Detection of in vivo hepatitis B virus surface antigen mutations-A comparison of four routine screening assays.

An important requirement for a state-of-the-art hepatitis B surface antigen (HBsAg) screening assay is reliable detection of mutated HBsAg. Currently, there is a striking shortage of data regarding the detection rates of in vivo HBsAg mutations for these clinically important assays. Therefore, we compared the detection rates of four commercial HBsAg screening assays using a global cohort of 1553 patients from four continents with known HBV genotypes. These samples, which represent the broadest spectrum of known and novel HBsAg major hydrophilic region (MHR) mutations to date, were analyzed for the presence of HBsAg using the Roche Elecsys® HBsAg II Qualitative, Siemens ADVIA Centaur XP HBsAg II, Abbott Architect HBsAg Qualitative II and DiaSorin Liaison® HBsAg Qualitative assays, respectively. Of the 1553 samples, 1391 samples could be sequenced; of these, 1013 (72.8%) carried at least one of the 345 currently known amino acid substitutions (distinct HBsAg mutation) in the HBsAg MHR. All 1553 patient samples were positive for HBsAg using the Elecsys® HBsAg II Qual assay, with a sensitivity (95% confidence interval) of 99.94% (99.64%-100%), followed by the Abbott Architect 99.81% (99.44%-99.96%), Siemens ADVIA 99.81% (99.44%-99.96%) and DiaSorin Liaison® 99.36% (98.82%-99.69%) assays, respectively. Our results indicate that the Elecsys® HBsAg II Qual assay exhibits the highest sensitivity among the commercial HBsAg screening assays, and demonstrate that its capacity to detect HBV infection is not compromised by HBsAg MHR mutants.

Long-term monitoring drug resistance by ultra-deep pyrosequencing in a chronic hepatitis B virus (HBV)-infected patient exposed to several unsuccessful therapy schemes.

The aim of this study was to analyze the spectrum and dynamics of low-prevalent HBV mutations in the reverse transcriptase (rt) and S antigen by ultra-deep pyrosequencing (UDPS). Samples were obtained from a chronically infected patient who was followed throughout a thirteen-year period. This technology enabled simultaneous analysis of 4084 clonally amplified fragments from the patient allowing detecting low prevalent (<1%) mutations during the follow-up. At baseline, HBV sequences were predominately wild-type. Under sequential HBV monotherapies including lamivudine, adefovir and entecavir, a high frequency of rtM204I mutation was detected initially as unique and then coexisting with rtM204V. Both mutations were statistically associated with rtA200V and rtV207I, respectively. Once the entecavir and tenofovir combined therapy was started, polymerase and consequently envelope gene mutations appeared at several positions at a higher frequency than before, including the entecavir resistance-associated mutation rtT184L.

Analysis of the PKR-eIF2alpha phosphorylation homology domain (PePHD) of hepatitis C virus genotype 1 in HIV-coinfected patients by ultra-deep pyrosequencing and its relationship to responses to pegylated interferon-ribavirin treatment.

Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among the greatest challenges facing public health worldwide. In this population, the response to hepatitis C therapy by treatment with pegylated interferon plus ribavirin (PEG-IFN+RBV) is lower than in HCV-monoinfected patients, particularly in those infected by HCV genotype 1. A PKR/eIF-2α phosphorylation homology domain (PePHD) within the E2 protein has been found to interact with PKR and inhibit PKR in vitro, suggesting a possible mechanism for HCV to evade the antiviral effects of IFN. The aim of this work was to analyze the amino acid conservation in the HCV-E2-PePHD and quasispecies diversity among HCV-HIV-coinfected patients exhibiting sustained virological response, non-response, or partial response with viral relapse to PEG-IFN+RBV by ultra-deep pyrosequencing. For this purpose, HCV-E2-PePHD PCR products were generated and sequenced directly for four patients with a sustained response, seven patients with no virological response, and four patients with viral relapse before and after treatment with PEG-IFN+RBV. HCV-E2-PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment (24 h, 4 weeks, and 12 weeks). Quasispecies analysis of the HCV-E2-PePHD and flanking genomic regions was performed using 454/Roche pyrosequencing, analyzing 39,364 sequence reads in total. The HCV-E2-PePHD sequence at the amino acid and nucleotide level was highly conserved among HCV genotype 1 strains, irrespective of the PEG-IFN+RBV response. This high degree of amino acid conservation and sporadic mutations in the HCV-E2-PePHD domain do not appear to be associated with treatment outcome. The HCV-E2-PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in patients coinfected with HCV genotype 1 and HIV.

Biobehavioural correlates of hand preference in free-ranging female primates.

In this research we examined biological and behavioural correlates of handedness in a subject cohort of 41 free-ranging young female rhesus macaques (Macaca mulatta). Specifically, we examined relationships between handedness and cerebrospinal fluid (CSF) concentrations of the monoamine metabolites 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA), plasma concentrations of the hormones cortisol and adrenocorticotropin (ACTH), prolactin, and multiple indices of social behaviour, including proximity to other animals, grooming, submission, and aggression. Handedness was determined through systematic observation of animals reaching for food in their unrestricted home environment. We found a population-level bias for left-hand use in this cohort of young females. The frequency of right versus left hand use was positively correlated with CSF 5-HIAA, plasma cortisol concentrations, the frequency of submissive behaviour, and with the frequency of bouts in which animals received low-level aggression. The positive correlation between right versus left hand use, submissive behaviour, and received aggression found here in females contrasts with the negative correlation among these same variables that we have previously reported in rhesus males. We conclude that these results may be explicable in terms of sex-based differences in rhesus life-history patterns, and that the influence of the serotonergic system on patterns of male aggression, social behaviour, and handedness, and the associations between handedness and social behaviour found previously among males may not be generalised to female rhesus macaques.

Throwing behavior and mass distribution of stone selection in tufted capuchin monkeys (Cebus apella).

Cannell [Journal of Archaeological Science 29:335-339, 2002] argued that sex-based differences among humans in terms of the mass of chosen throwing stones could be used to infer body mass and patterns of sexual dimorphism in early hominids from Olduvai and Koobi Fora by examining the mass distributions of unaltered stone tools at those sites. We examined this hypothesis in tufted capuchin monkeys using a comparative approach, by investigating the relationships among body mass, sex, stone weight preference, and accuracy in a throwing task. The subject sample consisted of nine monkeys trained to perform an aimed-throwing task in which a food reward could be obtained by throwing a stone into a bucket. We found that 1) the subjects showed a strong mean stone mass preference; 2) the females chose heavier stones than the males, in terms of absolute mean selected stone mass and selected stone mass relative to body mass; 3) subjects threw more accurately when they used stones of preferred mass vs. stones of nonpreferred mass; and 4) overall, the males were more accurate in the throwing task than the females. We conclude that capuchins are highly selective when choosing throwing stones, and that this confers an advantage for throwing accuracy. Our results indicate that the sexually dimorphic pattern in stone mass preference observed among humans does not generalize to Cebus apella. We suggest that researchers examining this pattern in humans in an attempt to explain early hominid patterns of dimorphism and behavior should take into account not only stone weight preference, but also its adaptive advantage.

CSF 5-HIAA concentration as an early screening tool for predicting significant life history outcomes in female specific-pathogen-free (SPF) rhesus macaques (Macaca mulatta) maintained in captive breeding groups.

We examined relationships among cerebrospinal fluid (CSF) concentrations of the major serotonin metabolite (5-HIAA, 5-hydroxyindoleacetic acid) and significant medical and behavioral outcomes for female rhesus macaques. Based on earlier findings with males we predicted that low CSF 5-HIAA concentrations would be associated with a range of negative life history outcomes in our captive specific-pathogen-free (SPF) breeding colony. We found that the mean CSF 5-HIAA concentration among animals that died over the course of the study period was significantly lower than among animals that survived. Further examination indicated an inverse relationship between CSF 5-HIAA concentration and number of treatments for illness, further suggesting a link between serotonergic functioning and overall animal health. Examination of behavioral data indicated that individuals with low CSF 5-HIAA concentrations were more often the targets of aggressive bouts than were individuals with high CSF 5-HIAA concentrations. Finally, we found a positive relationship between CSF 5-HIAA concentration and infant survivorship. These results suggest negative life history consequences of impaired serotonergic functioning in captive female rhesus macaques, and indicate that CSF 5-HIAA concentration sampled early in life may provide a useful tool in facilitating colony management decisions concerning utilization of scarce and increasingly valuable non-human primate resources.

Left-handedness is correlated with CSF monoamine metabolite and plasma cortisol concentrations, and with impaired sociality, in free-ranging adult male rhesus macaques (Macaca mulatta).

In this research we examined biological and behavioural correlates of handedness in free-ranging adult male rhesus macaques (Macaca mulatta). Specifically, we examined relationships between handedness and cerebrospinal fluid (CSF) concentrations of the monoamine metabolites 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylglycol (MHPG), plasma concentrations of the hormones cortisol and adrenocorticotropin (ACTH), and multiple indices of social behaviour, including occurrences of proximity to other animals, grooming, submission, and aggression. We determined handedness through systematic observation of animals reaching for food in their unrestricted home environment. The frequency of right- versus left-hand use was significantly positively correlated with CSF 5-HIAA, CSF MHPG, and plasma cortisol concentrations, and with social proximity and the frequency and duration of bouts in which animals received grooming. The frequency of right- versus left-hand use was significantly negatively correlated with the frequency of submissive behaviour, and with the frequency and intensity of bouts in which animals received aggression. We conclude that handedness is associated with an array of biological and behavioural processes in free-ranging adult male rhesus macaques and that left-handedness may be used to identify individuals at increased risk for impaired functioning of the serotonin, norepinephrine, and hypothalamic-pituitary-adrenal systems, and for social isolation and susceptibility to violent attack.

Handedness is associated with immune functioning and behavioural reactivity in rhesus macaques.

In the present study we examined the relationship among handedness, immune functioning, and behavioural reactivity in rhesus macaques. We used the absolute number of CD4+ (T-helper) and CD8+ (T-suppressor) cells as dependent measures of immune functioning. We derived reactivity profiles from behavioural responses to a threat, and hand preference profiles from a quadrupedal food-reaching test. The results indicate positive correlations between the frequency of right versus left hand reaches and the absolute number of CD4+ cells, and between the frequency of right versus left hand reaches and the degree of human-directed aggression in response to an invasive threat. Immune measures were not associated with the strength of hand preference. These results are consistent with and extend previous findings obtained with rodents to nonhuman primates and provide further support for the view that behavioural lateralisation is associated with immune functioning and behavioural reactivity.

Between-species variation in the development of hand preference among macaques.

This research examined between-species variation in the development of hand preference among Macaca. Specifically, we examined hand preference using juveniles and adults of three macaque species that differ in social and reactive tendencies in order to examine whether the correlation between temperament and handedness that has been noted within Macaca mulatta occurs between closely related species. Each of the species studied exhibited a different pattern of hand preference development. Both juvenile and adult M. mulatta exhibited group-level left-hand bias. Juvenile Macaca nemestrina were not biased towards either hand at the group-level, whereas adults exhibited a group-level left-hand bias. Neither juvenile nor adult Macaca fascicularis exhibited manual bias at the group-level. Analysis of variance indicated statistically significant main effects of species and age class on hand preference measures. Post-hoc analysis indicated greater use of the left- versus right-hand, and greater hand preference strength independent of direction, among M. mulatta and M. nemestrina than among M. fascicularis, and among adults than among juveniles. These results indicate significant between-species variation in the development of hand preference within the genus Macaca, and are inconsistent with any one single-factor theory yet offered to explain the etiology of primate laterality. We hypothesize that the relationship between handedness and temperament that has been shown within M. mulatta may generalize across closely related primate species.

Stress correlates of hand preference in rhesus macaques.

In this research we examined stress-related correlates of hand preference in monkeys. Specifically, we tested the hypothesis that stress reactivity and plasma levels of the stress hormone cortisol are developmentally related to handedness in rhesus macaques (Macaca mulatta). We found a significant positive correlation between cortisol levels sampled in juveniles and the frequency of right- versus left-hand use sampled in these same animals during adulthood. Right-hand preference was negatively correlated with stress reactivity. These data are consistent with the view that stress functioning and reactivity are associated with the development of hemispheric specialization in primates.

Plasma cortisol is associated with handedness in infant rhesus monkeys.

In this research we examined the relationship between plasma cortisol and handedness in infant rhesus macaques (Macaca mulatta). Specifically, we tested the hypothesis that stress functioning is related to hemispheric specialization and manifested in a positive correlation between cortisol levels and the frequency of right- versus left- hand use. We found a significant positive relationship between cortisol levels sampled at ages 1 and 3 months and lateral bias toward greater use of the right hand versus left hand sampled between ages 4 and 11 months. Further, we found a significant negative relationship between cortisol sampled at age 5 months and strength of lateral bias (independent of direction). These data suggest an early developmental influence of stress functioning on hemispheric specialization for manual control in infant monkeys.

Familial influences on hand preference: genotypic variation between closely related primate species.

The emergence of hemispheric specialization has important implications for the development of higher order cognitive processes, including language and spatial skills. In this research we sought to further understand psychobiological processes associated with the development of hemispheric specialization by examining and comparing familial influences on hand preference in two closely related macaque species: rhesus macaques (Macaca mulatta) and pigtailed macaques (Macaca nemestrina). The results of our study indicate contrasting patterns of familial influence on hand preference in each species. For the rhesus macaque we found a positive correlation in the direction of hand preference between mothers and their juvenile offspring, and for the pigtailed macaque we found a negative mother-offspring correlation in the direction of hand preference. Fathers did not contribute significantly to the direction of hand preference in either species. There was a trend toward a positive correlation for strength and consistency of hand preference between parents and offspring in rhesus macaques but not in pigtailed macaques. These findings indicate that maternal influences on offspring hand preference vary between closely related primate species and lead us to question the generalizability of universal single-factor theories used to explain intergenerational transmission of hand preference in humans.

Hormonal correlates of hand preference in free-ranging primates.

In this research we examined hormonal correlates of hand preference in free-ranging primates. Specifically, we tested the hypothesis that levels of the stress hormone cortisol and the male sex hormone testosterone are correlated with handedness in male rhesus macaques (Macaca mulatta). We found significant positive relationships between cortisol and testosterone levels sampled during adolescence and the frequency of right- versus left-hand use sampled during adulthood. These data indicate that adolescent measures of cortisol and testosterone are correlated with hemispheric specialization in adult free-ranging primates.

A comparative study of aimed throwing by monkeys and humans.

This research examined hand preference and postural characteristics of aimed throwing in capuchin monkeys and humans. We sought to directly compare the throwing performances of these primates, particularly the extent to which target distance influences hand preference, throwing posture, and throwing accuracy. For both species we found positive correlations between target distances for throwing accuracy, direction and strength of hand preference, percentage of bipedal vs tripedal throws, and percentage of overarm vs underarm throws. Throwing accuracy did not vary as a function of right vs left hand use although for monkeys throwing accuracy was positively associated with hand preference strength. We noted a sex difference among humans as males threw more accurately than did females. Between-species analysis indicated that humans exhibited greater right- vs left-hand use, greater hand preference strength, a greater relative percentage of bipedal vs tripedal throws, and a lower relative percentage of overarm vs underarm throws than did monkeys. We believe that the capuchin monkey is an informative nonhuman primate model of aimed throwing in humans and that research examining the throwing behavior of capuchins provides insight into the neurological and behavioral characteristics that underlie coordinated multi-joint movements across the primate order.

Handedness and cortisol in tufted capuchin monkey infants.

In this research, we examined the relationship between handedness and levels of plasma cortisol in infant monkeys (Cebus apella). Specifically, we sought to test the hypothesis that stress functioning is related to hemispheric specialization and is manifested in a positive correlation between cortisol levels and the frequency of right- versus left-hand use. We found a significant relationship between stress cortisol at age 6 months and lateral bias towards greater use of the right versus left hand at ages 6 and 12 months. These data suggest an early developmental influence of stress reactivity on the emergence of hemispheric specialization for manual control in infant monkeys through the 1st year of postnatal life.

CSF 5-HIAA and nighttime activity in free-ranging primates.

Men with low CNS serotonin turnover, as measured by cerebrospinal fluid 5-hydroxyindoleacetic acid (CSF 5-HIAA) concentrations, exhibit aberrant circadian activity patterns characterized by disrupted sleep rhythms and daytime hyperactivity. To assess whether similar patterns are found in nonhuman primates we examined the relationships between CSF 5-HIAA and nighttime activity in free-ranging monkeys. CSF samples were obtained from 16 adult male rhesus macaques living on a 475 acre, heavily forested sea island. Each subject was captured, fitted with a radio-telemetry motion-detector collar, and then released back into its group. A receiver placed near the sleeping trees of the study subjects recorded activity between 2100 hrs and 0600 hrs. Trained observers recorded behavioral data during the day. The animals followed a typical diurnal activity pattern, as they were active 74% of the sampled time during the day and 37% of the sampled time during the night. CSF 5-HIAA concentrations were inversely correlated with total duration of nighttime activity as well as mean duration of all active events. Nighttime activity was inversely correlated with daytime activity. CSF 3-methoxy-hydroxyphenylglycol (MHPG) concentrations were positively correlated with total nighttime activity, and inversely correlated with daytime sleep frequency. We conclude that male rhesus with low CSF 5-HIAA concentrations have higher total nighttime activity, longer mean periods of nighttime activity, and sleep more during the day than do males with high CSF 5-HIAA concentrations. This suggests that low serotonergic neurotransmission is associated with aberrant diurnal activity, as evidenced by a disruption of nighttime sleep patterns and a compensatory higher rate of inactivity during the day.

Reproductive performance of rhesus macaques (Macaca mulatta) in two outdoor housing conditions.

This study examined the reproductive performance of rhesus macaques maintained in two different housing conditions: high-density semi-sheltered gang cages and low-density outdoor corrals. Two hundred sixteen subjects were housed in 49 gang cages, each of which contained one breeding male and between one and eight breeding females. Two hundred seven subjects were housed in 13 corrals, each of which contained between two and four breeding males and between 9 and 26 breeding females. Over a 3-year period, pregnancy, live birth, and production rates were significantly greater for females in corrals than for females in gang cages. Fetal death rate was lower in corrals than in gang cages, while neonatal death rates did not differ between housing conditions. These differences did not result from potential confounds such as differential age structures or virological statuses between housing conditions. We conclude that, for rhesus macaques, outdoor corral housing leads to better reproductive performance than does semi-sheltered gang housing, probably as a result of increased individual space and relaxation of intense social stressors.

Rhesus macaque (Macaca mulatta) group formation and housing: wounding and reproduction in a specific pathogen free (SPF) colony.

In the present report, we examined the effects of group formation strategy and corral design on wounding and reproduction rates in rhesus macaques. Specifically, we examined group formation using a staged strategy, in which small groups of animals were introduced incrementally over a period of weeks, and a rapid formation strategy, in which all animals were introduced in 1 day. We also examined group formation using a divided corral design that facilitated visual and social separation of individuals, and an undivided corral design that did not facilitate visual or social separation. Dependent measures were wounding and reproductive rates over each of the 2 years that followed group formation. Results indicate that incrementally releasing subgroups of animals, and using a corral design that provides for visual and social separation of individuals, are effective strategies for reducing rates of traumatic wounding when forming multimale-multifemale rhesus macaque breeding groups. However, it must be noted that differences in formation strategy and corral design did not lead to higher reproductive rates. We conclude that incrementally releasing animals in hierarchical subgroups, and using a divided vs. undivided housing design, reduced intra-group wounding and associated demands on veterinary and animal management resources following formation of rhesus macaque breeding groups.

CSF 5-HIAA and aggression in female macaque monkeys: species and interindividual differences.

RATIONALE: While the relationship among CSF 5-HIAA, impulsivity, and aggression is well characterized in males, its investigation in females is limited, and no studies have assessed its generalizability across primates by making simultaneous comparisons between and within closely-related species. OBJECTIVES: We tested three hypotheses. First, that female rhesus macaques would have lower CSF 5-HIAA concentrations and be more aggressive than would female pigtailed macaques. Second, that females of both macaque species would exhibit an inverse relationship between interindividual differences in CSF 5-HIAA concentrations and rates of severe aggression. Third, that subjects with high CSF 5-HIAA concentrations would be higher in social dominance within their respective groups than would subjects with low CSF 5-HIAA concentrations. METHODS: We obtained CSF samples from 61 individually housed female primates of two closely related species: rhesus macaques (Macaca mulatta) and pigtailed macaques (Macaca nemestrina). We later placed subjects in unisex social groups, and correlated interindividual differences in CSF 5-HIAA with aggression, wounding, and acquisition of social dominance rank. RESULTS: Between-species analyses indicated higher CSF 5-HIAA concentrations in pigtailed macaques, and higher rates of high-intensity aggression, escalated aggression, and wounds requiring medical treatment in rhesus macaques. Within-species analyses indicated that interindividual differences in CSF 5-HIAA concentrations were inversely correlated with escalated aggression and positively correlated with social dominance rank. CONCLUSIONS: These findings show that agonistic and social differences between closely-related species are correlated with CNS serotonin activity, as species that show relatively high rates of severe aggression also tend to have low concentrations of CSF 5-HIAA. We conclude that serotonergic functioning plays an important role in controlling impulses that regulate severe aggression and social dominance relationships in both male and female primates, and that between-species differences in agonistic temperament can be predicted by species typical CNS serotonin functioning.

Left-handedness and longevity in primates.

In the present research we used the rhesus macaque to model the decline in the incidence of left-handedness that has been noted in the aged human population. We found a significant group-level bias towards use of the left hand among young-adult macaques, and a significant group-level bias towards use of the right hand among aged macaques. The distribution of hand preference across age classes cannot be explained through simple elimination of left-handed subjects in the aged population. Rather, our data are consistent with a maturational view positing increased use of the right hand with increased age. Similar findings across phylogenetically diverse primate taxa suggest that this phenomenon is an evolutionarily ancient trait. Greater use of the left hand in male versus female macaques indicates that sex differences in hand preference are also deeply rooted in our primate origins.