Fluoroquinolone heteroresistance in Mycobacterium tuberculosis: detection by genotypic and phenotypic assays in experimentally mixed populations.

Heteroresistance - the simultaneous presence of drug-susceptible and -resistant organisms - is common in Mycobacterium tuberculosis. In this study, we aimed to determine the limit of detection (LOD) of genotypic assays to detect gatifloxacin-resistant mutants in experimentally mixed populations. A fluoroquinolone-susceptible M. tuberculosis mother strain (S) and its in vitro selected resistant daughter strain harbouring the D94G mutation in gyrA (R) were mixed at different ratio's. Minimum inhibitory concentrations (MICs) against gatifloxacin were determined, while PCR-based techniques included: line probe assays (Genotype MTBDRsl and GenoScholar-FQ + KM TB II), Sanger sequencing and targeted deep sequencing. Droplet digital PCR was used as molecular reference method. A breakpoint concentration of 0.25 mg/L allows the phenotypic detection of ≥1% resistant bacilli, whereas at 0.5 mg/L ≥ 5% resistant bacilli are detected. Line probe assays detected ≥5% mutants. Sanger sequencing required the presence of around 15% mutant bacilli to be detected as (hetero) resistant, while targeted deep sequencing detected ≤1% mutants. Deep sequencing and phenotypic testing are the most sensitive methods for detection of fluoroquinolone-resistant minority populations, followed by line probe assays (provided that the mutation is confirmed by a mutation band), while Sanger sequencing proved to be the least sensitive method.

Evaluation of a novel line probe assay to detect resistance to pyrazinamide, a key drug used for tuberculosis treatment.

OBJECTIVES: The development of rapid molecular diagnostic assays for pyrazinamide (PZA) resistance is considered technically challenging as mutations are highly diverse, scattered along the full length of the pncA gene and not all are associated with PZA resistance. We evaluated the performance of the novel Genoscholar PZA-TB II line probe assay (PZA-LPA2; NIPRO Corporation, Japan). METHODS: To evaluate the applicability of the PZA-LPA2 in clinical settings, we compared the performance of the PZA-LPA2 to a composite reference standard pncA Sanger and Illumina sequencing plus phenotypic susceptibility testing on a panel of 87 Mycobacterium tuberculosis isolates from World Health Organization (WHO) drug resistance surveys, harbouring mutations previously classified as associated or not associated with resistance according to data from peer-reviewed literature. In addition, the PZA-LPA2 was challenged against a selection of isolates with lineage-specific and non-resistance-associated mutations, for which the frequency among clinical isolates is unknown, and tested directly on 59 sputum extracts. RESULTS: For the survey isolates, the PZA-LPA2 reached an overall agreement with the composite reference of 97.6% (80/82) or 94.3% (82/87) excluding or including heteroresistance, respectively. The PZA-LPA2 failed on 8.5% (5/59) of clinical samples; among valid results, 100% (14/14) sensitivity and 100% (7/7) specificity was reached relative to pncA Sanger sequencing. CONCLUSIONS: The PZA-LPA2 represents a valid and rapid alternative for indirect PZA susceptibility testing. Preliminary findings on clinical samples show promise for direct testing. Further studies are needed to assess the clinical risk of missing heteroresistance and falsely detecting lineage-specific, silent and nonassociated mutations.

Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.

Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents.

A comprehensive meta-analysis of the risk of suicide in eating disorders.

OBJECTIVE: Past meta-analyses on suicide in eating disorders included few available studies. METHOD: PubMed/Medline search for papers including sample n ≥40 and follow-up ≥5 years: 40 studies on anorexia nervosa (AN), 16 studies on bulimia nervosa (BN), and three studies on binge eating disorder (BED) were included. RESULTS: Of 16,342 patients with AN, 245 suicides occurred over a mean follow-up of 11.1 years (suicide rate=0.124 per 100 person-years). Standardized mortality ratio (SMR) was 31.0 (Poisson 95% CI=21.0-44.0); a clear decrease in suicide risk over time was observed in recent decades. Of 1768 patients with BN, four suicides occurred over a mean follow-up of 7.5 years (suicide rate=0.030 per 100 person-years): SMR was 7.5 (1.6-11.6). No suicide occurred among 246 patients with BED (mean follow-up=5.3 years). CONCLUSION: AN and BN share many risk factors for suicide: the factors causing lower suicide rates per person-year in BN compared to AN should be investigated.

Suicide ideation and social desirability among school-aged young people.

A mixed male-female sample of 950 school-aged adolescents, corresponding to 10% of the young population aged 15-19 living in a rural district of Northeast Italy, was investigated with self-reported questionnaires about the links between social desirability and suicide ideation. On the whole 30.6% of females and 23.9% of males reported suicidal ideation of some degree, with 5% in both genders reporting suicide ideation of a high degree (i.e. quite a lot/extremely often). Those who scored higher at the Marlowe-Crowne Social Desirability Scale (SDS) reported lower levels of psychiatric symptoms on the Symptom checklist 90R (SCL-90R), with the "denial" subscale of the SDS resulting specifically associated to lower scores on SCL-90R items measuring hopelessness, thoughts about death and suicide ideation. The personality traits measured by the Marlowe-Crowne SDS, such as defensiveness, denial and self-deception, might be conceived as a protection against psychiatric disorders and suicide ideation.

Suicide in classical mythology: cues for prevention.

OBJECTIVE: To compare well established antecedents and correlates of completed suicide with the motives and the mechanics reported in Greek mythology. METHOD: A well-known collection of Greek myths, the Book of fables by Hyginus, was explored to investigate the mechanics driving an individual to imagine, design and carry out a suicide attempt. RESULTS: Females outnumber males in the mythographer's list, their favourite methods to die being drowning, hanging, self-burning and throwing themselves down from on high. Some kind of familial recurrence of suicide was accounted for, and a large percentage of these suicides was connected to incest. Shame, sense of guilt and grief for the death of a loved one are the most frequently reported psychological correlates of the act, whereas defeat, failure or a catastrophic change in living conditions and, among females, an unfortunate love affair figure as the main antecedents of suicide. CONCLUSION: Negative life events and emotional reactions to the severing of social ties frequently occur as antecedents of suicide in Greek mythology.

Eating disorders and aggressiveness among adolescents.

OBJECTIVE: To explore the links between propensity to aggression and eating disorders in a sample of school-aged adolescents in a northeastern area of Italy. METHOD: In a mixed male-female sample of 1000 adolescents (10% of the district's population aged 15-19 years) we administered the Eating Attitudes Test, the Bulimic Investigatory Test of Edinburgh and the Body Attitudes Test, as measures of abnormal eating attitudes and behaviours, and the Aggression Questionnaire, as a measure of the propensity to aggression. RESULTS: Females scored significantly higher than males at all eating disorders inventories (P < 0.0001). Males scored higher than females at the Aggression Questionnaire. In both genders, there was a positive correlation between scores at any of the eating disorders inventories and those at the Aggression Questionnaire (P < 0.0001). CONCLUSION: Overtly expressed aggression might have a negative impact on the course of eating disorders and on the compliance with treatment, also enhancing the risk of suicide.

Deaths by unintentional illicit drug overdose in Italy, 1984-2000.

AIM: To determine whether there has been an increase in deaths by overdose in Italy, as elsewhere in the Western world, over the past fifteen years. METHOD: This study's conclusions are based on analysis of official data on overdose deaths attributed to illicit drug addiction and abuse (ICD-9 codes 304 and 305) from 1984 to 2000, drawn from two archives of drug abuse information: the Direzione Centrale per i Servizi Antidroga (DADE) of the Italian Ministry of the Interior (1984-2000), and the Health Statistics held at the Italian Central Statistics Institute (ISTAT) (1984-1997). Mortality rates have been calculated for both genders in the following age groups: 15-24, 25-34, and 35-44 years. RESULTS: Official data indicate that there has been a steady increase in the number of deaths by overdose in Italy over recent 15 years. This trend has affected both genders, but is more evident among males. Over the whole period females had consistently lower overdose rates than males. In both genders the age group 35-44 was subject to the highest mortality rate increase over the study period, however, the highest overdose rates for both males and females were observed in the 25-34 age group. Consistently higher rates were witnessed in the northern regions of Italy with an overall increase across all latitudes. However, the greatest increase over the study period occurred in the South. In 5190 cases evaluated by the Italian Forensic Toxicology Group, 95.9% of deaths were attributed to heroin, but in about half of these, mixtures of three or more substances (heroin, benzodiazepines, cannabinoids, cocaine, methadone) were found in the deceased at doses that were likely to have contributed to death. CONCLUSIONS: The increase observed in the rates of death by overdose is likely to be a reflection of increased use of illicit drugs in the general population. Reporting practice by forensic pathologists might explain the extent of attribution of cause of death to heroin. Drop-out from addiction treatment is a commonly observed antecedent of fatal opioid overdose, therefore, caution is required when establishing treatment protocol for patients. Interventions that aim specifically at improving patient compliance with medical and psychiatric therapies should be favoured.

Social desirability and eating disorders. A community study of an Italian school-aged sample.

OBJECTIVE: To explore the links between social desirability and eating disorders in a sample of adolescents in a north-east area of Italy. METHOD: A mixed male-female sample of 1000 school-aged adolescents, corresponding to 10% of the young population aged 15-19 years living in the district, were investigated with self-reported questionnaires, including the Eating Attitudes Test (EAT), the Bulimic Investigatory Test of Edinburgh (BITE), the Body Attitudes Questionnaire (BAT), and an Italian version of the Marlowe-Crowne Social Desirability Scale (MC-SDS). RESULTS: Females scored higher than males at all eating disorder inventories. In both genders there was a negative relationship (in all cases P < 0.01) between scores at the eating disorder inventories and those at the MC-SDS. When analysing eating disorder "caseness", as measured by cut-off, "cases" reported significantly lower scores than "non-cases" at the MC-SDS in both genders. CONCLUSION: Personality traits measured by the MC-SDS, such as defensiveness, self-esteem, and dependence from approval, might contribute to the development of abnormal eating patterns at risk of eating disorders.

Diurnal variations in suicide by age and gender in Italy.

BACKGROUND: Recent Italian statistics on suicide distribution by time of day also report data on gender and age of victims, factors which have been shown to influence the seasonal distribution of suicide and which could also affect the influence of biological circadian rhythms on suicidal behaviour. This study aims to identify and evaluate any diurnal variations that may be present in suicide occurrence by age and gender in Italy, considering data from 1994 to 1997. METHODS: The null hypothesis that there are no variations in the distribution of suicides by time of day (or over the three major periods of the day: morning, afternoon, evening/night) was tested with the chi2 goodness-of-fit test and with ANOVA. RESULTS: A clear diurnal variation in the distribution of suicides over time can be observed for both genders, with a peak in the late morning (08:00-11:00 h), and a subsequent decrease to a trough in the night hours. This trend varies with age for both genders: in particular, the age groups 45-64 and 65+ show a clear suicide peak in the morning (08:00-11:00 h), whereas younger people have a peak number of suicides in the late afternoon (16:00-19:00 h). Adults (25-44 years old) show an intermediate trend, with a less pronounced peak between the morning and early afternoon hours. The observed trend is more marked among males; however, the distribution of suicides by time of day is clearly congruent by age between both genders. CONCLUSIONS: Diurnal variation in suicide occurrence by age group may be affected by factors distributed unevenly across age groups. In particular, age distribution of disorders leading to suicidal ideation, and the sensitivity of biological systems of different age groups to environmental cues may affect each group's risk of suicide. Socio-relational factors are also likely to contribute to diurnal variation in suicide risk by age and gender. Children and adolescents can generally be presumed to be at school during the morning, therefore their opportunity for self-harm is restricted to afternoon hours. The elderly, on the other hand, may find themselves alone in the morning, when family and friends spend more time away from home due to daily work activities. LIMITATIONS: Data are based on time of death and not on presumed time of the suicidal act. For suicides committed by certain methods (e.g., poisoning) there may be a considerable difference between time of act and time of death. CLINICAL RELEVANCE: The existence of a temporal window in suicide risk implies an improvement in the surveillance of people at risk of suicide and greater attention to chronobiological factors affecting those suffering from mental disorders leading to suicide ideation.

Musical creativity and suicide.

The different abilities involved in artistic creativity may be mirrored by differences among mental disorders prevalent in each artistic profession, taking poets, painters, and composers as examples. Using suicide rates as a proxy for the prevalence of mental disorders in groups of artists, we investigated the percentage of deaths by suicide in a sample of 4,564 eminent artists who died in the 19th and 20th centuries. Of the sample, 2,259 were primarily involved in activities of a linguistic nature, e.g., poets and writers; 834 were primarily visual artists, such as painters and sculptors; and 1,471 were musicians (composers and instrumentalists). There were 63 suicides in the sample (1.3% of total deaths). Musicians as a group had lower suicide rates than literary and visual artists. Beyond socioeconomic reasons, which might favour interpretations based on effects of health selection, the lower rate of suicides among musicians may reflect some protective effect arising from music.

Obstetric complications in patients with depression--a population-based case-control study.

BACKGROUND: To examine whether sufferers of affective disorders are more likely to be subject to obstetric complications than normal healthy people. METHOD: Data based on prospectively recorded birth case-notes for patients with a diagnosis of depression (or related disorders) with early onset were compared to those of normal healthy controls, individually matched by gender, time and parity of birth, maternal age and marital status. RESULTS: Forty-one case-controls pairs born between 1964 and 1978 were compared. No differences between cases and controls in gestational age or birthweight were significant, though depressive patients on average weighed 200 g less than controls at birth. Patients were more likely than controls to be small for their gestational age (22 vs. 1: chi(2)=4.34, P=0.03). They were significantly more likely than controls to have suffered at least one obstetric complication: 35 (85%) vs. 25 (60%), chi(2)=5.03, P=0.02; or more than one (two on average, as opposed to one on average among controls). No obstetric complication was seen significantly more among cases than controls, apart from bleeding during gestation, which was observed for four cases and no controls. The prevalence of complications with a clear brain damaging potential did not differ significantly between cases and controls: 11 (26%) vs. 8 (19%). CONCLUSIONS: A developmental deficit, as indicated by lower birthweight and gestational age, may contribute to the risk of depressive breakdowns and affective disorders in later life. Severe, brain damaging obstetric complications are unlikely to be a significant risk factor for affective disorders, though some early onset cases may be accounted for by prenatal brain lesions. LIMITATIONS: Sample size limits statistical power for isolation of a rare, single risk factor.

Risk for obstetric complications and schizophrenia.

The goal of this study was to determine whether cases with schizophrenia or related disorders show a history of obstetric complications significantly more often than control subjects and, if so, whether the enhanced risk of a negative pregnancy outcome also extends to the non-schizophrenic offspring of cases. Data based on the obstetric birth case-notes of patients with diagnosed schizophrenia or related disorders were compared to those of normal 'healthy' control subjects; each case/control pair was individually matched by gender, time and parity of birth, maternal age and marital status. Forty-four case/control pairs born in Padova (Italy) between 1964 and 1978 were assessed for prenatal and perinatal complications, including abnormal gestational age or birthweight. No significant differences were observed between cases and control subjects in the general characteristics of birth; gestational age and birthweight in particular were strictly comparable between cases and control subjects. The schizophrenia spectrum patients (75%) were more likely than control subjects (59%) to have experienced at least one definite obstetric complication: odds ratio=2.07 (95% CI: 0.83-5. 15). Cases also suffered more complications per birth than control subjects (average 2:1). In particular, obstetric complications involving a clear damaging potential were seen significantly more often among cases than control subjects: 34% vs. 9%, Fisher's exact test, P=0.008 (odds ratio=5.17, 95% CI: 1.55-17.21). Moreover, severe obstetric complications were noted more often among males (n=13, 41%) than females (n=2, 15%). When any previous pregnancies of the mothers of patients were compared with those of the mothers of control subjects, mothers of cases were seen to have suffered unfavorable pregnancy outcomes significantly more often. In particular mothers of cases were seen to have had more miscarriages (OR=4.66), and pre-term births (OR=2.58) than control subects' mothers. Severe, brain-damaging obstetric complications would seem to be a possible antecedent to a diagnosis of schizophrenia or a related disorder in adulthood. Indeed, some early onset cases may be accounted for by prenatal brain lesions. This enhanced risk of negative pregnancy outcome may be under genetic control, contributing to the persistence of schizophrenia in the general population. The 'healthy' status of control subjects was ascertained indirectly, not by individual assessment of the subjects. The sample size limits the statistical power of calculations.

Season and suicide: recent findings from Italy.

Seasonal asymmetry in yearly suicide occurrence is a long-observed phenomenon in psychiatric, suicidological, and sociological research, and the effects of seasonal factors on suicidal behavior have been the focus of a number of earlier studies. Taking into account limitations of data and methods, these studies have generally favored interpretations based on psychosocial factors. Recent studies have challenged the widely held notion that seasonal effects on suicide are a unitary phenomenon, not influenced by age, gender, or circumstances of the act. In particular, the seasonal occurrence of suicides has been found to differ significantly between the young and the elderly, and differences have also been found between male and female cycles of occurrence. Suicides using violent methods have been shown to follow clearer seasonal patterns than suicides by less violent methods (such as drug or gas poisoning), possibly reflecting the greater impulsive component involved in the choice of a violent lethal means. In this paper, findings from Italy are used to illustrate the clinical implications of studies into the topic of season and suicide, with the aim of developing more effective preventative strategies.