DIFFERENCES IN CULTURAL YIELD OF MYCOBACTERIUM TUBERCULOSIS ON MEDIA PREPARED USING COMMERCIAL AND HOUSEHOLD EGGS.

BACKGROUND: Mycobacterial culture is considered as the gold standard for TB diagnosis. It is performed on egg-based media using commercially available eggs to grow Mycobacteria from clinical samples. These eggs are known to contain high concentration of antibiotics, including fluoroquinolones, given to chicken to prevent early mortality. This study was performed to compare Mycobacterial growth on media prepared from commercial and antibiotic free household eggs. METHODS: Sputum samples from negative (No bacilli in 100 oil immersion field), scanty (1-9 AFB in 100 fields), 1+ (10-99 bacilli per field), 2+ (1-10 bacilli per field) and 3+ (>10 bacilli per field) were inoculated dually on Ogawa medium prepared from commercial and household eggs. Tubes were inspected every fourth day for the appearance of colonies till 60 days. Data tabulations and statistical analysis (F test for variation and unpaired Student's t test) were performed on Microsoft Excel. RESULTS: One microscopically negative sample showed growth on media prepared from household eggs, while all were negative on that prepared from commercial eggs. There were significant differences in time to culture positivity for samples graded 1+ (p = 0.02), 2+ (p = 0.002) and 3+ (p = 0.0003). CONCLUSION: Commercial eggs containing antibiotics can be a source of false negativity in cultures especially in microscopically negative samples. This can be of special concern in HIV patients who have high smear negativity. It is therefore important to either develop provision of antibiotic free eggs for media preparation or to develop and validate other laboratory investigations for smear negative TB patients.

Effects of Lower Limb Proprioceptive Training on Balance and Trunk Control Among the Adult Stroke Population.

Background and objective Balance and trunk control are major concerns among older adults during stroke rehabilitation. Loss of proprioception in the affected limb has a greater influence on motor learning and reeducation during balance training. Available studies stress the relevance of strength and functional training in regaining balance and trunk control. Proprioception training, in addition to available rehabilitation, can optimize the balance among this population. Our study aimed to find out the effects of proprioceptive training on balance and trunk control among the chronic stroke population. Methodology Out of 45 subjects enrolled at the Indian Head Injury Foundation, New Delhi, India, 30 subjects were selected based on selection criteria and randomized into two groups using the chit method, with 15 subjects in each group. The control group received conventional training, which included a range of motion, stretching, and strengthening exercises, while the intervention group received additional proprioceptive training five days per week for four consecutive weeks. Subjects were assessed on the Berg Balance Scale and Trunk Control Test for balance and trunk control on day one and after four weeks. A paired t-test was used to analyze the difference within the groups, and unpaired t-tests were used between the groups, keeping p < 0.05 as a significance level. Results After four weeks of intervention, statistically significant improvements were seen in the balance and trunk control groups, with p < 0.05 in both groups. However, a significant improvement in balance was observed in the experimental group when compared across groups (p = 0.001), whereas no statistically significant improvement in trunk control was found (p = 0.061). Conclusion We conclude that proprioceptive training and conventional physiotherapy both help to improve balance. Proprioceptive training is better for improving balance, but it has no significant effects on trunk control. It is likely that an extended intervention time or a different form of intervention may be required to achieve substantial gains in these areas. Future research might look at other outcome measures or the impacts of other types of therapies to see which ones are most helpful at increasing trunk control.

An analogue peptide from the Cancer/Testis antigen PASD1 induces CD8+ T cell responses against naturally processed peptide.

We have previously identified the novel Cancer/Testis antigen PASD1 by immunoscreening a testis library with pooled acute myeloid leukemia (AML) patient sera. To develop a cytotoxic T lymphocyte (CTL)-inducing vaccine, we have now investigated the carboxy-terminal region, known to contain serological determinants, for MHC class I (HLA-A⋆0201)-binding peptides. Algorithm-selected natural peptides failed to show detectable HLA-A⋆0201 binding in T2 assays. However, anchor-modified analogue peptides showed enhanced binding, with decreased off-rates. Analogue peptide-loaded antigen-presenting cells (APCs) induced IFN-γ production by T cells from normal donors and patients. In addition, peptide-specific T cells could be expanded from cancer patients by stimulation with the PASD1 analogue peptide Pa14. For clinical application, a DNA fusion gene vaccine encoding Pa14 was designed and tested in "humanized" mice. Splenocytes from vaccinated mice showed in vitro cytotoxicity against tumour cells, either exogenously loaded with the corresponding wild-type peptide (Pw8) or expressing endogenously processed PASD1 protein. We show for the first time that a DNA vaccine encoding an altered PASD1 epitope can induce CTLs to target the natural peptide expressed by human tumour cells.

Combined SARS-CoV-2 nucleic acid amplification testing and respiratory virus panel RT-PCR on the Hologic Panther Fusion system.

BACKGROUND: Significant overlap exists between the symptoms of SARS-CoV-2 and other respiratory viruses. This poses a serious challenge to clinical diagnosis, laboratory testing, and infection control programs. OBJECTIVES: To evaluate the performance of the Hologic Panther Fusion Respiratory Assays (RA) compared to the GenMark ePlex Respiratory Pathogen Panel (RPP) and to assess the ability of the Panther Fusion to perform parallel testing of SARS-CoV-2 and other respiratory viruses from a single sample. STUDY DESIGN: A diagnostic comparison study was carried out using 375 clinical nasopharyngeal specimens. Assay performance was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient. RESULTS: Overall agreement between the Fusion RA and ePlex RPP was 97.3 % (95 % CI 96.3-98.0), positive percent agreement was 97.2 % (95 % CI 93.0-99.2), negative percent agreement was 97.3 % (95 % CI 96.3-98.0), and the kappa coefficient was 0.85 (95 % CI 0.81-0.89). Forty additional viruses in 30 specimens were detected by Fusion that were not detected by ePlex. The maximum specimen throughput for parallel testing of the Fusion Respiratory Assays with SARS-CoV-2 was 275 samples in 20.7 h for Fusion SARS-CoV-2 and 350 samples in 20.0 h for Aptima Transcription Mediated Amplification SARS-CoV-2. CONCLUSION: Fusion RA demonstrated substantial agreement compared to the ePlex RPP. However, the Fusion detected respiratory viruses not identified by ePlex, consistent with higher clinical sensitivity. Workflows for parallel testing of respiratory pathogens and SARS-CoV-2 demonstrate that the Panther Fusion instrument provides a flexible, moderate to high throughput testing option for pandemic and seasonal respiratory viruses.

Antigenic Targets for the Immunotherapy of Acute Myeloid Leukaemia.

One of the most promising approaches to preventing relapse is the stimulation of the body's own immune system to kill residual cancer cells after conventional therapy has destroyed the bulk of the tumour. In acute myeloid leukaemia (AML), the high frequency with which patients achieve first remission, and the diffuse nature of the disease throughout the periphery, makes immunotherapy particularly appealing following induction and consolidation therapy, using chemotherapy, and where possible stem cell transplantation. Immunotherapy could be used to remove residual disease, including leukaemic stem cells from the farthest recesses of the body, reducing, if not eliminating, the prospect of relapse. The identification of novel antigens that exist at disease presentation and can act as targets for immunotherapy have also proved useful in helping us to gain a better understand of the biology that belies AML. It appears that there is an additional function of leukaemia associated antigens as biomarkers of disease state and survival. Here, we discuss these findings.

Urine Biomarkers for the Early Detection of Ovarian Cancer - Are We There Yet?

Ovarian cancer affects around 7500 women in the United Kingdom every year. Despite this, there is no effective screening strategy or standard treatment for ovarian cancer. If diagnosed during stage I, ovarian cancer has a 90% 5-year survival rate; however, there is usually a masking of symptoms which leads to an often late-stage diagnosis and correspondingly poor survival rate. Current diagnostic methods are invasive and consist of a pelvic examination, transvaginal ultrasonography, and blood tests to detect cancer antigen 125 (CA125). Unfortunately, surgery is often still required to make a positive diagnosis. To address the need for accurate, specific, and non-invasive diagnostic methods, there has been an increased interest in biomarkers identified through non-invasive tests as tools for the earlier diagnosis of ovarian cancer. Although most studies have focused on the identification of biomarkers in blood, the ease of availability of urine and the high patient compliance rates suggest that it could provide a promising resource for the screening of patients for ovarian cancer.

Use of Prescription Assistance Programs After the Affordable Health Care Act.

BACKGROUND: Insurance coverage in the United States seems to be in a state of unrest. The 2010 passage of the Patient Protection and Affordable Health Care Act (ACA) extended health insurance coverage to roughly 32 million people. An increase in the number of people with health insurance benefits raised the question of whether prescription assistance programs (PAPs) would still be used after ACA implementation. OBJECTIVE: To evaluate the use of PAPs following the implementation of the ACA insurance mandate. METHODS: Health insurance was not required by the ACA until January 2014, so we retrospectively examined the use of drug company-sponsored PAPs before and after the ACA implementation. Since each PAP had its own qualifying criteria, any person who used a PAP through the assistance of NeedyMeds and its PAPTracker between the years of 2011 and 2016 were included for analysis. Data were pulled by NeedyMeds from the PAPTracker software, which produces completed PAP applications from drug manufacturer forms for PAPs. The number of PAP orders, number of unique patient orders, and annual patient prescription savings were assessed. RESULTS: Between 2011 and 2013, there was an average of 4.2 annual PAP orders per patient; however, annual PAP orders decreased to 3.1 per patient between 2014 and 2016 (P < 0.001). PAP orders declined by an average of 3.0% per month between 2014 and 2016 (P < 0.001), and average prescription savings per order increased from $870.40 before the ACA to $1,086.40 after ACA implementation (P = 0.0024). Patients saved an average of over $3,000 on prescriptions annually with the use of PAPs after the ACA mandate. CONCLUSIONS: Although health care reform is inevitable, our study showed that PAPs remain important to help cover prescription drug costs for eligible patients, even with invariable changes to health insurance, including a health insurance requirement. While the ACA may have been an important step forward in extending health insurance coverage to millions, PAPs are still used to help U.S. patients obtain their medications at no cost or very low cost. These programs will most likely remain relevant until other approaches are taken to help alleviate the effects of increasing drug prices in the United States. DISCLOSURES: No outside funding supported this research. The authors have no relevant financial or nonfinancial relationships to disclose. Study concept and design were contributed by Khan, Lerchenfeldt, and Karabon. Khan collected the data, and all authors participated in data analysis. The manuscript was primarily written by Lerchenfeldt, along with Khan and Karabon, and revised by Lerchenfeldt, along with Karabon and Khan.

Infrequent Expression of the Cancer-Testis Antigen, PASD1, in Ovarian Cancer.

Ovarian cancer is very treatable in the early stages of disease; however, it is usually detected in the later stages, at which time, treatment is no longer as effective. If discovered early (Stage I), there is a 90% chance of five-year survival. Therefore, it is imperative that early-stage biomarkers are identified to enhance the early detection of ovarian cancer. Cancer-testis antigens (CTAs), such as Per ARNT SIM (PAS) domain containing 1 (PASD1), are unique in that their expression is restricted to immunologically restricted sites, such as the testis and placenta, which do not express MHC class I, and cancer, making them ideally positioned to act as targets for immunotherapy as well as potential biomarkers for cancer detection where expressed. We examined the expression of PASD1a and b in a number of cell lines, as well as eight healthy ovary samples, eight normal adjacent ovarian tissues, and 191 ovarian cancer tissues, which were predominantly stage I (n = 164) and stage II (n = 14) disease. We found that despite the positive staining of skin cancer, only one stage Ic ovarian cancer patient tissue expressed PASD1a and b at detectable levels. This may reflect the predominantly stage I ovarian cancer samples examined. To examine the restriction of PASD1 expression, we examined endometrial tissue arrays and found no expression in 30 malignant tumor tissues, 23 cases of hyperplasia, or 16 normal endometrial tissues. Our study suggests that the search for a single cancer-testes antigen/biomarker that can detect early ovarian cancer must continue.

Application of the pMHC Array to Characterise Tumour Antigen Specific T Cell Populations in Leukaemia Patients at Disease Diagnosis.

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms' Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8-1.4 x 10(6)). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1(126-134) (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1(950-958) epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.