Role of ERG11 and MDR1 genes in cycloheximide and multidrug resistance in Candida species.

Candida species are amongst the commensals of the mucosal surfaces of the human body which include the oral cavity, vagina, and intestinal mucosa. Fungal infections are on the rise worldwide. The overall burden of infections due to fungi is difficult to estimate because the majority of them remain undiagnosed. The present study aims to determine the burden of antifungal resistance in low socioeconomic country, Pakistan and the frequency of ERG11 and MDR1 genes involved. A total of 636 Candida isolates were obtained from various tertiary care institutions in Lahore in the form of culture on various culture plates. Sabouraud agar culture plates were used to culture the Candida spp. Antifungal resistance was determined against Fluconazole, Itraconazole, Ketoconazole, and Nystatin via disk diffusion technique. Most resistance was observed against Fluconazole followed by Itraconazole, Ketoconazole, and Nystatin. The Candida isolates recovering from CVP tip and tissue have a high resistance profile. Candida species resistant to at least two antifungals were chosen for further ERG11 and MDR1 detection through real-time PCR. Among 255 Candida isolates, 240 contained ERG11 gene while MDR1 gene is present in 149 Candida isolates. The isolates carrying both genes were tested by the broth microdilution technique for the susceptibility against cycloheximide, all of them were able to grow in cycloheximide. The genetic determinants of antifungal resistance such as ERG11 and MDR1 are as important in the multidrug resistance against a variety of compounds and antifungal drugs.

Antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure.

Tuberculosis stands as a prominent cause of mortality in developing countries. The treatment of tuberculosis involves a complex procedure requiring the administration of a panel of at least four antimicrobial drugs for the duration of six months. The occurrence of treatment failure after the completion of a standard treatment course presents a serious medical problem. The purpose of this study was to evaluate antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure. Additionally, it aimed to evaluate the effectiveness of second line drugs such as amikacin, linezolid, moxifloxacin, and the efflux pump inhibitor verapamil against M. tuberculosis isolates associated with treatment failure. We monitored 1200 tuberculosis patients who visited TB centres in Lahore and found that 64 of them were not cured after six months of treatment. Among the M. tuberculosis isolates recovered from the sputum of these 64 patients, 46 (71.9%) isolates were simultaneously resistant to rifampicin and isoniazid (MDR), and 30 (46.9%) isolates were resistant to pyrazinamide, Resistance to amikacin was detected in 17 (26,5%) isolates whereas resistance to moxifloxacin and linezolid was detected in 1 (1.5%) and 2 (3.1%) isolates respectively. Among MDR isolates, the additional resistance to pyrazinamide, amikacin, and linezolid was detected in 15(23.4%), 4(2.6%) and 1(1.56%) isolates respectively. One isolate simultaneously resistant to rifampicin, isoniazid, amikacin, pyrazinamide, and linezolid was also identified. In our investigations, the most frequently mutated amino acid in the treatment failure group was Serine 315 in katG. Three novel mutations were detected at codons 99, 149 and 154 in pncA which were associated with pyrazinamide resistance. The effect of verapamil on the minimum inhibitory concentration of isoniazid and rifampicin was observed in drug susceptible isolates but not in drug resistant isolates. Rifampicin and isoniazid enhanced the transcription of the efflux pump gene rv1258 in drug susceptible isolates collected from the treatment failure patients. Our findings emphasize a high prevalence of MDR isolates linked primarily to drug exposure. Moreover, the use of amikacin as a second line drug may not be the most suitable choice in such cases.

Long-Term Disease Control of Locally Invasive Epithelial-Myoepithelial Carcinoma of Parapharyngeal Salivary Glands With Definitive Radiotherapy.

Epithelial-myoepithelial carcinoma (EMC) is a rare clinical entity that affects glandular tissues, most commonly salivary glands. EMC of parapharyngeal space is exceedingly rare. Surgery is the mainstay of treatment with or without chemotherapy, radiotherapy, or both. Due to the rarity of the disease, select cases where surgery is not possible present a management conundrum. We present a case of locally advanced, stage IVa EMC of parapharyngeal space that was treated with upfront definitive radiotherapy. Radiotherapy treatment alone led to long-term disease control in both clinical and radiological follow-ups. The patient was followed for more than eight years posttreatment with no disease recurrence, enjoying the normal activities of life with no late toxicities including xerostomia. This case report highlights the role of radiotherapy in the management of such patients, and more studies are required in this context for surgical candidates with positive disease margins.

A Case Report on Rare Case of Pancreatic Metastasis from Primary Lung Adenocarcinoma: Treated Through a Non-surgical Approach.

Introduction: Most frequent sites of metastasis from lung cancer are the liver, brain and adrenal. Pancreas is an infrequent site of solitary metastasis from the lung primary with limited treatment options. There is insufficient data on the prognosis and optimal management of such cases. Case Description: We report a case of 44-year-old gentleman diagnosed with locally advanced lung adenocarcinoma Stage T4N3 who was treated radically with chemoradiation therapy, followed by a relapse of solitary pancreatic metastasis, which was treated with targeted therapy, erlotinib, due to the presence of epidermal growth factor receptor (EGFR) mutation. Practical Implications: This case reports an excellent radiological and symptomatic response in a patient who received erlotinib for advanced non-small-cell lung cancer (NSCLC). The use of EGFR-tyrosine kinase inhibitors has led to better prognosis and longer progression-free survival for patients with advanced NSCLC. However, the long-term survival of patients with metastatic NSCLC is limited.

Anticancer genes (NOXA, PAR-4, TRAIL) are de-regulated in breast cancer patients and can be targeted by using a ribosomal inactivating plant protein (riproximin).

BACKGROUND: Anticancer genes are an endogenous defense against transformed cells as they impose antineoplastic effects upon ectopic expression. Profiling the expression of these genes is fundamental for exploring their prognostic and therapeutic relevance in cancers. Natural compounds can upregulate anticancer genes in malignant cells and thus be useful for therapeutic purposes. In this study, we identified the expression levels of anticancer genes in breast cancer clinical isolates. In addition, the purified and sequenced plant protein (riproximin) was evaluated for its potential to induce anticancer genes in two breast cancer cell lines. METHODOLOGY: Expression profiles of three anticancer genes (NOXA, PAR-4, TRAIL) were identified by immunohistochemistry in 45 breast cancer clinical isolates. Breast cancer cells were exposed to riproximin and expression of the anticancer genes was determined by microarray, real-time PCR and western blot methodologies. Lastly, a bioinformatic approach was adopted to highlight the molecular/functional significance of the anticancer genes. RESULTS: NOXA expression was evenly de-regulated among the clinical isolates, while PAR-4 was significantly down-regulated in majority of the breast cancer tissues. In contrast, TRAIL expression was increased in most of the clinical samples. Expression levels of the anticancer genes followed a distinct trend in accordance with the disease severity. Riproximin showed a substantial potential of inducing expression of the anticancer genes in breast cancer cells at transcriptomic and protein levels. The bioinformatic approach revealed involvement of anticancer genes in multiple cellular functions and signaling cascades. CONCLUSION: Anticancer genes were de-regulated and showed discrete expression patterns in breast cancer patient samples. Riproximin effectively induced the expression of selected anticancer genes in breast cancer cells.

Molecular Characterization of Extensively Drug Resistant Salmonella Enterica Serovar Typhi Clinical Isolates from Lahore, Pakistan.

Background: The emergence of extensively drug-resistant (XDR) typhoid in Pakistan has endangered the treatment options available to manage this infection. Third generation cephalosporin were the empiric choice to treat typhoid fever in Pakistan, but acquisition of ESBLs have knocked them out of the arsenal. The current empiric choice is azithromycin which is vulnerable to resistance too. This study aimed to assess the burden of XDR typhoid and the frequency of resistance determinants in blood culture samples collected from different hospitals in Lahore, Pakistan. Methods: A total of 835 blood cultures were collected from different tertiary care hospitals in Lahore during January 2019 to December 2021. Among 835 blood cultures, 389 Salmonella Typhi were identified, and 150 were XDR S. Typhi (resistant to all recommended antibiotics). Antibiotics resistance genes of the first-line drugs (blaTEM-1, catA1, sul1, and dhfR7) and second line drugs (gyrB, gyrA, qnrS, ParC and ParE) were investigated among XDR S. Typhi. There were different CTX-M genes isolated using the specific primers, blaCTX-M-U, blaCTX-M-1, blaCTX-M-15, blaCTX-M-2, blaCTX-M-8 and blaCTX-M-9. Results: Antibiotic resistant genes of the first-line drugs were isolated with different frequency, blaTEM-1 (72.6%), catA1 (86.6%), sul1 (70%), and dhfR7 (56%). Antibiotics resistance genes of second-line drugs were isolated as: gyrB (60%), gyrA (49.3%), qnrS (32.6%), parC (44%) and parE (28%). Among CTX-M genes, blaCTX-M-U (63.3%) was the most frequent followed by blaCTX-M-15 (39.3%) and blaCTX-M-1 (26%). Conclusion: Our study concluded that XDR isolates circulating in Pakistan have acquired first-line and second-line antibiotic resistant genes quite successfully along with CTX-M genes (ESBLs) rendering them resistant to the third generation cephalosporins as well. Emergence of azithromycin resistance in XDR S. Typhi which is currently used as an empiric treatment option is worrisome and needs to be monitored carefully in endemic countries like Pakistan.

Expression profiling of anticancer genes in colorectal cancer patients and their in vitro induction by riproximin, a ribosomal inactivating plant protein.

BACKGROUND: Ectopic expression of anticancer genes (ACGs) imposes antineoplastic effects on transformed cells. Clinically, reduced expression of these genes has been linked with poor prognosis, metastasis and chemo/radiotherapy resistance in cancers. Identifying expression pattern of ACGs is crucial to establish their prognostic and therapeutic relevance in colorectal cancer (CRC). In addition to the clinical perspective, naturally occurring compounds can be explored in parallel for inducing ACGs to achieve cancer cell-specific death. METHODOLOGY: Expression profiles of three ACGs (NOXA, PAR-4, TRAIL) were identified via real-time PCR in CRC clinical isolates. Time lapse-based expression modifications in ACGs were studied in a CRC liver metastasis animal model using microarray methodology. Effects of a purified plant protein (riproximin) on selected ACGs were identified in three primary and metastatic CRC cell lines by real-time PCR. Lastly, importance of the ACGs in a cellular environment was highlighted via bioinformatic analysis. RESULTS: ACGs (except NOXA) were persistently downregulated in clinical isolates when comparing the overall mean expression values with normal mucosa levels. In vivo studies showed a prominent inhibition of NOXA and PAR-4 genes in implanted CRC cells during rat liver colonization. TRAIL showed deviation from this theme while showing marked induction during the early period of liver colonization (days 3 and 6 after CRC cell implantation). Riproximin exhibited substantial potential of inducing ACGs at transcriptome levels in selected CRC cell lines. Bioinformatic analysis showed that vital molecular/functional aspects of a cell are associated with the presence of ACGs. CONCLUSION: ACGs are downregulated in primary and metastatic phase of CRC. Riproximin effectively induces ACGs in CRC cells and can be exploited for clinical investigations over time.

Epidemiology of young breast cancer patients at Gujranwala: A single institution based study.

The objective of this study was to illustrate the statistical and medical characteristics of females younger than 40 years presenting with breast cancer at GINUM. A descriptive case series in which 235 patients who fulfilled the inclusion and exclusion criteria were included. The majority of the patients had advanced disease at presentation. Family history was positive in 59 (25.11%) patients. Patients having grade III and II disease were 142(60.42%) and 89 (37.87%) respectively. Thirteen (5.5%) patients had Right-sided breast cancers in our series. No significant association of null parity with breast cancer was found. Receptor status studies revealed only 2.55% increased oestrogen receptor/progesterone receptor (ER/PR) positive patients as compared to oestrogen receptor/progesterone receptor (ER/PR) negative patients. Our population shares slightly different features as compared to Western population because there were slightly increased right-sided tumours and majority of our patients were parous.

Blood-based gene expression profile of oxidative stress and antioxidant genes for identifying surrogate markers of liver tissue injury in chronic hepatitis C patients.

Oxidative stress is the process by which reactive molecules and free radicals are formed in cells. In this study, we report the blood-based gene expression profile of oxidative stress and antioxidant genes for identifying surrogate markers of liver tissue in chronic hepatitis C (CHC) patients by using real-time PCR. A total of 144 untreated patients diagnosed with CHC having genotype 3a and 20 healthy controls were selected for the present study. Liver biopsy staging and grading of CHC patients were performed using the METAVIR score. Total RNA was extracted from liver tissue and blood samples, followed by cDNA synthesis and real-time PCR. The relative expression of genes was calculated using the ΔΔCt method. The expression profile of 84 genes associated with oxidative stress and antioxidants was determined in liver tissue and blood samples. In liver tissue, 46 differentially expressed genes (upregulated, 27; downregulated, 19) were identified in CHC patients compared to normal samples. In blood, 61 genes (upregulated, 51; downregulated; 10) were significantly expressed in CHC patients. A comparison of gene expression in liver and whole blood showed that 20 genes were expressed in a similar manner in the liver and blood. The expression levels of commonly expressed liver and blood-based genes were also correlated with clinical factors in CHC patients. A receiver operating curve (ROC) analysis of oxidative stress genes (ALB, CAT, DHCR24, GPX7, PRDX5, and MBL2) showed that infections in patients with CHC can be distinguished from healthy controls. In conclusion, blood-based gene expression can reflect the behavior of oxidative stress genes in liver tissue, and this blood-based gene expression study in CHC patients explores new blood-based non-invasive biomarkers that represent liver damage.

Current Status of Direct Acting Antiviral Agents against Hepatitis C Virus Infection in Pakistan.

In Pakistan, the burden of the hepatitis C virus (HCV) infection is the second highest in the world with the development of chronic hepatitis. Interferon-based combination therapy with ribavirin was the only available treatment until a few years back, with severe side-effects and high failure rates against different genotypes of HCV. Interferon-free all-oral direct-acting antiviral agents (DAAs) approved by the FDA have revolutionized the HCV therapeutic landscape due to their efficiency in targeting different genotypes in different categories of patients, including treatment naïve, treatment failure and relapsing patients, as well as patients with compensated and decompensated cirrhosis. The availability and use of these DAAs is limited in the developing world. Sofosbuvir (SOF), a uridine nucleotide analogue and inhibitor of HCV encoded NS5B polymerase, is now a widely available and in-use DAA in Pakistan; whereas daclatasvir was recently added in the list. According to the documented results, there is hope that this disease can be effectively cured in Pakistan, although a few concerns still remain. The aim of this article is to review the effectiveness of DAAs and the current status of this treatment against HCV genotype 3 infection in Pakistan; various factors associated with SVR; its limitations as an effective treatment regime; and future implications.

Primary Mammary Small Cell Carcinoma.

Neuroendocrine breast cancer was defined in 2003 by WHO as a separate and unique breast cancer subtype. Primary small cell carcinoma of the breast, an exceptionally uncommon and aggressive tumor, is frequently characterised by early progression and worse outcome. Moreover, it is essential to distinguish between small cell carcinoma arising primarily in the breast and the metastatic disease to the breast. We had a patient of primary small cell carcinoma of breast. As her initial metastatic workup was negative for disease elsewhere, so she was started on neoadjuvant chemotherapy to which she responded well. Her modified radical mastectomy (MRM) was done followed by completion of chemotherapy up to 6 cycles and local radiotherapy of chest wall. However, the disease behaved aggressively afterwards as she developed recurrence twice at 9 and 19 months interval, respectively, for which she was considered for second and third line chemotherapy. An accurate management of the primary small cell carcinoma of the breast still lacks a consensus. Relevant studies were also reviewed to enhance knowledge and expertise in diagnosis, clinicopathologic features, management, and outcome of this tumor.

Association of TP53 codon 72 polymorphism in women suffering from endometriosis from Lahore, Pakistan.

OBJECTIVE: To investigate TP53 gene codon 72 polymorphism in women with endometriosis and compare it with healthy samples. METHODS: This case-control study was carried out at Jinnah Hospital, Services Hospital and Sheikh Zayed Hospital, Lahore, Pakistan, from 2014 to 2016, and comprised patients with endometriosis and healthy controls. SPSS 21 was used for statistical analysis. RESULTS: Of the 176 participants, 88(50%) were healthy controls and 88(50%) were endometriosis patients. The observed genotype frequencies for controls and patients were 14(15.9%) and 31(35.3%) for proline/proline, 46(52.3%) and 35(39.8%) for proline/arginine, and 28(31.8%) and 22(25%) for arginine/arginine, respectively. The association of different genotypes was not significant in patients with moderate-to-severe endometriosis (p=0.574). The presence of pro/pro genotype enhanced the chances/odds of getting the disease (p<0.05). However, the risk further increased with the advancement of age, particularly in the 27-46 age group (p<0.05). CONCLUSIONS: In Pakistani women the association of TP53 gene codon 72 arginine/proline polymorphism was present..

Distribution of IL28B and IL10 polymorphisms as genetic predictors of treatment response in Pakistani HCV genotype 3 patients.

There are over 10 million hepatitis C virus (HCV)-infected patients in Pakistan. For these patients, a combination of interferon with ribavirin is the most economical and easily available treatment. Single-nucleotide polymorphisms in interleukin genes have been reported to be associated with the pathogenesis and clearance of HCV, and sustained virologic response (SVR). An interleukin 28B (IL28B) gene polymorphism has been shown to modify treatment outcomes, but the effects of interleukin 10 (IL10) polymorphisms have not been previously assessed in the Pakistani population. The present study was conducted with 302 subjects categorized into two groups: 100 healthy volunteers (Group I) and 202 patients with chronic HCV (Group II). Patients within Group II were further divided into two subgroups according to therapeutic response: SVR (responders = 132) and NR (non-responders/relapsers = 70). IL28B (rs8099917, rs12979860) and IL10 (rs1800872, rs1800871, rs1800896) gene polymorphisms were studied in all subjects. A significant difference in the distribution of IL28B rs12979860C/T genotypes between the two groups (p<0.05) was observed, while of the three IL10 polymorphisms, a significant difference was only shown for rs1800896 A/G. Haplotype analysis (IL28B and IL10) showed a significant association of TTGTC and TTGTA when comparing the groups. There was a strong association of the favorable alleles rs8099917T and rs12979860C in the SVR group as compared with the NR group (p<0.05), and rs1800896 also showed an association with the SVR group as compared to the NR group (p<0.004). Haplotype analysis showed significant associations when comparing the SVR and NR subgroups, i.e. TCATC (p=0.009), TTGTA (p=0.005), TCATA (p<0.0005), TCACA (p=0.002), GTGCC (p=0.002) and TCGTC (p=0.005). IL28B (rs8099917 and rs12979860) and IL10 (rs1800896) polymorphisms alone, or in combination, are good predictors of therapeutic response in HCV-3a patients.

SOCS3 mRNA expression and polymorphisms as pretreatment predictor of response to HCV genotype 3a IFN-based treatment.

AIM: Suppressor of Cytokine Signaling 3 (SOCS3) gene belongs to SOCS family as one of the negative regulators of cytokine signaling and IFN response that function via the JAK-STAT pathway in antiviral response. SOCS3 expression and genetic polymorphism influences the pathogenesis and outcome of antiviral treatment in hepatitis C virus (HCV) infected patients. This study was designed for analysis of SOCS3 gene expression and polymorphism in Pakistani HCV patients. METHODS: This descriptive study was conducted on 250 diagnosed HCV genotype 3a infected subjects. The study population was divided into two major groups on the basis of therapeutic response i.e. sustained virological response (SVR) and non-responders/relapsers (NR). SOCS3 gene mRNA expression analysis was done by using Real time PCR technique, whereas ARMS PCR technique was used for analysis of SOCS3 gene polymorphisms i.e. 8464 A/C (rs12952093), -4874 A/G (rs4969170) and -1383 A/G, (rs4969168). RESULTS: Gene expression analysis of SOCS3 showed that there was statistically significant increase of 2.275-fold and 3.72-fold in relative gene expression for SVR and NR as compared to normal healthy samples (p < 0.001). The distribution of rs4969168, rs4969170 and rs12952093 genotype frequencies between SVR versus NR group were not statistically significant, only the allelic frequency of rs4969170 was statistically significant (p ≤ 0.0001) with therapeutic response. CONCLUSION: The gene expression analysis of SOCS3 showed a clear difference in mRNA expression of SOCS3 as a possible indicator of therapeutic response rather than polymorphism of SOCS3 gene in our studied population.

Association of FcγRIIa Polymorphism with Clinical Outcome of Dengue Infection: First Insight from Pakistan.

Dengue illness has been a major health concern in Pakistan during the last decade. Dengue infection can result in a spectrum of clinically distinct outcomes, ranging from asymptomatic infection to potentially life-threatening forms of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). A single-nucleotide polymorphism in FcγRIIa (rs1801274) results in altered affinity of the receptor for different subclasses of immunoglobulin G, and is a key player in determining the susceptibility to or protection from severe clinical infection of dengue. In this study, we analyzed the allelic and genotypic distribution of rs1801274 in subjects of Pakistani origin with subclinical dengue infection (n = 40), dengue fever (DF) (n = 40), and DHF/DSS (n = 30). We found that HH homozygotes and heterozygotes were significantly more likely to develop clinical dengue (odds ratio [OR] = 3.21, 95% confidence interval [CI] = 1.29-7.97, P = 0.009), either DF (OR = 2.82, 95% CI = 1.00-7.97, P = 0.045) or DHF/DSS (OR = 3.90, 95% CI = 1.13-13.07, P = 0.024) than the asymptomatic dengue infection. Results of allelic distribution comparisons and logistic regression analysis also supported the same relationship. The results suggest complex nature of interacting factors in determining the course for severe dengue illness.

Coexisting tertiary hyperparathyroidism and severe hypothyroidism in an end-stage renal disease patient on hemodialysis.

INTRODUCTION: The clinical syndrome of uremia is a bedside diagnosis which mimics a wide spectrum of other clinical disorders, most commonly thyroid disease. End-stage renal disease (ESRD), as a disorder, frequently alters thyroid hormone metabolism, and this is not significantly normalized by dialysis. Although the thyroid and parathyroid glands are considered independent organs, their anatomical juxtaposition in the neck, as well as sharing a common embryological origin, might play a role in some of the possible association between thyroid and parathyroid disease. It has been demonstrated in experimental animals that changes in the thyroid gland might lead to pathological changes in the parathyroids and vice versa. CASE PRESENTATION: An incidence of as high as 25% of hypothyroidism has been reported in patients with ESRD on dialysis. We report a patient with ESRD on maintenance hemodialysis (MHDx) who has had a combination of profound tertiary hyperparathyroidism (HPTH) and severe hypothyroidism. CONCLUSIONS: Literature search revealed an increased prevalence of hypothyroidism with secondary HPTH from renal failure. Although there is increased prevalence of hypothyroid state in secondary HPTH from renal failure, the association appears much weaker in primary HPTH and again no conclusive pathological relation has been identified between the two endocrine glands. A closer look and perhaps long-term prospective studies are required in the future to determine this association.

Colors as catalysts in enzymatic reactions.

We studied the effects of visible range irradiation (in vitro) on the enzyme solutions (glucose oxidase, cholesterol oxidase + cholesterol esterase and lipase) in order to infer the changes produced in the human body after chromotherapy. The glucose oxidase showed enhanced activity to the color purple (464 nm), while the activity of the other enzymes, cholesterol esterase + cholesterol oxidase and lipase, increased when exposed to dark violet (400 nm). Purple is being used in conventional chromotherapy for diabetes, as supported by the experimental observation in which purple enhanced the activity of enzymes responsible for the oxidation of glucose. Specific wavelengths regulate living processes by acting as catalysts in enzyme activity, while some wavelengths may reduce enzyme activity. The irradiation of specific wavelengths effect enzymatic processes, which as a consequence, accelerated biochemical reactions. This particular frequency when provided to the enzymes (in vitro) lead to changes which may well be occurring in vivo.