A rare case of cerebral phaeohyphomycosis caused by Fonsecaea species in a renal transplant patient.

Cerebral phaeohyphomycosis (CP) is a serious form of phaeohyphomycosis. We report a case of CP caused by Fonsecaea species in a 66-year-old immunocompromised renal transplant recipient female. Craniotomy was performed on an irregularly enhancing right cerebellar hemisphere lesion and abscess and tissue samples collected for microbiological and histological evaluation, showing fungal elements and Fonsecaea species was isolated. Antifungal treatment with voriconazole & liposomal amphotericin B was initiated with a temporary improvement in the patient's condition. Deep vein thrombosis jeopardized patient's prognosis. Despite aggressive surgical and medical intervention, our patient succumbed to the disease. Historically, CP has been linked with fatality rates as high as 65 %, despite surgical intervention and systemic antifungal medication.

Updates on Epstein-Barr Virus (EBV)-Associated Nasopharyngeal Carcinoma: Emphasis on the Latent Gene Products of EBV.

Nasopharyngeal carcinoma (NPC) is an uncommon type of malignancy/cancer worldwide. However, NPC is an endemic disease in southeast Asia and southern China and the reasons behind the underlying for such changes are unclear. Even though the Epstein-Barr infection (EBV) has been suggested as an important reason for undistinguishable NPC, the EBV itself is not adequate to source this type of cancer. The risk factors, for example, genetic susceptibility, and environmental factors might be associated with EBV to undertake a part in the NPC carcinogenesis. Normal healthy people have a memory B cell pool where the EBV persists, and any disturbance of this connection leads to virus-associated B cell malignancies. Less is known about the relationship between EBV and epithelial cell tumors, especially the EBV-associated nasopharyngeal carcinoma (EBVaNPC) and EBV-associated gastric carcinoma (EBVaGC). Currently, it is believed that premalignant genetic changes in epithelial cells contribute to the aberrant establishment of viral latency in these tumors. The early and late phases of NPC patients' survival rates vary significantly. The presence of EBV in all tumor cells presents prospects for the development of innovative therapeutic and diagnostic techniques, despite the fact that the virus's exact involvement in the carcinogenic process is presently not very well known. EBV research continues to shed light on the carcinogenic process, which is important for a more comprehensive knowledge of tumor etiology and the development of targeted cancer therapeutics. In order to screen for NPC, EBV-related biomarkers have been widely used in a few high-incidence locations because of their close associations with the risks of NPC. The current review highlights the scientific importance of EBV and its possible association with NPC.

CLINICAL VALIDATION OF ANKRD36 MUTATIONS AS A NOVEL BIOMARKER FOR MONITORING EARLY PROGRESSION AND TIMELY CLINICAL INTERVENTIONS IN BLAST CRISIS CML.

Background: Chronic Myeloid Leukemia (CML) is initiated in the bone marrow due to the chromosomal translocation t(9;22), resulting in the fusion oncogene BCR-ABL. Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL have transformed fatal CML into an almost curable disease. However, TKIs lose efficacy during disease progression, and the mechanism of CML progression remains to be fully understood. Additionally, common molecular biomarkers for CML progression are lacking. Our studies previously detected ANKRD36 (c.1183_1184 delGC and c.1187_1188 dupTT) associated exclusively with advanced phase CML. However, clinical validation of this finding was pending. Therefore, this study aimed to clinically validate mutated ANKRD36 as a novel biomarker of CML progression. Materials and Methods: The study enrolled 124 patients in all phases of CML, recruited from Mayo Hospital and Hameed Latif Hospital in Lahore, Punjab, between January 2019 and August 2021. All response criteria were adopted from the European LeukemiaNet guideline 2020. Informed consent was obtained from all study subjects. The study was approved by scientific and ethical review committees of all participating centers.Sanger sequencing was employed to detect ANKRD36 mutations in CML patients in accelerated phase (AP) (n=11) and blast crisis (BC) (n=10), with chronic-phase CML (CP-CML) patients as controls (n=103). Samples were processed using Big Dye Terminator Cycle Sequencing Ready Reaction kits and sequenced using ABI Prism 3730 Genetic Analyzer, and sequencing using forward and reverse primers for ANKRD36. Results: During our study, 17% of CML patients progressed to advanced phases AP-CML n=11 (8.9%) and BC-CML n=10 (8.1%). The chronic- and advanced-phase patients showed significant difference with respect to male-to-female ratio, hemoglobin level, WBC count, and platelet count. Sanger sequencing detected ANKRD36 mutations c. 1183 1184 delGC and c. 1187 1185 dupTT exclusively in all AP- and BC-CML patients but in none of the CP-CML patients. Nevertheless, mutations status was not associated with male-to-female ratio, hemoglobin level, WBC count, and platelet count, which makes ANKRD32 as an independent predictor of early and terminal disease progression in CML. Conclusions: The study confirms ANKRD36 as a novel genomic biomarker for early and late CML progression. Further prospective studies should be carried out in this regard. ANKRD36, although fully uncharacterized in humans, shows the highest expression in bone marrow, particularly myeloid cells. Functional integrated genomic studies are recommended to further explore the role of ANKRD36 in the biology and pathogenesis of CML.

Integrated Genomic Analysis Identifies ANKRD36 Gene as a Novel and Common Biomarker of Disease Progression in Chronic Myeloid Leukemia.

Background: Chronic myeloid leukemia (CML) is initiated in bone marrow due to chromosomal translocation t(9;22) leading to fusion oncogene BCR-ABL. Targeting BCR-ABL by tyrosine kinase inhibitors (TKIs) has changed fatal CML into an almost curable disease. Despite that, TKIs lose their effectiveness due to disease progression. Unfortunately, the mechanism of CML progression is poorly understood and common biomarkers for CML progression are unavailable. This study was conducted to find novel biomarkers of CML progression by employing whole-exome sequencing (WES). Materials and Methods: WES of accelerated phase (AP) and blast crisis (BC) CML patients was carried out, with chronic-phase CML (CP-CML) patients as control. After DNA library preparation and exome enrichment, clustering and sequencing were carried out using Illumina platforms. Statistical analysis was carried out using SAS/STAT software version 9.4, and R package was employed to find mutations shared exclusively by all AP-/BC-CML patients. Confirmation of mutations was carried out using Sanger sequencing and protein structure modeling using I-TASSER followed by mutant generation and visualization using PyMOL. Results: Three novel genes (ANKRD36, ANKRD36B and PRSS3) were mutated exclusively in all AP-/BC-CML patients. Only ANKRD36 gene mutations (c.1183_1184 delGC and c.1187_1185 dupTT) were confirmed by Sanger sequencing. Protein modeling studies showed that mutations induce structural changes in ANKRD36 protein. Conclusions: Our studies show that ANKRD36 is a potential common biomarker and drug target of early CML progression. ANKRD36 is yet uncharacterized in humans. It has the highest expression in bone marrow, specifically myeloid cells. We recommend carrying out further studies to explore the role of ANKRD36 in the biology and progression of CML.

RUNX1 mutation and elevated FLT3 gene expression cooperates to induce inferior prognosis in cytogenetically normal acute myeloid leukemia patients.

BACKGROUND: Acute myeloid leukemia (AML) is a bone marrow malignancy having multiple molecular pathways driving its progress. In recent years, the main causes of AML considered all over the world are genetic variations in cancerous cells. The RUNX1 and FLT3 genes are necessary for the normal hematopoiesis and differentiation process of hematopoietic stem cells into mature blood cells, therefore they are the most common targets for point mutations resulting in AML. METHODS: We screened 32 CN-AML patients for FLT3-ITD (by Allele-specific PCR) and RUNX1 mutations (by Sanger sequencing). The FLT3 mRNA expression was assessed in all AML patients and its subgroups. RESULTS: Eight patients (25%) carried RUNX1 mutation (K83E) while three patients (9.37%) were found to have internal tandem duplications in FLT3 gene. The RUNX1 mutation data were correlated with clinical parameters and FLT3 gene expression profile. The RUNX1 mutations were observed to be significantly prevalent in older males. Moreover, RUNX1 and FLT3-mutated patients had lower complete remission rate, event-free survival rate, and lower overall survival rate than patients with wild-type RUNX1 and FLT3 gene. The RUNX1 and FLT3 mutant patients with up-regulated FLT3 gene expression showed even worse prognosis. Bradford Assay showed that protein concentration was down-regulated in RUNX1 and FLT3 mutants in comparison to RUNX1 and FLT3 wild-type groups. CONCLUSION: This study constitutes the first report from Pakistan reporting significant molecular mutation analysis of RUNX1 and FLT3 genes including FLT3 expression evaluation with follow-up. This provides an insight that aforementioned mutations are markers of poor prognosis but the study with a large AML cohort will be useful to further investigate their role in disease biology of AML.

Phenotypic and molecular characterization of virulence factors of extra-intestinal pathogenic Escherichia coli isolated from patients of Peshawar, Pakistan.

Extra-Intestinal Escherichia coli (ExPEC) are important cause of Urinary Tract Infections (UTIs) and systemic infections. The purpose of this study was to investigate numerous ExPEC bacterial isolates for phenotypic virulence characteristics including hemolytic activity and resistance pattern and to observe their association with genetic traits via Polymerase Chain Reaction (PCR). A total of 367 ExPEC isolates were collected from patients admitted in Khyber Teaching Hospital (KTH) Peshawar, Pakistan. Standard techniques were used for identification of isolates, determination of hemolytic potential and antimicrobial susceptibility testing. PCR was used for screening of virulence genes using specific primers. A total of 367 ExPEC isolates were characterized, among which 62.7, 24.3, 7.1 and 6% were isolated from urine, pus, sputum and wound specimens, respectively. Majority of the isolates (82.8%) were hemolysin positive. Multi drug resistance pattern was shown by 41% of the isolates and harbored at least one virulence gene (71.7%), of which sat was the most prevalent (64.3%). The highest resistance was found to cefotaxime (99.2%), ampicillin (97.5%) and aztreonem (89.6%). 15 different virulence genes combinations were observed in the current study. A total of 16 virotypes (15 of positive virulence genes and one of no virulence gene) were observed in the current study. The current investigation showed a high prevalence of sat and hlyA genes among ExPEC isolate, suggesting a role of these genes in the pathogenesis of ExPEC.

Whole exome sequencing identifies a novel FANCD2 gene splice site mutation associated with disease progression in chronic myeloid leukemia: Implication in targeted therapy of advanced phase CML.

Tyrosine Kinase Inhibitors (TKIs) have significantly improved the clinical outcome of BCR-ABL+ Chronic Phase-Chronic Myeloid Leukemia (CP-CML). Nonetheless, approximately one-third of the CP-CML patient's progress to advanced phases of CML (accelerated and blast phase). Impaired DNA repair including mutations in Fanconi anemia (FA) pathway genes are responsible for progression of many cancers. Nevertheless, FA-pathways genes have never been reported in myeloid cancers. Hence, this study was aimed to discover DNA repair genes associated with CML progression. AP-CML patients were subjected to whole exome sequencing along with appropriate controls. A novel splice site FANCD2 mutation was detected. FANCD2 is a well-known FA-pathway gene with established role in DNA repair. This is first report of FA-pathway DNA repair genes in myeloid cancers that can serve as a novel marker of CML progression to clinically intervene CML progression. Further studies are needed to establish the functional role of FANCD2 in CML progression that can provide novel insights into CML pathogenesis. This study also indicates that a combination TKIs and Poly (ADP-ribose) polymerase (PARP) inhibitors like Olaparib (FDA approved anti-cancer drug for FA-pathway gene mutations) could improve the clinical outcome CML patients in accelerated and blast-crisis phases of the disease.

Discovery and Protein Modeling Studies of Novel Compound Mutations Causing Resistance to Multiple Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia.

OBJECTIVE: BCR-ABL fusion oncogene is the hallmark of chronic myeloid leukemia (CML), causing genomic instability which leads to accumulation of mutations in BCR-ABL as well as other genes. BCR-ABL mutations are the cause of tyrosine kinase inhibitors (TKIs) resistance in CML. Recently, compound BCR-ABL mutations have been reported to resist all FDA approved TKIs. Therefore, finding novel compound BCR-ABL mutations can help and clinically manage CML. Therefore, our objective was to find out novel drug-resistant compound BCR-ABL mutations in CML and carry out their protein modelling studies. METHODOLOGY: Peripheral blood samples were collected from ten imatinib resistant CML patients receiving nilotinib treatment. BCR-ABL transcript mutations were investigated by employing capillary sequencing. Patient follow-up was carried out using European LeukemiaNet guidelines. Protein modeling  studies were carried out for new compound mutations using PyMol to see the effects of mutations at structural level. RESULTS: A novel compound mutation (K245N mutation along with G250W mutation) and previously known T351I utation was detected in two of the nilotinib resistance CML patients respectively while in the rest of 8 nilotinib responders, no resistant mutations were detected. Protein modelling studies indicated changes in BCR-ABL mutant protein which may have negatively impacted its binding with nilotinib leading to drug resistance. CONCLUSION: We report a novel nilotinib resistant BCR-ABL compound mutation (K245N along with G250W mutation) which impacts structural modification in BCR-ABL mutant protein leading to drug resistance. As compound mutations pose a new threat by causing resistance to all FDA approved tyrosine kinase inhibitors in BCR-ABL+ leukemias, our study opens a new direction for in vitro characterization of novel BCR-ABL compound mutations and their resistant to second  generation and third generation TKIs.

Long term outcome of chronic myeloid leukemia patients treated with imatinib: Report from a developing country.

The outcome of chronic myeloid leukemia has been greatly improved by the use of Imatinib (IM), a selective BCR/ABL kinase inhibitor. The aim of present study was to report long term follow-up & outcome of IM-treated CML patients along with their clinicopathological features, risk group stratification, adverse events and to compare it with CML patients reported from western countries. The mean follow-up of 123 CML patients was 5.5 years in present study, who were treated with frontline IM 400mg daily in a tertiary care hospital in Pakistan. Risk stratification scores, response to treatment (ELN guidelines) and survival outcomes estimated by Kaplan-Meier analysis. Mean age: 35 years (9-67 years) and M: F: 1.5:1, mean follow up time: 5.5 years (1-15 years). Overall survival (OS): at 5.5, 8, 10 and 12 years were 93%, 88%, 81% and 73%, respectively. Progressions free survival (PFS) was 95%, 83%, 83% and 78% at 5.5, 8, 10 and 12 years, respectively. OS estimate by Sokal score was significant (P-value: 0.0019). Additional chromosomal aberrations: 1.6%. Eighteen (14.6%) patients progressed to AP/BC. Adverse events were moderate and tolerable. We present findings from a long term follow up of CML patients treated with IM in a developing country. CML mean age at onset was considerably lower than the western populations. Furthermore, 5.5 years OS are comparable to western CML population. IM in our patients as frontline choice proved to be very effective. IM was found to be well tolerated, safe with manageable moderate side effects.

Thymic Stromal Lymphopoietin (TSLP) gene variant rs1837253 is significantly associated with Asthma prevalence in Pakistani Pashtun women.

Asthma is a chronic inflammatory disease of the airways characterized by airway hyperresponsiveness and remodeling. Thymic stromal lymphopoietin (TSLP), a member of the interleukin-2 family of cytokines, is produced by activated lung and intestinal epithelial cells, mast, and other immune cells. Population-based studies identified associations between SNPs in the TSLP promoter region and asthma pathogenesis. In this study, we analyzed the genotypic association of TSLP rs1837253 with asthma predisposition in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. Target DNA sequence of 250 asthmatics and an equal number of healthy individuals was PCR amplified, and allelic determination was performed by Sanger sequencing. Statistical analysis was conducted using chi-square tests and logistic regression analysis. Homozygous T/T genotype was frequent in the asthmatic subjects with a statistically significant level (P<0.05). Genetic models, including recessive, dominant, co-dominant, over-dominant, and additive were tested while adjusting allele frequencies with covariates (gender and age). Combined C/T and T/T individuals had higher odds ratios of 3.00, 1.91, and 1.73 in co-dominant, dominant, and additive models with statistically significant P-values of 0.029*, 0.022*, and 0.02*, respectively. T allele of rs1837253 was associated with increased susceptibility to asthma among Pashtuns, particularly in females, and we corroborate rs1837253 as a SNP of interest with a potential functional role.

Designing of potent inhibitors for metallo-beta-lactamases producing Escherichia coli in molecular specification hereto.

The rapid spread of Metallo-ß-Lactamases producing Gram-negative bacteria in Pakistan is alarming and novel inhibitors with multi inhibition potential are required. In the current study, an effort was made to identify the resistance genes of MBLs producing E. coli and single inhibitor was designed having the potential to block all resistant proteins. Results showed that out of 573 clinical isolates, 14.1% MBLs producers have NDM-1 (27.2%) and VIM (13.6%) gene. The isolates were resistant to MEM, AMP, AMC, FEP, CTX, LEV and ATM, while effective antibiotics were TGC, CO, FOS and AK with MICs ranging from 4 to >32µg/ml. RECAP synthesis was used for de-novo discovery of 1000 inhibitors and protein crystal structures were retrieved from PDB. Active sites were identified in each protein and to improve ADMET properties, Lipinski's rules of five was applied. Placement of the ligand was done by London dG algorithm implemented in MOE. For final refinement, GBVI/WSA dG algorithm was used. Based on docking score, visual inspection of ligands interaction with key residues, binding affinity and binding energy of ligands with proteins, 10 compounds were selected for MBLs proteins which presented best ADMET properties, binding energy and affinity than the reported ones.

In-vitro susceptibility of methicillin-resistant Stayphylococcus aureus to honey.

Wound infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is becoming much complicated and costly to treat as antimicrobial resistance is quite common. Twenty five MRSA strains isolated from infected wounds and three ATCC reference strains were evaluated for their susceptibility to locally produced black seed (Nigella sativa), beri (ZiziphusJujuba) and shain honey (Plectranthus rugosus wall) by agar incorporation assay. Medically graded manuka honey (UMF 21+) was included as control. Locally produced black seed honey inhibited all clinical isolates at mean MIC of 5.5% (v/v), whereas manuka honey at mean MIC of 4.4% (v/v). The other two locally produced honey; beri and shain honey inhibited these isolates at 6.4% and 10.4% (v/v) respectively. The result of the study has demonstrated that indigenous black seed honey has comparable antibacterial activity to manuka honey and thus offers a good new addition to the existing honey resource for the treatment of wound infections.

Comparison of Molecular and Phenotypic Methods for the Detection and Characterization of Carbapenem Resistant Enterobacteriaceae.

In recent years, there has been a rapid dissemination of carbapenem resistant Enterobacteriaceae (CRE). This study aimed to compare phenotypic and molecular methods for detection and characterization of CRE isolates at a large tertiary care hospital in Saudi Arabia. This study was carried out between January 2011 and November 2013 at the King Khalid University Hospital (KKUH) in Saudi Arabia. Determination of presence of extended-spectrum beta-lactamases (ESBL) and carbapenem resistance was in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Phenotypic classification was done by the MASTDISCS(TM) ID inhibitor combination disk method. Genotypic characterization of ESBL and carbapenemase genes was performed by the Check-MDR CT102. Diversilab rep-PCR was used for the determination of clonal relationship. Of the 883 ESBL-positive Enterobacteriaceae detected during the study period, 14 (1.6%) isolates were carbapenem resistant. Both the molecular genotypic characterization and phenotypic testing were in agreement in the detection of all 8 metalo-beta-lactamases (MBL) producing isolates. Of these 8 MBL-producers, 5 were positive for blaNDM gene and 3 were positive for blaVIM gene. Molecular method identified additional blaOXA gene isolates while MASTDISCS(TM) ID detected one AmpC producer isolate. Both methods agreed in identifying 2 carbapenem resistant isolates which were negative for carbapenemase genes. Diversilab rep-PCR analysis of the 9 Klebsiella pneumoniae isolates revealed polyclonal distribution into eight clusters. MASTDISCS(TM) ID is a reliable simple cheap phenotypic method for detection of majority of carbapenemase genes with the exception of the blaOXA gene. We recommend to use such method in the clinical laboratory.

Low Frequency of ETV6-RUNX1 (t 12; 21) in Saudi Arabian Pediatric Acute Lymphoblastic Leukemia Patients: Association with Clinical Parameters and Early Remission.

BACKGROUND: Pediatric acute lymphoblastic leukemia (pALL) patients at King Abdulaziz Medical City represent a pure Saudi Arabian population. ETV6-RUNX1 positive pALL patients have good prognosis as compared to ETV6-RUNX1 negative counterparts. Therefore, frequencies of these two patient groups have a huge consideration in treatment strategies of pALL in a given population. Different geographical locations have been reported to have different frequencies of ETV6-RUNX1 ranging from 10% in Southeast Asia to 30% in Australia. AIM: Therefore, the objective of this study was to establish the ETV6-RUNX1 status of Saudi Arabian pALL patients and its association with clinical parameters and early remission. MATERIALS AND METHODS: Clinical parameters and ETV6-RUNX1 status (using FISH technique) of pALL patients attending the Pediatric Oncology Clinic, King Abdulaziz Medical City, Riyadh from 2006 to 2011 were studied. Comparisons between ETV6-RUNX1 positive and negative groups were accomplished using chi-square test or Fisher's exact test. All statistical analyses were performed using SAS version 9.2 (SAS Institute, Inc., Cary, NC). RESULTS: Out of 54 patients, 33 were male and 21 were females (ratio 1.57:1). B- and T-cell lineages were found in 47 (87%) and 7 (13%) patients respectively. Only 5 (9.3%) patients were ETV6-RUNX1 positive while 49(80.7%) were ETV6-RUNX1 negative. All ETV6-RUNX1 patients (100%) were of B-cell lineage and 80% (4/5) were in the 3-7 year age group. None of the ETV6-RUNX11 patients had ≥ 5% blasts (no remission) at day 14 as compared with 9% in the ETV6-RUNX1 negative group (Figure 1). CONCLUSIONS: Frequency of ETV6-RUNX1 positive patients (less than 10%) in our pALL patients is much lower than reported for most European countries, North America, Australia and Japan while it is in accordance with ETV6-RUNX1 frequencies from Egypt (11.6%), Pakistan (10%), Spain (2%) and India (5-7%). This shows ethnic differences in genetics of pALL as well as higher frequencies of ETV6-RUNX1 positive pALL mostly in more industrialized countries, probably due to some industrial pollutants or westernized lifestyle.

High frequency and poor prognosis of late childhood BCR-ABL-positive and MLL-AF4-positive ALL define the need for advanced molecular diagnostics and improved therapeutic strategies in pediatric B-ALL in Pakistan.

BACKGROUND: Fusion oncogenes (FOs) resulting from chromosomal abnormalities have an important role in leukemogenesis in pediatric B cell acute lymphoblastic leukemia (ALL). The most common FOs are BCR-ABL, MLL-AF4, ETV6-RUNX1, and TCF3-PBX1, all of which have important prognostic and drug selection implications. Moreover, frequencies of FOs have ethnic variations. We studied Pakistani frequencies of FOs, clinical pattern, and outcome in pediatric B-ALL. METHODS: FOs were studied in 188 patients at diagnosis using reverse transcriptase-polymerase chain reaction (RT-PCR) and interphase fluorescent in situ hybridization (FISH). Data were analyzed using SPSS version 17 (SPSS Inc., Chicago, IL, USA). RESULTS: FOs were detected in 87.2 % of patients. Mean overall survival was 70.9 weeks, 3-year survival was 31.9 %, and 3-year relapse-free survival was 18.1 %. Four patients died of drug toxicities. ETV6-RUNX1 (19.14 %) had better survival (110.9 weeks; p = 0.03); TCF3-PBX1 (2.1 %) was associated with inferior outcome and higher central nervous system (CNS) relapse risk; MLL-AF4 (18.1 %) was more common in the 8- to 15-year age group (24/34; p = 0.001) and was associated with organomegaly, low platelet count, and poor survival; and BCR-ABL (47.9 %) was associated with older age (7-15 years, 52/90), lower remission rates, shorter survival (43.73 ± 4.24 weeks) and higher white blood cell count. Overall, MLL-AF4 and BCR-ABL were detected in 66 % of B-ALL, presented in later childhood, and were associated with poor prognosis and inferior survival. CONCLUSIONS: This study reports the highest ethnic frequency of BCR-ABL FO in pediatric ALL, and is consistent with previous reports from our region. Poor prognosis BCR-ABL and MLL-AF4 was detected in two-thirds of pediatric B-ALL and is likely to be the reason for the already reported poor survival of childhood ALL in South-East Asia. Furthermore, MLL-AF4, usually most common in infants, presented in later childhood in most of the ALL patients, which was one of the unique findings in our study. The results presented here highlight the need for mandatory inclusion of molecular testing for pediatric ALL patients in clinical decision making, together with the incorporation of tyrosine kinase inhibitors, as well as hematopoietic stem cell transplantation facilities, to improve treatment outcome for patients in developing countries.

ESBL-producing Escherichia coli and Klebsiella pneumoniae at a tertiary care hospital in Saudi Arabia.

INTRODUCTION: The increasing frequency and antibiotic resistance among extended-spectrum ß-lactamases (ESBLs)-producing bacteria are posing a serious threat. This study sought to investigate the frequency and antibiotic susceptibility of ESBL-producing E. coli and K. pneumoniae at a tertiary care hospital. METHODOLOGY: Data were collected from samples sent to the microbiology laboratory between 2006 and 2010 at King Khalid University Hospital, Riyadh. ESBLs were confirmed using Etest strips of cefotaxime/cefotaxime + clavulanic acid, ceftazidime/ceftazidime + clavulanic acid, and cefepime/cefepime + clavulanate. RESULTS: Out of 17,105 samples, 1,076 (6.3%) ESBL-producing isolates of E. coli (808) and K. pneumoniae (268) were confirmed. Among these, 680 (63.2%) isolates were found in urine samples, followed by 287 (26.7%) in superficial swabs, deep wounds swabs, tissues and sterile body fluids, 71 (6.6%) in respiratory, and 38 (3.5%) in blood samples. The overall frequency rates of ESBL E. coli and K. pneumoniae were 6.6% and 5.5%, respectively. The frequency of ESBL-producing E. coli and K. pneumoniae increased significantly during the study period. E. coli resistance against cotrimoxazole was 71.1%, followed by ciprofloxacin (68.2%) and gentamicin (47%). Similarly, 62.7% of K. pneumoniae isolates were resistant to gentamicin, 59.5% to cotrimoxazole, and 49.8% to ciprofloxacin. There was no statistically significant change in antimicrobial resistance over the study period. CONCLUSIONS: Although the frequency rates of ESBL-producing E. coli and K. pneumoniae increased, no change in the anti-microbial susceptibility was observed over the study period.

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.

Prognostically significant fusion oncogenes in Pakistani patients with adult acute lymphoblastic leukemia and their association with disease biology and outcome.

BACKGROUND AND OBJECTIVES: Chromosomal abnormalities play an important role in genesis of acute lymphoblastic leukemia (ALL) and have prognostic implications. Five major risk stratifying fusion genes in ALL are BCR-ABL, MLL-AF4, ETV6-RUNX11, E2A-PBX1 and SIL-TAL1. This work aimed to detect common chromosomal translocations and associated fusion oncogenes in adult ALL patients and study their relationship with clinical features and treatment outcome. METHODS: We studied fusion oncogenes in 104 adult ALL patients using RT-PCR and interphase-FISH at diagnosis and their association with clinical characteristics and treatment outcome. RESULTS: Five most common fusion genes i.e. BCR-ABL (t 9; 22), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (Del 1p32) were found in 82/104 (79%) patients. TCF3-PBX1 fusion gene was associated with lymphadenopathy, SIL-TAL positive patients had frequent organomegaly and usually presented with a platelets count of less than 50 x10(9)/l. Survival of patients with fusion gene ETV6-RUNX1 was better when compared to patients harboring other genes. MLL-AF4 and BCR-ABL positivity characterized a subset of adult ALL patients with aggressive clinical behaviour and a poor outcome. CONCLUSIONS: This is the first study from Pakistan which investigated the frequency of 5 fusion oncogenes in adult ALL patients, and their association with clinical features, treatment response and outcome. Frequencies of some of the oncogenes were different from those reported elsewhere and they appear to be associated with distinct clinical characteristics and treatment outcome. This information will help in the prognostic stratification and risk adapted management of adult ALL patients.

Antimicrobial susceptibility patterns of multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii against carbapenems, colistin, and tigecycline.

OBJECTIVE: To examine susceptibility of Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii ) against carbapenems along with colistin and tigecycline as alternative therapeutic options. METHODS: A total of 117 strains of multidrug-resistant (MDR) non-fermenting Gram negative bacteria isolated from non-duplicate samples were collected consecutively. We included one sample from each patient (84 isolates of A. baumannii and 33 isolates of P. aeruginosa isolated from patients seen at King Khalid University Hospital, Riyadh, Saudi Arabia, from June to December 2010). Isolates were identified by the MicroScan WalkAway 96 Plus system. The minimum inhibitory concentrations (MICs) were determined by E-test following the Clinical and Laboratory Standards Institute breakpoint recommendations. RESULTS: Most A. baumannii strains were resistant to imipenem (90.5%), meropenem (90.5%), and doripenem (77.4%). Whereas, a higher percentage of P. aeruginosa was resistant to imipenem (90.9%), and meropenem (81.8%), only 39.4% were resistant to doripenem. Colistin had excellent activity against both A. baumannii (100%) and P. aeruginosa (93.9%), while 89.3% of A. baumannii strains were susceptible to tigecycline. CONCLUSION: Among the carbapenems, doripenem was found to be the most potent antimicrobial agent against P. aeruginosa, whereas colistin proved to be an effective alternative antimicrobial agent for treatment of A. baumannii or P. aeruginosa. Tigecycline remains the best therapeutic option for MDR A. baumannii.

Five most common prognostically important fusion oncogenes are detected in the majority of Pakistani pediatric acute lymphoblastic leukemia patients and are strongly associated with disease biology and treatment outcome.

BACKGROUND AND OBJECTIVES: Acute lymphoblastic leukemia (ALL) is a complex genetic disease involving many fusion oncogenes (FO) having prognostic significance. The frequency of various FO can vary in different ethnic groups, with important implications for prognosis, drug selection and treatment outcome. METHOD: We studied fusion oncogenes in 101 pediatric ALL patients using interphase FISH and RT-PCR, and their associations with clinical features and treatment outcome. RESULTS: Five most common fusion genes i.e. BCR-ABL t (22; 9), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (del 1p32) were found in 89/101 (88.1%) patients. Frequency of BCR-ABL was 44.5% (45/101). BCR-ABL positive patients had a significantly lower survival (43.7±4.24 weeks) and higher white cell count as compared to others, except patients with MLL-AF4. The highest relapse-free survival was documented with ETV6-RUNX1 (14.2 months) followed closely by those cases in which no gene was detected (13.100). RFS with BCR-ABL, MLL-AF4, TCF3-PBX1 and SIL-TAL1 was less than 10 months (8.0, 3.6, 5.5 and 8.1 months, respectively). CONCLUSIONS: This is the first study from Pakistan correlating molecular markers with disease biology and treatment outcome in pediatric ALL. It revealed the highest reported frequency of BCR-ABL FO in pediatric ALL, associated with poor overall survival. Our data indicate an immediate need for incorporation of tyrosine kinase inhibitors in the treatment of BCR-ABL+ pediatric ALL in this population and the development of facilities for stem cell transplantation.