Duration of respiratory sample stability at -80ºC for SARS-CoV-2 PCR.

Objective: To determine the stability of respiratory samples for SARS-CoV-2 PCR at standard laboratory ultra-freezer temperatures (-80°C). Methods: Five hundred and sixty-five archived, SARS-CoV-2 PCR positive patient specimens received at the Pathology Department of the Indus Hospital & Health Network between January 2021 and June 2021 were retested in June 2021. Samples had been stored at -70°C or below throughout this duration. Sample integrity following storage was assessed as the percentage of samples with reproducible results, and as consistency of cycle threshold (Ct) values between the original testing and the repeat testing. Results: Of the 565 samples evaluated in this study, 86% gave reproducible results upon retesting. However, there was no correlation between the duration of storage and result reproducibility, though the majority (69% for PCR Target-I and 78% for PCR Target-II respectively) of non-reproducible results had Ct values above 30. Similarly, there was a consistent increase of Ct values upon storage at ultra-freezer temperatures, though the effect again was more contingent upon freezing the sample in the ultra-freezer rather than the duration of storage. Conclusion: SARS-CoV-2 positive respiratory specimens for PCR can be stored for up to six months at -70°C or below without loss of sample integrity, though there is some loss of PCR-detected viral targets as evidenced by an immediate increased in the PCR-generated Ct values. In addition, samples with initial Ct values above 30 are more likely to give non-reproducible results.

Concurrent adult pulmonary tuberculosis prevalence survey using digital radiography and Xpert MTB/RIF Ultra and child interferon-gamma release assay Mycobacterium tuberculosis infection survey in Karachi, Pakistan: a study protocol.

Background: Assessment of the effectiveness of tuberculosis control strategies requires the periodic measurement of M. tuberculosis transmission in populations, which is notoriously difficult. One well-established method is to measure the prevalence of infectious pulmonary tuberculosis in the population which is then repeated at a second time point after a period of 'intervention', such as scale up of the Search-Treat-Prevent strategy of the Zero TB Cities initiative, allowing for a 'before and after' comparison.  Protocol: The concurrent adult pulmonary tuberculosis prevalence survey (using digital radiography and Xpert MTB/RIF Ultra) and child M. tuberculosis infection survey (using QuantiFERON-TB® Gold Plus) will primarily provide a baseline measure of the burden of adult infectious tuberculosis in Karachi and assess whether a large-scale interferon gamma release assay survey in children aged 2 to 4 years is feasible. The target population for the prevalence survey is comprised of a stratified random sample of all adults aged 15 years and above and all children aged 2 to 4 years resident in four districts in Karachi. The survey procedures and analyses to estimate pulmonary tuberculosis prevalence are based on the World Health Organization methodology for tuberculosis prevalence surveys. Ethics and dissemination: The study protocol has been approved by the Interactive Research Development / The Indus Hospital Research Centre Research Ethics Committee in Karachi, Pakistan and the London School of Hygiene & Tropical Medicine Research Ethics Committee. Due to non-representative sampling in this setting, where a large proportion of the population are illiterate and are reluctant to provide fingerprints due to concerns about personal security, verbal informed consent will be obtained from each eligible participant or guardian. Results will be submitted to international peer-reviewed journals, presented at international conferences and shared with participating communities and with the Provincial and National TB programme.

Barriers to Implementation of Optimal Laboratory Biosafety Practices in Pakistan.

The primary goal of biosafety education is to ensure safe practices among workers in biomedical laboratories. Despite several educational workshops by the Pakistan Biological Safety Association (PBSA), compliance with safe practices among laboratory workers remains low. To determine barriers to implementation of recommended biosafety practices among biomedical laboratory workers in Pakistan, we conducted a questionnaire-based survey of participants attending 2 workshops focusing on biosafety practices in Karachi and Lahore in February 2015. Questionnaires were developed by modifying the BARRIERS scale in which respondents are required to rate barriers on a 1-4 scale. Nineteen of the original 29 barriers were included and subcategorized into 4 groups: awareness, material quality, presentation, and workplace barriers. Workshops were attended by 64 participants. Among barriers that were rated as moderate to great barriers by at least 50% of respondents were: lack of time to read biosafety guidelines (workplace subscale), lack of staff authorization to change/improve practice (workplace subscale), no career or self-improvement advantages to the staff for implementing optimal practices (workplace subscale), and unclear practice implications (presentation subscale). A lack of recognition for employees' rights and benefits in the workplace was found to be a predominant reason for a lack of compliance. Based on perceived barriers, substantial improvement in work environment, worker facilitation, and enabling are needed for achieving improved or optimal biosafety practices in Pakistan.

Knowledge and practices of laboratory workers on standardized antimicrobial susceptibility testing and biosafety practices to prevent the spread of superbugs in Pakistan.

A cross-sectional survey using structured questionnaire was conducted to assess practices of microbiological laboratories working with pathogens. Forty-eight laboratory workers (50%) agreed that laboratory methods to detect antimicrobial resistance are not standardized in Pakistan, and 6% of the laboratory workers were not aware of the standardization of antimicrobial susceptibility testing in Pakistan. Reported rates of awareness regarding the role of waste disposal, disinfection, and handwashing in limiting the spread of antimicrobial resistance were 75%, 42%, and 81%, respectively. Our results provide baseline data for planning programs to train, supervise, and improve the operational quality of microbiological laboratories nationwide to prevent the spread of superbugs.

Differential live Mycobacterium tuberculosis-, M. bovis BCG-, recombinant ESAT6-, and culture filtrate protein 10-induced immunity in tuberculosis.

The high prevalence of Mycobacterium tuberculosis makes it imperative that immune responses to evaluate could be predictive of infection. We investigated live Mycobacterium- and recombinant antigen-induced cytokine and chemokine responses in patients with active tuberculosis (TB) compared with those of healthy controls from an area where TB is endemic (ECs). M. tuberculosis-, M. bovis BCG-, ESAT6-, and culture filtrate protein 10 (CFP10)-induced responses were determined in peripheral blood mononuclear cells from patients with pulmonary TB (n = 38) and ECs (n = 39). The levels of the cytokines gamma interferon (IFN-gamma) and interleukin-10 (IL-10) and the chemokines CCL2, CCL3, and CXCL9 were measured. The levels of M. tuberculosis- and BCG-induced IFN-gamma secretion were significantly reduced (P = 0.002 and P < 0.01, respectively), while the amount of IL-10 induced by both virulent (P < 0.01) and avirulent (P = 0.002) mycobacteria was increased in patients with TB. The ESAT6-induced IFN-gamma responses were increased in the patients with TB (P = 0.013) compared with those in the EC group. When tuberculin skin test (TST)-negative (TST(-); induration, <10 mm) and TST-positive (TST(+)) donors were studied separately, both TST(-) and TST(+) individuals showed increased IFN-gamma responses to M. tuberculosis compared with the responses of the patients with TB (P = 0.037 and P = 0.006, respectively). However, only TST(+) ECs showed reduced IFN-gamma responses to ESAT6 (P = 0.008) compared with the responses of the patients with TB. The levels of M. tuberculosis-induced CCL2 (P = 0.006) and CXCL9 (P = 0.017) were greater in the patients with TB. The levels of CCL3 secretion in response to Mycobacterium and antigen stimulation were comparable between the two groups. While the levels of ESAT6-induced chemokines did not differ between the patients with TB and the ECs, the levels of CFP10-induced CCL2 (P = 0.01) and CXCL9 (P = 0.001) were increased in the patients. These data indicate differential host IFN-gamma, CXCL9, and CCL2 responses to live mycobacteria and mycobacterial antigens and have implications for the identification of potential biomarkers of infection which could be used for the diagnosis of TB.

Immune profiling of leprosy and tuberculosis patients to 15-mer peptides of Mycobacterium leprae and M. tuberculosis GroES in a BCG vaccinated area: implications for development of vaccine and diagnostic reagents.

Mycobacterium leprae (ML) GroES has been shown to induce strong T cell responses in tuberculoid as well as in exposed healthy contacts of leprosy patients, and therefore this antigen has been the focus of study as a potential vaccine candidate. Paradoxically, we have shown that ML GroES also induces extremely high titres of IgG1 antibody in leprosy patients across the disease spectrum, a response associated with disease progression. IgG1 antibodies in leprosy also show a negative association with interferon-gamma, a critical T cell cytokine responsible for macrophage activation and intracellular killing of mycobacteria. We therefore queried if antibody and T cell responses were being evoked by different epitopes in ML GroES proteins. To address the issue of epitope recognition in mycobacterial diseases, we have analysed 16 peptides (15-mer peptides) spanning the entire ML and M. tuberculosis GroES protein in leprosy (n = 19) and tuberculosis (n = 9) patients and healthy endemic controls (n = 8). Our analysis demonstrates clearly that the dominant peptides evokingT cell and IgG subclass antibodies were different. The target of both T and B cell responses were cross-reactive epitopes in all groups. Differences in disease and healthy states related to the strength (mean intensity) of the T cell and antibody response. IgG1 and IgG3 antibodies were associated with disseminated disease and IgG 2 and IgG4 with disease limitation. Such comprehensive immune profiling of antigen-specific responses is critical to understanding the disease pathogenesis and also if these reagents are to be exploited for either diagnostic or vaccine purposes.

Cytokine profiles using whole-blood assays can discriminate between tuberculosis patients and healthy endemic controls in a BCG-vaccinated population.

Whole-blood assays (WB) provide a simple tool for assessing immune cytokine profiles which may be useful laboratory predictors of early disease, aiding the evaluation of new tuberculosis (TB) vaccines and offering insights into disease pathogenesis. Although BCG does not provide protection against pulmonary disease in TB endemic areas, it does modulate immune responses to mycobacterial antigens. It is important, therefore, to evaluate any new tool in an endemic setting in both BCG vaccinees and patients with tuberculosis. We have assessed the optimal conditions in terms of dose and kinetics of those cytokines which are released early (TNF-alpha, IL6 and TGF-beta, IL10) or (interferon [IFN]-gamma and IL5) in WB cultures stimulated with mitogens and mycobacterial antigens. Responses were studied in parallel in untreated TB patients and endemic control groups. Optimal responses to LPS (predominantly monocyte-derived) occurred on days 1-2, whereas for PHA (predominantly T-cell-derived), they were on days 3-5. Secreted Mycobacterium tuberculosis culture filtrate proteins (CFP) provided a stronger stimulus for monocyte-derived cytokines compared to PPD, but both antigens were comparable for induction of T-cell cytokines. Using unpaired Student's t-tests, pulmonary tuberculosis patients (P.TB; n=11), in response to CFP, showed higher monocyte-derived IL6 (p=0.023) and IL10 (p=0.042) compared to endemic controls (EC; n=13), and significantly suppressed T-cell-derived IFN-gamma (p=0.028) and IL5 (p=0.012) secretion but increased IL10 (p=0.047) on day 5, indicating that CFP is a strong stimulus for IL10 secretion in pulmonary TB patients. Extrapulmonary TB patients (E.TB; n=6) showed no elevation of early monocyte-derived cytokines to either PPD or CFP, but showed a marked suppression of the T-cell-derived cytokines IFN-gamma (PPD, p=0.015; CFP, p=0.05) and IL5 (PPD, p=0.05; CFP, p=0.015). Cytokine analysis in WB cultures is, therefore, able to discriminate between active tuberculosis infection and nondiseased healthy controls.