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.

Improved catalytic efficiency of chimeric xylanase 10B from Thermotoga petrophila RKU1 and its synergy with cellulases.

TpXyl10B is a glycoside hydrolase family 10 xylanase of hyperthermophile Thermotoga petrophila RKU-1. This enzyme is of considerable importance due to its thermostability. However, in its native state, this enzyme does not possess any carbohydrate-binding module (CBM) for efficient binding to plant biomass. In this study CBM6 from Clostridium thermocellum was attached to the N- and C-termini of TpXyl10B, thereby producing the variants TpXyl10B-B6C and TpXyl10B-CB6, respectively. TpXyl10B-B6C showed 5-7 folds increased activity on Beechwood xylan and the different types of plant biomass as compared to that from the catalytic domain only. However, the activity of TpXyl10B-CB6 decreased 0.6-0.8 folds on Beechwood xylan and plant biomass compared to the catalytic domain. We explained these results through molecular modeling, which showed that binding residues of CBM6's cleft B, which were previously reported to show no contribution towards binding due to steric hindrance from a loop region, were exposed in a favorable position in TpXyl10B-B6C such that they efficiently bound the substrate. In contrast, these binding residues of CBM6 in TpXyl10B-CB6 were exposed opposite to the catalytic residues; thus, binding to the substrate resulted in decreased exposure of catalytic residues to the substrate. CD spectroscopy and thermostability assays showed that TpXyl10B-B6C was highly thermostable, having a melting point > 90 °C, which is relatively higher than that of the other variant, TpXyl10B-CB6. In addition, this xylanase variant showed synergism with cellulases for the hydrolysis of plant biomass. Therefore, TpXyl10B-B6C, an engineered xylanase in this study, can be a valuable candidate for industrial applications.

Identification of host biomarkers from dried blood spots for monitoring treatment response in extrapulmonary tuberculosis.

There is a lack of objective tools for monitoring treatment response in extrapulmonary tuberculosis (EPTB). This study aimed to explore the utility of inflammatory biomarkers from the dry blood spots (DBS) as a tool for monitoring treatment response in EPTB. In a prospective cohort study, 40 inflammatory biomarkers were investigated in DBS samples from 105 EPTB cases using a Luminex platform. The samples were taken before, and, at the end of the 2nd and 6th months of treatment. A total of 11 inflammatory host biomarkers changed significantly with treatment in all EPTB patients. CXCL9/MIG, CCL20, CCL23, CXCL10/IP-10, CXCL1, CXCL2, and CXCL8 significantly declined in our cohort of EPTB (48 TB pleuritis and 57 TB lymphadenitis) patients at both time points. A biosignature consisting of MIG, CCL23, and CXCL2, corresponded with the treatment response in 81% of patients in the 2nd month and 79% of patients at the end of treatment. MIG, CCL23, IP-10, and CXCL2 changed significantly with treatment in all patients including those showing partial clinical response at the 2nd month of treatment. The changes in the levels of inflammatory biomarkers in the DBS correspond with the treatment success and can be developed as a routine test in low-resource settings.

Spatial distribution and computational modeling for mapping of tuberculosis in Pakistan.

BACKGROUND: Tuberculosis (TB) like many other infectious diseases has a strong relationship with climatic parameters. METHODS: The present study has been carried out on the newly diagnosed sputum smear-positive pulmonary TB cases reported to National TB Control Program across Pakistan from 2007 to 2020. In this study, spatial and temporal distribution of the disease was observed through detailed district wise mapping and clustered regions were also identified. Potential risk factors associated with this disease depending upon population and climatic variables, i.e. temperature and precipitation were also identified. RESULTS: Nationwide, the incidence rate of TB was observed to be rising from 7.03% to 11.91% in the years 2007-2018, which then started to decline. However, a declining trend was observed after 2018-2020. The most populous provinces, Punjab and Sindh, have reported maximum number of cases and showed a temporal association as the climatic temperature of these two provinces is higher with comparison to other provinces. Machine learning algorithms Maxent, Support Vector Machine (SVM), Environmental Distance (ED) and Climate Space Model (CSM) predict high risk of the disease with14.02%, 24.75%, 34.81% and 43.89% area, respectively. CONCLUSION: SVM has a higher significant probability of prediction in the diseased area with a 1.86 partial receiver-operating characteristics (ROC) value as compared with other models.

Fusion peptide constructs from antigens of M. tuberculosis producing high T-cell mediated immune response.

Non availability of effective anti-TB vaccine impedes TB control which remains a crucial global health issue. A fusion molecule based on immunogenic antigens specific to different growth phases of Mycobacterium tuberculosis can enhance T-cell responses required for developing a potent vaccine. In this study, six antigens including EspC, TB10.4, HspX, PPE57, CFP21 and Rv1352 were selected for constructing EspC-TB10.4 (bifu25), TnCFP21-Rv1352 (bifu29), HspX-EspC-TB10.4 (trifu37), HspX-TnCFP21-Rv1352 (trifu44) and HspX-EspC-TB10.4-PPE57 (tetrafu56) fusion proteins. Th1-cell epitopes of EspC, PPE57 and Rv1352 antigens were predicted for the first time using different in silico tools. The fusion molecule tetrafu56, which consisted of antigens from both the replicating and the dormant stages of Mtb, induced a release of 397 pg/mL of IFN-γ from PBMCs of the active TB patients. This response was comparable to the response obtained with cocktail of the component antigens (396 pg/mL) as well as to the total of the responses obtained separately for each of its component antigens (388 pg/mL). However, PBMCs from healthy samples in response to tetrafu56 showed IFN-γ release of only 26.0 pg/mL Thus a previous exposure of PBMCs to Mtb antigens in TB plasma samples resulted in 15-fold increase in IFN-γ response to tetrafu56 as compared to the PBMCs from the healthy controls. Hence, most of the T-cell epitopes of the individual antigens seem to be available for T-cell interactions in the form of the fusion. Further investigation in animal models should substantiate the immune efficacy of the fusion molecule. Thus, the fusion tetrafu56 seems to be a potential candidate for developing an effective multistage vaccine against TB.

Enhanced serodiagnostic potential of a fusion molecule consisting of Rv1793, Rv2628 and a truncated Rv2608 of Mycobacterium tuberculosis.

Serodiagnosis of tuberculosis (TB) can be rapid, reliable and cost-effective if the issue of variable antibody responses of TB patients against different Mycobacterium tuberculosis (Mtb) antigens can be overcome by developing fusion proteins containing epitopes from multiple antigens of Mtb. In this study, Mtb antigens Rv1793, Rv2628, Rv2608 and a truncated variant produced by removing non-epitopic region from N-terminal of Rv2608 (tnRv2608), and the fusion protein Rv1793-Rv2628-tnRv2608 (TriFu64), were expressed in E. coli and purified. Plasma samples from TB patients characterized by sex, age and sputum/culture positivity, were used to compare the sensitivity of the single antigens with the fusion protein. Sensitivity of Rv1793, Rv2628 and Rv2608, was 27.8%, 39% and 36.3%, respectively. Truncation of Rv2608 increased sensitivity by approximately 35% in confirmed TB cases. Sensitivity of the fusion construct, TriFu64 increased to 66% with a specificity of 100%. Importantly, tnRv2608 was better able to detect sputum and culture negative patients, and this carried through to the fusion protein. We demonstrate that fusion of Mtb proteins ensures broad sensitivity across disease types, sex and age groups in a Pakistani population.

Host biomarkers for monitoring therapeutic response in extrapulmonary tuberculosis.

PURPOSE: The aim of this study was to explore the utility of inflammatory biomarkers in the peripheral blood to predict response to treatment in extrapulmonary tuberculosis (EPTB). METHODS: A Luminex xMAP-based multiplex immunoassay was used to measure 40 inflammatory biomarkers in un-stimulated plasma of 91 EPTB patients (48 lymphadenitis, and 43 pleuritis) before and at 2 and 6 months of treatment. RESULTS: Overall a significant change was observed in 28 inflammatory biomarkers with treatment in EPTB patients. However, MIG/CXCL9, IP-10/CXCL10, and CCL23 decreased in all patients' groups with successful treatment at both time points. At 2 months, 29/64 (45%) patients responded partially while 35/64 (55%) showed complete regress. Among good responders, a higher number of biomarkers (16/40) reduced significantly as compared to partial responders (1/40). Almost half (14/29) of partial responders required longer treatment than 6 months to achieve satisfactory response. The levels of MIG, IP-10, MIF, CCL22 and CCL23 reduced significantly among 80, 74, 60, 71, 51% good responders, as compared to 52, 52, 52, 59, 52% partial responders, respectively. A biosignature, defined by a significant decrease in any one of these five biomarkers, corresponded with satisfactory response to treatment in 97% patients at 2 month and 99% patients at 6 months of treatment. CONCLUSION: Change in inflammatory biomarkers correlates with treatment success. A five biomarker biosignature (MIG, IP-10, MIF, CCL22 and CCL23) could be used as an indicator of treatment success.

Serodiagnostic evaluation of fusion proteins from multiple antigens of Mycobacterium tuberculosis for active TB.

Tuberculosis (TB) is a global health problem, being prevalent in the developing countries. A rapid, reliable and cost effective diagnostic method would help in controlling TB in the endemic populations. Development of suitable fusion molecules detecting multiple antibodies produced against Mycobacterium tuberculosis antigens would enhance sensitivity of serodiagnostic assays. In this study, EspC, CFP7 and PPE57 antigens of M. tuberculosis were selected for constructing fusion molecules after prediction of B-cell epitopes using in silico tools. Fusion proteins EspC-CFP7, HspX-EspC-CFP7 and HspX-EspC-CFP7-PPE57 were expressed in E.coli (BL21). The serodiagnostic potential of the individual antigens and their fusions was analyzed by screening 230 plasma samples of pulmonary TB patients. The single antigens HspX, EspC, CFP7, PPE57 showed sensitivities of 30%, 31%, 22% and 35%, respectively. The fusion protein EspC-CFP7 showed sensitivity of 43%. Linking of HspX antigen to the N-terminus of EspC-CFP7 fusion molecule increased sensitivity to 58%, while joining PPE57 antigen to the C-terminus of HspX-EspC-CFP7 increased sensitivity to 69%. The fusion protein HspX-EspC-CFP7-PPE57 seems to be a promising molecule for use in the development of fusions with higher sensitivity.

Gut microbiome dysbiosis and correlation with blood biomarkers in active-tuberculosis in endemic setting.

Tuberculosis (TB) is the largest infectious disease with 10 million new active-TB patients and1.7 million deaths per year. Active-TB is an inflammatory disease and is increasingly viewed as an imbalance of immune responses to M. tb. infection. The mechanisms of a switch from latent infection to active disease is not well worked out but a shift in the immune responses is thought to be responsible. Increasingly, the role of gut microbiota has been described as a major influencer of the immune system. And because the gut is the largest immune organ, we aimed to analyze the gut microbiome in active-TB patients in a TB-endemic country, Pakistan. The study revealed that Ruminococcacea, Enetrobactericeae, Erysipelotrichaceae, Bifidobacterium, etc. were the major genera associated with active-TB, also associated with chronic inflammatory disease. Plasma antibody profiles against several M. tb. antigens, as specific biomarkers for active-TB, correlated closely with the patient gut microbial profiles. Besides, bcoA gene copy number, indicative of the level of butyrate production by the gut microbiome was five-fold lower in TB patients compared to healthy individuals. These findings suggest that gut health in TB patients is compromised, with implications for disease morbidity (e.g., severe weight loss) as well as immune impairment.

Designing fusion molecules from antigens of Mycobacterium tuberculosis for detection of multiple antibodies in plasma of TB patients.

Tuberculosis (TB) is amongst the deadliest diseases worldwide. For effective control of TB a rapid, reliable and sensitive method for its diagnosis is essential. Serodiagnosis detecting multiple antibodies against antigens of Mycobacterium tuberculosis (Mtb) in blood samples could prove beneficial. Based on the epitope position in the molecule, two truncated variants of Rv1984c, i.e., Tn1Rv1984c and Tn2Rv1984c were expressed in Escherichia coli. Screening of the Rv1984c, Tn1Rv1984c and Tn2Rv1984c against 231 sera samples from the culture positive TB patients showed sensitivities of 34.2%, 49.4% and 26.8%, respectively. Another antigen Rv1352 was analyzed for the location of epitopes, which had not been reported before. A fusion molecule consisting of Tn1Rv1984c and Rv1352, expressed in E. coli, showed enhanced sensitivity of 62.8%. Joining another antigen Rv2031c to the N-terminus of Tn1Rv1984c-Rv1352, improved sensitivity to 71.4%. The fusion construct Rv2031c-Tn1Rv1984c-Rv1352 showed comparatively higher sensitivity of 73.4% in the male group as compared to 67% in the female group. Data derived for the secondary structure analysis through Circular Dichroism (CD) spectroscopy and prediction on the basis of molecular modelling was also in agreement. This construct can be a potential base for producing constructs with greater sensitivity through fusion of epitopes from additional Mtb antigens.

Field evaluation of a blood based test for active tuberculosis in endemic settings.

Over 9 million new active tuberculosis (TB) cases emerge each year from an enormous pool of 2 billion individuals latently infected with Mycobacterium tuberculosis (M. tb.) worldwide. About 3 million new TB cases per year are unaccounted for, and 1.5 million die. TB, however, is generally curable if diagnosed correctly and in a timely manner. The current diagnostic methods for TB, including state-of-the-art molecular tests, have failed in delivering the capacity needed in endemic countries to curtail this ongoing pandemic. Efficient, cost effective and scalable diagnostic approaches are critically needed. We report a multiplex TB serology panel using microbead suspension array containing a combination of 11 M.tb. antigens that demonstrated overall sensitivity of 91% in serum/plasma samples from TB patients confirmed by culture. Group wise sensitivities for sputum smear positive and negative patients were 95%, and 88%, respectively. Specificity of the test was 96% in untreated COPD patients and 91% in general healthy population. The sensitivity of this test is superior to that of the frontline sputum smear test with a comparable specificity (30-70%, and 93-99%, respectively). The multiplex serology test can be performed with scalability from 1 to 360 patients per day, and is amenable to automation for higher (1000s per day) throughput, thus enabling a scalable clinical work flow model for TB endemic countries. Taken together, the above results suggest that well defined antibody profiles in blood, analyzed by an appropriate technology platform, offer a valuable approach to TB diagnostics in endemic countries.

Seasonality and trend analysis of tuberculosis in Lahore, Pakistan from 2006 to 2013.

Tuberculosis (TB) is a respiratory infectious disease which shows seasonality. Seasonal variation in TB notifications has been reported in different regions, suggesting that various geographic and demographic factors are involved in seasonality. The study was designed to find out the temporal and seasonal pattern of TB incidence in Lahore, Pakistan from 2006 to 2013 in newly diagnosed pulmonary TB cases. SPSS version 21 software was used for correlation to determine the temporal relationship and time series analysis for seasonal variation. Temperature was found to be significantly associated with TB incidence at the 0.01 level with p=0.006 and r=0.477. Autocorrelation function and partial autocorrelation function showed a significant peak at lag 4 suggesting a seasonal component of the TB series. Seasonal adjusted factor showed peak seasonal variation in the second quarter (April-June). The expert modeler predicted the Holt-Winter's additive model as the best fit model for the time series, which exhibits a linear trend with constant (additive) seasonal variations, and the stationary R(2) value was found to be 0.693. The forecast shows a declining trend with seasonality. A significant temporal relation with a seasonal pattern and declining trend with variable amplitudes of fluctuation was observed in the incidence of TB.