Evolution of COVID-19 infection in Punjab; trends during five waves of infection in the province of Punjab.

BACKGROUND: Pakistan witnessed five waves of COVID-19 infections during the pandemic. Punjab, the largest province of Pakistan, remained the epicentre due to a high infection rate. Administrative data for five waves of the pandemic was analyzed to determine the rate of infections and the significance of pharmacological and non-pharmacological interventions on the severity and duration of infection. METHODOLOGY: COVID-19 data from March 2020 to May 2023 was obtained from the Provincial Public Health Reference Laboratory (PPHRL), Punjab AIDS Control Program, Lahore. The data included samples from index cases, contacts, and recovered patients. A total of 36,252,48 cases were screened for COVID-19, and 90,923 (2.50%) were detected positive by RT-PCR, accounting for 5.69% of the cases reported positive throughout the country. RESULTS: Among the positive cases, 50.86% (n = 46,244) cases were new cases (registered for the first time), 40.41% (n = 36751) were the contact cases traced from the newly identified cases and 8.62% (n = 7842) repeated cases. The positivity rates among index cases were reported to be 2.37%, 2.34%, 4.61%, 2.09%, and 1.19%, respectively, for the five respective COVID-19 pandemic waves. Distribution by gender indicated that 64% of males and 35% of females were infected during the pandemic. The age factor demonstrated the most susceptibility to infection in women aged 19-29 years, whereas most males between the ages of 29-39 had an infection. Susceptibility to COVID-19 infection was observed to be equally likely between males and females; however, clinical outcomes indicated that infections in males were more severe and often resulted in fatalities as compared to those in females. This trend was also reflected in the viral titer as measured by the Ct values, where 40% of males had Ct values < 25 (an indicator of high viral titers) compared to 30% of females with Ct values < 25. CONCLUSION: Overall, our data indicated that infection rates remained stable throughout the pandemic except for 3rd wave, which showed a higher incidence of infection rate of 4%. Additionally, data showed a positive impact of masking, social distancing, and immunization, as indicated by the shorter window of high infection rates.

Sexually transmitted infections and associated risk factors among the transgender population of Pakistan.

BACKGROUND: Transgender (TG) people are key drivers for sexually transmitted infections (STIs) all over the world. There is substantial evidence that STIs are associated with an increased likelihood of risky sexual behavior however little is known about the prevalence of STIs (HIV, HBV, HCV, and Syphilis) among HIV infected transgender population in Pakistan. METHODS: The current study investigated the seroprevalence of four STIs and associated socio-demographic risk factors among TGs of Punjab, Pakistan from July 2019 to June 2021. The samples were tested serologically and final confirmation was done through PCR for HIV, HBV, and HCV. RESULTS: A total of 1,562 transgenders cross-sectional descriptive records of the Punjab AIDS Control Program (PACP) were reviewed during the period from July 2019 to June 2021. The serological results evidenced that 533 (34.1%) had one pathogen, 309 (19.8%) had two or more (multiple) infections. The most predominant mono-infection among the transgender population was Syphilis 324 (20.7%) followed by HCV 114 (7.3%), HIV 69 (4.4%), and HBV 26 (1.7%). The highest proportions of Infections were found in TG residing in urban areas (68.6%) as compared to rural areas (31.4%). The seropositivity of all STIs was predominantly increased in Sex worker TGs i-e 55%, 46.5%, 38.5%, and 41.8% in HIV, HBV, HCV, and Syphilis respectively. Among 280 HIV-infected Transgender, 177 (63.2%) had Syphilis co-infections. While 87 (31%) and 47 (16.8%) HIV-infected individuals had HC and HBV co-infection respectively. CONCLUSION: Transgender is neglected population group in society. All STIs were predominantly common among sex worker transgenders, Illiterate educational groups, and TGs residing in urban areas. There is a need to spread awareness about STIs, preventive strategies, and facilitation to health care programs in this high-risk population group.

Comparison of Postoperative Complications of Open Versus Laparoscopic Cholecystectomy According to the Modified Clavien-Dindo Classification System.

BACKGROUND: Though laparoscopic cholecystectomy has become a gold standard management technique for gallbladder diseases, an open approach can also be used for patients having complicated gallbladder disease. Post-cholecystectomy complications are well-documented in existing English scientific literature but are not well understood according to the grade of intervention required to treat those complications. OBJECTIVE: To compare the postoperative complications of laparoscopic versus open cholecystectomy according to the modified Clavien-Dindo classification (MCDC) system. MATERIALS AND METHODS:  A retrospective study was conducted at the Department of General Surgery, Unit - III, Lahore General Hospital, Lahore, comprising the data of patients operated between July 01, 2021, and December 31, 2021, after departmental approval # SU-III/73/LGH, dated April 1, 2022. Patients with the definitive diagnosis of acute cholecystitis, chronic cholecystitis, cholelithiasis, and cholecysto-duodenal fistula were included, while cases of choledocholithiasis and, gall bladder carcinoma were excluded from this study. Eighty patients met the inclusion criteria, with 40 patients in each group of open and laparoscopic cholecystectomy. Information for the data set of age, gender, history of surgical procedure, immediate and late outcome, length of surgery, and MCDC grade were collected. Low-grade complications were Grade I and Grade II, while Grades III to V were high-grade. RESULTS: The mean age of included patients was 42.52 ± 8.76 and 40.025 ± 8.12 years, in the open and laparoscopic group, with 80% and 90% female preponderance, respectively. Grade I and Grade II complications occurred in both groups of patients, with Grade III only in patients who underwent open cholecystectomy. None of the patients from each group developed Grade IV or Grade V complications. Among 40 patients who underwent laparoscopic cholecystectomy, 35% of the patients developed low-grade complications, whereas 40% of the patients developed low-grade complications after open cholecystectomy, with respiratory complications being the most common. High-grade complications after open cholecystectomy were found among 2.5% of patients, whereas no patients developed high-grade complications following the laparoscopic approach. CONCLUSION: Patients who underwent laparoscopic cholecystectomy are less prone to develop complications than patients undergoing open cholecystectomy, hence requiring low-grade interventions of surgical and non-surgical types. MCDC is a valuable tool for assessing surgical complications and can help improve patient outcomes by providing a standardized method for reporting and comparing complication rates.

Diagnosis of Acute Dengue Virus Infection Using Enzyme-Linked Immunosorbent Assay and Real-Time PCR.

Dengue fever is a viral infection caused by the dengue virus and is a growing concern for public health worldwide, particularly in tropical and subtropical regions. This study aimed to assess the diagnostic accuracy of a commercially available NS1 ELISA kit for dengue fever in Pakistan using multiplex qRT-PCR as the gold standard. The study recruited 1236 suspected cases of dengue fever admitted to public sector hospitals in Lahore, Pakistan. Of the suspected cases, 610 (49.3%) were confirmed positive for DENV infection through qRT-PCR, with all four serotypes detected. DENV-2 was the most prevalent serotype, detected in 95.7% of cases. The NS1 ELISA kit detected 71.1% of the positive cases. However, the diagnostic accuracy of the NS1 ELISA kit was found to be only 64.89%. Of the 610 confirmed cases, 68% were male and 32% were female, with a median age of 30 years. Dengue fever was diagnosed in 91.8% of cases, while 8.2% were diagnosed with dengue hemorrhagic fever (DHF). DHF patients had a higher prevalence of abdominal pain, hemorrhagic manifestations, and thrombocytopenia. The cocirculation of all four DENV serotypes in Lahore is concerning and could lead to more severe forms of the disease, such as DHF or dengue shock syndrome, in the future. The study highlights the low diagnostic accuracy of commercially available NS1 ELISA kits and emphasizes the importance of using molecular methods to confirm acute dengue infections. Given the increasing prevalence of dengue fever in developing countries like Pakistan, more accurate and reliable diagnostic tools are needed for effective disease management and control.

Association of common BRCA1 variants with predisposition to breast tumors in Pakistan.

BRCA1 variants are extensively associated with increased risk of breast cancer. Early detection and screening of variants is still rare in developing countries. Here, we investigated six BRCA1 variants in 300 subjects from Pakistani population using tetra amplification-refractory mutation system (T-ARMS) PCR. Our results indicate significant association of BRCA1 variants rs8176237 (AA; OR 8.2, 95% CI 3.02-22.64, p < 0.0001), rs1060915 (CC; OR 4.29, 95% CI 1.94-9.48, p = 0.0003), and rs799912 (TT; OR 3.16, 95% CI 1.44-6.94, p = 0.004) with up to 8-fold increased odds of breast cancer under recessive model. Furthermore, BRCA1 haplotypes AGCACG and AGCCCT were associated with up to 18% breast cancer cases (p < 0.05). Additionally, we found association of these variants with up to 11-fold increased odds of benign breast tumors. Linkage disequilibrium (LD) block-wise analysis revealed haplotypes GCAC and ATAC were associated with significantly increased risk. To our knowledge, this is the first study that identifies the association of these BRCA1 variants with breast tumors in Pakistani population. In conclusion, BRCA1 variants investigated in the present study are associated with high odds of benign- and malignant breast tumors. Studies with bigger sample size may help early detection and screening to reduce the odds of breast cancer.

Significance of Histopathology of Appendectomy Specimens: Analysis From a Teaching Hospital of Pakistan.

Background Histopathology of a tissue specimen plays a crucial role in formulating the final diagnosis of any disease. It confirms whether the histopathological findings are in correspondence with the clinical diagnosis and thus suggests an optimal management plan. Standard surgical practices guide that every human tissue specimen must undergo postoperative tissue analysis unless indicated otherwise. Objective To determine the significance of histopathology in determining the final diagnosis of appendectomy specimens. Materials and methods This retrospective clinical study conducted in May 2022 included 100 patients operated for appendectomy from January 1, 2021, to December 31, 2021, in the emergency room of the Department of General Surgery, Unit-III, Lahore General Hospital, Lahore. Data were retrieved from patients' records and the picture archiving and communication system (PACS). A Google Forms-based pro forma (Google, Mountain View, CA) was generated to include the demographic details, clinical manifestations, and histopathology reports of the patients. Descriptive analysis was completed using a Microsoft Excel spreadsheet (Microsoft Corporation, Redmond, WA). Results Fifty-two patients were females out of the total 100. The mean age at presentation was 23.02 ± 12.02 years. Of the samples, 54% were not sent for histopathology. Among the remaining ones, 27% of cases were proven to be acute appendicitis. Alvarado score was 7-10 in 50% of patients. Other lesions proven by histopathology were appendiceal phlegmon (4%), perforated appendix (4%), mucocele (1%), carcinoid tumor (1%), tuberculosis (1%), and adenocarcinoma (1%). Conclusions Histopathological analysis is the gold standard for the tissue diagnosis of a disease. The high percentage of the samples not sent for histopathology is alarming since the appendix is not only a site for inflammatory pathologies but for neoplastic lesions as well. This practice depicts that the incidence of non-inflammatory pathologies is being ignored by healthcare professionals and there is a dire need to emphasize the significance of acquiring histopathology reports for the specimens of appendectomy in all circumstances.

Chromosomal region 1q24.1 is associated with increased risk of schizophrenia in Pakistani population.

Schizophrenia is a stressful, chronic and in many cases incorrigible psychological disorder. Till now no biomarker is available for diagnosis of this condition and diagnosis is done purely on psychiatric bases. A strong genetic association of human genome region 1q24.1 is implicated with onset of schizophrenia in many studies. Present study is first from Pakistan to report effect of this locus in transmission and liaison of schizophrenia in Pakistani population. For this analysis 300 samples were genotyped of four SNPs, rs1417584, rs1954175, rs821616 and rs113012343 that were selected on basis of minor allele frequency and effects on schizophrenia. Haplotype and transmission disequilibrium analysis was also performed on data. Association of SNPs revealed a significant relation between MAF of rs1417584 and schizophrenia in given samples (χ2 5.57; p 0.02). Haplotype association led to formation of three significant blocks TCAG (OR 20.06), TTAG (OR 4.65) and CCTG (OR 26.17) for rs1417584, rs1954175, rs821616 and rs113012343 that were expected to cause schizophrenia in said combinations. rs1417584, rs1954175 and rs821616 were found to be in a linkage block based on D' value (p < 0.0001) with 22% co inheritance alongside disease onset. This block was represented by 325 kb on chromosome 1. It is concluded from this study that this 325 Kb region can be considered prognostic marker for schizophrenia development in Pakistani population.

Breast cancer risk associated with BRCA1/2 variants in the Pakistani population.

BACKGROUND: Majority of the BRCA1 and BRCA2 mutations are associated with the risk of sporadic and familial breast cancer. Since these genes are significant in DNA repair mechanisms, we focused homology-directed DNA repair (HDDR) and BRCA complex. METHODS: We selected BRCA1 variant (rs80356932, 4491C/T) and BRCA2 variant (rs80359182, 319T/C) from the interaction region of BRCA complex and studied in 100 breast cancer patients and 100 controls using tetra-ARMS-PCR. RESULTS: Here we show that BRCA1 and BRCA2 variants are significantly associated with high breast cancer risk (BRCA1 rs80356932; Genotype T/T OR 8.66, 95% CI 3.16-23.71, p < 0.0001; Allele-T, OR 2.48, 95% CI 1.62-3.81, p < 0.0001 and BRCA2 rs80359182; Genotype C/C OR 4.32, 95% CI 1.95-9.53, p = 0.0001; Allele-C, OR 2.19, 95% CI 1.43-3.34, p = 0.0002). Additionally, bioinformatics analysis showed that BRCA2-tryptophan > arginine substitutions result in altered interaction of BRCA1/PALB2/BRCA2/protein complex and impaired HDDR pathway. We also observed that breast cancer risk was significantly increased in over-weighted and obese women. CONCLUSIONS: Our results indicate that high risk of breast cancer is significantly associated with BRCA1 and BRCA2 variants, and mutations may alter the protein interactions of BRCA complex that results in tumor genesis.

Breast cancer risk associated with genes encoding DNA repair MRN complex: a study from Punjab, Pakistan.

BACKGROUND: Variants of DNA repair genes are extensively reported to cause genetic instability and increase the risk of breast cancer. In combination with NBS1, MRE11 and RAD50 constitute an MRN (MRE11-RAD50-NBS1) complex that repairs DNA damage. However, certain genetic alterations in MRE11 and RAD50 produce abnormal protein that affects the repairing process and may result in malignancy. We aimed to investigate the association of MRE11 and RAD50 polymorphisms with breast risk in the female population of Punjab, Pakistan. METHODS: We collected blood samples of 100 breast cancer patients and 100 tumor-free females selected as controls. Extracted DNA was genotyped by tetra ARMS-PCR followed by gel electrophoresis. Results were analyzed by SPSS and SNPstats to analyze the association of different clinical factors and SNPs (single nucleotide polymorphisms) with the risk of breast cancer. RESULTS: We found that the increased risk of breast cancer is associated with MRE11 variant rs684507 (odds ratio-OR 3.71, 95% confidence interval-CI 1.68-8.18, p value < 0.0001), whereas, RAD50 variant rs28903089 appeared to have protective effect (OR 0.55, CI 0.29-1.02, p value = 0.003). Additionally, clinical factors such as positive family history, life style, and marital status also play significant roles in breast cancer development. CONCLUSION: In the present study, strong risk of breast cancer was associated with MRE11 gene. However, RAD50 showed protective effect. Additionally, clinical factors are also pivotal in risk assessment. We anticipate that targeting specific genetic variations confined to ethnic groups would be more effective in future therapeutic approaches for prevention and treatment of breast cancer.

Screening for Del 185 AG and 4627C>A BRCA1 Mutations in Breast Cancer Patients from Lahore, Pakistan.

Breast cancer contributes to approximately 23% of the cancer cases identified and 14% of cancer related deaths worldwide. Including a strong association between genetic and environmental factors, breast cancer is a complex and multi factorial disorder. Two high penetration breast cancer susceptibility genes (BRCA1 and BRCA2) have been identified, and germ line mutations in these are thought to account for between 5% and 10% of all breast cancer cases. The human BRCA1 gene, located on 17q, is involved in the regulation of cell proliferation by aiding in DNA repair, transcriptional responses to DNA damage and cell cycle check points. Mutations in this gene enhance cell proliferation and facilitate formation of tumors. Two mutations, the 185 deletion of AG and the 4627 substitution from C to A, are founder mutations in the BRCA1 gene for breast cancer in Asian populations. Allele specific PCR was performed to detect these selected mutations in 120 samples. No mutation of 4627 C to A was detected in the samples and only one of the patients had the 185 del AG mutation in the heterozygous condition. Our collected samples had lower consanguinity and family history indicating the greater involvement of environmental as compared to genetic factors.

Impact of a Breast Health Awareness Activity on the Knowledge Level of the Participants and its Association with Socio- Demographic Features.

The developing countries have higher mortality rates for breast cancer. A reason for this is presentation at advanced stages due to low levels of public awareness. Activities are arranged by health authorities of developing countries to increase the knowledge of women but their effectiveness has not been evaluated in detail. A multiple choice questionnaire with questions about socio-demographic profile and questions about breast cancer knowledge was designed in local language Urdu, to evaluate the knowledge of the participants before and after an audio visual educational activity in Lahore, Pakistan. Scores of 0-2, 3-5 and 6-8 were ranked as poor, fair and good, respectively. Among 146 participants these scores were achieved by 1%, 55% and 45% before activity and 0%, 16% and 84% after the activity. Overall 66% of participants increased their knowledge score. Younger age, higher education, reliance on television as source of information and being a housewife were associated with better impact of the awareness activity. For the six knowledge related questions 3%, 5%, 11%, 23%, 33% and 44% more participants gave correct answers after the activity. However 6% and 7% fewer participants answered correctly for 2 questions related to the cause and the best prevention for breast cancer. The study indicated that awareness activities are effective to increase the knowledge of women and better impact is associated with higher education and younger age of women. The component analysis showed that the questions and related presentations using medical terms have a negative impact and should not therefore be used. Analysis of activity therefore leads to identification of deficiencies which can be remedied in future.

Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia.

Circadian rhythms are endogenous and self-sustained oscillations of multiple biological processes with approximately 24-h rhythmicity. Circadian genes and their protein products constitute the molecular components of the circadian oscillator that form positive/negative feedback loops and generate circadian rhythms. The circadian regulation extends from core clock genes to various clock-controlled genes that include various cell cycle genes. Aberrant expression of circadian clock genes, therefore, may lead to genomic instability and accelerated cellular proliferation potentially promoting carcinogenesis. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of serum melatonin levels in peripheral blood samples of 37 CLL (chronic lymphocytic leukemia) patients and equal number of age- and sex-matched healthy controls in order to indicate association between deregulated circadian clock and manifestation of CLL. Results showed significantly down-regulated expression of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc and Cyclin D1 (P < 0.0001) in CLL patients as compared to healthy controls. When expression of these genes was compared between shift-workers and non-shift-workers within the CLL group, the expression was found more aberrant in shift-workers as compared to non-shift-workers. However, this difference was found statistically significant for Myc and Cyclin D1 only (P < 0.05). Serum melatonin levels were found significantly low (P < 0.0001) in CLL subjects as compared to healthy controls whereas melatonin levels were found still lower in shift-workers as compared to non-shift-workers within CLL group (P < 0.01). Our results suggest that aberrant expression of circadian clock genes can lead to aberrant expression of their downstream targets that are involved in cell proliferation and apoptosis and hence may result in manifestation of CLL. Moreover, shift-work and low melatonin levels may also contribute in etiology of CLL by further perturbing of circadian clock.

Screening of intron 1 inversion and three intragenic factor VIII gene polymorphisms in Pakistani hemophilia A families.

Indirect linkage analysis using highly informative polymorphic markers is the method of choice for carrier detection of hemophilia A in developing countries because direct DNA or mRNA sequence analysis is manifold costly and difficult than indirect gene tracking. Worldwide populations have revealed marked variation in the informativeness of polymorphic markers because of which each country has to select its own panel of markers for linkage analysis in hemophilia A families. The present study aimed at determining the informativeness of three factor VIII gene polymorphisms [intron 13(CA)n repeats, HindIII and AlwNI] in the Pakistani population. One hundred and forty-three individuals from 32 hemophilia A families and 68 unrelated anonymous females from the general population were screened for these polymorphisms using PCR and RFLP techniques. An inversion in intron 1 of the factor VIII gene causing 2-5% of severe hemophilia A cases was also screened in 128 Pakistani hemophilia A patients. None of the affected individuals carried the intron 1 inversion at least in peripheral blood leucocytes. The informativeness of intron 13 repeats, HindIII and AlwNI was 59.1% (13/22 hemophilia A families revealing five different alleles), 40.6% (13/32 hemophilia A families) and 6.25% (2/32 hemophilia A families), respectively. The cumulative informativeness of intron 13 repeats and HindIII was 63.6% (14/22 hemophilia A families), revealing strong linkage disequilibrium between these two polymorphic markers. These results suggest that there is a need to determine the informativeness of other polymorphic markers of the factor VIII gene to achieve 100% success rate for carrier detection of hemophilia A in Pakistan.

Leptin deficiency and leptin gene mutations in obese children from Pakistan.

BACKGROUND: Congenital leptin deficiency is a rare human genetic condition clinically characterized by hyperphagia and acute weight gain usually during the first postnatal year. The worldwide data on this disorder includes only 14 cases and four pathogenic mutations have been reported in the leptin gene. STUDY OBJECTIVE: The objectives of this study were to measure serum leptin levels in obese children and to detect leptin gene mutations in those found to be leptin deficient. PATIENTS AND RESULTS: A total of 25 obese children were recruited for the study. Leptin deficiency was detected in nine of them. Leptin gene sequencing identified mutations in homozygous state in all the leptin deficient children. Two cases carried novel mutations (c.481_482delCT and c.104_106delTCA) and each of the remaining seven the previously reported frameshift mutation (c.398delG). CONCLUSION: The results suggest that leptin deficiency caused by mutations in the leptin gene may frequently be seen in obese Pakistani children from Central Punjab.