Evaluation of molecular diagnostic test for detection of adult pulmonary tuberculosis: A generic protocol.

BACKGROUND OBJECTIVES: Tuberculosis (TB) continues to be the second most-leading cause of death due to a single infectious agent as of 2022 after COVID-19. Many affordable new molecular diagnostic tools are being developed for early and more accurate diagnosis, especially for low-resource settings in low- and middle-income countries. In this context, there is a need to develop a standardized protocol for validation of new diagnostic tools. Here, we describe a generic protocol for multi-centric clinical evaluation of molecular diagnostic tests for adult pulmonary TB. METHODS: This protocol describes a cross-sectional study in TB reference laboratories in India. Adults (>18 yr) visitng the chest clinics or outpatient departments with symptoms of TB need to be enrolled consecutively till the required sample size of 150 culture positives and 470 culture negatives are met. Mycobacterium tuberculosis (Mtb) culture (mycobacteria growth indicator tube liquid culture) to be used under this protocol as the gold standard and Xpert MTB/RIF molecular test will be used as the comparator. The sputum samples will be tested by smear microscopy, Mtb culture, Xpert MTB/RIF and index molecular test as per the proposed algorithm. The specificity sensitivity, and positive/ negative predictive values are to be calculated for the index test with reference to the gold standard. DISCUSSION: TB diagnosis poses many challenges as it differs with type of disease, age group, clinical settings and type of diagnostic tests/kits used. Globally, different protocols are used by several investigators. This protocol provides standard methods for the validation of molecular tests for diagnosis of adult pulmonary TB, which can be adopted by investigators.

Human anthrax in India in recent times: A systematic review & risk mapping.

The disease anthrax occurs generally in herbivores and the causative organism (Bacillus anthracis) infects humans who come in contact with infected animals or their products. The persistence of anthrax spores for decades and its lethality contribute to its biowarfare potential. We conducted this systematic review along with risk mapping to investigate the spatio-temporal distribution, clinico-epidemiological, socio-behavioural and programmatic issues pertaining to anthrax in India over the last two decades. Peer reviewed quantitative and qualitative studies and grey literature comprising weekly reports of the 'Integrated Disease Surveillance Program' (IDSP), were accessed for extracting data. IDSP data were used for geo-referencing of the villages of anthrax cases; Pseudo-absence was generated to fit a Bayesian Additive Regression Trees (BART) model to develop anthrax risk map. The case fatality rate of cutaneous anthrax ranged from 2% to 38%, while the gastrointestinal and inhalational types were 100% fatal. Our synthesis revealed that human anthrax outbreaks in India were clustered around the eastern coastal regions. The states of Odisha, West Bengal, Andhra Pradesh and Jharkhand reported maximum number of outbreaks. Odisha reported a maximum number of 439 human anthrax cases since 2009, of which Koraput district contributed to 200 cases (46%). While handling or consumption of infected animal product were proximal drivers of these events, poverty, lack of awareness, traditional beliefs and local practices served as facilitatory factors. Other structural determinants were wild life-livestock interface, historical forest loss, soil pH, soil-water balance, organic carbon content, temperature, rainfall and humidity. The programmatic issues identified through this review were lack of active surveillance, non-availability of diagnostic facility at the periphery, delayed reporting, absence of routine livestock vaccination and lack of adequate veterinary services. Interventions based on One-health approach in the country merit immediate policy and program attention; high risk zones for anthrax identified during present investigation, should be prioritized.

Curcumin augments therapeutic efficacy of TRAIL-based immunotoxins in leukemia.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been perceived as a promising anti-cancer agent because of its unique ability to kill cancer cells while sparing normal cells. However, translation of TRAIL to clinical studies was less successful as a large number of cancer cells acquire resistance to TRAIL-based monotherapies. An ideal strategy to overcome TRAIL resistance is to combine it with potential sensitizing agents. OBJECTIVE: To investigate the TRAIL-sensitizing effect of curcumin in leukemia. METHODS: The mechanism underlying TRAIL sensitization by curcumin was studied by flow cytometric analysis of TRAIL receptors in leukemic cell lines and patient samples, and immunoblot detection of TRAIL-apoptosis signaling proteins. RESULTS: Curcumin augments TRAIL-apoptotic signaling in leukemic cells by upregulating the expression of DR4 and DR5 along with suppression of cFLIP and anti-apoptotic proteins Mcl-1, Bcl-xl, and XIAP. Curcumin pre-treatment significantly (p < 0.01) enhanced the sensitivity of leukemic cell lines to TRAIL recombinant proteins. IL2-TRAIL peptide in the presence of curcumin induced potent apoptosis (p < 0.001) as compared to TRAIL and IL2-TRAIL protein in leukemic cell lines with IC50 < 0.1 µΜ. Additionally, the combination of IL2-TRAIL peptide and curcumin showed significant cytotoxicity in patient peripheral blood mononuclear cells (PBMCs) with an efficacy of 90% in acute myeloid leukemia (AML), but 100% in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and chronic myelomonocytic leukemia (CMML). CONCLUSION: Overall, our results suggest that curcumin potentiates TRAIL-induced apoptosis through modulation of death receptors and anti-apoptotic proteins which significantly enhances the therapeutic efficacy.

Anticancer effect of acid ceramidase inhibitor ceranib-2 in human breast cancer cell lines MCF-7, MDA MB-231 by the activation of SAPK/JNK, p38 MAPK apoptotic pathways, inhibition of the Akt pathway, downregulation of ERα.

Acid ceramidase is the key enzyme of the ceramide metabolic pathway, which plays a vital role in regulating ceramide - sphingosine-1-phosphate rheostat. Ceramide acts as a proapoptotic molecule, but its metabolite sphingosine-1-phosphate, in contrast, signals for cell proliferation, cell survival, and angiogenesis. Acid ceramidase is highly upregulated in breast tumors and treatment with an acid ceramidase inhibitor, ceranib-2, significantly induced apoptosis in human breast cancer cell lines. However, the mechanisms underlying the induction of apoptosis remain ambiguous to date. Hence, in the present study, we have explored ceranib-2-mediated apoptotic signaling pathways in human breast cancer cell lines. MCF-7 and MDA MB-231 cells were treated with IC50 doses of ceranib-2 and tamoxifen. Nuclear changes showed the apoptotic effect of ceranib-2 in both the cell lines. Loss in the mitochondrial membrane potential was observed only in ceranib-2-treated MCF-7 cells. Ceranib-2 activated intrinsic and extrinsic apoptotic pathways in MCF-7 cells, but only the extrinsic apoptotic pathway was activated in MDA MB-231 cells. Further, ceranib-2 induced apoptosis by activating SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), p38 MAPK (mitogen-activated protein kinase) apoptotic pathways and by inhibiting the Akt (antiapoptotic) pathway in both the cell lines. Most importantly, ERα (estrogen receptor-α) expression was highly downregulated after ceranib-2 treatment and a docking study predicted the highest binding affinity of ceranib-2 than tamoxifen with ERα in MCF-7 cells. Hence, ceranib-2 may have potential as a chemotherapeutic drug of breast cancer.