Transplacental Transfer of SARS-CoV-2 Receptor-Binding Domain IgG Antibodies from Mothers to Neonates in a Cohort of Pakistani Unvaccinated Mothers.

The presence of COVID-19 antibodies in the maternal circulation is assumed to be protective for newborns against SARS-CoV-2 infection. We investigated whether maternal COVID-19 antibodies crossed the transplacental barrier and whether there was any difference in the hematological parameters of neonates born to mothers who recovered from COVID-19 during pregnancy. The cross-sectional study was conducted at the Saidu Group of Teaching Hospitals, located in Swat, Khyber Pakhtunkhwa. After obtaining written informed consent, 115 healthy, unvaccinated mother-neonate dyads were included. A clinical history of COVID-19-like illness, laboratory-confirmed diagnosis, and contact history were obtained. Serum samples from mothers and neonates were tested for SARS-CoV-2 anti-receptor-binding domain (anti-RBD) IgG antibodies. Hematological parameters were assessed with complete blood counts (CBC) and peripheral blood smear examinations. The study population consisted of 115 mothers, with a mean age of 29.44 ± 5.75 years, and most women (68/115 (59.1%)) were between 26 and 35 years of age. Of these mothers, 88/115 (76.5 percent) tested positive for SARS-CoV-2 anti-RBD IgG antibodies, as did 83/115 (72.2 percent) neonatal cord blood samples. The mean levels of SARS-CoV-2 IgG antibodies in maternal and neonatal blood were 19.86 ± 13.82 (IU/mL) and 16.16 ± 12.90 (IU/mL), respectively, indicating that maternal antibodies efficiently crossed the transplacental barrier with an antibody transfer ratio of 0.83. The study found no significant difference in complete blood count (CBC) parameters between seropositive and seronegative mothers, nor between neonates born to seropositive and seronegative mothers.

Aerobic Bacterial Contamination In Platelet Concentrates At The Regional Blood Centre, Peshawar.

BACKGROUND: Platelet concentrates play a crucial role in transfusion medicine, aiding in the management of various medical conditions, including haemorrhage, thrombocytopenia, and platelet dysfunction. However, their storage conditions at 22° C present an optimal environment for bacterial growth, making them susceptible to contamination. Of particular concern is the transmission of microorganisms from the skin flora during the phlebotomy process, which can lead to the transfusion of contaminated platelet concentrates. Such contamination poses significant risks to patients, potentially resulting in morbidity and mortality. Determining the frequency and identifying causative organisms of bacterial contamination in platelet concentrates. METHODS: It was a descriptive cross-sectional study conducted at the Institute of Pathology and Diagnostic Medicine, Khyber Medical University, and the Regional Blood Center in Peshawar from May to October 2021, spanning a duration of six months. The study included 500 participants aged between 18 and 50 years (mean: 28.13±7.67 years. A simple convenient sampling technique was employed. Blood products underwent screening for Hepatitis B, Hepatitis C, HIV, Syphilis, and Malaria. Leaked units were excluded from the study. Platelets were prepared using a Cryofuge and subsequently subjected to culture media. RESULTS: The mean age of the participants included in the study was 28.13±7.67 years, with an age range of 18 to 50 years. Out of the total sample size of 500, there were 483 (96.6%) male participants and 17 (3.4%) female participants. Among the collected samples, bacterial growth was observed in only 11 (2.2%) platelet concentrates. The isolated organisms were Staphylococcus epidermidis, found in 7 (1.4%) platelet concentrates, and Staphylococcus aureus, found in 4 (0.8%) platelet concentrates. CONCLUSIONS: Bacterial contamination of platelet bags is higher compared to developed countries. Therefore, implementing quality control procedures is necessary to reduce the risk of bacterial contamination in platelet concentrates. Additionally, employing enhanced skin disinfection techniques at the phlebotomy site can significantly minimize bacterial contamination.

Effectiveness Of Pcr Pool Testing For Screening Of Covid-19 Infection In Pakistan.

BACKGROUND: We tested the utility of mini-pool PCR testing for the rational use of PCR consumables in screening for CoViD-19. METHODS: After pilot experiments, 3-samples pool size was selected. One step RT-PCR was performed. The samples in the mini-pool having COVID gene amplification were tested individually. RESULTS: 1548 samples tested in 516 mini-pools resulted 396 mini-pools as negative and 120 as positive. Upon individual testing, 110 samples tested positive and 9 were inconclusive. 876 PCR reactions were performed to test 1548 samples, saving 43% PCR reagents. Centres with low prevalence resulted in most saving on reagents (50%), while centres with high prevalence resulted in more test reactions. Testing of individual samples resulted in delays in reporting. CONCLUSIONS: Pooling can increase lab capacity, however, pooling delays results and cause degradation of samples.

Determinants of Response at 2 Months of Treatment in a Cohort of Pakistani Patients with Pulmonary Tuberculosis.

Mycobacterium tuberculosis infection continues to be a major global challenge. All patients with pulmonary tuberculosis are treated with a standard 6-month treatment regimen. Historical data suggest that even with shortened treatment, most patients achieve long-term remission. Risk stratification is a goal for reducing potentially toxic prolonged treatment. This study aimed to determine the factors associated with the early clearance of sputum acid-fast bacilli (AFB). A total of 297 freshly diagnosed patients with pulmonary tuberculosis were included and enrolled in this study. Information related to their ethno-demographic and anthropometric characteristics was collected. We also assessed their complete blood counts, and blood iron, folate, and vitamin B12 levels. We found that the presence of higher levels of acid-fast bacilli (AFB) in diagnostic sputum microscopy was the single most significant prognostic factor associated with early clearance of sputum AFB after 2 months of treatment. All of our patients achieved treatment success after 6 months of treatment and were disease free. Our results support the data obtained from previous studies indicating that AFB clearance at 2 months is unlikely to be a clinically useful biomarker or indicator for therapeutic stratification. Furthermore, demographic, anthropometric, and nutritional factors are not clinically useful biomarkers.

Deep Learning Assisted Automated Assessment of Thalassaemia from Haemoglobin Electrophoresis Images.

Haemoglobin (Hb) electrophoresis is a method of blood testing used to detect thalassaemia. However, the interpretation of the result of the electrophoresis test itself is a complex task. Expert haematologists, specifically in developing countries, are relatively few in number and are usually overburdened. To assist them with their workload, in this paper we present a novel method for the automated assessment of thalassaemia using Hb electrophoresis images. Moreover, in this study we compile a large Hb electrophoresis image dataset, consisting of 103 strips containing 524 electrophoresis images with a clear consensus on the quality of electrophoresis obtained from 824 subjects. The proposed methodology is split into two parts: (1) single-patient electrophoresis image segmentation by means of the lane extraction technique, and (2) binary classification (normal or abnormal) of the electrophoresis images using state-of-the-art deep convolutional neural networks (CNNs) and using the concept of transfer learning. Image processing techniques including filtering and morphological operations are applied for object detection and lane extraction to automatically separate the lanes and classify them using CNN models. Seven different CNN models (ResNet18, ResNet50, ResNet101, InceptionV3, DenseNet201, SqueezeNet and MobileNetV2) were investigated in this study. InceptionV3 outperformed the other CNNs in detecting thalassaemia using Hb electrophoresis images. The accuracy, precision, recall, f1-score, and specificity in the detection of thalassaemia obtained with the InceptionV3 model were 95.8%, 95.84%, 95.8%, 95.8% and 95.8%, respectively. MobileNetV2 demonstrated an accuracy, precision, recall, f1-score, and specificity of 95.72%, 95.73%, 95.72%, 95.7% and 95.72% respectively. Its performance was comparable with the best performing model, InceptionV3. Since it is a very shallow network, MobileNetV2 also provides the least latency in processing a single-patient image and it can be suitably used for mobile applications. The proposed approach, which has shown very high classification accuracy, will assist in the rapid and robust detection of thalassaemia using Hb electrophoresis images.

Seroprevalence of Sars-Cov-2 antibodies among eligible blood donors of Peshawar, Pakistan.

BACKGROUND AND OBJECTIVES: To determine the seroprevalence of SARS-CoV-2 antibodies and the associated risk factors among healthy blood donors from Peshawar Pakistan, during the second and third waves of the COVID-19 pandemic. METHODS: The study was conducted on 4047 healthy (with no history or symptoms of COVID-19) blood donors attending regional blood center Peshawar between Nov 2020 and June 2021. Demographic data was collected and donors were screened for the presence of anti-SARS-CoV-2 antibodies using electrochemiluminescence immunoassay (ECLIA). RESULTS: The mean age of the participants was 27.27±7.13 and the majority (99%) were males. Overall, 59% (2391/4047) of the blood donors were reactive for SARS-CoV-2 antibodies. An increasing trend in seropositivity was observed from 45.5% to 64.8% corresponding to the second and third wave of the pandemic in Pakistan. Logistic regression analysis revealed significantly higher odds of seropositivity among male donors compared to females. Similarly, in multivariable analysis, the odds ratio for seropositivity among blood types AB, A, and B were, 1.6, 1.4, and 1.3 (CI 95%) times higher compared to blood group O (P-value ≤0.0001). CONCLUSIONS: Seropositivity of SARS-CoV-2 antibodies among blood donors gradually increased during the second and third wave of the pandemic in Pakistan indicating a widespread prevalence of Covid-19 in the general population. Susceptibility to SARS-CoV-2 varies with ABO blood types, with blood group O associated with low risk of infection.

Development of a Real-Time qPCR Assay for Detection of Common MPL Mutations in Myeloproliferative Neoplasms (MPNS).

Myeloproliferative neoplasms (MPNs) are blood cell disorders, characterized by overproduction of abnormal cells in bone marrow due to stem cell mutation. The proliferations of blood cell are controlled by many genes particularly MPL gene which encodes thrombopoietin receptor, a hematopoietic growth factor involved in the production and regulation of the platelets and multipotent hematopoietic progenitor cells. Acquired mutations including (W515L and W515K) in this gene have been observed in patients with primary myelofibrosis or essential thrombocythemia lacking JAK2 (V617F) mutations. MPL mutation detection is important for MPNs diagnosis, but due to low frequency of mutant allele burden (< 15%) may be missed by already available common assays such as Sanger sequencing. Furthermore, these techniques are costly, time-consuming, and less sensitive. In present study, we aimed to develop sensitive, less time-consuming, and cost-effective real-time PCR assay for the detection of MPL mutations that is based on TaqMan fluorescent probes. DNA was extracted from blood sample of 128 MPNs patients collected and further analysis was performed on TaqMan RT-PCR. Reference curve was obtained for amplified product of MPL gene containing mutated sequence. The predicted sensitivity level was at least 5% mutant allele burden by our developed assay that is much higher than sequencing output. Out of 128, 2 (1.56%) patients harbored W515L mutation and 1 (0.78%) harbored W515K mutation. It was concluded that TaqMan qRT-PCR assay is an efficient, sensitive, cost-effective, and less time-consuming method capable of detecting MPL mutation in MPNs patients. We suggested that this assay might be helpful in investigating mutant allele load in MPNs patients.

Repeated Covid-19 Infection Exists: A Case Series From Pakistan.

BACKGROUND: Protection against SARS-CoV-2 in infected individuals of COVID-19 is lacking. We report a case series of repeated infections of SARS-CoV-2. METHODS: A total of 12 patients were identified with repeated infections for SARS-CoV-2 from 25 April 2020 to 16 March 2021 from Pakistan. Repeated infection was defined as diagnosis of the SARS-CoV-2 with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) tests in the first and second phase of infection after complete recovery from the first phase of infection with a negative RT-PCR. RESULTS: Of the 12 participants, 75% (n=9) were male and mean age of the participants were 40.1±9.7 years. Mean duration between the first and second phase of infection was 184±68.9 days. Patients presenting with mild infection in the first phase largely developed moderate to severe infection in the second phase. None of them were vaccinated. CONCLUSION: The pandemic of COVID-19 is on the rise and repeated infection from SARS-CoV-2 is occurring.

Clinical and laboratory relevance of JAK2 V617F and BCR-ABL co-existence in Philadelphia positive CML patients.

Chronic Myeloid Leukaemia (CML) is characterized by BCR-ABL1 mutation. A number of research studies have published reports of concomitant JAK2-V617F mutation in BCR-ABL positive Chronic Myeloid Leukaemia. This study aims to investigate the frequency of JAK2-V617F mutation in BCR-ABL positive CML cases. After approval from ethical committee, participants were enrolled in the study. A total of 103 samples from CML patients were analysed for the presence of JAK2-V617F mutation using real-time polymerase chain reaction. Patients were monitored for treatment response using real-time quantitative PCR for BCR-ABL1 mutation. Out of 103 samples analysed, 2 patients tested positive for JAK2-V617F mutation. These two patients when treated with standard Tyrosine Kinase Inhibitors (TKI) therapy achieved molecular response and normalized the haemoglobin and white cell counts. However, one patient has sustained thrombocytosis. JAK2 remained positive throughout the treatment course. We could not follow the second patient till the end of the study. JAK2 mutation in BCR-ABL1 mutated CML appears to be rare. Treatment with TKI does not appear to reduce JAK2 mutation burden despite a decrease in BCR-ABL1 copy numbers.

Effects of transfusion of stored blood in patients with transfusion-dependent thalassemia.

OBJECTIVES: The aim of this study was to investigate the hematological and biochemical effects of stored blood transfusion on patients with transfusion-dependent thalassemia (TDT). METHODS: In this quasi-experimental study, 20-patients with TDT were enrolled. Each participant received on first visit, freshly collected red cell concentrate (RCC) (<2-days storage) and 15-days later on second visit, 7-days stored blood. Blood samples were obtained immediately before and 24-hours after each transfusion. Differences in the Complete blood counts, bilirubin, LDH, C-Reactive protein, ferritin, and iron levels in the pre- and post-transfusion samples were compared between the first and second transfusion. RESULTS: Fresh blood transfusion resulted in a higher (but non-significant) increase in hemoglobin and other red cell parameters. Notably, a significant increase in white cell counts (WCC) was seen in 7-days stored blood vs fresh blood (1.82×109/l vs 1.01×109/l, P=0.002). No statistically significant difference was found in LDH, direct and indirect bilirubin, creatinine, blood glucose, serum uric acid, serum ferritin, and serum Iron levels. There was a statistically significant rise in C-reactive protein levels in stored (6.43±7.46 mg/dl) versus fresh RCC (1.89±2.38 mg/dl), p-value =0.012. CONCLUSIONS: We show that in patients with chronic TDT, an increase in inflammation-associated markers (WCC and CRP) is observed. Further studies to assess the extent and duration of this increase are needed.

Development of the Next Generation Sequencing-Based Diagnostic Test for ß-Thalassemia and its Validation in a Pashtun Family.

ß-Thalassemia (ß-thal) is a common monogenic disease with ethnic-specific mutations on the HBB gene throughout the world. The reported mutations either reduce the expression or completely inactivate the HBB gene. In Pakistan, the prevalence of ß-thal is high due to consanguineous marriages. Accurate identification of mutations in carriers is imperative for prevention of ß-thal in subsequent generations. To overcome the limitations of traditional testing methods for ß-thal, a next-generation sequencing (NGS)-based diagnostic test was designed and validated by sequencing the entire HBB gene. The primer set covering the entire HBB gene was designed and validated in a Pashtun ß-thalassemic family. The polymerase chain reaction (PCR) product was sequenced using an Illumina MiSeq platform. A homozygous pathogenic insertion of A>AC/AC (rs35699606) was detected in an affected member of the family, while unaffected members were heterozygous for it. In addition, all family members were homozygous for the synonymous variant, A>G/G (rs713040), except the father who was heterozygous for it. We sequenced the entire HBB gene using the NGS-based test, which is highly sensitive, robust and specific for the diagnosis and screening of ß-thal in Pakistan, especially for families practicing consanguineous marriages.

First documented reinfection of SARS-COV-2 in second wave from Pakistan.

Little is known about the pathophysiology and immunology of Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2). It is not yet clear whether SARS-CoV-2 infection produces long-term immune protection, or a short-lived immunity that would fade with the passage of time. We report a first case of SARS-CoV-2 reinfection in second wave from Pakistan. A 41-year-old male, health care worker developed flu-like illness, tested positive on 6th June 2020 and became PCR negative on 19th June 2020 with reactive antibodies. After 4 months and 13 days of negative PCR, he developed symptoms again and tested positive for SARS-CoV-2 with non-reactive antibodies.

Time dependent changes in red blood cells during storage in the local blood banks of Khyber Pakhtunkhwa.

OBJECTIVE: This project aimed at determining time-dependent ultrastructural and haematological changes taking place in blood stored in local blood banks of Khyber Pakhtunkhwa. METHODS: It was a longitudinal study with repeated measures design. Twenty healthy blood donors participated in this study. An amount of 250ml blood was collected from each donor and stored in Citrate Phosphate Dextrose Adenine-1 (CPDA-1)- containing blood bags. Within first four hours, baseline samples were taken while subsequent samples were obtained at 5 days interval till day 20th. Structural changes in RBCs were observed under light and scanning electron microscope (SEM) at different intervals. Furthermore, haematological parameters and osmotic fragility were also determined. RESULTS: Remarkable alterations were seen in RBCs morphology. From 5th day onwards, multiple visible spicules were observed on the RBC's outer membrane and more than 2/3rd cells were abnormal at day 20. There was a significant reduction in RBCs count and haemoglobin concentration while the remaining parameters remained unchanged. Osmotic fragility increased significantly over time, with <1% haemolysis noted in baseline samples as compared to 2.4% haemolysis on day 20th (p≤0.0001). CONCLUSIONS: Prolonged storage of blood results in distorted RBCs morphology and increased fragility. Transfusion of such cells would potentially result in rapid lysis in patients with hepatosplenomegaly and conditions requiring multiple blood transfusions.

Effectiveness of Cardiac Rehabilitation on Health-related Quality of Life in Patients with Myocardial Infarction in Pakistan.

OBJECTIVE: To find out the effectiveness of cardiac rehabilitation in patients with myocardial infarction in Pakistan. STUDY DESIGN: Randomised controlled trial. PLACE AND DURATION OF STUDY: Cardiac Rehabilitation Unit, Lady Reading Hospital, Peshawar, Pakistan, from July to December 2016. METHODOLOGY: Patients suffering first myocardial infarction (MI) were randomly allocated to usual care or cardiac rehabilitation in a 1:1 ratio. Cardiac rehabilitation comprised two phases: 1-2 weeks during hospital stay followed by 6-7 weeks outpatient structured exercise programme. Two generic health related quality of life (HRQoL) outcomes (General Health Questionnaire (GHQ) and Self-Rated Health (SRH)) and one post-MI specific tool (MacNew QLMI) were measured at baseline and at 8 weeks follow-up among both groups. Lower SRH and GHQ scores and higher MacNew QLMI scores indicate better health status. Data were analysed using STATA 14. RESULTS: Out of 206 participants, 195 (94.6%) were analysed at the end of trial. The mean age was 53 +8.3 years. In the cardiac rehabilitation group, the mean SRH score changed from 3.97 +0.9 at baseline to 2.36 +0.8 at follow-up (p<0.001). The mean GHQ of the cardiac rehabilitation group was 21.26 +5.5 at baseline and it decreased significantly to 7.43 +4.2 at follow-up (p<0.001). The MacNew QLMI of the cardiac rehabilitation group increased from 3.61 +1.07 to 5.62 +0.5 (p<0.001). The multivariate regression of all three HRQoL measures confirmed better HRQoL following cardiac rehabilitation compared with usual care (all p<0.001). CONCLUSION: Cardiac rehabilitation following MI was effective in terms of improving HRQoL and can be implement in Pakistan as it produced significant improvements in HRQoL.

Co-existing iron deficiency/overload in beta-thalassemia trait.

OBJECTIVE: To identify the co-existence of iron deficiency and iron overload in individuals with beta thalassaemia trait. METHODS: The cross-sectional study was conducted at Rehman Medical Institute and Khyber Medical University, Peshawar, Pakistan, September 1, 2015, to December 31, 2017, and comprised individuals with hypochromic microcytic blood picture. Haemoglobin electrophoresis was performed on their blood samples. Serum ferritin levels of subjects with Haemoglobin Subunit Alpha 2 levels between 3.5% and 7% were checked. Data were analysed using analysed using GraphPad Prism v6. RESULTS: Of the 292 subjects, 159(54.5%) were males and 133(45.5%) were females. Of these, 240 (82.2%) were anaemic and 52 (17.8%) had haemoglobin within the normal range. Serum ferritin level of 55(18.8%) subjects was low and 207(70.9%) were iron-replete. Notably, 30(10.3%) subjects had serum ferritin levels higher than the reference range, and this was more common among adults (p<0.001). CONCLUSIONS: Ferritin levels in beta thalassaemia trait can be low, normal or higher than the normal values..

Novel deleterious mutation in MYO7A, TH and EVC2 in two Pakistani brothers with familial deafness.

OBJECTIVE: In Pakistan, 74% of consanguineous marriages are among the first cousins. Continuity of consanguineous marriages over generations increases the risk of recessive diseases such as deafness. The objective of this study was to investigate genetic origin of Pakistani deaf brothers with parents of consanguineous marriage. METHODS: DNA was extracted from the blood through Qiagen kit. Paired-end sequencing library was prepared according to protocol of Illumina's TruSight Rapid Capture kit and TruSight Inherited Disease Panel. Library was normalized and used for Next Generation Sequencing through MiSeq. NGS data were analyzed using various bioinformatics tools. RESULTS: Both brothers were found to have novel deleterious mutation in MYO7A (c.2476G>A) while the younger brother had additional novel deleterious mutation in TH (c.43C>T) and EVC2 (c.2614C>T) genes. CONCLUSION: It is concluded that in addition to novel mutations in MYO7A, TH and EVC2, the CDH23 and GJB2 can also be responsible for deafness in the family with consanguineous marriages.

Mutational Analysis Of Beta Thalassaemia By Multiplex Arms-Pcr In Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Beta thalassaemia is one of the commonest genetic conditions in the world. More than 200 different mutations have been reported in the beta globin chain genes. Notably, regional and ethnic variations in most common mutations in beta-thalassaemia have been identified. It is therefore imperative that region- and ethnicity- specific commonest mutations be identified for cost-effective molecular diagnosis of ß-thalassaemia mutations. The objective of this study was to determine the molecular mutations in ß-globin chain gene in patients with thalassemia in Khyber Pakhtunkhwa (KP) using multiplex- Amplification Refractory Mutation System (ARMS) PCR. METHODS: It was a cross sectional descriptive study. Blood samples from newly diagnosed ß thalassemia patients was collected and used as source for DNA isolation. ARMS PCR was performed for detection of mutations in ß-globin gene. SDS-PAGE was conducted for visualization of the amplicon. RESULTS: Prominent mutations were Fr 8-9 (+G), CD 5 (-CT) and Fr 41-42 (-TTCT). Congenital marriages and lack of awareness are largest contributing factor for increasing the disease burden. Organomegaly being a serious clinical complication which contributes to morbidity was proportional to age and disease progression. Fr 8-9 (+G) & CD 5 (- CT) were the most frequent mutation prevalent among different ethnic groups residing in KP. CONCLUSIONS: Multiplex-ARMS PCR is capable of assessing for multiple mutations in a single tube. Regional- and ethnic- variations in the commonest mutations in KP are noted. Any mutational diagnostic strategy should consider costs and genetic variations in a particular setting..