Association of Seroconversion Status with Outcome in Admitted Covid-19 Patients.

OBJECTIVE: To determine the association between seroconversion status and outcome in admitted COVID-19 patients and compare inflammatory markers amongst them. STUDY DESIGN: Single cohort observational study. PLACE AND DURATION OF STUDY: Indus Hospital and Health Network between 10th May and 10th July 2020. METHODOLOGY: All admitted patients were tested serially for anti-COVID-IgM and IgG until their sera showed positive results. This was continued until their expiry or discharge. Those patients who remained negative for both anti-COVID-19-IgG and IgM were labeled as non-seroconverts. Demographics, comorbidities, inflammatory marker levels and outcome (alive/expired) were compared between seroconverts and non-seroconverts. RESULTS: In 224 admitted patients, the median seroconversion time of IgM and IgG was six and seven days in survivors and non-survivors respectively. Expired patients displayed higher levels of procalcitonin (maximum), C-reactive protein, and Interleukin-6 (baseline and maximum). Of 34 non-seroconverts, 17 (50%) expired. Non-seroconverts significantly failed to develop fever and had lower levels of ferritin, CRP, and LDH. CONCLUSION: Non-seroconversion in hospitalised COVID-19 infected patients indicated muted immune and acute phase response and was associated with poor outcomes. Hence these patients need to be carefully evaluated and managed. KEY WORDS: Antibody response, Corticosteroids, Immunosuppression, SARS-Cov-2, Seroconversion.

Frequency, Distribution Of Subtypes And Immunohistochemical Profile Of Non-Hodgkin Lymphoma In Paediatric And Adolescent Patients.

BACKGROUND: Non-Hodgkin lymphoma is a common malignant disorder in paediatric and adolescent age group. There is a need of large-scale studies to understand the disease pattern in Pakistan as no official registry exist in most of the developing countries. This study comprised a large cohort of 223 patients, spanned over a decade from January 2008-December 2019 and aimed to report the prevalence of subtypes, demographics and immunohistochemical profile from this region. METHODS: Retrospective study, conducted at Indus hospital and health network and Ziauddin university hospital, Karachi, Pakistan. Sequential data analysis was carried out on all consecutive samples including both needle and excisional biopsies of patients below 18 years of age. Morphological examination of H&E stained sections along with immunohistochemistry is performed in order to identify subtypes and immunophenotypic patterns using an extensive panel of markers. RESULTS: Our results demonstrate 66% B-cell lymphomas while 34% T-cell lymphomas. Overall male to female ratio was 3.3:1 with median age 8 years (1.1-17 years). Among B-cell lymphoma, Burkitt lymphoma is most common while in T-cell, T-lymphoblastic lymphoma is the most common subtype. In anaplastic large cell lymphoma category, null cell phenotype was predominant, i.e., 65%. T-NHL frequency is found to be higher in our population. However, results of immunohistochemistry are similar to published literature. CONCLUSIONS: The study will help to identify disease patterns in terms of subtypes of NHL and its immunohistochemical profile that plays a vital role in diagnostic, prognostic and therapeutic implications.

High risk histopathological factors in retinoblastoma in upfront enucleated eyes: An experience from a tertiary care centre of Pakistan.

Background & Objectives: The assessment of histopathological risk factors (HRFs) in retinoblastoma in upfront enucleated eyes is important in deciding treatment protocols. Limited data is available from the developing countries as very few studies were conducted on retinoblastoma. The study aims to report this data from Pakistan. Methods: This cross-sectional study included treatment naïve retinoblastoma patients who underwent upfront enucleation between 2017 to 2021. Various tumor characteristics i.e. laterality, size, histologic grade, anaplasia grade, growth pattern, extent and length of optic nerve invasion, pathologic staging, tumor involvement of ocular structures were assessed. High-risk factors such as involvement of anterior chamber, choroidal, scleral, extrascleral, and optic nerve were also noted. Results: A total number of 54 patients were enrolled, out of which 53.7% were females while remaining were males. Median age at presentation was 24 months. Unilateral tumor was seen in 92.6% cases. Most frequent histologic grade was G2 (64.7%) and moderate anaplasia was observed in 59.2% cases. Vitreous involvement was seen in (86.5%). Pathologic staging of most of the tumors was pT1 (39.2%). Assessment of high-risk factors revealed that optic nerve involvement (35.1%) was the most common finding with retrolaminar tumor invasion seen in 75% cases. Choroidal invasion (≤3mm) was seen in 55.6% of patients. Limited involvement of anterior chamber (3.8%), sclera (7.4%), and extrascleral (3.8%) tissue was also observed. Conclusion: The presence of high risk histopathological factors in enucleated eyes diagnosed with retinoblastoma are known to have a profound impact on the risk stratification as well as decision of future treatment plan.

Chemotherapy induced histopathological changes in retinoblastoma, assessment of high risk predictive factors & its correlation with comorbid conditions.

Background & Objectives: Retinoblastoma is a malignant intraocular tumor and its treatment requires a multidisciplinary approach. Chemotherapy is an important modality in treatment of retinoblastoma. The purpose of this study was to assess the histopathological changes in retinoblastomas treated with chemotherapy along with correlation of comorbid conditions with high risk histopathological factors (HRF). Methods: All post-chemotherapy enucleated eye specimens received in the pathology department between 2017 to 2021 were included in the study. Slides were retrieved and reviewed for chemotherapeutic effects, tumor regression, and for assessment of HRF. Patient demographic data, information regarding chemotherapy and co-morbid conditions were retrieved through the hospital database. Chi-square was used to analyze the relation between comorbid conditions and HRF. Results: Chemotherapeutic effects were seen in all eyes with varying degrees of responses. Necrosis, calcification, and gliosis were the most common findings. The majority of eyes showed tumor occupying less than 50% of the eye whereas complete regression was noted in one eye only. Retinal detachment, glaucoma, and buphthalmos were the most common comorbid conditions at the time of diagnosis. Patients with glaucoma were more likely to have ciliary body invasion. Kaplan Meier analysis showed that patients with more than two HRF had decreased survival rates in comparison to those with one or no HRF. Conclusion: Histopathological evaluation of chemotherapy-treated eyes shows varying degrees of response to chemotherapy. Post enucleation histopathological evaluation of the globe plays an important role in assessing disease activity and guiding further treatment to prevent metastasis.

Correlation of clinical and radiological predictors of retinoblastoma with high-risk histopathological features.

INTRODUCTION: Retinoblastoma (RB) tumors having high-risk histopathologic features (HRFs) have an increased risk of metastasis and disease relapse. However, RB has not been studied widely in Pakistan. Therefore, we evaluated the association of clinical, histopathologic, and radiological findings with HRFs in patients with RB who were treated at the Indus Health & Hospital Network in Karachi, Pakistan. METHODS: We enrolled treatment-naïve patients with RB who received upfront enucleation from September 2017 to February 2021. We evaluated enucleated eyes with the Intraocular Classification of Retinoblastoma system and classified HRFs as invasion of the anterior chamber, including the iris and ciliary body, or massive invasion of the choroid, sclera, or optic nerve (postlaminar and/or up to the transection line). RESULTS: Of 117 patients with RB treated at our institution during the study period, 54 received upfront enucleation. Unilateral disease was present in 92.6% of cases. The most frequent disease signs and symptoms included the presence of vitreous seeds (30.6%) and leukocoria (100%), respectively. The most frequent HRFs and radiological findings comprised massive choroidal invasion (15.1%) and anterior chamber enhancement (66.7%), respectively. The majority (62.9%) of patients did not exhibit any HRFs. Female sex, pseudohypopyon, iris neovascularization, buphthalmos, and glaucoma had significant predictive ability for HRF occurrence. CONCLUSION: Pseudohypopyon, iris neovascularization, buphthalmos, and glaucoma are important clinical factors that should be taken into consideration before the management of RB. Early recognition of high-risk histopathological and radiological features is essential for appropriate treatment of RB.

P-Loop mutations-Negative prognosticators in tyrosine kinase inhibitors resistant chronic myeloid leukemia patients.

INTRODUCTION: P-Loop mutations in CML patients prevent the conformational change in BCR-ABL1 necessary for drug binding. The present study aimed to evaluate the impact of mutations in this domain on the prognosis of the disease and also to associate the baseline Sokal relative risk score with the overall survival in non-responding CML patients. METHODS: Blood samples were analyzed using ARMS-PCR and then an association was assessed between presence/absence of mutations, hematological and molecular response, disease progression, overall survival, and Sokal score. RESULTS: Of the total 250 CML patients, 102 were found to be treatment-resistant. Fifty-three patients harbored P-Loop mutations with G250E (12.7%) being most frequent. Complete hematological response and major molecular response were achieved by only 27.7% and 5.7 patients, respectively. Worst survival (57.1%) was observed in Y253H positive patients while according to Sokal score in high-risk patients harboring Y253F (50%) and E255V (50%). CONCLUSION: The presence of P-Loop domain mutations negatively impacted the prognosis of the disease in terms of disease advancement and overall survival. So, the timely performance of the BCR-ABL1 mutational analysis and the modifications in the treatment plan based on the mutation identified would help in a better outcome of the disease.

Sensitivity and Specificity of Anti-SARS-CoV-2 Detection Kits - Comparison and Agreement between Fifteen Different Assays.

Accurate and rapid diagnosis of coronavirus disease 2019 (COVID-19) is critical for proper care and identification of affected individuals. This led to early availability of many serological assays in the market, but with limited validation. In this study, we aimed to validate the serological assays based on different techniques. We evaluated 15 different assays based on four immunoassay techniques in 235 patients. The most sensitive kits employed were as follows: immunochromatography (Zybio severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] IgM/IgG Antibody Assay Kit - 83%), ELISA (Aeskulisa SARS-CoV-2 NP IgG -88.1%), chemiluminescence (Alinity SARS-CoV-2 IgG - 82.2%), and immunofluorescence (Lifotronic FA160 (Shenzhen SARS-CoV-2 Assay Kit [IgG]) - 88.9%). The kits by Uniper (Singuway Biotec COVID-19 IgM/IgG Presumptive Kit), Genrui 2019-nCoV IgM/IgG Test Kit, Wondfu SARS-CoV-2 Antibody Test, and Aeskulisa SARS-CoV-2 NP IgG exhibited 100% specificity, whereas IgG assay using Lifotronic FA160 (Shenzhen SARS-CoV-2 Assay Kit) exhibited the lowest specificity at 58%. Maximum agreement was observed between Aeskulisa SARS-CoV-2 NP IgG and Alinity SARS-CoV-2 IgG at 94%. Serological tests are practical alternatives, but their reliability depends on critical validation. The COVID-19 pandemic warranted investment in healthcare research at both the national and international levels.

A Pragmatic Scoring Tool to Predict Hydroxyurea Response Among ß-Thalassemia Major Patients in Pakistan.

Despite high prevalence and incidence of ß-thalassemia in Pakistan, there is very limited work on the use of hydroxyurea (HU) in thalassemia patients in the country. This is the first insight regarding genetic profiling of BCL11A and HU responses in Pakistani ß-thalassemia. It correlates single-nucleotide polymorphisms on BCL11A (rs4671393, rs766432) and HBG2 (XmnI), age at first transfusion, and ß-globin mutations with HU response in ß-thalassemia major (BTM). Of 272 patients treated with HU, 98 were complete responders, 55 partial responders, and 119 nonresponders. Our analysis shows that HU response was significantly associated with patients having IVSI-1 or CD 30 mutation (P<0.001), age at first transfusion >1 year (P<0.001), and with the presence of XmnI polymorphism (P<0.001). The single-nucleotide polymorphisms of BCL11A were more prevalent among responders, but could not show significant association with HU response (P>0.05). Cumulative effect of all 5 predicting factors through simple binary scoring indicates that the likelihood of HU response increases with the number of primary and secondary genetic modifiers (P<0.001). Predictors scoring is a pragmatic tool to foresee HU response in patients with BTM. The authors recommend a score of ≥2 for starting HU therapy in Pakistani patients with BTM.

Sigma metrics of Alinity ci system - a study on thirty-nine clinical chemistry and immunoassay parameters.

Objectives: Sigma metrics in an invaluable and inexpensive tool used in laboratories to monitor analytical quality of the assays. Alinity ci platform is a relatively recent analytical system launched by Abbott Diagnostics, and as such performance studies on it are few. We have calculated sigma metrics of 39 clinical chemistry and immunoassay analytes on two Alinity ci systems. Methods: Sigma metrics were calculated using results of method validation studies. Coefficient of variation (CV) was calculated according to CLSI EP 15 guidelines. Bias was calculated using three different methods i.e., proficiency testing material, alternate method comparison with existent analyzers and linearity experiment. Total allowable error limits were kept similar to or less than the ones used in reference studies. Results: All analytes except blood urea nitrogen (BUN) demonstrated greater than six sigma value across one or more levels and methods. No analyte amongst clinical chemistry and immunoassays was at below three sigma class. Amongst electrolytes, sodium was below three sigma class at two levels by proficiency testing method, although it was above four sigma class by other two methods. Sigma levels obtained were comparable to those reported in previously published studies. Conclusions: Acceptable sigma metrics were achieved for all clinical chemistry, immunoassays and electrolytes on Alinity ci. Sigma metrics is an objective and well established cost effective tool to tailor internal quality control practices. This study determines sigma metrics for a wide range of high throughput assays. Long term assay performance needs to be monitored.

A distinct molecular mutational profile and its clinical impact in essential thrombocythemia and primary myelofibrosis patients.

BACKGROUND: Classical MPNs including ET and PMF have a chronic course and potential for leukaemic transformation. Timely diagnosis is obligatory to ensure appropriate management and positive outcomes. The aim of this study was to determine the mutational profile, clinical characteristics and outcome of ET and PMF patients in Pakistani population. METHODS: This was a prospective observational study conducted between 2012 and 2017 at NIBD. Patients were diagnosed and risk stratified according to international recommendations. Response to treatment was assessed by IWG criteria. RESULTS: Of the total 137 patients analysed, 75 were ET and 62 were PMF. JAK2 positivity was seen in 51 cases (37.2%), CALR in 41 cases (29.9%), while triple-negative in 17 (12.4%) cases. None of the patients in the present study were MPL positive. Overall survival for patients with ET and PMF was 92.5 and 86.0% respectively and leukaemia free survival was 100 and 91.6% respectively, at a median follow-up of 12 months. Leukaemic transformation occurred in 6.5% of MF patients; among them, JAK2 mutation was frequently found. Molecular mutations did not influence the OS in ET whereas in PMF, OS was shortest in the triple-negative PMF group as compared to the JAK2 and CALR positive patient groups. CONCLUSION: This study shows a different spectrum of molecular mutations in ET and PMF patients in Pakistani population as compared to other Asian countries. Similarly, the risk of leukaemic transformation in ET and PMF is relatively lower in our population of patients. The factors responsible for these phenotypic and genotypic differences need to be analysed in large scale studies with longer follow-up of patients.

Pattern of Antimicrobial Sensitivity in Microbiologically Documented Infections in Neutropenic Patients with Haematological Malignancies: A single Center Study.

Febrile neutropenia is a medical emergency that complicates the clinical course and treatment of haematological malignancies, significantly enhancing the financial burden and worsening the overall outcome. This study was carried out to evaluate the efficacy of institution's current first-line antibiotic regimen for febrile neutropenia in view of recent spectrum of institution's local flora and its susceptibility pattern. 163 episodes of microbiologically documented infections in 110 adult patients were studied over a period of 1 year. Of 110 patients, 61 patients were male. The mean age of the patient population, mean absolute neutrophil count and temperature as documented were 30.1 years (SD ± 16.8), 450 cells/ul, and 101.9 °C respectively. Gram-negative and gram-positive organisms accounted for 79% and 21% of the febrile neutropenic infections respectively. E. coli and Staphylococcus aureus were the most common gram positive and gram negative pathogens respectively. A susceptibility pattern of > 60% was documented for all the gram negative pathogen's associated febrile neutropenic infections for the current first-line antibiotic combination of Piperacillin/Tazobactum and Amikacin. Comparative analysis of results with the institutional data of 2015 study revealed no statistically significant difference in the resistance pattern of the organisms hence, validating the persistent use of Piperacillin/Tazobacum and Amikacin combination as a potent and efficacious therapy for febrile neutropenia patients with haematological malignancies. However, continuous surveillance remains prudent for the emerging changes in the spectrum and resistance pattern of local flora so that timely revision of empirical antibiotic regimens can save the added financial burdens and associated high morbidity and mortality.

Early and sustained deep molecular response achieved with nilotinib in high Sokal risk chronic myeloid leukemia patients.

BACKGROUND: Nilotinib (Tasigna®) is a second-generation tyrosine kinase inhibitor that shows faster and deeper molecular responses (MR) in comparison to Imatinib as initial therapy in chronic phase chronic myeloid leukemia (CML). Efficacy and safety data for nilotinib in the Asian population is scarce, particularly in Pakistan. We aimed to determine the MR to nilotinib and its safety profile in patients with chronic phase CML. PATIENTS AND METHODS: This observational study was conducted among 173 patients with newly diagnosed CML presenting in the chronic phase. Most patients (50.1%) had a high Sokal score at diagnosis. All patients received nilotinib 600 mg/day. The hematological and molecular responses were assessed at 3 and 6 months respectively and thereafter at 6-monthly intervals. Long-term event free survival (EFS), transformation free survival (TFS), overall survival (OS) and adverse events were observed. RESULTS: Cumulative incidence of major MR (MMR) was 86% and deep MR (DMR ie MR 4.0 and MR4.5) was 39%. Early MMR and DMR after 6 months of therapy were achieved by 74.9% and 37% of patients, respectively. Two-year EFS, TFS and OS rates for all patients were 91.9%, 92% and 92.3%, respectively. At median follow-up of 24 months, 81% and 49% of patients sustained MMR and DMR, respectively. The main adverse events were weight gain (4.6%) and abdominal pain (4%). CONCLUSION: This study showed promising results in terms of achievement of early and sustained DMR in chronic phase CML, therefore, we recommend nilotinib as frontline treatment in Pakistani population.

Laboratory diagnosis for thalassemia intermedia: Are we there yet?

BACKGROUND: Differentiation between thalassemia major and thalassemia intermedia at presentation is not uniformly characterized, for which an absolute criteria needs to be developed. This study investigated the primary and secondary genetic modifiers to develop a laboratory finding by forming different genetic mutational combinations seen among thalassemia intermedia patients and comparing them with thalassemia major. METHODS: This cross-sectional study analyzed 315 thalassemia intermedia patients. One hundred and five thalassemia major patients were recruited on the basis of documented evidence of diagnosis and were receiving blood transfusion therapy regularly. Various mutational combinations were identified, and comparison was performed between thalassemia intermedia and major using statistical software STATA 11.1. RESULTS: The mean age of the total population was 5.9 ± 5.32 years of which 165 (52%) were males. Of the two groups (thalassemia intermedia and thalassemia major), IVSI-5, IVSI-1, and Fr 8-9 were more prevalent among the thalassemia intermedia cohort. When comparison was performed between the thalassemia intermedia and thalassemia major patients, it showed significant results for the presence of Xmn-1 polymorphism. CONCLUSION: The presence of IVSI-5 homozygous with Xmn-1, IVSI-5 heterozygous with Xmn-1, Cd 30 homozygous with or without Xmn-1 and IVSI-1 homozygous or heterozygous either with or without Xmn-1 prove to be strong indicators towards diagnosis of thalassemia intermedia.

Chronic Myeloid Leukemia--Prognostic Value of Mutations.

Chronic myeloid leukemia (CML) is a stem cell disorder characterized by unrestricted proliferation of the myeloid series that occurs due to the BCR-ABL fusion oncogene as a result of reciprocal translocation t(9;22) (q34;q11). This discovery has made this particular domain a target for future efforts to cure CML. Imatinib revolutionized the treatment options for CML and gave encouraging results both in case of safety as well as tolerability profile as compared to agents such as hydroxyurea or busulfan given before Imatinib. However, about 2-4% of patients show resistance and mutations have been found to be one of the reasons for its development. European Leukemianet gives recommendations for BCR-ABL mutational analysis along with other tyrosine kinase inhibitors (TKIs) that should be administered according to the mutations harbored in a patient. The following overview gives recommendations for monitoring patients on the basis of their mutational status.