Gold Standard Cholera Diagnostics Are Tarnished by Lytic Bacteriophage and Antibiotics.

A fundamental, clinical, and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. Cholera was chosen as a model disease to investigate this important question, because cholera outbreaks enable large enrollment, field methods are well established, and the predatory relationship between lytic bacteriophage and the etiologic agent Vibrio cholerae share commonalities across bacterial taxa. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection and exposure to the first-line antibiotic azithromycin, detected in stool samples by mass spectrometry. Among diarrheal samples positive by nanoliter quantitative PCR (qPCR) for V. cholerae (n = 78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (odds ratio [OR], 0.108; 95% confidence interval [CI], 0.002 to 0.872) and 87% (OR, 0.130; 95% CI, 0.022 to 0.649), respectively, when lytic bacteriophage were detected. The odds that an RDT or qPCR was positive was reduced by more than 99% (OR, 0.00; 95% CI, 0.00 to 0.28) and 89% (OR, 0.11; 95% CI, 0.03 to 0.44), respectively, when azithromycin was detected. Analysis of additional samples from South Sudan found similar phage effects on RDTs; antibiotics were not assayed. Cholera burden estimates may improve by accommodating for the negative effects of lytic bacteriophage and antibiotic exposure on diagnostic positivity. One accommodation is using bacteriophage detection as a proxy for pathogen detection. These findings have relevance for other diagnostic settings where bacterial pathogens are vulnerable to lytic bacteriophage predation.

Toxins and virulence factors of enterotoxigenic Escherichia coli associated with strains isolated from indigenous children and international visitors to a rural community in Guatemala.

Diarrhoea remains a common cause of illness in Guatemala, with children suffering most frequently from the disease. This study directly compared the frequency, enterotoxin, and colonization factor (CF) profiles of enterotoxigenic Escherichia coli (ETEC) strains isolated from children living in a rural community in Guatemala and from Western visitors to the same location during the same seasons, using similar detection methodologies. We found that ETEC accounted for 26% of severe cases of diarrhoea in children requiring hospitalization, 15% of diarrhoea in the community, and 29% of travellers' diarrhoea in visitors staying ⩾2 weeks. The toxin and CF patterns of the ETEC strains isolated from both groups differed significantly (P < 0·0005) as determined by χ 2 = 60·39 for CFs and χ 2 = 35 for toxins, while ETEC phenotypes found in Guatemalan children were comparable to those found in children from other areas of the world.

Epidemiology of rotavirus and cholera in children aged less than five years in rural Bangladesh.

Despite the known presence of rotavirus-associated diarrhoea in Bangladesh, its prevalence, including records of hospitalization in rural health facilities, is largely unknown. In a systematic surveillance undertaken in two government-run rural health facilities, 457 children, aged less than five years, having acute watery diarrhoea, were studied between August 2005 and July 2007 to determine the prevalence of rotavirus. Due to limited financial support, the surveillance of rotavirus was included as an addendum to an ongoing study for cholera in the same area. Rotavirus infection was detected in 114 (25%) and Vibrio cholerae in 63 (14%) children. Neither rotavirus nor V cholerae was detected in 280 (61%) samples; these were termed 'non-rotavirus and non-cholera' diarrhoea. Both rotavirus and cholera were detected in all groups of patients (<5 years). The highest proportion (41%; 47/114) of rotavirus was in the age-group of 6-11 months. In children aged less than 18 months, the proportion (67%; 76/114) of rotavirus was significantly (p < 0.001) higher than that of cholera (16%; 10/63). By contrast, the proportion (84%; 53/63) of cholera was significantly (p < 0.001) higher than that of rotavirus (33%; 38/114) in the age-group of 18-59 months. During the study period, 528 children were hospitalized for various illnesses. Thirty-eight percent (202/528) of the hospitalizations were due to acute watery diarrhoea, and 62% were due to non-diarrhoeal illnesses. Rotavirus accounted for 34% of hospitalizations due to diarrhoea. Severe dehydration was detected in 16% (74/457) of the children. The proportion (51%; 32/63) of severe dehydration among V cholerae-infected children was significantly higher (p < 0.001) compared to the proportion (16%; 18/114) of rotavirus-infected children. The study revealed that 12-14% of the hospitalizations in rural Bangladesh in this age-group were due to rotavirus infection, which has not been previously documented.

El Tor cholera with severe disease: a new threat to Asia and beyond.

During epidemics of cholera in two rural sites (Bakerganj and Mathbaria), a much higher proportion of patients came for treatment with severe dehydration than was seen in previous years. V. cholerae O1 isolated from these patients was found to be El Tor in its phenotype, but its cholera toxin (CT) was determined to be that of classical biotype. Whether the observed higher proportion of severe dehydration produced by the El Tor biotype was due to a shift from El Tor to classical CT or due to other factors is not clear. However, if cholera due to strains with increased severity spread to other areas where treatment facilities are limited, there are likely to be many more cholera deaths.

Peruvian Vibrio cholerae O1 El Tor strains possess a distinct region in the Vibrio seventh pandemic island-II that differentiates them from the prototype seventh pandemic El Tor strains.

A collection of environmental and clinical strains of Vibrio cholerae O1 isolated from the beginning of the Latin American epidemic of cholera in 1991 to 2003 from multiple locations in Peru were characterized and compared with V. cholerae O1 El Tor strains of the seventh pandemic from the rest of the world (Asia, Africa, Australia and Europe) using a multilocus virulence gene profiling strategy and DNA sequencing. Peruvian strains differed from El Tor strains from the rest of the world by the failure of PCR to amplify genes VC0512, VC0513, VC0514 and VC0515 in the Vibrio seventh pandemic island-II (VSP-II) gene cluster. Sequencing of the VSP-II gene cluster and its flanking regions in one Peruvian strain (PERU-130) confirmed the PCR results, indicating that the Peruvian strain had low DNA homology (46.6 %) compared to the reference strain N16961 within the VSP-II region encompassing genes VC0511 to VC0515. Based on these differences in VSP-II, and based on the overall similarity between the pulsotypes of the Peruvian strains and the El Tor reference strain N16961, we concluded that the Peruvian, Eurasian and African strains belonged to the same clonal complex, and that the Peruvian strains represented variants that had independently evolved for a relatively short time. Since these ORFs in VSP-II of Peruvian strains are unique and conserved, they could form the basis for tracking the origin of the Peruvian strains and therefore of the Latin American pandemic.

Zinc supplementation in the management of shigellosis in malnourished children in Bangladesh.

OBJECTIVE: To assess the impact of zinc supplementation on clinical recovery, weight gain and subsequent growth and morbidity in moderately malnourished children with shigellosis. DESIGN: A randomized, double-blind, controlled trial. SETTING: Dhaka hospital of ICDDR,B: Centre for Health and Population Research, Dhaka, Bangladesh. SUBJECTS: Fifty-six moderately malnourished children, aged 12-59 months with culture-proven shigellosis. METHODS: Subjects were randomly allocated to receive zinc (20 mg/day elemental) in multivitamin syrup (intervention) or multivitamin syrup without zinc (control) in two equally divided doses daily for 2 weeks. All children received pivmecillinam in a dose of 15 mg/kg every 6 h for 5 days. After supplementation, children were followed in their respective homes every 2 weeks for 6 months. RESULTS: Children receiving zinc recovered from acute illness significantly faster than the control children (P<0.05). The medians time (days) to recovery and disappearances of blood and mucous were significantly 50% shorter in the zinc-supplemented group compared to the control group. The mean body weight of zinc supplemented children increased significantly from 8.8 kg on admission to 9.2 kg (P<0.01) at recovery, which was not observed in the control children (from 9.3 to 9.6 kg; P=0.12). During the 6-month follow-up period, zinc-supplemented children had significantly fewer mean episodes of diarrhoea compared to the control children (2.2 vs 3.3; P=0.03). CONCLUSION: Zinc supplementation significantly shortens the duration of acute shigellosis, promotes better weight gain during recovery and reduces diarrhoeal morbidity during the subsequent 6 months.

Characterization of enterotoxigenic Escherichia coli from diarrhoeal patients in Bangladesh using phenotyping and genetic profiling.

A total of 99 isolates out of 370 colonization factor (CF)-positive, well-characterized enterotoxigenic Escherichia coli (ETEC) strains belonging to 13 different CF types isolated from diarrhoeal patients admitted to the hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, were tested. The isolates were selected at random based on expression of the major CFs prevailing in Dhaka, Bangladesh, from 1996 to 1998. These isolates were characterized by O-antigenic serotyping, randomly amplified polymorphic DNA (RAPD) analysis and biochemical fingerprinting using the PhenePlate (PhP) system. The 99 ETEC isolates belonged to 10 O serogroups, the predominant ones being O6 (n=28), O115 (n=20) and O128 (n=20). Most isolates of serogroup O6 (CS1+CS3, 11/14; CS2+CS3, 5/8) belonged to the same PhP/RAPD type (H/f), whereas other isolates of serogroup O6 (n=12) belonged to different PhP/RAPD types (Si/f and F/c). Eleven serogroup O128 (CFA/I) isolates belonged to the same PhP/RAPD type (E/b), whereas the other O128 isolates formed different PhP/RAPD types. Fifteen (75%) serogroup O115 isolates (together with fourteen isolates from serogroups O25, O114, O142 and O159) demonstrated two closely related common groups by PhP typing (A and A1) and belonged to the same PhP/RAPD type (A/a). Three major clonal groups were identified among the ETEC strains in this study, largely based on O-antigenic type, CF expression pattern and toxin profile.

Shiga toxin-producing Escherichia coli isolated from patients with diarrhoea in Bangladesh.

The prevalence of Shiga toxin-producing Escherichia coli (STEC) and its characteristics were determined among hospitalized patients with diarrhoea and children with diarrhoea in an urban slum community of Dhaka city using sensitive culture and PCR methods. Stool samples were collected from 410 patients with diarrhoea enrolled in the 2% surveillance system (every 50th patient attending the hospital with diarrhoeal disease is included) at the ICDDR,B hospital and from 160 children of 2-5 years of age with diarrhoea living in an urban slum in Dhaka, between September 2004 and April 2005. Shiga toxin genes (stx) were detected by multiplex PCR in the enrichment broth of nine samples (2.2%) from hospitalized patients and 11 samples (6.9%) from the community patients. STEC was isolated from five stool samples with positive PCR results using a colony patch technique. All five isolates were positive in the Vero cell assay and PCR fragments of stx genes were confirmed by sequencing. Two isolates were positive for the E. coli attaching-and-effacing (eae) gene and four were positive for the enterohaemolysin (hlyEHEC) gene and enterohaemolysin production. The five isolates belonged to five different serotypes:O32:H25, O2:H45, O76:H19, ONT:H25 and ONT:H19. It can be concluded that STEC is not a common pathogen in Bangladesh among hospitalized patients with diarrhoea nor among mild cases of diarrhoea in the community.

Increasing spectrum in antimicrobial resistance of Shigella isolates in Bangladesh: resistance to azithromycin and ceftriaxone and decreased susceptibility to ciprofloxacin.

Antimicrobial resistance of Shigella isolates in Bangladesh, during 2001-2002, was studied and compared with that of 1991-1992 to identify the changes in resistance patterns and trends. A significant increase in resistance to trimethoprim-sulphamethoxazole (from 52% to 72%, p < 0.01) and nalidixic acid (from 19% to 51%, p < 0.01) was detected. High, but unchanged, resistance to tetracycline, ampicillin, and chloramphenicol, low resistance to mecillinam (resistance 3%, intermediate 3%), and to emergence of resistance to azithromycin (resistance 16%, intermediate 62%) and ceftriaxone/cefixime (2%) were detected in 2001-2002. Of 266 recent isolates, 63% were resistant to > or =3 anti-Shigella drugs (multidrug-resistant [MDR]) compared to 52% of 369 strains (p < 0.007) in 1991-1992. Of 154 isolates tested by E-test in 2001-2002, 71% were nalidixic acid-resistant (minimum inhibitory concentration [MIC] > or =32 microg/mL) and had 10-fold higher MIC90 (0.25 microg/mL) to ciprofloxacin than that of nalidixic acid-susceptible strains exhibiting decreased ciprofloxacin susceptibility, which were detected as ciprofloxacin-susceptible and nalidixic acid-resistant by the disc-diffusion method. These strains were frequently associated with MDR traits. High modal MICs were observed to azithromycin (MIC 6 microg/mL) and nalidixic acid (MIC 128 micdrog/mL) and low to ceftriaxone (MIC 0.023 microg/mL). Conjugative R-plasmids-encoded extended-spectrum beta-lactamase was responsible for resistance to ceftriaxone/cefixime. The growing antimicrobial resistance of Shigella is worrying and mandates monitoring of resistance. Pivmecillinam or ciprofloxacin might be considered for treating shigellosis with caution.

Neuroendocrine response to 5-hydroxytryptophan in seasonal affective disorder.

A double-blind random-ordered comparison of the effects of placebo and 5-hydroxytryptophan (200 mg, orally) in ten depressed patients with seasonal affective disorder (SAD) and ten controls disclosed slightly but significantly higher basal levels of serum prolactin and a trend toward higher basal levels of serum cortisol in the patients with SAD compared with controls. After administration of 5-HTP, the cortisol level significantly increased and the prolactin level significantly decreased in both patients and controls. No differences in the melatonin level, growth hormone level, blood pressure, or pulse rate and no side effects were noted between patients and controls in the two study conditions; the timing of basal and 5-hydroxytryptophan-stimulated hormonal secretions was similar for both groups. These results are discussed with reference to current hypotheses of the cause of SAD.

The diurnal variation in plasma homovanillic acid level persists but the variation in 3-methoxy-4-hydroxyphenylglycol level is abolished under constant conditions.

Plasma concentrations of homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were determined every two hours for two consecutive days in seven depressed patients and seven matched normal controls. On the first day subjects followed their regular ward routine. On the second day they were placed on a regimen in which activity, posture, diet, and wakefulness were held constant. There were significant diurnal variations in both MHPG and HVA concentrations on the baseline day, whereas on the constant routine, a diurnal variation was present only in HVA. We conclude that diurnal variations in plasma MHPG are evoked by changes in physical activity, posture, or other factors controlled on the constant routine, and that a major component of the diurnal variation in plasma HVA concentrations is regulated by a circadian oscillator that is independent of sleep or activity.

Phototherapy of seasonal affective disorder. Time of day and suppression of melatonin are not critical for antidepressant effects.

Seasonal affective disorder is characterized by recurring cycles of fall-winter depression and spring-summer hypomania (or euthymia). In winter, depressed patients with seasonal affective disorder respond to daily treatments with five to six hours of bright artificial light in two to three days. They relapse two to three days after light is withdrawn. In this study carefully controlled experimental conditions were used to determine whether phototherapy acts via a photoperiodic mechanism in which the timing of light is critical for its therapeutic effect. Photoperiodism is a common regulatory mechanism in animal seasonal rhythms and depends for its effect on light-induced changes in the pattern of nocturnal melatonin secretion. The results reported herein of "skeleton photoperiod" experiments indicate that the efficacy of phototherapy may not depend on its timing or its effect on melatonin secretion.

Seasonal affective disorder. A description of the syndrome and preliminary findings with light therapy.

Seasonal affective disorder (SAD) is a syndrome characterized by recurrent depressions that occur annually at the same time each year. We describe 29 patients with SAD; most of them had a bipolar affective disorder, especially bipolar II, and their depressions were generally characterized by hypersomnia, overeating, and carbohydrate craving and seemed to respond to changes in climate and latitude. Sleep recordings in nine depressed patients confirmed the presence of hypersomnia and showed increased sleep latency and reduced slow-wave (delta) sleep. Preliminary studies in 11 patients suggest that extending the photoperiod with bright artificial light has an antidepressant effect.

Nocturnal TSH and prolactin secretion during sleep deprivation and prediction of antidepressant response in patients with major depression.

In order to test the hypothesis that changes in the hypothalamic-pituitary axis during sleep deprivation are related to the antidepressant effects of this procedure, we measured thyroid-stimulating hormone (TSH) and prolactin levels in 32 depressed patients at 2:00 AM during a night before, during, and after total sleep deprivation (TSD). TSH levels increased significantly (p less than 0.05) during TSD, and prolactin levels decreased significantly (p less than 0.0001). When we divided the patients into responder and nonresponder groups based on a 30% reduction in the Hamilton Rating Scale, there was no difference between the two groups in their hormone levels on the baseline, TSD, or recovery nights. Changes in prolactin or TSH were not correlated with clinical improvement when the two groups were considered together or in the responder/nonresponder groups separately. Baseline values of both hormones were significantly (p less than 0.01) correlated with their respective levels during TSD and recovery sleep. These findings indicate that the relative levels of nocturnal TSH and prolactin are stable even within acutely depressed individuals and that changes in their levels are not related to the clinical response to sleep deprivation.

Sleep reduction as a final common pathway in the genesis of mania.

Diverse psychological, interpersonal, environmental, and pharmacological factors that appear to trigger the onset of mania could act via their capacity to cause sleep deprivation, a mechanism that has been shown in experiments with bipolar patients to induce transient or sustained switches into mania. Since mania in turn causes insomnia, the development of mania is potentially self-reinforcing and could become autonomous after being initiated by precipitating factors. The sleep reduction model is based on experimental evidence and is a parsimonious explanation for the precipitation of manic episodes by a wide variety of factors. Furthermore, this model has clear implications for the prevention and treatment of mania and provides a conceptual focus and an experimental paradigm for psychological investigations of the causes of mania.

Seasonal affective disorder with summer depression and winter hypomania.

The authors describe 12 patients who regularly became depressed in summer. This pattern is opposite to one the authors previously described, in which patients became depressed in winter and responded to treatment with light. Temperature may influence some summer depressions.

Eye versus skin phototherapy of seasonal affective disorder.

In winter, depressed patients with seasonal affective disorder respond to treatment with bright artificial light (phototherapy). The authors found that the antidepressant effects of phototherapy were much greater for 10 patients when light was applied to the eyes than when it was applied to the skin, suggesting that its effects may be mediated by the eyes. The identification of a probable anatomical route of entry is clinically relevant and an important clue for further investigations of the mechanism of phototherapy. However, patients' expectations nearly always predicted the outcome, leaving open the possibility that expectations were responsible for their responses.

Morning versus midday phototherapy of seasonal affective disorder.

Sixteen depressed patients with seasonal affective disorder participated in a double-blind crossover study comparing the antidepressant effects of 2 hours of early morning and 2 hours of early afternoon therapy with bright light. They responded equally well to both treatments. These results suggest that the antidepressant effects of phototherapy in seasonal affective disorder do not depend on its capacity to extend day length (photoperiod) and are not likely to be due to a shift in the timing of circadian rhythms. These findings have practical implications for the administration of phototherapy in the treatment of seasonal affective disorder.

Twenty-four-hour profiles of body temperature and plasma TSH in bipolar patients during depression and during remission and in normal control subjects.

The 24-hour patterns of body temperature and plasma thyrotropin (TSH) were measured in eight bipolar patients in both depressed and recovered (after 3 weeks of treatment) states and in eight normal control subjects. Clear circadian patterns were detected for both temperature and TSH. Nocturnal body temperature was increased and the nocturnal surge of TSH was blunted during depression; these abnormalities were corrected after recovery. The inverse relationship between changes in body temperature and TSH levels at night suggests that changes in thermoregulation may be responsible for the neuroendocrine disturbance and may play a role in the pathophysiology of depression.

Rapid cycling affective disorder: contributing factors and treatment responses in 51 patients.

For 51 patients with rapid cycling affective disorder, clinical and family history data indicated that the illness was phenotypically and genetically related to more typical forms of affective disorder, was characterized by a bipolar course (100%), and was more common in women (92%). Manic-depressive cycles were separate from menstrual cycles. At the time of onset of rapid cycling, 73% of the patients were taking antidepressant drugs; the continuation of rapid cycling was associated with antidepressant drug therapy in 51% of the patients. Although most patients had been referred to a research ward because they were considered to be refractory to treatment, 37% attained essentially complete remissions, usually during treatment with lithium and/or monoamine oxidase inhibitors.