Detection of BCR-ABL kinase domain mutations in CD34+ cells from newly diagnosed chronic phase CML patients and their association with imatinib resistance

BCR-ABL kinase domain (KD) mutations, the most common cause of imatinib resistance, are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CP-CML) patients. Recent studies indicate pre-existing mutations (PEMs) can be detected in a higher percentage of CML patients using CD34+ stem/progenitor cells, and these mutations may correlate with imatinib resistance. We investigated KD mutations in CD34+ stem cells from 100 CP-CML patients by multiplex ASO-PCR and sequencing ASO-PCR products at the time of diagnosis. PEMs were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8-48), all patients with PEMs exhibited imatinib resistance. Of 68 patients without PEMs, 24 developed imatinib resistance. Mutations were detected in 21 of these patients by ASO-PCR and KD sequencing. All 32 patients with PEMs had the same mutations. In imatinib-resistant patients without PEMs, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients without T315I and Y253F mutations responded to imatinib dose escalation. In conclusion, BCR-ABL PEMs can be detected in a substantial number of CP-CML patients when investigated using CD34+ stem/progenitor cells. These mutations are associated with imatinib resistance, and mutation testing using CD34+ cells may facilitate improved, patient-tailored treatment

Upwind skewed radial basis functions (USRBF) for solution of highly convective problems over meshfree nodes

An upwind skewed radial basis function (USRBF)-based solution scheme is presented for stabilized solutions of convection-dominated problems over meshfree nodes. The conventional, radially symmetric radial basis functions (RBFs) are multiplied with an upwinding factor which skews the RBFs toward the upwind direction. The upwinding factor is a function of flow direction, intensity of convection, size of local support domain, and nodal distribution. The use of USRBFs modifies the weight values such that the necessary artificial diffusion is added only along the flow direction, whereas the crosswind diffusion is avoided. Subsequently, these skewed radial basis functions are employed in finite difference mode (RBF-FD) for derivative approximation. The performance and accuracy of the proposed scheme is studied by solving convection–diffusion problems over uniform and random distribution of meshfree nodes with various convection intensities. The upwinding effectively suppresses non-physical perturbation in numerical solution of convection-dominated problems. The results show that significant improvement in accuracy can be achieved by using the proposed USRBF-based solution scheme, particularly at higher convection intensities

The Anticipated Future of Public Health Services Post COVID-19: Viewpoint

In March 2020, the World Health Organization declared COVID-19 as a global pandemic. The COVID-19 pandemic has affected various public health functions and essential services in different ways and magnitudes. Although all countries have witnessed the effect of COVID-19, the impact differed based on many factors including the integrity and resiliency of the countries’ health systems. This paper presents opinions and expectations of the authors about the anticipated changes in the future of public health at the global, regional, and national levels. The viewpoint is based on the current efforts and challenges that various stakeholders have carried out to control COVID-19 and the contribution from the literature on the future of public health. Numerous agencies and actors are involved in the fight against COVID-19 with variations in their effectiveness. The public health services showed weaknesses in most of the countries, in addition to the lack of adequate curative medicine settings. The pandemic highlighted the need for better governance and stronger and more resilient health systems and capacities. The COVID-19 experience has also emphasized the importance of coordination and collaboration among the countries and stakeholders. The COVID-19 pandemic might lead to a wide discussion to improve international and national approaches to prepare for and respond to similar events in terms of preparedness and response mechanisms and tools. Public health will not be the same as before COVID-19. New health priorities, approaches, and new agendas will be on the table of the global platforms and initiatives. More investment in research and technology to meet the demand for new vaccines and medicines, innovative methods like distance learning and working, more respect and remuneration to health professionals, and normalization of the public health and social measures that were induced during the COVID-19 pandemic are expected to be seen in future

Point Prevalence Survey of Antimicrobial Use in Selected Tertiary Care Hospitals of Pakistan Using WHO Methodology: Results and Inferences

Background and objectives: The inappropriate use of antibiotics in hospitals can potentially lead to the development and spread of antibiotic resistance, increased mortality, and high economic burden. The objective of the study was to assess current patterns of antibiotic use in leading hospitals of Pakistan. Moreover, the information collected can support in policy-making and hospital interventions aiming to improve antibiotic prescription and use. Methodology and materials: A point prevalence survey was carried out with data abstracted principally from patient medical records from 14 tertiary care hospitals. Data were collected through the standardized online tool KOBO application for smart phones and laptops. For data analysis, SPSS Software was used. The association of risk factors with antimicrobial use was calculated using inferential statistics. Results: Among the surveyed patients, the prevalence of antibiotic use was 75% on average in the selected hospitals. The most common classes of antibiotics prescribed were third-generation cephalosporin (38.5%). Furthermore, 59% of the patients were prescribed one while 32% of the patients were prescribed two antibiotics. Whereas the most common indication for antibiotic use was surgical prophylaxis (33%). There is no antimicrobial guideline or policy for 61.9% of antimicrobials in the respected hospitals. Conclusions: It was observed in the survey that there is an urgent need to review the excessive use of empiric antimicrobials and surgical prophylaxis. Programs should be initiated to address this issue, which includes developing antibiotic guidelines and formularies especially for empiric use as well as implementing antimicrobial stewardship activities

Mortality Analysis of COVID-19 Confirmed Cases in Pakistan

Introduction: COVID-19, a novel disease, appeared in December 2019 in China and rapidly spread across the world. Till the second week of April 2020, high incidence (2.9/100,000) and cases fatality rates (1.7%) were observed in Pakistan. This study was conducted to determine the temporal and spatial distribution of the first 100 deaths attributed to COVID-19 in Pakistan and their associated demographic factors. Method: A record review of the first 100 deaths reported among RT-PCR confirmed COVID-19 cases was conducted. Demographic, epidemiological, and risk factors information was obtained associated comorbidities and clinical signs and symptoms were recorded and frequencies were determined. Results: A total of 100 mortalities with an overall case fatality rate of 1.67% (CFR) were analyzed. The median age of patients was 64.5 years (IQR: 54-70) with 75% (n=75) males. Among all deaths reported, 71 (71%) cases had one or more documented comorbidities at the time of diagnosis.  The most frequently reported co-morbidities were: hypertension (67%), followed by Diabetes Mellitus (45%) and Ischemic Heart Diseases (27%). The most frequent presenting symptoms were shortness of breath (87%) and fever (79%). The median duration of illness was eight days (IQR: 4-11 days), the median delay reaching hospital to seek health care was three days (IQR: 0-6 days) while the median duration of hospital stay was also three days (IQR: 1-7 days). Among all, 62% had no history of international travel. The most affected age group was 60-69 years while no death reported in the age group below 20 years

Sensitive detection of pre-existing BCR-ABL kinase domain mutations in CD34+ cells of newly diagnosed chronic-phase chronic myeloid leukemia patients is associated with imatinib resistance: implications in the post-imatinib era.

BACKGROUND: BCR-ABL kinase domain mutations are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CML) patients. Recent studies indicate the presence of pre-existing BCR-ABL mutations in a higher percentage of CML patients when CD34+ stem/progenitor cells are investigated using sensitive techniques, and these mutations are associated with imatinib resistance and disease progression. However, such studies were limited to smaller number of patients. METHODS: We investigated BCR-ABL kinase domain mutations in CD34+ cells from 100 chronic-phase CML patients by multiplex allele-specific PCR and sequencing at diagnosis. Mutations were re-investigated upon manifestation of imatinib resistance using allele-specific PCR and direct sequencing of BCR-ABL kinase domain. RESULTS: Pre-existing BCR-ABL mutations were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8-48), all patients with pre-existing BCR-ABL mutations exhibited imatinib resistance. Of the 68 patients without pre-existing BCR-ABL mutations, 24 developed imatinib resistance; allele-specific PCR and BCR-ABL kinase domain sequencing detected mutations in 22 of these patients. All 32 patients with pre-existing BCR-ABL mutations had the same mutations after manifestation of imatinib-resistance. In imatinib-resistant patients without pre-existing BCR-ABL mutations, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients except T315I and Y253F mutations responded to imatinib dose escalation. CONCLUSION: Pre-existing BCR-ABL mutations can be detected in a substantial number of chronic-phase CML patients by sensitive allele-specific PCR technique using CD34+ cells. These mutations are associated with imatinib resistance if affecting drug binding directly or indirectly. After the recent approval of nilotinib, dasatinib, bosutinib and ponatinib for treatment of chronic myeloid leukemia along with imatinib, all of which vary in their effectiveness against mutated BCR-ABL forms, detection of pre-existing BCR-ABL mutations can help in selection of appropriate first-line drug therapy. Thus, mutation testing using CD34+ cells may facilitate improved, patient-tailored treatment

Comparison of the frequencies of pre-existing BCR-ABL KD mutations and mutations detected after manifestation of imatinib resistance in CML patients.

<p>Comparison of the frequencies of pre-existing BCR-ABL KD mutations and mutations detected after manifestation of imatinib resistance in CML patients.</p

Clinical, cytogenetic, and molecular follow-up studies of CML patients with and without BCR-ABL PEMs who received imatinib treatment.

<p>N: number of patients, PEMs: pre-existing mutations; IM: imatinib; CHR: complete hematological response; PHR: partial hematological response; CCyR: complete cytogenetic response; MCyR: major cytogenetic response; minor CyR: minor cytogenetic response; MMR: major molecular response.</p

Patients’ characteristics.

<p>Ph+<b> = </b>Philadelphia chromosome positive.</p><p>PEM = pre existing mutations.</p

Sequences of ASO primers and corresponding annealing temperatures (bold nucleotides in the primers denote nucleotide changes corresponding to mutations).

*<p>Substitutions of amino acids; positions according to GenBank no. AAB60394for ABL type 1a.</p>**<p>Changes of nucleotide; positions according to GenBank no.M14752.</p>$<p>L (bp) = Primer length in base pairs.</p>#<p>A Tm =  Annealing temperature in degree Celsius.</p