Molecular characterization of recombinant premembrane protein of dengue virus serotype-2 for development of diagnostic assay.

Dengue is an acute arboviral infection common in tropical and subtropical countries. Dengue has been highlighted as a public health concern in the last five decades, affecting almost 50% of the population in developing nations. Dengue infection results in a complex symptomatic disease that ranges from headache, fever, and skin rash to extreme hemorrhage fever and liver dysfunction. The diagnosis of the disease is essential for effective treatment. The early onset of the infection can be detected through viral structural peptides that act as markers for detection, including Pre-Membrane (Pre-M) protein. In the currently proposed research, the structural gene obtained from local isolates was targeted for studies. For this purpose, recombinant structural protein Pre-M was amplified, cloned, and expressed in the bacterial expression system. The expression of the structural protein (Pre-M) was scrutinized by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and validated by western blot and dot blot, and afterwards, the antigen was purified. The purified Pre-M protein carries the potential for the development of in-house diagnostic assay as well as for vaccine production. This study aimed to develop a highly specific, sensitive, and cost-effective in-house enzyme-linked immunoassay (ELISA) for the detection of antibodies of Pakistani most prevalent dengue virus serotype 2 (DENV-2). The success of this research would also pave the way toward developing novel vaccines for the future prevention of dengue infection.

HSV-1 Infection: Role of Viral Proteins and Cellular Receptors.

The interaction between herpes simplex virus type 1 (HSV-1) and its host starts with the attachment of the virus for entry and spreading into host cells involving viral glycoproteins and host receptors. Once entered, it remains persistent as a latent infection throughout the host's life as it cannot be cleared completely by the immune system. Viral regulatory proteins and host factors determine whether the virus will enter into the acute or latent mode of infection. Acute viral infection is usually asymptomatic and self-limiting whereas latent infection may remain in the trigeminal ganglion of oropharyngeal mucosa, where it can be activated at any time depending upon the stimulus. Host innate and adaptive immune elements play important roles in limiting HSV-1 infection by interfering with viral replication but are unable to remove the virus completely. In this review, we update how the major proteins involved in entry and pathogenesis of viruses and immune responses against infection.

Distribution of CTX-M group I and group III ß-lactamases produced by Escherichia coli and klebsiella pneumoniae in Lahore, Pakistan.

Extended-spectrum-lactamases (ESBLs) of the CTX-M type is worrisome issue in many countries of the world from past decade. But little is known about CTX-M beta-lactamase producing bacteria in Pakistan. Therefore, this study was carried out to investigate the distribution of CTX-M beta-lactamase producing E. coli and Klebsiella pneumoniae using phenotypic and molecular techniques. A total of 638 E. coli and 338 Klebsiella pneumoniae were isolated from patients attending two hospitals and one diagnostic Centre in Pakistan during 2013-2015. ESBL production was screened by double disc synergism, combination disc (cefotaxime and ceftazidime with clavulanic acid) and E-test. These strains were further characterized by PCR (CTX-M I, CTX-M III) and sequencing. After ribotyping of strains accession numbers were obtained. These isolates were highly resistant to cephalosporins, ceftazidime, cefotaxime, aztreonam, and cefuroxime but susceptible to carbapenems, sulfzone, amikacin and tazocin. Multiple antibiotic resistances index (MAR) revealed that 51% of E. coli strains fell in the range of 0.61-0.7 and 39% of Klebsiella pneumoniae strains fell in the range of 0.71-0.8. 64% Double disc synergism (DDS), 76.4% combination disc (CD), 74% E-test showed ESBL positivity in strains. In E. coli ESBL genes blaCTX-M-I and blaCTX-M-III were detected in 212 (72.1%) and 25 (8.5%) respectively. In Klebsiella pneumoniae ESBL genes blaCTX-M-I and blaCTX-M-III were detected in 89 (82.4%) and 10 (9.2%). Combination of both genes blaCTX-M-I and blaCTX-M-III were found in 16 (5.4%) of E. coli strains and 5 (4.6%) of Klebsiella pneumoniae strains. Sequencing revealed that CTXM-15 was predominately present in the CTX-M-I group. The prevalence of ESBL producing E. coli and Klebsiella pneumoniae isolates was high and the majority of them positive for blaCTX-M-I as compared to blaCTX-M-III. These findings highlight the need to further investigate the epidemiology of other CTX-M beta-lactamases in Pakistan.

Genotypic distribution of human papillomavirus and phylogenetic analysis of E6 and E7 gene of HR-HPV variants isolated from Pakistani population.

High-risk-human papillomavirus (HR-HPV)-induced cervical cancer is the second most common cause of death among females worldwide. HPV16 is the most prevalent HR-HPV infection worldwide. This study found the genotypic distribution of HR-HPV in the local population and investigated the sequence variations among the E6 and E7 oncogenes of the local HPV16 genotype to the E6 and E7 oncogenes of the foreign HPV16 genotypes and constructed a phylogenetic relationship based on nucleotide sequence comparison among the variants identified in our study along with previously reported isolates that were obtained from different regions of the world. The samples were collected from patients with cervical cancer. Genomic DNA was extracted, and HR-HPV genotypes were determined using real-time PCR. The HPV16 E6 and E7 genes were amplified and sequenced. A HPV16 phylogenetic tree was constructed using the maximum likelihood method with MEGA 7. HPV16 was the most prevalent human papillomavirus (HPV) type identified in the present study. HPV16 isolates belonged to the A1 sublineage of the European branch. Twenty-one nucleotide sequences were included in this analysis. The first, second, and third codon positions are also included. The final dataset included 776 positions.

Assessment of biosafety implementation in clinical diagnostic laboratories in Pakistan during the COVID-19 pandemic.

Laboratory diagnostic capacity is crucial for an optimal national response to a public health emergency such as the COVID-19 pandemic. Preventing laboratory-acquired infections and the loss of critical human resources, especially during a public health emergency, requires laboratories to have a good biorisk management system in place. In this study, we aimed to evaluate laboratory biosafety and biosecurity in Pakistan during the COVID-19 pandemic. In this cross-sectional study, a self-rated anonymous questionnaire was distributed to laboratory professionals (LPs) working in clinical diagnostic laboratories, including laboratories performing polymerase chain reaction (PCR)-based COVID-19 diagnostic testing in Punjab, Sindh, Khyber Pakhtunkhwa, and Gilgit-Baltistan provinces as well as Islamabad during March 2020 to April 2020. The questionnaire assessed knowledge and perceptions of LPs, resource availability, and commitment by top management in these laboratories. In total, 58.6% of LPs performing COVID-19 testing reported that their laboratory did not conduct a biorisk assessment before starting COVID-19 testing in their facility. Only 31% of LPs were aware that COVID-19 testing could be performed at a biosafety level 2 laboratory, as per the World Health Organization interim biosafety guidelines. A sufficiently high percentage of LPs did not feel confident in their ability to handle COVID-19 samples (32.8%), spills (43.1%), or other accidents (32.8%). These findings demonstrate the need for effective biosafety program implementation, proper training, and establishing competency assessment methods. These findings also suggested that identifying and addressing gaps in existing biorisk management systems through sustainable interventions and preparing LPs for surge capacity is crucial to better address public health emergencies.