Nucleic Acid Template and the Risk of a PCR-Induced HIV-1 Drug Resistance Mutation

Background

The HIV-1 nucleoside RT inhibitor (NRTI)-resistance mutation, K65R confers intermediate to high-level resistance to the NRTIs abacavir, didanosine, emtricitabine, lamivudine, and tenofovir; and low-level resistance to stavudine. Several lines of evidence suggest that K65R is more common in HIV-1 subtype C than subtype B viruses.

Methods and Findings

We performed ultra-deep pyrosequencing (UDPS) and clonal dideoxynucleotide sequencing of plasma virus samples to assess the prevalence of minority K65R variants in subtype B and C viruses from untreated individuals. Although UDPS of plasma samples from 18 subtype C and 27 subtype B viruses showed that a higher proportion of subtype C viruses contain K65R (1.04% vs. 0.25%; p<0.001), limiting dilution clonal sequencing failed to corroborate its presence in two of the samples in which K65R was present in >1.5% of UDPS reads. We therefore performed UDPS on clones and site-directed mutants containing subtype B- and C-specific patterns of silent mutations in the conserved KKK motif encompassing RT codons 64 to 66 and found that subtype-specific nucleotide differences were responsible for increased PCR-induced K65R mutation in subtype C viruses.

Conclusions

This study shows that the RT KKK nucleotide template in subtype C viruses can lead to the spurious detection of K65R by highly sensitive PCR-dependent sequencing techniques. However, the study is also consistent with the subtype C nucleotide template being inherently responsible for increased polymerization-induced K65R mutations in vivo.     

Prolongation of the QT interval and post-extrasystolic augmentation of the TU-wave during emotional stress

We present a case of a 25-year-old woman with multiple blackouts and no structural heart disease, with abnormal T-U waves and borderline QT interval on her resting electrocardiogram. During emotional stress she developed frequent monomorphic ventricular premature beats, with characteristic changes of the sinus complexes immediately following the premature beats, namely augmentation and greater degree of merging of the T and U waves and QTc interval prolongation. The changes alert about the possibility of congenital long QT syndrome, specifically genotype 2 or 1.     

Adequacy of control of cardiovascular risk factors in ambulatory patients with type 2 diabetes attending diabetes out-patients clinic at a county hospital,Kenya

Background

Type 2 diabetes is associated with substantial cardiovascular morbidity and mortality arising from the high prevalence of cardiovascular risk factors such as hypertension, dyslipidaemia, obesity, poor glycaemic control and albuminuria. Adequacy of control of these risk factors determines the frequency and outcome of cardiovascular events in the patients. Current clinical practice guidelines emphasize primary prevention of cardiovascular disease in type 2 diabetes. There is scarce data from the developing countries, Kenya included, on clinical care of patients with type 2 diabetes in the regions that are far away from tertiary health facilities. So we determined the adequacy of control of the modifiable risk factors: glycaemic control, hypertension, dyslipidemia, obesity and albuminuria in the study patients from rural and peri-urban dwelling.

Methods

This was a cross-sectional study on 385 randomly selected ambulatory patients with type 2 diabetes without overt complications. They were on follow up for at least 6 months at the Out-patient diabetes clinic of Nyeri County Hospital, a public health facility located in the central region of Kenya.

Results

Females were 65.5%. The study subjects had a mean duration of diabetes of 9.4 years, IQR of 3.0–14 years. Their mean age was 63.3 years, IQR of 56-71 years.Only 20.3% of our subjects had simultaneous optimal control of the three (3) main cardiovascular risk factors of hypertension, high LDL-C and hyperglycaemia at the time of the study. The prevalence of cardiovascular risk factors were as follows: HbA1c above 7% was 60.5% (95% CI, 55.6–65.5), hypertension, 49.6% of whom 76.6% (95% CI, 72.5–80.8) were poorly controlled. High LDL-Cholesterol above 2.0 mmol/L was found in 77.1% (95% CI 73.0–81.3) and Albuminuria occurred in 32.7% (95% CI 27.8–37.4). The prevalence of the other habits with cardiovascular disease risk were: excess alcohol intake at 26.5% (95% CI 27.8–37.4) and cigarette-smoking at 23.6%.A modest 23.4% of the treated patients with hypertension attained target blood pressure of <140/90 mmHg. Out of a paltry 12.5% of the statin-treated patients and others not actively treated, only 22.9% had LDL-Cholesterol of target <2.0 mmol/L.There were no obvious socio-demographic and clinical determinants of poor glycaemic control. However, old age above 50 yrs., longer duration with diabetes above 5 yrs. and advanced stages of CKD were significantly associated with hypertension. Female gender and age, statin non-use and socio-economic factor of employment were the significant determinants of high levels of serum LDL-cholesterol.

Conclusion

The majority of the study patients attending this government-funded health facility had high prevalence of cardiovascular risk factors that were inadequately controlled. Therefore patients with type 2 diabetes should be risk-stratified by their age, duration of diabetes and cardiovascular risk factor loading. Consequently, composite risk factor reduction strategies are needed in management of these patients to achieve the desired targets safely. This would be achieved through innovative care systems and modes of delivery which would translate into maximum benefit of primary cardiovascular disease prevention in those at high risk. It is a desirable quality objective to have a higher proportion of the patients who access care benefiting maximally more than the numbers we are achieving now.

     

The incidence of Campylobacter spp. on processed turkey from processing plants in the midwestern United States

AIM: The primary aim of this study was to determine the incidence of Campylobacter spp. on turkey, presented for processing at participating production plants located in the midwest region of the United States. METHODS AND RESULTS: The two participating plants were visited on a monthly basis for a period of 1 year. Sampling of carcasses was carried out using a surface swab technique. Swabs were obtained from carcasses at two points on the production line - prechill and postchill. In addition, samples of chill water were also obtained for examination. Isolation and detection of Campylobacter was carried out using enrichment in Preston broth with recovery of the organism on blood free Campylobacter selective agar (CCDA). Isolates recovered were screened and identified using the API Campy identification system. The study found that 34.9% of all samples tested were positive for Campylobacter spp. The overall, contamination rates observed for both plants were relatively similar (39.2% for plant A and 30.6% for plant B). Differences were observed in the incidence of Campylobacter spp. on prechill vs postchill carcasses (i.e. 40.8% prechill vs 37.6% postchill for plant A and 41.8% prechill vs 19.8% postchill for plant B). Campylobacter species most often isolated included Camp. jejuni and Camp. coli. Other species recovered were Camp. fetus fetus, Camp. upsaliensis and Camp. lari. CONCLUSIONS: The incidence of Campylobacter spp. on processed poultry was relatively common. Factors such as the processing plant examined, season and the farms presenting birds for processing influenced the incidence of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences were observed in the prevalence of Campylobacter spp. isolated from the two plants examined. The study suggests a seasonal prevalence of Campylobacter in the cooler months with processing conditions also influencing the overall occurrence of the organism. The incidence, isolation and detection of Campylobacter spp. from processed poultry are discussed.     

The incidence of antimicrobial-resistant Salmonella spp on freshly processed poultry from US Midwestern processing plants

AIMS: To determine the incidence of antimicrobial-resistant Salmonella spp. on processed poultry (turkey) at Midwestern poultry plants. METHODS AND RESULTS: Two participating plants were visited at monthly intervals for a period of 1 year. Surface swabs were obtained from carcasses at two selected points on the production line, pre- and post-chill. In addition, samples of the chill water from chill tanks were also examined. Isolation and detection of Salmonella spp. from carcass swabs and chill water was carried out using standard enrichment techniques. Immunomagnetic separation was used to enhance the recovery of the pathogen. Salmonella isolates recovered were identified, serotyped and their antimicrobial resistance profiles determined using the National Antimicrobial Resistance Monitoring System. Results from the study indicated that the overall incidence of Salmonella was approx. 16.7%, with a greater incidence of the pathogen observed on pre-chill than post-chill carcasses. Salmonella isolates recovered displayed resistance to an average of four different antimicrobials. Approximately 15 different serotypes of Salmonella spp. were recovered, with Salmonella serotype Agona, Salmonella serotype Hadar, Salmonella serotype Heidelberg and Salmonella serotype Senftenberg being the most common. CONCLUSIONS: The incidence of Salmonella spp. was relatively low and isolates recovered showed significant degrees of antimicrobial resistance. Factors such as the processing plant examined, the season and farms that were presenting animals for processing influenced the incidence of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences were observed in the serotypes of Salmonella recovered and the types of antimicrobial resistance found at the two plants. The study suggests that the use of antimicrobials at the farm level influences the creation of an environment that promotes the selection of antimicrobial-resistant Salmonella spp. The incidence, isolation and detection of Salmonella spp. on processed poultry are discussed.     

Incidence of Appendicitis over Time: A Comparative Analysis of an Administrative Healthcare Database and a Pathology-Proven Appendicitis Registry

ImportanceAt the turn of the 21^st century, studies evaluating the change in incidence of appendicitis over time have reported inconsistent findings.ObjectivesWe compared the differences in the incidence of appendicitis derived from a pathology registry versus an administrative database in order to validate coding in administrative databases and establish temporal trends in the incidence of appendicitis.DesignWe conducted a population-based comparative cohort study to identify all individuals with appendicitis from 2000 to2008.ResultsThe administrative database (PPV = 83.0%) overestimated the incidence of appendicitis (100.3 per 100,000) when compared to the pathology registry (84.2 per 100,000). Codes for perforated appendicitis were not reliable (PPV = 52.4%) leading to overestimation in the incidence of perforated appendicitis in the administrative database (34.8 per 100,000) as compared to the pathology registry (19.4 per 100,000). The incidence of appendicitis significantly increased over time in both the administrative database (APC = 2.1%; 95% CI: 1.3, 2.8) and pathology registry (APC = 4.1; 95% CI: 3.1, 5.0).

Conclusion & Relevance

The administrative database overestimated the incidence of appendicitis, particularly among perforated appendicitis. Therefore, studies utilizing administrative data to analyze perforated appendicitis should be interpreted cautiously.     

Weed species in tomato production and their role as alternate hosts of Tomato spotted wilt virus and its vector Frankliniella occidentalis

Tomato spotted wilt virus (TSWV) is an important plant virus that infects a wide range of hosts including weeds making its management difficult. A survey was undertaken to establish the occurrence of weed species in tomato production systems in Kenya and their role as hosts of TSWV and its vectors. Selected weed species were further evaluated for their reaction to TSWV, transmission efficiency by Frankliniella occidentalis and ability to support thrips reproduction. Of the 43 weed species identified in the field, 29 species had been reported as hosts of TSWV, two were non‐hosts and 11 had no record of their status. Among the more common species, Amaranthus hybridus, Solanum nigrum, Tagetes minuta and Datura stramonium were susceptible to the virus and supported high levels of thrips reproduction. The TSWV could not be transmitted to Galinsoga parviflora and Sonchus oleraceus by F. occidentalis despite them being highly susceptible in mechanical transmission tests. There was a significant correlation between feeding damage and number of larvae of F. occidentalis on different weeds. Occurrence of weeds that support thrips reproduction and are good hosts of TSWV is a clear indicator of their role in epidemiology and the importance of their management for disease control.     

The evolution of vertebrate eye size across an environmental gradient: phenotype does not predict genotype in a Trinidadian killifish

Vertebrates exhibit substantial variation in eye size. Eye size correlates positively with visual capacity and behaviors that enhance fitness, such as predator avoidance. This foreshadows a connection between predation and eye size evolution. Yet, the conditions that favor evolutionary shifts in eye size, besides the well‐known role for light availability, are unclear. We tested the influence of predation on the evolution of eye size in Trinidadian killifish, Rivulus hartii. Rivulus are located across a series of communities where they coexist with visually oriented piscivores ("high predation" sites), and no predators (“Rivulus‐only” sites). Wild‐caught Rivulus from high predation sites generally exhibited a smaller relative eye size than communities that lack predators. Yet, such differences were inconsistent across rivers. Second‐generation common garden reared fish revealed repeatable decreases in eye size in Rivulus from high predation sites. We performed additional experiments that tested the importance of light and resources on eye size evolution. Sites that differ in light or resource availability did not differ in eye size. Our results argue that differences in predator‐induced mortality underlie genetically‐based shifts in vertebrate eye size. We discuss the drivers of eye size evolution in light of the nonparallel trends between the phenotypic and common garden results.     

Parameter Identification in a Generalized Time-Harmonic Rayleigh Damping Model for Elastography

The identifiability of the two damping components of a Generalized Rayleigh Damping model is investigated through analysis of the continuum equilibrium equations as well as a simple spring-mass system. Generalized Rayleigh Damping provides a more diversified attenuation model than pure Viscoelasticity, with two parameters to describe attenuation effects and account for the complex damping behavior found in biological tissue. For heterogeneous Rayleigh Damped materials, there is no equivalent Viscoelastic system to describe the observed motions. For homogeneous systems, the inverse problem to determine the two Rayleigh Damping components is seen to be uniquely posed, in the sense that the inverse matrix for parameter identification is full rank, with certain conditions: when either multi-frequency data is available or when both shear and dilatational wave propagation is taken into account. For the multi-frequency case, the frequency dependency of the elastic parameters adds a level of complexity to the reconstruction problem that must be addressed for reasonable solutions. For the dilatational wave case, the accuracy of compressional wave measurement in fluid saturated soft tissues becomes an issue for qualitative parameter identification. These issues can be addressed with reasonable assumptions on the negligible damping levels of dilatational waves in soft tissue. In general, the parameters of a Generalized Rayleigh Damping model are identifiable for the elastography inverse problem, although with more complex conditions than the simpler Viscoelastic damping model. The value of this approach is the additional structural information provided by the Generalized Rayleigh Damping model, which can be linked to tissue composition as well as rheological interpretations.