be useful in eye color prediction (4,8,9,17,23,24). Here we used six IrisPlex predictors, which were selected by Liu et al (4) from a larger set of polymorphisms potentially influencing pigmentation in humans and included into the IrisPlex prediction system (4,13,17). The IrisPlex prediction model is based on a multinomial logistic regression method and uses phenotype and genotype data from 3804 Dutch individuals. Based on these data the model gives three probabilities for blue, brown, and intermediate eye color (13). From the obtained probabilities, the most probable iris color is predicted based on recommendations given in Walsh et al (13).     

The construction of the Split Sleep Questionnaire on sleep habits during the COVID-19 pandemic in the general population

AimTo construct a single-format questionnaire on sleep habits and mood before and during the COVID-19 pandemic in the general population.MethodsWe constructed the Split Sleep Questionnaire (SSQ) after a literature search of sleep, mood, and lifestyle questionnaires, and after a group of sleep medicine experts proposed and assessed questionnaire items as relevant/irrelevant. The study was performed during 2021 in 326 respondents distributed equally in all age categories. Respondents filled out the SSQ, the Pittsburgh Sleep Quality Index (PSQI), and State Trait Anxiety Inventory (STAI), and kept a seven-day sleep diary.ResultsWorkday and work-free day bedtime during the COVID-19 pandemic assessed with SSQ were comparable to the sleep diary assessment (P = 0.632 and P = 0.203, respectively), as was the workday waketime (P = 0.139). Work-free day waketime was significantly later than assessed in sleep diary (8:19 ± 1:52 vs 7:45 ± 1:20; P < 0.001). No difference in sleep latency was found between the SSQ and PSQI (P = 0.066). Cronbach alpha for Sleep Habits section was 0.819, and 0.89 for Mood section. Test-retest reliability ranged from 0.45 (P = 0.036) for work-free day bedtime during the pandemic to 0.779 (P < 0.001) for sleep latency before the pandemic.ConclusionThe SSQ provides a valid, reliable, and efficient screening tool for the assessment of sleep habits and associated factors in the general population during the COVID-19 pandemic.

The COVID-19 pandemic, along with its multiple adverse effects on various aspects of mental health, has significantly affected sleep. Sleep habits alterations and newly developed sleep disturbances during the COVID-19 pandemic may influence the overall well-being and health (1). Since the beginning of the pandemic, several studies reported a delay in bedtimes and waketimes, and an associated shift in chronotype toward eveningness (2-5).Even though actigraphy and sleep diaries provide a valid and reliable assessment of sleep habits (6,7), to achieve the highest reliability and validity, these methods require an assessment during seven consecutive days including weekends (8). Daily reporting may be perceived by the respondents as an additional burden (6,9), a limitation that may be overcome by the use of single-administration questionnaires (9,10). Since sleep disturbances recognized in the first pandemic outbreak remained stable during new waves of the COVID-19 pandemic (5), single-administration questionnaires may enable screening of large population groups and an extended assessment of sleep disturbances during the pandemic.So far, validated sleep questionnaires have most often aimed at sleep disorders or symptoms associated with sleep disorders (9). Studies commonly report the Pittsburgh sleep Quality Index (PSQI) (11), which provides data on sleep duration, sleep disturbances, and sleep latency during the previous month. However, PSQI reflects mainly sleep quality on workdays (12), while not collecting information on sleep habits on weekends. The Sleep Timing Questionnaire (STQ) has been developed as an alternative to the sleep diary for the healthy adult population, showing good reliability and validity (10). Still, although sleep habits are associated with mood (13), social media use (14-16), learning time in students (17-19), sports or exercise (20), and symptoms of insomnia (21), the STQ does not assess variables such as mood and lifestyle habits.Large studies objectively assessing sleep with wearable devices have recognized sleep timing and sleep duration to be modifiable risk factors for adverse mental health during the current pandemic (22). Young adults are especially at risk for increased mood disorder symptoms, higher levels of perceived stress, and more common alcohol use during the pandemic (23). Even though mood disorders are often reported in pandemic studies on sleep habits, mood itself has been less commonly measured and associated with sleep parameters (24). A review of the literature showed a transactional relationship between mood and emotion (25), indicating that mood is characterized by longer duration than emotion (26). Mood is often assessed with the Brief Mood Introspection Scale (27), the Profile of Mood States (28), or the Visual Analogue Mood Scale (29). A relevant aspect of mood measurement is a hierarchical structure with two broad dimensions in positive and negative affect, and multiple specific states (30). Commonly used mood assessment scales evaluate the basic negative mood of fear/anxiety, sadness/depression, and anger/hostility, as well as at least one positive mood. Therefore, it has been strongly recommended that mood researchers assess a broad range of both positive and negative emotions (30).Linking mood changes and lifestyle habits during the pandemic has been relevant in order to recognize possible predictors of mood changes, especially due to a reported increase in depression (31). Since sleep is often intertwined with mood and lifestyle changes (31), we assumed that a single-format questionnaire comprehensively assessing these variables and sleep may be applicable and timely.The aim of this study was to construct a single-format Split Sleep Questionnaire (SSQ) comprehensively assessing sleep habits, lifestyle habits, and mood changes, as well as to evaluate its reliability and validity in the general population. Sleep habits were validated by using standard instruments such as sleep diary, PSQI, and STAI questionnaires as the measures of construct validity. Additionally, we aimed to assess the psychometric properties of the Mood section and to explore the effects of the COVID-19 pandemic on sleep habits and mood alterations in the general population of Croatia.     

SIRT1 and Klotho expression in the heart and kidneys of rats with acute and chronic renovascular hypertension

AimTo evaluate Klotho and SIRT1 expression in the heart and kidneys of rats with acute and chronic renovascular hypertension.MethodsFour and sixteen weeks after the induction of renovascular hypertension by clipping the left renal artery, systemic blood pressure, serum angiotensin II level, and the expression of Klotho and SIRT1 proteins and oxidative stress indices in the heart and kidneys were assessed.ResultsSIRT1 level was significantly reduced in the ischemic (left) kidney in acute and chronic phases of hypertension. In the heart, it decreased in the acute phase, but increased in the chronic phase. Klotho levels in the heart and kidneys did not change significantly in either hypertension phase. Superoxide dismutase (SOD) activity in the heart significantly decreased, and SOD, total antioxidant capacity, and malondialdehyde in the ischemic kidney significantly increased during the development of hypertension. Serum angiotensin II level significantly increased in the acute phase of hypertension.ConclusionDevelopment of renovascular hypertension was associated with a reduction of SIRT1 expression in the heart and ischemic kidney. As angiotensin II and SIRT1 counteract each other''s expression, a SIRT1 reduction in the heart and kidney, along with the influence of systemic/local angiotensin II, seems to be partly responsible for hypertension development. A combination of SIRT1 agonists and angiotensin II antagonists may be considered for use in the treatment of renovascular hypertension.

Hypertension is one of the leading causes of disease burden worldwide, doubling the risk of coronary artery diseases (1). The prevalence of hypertension in US adults in the 2013-2016 period ranged from 26.1% in the age group 20-44 to 78.2% among people older than 65 years (2). Despite antihypertensive treatment, blood pressure of more than half of American adults is not controlled (3). Thus, to be able to produce more effective drugs, the underlying mechanisms of hypertension should be investigated.The most common cause of death in hypertensive patients is hypertensive heart disease, which results from functional and structural adaptation of the heart to high blood pressure (1). Secondary hypertension is most frequently a result of primary kidney disease. On the other hand, hypertension is a risk factor for kidney damage and end-stage renal disease (1).Hypertension and related cardiovascular diseases are age-dependent (4,5). The aging of the cardiovascular system is an important process determining longevity (6).Sirtuins are a family of enzymes encoded by SIRT1 to SIRT7 in mammals that play important roles in longevity (7). These enzymes are abundantly expressed in the nucleus and cytoplasm of several tissues, including the heart and vascular endothelium (8). The most well-known member of the sirtuin family is SIRT1, which plays beneficial roles in age-associated metabolic, inflammatory, and cardiovascular diseases (9). SIRT1 has anti-oxidant, anti-inflammatory, and anti-apoptotic effects in the endothelium and prevents endothelial senescence and dysfunction (10,11). Several studies showed that SIRT1 protected against atherosclerosis (10-13). Increasing SIRT1 expression in mice improved vascular remodeling and hypertension caused by angiotensin II (14). In addition, through reducing SIRT1 expression, hyperglycemia causes vascular damage (15).Klotho is a membrane-bound protein that exerts anti-aging function (16). Klotho deficiency leads to premature aging phenotype and shortens the lifespan (17), while its increased gene expression increases life expectancy (18). Klotho is involved in the prevention of arteriosclerosis, inducing its effects even in tissues that do not express it, which indicates its endocrine role (16). A recent study on Klotho haplodeficient mice showed that Klotho deficiency led to arteriosclerosis and hypertension, but these effects were diminished by increasing SIRT1 activity (19).One of the experimental models to evaluate secondary hypertension is 2-kidney-1-clip (2K1C) hypertension (20). In this model, a clamp is placed on one of the renal arteries to induce ischemia, while the other renal artery remains intact. This procedure steadily increases blood pressure due to an increased activity of the renin-angiotensin system in the acute phase, and sodium and water retention in the chronic phase (20,21). As SIRT1 and Klotho play a role in blood pressure regulation, and the kidneys play a role in secondary hypertension, we hypothesized that these two proteins may partake in the development of acute and chronic renovascular hypertension. Therefore, the aim of this study was to assess the expression of these two proteins in the heart and in the ischemic and non-ischemic kidneys of 2K1C rats. On the other hand, it has been shown that angiotensin II infusion increases oxidative stress and blood pressure, and that the deleterious effects of angiotensin II on blood pressure and the kidneys can be prevented by an inhibition of reactive oxygen species after angiotensin II infusion (22) and in 2K1C rats (23). Furthermore, it has been shown that SIRT1 exerts its beneficial effects by reducing oxidative stress (11,24). Therefore, the amount of oxidative stress in the heart and kidneys of the experimental animals was also assessed.     

Comparison of C-reactive protein and procalcitonin as predictors of postoperative infectious complications after elective colorectal surgery

Aim

To assess diagnostic value of perioperative procalcitonin (PCT) levels compared to C-reactive protein (CRP) levels in early detection of infectious complications following colorectal surgery.

Methods

This prospective observational study included 79 patients undergoing elective colorectal surgery. White blood cell count, CRP, and PCT were measured preoperatively and on postoperative days (POD) 1, 2, 3, 5, and patients were followed for postoperative complications. Diagnostic accuracy of CRP and PCT values on each day was analyzed by the receiver operating characteristics (ROC) curve, with infectious complications as an outcome measure. ROC curves with the largest area under the curve for each inflammatory marker were compared in order to define the marker with higher diagnostic accuracy.

Results

Twenty nine patients (36.7%) developed infectious complications. CRP and PCT concentrations increased in the early postoperative period, with a significant difference between patients with and without complications at all measured postoperative times. ROC curve analysis showed that CRP concentrations on POD 3 and PCT concentrations on POD 2 had similar predictive values for the development of infectious complications (area under the curve, 0.746 and 0.750, respectively) with the best cut-off values of 99.0 mg/L for CRP and 1.34 µg/L for PCT. Diagnostic accuracy of CRP and PCT was highest on POD 5, however the cut-off values were not considered clinically useful.

Conclusion

Serial postoperative PCT measurements do not offer an advantage over CRP measurements for prediction of infectious complications following colorectal surgery.Colorectal surgery leads to high rates of postoperative complications, varying between 28% and 38% (1-3). Early diagnosis and prompt treatment of complications is crucial for a favorable outcome. However, surgical trauma induces systemic inflammatory response syndrome (SIRS), which can hinder the diagnosis of postoperative infections (4). Therefore, it would be useful to find a biochemical marker that could accurately differentiate between infectious and non-infectious SIRS.C-reactive protein (CRP), the first of the acute phase proteins to be described, was discovered in 1930 and named for its capacity to precipitate a non-protein somatic fraction (Fraction C) of Streptococcus pneumoniae. It is a sensitive systemic marker of inflammation and tissue damage, but is not specific for infection (5,6). Surgical trauma induces a significant increase in CRP levels, which can reduce its predictive value for the diagnosis of infection in the early postoperative period (7-9). Despite this, an interest in CRP as an infection monitoring tool in the perioperative setting has increased since it was reported that in values higher than 140 mg/L on the postopertaive day (POD) 3-4 it well predicted infectious complications after colorectal surgery (10). Since then, several studies have found it to be a useful predictor of septic complications following colorectal and esophagogastric resections (11-15).Procalcitonin (PCT), the prohormone of calcitonin, was first described as a biochemical marker of infection in 1993 (16). Bacterial endotoxins are potent stimuli for PCT synthesis, which exhibits faster kinetics than CRP. PCT is released into the circulation 3-4 hours after an injection of endotoxin, reaching peak levels after 8-24 hours, while CRP peaks at 36-50 hours after stimulus (5,17). This would make PCT more suitable as an infection monitoring tool in the perioperative setting (18,19). A meta analysis of 33 studies, which included adults in intensive care units or after surgery and trauma, showed that PCT was a good diagnostic marker of sepsis, with greater diagnostic accuracy than CRP (20). Recent studies in surgical patients have also shown that after orthopedic, cardiac, and thoracic surgery PCT was better for detecting postoperative infections than CRP (21-23).To the best of our knowledge, no studies have compared the diagnostic accuracy of CRP and PCT for early detection of postoperative complications in patients undergoing colorectal surgery. The aim of this study was to assess the predictive value of serial postoperative determinations of CRP, PCT, and white blood cell (WBC) count for infectious complications after elective colorectal surgery and to compare the diagnostic accuracy of CRP and PCT.     

Glutathione S-transferase and microsomal epoxide hydrolase gene polymorphisms and risk of chronic obstructive pulmonary disease in Slovak population

Aim

To determine the risk of chronic obstructive pulmonary disease (COPD) associated with polymorphisms in the glutathione S-transferase (GST) M1, GST T1, and microsomal epoxide hydrolase (EPHX1) genes in a cohort of Slovak population.

Methods

Two hundred and seventeen patients with the diagnosis of COPD and 160 control subjects were enrolled in the study. Blood samples were collected from all subjects and the DNA from peripheral blood lymphocytes was used for subsequent genotyping assays, using polymerase chain reaction and restriction fragment-length polymorphism methods.

Results

In an unadjusted model, an increased risk for COPD was observed in subjects with EPHX1 His113-His113 genotype (odds ratio [OR], 2.32; 95% confidence interval [CI], 1.20-4.69; P = 0.008), compared with the carriers of the Tyr113 allele. However, after the adjustments for age, sex, and smoking status, the risk was not significant (adjusted OR, 1.79; 95% CI, 0.91-3.53; P = 0.093). In a combined analysis of gene polymorphisms, the genotype combination EPHX1 His113-His113/GSTM1 null significantly increased the risk of COPD in both, unadjusted (OR, 5.08; 95% CI, 1.70-20.43; P = 0.001) and adjusted model (OR, 4.87; 95% CI, 1.57-15.13; P = 0.006).

Conclusion

Although none of the tested gene polymorphisms was significantly related to an increased risk of COPD alone, our results suggest that the homozygous exon 3 mutant variant of EPHX1 gene in the combination with GSTM1 null genotype is a significant predictor of increased susceptibility to COPD in the Slovak population. The findings of the present study emphasize the importance of detoxifying and antioxidant pathways in the pathogenesis of COPD.Chronic obstructive pulmonary disease (COPD) represents a major public health care problem worldwide due to its increasing prevalence, morbidity, and mortality (1). Generally, COPD is characterized by progressive and only partially reversible airflow limitation (2). Although cigarette smoking is the most important risk factor for COPD, only 20%-30% of chronic smokers develop severe impairment of lung function associated with COPD (3). Besides smoking, other environmental and genetic factors and gene-environment interactions influence the development of COPD (4).Severe α-1-antitrypsin deficiency is a well established genetic risk factor for COPD that has provided a basis for the protease-antiprotease hypothesis in the pathogenesis of COPD (5,6). Other candidate genes that might play a role in the development of COPD are involved in endogenous protease/antiprotease imbalance, inflammatory processes, metabolism of mutagens and carcinogens in tobacco smoke, and in mucocilliary clearance (7). Interindividual differences in the polymorphisms of enzymes metabolizing the xenobiotic substances and free radicals contained in the cigarette smoke may play a role in the individual susceptibility to the decrease in lung functions in smokers (8).Microsomal epoxide hydrolase (EPHX1) is generally considered to be a protective enzyme involved in the defense from oxidative damage (9,10). Two common polymorphic sites in the EPHX1 gene that influence the enzyme activity can be detected (11). An exon 3 thymine-to-cytosine mutation changes Tyr residue 113 to His, thus reducing the enzyme activity by about 50%. The second mutation, an adenine-to-guanine transition in exon 4 of the gene, changes His residue 139 to Arg and results in the production of EPHX1 with the activity increased by about 25% (11). The combination of these polymorphisms leads to a formation of several functional phenotypes of EPHX1. The slow metabolizing type of EPHX1 was associated with emphysema and COPD (9). In another study, an association of slow metabolizing EPHX1 phenotype with an accelerated deterioration of lung function in smokers was observed (12). In addition, several studies conducted in different populations have suggested that the EPHX1 genotype may influence individual susceptibility to COPD (9,13-15). Nevertheless, other investigators failed to confirm an association between the EPHX1 gene polymorphisms and COPD (16-18).Glutathione S-transferases (GST) play a role in the detoxification of carcinogenic compounds contained in cigarette smoke and in the antioxidant protection (19,20). Recently, the GSTM1 and the GSTT1 gene polymorphisms have been excessively studied with respect to their potential contribution to the risk of COPD (8,17,21,22). The deficiency in the activity of GSTM1 and GSTT1 enzymes is caused by the inherited homozygous absence of the GSTM1 or GSTT1 gene, respectively (ie, GSTM1 null or GSTT1 null genotype). Previously, the homozygous GSTM1 null genotype has been associated with lung cancer (23), emphysema (21), and reductions in the lung function in Caucasian smokers with non-small-cell lung cancer (22). However, another study conducted in Koreans found no differences in the frequencies of polymorphic genotypes of GSTM1 and GSTT1 genes between patients with COPD and healthy smokers (17).Since current data on the potential associations between an increased COPD risk and genes encoding the enzymes metabolizing xenobiotic substances are inconsistent, the aim of our study was to analyze the relation between COPD and gene polymorphisms of EPHX1, GSTM1, and GSTT1 genes in a sample of Slovak population.     

Frequency of tuberculosis at autopsies in a large hospital in Zagreb, Croatia: a 10-year retrospective study

Aim

To assess the frequency and forms of pulmonary tuberculosis at autopsy in a high-traffic hospital in the capital city of a country with a low tuberculosis incidence.

Methods

We performed a retrospective search of autopsy data from the period 2000 to 2009 at Sestre Milosrdnice University Hospital Center, Zagreb, Croatia. We also examined patients’ records and histological slides.

Results

Of 3479 autopsies, we identified 61 tuberculosis cases, corresponding to a frequency of 1.8%. Active tuberculosis was found in 33 cases (54%), 23 of which (70%) were male. Of the 33 active cases, 25 (76%) were clinically unrecognized and 19 (76%) of these were male.

Conclusion

Clinically undiagnosed tuberculosis accounted for a substantial proportion of active tuberculosis cases diagnosed at autopsy. Autopsy data may be an important complement to epidemiological data on tuberculosis frequency.Each year, there are nearly 9 million new tuberculosis cases globally and nearly 2 million tuberculosis-related deaths (1,2). Tuberculosis occurs throughout the world, but its incidence varies greatly (3). Preventing infection through contact between healthy individuals and patients is the best measure to fight tuberculosis. The new World Health Organization strategy to fight tuberculosis, Stop TB Strategy (2006-2015), deals with the human immunodeficiency virus epidemic that has increased the incidence of tuberculosis (4). The European Centre for Disease Prevention and Control in 2008 created a strategy against tuberculosis called the “Framework Action Plan to Fight Tuberculosis in the European Union” (5). The long-term goal of the Stop TB Strategy and TB Framework Action Plan is to control and ultimately eliminate tuberculosis in the world based on four basic principles: ensure prompt and quality care for all; strengthen the capacity of health systems; develop new tools; and build partnerships and collaboration with countries and stakeholders (4,5).Croatia has a low incidence of tuberculosis, which has been steadily decreasing for the last five decades (6). The peak of the epidemic was at the turn of the 19th and 20th century, when more than 400 deaths per 100 000 people occurred as a direct result of tuberculosis (6). In the mid-20th century, the incidence of new tuberculosis cases was 20 000 per 100 000 people (6). In 2009, the incidence of new tuberculosis cases was 20 per 100 000 people (7) and in 2006 nearly all reported cases showed low levels of multidrug resistance (2,6,7). In accordance with international and European efforts, Croatia has its own guidelines for the fight against tuberculosis, with the following goals: to cure at least 85% of cases; to detect at least 70% of tuberculosis patients, and to decrease the incidence of the disease to 10 per 100 000 people (6-8).Although tuberculosis can affect any organ, 70%-80% of cases suffer from pulmonary tuberculosis (2). Generally, it is possible to detect tuberculosis infection 8-10 weeks after exposure based on a positive tuberculin skin test or an interferon-gamma release assay (9). The rest of the cases have latent tuberculosis infection (LTBI), which is an asymptomatic condition, and cannot transmit the disease (1,2). However, transmission becomes possible under certain conditions such as stress or immune suppression (6,10,11). It is believed that individuals with LTBI account for most infections in low-incidence countries like Croatia, and that this problem is compounded by migration and increasing numbers of homeless persons, alcoholics, and drug addicts (6,10,12).Statistics about tuberculosis prevalence may underestimate the number of infected people, since as many as half of the cases of pulmonary tuberculosis seen at autopsy were previously undiagnosed (12,13). In fact, few studies have examined the relationship between tuberculosis diagnoses at autopsy and reported tuberculosis prevalence in the population (14). This information may help assess whether clinically unrecognized tuberculosis poses a significant public health threat. The present study examined 3479 autopsies performed from 2000 through 2009, to assess the frequency and forms of pulmonary tuberculosis in a country with a low tuberculosis incidence. The results were compared with the number of tuberculosis patients in Croatia recorded in the Croatian Health Service Yearbook for the same period (7,8).     

Detection of Large Numbers of Pneumococcal Virulence Genes in Streptococci of the Mitis Group

Seven streptococcal isolates from the mitis group were analyzed for the presence of pneumococcal gene homologues by comparative genomic hybridization studies with microarrays based on open reading frames from the genomes of Streptococcus pneumoniae TIGR4 and R6. The diversity of pneumolysin (ply) and neuraminidase A (nanA) gene sequences was explored in more detail in a collection of 14 S. pseudopneumoniae and 29 mitis group isolates, respectively. The mitis group isolates used in the microarray experiments included a type strain (NCTC 12261), two S. mitis isolates from the nasopharynxes of children, one S. mitis isolate from a case of infective endocarditis, one S. mitis isolate from a dental abscess, and one S. oralis isolate and one S. pseudopneumoniae isolate from the nasopharynxes of children. The results of the microarray study showed that the 5 S. mitis isolates had homologues to between 67 and 82% of pneumococcal virulence genes, S. oralis hybridized to 83% of pneumococcal virulence genes, and S. pseudopneumoniae hybridized to 92% of identified pneumococcal virulence genes. Comparison of the pneumolysin, mitilysin (mly), and newly identified pseudopneumolysin (pply) gene sequences revealed that mly and pply genes are more closely related to each other than either is to ply. In contrast, the nanA gene sequences in the pneumococcus and streptococci from the mitis group are closely clustered together, sharing 99.4 to 99.7% sequence identity with pneumococcal nanA alleles.Streptococcus pneumoniae, also known as the pneumococcus, is an important human pathogen causing high levels of mortality worldwide. Diseases caused include meningitis, pneumonia, and otitis media. S. pneumoniae is a member of the viridans group streptococci (VGS) and is closely related to Streptococcus mitis and Streptococcus oralis, sharing >99% homology by 16S rRNA gene analysis (13, 23). Recently, a subset of “atypical” pneumococci were identified that were distinct from both S. mitis and S. pneumoniae by housekeeping gene analysis (48). It is likely that these isolates belonged to the species Streptococcus pseudopneumoniae, which has since been identified, with isolates appearing phenotypically similar to S. mitis but undistinguishable from S. pneumoniae in genotypic tests (2). As a result of the close relationships between these species, clear identification can be problematic. A recent study showed that definitive species identification can be achieved by phylogenetic analysis of 4 housekeeping genes. Furthermore, although it has been reported that comparison of superoxide dismutase genes (sodA) was sufficient to identify most species, this did not distinguish between S. pneumoniae and S. pseudopneumoniae isolates (19).Despite the close genetic relationships between these species, while S. pneumoniae can be a severe pathogen, S. oralis and S. mitis are usually commensal organisms of mucous membranes. However, on rare occasions, they are able to cause opportunistic disease in immunocompromised patients, particularly those with replacement heart valves, organ transplants, or cancer (4, 11, 28).The reasons for the dramatic difference in pathogenicity observed between these species are not fully known, although the pneumococcus has a large number of well-characterized virulence factors with known roles in pathogenesis, and these may account for the differences in virulence between these species. However, VGS are naturally transformable, which allows exchange of genetic information between species by homologous recombination (16, 30). Indeed, an increase in penicillin resistance in the pneumococcal population has been attributed to acquisition of resistant penicillin-binding protein variants from S. mitis and S. oralis, resulting in a “pool” of penicillin resistance genes being transferred between these species (9, 12, 38, 41). Furthermore, homologous recombination with related VGS species has been observed in the pneumococcal virulence genes encoding IgA protease and neuraminidase A (nanA) (25, 37), and homologues of pneumococcal virulence factors such as pneumolysin (Ply) and autolysin A (LytA) have been identified in related VGS species (20, 32, 48).Many pneumococcal virulence factors are currently being considered vaccine candidates for a protein-based pneumococcal vaccine affording cross-serotype protection. However, the presence of homologues of pneumococcal virulence genes in related species may allow evasion of a vaccine targeting a particular virulence factor by acquisition of a gene variant by homologous recombination (6, 36). As a result, it is important not only to increase our understanding of the biological differences between closely related commensals and invasive isolates but also to provide insight into selection of appropriate targets for future vaccine design.The aim of this study was to assess the presence of pneumococcal virulence factors in a cohort of streptococci from the mitis group at a genome-wide level, using comparative genomic hybridizations. Furthermore, the diversity and phylogenetic relatedness present in homologues of the pneumococcal virulence genes and vaccine candidates Ply and NanA were explored.     

The vicious interplay between disrupted sleep and malignant brain tumors: a narrative review

Malignant brain tumors are among the most aggressive human neoplasms. One of the most common and severe symptoms that patients with these malignancies experience is sleep disruption. Disrupted sleep is known to have significant systemic pro-tumor effects, both in patients with other types of cancer and those with malignant brain lesions. We therefore provide a review of the current knowledge on disrupted sleep in malignant diseases, with an emphasis on malignant brain tumors. More specifically, we review the known ways in which disrupted sleep enables further malignant progression. In the second part of the article, we also provide a theoretical framework of the reverse process. Namely, we argue that due to the several possible pathophysiological mechanisms, patients with malignant brain tumors are especially susceptible to their sleep being disrupted and compromised. Thus, we further argue that addressing the issue of disrupted sleep in patients with malignant brain tumors can, not just improve their quality of life, but also have at least some potential of actively suppressing the devastating disease, especially when other treatment modalities have been exhausted. Future research is therefore desperately needed.

The annual incidence of tumors of the central nervous system (CNS) is little over 22 per 100 000 in the general population (1). Around a third of these lesions are malignant. Among the malignant tumors, gliomas are by far the most common type, constituting over 80% of the number. Among gliomas, the most aggressive type (glioblastoma) is the most common one, making up over a half of all newly diagnosed gliomas (2,3). The five-year survival of patients with malignant CNS tumors is around 30%, with patients being diagnosed a glioblastoma having a five-year survival rate of less than 5%. All this goes to show how malignant CNS tumors are some of the most aggressive human malignancies today. It also shows how the vast accumulated knowledge on the disease origin and progression still has not translated into significant improvement of the overall survival of these patients. New treatment modalities are therefore desperately needed.Besides the devastating diagnosis of a malignant brain tumor, these patients often experience a wide variety of severe symptoms, which significantly diminish their quality of life (4). There has been an increasing awareness of the importance of supportive and palliative care in patients suffering from malignant brain tumors, especially those in whom other treatment modalities have been exhausted (5-7). One of the most commonly reported symptoms is sleep disturbance (4,8-12).Sleep is a recurrent, physiological phenomenon, which consists of many measurable factors (12) and is ubiquitous throughout the natural world (13-16). It is a highly active, easily reversible process, which is crucial not only for the physical and mental well-being of all living organisms, but also for the very concepts we as humans have of ourselves and the world around us (17). There are many theories regarding the possible function of sleep, ranging from the physiological explanations such as rest of individual cells (18) to behavioral explanations of why a biological system needs periodic inactivity (19). There is a growing understanding of how the modern lifestyle disrupts the natural circadian rhythm in humans, consequences of which are still not sufficiently explored (20).Sleep disruption has a well known detrimental role for an organism. Indeed, patients with disrupted sleep have been found to have a higher prevalence of several diseases, such as cardiovascular disorders (21), cognitive impairment (22), various metabolic disorders and obesity (23,24), and systemic and local inflammation (25,26). Furthermore, sleep can be impaired in many ways. The current classification of sleep disorders consists of several clinical entities such as insomnia, parasomnia, hyper-somnolence, sleep-related movement disorders, etc (27). However, this article refers to all of this broad pathology as “sleep disturbance,” primarily for clarity and simplicity sake. In addition, research on disrupted sleeping patterns in patients with malignant lesions usually also encompasses all of these entities into this broader term (28,29).     

Sex-specific chronic stress response at the level of adrenal gland modifies sexual hormone and leptin receptors

Aim

To compare cardiometabolic risk-related biochemical markers and sexual hormone and leptin receptors in the adrenal gland of rat males, non-ovariectomized females (NON-OVX), and ovariectomized females (OVX) under chronic stress.

Methods

Forty six 16-week-old Sprague-Dawley rats were divided into male, NON-OVX, and OVX group and exposed to chronic stress or kept as controls. Weight, glucose tolerance test (GTT), serum concentration of glucose, and cholesterol were measured. Adrenal glands were collected at the age of 28 weeks and immunohistochemical staining against estrogen beta (ERβ), progesterone (PR), testosterone (AR), and leptin (Ob-R) receptors was performed.

Results

Body weight, GTT, serum cholesterol, and glucose changed in response to stress as expected and validated the applied stress protocol. Stressed males had significantly higher number of ERβ receptors in comparison to control group (P = 0.028). Stressed NON-OVX group had significantly decreased AR in comparison to control group (P = 0.007). The levels of PR did not change in any consistent pattern. The levels of Ob-R increased upon stress in all groups, but the significant difference was reached only in the case of stressed OVX group compared to control (P = 0.033).

Conclusion

Chronic stress response was sex specific. OVX females had similar biochemical parameters as males. Changes upon chronic stress in adrenal gland were related to a decrease in testosterone receptor in females and increase in estrogen receptor in males.Maintaining homeostasis is often challenged by different types of stressors (1). Homeostasis is regulated by a complex endocrine processes engaging the hypothalamic-pituitary-adrenal axis (HPA) and sympathetic autonomic system (2-4). Stress can occur either in acute or chronic form with different consequences – the acute stress mostly induces the ˝fight or flight˝ response, while chronic stress promotes long term changes, which can lead to a variety of diseases (5,6). If stress is of sufficient magnitude and duration, the action of HPA is unsuppressed and results in prolonged elevation of cortisol (7), induced production of energy, vasoconstriction, lipolysis, proteolysis, immunosuppression, and suppression of reproductive function to save energy and retain overall homeostasis (8). Women are generally less susceptible to chronic stress up to the period of menopause, when the loss of protective hormones, estrogen and progesterone, occurs and thus they become prone to development of depression, anxiety, or schizophrenia (9). In contrast, men are generally more susceptible and sensitive to chronic stress, showing changes in feeding habits and decreased body weight (10,11).Chronic stress can cause the development of cardiovascular disorder, obesity, and diabetes, which can be reflected in serum cholesterol, glucose, and decreased glucose tolerance (12-14). There is a strong correlation between stress and sexual hormones, but the mechanisms by which estrogen, testosterone, and progesterone exert their possible protective role under stress conditions are not fully explored. Sexual hormones affect stress outcome and stress hormones affect the levels of sexual hormones (15-17). Testosterone is activated during stress response in rats and humans (18,19) and tends to increase more in men than women (20). Estrogen lowers the stress-induced response in women and men (9,21). Estrogens and progesterone are produced even after ovariectomy by adrenal glands (22) but it is not known if such compensation can withstand additional challenge like stress. Another possible player in stress response is leptin (Ob), hormone responsible for maintaining body weight, which is synthesized and secreted by adipose tissue (23), exerting its effects through the leptin receptor (Ob-R) (24). Chronic stress models imply a direct link between stress response and leptin (25,26). Receptors for leptin are present in the adrenal gland (27). The aim of this study was to investigate cardiovascular risk parameters and changes in leptin and sexual hormone receptors in adrenal gland during chronic stress. There is a clinically relevant change in the onset of cardiometabolic risk between healthy women and women with premature ovarian failure (28) and because of that ovariectomized female rats were included in the study.     

Comparison of peritonsillar infiltration effects of ketamine and tramadol on post tonsillectomy pain: a double-blinded randomized placebo-controlled clinical trial

Aim

To assess the effect of peritonsillar infiltration of ketamine and tramadol on post tonsillectomy pain and compare the side effects.

Methods

The double-blind randomized clinical trial was performed on 126 patients aged 5-12 years who had been scheduled for elective tonsillectomy. The patients were randomly divided into 3 groups to receive either ketamine, tramadol, or placebo. They had American Society of Anesthesiologists physical status class I and II. All patients underwent the same method of anesthesia and surgical procedure. The three groups did not differ according to their age, sex, and duration of anesthesia and surgery. Post operative pain was evaluated using CHEOPS score. Other parameters such as the time to the first request for analgesic, hemodynamic elements, sedation score, nausea, vomiting, and hallucination were also assessed during 12 hours after surgery.

Results

Tramadol group had significantly lower pain scores (P = 0.005), significantly longer time to the first request for analgesic (P = 0.001), significantly shorter time to the beginning of liquid regimen (P = 0.001), and lower hemodynamic parameters such as blood pressure (P = 0.001) and heart rate (P = 0.001) than other two groups. Ketamine group had significantly greater presence of hallucinations and negative behavior than tramadol and placebo groups. The groups did not differ significantly in the presence of nausea and vomiting.

Conclusion

Preoperative peritonsillar infiltration of tramadol can decrease post-tonsillectomy pain, analgesic consumption, and the time to recovery without significant side effects.Registration No: IRCT201103255764N2Postoperative pain has not only a pathophysiologic impact but also affects the quality of patients’ lives. Improved pain management might therefore speed up recovery and rehabilitation and consequently decrease the time of hospitalization (1). Surgery causes tissue damage and subsequent release of biochemical agents such as prostaglandins and histamine. These agents can then stimulate nociceptors, which will send the pain message to the central nervous system to generate the sensation of pain (2-4). Neuroendocrine responses to pain can also cause hypercoagulation state and immune suppression, leading to hypoglycemia, which can delay wound healing (5).Tonsillectomy is a common surgery in children and post-tonsillectomy pain is an important concern. Duration and severity of pain depend on the surgical technique, antibiotic and corticosteroid use, preemptive and postoperative pain management, and patient’s perception of pain (6-9). There are many studies that investigated the control of post tonsillectomy pain using different drugs such as intravenous opioids, non-steroidal anti-inflammatory drugs, steroids, ketamine, as well as peritonsillar injection of local anesthetic, opioid, and ketamine (6,7,10-14).Ketamine is an intravenous anesthetic from phencyclidin family, which because of its antagonist effects on N methyl-D-aspartate receptors (that are involved in central pain sensitization) has regulatory influence on central sensitization and opium resistance. It can also band with mu receptors in the spinal cord and brain and cause analgesia. Ketamine can be utilized intravenously, intramuscularly, epidurally, rectally, and nasaly (15,16). Several studies have shown the effects of sub-analgesic doses of ketamine on postoperative pain and opioid consumption (7,13,15-17). Its side effects are hallucination, delirium, agitation, nausea, vomiting, airways hyper-secretion, and increased intra cerebral pressure and intra ocular pressure (10,11,15,16).Tramadol is an opium agonist that mostly effects mu receptors, and in smaller extent kappa and sigma receptors, and like anti depressant drugs can inhibit serotonin and norepinephrine reuptake and cause analgesia (6,12,18). Its potency is 5 times lower than morphine (6,12), but it has lower risk of dependency and respiratory depression, without any reported serious toxicity (6,12). However, it has some side effects such as nausea, vomiting, dizziness, sweating, anaphylactic reactions, and increased intra-cerebral pressure. It can also lower the seizure threshold (6,12,18,19).Several studies have confirmed the efficacy of tramadol and ketamine on post-tonsillectomy pain (6,10-12,20). In previous studies, effects of peritonsillar/ IV or IM infiltration of tramadol and ketamine were compared to each other and to placebo, and ketamine and tramadol were suggested as appropriate drugs for pain management (6,7,10-19,21). Therefore, in this study we directly compared the effect of peritonsillar infiltration of either tramadol or ketamine with each other and with placebo.     

Intermittent tramadol vs tramadol administration via patient-controlled pump after lumbar discectomy: a randomized controlled trial

AimTo compare the effect of intermittent tramadol dosing vs tramadol administration via patient-controlled pump on pain after lumbar discectomy.MethodsThis randomized prospective study enrolled 100 patients who underwent elective LIV-LV lumbar discectomy in the neurosurgery department at Sestre Milosrdnice University Hospital Center from May 2016 to July 2017. Patients were randomized to receive either tramadol (600 mg daily) via a patient-controlled analgesia (PCA) pump or intermittently. Pain was evaluated by the Croatian version of Short-Form McGill Pain Questionnaire.ResultsForty percent of patients were women. The median (interquartile range) age of the patients was 51 (40-61) years. The groups did not differ in pain at 7 pm on the day of discectomy. However, in the morning and evening on the first postoperative day and in the morning and evening of the second postoperative day, the PCA group had significantly lower pain (P = 0.023, P < 0.001, P < 0.001, P = 0.026, respectively).ConclusionThis is the first study that used the Short Form McGill Pain Questionnaire to compare the effect of tramadol administration via PCA pump and intermittent administration on pain after LIV-LV discectomy in a neurosurgery department. Tramadol showed a good analgesic efficacy in lumbar spine surgery; tramadol via PCA controlled pain more effectively than intermittently administered tramadol.Australian New Zealand Clinical Trials Registry: ANZCTR12618001893291.

Lower back pain due to lumbar disc herniation is a common complaint. In more than two-thirds of patients, non-medical pain relief methods did not decrease pain or even increased it (1). Lumbar discectomy is one of the most common spinal surgical procedures (2-4). The treatment of pain after lumbar discectomy includes specific analgesia administered according to pain intensity and balanced use of medications. Therefore, a successful treatment requires an understanding of neuroanatomy and of the complex pathological and physiological pain mechanisms.Adequate pain management in the postoperative period demands an interdisciplinary approach and is associated with better functional outcomes, early patient mobilization, early hospital discharge, and preventing chronic pain occurrence (5,6). Unimodal analgesia implies administering one analgesic from a specific group (nonsteroidal anti-inflammatory drugs, opioids, non-opioids), requiring relatively high doses of analgesics to achieve a better pain relief.Tramadol is an antinociceptive drug with low affinity for opioid receptors that achieves analgesia by combining indirect postsynaptic activation of two adrenergic receptors and opioid activity. It is administered at doses of up to 600 mg daily (7-13). Tramadol shows good analgesic efficacy and potency, comparable to codeine and morphine (14,15). It is associated with a lower risk of sedation and opioid addiction due to its low potency and weak affinity for μ receptors and blocking of repeated noradrenalin storage (16,17). A meta-analysis of 18 placebo-controlled studies confirmed the safety and efficacy of tramadol in treating moderate to severe postoperative pain, depending on the dose (18). However, some authors suggest that the relative “safety” of tramadol or opioids in general cannot be determined (19). Postoperative pain can be differently affected by different methods of unimodal analgesic administration. In some studies, patient-controlled analgesia (PCA), compared with conventional opioid analgesia, resulted in better pain control and increased patient satisfaction in postoperative pain management (20,21). While some studies indicated equal efficacy of intermittent and PCA administration in pain assessment (22,23), others showed PCA superiority (24). Unimodal administration of tramadol by two different methods has not been investigated in postoperative pain management after lumbar discectomy. The aim of this study was to investigate intermittent tramadol dosing vs tramadol administration via patient-controlled pump after lumbar discectomy.     

Serotype and Genotype Replacement among Macrolide-Resistant Invasive Pneumococci in Adults: Mechanisms of Resistance and Association with Different Transposons

The aim of this study was to analyze trends in adult invasive pneumococcal disease (IPD) due to macrolide-resistant strains and to study the evolution of serotypes, genotypes, and macrolide-resistant determinants of strains collected in a prospective study between 1999 and 2007 in Barcelona, Spain. IPD due to macrolide-resistant strains of serotypes included in the 7-valent conjugate vaccine (PCV7) decreased from 2.16/100,000 (pre-PCV7 period, 1999 to 2001) to 0.80/100,000 (late-PCV7 period, 2005 to 2007) (P = 0.001), whereas IPD due to macrolide-resistant strains of non-PCV7 serotypes increased from 1.08/100,000 to 2.83/100,000 (P < 0.001). These changes were related to a fall of clones of PCV7 serotypes (ST81 [P < 0.05], ST90, ST315, and ST17) and an increase in new clones of serotypes 19A and 24F (ST230) and 33F (ST717) in the late-PCV7 period. The most common phenotype was MLS_B (90.9%), related to the erm(B) gene. The frequent association between MLS_B phenotype and tetracycline resistance [tet(M) gene], was related to transposons of the Tn916-family such as Tn6002 or Tn3872. In conclusion, overall adult IPD rates due to macrolide-resistant pneumococci stabilized between 1999 and 2007 in Barcelona. The decrease in macrolide-resistant PCV7 pneumococci was balanced by the increase in macrolide-resistant non-PCV7 pneumococci.The worldwide increase in the prevalence of macrolide resistance (MR) in Streptococcus pneumoniae is a matter for concern, because of the risk of treatment failure in infections caused by these microorganisms (16). A global international surveillance project (PROTEKT, 2003 to 2004) found that the overall rate of erythromycin resistance was 37.2%, although significant geographical differences in these rates were observed (11). MR in S. pneumoniae is mediated mainly by two major mechanisms, namely, target site modification [encoded by the erm(B) gene and related to the MLS_B phenotype] and an efflux pump (encoded by mef genes and related to the M phenotype). Other, less-common resistance mechanisms are mutations in the 23S rRNA and/or alterations in ribosomal proteins (L4 and L22) (18). Most macrolide-resistant S. pneumoniae strains are also resistant to tetracycline. This association is due to the insertion of the erm(B) gene into conjugative and composite transposons of the Tn916 family (Tn1545, Tn3872, Tn6002, and Tn6003) which carry the tet(M) gene (32).Since the introduction of the pediatric heptavalent pneumococcal conjugate vaccine (PCV7), major changes in the epidemiology of invasive pneumococcal disease have been described. In the United States, the incidence of invasive pneumococcal disease (IPD) has decreased since PCV7 introduction, even in the nontarget population due to herd immunity (33). Since most PCV7 serotypes (6B, 9V, 14, 19F, and 23F) are usually penicillin and multidrug resistant, the rates of penicillin-resistant disease have decreased (17). Moreover, a decrease in macrolide-resistant IPD was observed in the United States (29), mainly due to a reduction of serotype 14 pneumococci harboring mef(E).In Spain, since the introduction of voluntary PCV7 vaccination in June 2001, the rates of IPD due to PCV7 serotypes disease have decreased in both children and adults (13). However, an overall increase in IPD due to non-PCV7 serotypes was observed between 2005 and 2007 in our geographical area (2, 24).The aim of the present study was to analyze trends in macrolide-resistant IPD and their relationship to serotypes, genotypes, and MR genes.     

Clinical and pathohistological characteristics of Alport spectrum disorder caused by COL4A4 mutation c.193-2A>C: a case series

AimTo present the pathohistological and clinical characteristics of five Croatian families with Alport spectrum disorders caused by splice acceptor pathogenic variant c.193-2A>C in COL4A4 at the genomic position chr2:227985866.MethodsThe study enrolled five probands with kidney biopsy analysis and five family members. Mutation screening was performed with Illumina MiSeq platform. The pathogenic variant was confirmed with standard dye-terminator sequencing.ResultsThe only homozygous patient, aged two, had proteinuria and hematuria with preserved kidney function and no extrarenal manifestations. This patient had changes characteristic for Alport syndrome observed on electron microscopy of the kidney biopsy. In the heterozygous group, six patients had hematuria, four biopsied probands had proteinuria, and only one had moderately reduced kidney function. Heterozygous probands had variable kidney biopsy findings. Three patients had thin glomerular basement membrane nephropathy visible on electron microscopy and focal segmental glomerulosclerosis on light microscopy, two of them with focal lamellation on electron microscopy. One heterozygous patient had changes characteristic for Alport syndrome on electron microscopy without focal segmental glomerulosclerosis.ConclusionThe homozygous patient had hematuria and proteinuria with preserved kidney function. The heterozygous patients presented with reasonably mild clinical phenotype and variable pathohistological findings.

Alport syndrome (AS) is a structural disorder of the glomerular basement membrane (GBM). Its genetic basis lies in the diverse mutations of COL4A3, COL4A4, and COL4A5 genes and it phenotypically manifests as a progressive nephropathy with hematuria, ultrastructural changes of the GBM, sensorineural hearing impairment, and eye abnormalities (1-5). The most frequent mutations (85%) are COL4A5 mutations, resulting in X-linked AS (6). Individuals with autosomal recessive AS (ARAS), caused by two mutations in COL4A3 and/or COL4A4, have similar clinical features to men with X-linked AS (7,8). The type of mutation affects disease phenotype and manifestation. The phenotype is usually severe both in men and women, with early onset of end-stage renal disease (ESRD) and frequent extrarenal disorders (9,10).The spectrum of AS disorders has recently been expanded (11). Naming and describing individuals with heterozygous COL4A3 and COL4A4 mutations is still a matter of debate (10,12,13). A number of studies showed a correlation between thin glomerular basement membrane nephropathy (TBMN) with the heterozygosity for COL4A3 or COL4A4 mutation and benign familial hematuria (2,14-25). However, a variable proportion of COL4A3 or COL4A4 carriers progress to proteinuria, hypertension, and ESRD, which raises the question of the nomenclature of autosomal dominant AS (ADAS) (14,25-31). Some scientists advocate the use of the term ADAS, others continue to use the term TBMN, while a Cyprus research group uses the term late-onset Alport nephropathy (10,12,13,32). The rationale behind ADAS nomenclature for heterozygous COL4A3 or COL4A4 patients lies in the presence of thin GBM in the kidney biopsy specimens of patients with X-linked AS and ARAS and the heterozygous carriers of COL4A3 or COL4A4 mutation (10). The authors suggest that this approach would improve clinical and diagnostic evaluation, with the possibility of ESRD rate reduction and treatment optimization (10). There are also rationales behind the use of the term TBMN. Savige et al (13) stated that most of heterozygous COL4A3 and COL4A4 carriers show either no decline in kidney function or show only mild decline with inconstant progression to ESRD and hearing impairment. The authors also argue that there is no unmistakable evidence that one mutation in COL4A3 or COL4A4 gene without disease modifying factors can be responsible for the characteristic ultrastructural signs of AS, hearing impairment, or eye abnormalities (13). Furthermore, in other genetic diseases autosomal dominant (AD) term is not used for the carriers of autosomal recessive (AR) disease because it can lead to the diagnosis of AD and AR disease in different members of the same family (13). However, there are emerging reports of autosomal dominant Alport spectrum disorders, especially in the cases that are hard to diagnose clinicopathologically (33). Here, we present the pathohistological and clinical characteristics of disorders caused by splice site mutation c.193-2A>C in COL4A4 at the genomic position chr2:227985866.     

Streptococcus pneumoniae DNA Load in Blood as a Marker of Infection in Patients with Community-Acquired Pneumonia

Direct detection of Streptococcus pneumoniae DNA in blood adds to culture results in the etiological diagnosis of patients with community-acquired pneumonia (CAP). Quantification of the amount of DNA, the bacterial DNA load (BDL), provides a measurement of DNAemia that may increase the understanding of the clinical relevance of S. pneumoniae DNA in blood. We evaluated the S. pneumoniae BDL as a diagnostic tool in adult patients with CAP. The BDL was determined in whole-blood samples collected simultaneously with blood for culture from 45 adult patients with CAP. After DNA extraction, S. pneumoniae DNA was detected with specific real-time PCR amplification, and the BDL was calculated with a standard curve. PCR and microbiological results were compared, and the BDL was related to clinical and laboratory parameters. S. pneumoniae DNA was detected in 10/13 patients with positive blood cultures and in 67% of patients with microbiologically confirmed pneumococcal pneumonia. The positive predictive values of the receiver operating characteristic curves for the BDLs for pneumococcal infection (100%) and pneumococcal bacteremia (69%) were higher than those for the level of C-reactive protein (CRP; 43% and 23%, respectively) and the white blood cell count (WBC; 42% and 35%, respectively); the negative predictive values of these three parameters were in the same range (±90 and ±97%, respectively). The BDL was higher in patients presenting with systemic inflammatory response syndrome and in patients with bacteremia. Positive correlations were observed for the BDL with WBC, CRP level, and length of stay. We conclude that the BDL supports the diagnosis of S. pneumoniae infection in patients with CAP and provides a putative marker of the severity of disease.Streptococcus pneumoniae is the most prevalent cause of community-acquired pneumonia (CAP) in adults (1, 10). Identification of S. pneumoniae as a cause of CAP can be achieved with Gram stain microscopy, culture of respiratory tract samples, blood culture, PCR, and antigen detection testing (26). A positive blood culture result provides a definite diagnosis. However, blood cultures have a low sensitivity for the confirmation of the pneumococcal etiology of CAP. This is due to the low prevalence of bacteremia in CAP, especially in uncomplicated cases, and the use of antibiotics prior to drawing blood samples for culture (11, 18, 32). In addition, blood cultures usually require 1 or 2 days before results are available and thus may have limited impact on the initial choice of empirical antimicrobial treatment (6, 29). PCR and antigen detection provide non-culture-based tools for the rapid identification of S. pneumoniae in respiratory tract specimens, pleural aspirate samples, and blood samples (7, 8, 13). These tests can add to culture methods in the etiological diagnosis of patients with CAP, particularly for patients who received antimicrobial treatment prior to sampling for culture (13, 26, 33).Several studies have evaluated the use of real-time PCR assays for the detection of S. pneumoniae DNA in blood samples from patients suspected of infection with invasive pneumococcal disease, with variable results (5, 14, 24, 31). These studies all used blood culture as the reference standard. However, positive PCRs relate not only to bacteremia but also to detection of DNA from nonviable bacteria, i.e., within phagocytes or due to the use of antibiotics. As such, it is understandable that most studies found cases with positive S. pneumoniae DNAemia and negative blood cultures. In this study, we used microbiologically documented S. pneumoniae infection as the gold standard for evaluation of PCR results and hypothesized that the bacterial DNA load (BDL) can be used to discriminate between patients with invasive infection and those with localized infection.The magnitude of bacteremia, as based on quantitative blood cultures, relates to the severity of S. pneumoniae infection (2). A high bacterial count in blood samples from children with bacteremia is associated with an increased risk of development of more-serious disease (27). Also, a shorter amount of time needed to detect positivity for blood cultures, supposedly reflecting a higher initial bacterial load, is associated with higher severity of disease (20). This relation between bacterial concentration and severity of disease may also hold true for the level of S. pneumoniae DNA in blood samples: the S. pneumoniae BDL was higher in human immunodeficiency virus-infected children who died from invasive pneumococcal disease than in survivors (5).In this study, we evaluated the S. pneumoniae BDL as a putative diagnostic tool and clinical marker of infection in adult patients with CAP.