Comparison of immune response to SARS-COV-2 vaccine in COVID-recovered versus non-infected Individuals.

To determine the antibody levels at 6 months in SARS-CoV-2 vaccinated individuals in COVID-recovered versus non-infected groups to determine the need to administer booster COVID vaccine in each group. Prospective longitudinal study. Pathology Department, Combined Military Hospital, Lahore for a period of eight months from July 2021 to February 2022. Two hundred and thirty three study participants in both COVID recovered and non-infected groups (105 participants in infected group, 128 participants in non-infected group) were subjected to blood sampling at 6 months post-vaccination. Anti-SARS-CoV-2 IgG antibody test was done using Chemiluminescence method. Comparison of antibody levels between COVID-recovered and non-infected groups was made. Results were compiled and statistically analyzed using SPSS version 21. Out of 233 study participants, males were 183 (78%) while females were 50 (22%), mean age being 35.93 years ± 8.298. Mean Anti-SARS-CoV-2 S IgG levels among COVID-recovered group was 1342 U/ml and among non-infected group was 828 U/ml at 6 months post-vaccination. Mean antibody titers in COVID-19 recovered group are higher than in non-infected group at 6 months post-vaccination in both groups.

Characterization of rotavirus strains isolated from children with acute gastroenteritis in Rawalpindi, Pakistan.

Diarrhea is the leading cause of childhood morbidity and mortality particularly in developing countries and rotavirus has been identified as the major pathogen associated with diarrheal infections. This study was conducted to detect genotypic distribution of predominant rotavirus strains circulating in children suffering from acute gastroenteritis in Rawalpindi, Pakistan. Stool specimens were collected from children ≤5 years of age, visiting Military Hospital, Rawalpindi, with signs and symptoms of acute gastroenteritis. Two hundred and eighty-four specimens were collected during the period from April 2017 to March 2018. Enzyme immunoassay was performed for detection of rotavirus and reverse transcription-PCR (RT-PCR) was carried out for amplification of VP7 and VP4 gene segments followed by multiplex PCR using genotype-specific primers. Out of 284 children, 71 were found rotavirus positive and among them, 54% were females and 46% males. Our findings showed 92% of infection among children ≤2 years of age, while, the peak age of rotavirus incidence was found to be 6-12 months. Although, rotavirus infection was observed throughout the year but frequency increased in winter. Subtype G1P[8] was more prevalent followed by G2P[4], G3P[8], and G4P[6] subtypes. The results of this study provide insight into the disease burden as well as information on rotavirus diversity which will be useful to develop future strategies to control and prevent diarrheal infections among children.

BK virus associated haemorrhagic cystitis in related donor haematopoietic stem cell transplant recipients: A developing country experience.

A retrospective cross sectional study was conducted at the Virology Department, Armed Forces Institute of Pathology (AFIP) and Armed Forces Bone Marrow Transplant Centre (AFBMTC), Rawalpindi, from January 2016 to July 2018. Medical records of 193 patients were examined to determine the number of patients developing Haemorrhagic Cystitis associated with BK virus (BKV). BKV PCR testing was done on the patients' urine samples. Cytomegalovirus reactivation was also assessed weekly from day 30 to day 100, by CMV quantitative PCR testing on blood samples. Out of 193 patients, 11 (5.6%) developed haemorrhagic cystitis and all these patients were positive for BKV on urine samples. The maximum number of positive cases, i.e. 5 (2.6%) was in the age group three months to 10 years. Primary disease in seven out of 11 cases was Beta-Thalassemia Major.

Coding-Complete Genome Sequence of a SARS-CoV-2 Strain Isolated in Gilgit, Pakistan.

Here, we report the coding-complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate obtained from a nasopharyngeal swab from the first patient with COVID-19 in Gilgit, Pakistan.

Phylogenetic and sequencing analysis of Chikungunya virus strains from local Pakistani population.

OBJECTIVE: To outline the genotype of Chikungunya virus strains and to form their phylogenetic tree. METHODS: The cross-sectional study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from August 2016 to September 2017, and comprised suspected Chikungunya samples. The viral ribonucleic acid was extracted and amplified. The purified product was sequenced using Sanger dideoxy method. Phylogenetic tree was constructed. Sequences generated were checked for alignment with sequences reported globally. Structural prediction and analysis was performed using Phyre2 and the models were visualised in PyMol2.2. RESULTS: In the 11 suspected samples, 7(63.6%) were found to be positive for Chikungunya. Of them, 5(71.4%) sequences generated were found to be aligned with other full structural polyprotein sequences, having 99% similarity with amino acid sequences. CONCLUSIONS: The Chikungunya strains in the study belonged to the East/Central/South African genotype.

JC Virus Infection: A Clinical Conundrum.

John Cunningham virus (JCV), a member of polyomaviridae family, has been described as a cause of the progressive multifocal leukoencephalopathy (PML). It is a potentially fatal, disabling demyelinating infection of the brain occurring mostly in the setting of immunosuppression. A few cases of JCV-associated meningitis and encephalitis have been described in literature. We report a case presenting with laboured breathing and reduced conscious level, who after thorough investigations was diagnosed to be a case of PML.

Influenza Virus Subtyping by Multiplex PCR during Winter Months of 2017-2018.

OBJECTIVE: To determine the frequency of infections caused by Influenza viruses, i.e. Influenza A (H1N1) pdm09, Influenza A (H3N2) and Influenza B in patients presenting with respiratory tract infections, i.e. influenza-like illness (ILI) and severe acute respiratory illness (SARI). STUDY DESIGN: Descriptive, cross-sectional study. PLACE AND DURATION OF STUDY: Department of Virology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from October 2017 to February 2018. METHODOLOGY: A total of 624 samples from patients with respiratory tract infections (both ILI and SARI) were included in the study. Specimens collected from the patients included nasal swabs and throat swabs, which were transported in viral transport medium (VTM) to Virology Department, AFIP. Multiplex PCR was done for Influenza A (H1N1) pdm09, Influenza A (H3N2) and Influenza B. RESULTS: A total of 200 (32%) samples were found to be positive for Influenza viruses. Out of total 624 samples analysed, 220 (35.3%) were from females and 404 (64.7%) from males. Among these, 510 (81.7%) presented with ILI and 114 (18.3%) with SARI. Among positive samples, 120 (19.2%) samples were positive for H1N1, 61 (9.8%) for H3N2 and 19 (3%) were positive for Influenza B. Highest number of positive cases occurred in the month of January, i.e. 148 (74%) cases. Only 3 (2.5%) patients out of 120 infected with H1N1 were in age group-I (0-5 years). While in age group-II (6-30 years), age group-III (31-60 years), and age group-IV (>60 years); 39 (32.5%), 63 (52.5%) and 15 (12.5%) patients were infected by H1N1, respectively. Maximum patients with H3N2 infection were in age group-III; 30 (49.2%) of the total 61. Commonest Influenza subtype in age group-IV was H3N2 found in 20 (32.8%) patients, followed by H1N1 in 15 (12.5%) patients. CONCLUSION: The dominant subtype in our set-up, during winter of 2017-2018, was Influenza A (H1N1) pdm09. Highest numbers of positive cases were recorded in the month of January. People with ILI and SARI should be tested for Influenza viruses to avoid unnecessary use of antibiotics.

Detection of West Nile virus lineage 1 sequences in blood donors, Punjab Province, Pakistan.

OBJECTIVES: This study was performed to determine the presence of West Nile virus (WNV) in mosquito specimens and human blood donors in Pakistan. METHODS: A total of 4150 mosquito specimens were collected using CO2-baited traps from five selected districts of Punjab Province, Pakistan. The mosquitoes were taxonomically identified using standard morphological keys, resulting in 166 pools. In addition, 1070 serum samples were collected from human blood donors. RNA was extracted from mosquito and human samples and screened for WNV using a reverse transcriptase PCR (RT-PCR). RESULTS: None of the mosquito pools tested positive for WNV, whereas three samples from asymptomatic humans tested positive. To determine the WNV strains, partial sequences were compared against a global representation of 23 WNV sequences. The study strains were determined to come from WNV lineage 1. CONCLUSIONS: This study is novel in reporting the circulation of lineage 1 WNV in Pakistan. Given its ability to transmit from human to human via blood transfusion, this highlights the urgent need for nationwide surveillance to assess the distribution and impact of WNV in Pakistan. Determining the source of human infection will require more extensive mosquito sampling.

Comparison of chitinase-3-like protein 1, aspartate aminotransferase-to-platelet ratio index, and fibrosis-4 index with shear-wave elastography.

BACKGROUND: In the past, there has been an exponential increase in the potential biomarkers that can be used for staging of liver fibrosis. In light of intraobserver and intralobular variations, criticism has been directed at liver biopsy, and its efficacy has been challenged. Shear-wave elastography (SWE) has become a routine method for pre-assessment of liver fibrosis. Serum markers such as chitinase-3-like protein 1 (CHI3L1) also known as YKL-40, aspartate aminotransferase-to-platelet ratio index, and fibrosis-4 (Fib-4) index have been researched as potential alternates to detect liver fibrosis. STUDY: A total of 150 enrolled patients with chronic hepatitis underwent serum analysis to estimate CHI3L1 or YKL-40 level, aspartate aminotransferase-to-platelet ratio index, and Fib-4 index. These patients also underwent SWE. RESULTS: The distribution of fibrosis grade according to SWE was F0: 46 patients, F1: 31 patients, F2: 16 patients, F3: four patients, and F4: 53 patients. Receiver operating characteristic curve analysis for F0-F1 versus F2-F3, F0-F1 versus F4, and F2-F3 versus F4 gave area under curve values of 0.56 (P>0.05), 0.76 (P<0.01), and 0.75, respectively (P<0.01) for aspartate aminotransferase-to-platelet ratio index; of 0.65 (P<0.05), 0.78 (P<0.01), and 0.7, respectively (P<0.05) for Fib-4 index; and 0.98, 0.99, and 0.95, respectively (P<0.01 for all) for CHI3L1. CONCLUSION: CHI3L1 could be used as a preliminary tool to assess mild/absent fibrosis from significant fibrosis and cirrhosis.

HCV-RNA Pcr Positivity In Hcv Antibody Negative Patients Undergoing Haemodialysis.

BACKGROUND: It's estimated that almost 2.2% of the world's inhabitants suffer from hepatitis C virus (HCV). The most common cause of chronic liver disease in haemodialysis centres is due to HCV. In 1993, it was first described by Bukh and colleagues that HCV viremia can occur without any detectable antibodies to the HCV. Keeping this in mind the purpose of this cross-sectional study was to assess the frequency of HCV in haemodialysis patients by PCR who are serologically negative for HCV. METHODS: This cross-sectional study was conducted from 1st June to 31st December 2016 on all haemodialysis patients at MH Rawalpindi. Epidemiological data for gender, age, duration on haemodialysis, cause of chronic renal failure and any associated risk factor for acquiring hepatitis C infection was asked. Patients undergoing haemodialysis were investigated by fourth generation ELISA for Anti HCV antibodies, HCV DNA polymerase chain reaction, HCV genotype (where required) and liver function test were also done. RESULTS: A total of 201 patients were undergoing haemodialysis. Among these patients 73 were hepatitis "C" negative and 128 were hepatitis "C" positive. Among the 73 patients who were hepatitis C negative by ELISA method 17 (23%) were PCR positive. Of the 17 patients 13 (76.5%) were men and 4 (23.5%) were women. The mean age of the patients was 49.7±18.0 years and mean duration of haemodialysis was 4.4±4.1 months. The most common cause of CKD requiring haemodialysis was hypertension (64.7%). The most common genotype was type 1 (58.8%) followed by genotype 3 (41.2%). The mean viral load was 23583615.70 IU. CONCLUSIONS: HCV-RNA detection by PCR should be used as standard of care to detect HCV infection in patients undergoing haemodialysis.

Multiplex polymerase chain reaction-based serotype analysis of dengue virus during 2015 dengue outbreak in Pakistan.

Dengue is one of the most important arthropod-borne viral diseases. It is endemic in > 125 countries including Pakistan, with a global incidence of 50-200 million. We determined the frequency of different serotypes of dengue virus to highlight its hyperendemicity in Rawalpindi, Pakistan. Between May and October 2015 we analysed the serum samples of 140 patients with a suspicion of dengue, using ELISA and multiplex polymerase chain reaction. One hundred and eight were infected with serotype 2, 16 with serotype 3, 7 with serotype 4 and 3 with serotype 1. Three patients were infected with serotypes 1 and 2, and 1 each with serotypes 1 and 4 and serotypes 2 and 3. Incidence of dengue has increased many fold in the past 50 years and has expanded to areas that were previously free from the disease. Serotype 2 was predominant in our population followed by serotype 3. There is currently no specific treatment for dengue, and vector control and vaccination are the only effective methods to prevent future outbreaks.

Cytomegalovirus Retinitis in Immunocompetent Young Male.

Cytomegalovirus (CMV) retinitis is a sight-threatening form of posterior uveitis affecting patients with Acquired Immunodeficiency Syndrome (AIDS), especially those with CD4 count <50 cells/mm3. There are few reported cases of CMV retinitis in patients with CD4 count >100 cells/mm3. Avirostatic agent like Ganciclovir has good response rate when given as intravitreal injection. Here, we report a case of CMV retinitis in a young immunocompetent male who presented with history of progressive loss of vision in both eyes despite receiving oral and intra-vitreal steroids. At the time of diagnostic testing, there was no history of high dose immunosuppressant therapy. CMV infection was confirmed by Polymerase Chain Reaction (PCR) for viral deoxyribonucleic acid (DNA) testing. Physicians treating such cases should take into account infectious causes of retinal vasculitis before starting anti-inflammatory therapy. Proper diagnosis should precede the treatment as far as possible.

Nucleic Acid Amplification Test For Detection Of West Nile Virus Infection In Pakistani Blood Donors.

BACKGROUND: The study was planned to determine the presence of West Nile Virus (WNV) infection in Pakistani blood donors, using Nucleic Acid Amplification Test (NAT). METHODS: The blood donors for study were selected on the basis of the standard questionnaire and routine screening results. Six donors were pooled using an automated pipettor and NAT for WNV was performed on Roche Cobas s 201 NAT system. The reactive pools were resolved in Individual Donation-NAT (ID-NAT) format and a sample from FFP bags of reactive donations was retrieved. NAT was again performed on retrieved plasma bag (RPB) sample to confirm the reactive donations. The donors were also recalled and interviewed about history of illness related to recent WNV infection. RESULTS: After serological screening of 1929 donors during the study period, 1860 donors were selected for NAT test for WNV detection. The mean age of the donors was 28±8.77 (range: 18-57 years). 1847 (99.3%) donors were male and 13 (0.7%) were female. NAT for WNV identified six initially reactive pools (0.32%). On follow-up testing with RPB samples, 4 donors (0.21%) were found confirmed reactive for WNV RNA (NAT yield of 1 in 465 blood donors). CONCLUSIONS: WNV is a threat to safety of blood products in Pakistan. A screening strategy can be implemented after a large-scale study and financial considerations. One of the reduced cost screening strategies is seasonal screening of blood donors for WNV, with pooling of samples.

Role of interferon gamma and tumor necrosis factor-related apoptosis-inducing ligand receptor 1 single nucleotide polymorphism in natural clearance and treatment response of HCV infection.

Hepatitis C virus (HCV) pathogenesis and treatment outcomes are multifactorial phenomena involving both viral and host factors. This study was designed to determine the role of tumor necrosis factor-related apoptosis-inducing ligand receptor 1(TRAIL-R1) and interferon gamma (IFN-γ) genetic mutations in susceptibility and response to interferon-based therapy of hepatitis C virus (HCV) infection. The detection of TRAIL-R1 rs4242392 and IFN-γ rs2069707 single nucleotide polymorphisms was completed in 118 chronic HCV patients and 96 healthy controls by allele-specific polymerase chain reaction and restriction fragment length polymorphisms polymerase chain reaction. Patients were further categorized into sustained virological responder (SVR) and nonresponder (NR) groups on the basis of their response to interferon-based therapy for HCV infection. Real-time PCR was used for HCV quantification. HCV genotyping was performed by Ohno's method. The results demonstrated that the distribution of the TRAIL-R1 rs4242392TT genotype was significantly higher in the SVR group (78%) compared to the NR group (36%). It showed that chronic HCV patients possessing the TRAIL-R1 rs4242392TT genotype are better responders to interferon-based therapy (p<0.05). The prevalence of the TRAIL-R1 rs4242392TT genotype in healthy controls and chronic HCV patients was 56% and 65% respectively. It indicated that there is the TRAIL-R1 rs4242392 genetic variation plays no role in the spontaneous clearance of HCV infection (p>0.05). The distribution of IFN-γ rs2069707 was the opposite to TRAIL-R1 rs4242392 prevalence, that is, there was high distribution of the IFN-γ rs2069707GG genotype in patients and healthy controls (p<0.05), while the prevalence of IFN-γ rs2069707GG in SVR and NR groups was comparable (p>0.05). In conclusion, genetic variation of TRAIL-R1 rs4242392 is linked with response to interferon-based therapy for HCV infection, and genetic variation IFN-γ rs2069707 is associated with natural clearance of HCV infection.

Impact of nucleic acid amplification test on screening of blood donors in Northern Pakistan.

BACKGROUND: The Armed Forces Institute of Transfusion located in Rawalpindi, Northern Pakistan, acts as a regional blood center with more than 50,000 donations collected annually. Nucleic acid amplification testing (NAT) was introduced in our institution in September 2012 for screening all seronegative blood donors. STUDY DESIGN AND METHODS: The study was conducted from September 21, 2012, to September 20, 2013. Samples from the seronegative donors were run on cobas s 201 platform (Roche) in pools of six. Reactive donors were followed up for further confirmatory testing to rule out false-positive results. Viral load estimation was done for all NAT-reactive donors. RESULTS: After serologic screening of 56,772 blood donors, 2334 were found to be reactive; 719 (1.27%) were reactive for hepatitis B surface antigen, 1046 (1.84%) for antibody to hepatitis C virus (anti-HCV), 12 (0.02%) for antibody to human immunodeficiency virus, and 557 (0.98%) for syphilis antibodies. A total of 27 NAT-reactive donors were confirmed after testing 54,438 seronegative donors, with an overall NAT yield of one in 2016 donors: 23 for hepatitis B virus (HBV) DNA (HBV NAT yield, 1:2367) and four for HCV RNA (HCV NAT yield, 1:13,609). The residual risk after NAT implementation, calculated for the first-time blood donors, was 62.5 and 4.4 per million donors for HBV and HCV, respectively. CONCLUSIONS: NAT has improved the safety of blood products at our transfusion institution. Confirmation of NAT results must always be done either on follow-up samples or on samples from the retrieved frozen plasma bag.

End-of-Treatment-Response in patients treated for hepatitis C virus with standard interferon and ribavirin based on viral load.

OBJECTIVE: To determine the End-of-Treatment-Response (ETR) to standard interferon and ribavirin based regimen in patients of chronic hepatitis C and to compare the ETR response in low and high viral load groups. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Virology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from March 2012 to May 2013. METHODOLOGY: Patients with chronic hepatitis C virus infection were included in the study. Pre-treatment viral load was determined by RoboGene Quantification kit. Based on viral load, the 400 patients were divided into two equal groups of low viral load (< 800,000 IU/ml) and high viral load (> 800,000 IU/ml). The patients were treated with standard interferon alpha (3 million units subcutaneously thrice weekly) and ribavirin (10.6 mg/kg body weight) for 6 months. ETR was measured using Sacace Biotechnologies Qualitative kit. Chi-square test was used to compare the ETR in the two viral load groups. P-value < 0.05 was considered as significant. RESULTS: Out of 400 patients, 206 (51.5%) were males and 194 (48.5%) were females. Two hundred seventy (67.5%) patients achieved ETR and 130 (35.5%) failed to do so. In low viral load group, 145 (72.5%) patients achieved and 55 (27.5%) patients did not achieve ETR. In high viral load group, 123 (61.5%) patients achieved and 77 (38.5%) did not achieve ETR. The difference in ETR between low and high viral load groups was statistically significant (p=0.019). CONCLUSION: End-of-treatment-response in patients treated for hepatitis C virus with standard interferon and ribavirin was greater in patients with low viral load as compared to patients with high viral load.

Molecular and serological assessment of chronic HBV carriers and additional burden of applying updated guidelines in Pakistan.

OBJECTIVE: To assess the additional burden of the patients eligible for treatment, based on recommendations on viral load, in the light of 2009 version of AASLD guidelines, as compared to 2004 guidelines and to determine the frequency of HBeAg in chronic HBV carriers. STUDY DESIGN: Descriptive cross-sectional study. PLACE AND DURATION OF STUDY: Virology Department, Armed Forces Institute of Pathology, Rawalpindi, from November 2010 to January 2012. METHODOLOGY: Persons with chronic HBV infection, reporting for HBV DNA PCR test, were included in the study and blood samples were collected. HBV DNA load was determined by Real Time PCR. HBsAg and HBeAg were tested by ELISA. RESULTS: Out of the 801 subjects positive for HBsAg, 74 (9.24%) were positive for HBeAg. Out of them, 113 (14.1%) had HBV DNA load > 100,000 copies/ml and were eligible for treatment according to AASLD 2004 guidelines. Forty one (5.1%) had HBV load between 10,000 and 100,000 copies/ml, and were additionally eligible for treatment as per AASLD 2009 guidelines. The 5.1% of 4.5 million estimated HBV carries in Pakistan comes to 229500. CONCLUSION: There was a low HBeAg positivity and HBV DNA positivity in our chronic HBV infected persons. Moreover, there is an increase of 229500 potential candidates for HBV treatment in Pakistan based on viral load testing, according to the AASLD 2009 guidelines when compared with 2004 guidelines. The increase in the number of candidates for treatment may require an additional expenditure of tens of billions of rupees.

Hepatitis A virus infection -- shifting epidemiology.

OBJECTIVE OF THE STUDY: To determine the age distribution in HAV infection and seasonal variations in the prevalence of acute viral hepatitis caused by hepatitis A virus. STUDY DESIGN: A descriptive study. PLACE AND DURATION: The study was carried out on the patients reporting at Virology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, for determination of hepatitis A virus (HAV) IgM antibody, from July 2003 to June 2004. PATIENTS AND METHODS: Altogether 626 patients with clinical suspicion of hepatitis A virus infection were referred to AFIP Rawalpindi for this test. Blood samples were collected and sera were separated and transferred to plastic aliquots that were stored at -20 degrees C in a retrievable fashion until utilized in testing. The testing for ant-HAV IgM was carried out with the help of a commercial Enzyme Linked Immunosorbant Assay (ELISA) using reagent kits of DiasSorin (Germany) for HAV IgM antibodies. RESULTS: The HAV IgM positive rate was 40.57% (252/626). Those tested included the sporadic cases as well as the patients from outbreak in two schools of Nowshera cantonment. The age of patients testing positive for HAV IgM, ranged from 03 to 27 years. There was a statistically significant seasonal difference in rate of positivity in different months of the calendar year. An outbreak of HAV infection was seen in the children of two neighboring schools of a cantonment, in which 44 children in different classes developed clinical jaundice. CONCLUSION: HAV infection occurs in a significant proportion of young people with a clinical suspicion of HAV infection. There is a changing trend of developing hepatitis A in the age beyond 18 years and in outbreaks, which was not there in our patients previously due to universal immunity found against HAV by the age of 18. It was because of chances of consumption of polluted food.

Winter peak of respiratory syncytial virus in Islamabad.

A study was carried out on 391 cases of bronchiolitis and pneumonia from different paediatric units in Rawalpindi/Islamabad, Pakistan. A clear winter spike of respiratory syncytial virus (RSV) was noted. It was found that there was a substantial increase of 30-50% in the positivity of RSV from December to February.