The Role of Biofilms, Bacterial Phenotypes, and Innate Immune Response in Mycobacterium avium Colonization to Infection.

Mycobacterium avium complex (MAC), is known for colonizing and infecting humans following inhalation of the bacteria. MAC pulmonary disease is notoriously difficult to treat and prone to recurrence. Both the incidence and prevalence MAC pulmonary disease have been increasing globally. MAC is well known to form biofilms in the environment. In vitro, these biofilms have been shown to aid MAC in epithelial cell invasion, protect MAC from phagocytosis, and cause premature apoptosis in macrophages. In vivo, the system of interactions between MAC, biofilms and host macrophages is complex, difficult to replicate in vitro and in animal models, has not been fully characterized. Here we present a three-dimensional agent-based model of a lung airway to help understand how these interactions evolve in the first 14 days post-bacterial inhalation. We parameterized the model using published data and performed uncertainty analysis to characterize outcomes and parameters' effects on those outcomes. Model results show diverse outcomes, including wide ranges of macrophage recruitment levels, and bacterial loads and phenotype distribution. Though most bacteria are phagocytosed by macrophages and remain intracellular, there are also many simulations in which extracellular bacteria continue to drive the colonization and infection. Initial parameters dictating host immune levels, bacterial loads introduced to the airway, and biofilm conditions have significant and lasting impacts on the course of these results. Additionally, though macrophage recruitment is key for suppressing bacterial loads, there is evidence of significant excess recruitment that fail to impact bacterial numbers. These results highlight a need and identify a path for further exploration into the inhalation events in MAC infection. Early infection dynamics could have lasting impacts on the development of nodular bronchiectatic or fibrocavitary disease as well as inform possible preventative and treatment intervention targeting biofilm-macrophage interactions.

Active and latent tuberculosis infections in patients treated with immune checkpoint inhibitors in a non-endemic tuberculosis area.

BACKGROUND: Immune checkpoint inhibitors (ICIs) blocking inhibitory immune pathways (e.g., programmed cell death protein-1/-ligand1 [PD-1/PD-L1]) have revolutionized cancer therapy for numerous malignancies. There have been an increasing number of cases of active tuberculosis (TB) reported in association with ICI use, and recent data suggest alterations in immune responses in TB by ICI. The aim of this study was to characterize the frequency of latent tuberculosis infection (LTBI) and active TB in a large cohort of ICI-treated patients in a low TB incidence area. METHODS: We conducted a retrospective review of all ICI-treated patients tested for TB between January, 1997 and August, 2018. Data extracted included patient demographics, TB risk factors, latent/active TB diagnosis and treatment, tumor type, ICI used, immunosuppressive medications, and mortality related to TB. RESULTS: We identified 1844 ICI-treated patients, including 30 abnormal TB test results. Two patients were diagnosed with active TB, both prior to starting ICI therapy. One patient was treated for TB prior to starting ICI and the other patient was successfully treated concurrently. Seven patients were diagnosed with LTBI and none developed active TB. Twenty patients had indeterminate interferon gamma release assays (IGRA). CONCLUSION: Despite recent reports of TB in patients taking ICI, we found no patients developing TB during ICI therapy in our large retrospective cohort of ICI-treated cancer patients in a non-endemic TB area. The high rate of indeterminate IGRA results suggests the need for prospective research with better diagnostics to quantify the actual risk of TB in this patient population.

Antifungal prophylaxis in lung transplant: A survey of United States' transplant centers.

BACKGROUND: Antifungal prophylaxis strategies for lung transplant recipients vary without consensus or standard of care. Our current study aims to identify antifungal prophylaxis practices in the United States. METHODS: From November 29, 2018, to February 15, 2019, we emailed surveys to medical directors of adult lung transplant centers. An alternate physician representative was approached if continued non-response after three survey attempts. Descriptive statistics were used to report findings. RESULTS: Forty-four of 62 (71.0%) eligible centers responded. All Organ Procurement and Transplantation Networks were represented. Only four (9.1%) centers used pre-transplant prophylaxis for prevention of tracheobronchitis (3 of 4) and invasive fungal disease (4 of 4). Thirty-nine of forty (97.5%) centers used post-transplant prophylaxis: 36 (90.0%) universal and 3 (7.5%) pre-emptive/selective prophylaxis. Most centers used nebulized amphotericin with a systemic agent (26 of 36, 72.2%). Thirty-two of thirty-six (88.9%) centers continued universal prophylaxis beyond the hospital setting. Duration of prophylaxis ranged from the post-transplant hospitalization to lifelong with most centers (25 of 36, 69.4%) discontinuing prophylaxis 6 months or less post-transplant. CONCLUSION: Most United States' lung transplant centers utilize a universal prophylaxis with nebulized amphotericin and a systemic triazole for 6 months or less post-transplant. Very few centers use pre-transplant antifungal prophylaxis.

Comparison of the QuantiFERON-TB Gold Plus and QuantiFERON-TB Gold In-Tube Interferon Gamma Release Assays in Patients at Risk for Tuberculosis and in Health Care Workers.

The QuantiFERON-TB Gold Plus (QFT-Plus; Qiagen, Germantown, MD) interferon gamma release assay (IGRA) received FDA clearance in 2017 and will replace the prior version of the assay, the QFT-Gold In-Tube (QFT-GIT). Here, we compared performances of the QFT-Plus assay and the QFT-GIT version in a diverse patient population, including patients undergoing evaluation for or follow-up of latent tuberculosis infection (LTBI; n = 39) or active TB infection (n = 3), and in health care workers (HCWs; n = 119) at Mayo Clinic (Rochester, MN). Compared to the QFT-GIT, the QFT-Plus assay showed 91.2% (31/34) positive, 98.4% (124/126) negative, and 96.6% (156/161) overall qualitative agreement among the 161 enrolled subjects, with a Cohen's kappa value of 0.91 (excellent interrater agreement). Among the 28 patients diagnosed with LTBI at the time of enrollment, the QFT-GIT and QFT-Plus assays agreed in 24 (85.7%) patients; in all four discordant patients, the positivity of the QFT-GIT or QFT-Plus IGRA was associated with low-level interferon gamma (IFN-γ) reactivity, ranging from 0.36 IU/ml to 0.66 IU/ml. Additionally, we document a high degree of correlation between IFN-γ levels in the QFT-GIT TB antigen tube and each of the two QFT-Plus TB antigen tubes, as well as between the QFT-Plus TB1 and TB2 tubes (Pearson's correlation coefficients [R] > 0.95). Overall, we show comparable results between the QFT-GIT and QFT-Plus assays in our study population composed of subjects presenting with a diverse spectrum of TB infections. Our findings suggest that the necessary transition to the QFT-Plus assay will be associated with a minimal difference in assay performance characteristics.

Combinatorial Immunoprofiling in Latent Tuberculosis Infection. Toward Better Risk Stratification.

RATIONALE: Most immunocompetent patients diagnosed with latent tuberculosis infection (LTBI) will not progress to tuberculosis (TB) reactivation. However, current diagnostic tools cannot reliably distinguish nonprogressing from progressing patients a priori, and thus LTBI therapy must be prescribed with suboptimal patient specificity. We hypothesized that LTBI diagnostics could be improved by generating immunomarker profiles capable of categorizing distinct patient subsets by a combinatorial immunoassay approach. OBJECTIVES: A combinatorial immunoassay analysis was applied to identify potential immunomarker combinations that distinguish among unexposed subjects, untreated patients with LTBI, and treated patients with LTBI and to differentiate risk of reactivation. METHODS: IFN-γ release assay (IGRA) was combined with a flow cytometric assay that detects induction of CD25(+)CD134(+) coexpression on TB antigen-stimulated T cells from peripheral blood. The combinatorial immunoassay analysis was based on receiver operating characteristic curves, technical cut-offs, 95% bivariate normal density ellipse prediction, and statistical analysis. Risk of reactivation was estimated with a prediction formula. MEASUREMENTS AND MAIN RESULTS: Sixty-five out of 150 subjects were included. The combinatorial immunoassay approach identified at least four different T-cell subsets. The representation of these immune phenotypes was more heterogeneous in untreated patients with LTBI than in treated patients with LTBI or unexposed groups. Patients with IGRA(+) CD4(+)CD25(+)CD134(+) T-cell phenotypes had the highest estimated reactivation risk (4.11 ± 2.11%). CONCLUSIONS: These findings suggest that immune phenotypes defined by combinatorial assays may potentially have a role in identifying those at risk of developing TB; this potential role is supported by risk of reactivation modeling. Prospective studies will be needed to test this novel approach.

Diagnosis of latent tuberculosis infection with T-SPOT(®).TB in a predominantly immigrant population with rheumatologic disorders.

PURPOSE: The objective of this study is to compare how likely positive tuberculin skin test (TST) and T-SPOT(®).TB (TSPOT) results predict risk factors for tuberculosis in a predominantly immigrant patient population at risk of latent TB infection (LTBI) and with rheumatologic conditions requiring immunomodulatory therapy (IMT). METHODS: Prospective study conducted at a referral rheumatology clinic. Inclusion criteria included patients on various IMT, including immunosuppressive drugs that could predispose to TB progression. We studied risk factors associated with LTBI, test results, and tests' agreement. RESULTS: We studied 101 patients. Eighty (79.2 %) were from countries where TB is prevalent and Bacille Calmette-Guérin vaccination is placed routinely. Seventy-four (73.3 %) had rheumatoid arthritis and 92 (90.7 %) were on IMT. Among patients with both TST and TSPOT results, 25 (30.9 %) were TST(+) and 20 (24.7 %) had TSPOT(+) results. Fifteen patients (18.5 %) had TST(+)/TSPOT(+) results, and 51 (63.0 %) had TST(-)/TSPOT(-) results (agreement = 81.5 %; kappa = .54 [95 % CI, .34-.74; P < .001]). Each TSPOT(+) and TST(+) results were independently associated with immigrant status and prior residence in a TB prevalent country after adjustment for immunosuppressive therapy: Adjusted OR(TSPOT+)=6.6 (95 % CI, 1.2-123.3; P = .027); and adjusted OR(TST+)=11.2 (95 % CI, 2.0-209.5; P = .003). Seven out of 10 TST(+)/TSPOT(-) cases had a TST ≥15 mm induration, including three cases with history of TST conversion. CONCLUSIONS: TST(+) and TSPOT(+) results predict risk factors associated with LTBI independent of immunosuppressive IMT. Some TST(+)/TSPOT(-) results were unlikely to be false-negatives. The combined use of TST and TSPOT appears to be a reasonable diagnostic strategy to evaluate for LTBI in this population.

Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease-A pilot study.

Clinical prediction of nontuberculous mycobacteria lung disease (NTM-LD) progression remains challenging. We aimed to evaluate antigen-specific immunoprofiling utilizing flow cytometry (FC) of activation-induced markers (AIM) and IFN-γ enzyme-linked immune absorbent spot assay (ELISpot) accurately identifies patients with NTM-LD, and differentiate those with progressive from nonprogressive NTM-LD. A Prospective, single-center, and laboratory technician-blinded pilot study was conducted to evaluate the FC and ELISpot based immunoprofiling in patients with NTM-LD (n = 18) and controls (n = 22). Among 18 NTM-LD patients, 10 NTM-LD patients were classified into nonprogressive, and 8 as progressive NTM-LD based on clinical and radiological features. Peripheral blood mononuclear cells were collected from patients with NTM-LD and control subjects with negative QuantiFERON results. After stimulation with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides), and control antigens, we performed IFN-γ ELISpot and FC AIM assays to access their diagnostic accuracies by receiver operating curve (ROC) analysis across study groups. Patients with NTM-LD had significantly higher percentage of CD4+/CD8+ T-cells co-expressing CD25+CD134+ in response to PPD stimulation, differentiating between NTM-LD and controls. Among patients with NTM-LD, there was a significant difference in CD25+CD134+ co-expression in MTB300-stimulated CD8+ T-cells (p <0.05; AUC-ROC = 0.831; Sensitivity = 75% [95% CI: 34.9-96.8]; Specificity = 90% [95% CI: 55.5-99.7]) between progressors and nonprogressors. Significant differences in the ratios of antigen-specific IFN-γ ELISpot responses were also seen for RD1-nil/PPD-nil and RD1-nil/anti-CD3-nil between patients with nonprogressive vs. progressive NTM-LD. Our results suggest that multiparameter immunoprofiling can accurately identify patients with NTM-LD and may identify patients at risk of disease progression. A larger longitudinal study is needed to further evaluate this novel immunoprofiling approach.

Anti-Inflammatory, Antipyretic, and Analgesic Potential of Chitin and Chitosan Derived from Cockroaches (Periplaneta americana) and Termites.

The chitin and chitosan biopolymers are extremely valuable because of their numerous industrial and pharmacological uses. Chitin and chitosan were extracted from the exoskeleton of Periplaneta americana (cockroaches) and termites using various acid and alkali techniques. The extraction process involves an initial demineralization step, during which integument dry powder was subjected to 500 mL (2.07 mol/L) of concentrated HCl at 100 degrees Celsius for 30 min, followed by meticulous rinsing with distilled water to restore the pH to its baseline. Deproteinization was conducted at 80 degrees Celsius using 500 mL (1 mol/L) of NaOH solution, which was repeated for 24 h. A total of 250 mL (0.06 mol/L) of NaOH was added at 100 degrees Celsius for 4 h to obtain chitosan, followed by extensive washing and subsequent drying. FTIR analysis was used to identify the functional groups in Periplaneta americana and termites. The crystallinity of these biopolymers, which have a face-centered cubic structure, was determined by X-ray diffraction analysis. This study assessed the analgesic properties of chitin and chitosan via an acetic-acid-induced writhing test in mice, revealing a significant reduction in writhing behavior following the chitin and chitosan extract. Notably, chitin exhibits the highest degree of analgesic activity compared to chitosan. Both chitin and chitosan show anti-inflammatory effects, with chitosan absorbing proton ions at sites of inflammation, while chitin effectively inhibits ear edema and elicits an analgesic response in mice. Furthermore, the present study revealed antipyretic activity, with termite chitin demonstrating the most significant effect at a concentration of 500 µL/mL, followed by chitosan and chitin at 100 µL/mL. These findings indicate the potential of using chitin and chitosan derived from termites and Periplaneta americana as natural anti-inflammatory compounds, implying prospective uses in anti-inflammatory, antipyretic, and analgesic capabilities.

Alarming multidrug resistance in Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis: Antibiotic resistance patterns and occurrence of selected resistance genes.

Bovine mastitis is a widespread and costly disease that affects dairy farming globally, characterized by mammary gland inflammation. Bovine intramammary gland infection has been associated with more than 135 different pathogens of which Staphylococcus aureus is the main etiology of sub-clinical mastitis (SCM). The current study was designed to investigate the prevalence, antibiotic resistance pattern, and the presence of antibiotic resistance genes (mecA, tetK, aacA-aphD and blaZ) in S. aureus isolated from the raw milk of cows with subclinical mastitis. A total of 543 milk samples were collected from lactating cows such as Holstein Friesian (n = 79), Sahiwal (n = 175), Cholistani (n = 107), and Red Sindhi (n = 182) from different dairy farms in Pakistan. From the milk samples microscopic slides were prepared and the somatic cell count was assessed to find SCM. To isolate and identify S. aureus, milk was streaked on mannitol salt agar (MSA) plates. Further confirmation was done based on biochemical assays, including gram staining (+ coccus), catalase test (+), and coagulase test (+). All the biochemically confirmed S. aureus isolates were molecularly identified using the thermonuclease (nuc) gene. The antibiotic resistance pattern of all the S. aureus isolates was evaluated through the disc diffusion method. Out of 543 milk samples, 310 (57.09%) were positive for SCM. Among the SCM-positive samples, S. aureus was detected in 30.32% (94/310) samples. Out of 94 isolates, 47 (50%) were determined to be multidrug resistant (MDR). Among these MDR isolates, 11 exhibited resistance to Cefoxitin, and hence were classified as methicillin-resistant Staphylococcus aureus (MRSA). The S. aureus isolates showed the highest resistance to Lincomycin (84.04%) followed by Ampicillin (45.74%), while the least resistance was shown to Sulfamethoxazole/Trimethoprim (3.19%) and Gentamycin (6.38%). Polymerase chain reaction (PCR) analysis revealed that 55.31% of the isolates carried blaZ gene, 46.80% carried tetK gene, 17.02% harbored the mecA gene, whereas, aacA-aphD gene was found in 13.82% samples. Our findings revealed a significant level of contamination of milk with S. aureus and half (50%) of the isolates were MDR. The isolated S. aureus harbored various antibiotic resistance genes responsible for the absorbed phenotypic resistance. The alarmingly high prevalence of MDR S. aureus isolates and MRSA strains in these cases possess a serious risk to public health, emphasizes the urgent need to address this issue to protect both human and animal health in Pakistan.

Antimicrobial resistance of Pseudomonas aeruginosa: navigating clinical impacts, current resistance trends, and innovations in breaking therapies.

Pseudomonas aeruginosa, a Gram-negative bacterium, is recognized for its adaptability and opportunistic nature. It poses a substantial challenge in clinical settings due to its complicated antibiotic resistance mechanisms, biofilm formation, and capacity for persistent infections in both animal and human hosts. Recent studies revealed a potential zoonotic transmission of P. aeruginosa between animals, the environment, and human populations which highlights awareness of this microbe. Implementation of the One Health approach, which underscores the connection between human, animal, and environmental health, we aim to offer a comprehensive perspective on the current landscape of P. aeruginosa management. This review presents innovative strategies designed to counteract P. aeruginosa infections. Traditional antibiotics, while effective in many cases, are increasingly compromised by the development of multidrug-resistant strains. Non-antibiotic avenues, such as quorum sensing inhibition, phage therapy, and nanoparticle-based treatments, are emerging as promising alternatives. However, their clinical application encounters obstacles like cost, side effects, and safety concerns. Effectively addressing P. aeruginosa infections necessitates persistent research efforts, advancements in clinical development, and a comprehension of host-pathogen interactions to deal with this resilient pathogen.

Trace metals and nutrient analysis of marine fish species from the Gwadar coast.

Trace metals are naturally occurring metals found in very small concentrations in the environment. In the context of fish flesh, metals such as copper, calcium, potassium, sodium, zinc, iron, and manganese are absorbed by fish and play vital roles in various physiological functions. However, if these metals exceed the recommended limits set by WHO/FAO, they are termed 'toxic metals' due to their harmful impacts on both the fish and its consumers. Therefore, the present study aims to analyze the levels of protein, lipids, and certain metals-Aluminum (Al), Sodium (Na), Zinc (Zn), Titanium (Ti), Iron (Fe), Copper (Cu), Potassium (K), and Calcium (Ca) in three commercially important marine fishes i.e. Rastrelliger kanagurta, Sardinella abella, and Otolithes ruber. The study also aims to assess their potential impact on human health. The macro-Kjeldhal method and Soxhlet apparatus were used to estimate protein and lipid contents, while atomic absorption spectroscopy (AAS) was used to estimate trace metals found in fishes. The study found that these fish species are valuable sources of protein, lipids, and certain essential minerals. The protein content (CP) in these three species ranged from 63.35 to 86.57%, while lipid content was from 21.05 to 23.86%. The overall results of the trace metal concentrations analyzed in the present study revealed that Aluminum (Al), Sodium (Na), Zinc (Zn), Titanium (Ti), Copper (Cu), Potassium (K), and Calcium (Ca) were found in low concentration or traces and also within suitable ranges as set by WHO/FAO. However, Iron (Fe) was absent in all three species. Moreover, both copper and potassium were found in all three species, while Zinc was present in Rastrelliger kanagurta and Sardinella abella, calcium in Sardinella abella, and sodium in Otolithes ruber only. Titanium was recorded for the first time in S. abella. However, the total health risk assessment associated with these fish food consumption was measured by THQ and TTHQ and found to be less than 1, which shows no potential risk related to trace metals found in these fishes on human health upon their consumption. In conclusion, these commercially important marine fish species were found valuable sources of protein, lipids, and essential trace minerals that are necessary for human health. Thus, the current study provides useful information for the local population to make informed decisions about their daily diets and highlights the importance of sustainable fishing practices to maintain these valuable marine resources by periodical monitoring of their ecosystem.

Agent-based model indicates chemoattractant signaling caused by Mycobacterium avium biofilms in the lung airway increases bacterial loads by spatially diverting macrophages.

Incidence and prevalence of MAC infections are increasing globally, and reinfection is common. Thus, MAC infections present a significant public health challenge. We quantify the impact of MAC biofilms and repeated exposure on infection progression using a computational model of MAC infection in lung airways. MAC biofilms aid epithelial cell invasion, cause premature macrophage apoptosis, and limit antibiotic efficacy. In this computational work we develop an agent-based model that incorporates the interactions between bacteria, biofilm, and immune cells. In this computational model, we perform virtual knockouts to quantify the effects of the biofilm sources (deposited with bacteria vs. formed in the airway), and their impacts on macrophages (inducing apoptosis and slowing phagocytosis). We also quantify the effects of repeated bacterial exposures to assess their impact on infection progression. Our simulations show that chemoattractants released by biofilm-induced apoptosis bias macrophage chemotaxis towards pockets of infected and apoptosed macrophages. This bias results in fewer macrophages finding extracellular bacteria, allowing the extracellular planktonic bacteria to replicate freely. These spatial macrophage trends are further exacerbated with repeated deposition of bacteria. Our model indicates that interventions to abrogate macrophages' apoptotic responses to bacterial biofilms and/or reduce frequency of patient exposure to bacteria will lower bacterial load, and likely overall risk of infection.

Risk factors and outcomes of non-tuberculous mycobacteria infection in lung transplant recipients: A systematic review and meta-analysis.

BACKGROUND: Patients with structural lung disease and immunocompromised status are at increased risk of pulmonary non-tuberculous mycobacteria (NTM) infection. However, literature on NTM in lung transplant recipients (LTR) is limited. We sought to systematically review the literature and perform a meta-analysis to examine associations with NTM disease and isolation in LTRs and their influence on mortality and chronic lung allograft dysfunction (CLAD). METHODS: A literature search of MEDLINE and Embase was performed on February 23, 2022. NTM disease was defined according to international guidelines. Isolation was defined as any growth of NTM in culture. Odds ratios (OR) were pooled for risk factors of NTM disease or isolation, and hazard ratios (HR) were pooled for mortality or CLAD. RESULTS: Eleven studies totaling 3,371 patients were eligible for inclusion, 10 of which underwent meta-analysis. Cystic fibrosis (OR 1.84, 95% confidence interval [CI] 1.03-3.30; I2 = 0%) and pre-transplant NTM isolation (OR 2.40, 95% CI 1.20-4.83; I2 = 0%) were associated with NTM disease. Only male sex was associated with NTM isolation (OR 1.45, 95% CI 1.01-2.10; I2 = 0%). NTM disease was associated with increased mortality (HR 2.69, 95% CI 1.70-4.26; I2 = 0%) and CLAD (HR 2.11, 95% CI 1.03-4.35; I2 = 44%). NTM isolation was not associated with mortality in pooled analysis or CLAD in 1 included study. CONCLUSIONS: NTM disease, but not isolation, is associated with worse outcomes. Several factors were associated with development of NTM disease, including cystic fibrosis and pretransplant NTM isolation. Strategies to optimize prevention and treatment of NTM disease in lung transplant recipients are needed.

Convalescent Adaptive Immunity is Highly Heterogenous after SARS-CoV-2 Infection.

Optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, between April 23, 2020, to May 11, 2020, we recruited 30 COVID-19 unvaccinated convalescent donors and 7 unexposed asymptomatic donors. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG semiquantitative ELISA and T cell immunity against S1 and S2 subunits were studied by IFN-γ Enzyme-Linked Immune absorbent Spot (ELISpot), flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2 positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARSCoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Convalescent Adaptive Immunity Is Highly Heterogenous after SARS-CoV-2 Infection.

The optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, we recruited 30 unvaccinated convalescent donors who had previously been infected with COVID-19 and 7 unexposed asymptomatic controls. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG via semiquantitative ELISA, and T-cell immunity against S1 and S2 subunits were studied via IFN-γ enzyme-linked immunosorbent spot (ELISpot) and flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2-positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and the determination of optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Epidemiological analysis of anaplasmosis in cattle from Khyber Pakhtunkhwa, Pakistan.

Background and Aim: Anaplasmosis, a tick-borne disease affecting livestock caused by the bacteria Anaplasma, poses a global concern. This study aimed to estimate the prevalence, spatiotemporal variation, and associated risk factors of anaplasmosis in cattle from the Bannu and Lakki Marwat districts of Khyber Pakhtunkhwa, Pakistan. Materials and Methods: This study used 197 cattle exhibiting clinical symptoms of anaplasmosis in natural settings. Microscopic examination was used to estimate the prevalence. Potential risk factors, such as sampling regions and months, gender, breed, and age were studied. Results: The study revealed an overall anaplasmosis prevalence of 19.79%. Bannu district exhibited a higher occurrence at 22.10%, compared to Lakki Marwat district at 17.64%. Young cattle (<2 years) demonstrated a notably higher incidence of anaplasmosis (26.78%) compared to adults (>5 years), which had a prevalence of 12.35% (p < 0.05). Female cattle (22.36%) were more susceptible than male cattle (11.11%). Prevalence peaked in June (45.71%) and was lowest in February (3.57%). Crossbred cattle had a higher prevalence (23.52%) than purebred cattle (11.47%). Conclusion: Anaplasmosis can be effectively controlled using a comprehensive approach encompassing selective breeding for resilience, targeted care of young calves and females, effective tick control during warmer months, consistent use of insecticides, and proactive risk factor management. Raising awareness among farmers through diverse channels, including media, is pivotal to bolster tick-borne disease management strategies.

Toxoplasmosis-Awareness and Knowledge of Pregnant Women in Rural Areas of Malakand Region, Pakistan.

Background: The current study was carried out between October 2017 and October 2018 to explore knowledge, attitudes, practices, and information sources regarding toxoplasmosis among pregnant women in Malakand region, the northwestern part of Pakistan. The current study was carried out between October 2017 and October 2018. Methods: A structured questionnaire was used to interview the women after taking verbal informed consent. Graphpad version 5 was used to indicate the differences. Significant was considered as a P-value of less than 0.05. This study revealed poor knowledge regarding toxoplasmosis. Results: Overall, 31.2% of the respondents showed good knowledge, and 39.2% showed moderate knowledge. On the other hand, 29.5% of the participants showed poor knowledge about toxoplasmosis. The average knowledge score of pregnant women was 79 ± 12.2, which is considered to be within the scale of good knowledge. Number of children within the pregnant multipara women was significantly associated with knowledge about toxoplasmosis. Pregnant women who measured in number of childbirths within a women showed the highest mean score of 42.3 ± 13.3 with 57 (44.8%) displaying a good knowledge level. Pregnant women with more than one child had significantly higher (<0.0001) knowledge scores compared to women with one child or none child. The majority of pregnant women with one child used the social media, followed by mass media as sources of information about toxoplasmosis. Scientific sources of information were used more commonly by pregnant women with none of the child birth. Conclusion: Pregnant women knowledge regarding toxoplasmosis was poor as compared to attitudes and practices. Health workers and newspapers/magazines were the main sources of information.

Microplastic Quantification in Aquatic Birds: Biomonitoring the Environmental Health of the Panjkora River Freshwater Ecosystem in Pakistan.

Microplastic pollution has become a global concern, with potential negative impacts on various ecosystems and wildlife species. Among these species, ducks (Anas platyrhynchos) are particularly vulnerable due to their feeding habits and proximity to aquatic environments contaminated with microplastics. The current study was designed to monitor microplastic (MP) pollutants in the freshwater ecosystem of the Panjkora River, Lower Dir, Pakistan. A total of twenty (20) duck samples were brought up for four months and 13 days on the banks of the river, with no food intake outside the river. When they reached an average weight of 2.41 ± 0.53 kg, all samples were sacrificed, dissected, and transported in an ice box to the laboratory for further analysis. After sample preparation, such as digestion with 10% potassium hydroxide (KOH), density separation, filtration, and identification, the MP content was counted. A total of 2033 MP particles were recovered from 20 ducks with a mean value of 44.6 ± 15.8 MPs/crop and 57.05 ± 18.7 MPs/gizzard. MPs detected in surface water were 31.2 ± 15.5 MPs/L. The major shape types of MPs recovered were fragments in crop (67%) and gizzard (58%) samples and fibers in surface water (56%). Other types of particles recovered were fibers, sheets, and foams. The majority of these detected MP particles were in the size range of 300-500 µm (63%) in crops, and 50-150 µm (55%) in gizzards, while in water samples the most detected particles were in the range of 150-300 µm (61%). Chemical characterization by FTIR found six types of polymers. Low-density polyethylene (LDPE) had the greatest polymer detection rate (39.2%), followed by polyvinyl chloride (PVC) (28.3%), high-density polyethylene (HDPE) (22.7%), polystyrene (6.6%), co-polymerized polypropylene (2.5%), and polypropylene homopolymer (0.7%). This study investigated the presence of microplastics in the crops and gizzards of ducks, as well as in river surface water. The results revealed the significant and pervasive occurrence of microplastics in both the avian digestive systems and the surrounding water environment. These findings highlight the potential threat of microplastic pollution to wildlife and ecosystems, emphasizing the need for further research and effective mitigation strategies to address this pressing environmental concern.