67 research outputs found
Human Papillomavirus DNA extraction and genotype analysis by multiplex real time polymerase chain reaction from formalin fixed paraffin wax-embedded cervical carcinoma specimens
Introduction: Cervical squamous cell carcinoma is most commonly caused by persistent infection by high risk human papillomavirus (hrHPV) genotypes. The exact type of hrHPV varies geographically and is the basis for HPVâbased vaccination for cervical squamous cell carcinoma prevention. Little is known regarding local hrHPV genotypes within the Western Cape population of South Africa. Aims and objectives: This was a pilot study aiming to extract of high quality genomic DNA from archival FFPE cervical squamous cell carcinoma cases and identify hrHPV genotypes by multiplex real time PCR (RT-PCR). Materials and methods: A retrospective search identified a total of 57 cases of cervical squamous cell carcinoma for the period 2004-2014. This was reduced to a final number of 23 that exhibited sufficient tumour burden for DNA extraction. The most common age group was 40-49 years. HIV status was as follows: two HIV-positive, 14 HIV-negative and 7 HIV unknown. DNA was extracted from archival FFPE cervical squamous cell carcinoma samples using QIAGEN QIAampÂź DNA FFPE Tissue kit. Housekeeping genes were detected by endpoint PCR using standard primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequences to determine the quality and integrity of extracted DNA for downstream PCR amplification experiments. HrHPV DNA amplification was optimised using a touchdown PCR technique with L1 consensus gene GP5+/GP6+ primers. HrHPV genotypes were detected using a four colour multiplex hrHPV genotyping kit. Samples showing positive results in overlapping probe filter detection spectra were subjected to DNA Sanger sequencing for final confirmation of specific hrHPV genotype. Results: Standard xylene DNA extraction methods using QIAampÂź system yielded adequate amounts of DNA with average final concentration of 463.2 ng/l and A260/A280 ratio of 1.86. Housekeeping genes were successfully detected in all samples, confirming that no significant DNA degradation of target sequences occurred within the archival time range of 2004-2014. HPV L1 detection via GP5+/GP6+ primers with endpoint PCR was not achieved via standard cycling conditions and required the use of a touchdown technique with gradually decreasing annealing temperatures. This method successfully identified HPV L1 sequences in 22 out of 23 cases. Multiplex RT-PCR with four colour hydrolysis probes identified hrHPV genotypes in 22 of 23 cases with relative frequencies of HPV genotypes: 16>>18=39=45>33. Most cases showed infection with a single hrHPV genotype (HPV 16 and one case with HPV 33) with four cases demonstrating two genotypes (two with HPV 16&18, one with 16&33 and one with 39&45) and one case with three genotypes (HPV 16, 39, 45). Interestingly, none of the HIV-positive cases showed multiple hrHPV genotype infection. Four hrHPV cases with overlapping spectra for HPV 18/31 and 45/59 were subjected to Sanger sequencing for confirmation of genotype. Three of four cases showed 100% match for genotypes 18 and 45 with the final case demonstrating only co-infective HPV 16.Conclusion: Commercial DNA extraction kits yield adequate amounts of intact, amplifiable DNA in archival FFPE cervical carcinoma specimens. Touchdown PCR is necessary for HPV detection in extracted FFPE DNA cases using GP5+/GP6+ L1 primers. RT-PCR using multicolour hydrolysis probes is a rapid, sensitive technique for hrHPV genotype screening of cervical squamous cell carcinoma specimens. A three colour detection system rather than four colour kit is recommended for future studies in order to avoid extra cost in DNA sequencing cases with overlapping spectra. This pilot study demonstrates hrHPV genotype prevalence similar to that in other populations and suggests that vaccination with currently available formulations would provide a sufficiently wide coverage of HPV genotypes. Future studies will include application of the FFPE DNA extraction, endpoint PCR and RT-PCR techniques to the remainder of the cases in the original cohort
Exploration of the effect of protease inhibitor activity on snake venoms
Envenomation by snakes is a worldwide health crisis. Anti-venom, the current treatment standard, is a costly and imperfect treatment. Without proper ID of the snake species, the treatment provider is guessing at which anti-venom to use. Many snakes are also able to control venom release as they age, so not every bite by a venomous snake is envenoming. Treatment by anti-venom has the potential of severe side effects so treating a snake bite that hasn't resulted in envenoming could cause more problems than withholding treatment. Can protease inhibitors be used to block or decrease the gelatinase activity of snake venom? Established baseline venom activity and dose dependence of inhibition. Activity was measured with a fluorescein-labeled gelatin. NNGH is the enzyme inhibitor that was used. In dose-response experiments, there is significant gelatinase activity and over 50% inhibition by NNGH in Agkistrodon contortrix and complete inhibition in Crotalus atrox. We also see 50% inhibition of Cerastes cerastes venom by NNGH but the species has near half the initial gelatinase activity of C. atrox. These results provide substantial support that venoms of Crotalinae species, pit vipers, are inhibited by the protease inhibitor NNGH, supporting future research endeavors
Haemophagocytic lymphohistiocytosis: A fulminant syndrome associated with multiorgan failure and high mortality that frequently masquerades as sepsis and shock
Acquired haemophagocytic lymphohistiocytosis (HLH) is a condition involving cytokine overproduction by defective cytotoxic T lymphocytes and natural killer cells, resulting in life-threatening cytopenias and multiorgan infiltration and dysfunction. Triggers for acquired HLH vary and include viruses, malignancies and autoimmune conditions. Recent reports suggest that HLH may be underdiagnosed owing to variable clinical presentations, diagnostic criteria and a low level of awareness on the part of medical personnel, thus delaying prompt treatment and contributing to high mortality rates. Five patients in whom acquired HLH was diagnosed, following bone marrow investigations, for the period of May - September 2013 are presented. All were at an advanced stage of their disease at time of diagnosis. The three patients who were HIV-positive had a coexisting malignancy at the time of HLH diagnosis, which may have triggered HLH. A definite trigger was not identified in the remaining two HIV-negative patients despite early concerns regarding autoimmune disease. Two patients received timeous diagnosis, started chemotherapy and are currently improving. The remaining three succumbed to their illness. Adult acquired HLH may be more common in the acute care setting than currently appreciated. As awareness of this condition and its treatment is currently low, it may remain undiagnosed until the disease has evolved into multiorgan failure. Fever in the absence of infectious agents, marked hyperferritinaemia, unexplained cytopenias, organomegaly or liver dysfunction should raise the suspicion of HLH. Timeous introduction of therapy will improve outcomes
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Energy Systems and Population Health
It is well-documented that energy and energy systems have a central role in social and economic development and human welfare at all scales, from household and community to regional and national (41). Among its various welfare effects, energy is closely linked with people s health. Some of the effects of energy on health and welfare are direct. With abundant energy, more food or more frequent meals can be prepared; food can be refrigerated, increasing the types of food items that are consumed and reducing food contamination; water pumps can provide more water and eliminate the need for water storage leading to contamination or increased exposure to disease vectors such as mosquitoes or snails; water can be disinfected by boiling or using other technologies such as radiation. Other effects of energy on public health are mediated through more proximal determinants of health and disease. Abundant energy can lead to increased irrigation, agricultural productivity, and access to food and nutrition; access to energy can also increase small-scale income generation such as processing of agricultural commodities (e.g., producing refined oil from oil seeds, roasting coffee, drying and preserving fruits and meats) and production of crafts; ability to control lighting and heating allows education or economic activities to be shielded from daily or seasonal environmental constraints such as light, temperature, rainfall, or wind; time and other economic resources spent on collecting and/or transporting fuels can be used for other household needs if access to energy is facilitated; energy availability for transportation increases access to health and education facilities and allow increased economic activity by facilitating the transportation of goods and services to and from markets; energy for telecommunication technology (radio, television, telephone, or internet) provides increased access to information useful for health, education, or economic purposes; provision of energy to rural and urban health facilities allows increased delivery and coverage of 3 various health services and interventions such as tests and treatments, better storage of medicine and vaccines, disinfection of medical equipment by boiling or radiation, and more frequent and efficient health system encounters through mobile clinics or longer working hours; and so on. In fact, while the dominant view of development-energy-health linkages has been that improvements in energy and health are outcomes of the socioeconomic development process (e.g., the ''energy ladder'' framework discussed below), it has even been argued that access to higher quality energy sources and technologies can initiate a chain of demographic, health, and development outcomes by changing the household structure and socioeconomic relationships. For example, in addition to increased opportunities for food and income production, reduced infant mortality as a result of transition to cleaner fuels or increased coverage of vaccination with availability of refrigerators in rural clinics may initiate a process of ''demographic transition'' to low-mortality and low-fertility populations (14). Such a transition has historically been followed with further improvements in maternal and child health and increased female participation in the labor markets and other economic activities
The Evolving Spectrum of Kidney Histology in HIV-Positive Patients in South Africa
Introduction: Sub-Saharan Africa remains challenged by the highest burden of human immunodeficiency virus (HIV), an epidemic of tuberculosis (TB), and increasing number of people with HIV (PWH) on antiretroviral therapy (ART), all of which may result in kidney injury. Methods: This observational cohort study describes the spectrum of kidney disease in PWH in South Africa, between 2005 and 2020. Kidney biopsies were analyzed in 4 time periods as follows: early ART rollout (2005â2009), tenofovir disoproxil (TDF) introduction (2010â2012), TDF-based fixed dose combination (2013â2015), and ART at HIV diagnosis (2016â2020). Logistic regression was used to identify factors associated with HIV-associated nephropathy or focal segmental glomerulosclerosis (HIVAN/FSGS) and tubulointerstitial disease (TID). Results: We included 671 participants (median age 36, interquartile range, 21â44 years; 49% female; median CD4 cell count 162 [interquartile range, 63â345] cells/mm3). Over time, ART (31%â65%, PÂ < 0.001), rate of HIV suppression (20%â43%, PÂ < 0.001), nonelective biopsies (53%â72%, PÂ < 0.001), and creatinine at biopsy (242â449 ÎŒmol/l, PÂ < 0.001) increased. A decrease in HIVAN (45%â29% PÂ < 0.001) was accompanied by an increase in TID (13%â33%, PÂ < 0.001). Granulomatous interstitial nephritis accounted for 48% of TID, mostly because of TB. Exposure to TDF was strongly associated with TID (adjusted odds ratio 2.99, 95% confidence interval 1.89â4.73 PÂ < 0.001). Conclusion: As ART programs intensified and increasingly used TDF, the spectrum of kidney histology in PWH evolved from a predominance of HIVAN in the early ART era to TID in recent times. The increase in TID is likely due to multiple exposures that include TB, sepsis, and TDF as well as other insults
Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model
International audienceThe neglected tropical disease Buruli ulcer (BU) is an infection of subcu-taneous tissue with Mycobacterium ulcerans. There is no effective vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described Toll-like receptor 2 (TLR-2) agonist-based lipopeptide adjuvant, R 4 Pam 2 Cys. Mice were vaccinated and then challenged via tail inoculation with 14 to 20 CFU of a biolumines-cent strain of M. ulcerans. Mice receiving either the experimental ER vaccine or My-cobacterium bovis bacillus Calmette-GuĂ©rin (BCG) were equally protected, with both groups faring significantly better than nonvaccinated animals (P Ïœ 0.05). To explore potential correlates of protection, a suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were used to interrogate the immune response data to develop disease-prognostic models. High levels of interleukin 2 (IL-2) and low gamma interferon (IFN-â„) produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression revealed vaccine-specific profiles of protection. High ti-ters of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the ER vaccine. In contrast, high titers of IL-6, tumor necrosis factor alpha (TNF-âŁ), IFN-â„, and IL-10 in the DLN and low IFN-â„ titers in the spleen were associated with protection following BCG vaccination. This study suggests that an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection
corner.py: corner.py v2.0.0
Version 2 of corner.py is now tested, documented, and citable
Corner.Py: Corner.Py V2.0.0
Version 2 of corner.py is now tested, documented, and citable.</span
Trends in Diagnosis of Noninvasive Follicular Thyroid Neoplasm with Papillarylike Nuclear Features and Total Thyroidectomies for Patients with Papillary Thyroid Neoplasms
Importance: Increasing detection of early-stage papillary thyroid neoplasms without improvements in mortality has prompted development of strategies to prevent or mitigate overtreatment. Objective: To determine adoption rates of 2 recent strategies developed to limit overtreatment of low-risk thyroid cancers: (1) a new classification, noninvasive follicular thyroid neoplasm with papillarylike nuclear features (NIFTP), and (2) hemithyroidectomy for selected papillary thyroid carcinomas (PTCs) up to 4 cm in size. Design, Setting, and Participants: This is a cross-sectional analysis of 3368 pathology records of 2 cohorts of patients from 18 hospitals in 6 countries during 2 time periods (2015 and 2019). Participating hospitals were included from the US (n = 12), Canada (n = 2), Denmark (n = 1), South Korea (n = 1), South Africa (n = 1), and India (n = 1). The records of the first 100 patients per institution for each year who underwent thyroid-directed surgery (hemithyroidectomy, total thyroidectomy, or completion thyroidectomy) were reviewed. Main Outcomes and Measures: Frequency of diagnosis of NIFTP, PTCs, and thyroidectomies during the study period. Results: Of the 790 papillary thyroid neoplasms captured in the 2019 cohort, 38 (4.8%) were diagnosed as NIFTP. Diagnosis of NIFTP was observed in the US, South Africa, and India. There was minimal difference in the total proportion of PTCs in the 2015 cohort compared with the 2019 cohort (778 [47.1%] vs 752 [44.5%]; difference, 2.6% [95% CI, -16.9% to 22.1%]). The proportion of PTCs eligible for hemithyroidectomy but treated with total thyroidectomy in the 2 cohorts demonstrated a decreasing trend from 2015 to 2019 (341 of 453 [75.3%] vs 253 of 434 [58.3%]; difference, 17.0% [95% CI, -1.2% to 35.2%]). Conclusions and Relevance: Results of this cohort study showed that the 2 mitigation strategies for preventing overtreatment of early-stage thyroid cancer have had mixed success. The diagnosis of NIFTP has only been applied to a small proportion of thyroid neoplasms compared with expected rates. However, more patients eligible for hemithyroidectomy received it in 2019 compared with 2015, showing some success with this deescalation strategy
An optical biosensor-based immunoassay for the determination of bovine serum albumin in milk and milk products
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