The burden of tuberculosis among patients with non-small cell lung carcinoma in a tertiary care center.

BACKGROUND: Lung cancer and tuberculosis share similar risk factors, clinical spectrum, radiological features and it is difficult to differentiate but it is important to diagnose both conditions for targeted therapy and better outcome. AIMS: Our primary objective was to estimate the proportion of TB in primary biopsy proven non-small cell lung carcinoma (NSCLC) cases. MATERIAL & METHODS: This prospective observational study was conducted in the Departments of Medicine/Pulmonary Medicine/Medical Oncology and Microbiology at the All India Institute of Medical Sciences, New Delhi for a period of 2 years (January 2020-December 2021). Patients with biopsy proven, primary non-small cell lung cancer were recruited and sputum samples were subjected to microbiological investigations to confirm tuberculosis. Comparison was done in two groups of lung cancer patients with confirmed TB (Group A) and without confirmed tuberculosis (Group B). RESULTS: Total 75 patients with biopsy proven, primary NSCLC were recruited and 16 % (12/75) were diagnosed with confirmed TB. Adenocarcinoma (36.48 %) and Squamous cell carcinoma (33.44 %) were the two predominant histopathological subtypes of NSCLC. About 57 (76 %) of them were found to be in stage IV of Lung cancer at initial presentation itself (75 % in group A & 74.6 % in group B; p value < 0.80). A majority of patients (11/12 cases; 91 %) of group A were males with a mean age of 59 ± 7.5 years. The upper lobes of the lung were involved in 65 % (49/75) of the cases and showing a mass lesion on imaging (75 % in group A & 65 % in group B; p value < 0.52). Kaplan Meier survival revealed a median survival time of 11 months in subjects with only NSCLC and a median survival time of 4 months in the group with concomitant TB and NSCLC (p value < 0.44).

Diagnostics Evaluation of Smart Sure™ Mycobacterium tuberculosis Screening Kit and Smart Sure™ Multidrug-resistant Tuberculosis Detection Kit on Nonsputum Specimens at a Tertiary Care Center of North India.

BACKGROUND: Tuberculosis (TB) is caused due to the infection of Mycobacterium tuberculosis (MTB) and it can infect the various parts of the human body. The disease is highly prevalent and is the second most common cause of death worldwide after COVID-19. Apart from sputum specimen, it is exceedingly difficult to diagnose due to its paucibacillary nature. The current study was intended to evaluate the accuracy of Smart Sure™ MTB and multidrug-resistant-TB (MDR-TB) kits (Genetix Biotech Asia Pvt. Ltd., India) with Xpert ultra and Mycobacterium growth indicator tube (MGIT) culture on nonsputum specimens from TB suspects. METHODS: A total of 205 nonsputum specimens were received between October 2023 and May 2024 at Intermediate Reference Laboratory, Department of Medicine, All India Institute of Medical Sciences, New Delhi, India. Xpert ultra and Smart Sure™ MTB and MDR-TB tests were done directly on samples. However, processed specimens were used for MGIT culture and drug-susceptibility testing (DST). Invalid and MGIT contaminated specimens were excluded from the final calculation. RESULTS: Overall, sensitivity and specificity of Smart Sure™ MTB screening kit was 71.59% and 98.28%, respectively, with Xpert ultra and 68.35% and 90.83%, respectively, with MGIT culture. While comparing with both Xpert ultra and MGIT-DST to detect rifampicin (RIF) resistant, Smart Sure™ MDR-TB kits showed sensitivity of 75.0% and 100% of specificity. However, for isoniazid (INH) resistance, Smart Sure™ MDR-TB kits showed 100% of sensitivity and specificity with MGIT-DST. CONCLUSION: For the detection of MTB and its drug-resistance patterns (RIF and INH) in the specimens other than sputum, Smart Sure™ MTB and MDR-TB kits could play a vital role in TB endemic countries. While comparing the set-ups and skilled staffs, it required almost same as compared with previously approved WHO diagnostics used in resource-limited countries.

Early organism identification by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) decreases the time to appropriate antibiotic modifications for common bacterial infections.

Objective: MALDI-TOF-MS facilitates the identification of microorganisms from positive cultures in a timely and accurate manner. It eliminates the necessity for the application of biochemicals and operates on the principle of proteomics. It decreases the time required to report culture results. Prompt detection and notification of the pathogen, prior to the disclosure of antimicrobial susceptibilities, could potentially shorten the duration until the initial antibiotic adjustment is necessary, thereby influencing patients' clinical prognoses. Methodology: Fifty patients in the conventional arm and one hundred patients in the interventional arm were compared in a pre and post quasi-experimental study conducted at a tertiary care centre in North India. Patients with positive cultures from medical wards and intensive care units were included. Comparing the time to first antibiotic modification after culture positivity, MALDI-TOF-MS-based identification, and clinical outcomes in both arms was the primary objective. Antibiotic modifications, escalation, and de-escalation were all recorded. Results: The intervention arm exhibited a substantially shorter median time to first antibiotic modification (2010 mins vs 2905 mins, p=0.002) than the conventional arm. In the interventional group, a total of 44 out of 100 antibiotic modifications were implemented. Of these, 19 (43.3%) were determined solely by the MALDI report, without the anticipation of susceptibility assessments. De-escalation of antibiotics constituted the pre-dominant form of modification (47.4%). The difference between the 27% and 32% mortality rates in the intervention arm and the conventional arm was not statistically significant (p=0.52). Conclusion: MALDI-TOF-MS facilitates the modification of antibiotics early on. The primary benefit lies in the reduction of superfluous antibiotic usage. Early organism identification and reporting prior to the availability of susceptibility results did not result in any mortality benefit. This strategy, when combined with a strong antimicrobial stewardship programme, can aid in the reduction of antibiotic use.

Factors Impacting Outcome and Prognosis of Invasive Fungal Sinusitis: How Vital is Iron Metabolism?

Iron is an important micronutrient involved in cell biology through vital reactions. We examined the correlations between iron metabolism parameters and the course of invasive fungal sinusitis. Patients with invasive fungal sinusitis were enrolled. Serum iron and ferritin levels, total iron-binding capacity, and transferrin saturation were measured at the initiation of treatment. Patients were followed for 6 months, and the clinical course was categorised as improvement or worsening/death. A total of 35 patients were enrolled. The average ferritin levels in mucormycosis patients was 944.9 ng/ml, versus 110.7 ng/ml for aspergillosis patients. Iron levels were significantly lower in mucormycosis than in aspergillosis (29.14 µg/dl vs. 68.55 µg/dl). Total iron-binding capacity was significantly different between the two groups (16.76 µg/dl vs. 330.36 µg/dl). After 6 months, improvement, worsening, and death were noted for 18, 8, and 9 patients, respectively. Higher iron levels and lower ferritin levels were linked with improvement. Total iron-binding capacity was significantly higher in improved patients (2314 vs. 151). Iron metabolism parameters play significant roles in the preemptive judgment of the course of fungal sinusitis. Based on these findings, studies on drugs affecting iron metabolism should be conducted.

Evaluation of GeneNAT Real-Time Polymerase Chain Reaction Analyzer and Pre-loaded Chip-Based Mycobacterium tuberculosis and Multidrug-Resistant Tuberculosis Detection in the Diagnosis of Pulmonary Tuberculosis.

BACKGROUND: Tuberculosis (TB) is still the second causative agent of death worldwide after COVID-19. It is caused by Mycobacterium tuberculosis (MTB) infection. OBJECTIVE: The aim of the current study was to compare the performance of GeneNAT real-time polymerase chain reaction analyzer and pre-loaded chip-based MTB screening and multidrug-resistant tuberculosis (MDR-TB) detection kit (Smart SureTM MTB & MDR-TB, Genetix Biotech Asia Pvt. Ltd., New Delhi, India) against the established WHO-approved GeneXpert Ultra (MTB/rifampicin (RIF)), line probe assay (LPA), and mycobacteria growth indicator tube (MGIT) culture at point of care (POC) level. METHODS: A total of 450 pulmonary TB (PTB) suspect patients were enrolled from October 2023 to March 2024 at the Intermediate Reference Laboratory, Department of Medicine, All India Institute of Medical Sciences, New Delhi, India. GeneXpert and GeneNAT tests were done directly from sputum specimens. However, processed sputum specimens were used for both LPA (GenoType MTBDRplus) and liquid culture and drug susceptibility testing (MGIT culture and drug susceptibility testing (DST)). RESULTS: On comparing with GeneXpert, for the detection of MTB and rifampicin (RIF), Smart SureTM showed a sensitivity of 98.18% and 97.5% with a specificity of 100% and 98.92%, respectively. While comparing mutations in the rpoB gene with LPA, the Smart SureTM MDR-TB kit exhibited sensitivity and specificity of 96.77% and 99.12%, respectively. For katG and inhA genes, sensitivity and specificity were 97.6% & 85.71% and 98.66% & 98.01%, respectively, for both genes. Smart SureTM MDR-TB showed comparable results with MGIT-DST with sensitivity and specificity of 96.88% & 96.15% and 98.99% & 99.02%, respectively, for both RIF and isoniazid (INH) drugs. CONCLUSION: The GeneNAT system test may provide the status of RIF and INH resistance in PTB cases in a short time with the use of minimal specimens. It required very little infrastructure with less skilled laboratory staff in comparison with other WHO-approved diagnostics used in resource-limited countries with TB and drug-resistant TB burdens.

Clinico-mycological characteristics and outcomes of rare yeast infections.

Unusual fungi, encountered infrequently in practice, present a significant diagnostic challenge, leading to potential delays in diagnosis and treatment. This study aims to describe a number of cases, where infections were caused by rare yeast pathogens. Organisms isolated included rare Candida species, Geotrichum, Lodderomyces and Trichosporon species. The mean duration of the outcome of the patients from microbiological diagnosis was 20 days. A total of 3 patients succumbed to their illness. This study aims to shed light on the varied clinical presentation and outcome of infections caused by rare yeast pathogens.

Evaluation of Loop-Mediated Isothermal Amplification Assay in Diagnosing Tuberculous Meningitis.

BACKGROUND: Resource-limited settings like India need a simple, quick, and temperature-independent point-of-care diagnostic test that can diagnose tuberculous meningitis (TBM) at the earliest. METHODS: A prospective study was carried out at a tertiary care center in North India wherein 50 subjects suspected of TBM were recruited and followed up for six months between January 2019 and December 2020. The aim was to evaluate the performance of loop-mediated isothermal amplification (TB-LAMP) in diagnosing TBM as compared to a composite reference standard (CRS), mycobacteria growth indicator tube 960 (MGIT 960) culture, and GeneXpert®. RESULTS: Out of 50 patients, 32 were TBM cases (64%), and 18 were non-TBM cases (36%). The sensitivity of TB-LAMP and GeneXpert® for TBM diagnosis against CRS was 53% (17/32) for both, and the specificity was 78% (14/18) and 89% (16/18), respectively. On comparing TB-LAMP against GeneXpert® for TBM diagnosis, the two methods had almost perfect agreement (Cohen's kappa=0.83) with statistical significance (p<0.001). CONCLUSION: The performance of TB-LAMP assay is comparable to GeneXpert® in diagnosing TBM, and it may be used as a substitute for CSF GeneXpert® in resource-limited settings.

Metagenomic insights into fungal community composition of the nasopharyngeal region of COVID-19 associated mucormycosis patients from India.

Coronavirus disease 2019 (COVID-19) associated mucormycosis (CAM) was reported predominantly from India during the second wave of COVID-19  and has a high mortality rate. The present study aims to understand the fungal community composition of the nasopharyngeal region of CAM-infected individuals and compare it with severe COVID-19 patients and healthy controls. The fungal community composition was decoded by analyzing the sequence homology of the internal transcribed spacer-2-(ITS-2) region of metagenomic DNA extracted from the upper respiratory samples. The alpha-diversity indices were found to be significantly altered in CAM patients (p < 0.05). Interestingly, a higher abundance of Candida africana, Candida haemuloni, Starmerella floris, and Starmerella lactiscondensi was observed exclusively in CAM patients. The interindividual changes in mycobiome composition were well supported by beta-diversity analysis (p < 0.05). The current study provides insights into the dysbiosis of the nasal mycobiome during CAM infection. In conclusion, our study shows that severe COVID-19 and CAM are associated with alteration in mycobiome as compared to healthy controls. However, the sequential alteration in the fungal flora which ultimately leads to the development of CAM needs to be addressed by future studies.

Utility of an in-house real-time PCR in whole blood samples as a minimally invasive method for early and accurate diagnosis of invasive mould infections.

INTRODUCTION: Invasive mould infections (IMIs) are a leading cause of death in patients with compromised immune systems. Proven invasive mould infection requires detection of a fungus by histopathological analysis of a biopsied specimen, sterile culture, or fungal DNA amplification by PCR in tissue. However, the clinical performance of a PCR assay on blood samples taken from patients suspected of invasive mould disease has not been fully evaluated, particularly for the differential diagnosis of invasive aspergillosis (IA) and invasive Mucormycosis (IM). OBJECTIVES: To assess the diagnostic utility of our previously validated in-house real-time PCR in blood samples for diagnosis of invasive aspergillosis and mucormycosis in patients with suspected invasive mould infection. METHODS: All patients with suspected invasive mould infection were prospectively enrolled from May 2021 to July 2021. Conventional fungal diagnosis was performed using tissue and respiratory samples. In-house PCR was performed on blood samples and its diagnostic performance evaluated. RESULTS: A total of 158 cases of suspected invasive mould infection were enrolled in the study. The sensitivity and specificity of in-house PCR performed on blood samples was found to be 92.5% and 81.4% respectively for diagnosis of probable IA, and 65% and 84.62% respectively for diagnosis of proven and probable IM. It was also able to detect 3 out of 5 cases of possible IM where no other microbiological evidence of IM was obtained. CONCLUSIONS: This assay could be helpful in minimally invasive diagnosis of IMIs for patients in whom invasive sampling is not feasible, especially as a preliminary or screening test. It can help in early diagnosis, anticipating conventional laboratory confirmation by days or weeks. Possible correlation between fungal load and mortality can help in initiating aggressive treatment for patients with high initial fungal load.

Approach towards surveillance-based diagnosis of acute respiratory illness in India: Expert recommendations.

BACKGROUND: Emerging infectious diseases, often zoonotic, demand a collaborative "One-Health" surveillance approach due to human activities. The need for standardized diagnostic and surveillance algorithms is emphasized to address the difficulty in clinical differentiation and curb antimicrobial resistance. OBJECTIVE: The present recommendations are comprehensive diagnostic and surveillance algorithm for ARIs, developed by the Indian Council of Medical Research (ICMR), which aims to enhance early detection and treatment with improved surveillance. This algorithm shall be serving as a blueprint for respiratory infections landscape in the country and early detection of surge of respiratory infections in the country. CONTENT: The ICMR has risen up to the threat of emerging and re-emerging infections. Here, we seek to recommend a structured approach for diagnosing respiratory illnesses. The recommendations emphasize the significance of prioritizing respiratory pathogens based on factors such as the frequency of occurrence (seasonal or geographical), disease severity, ease of diagnosis and public health importance. The proposed surveillance-based diagnostic algorithm for ARI relies on a combination of gold-standard conventional methods, innovative serological and molecular techniques, as well as radiological approaches, which collectively contribute to the detection of various causative agents. The diagnostic part of the integrated algorithm can be dealt at the local microbiology laboratory of the healthcare facility with the few positive and negative specimens shipped to linked viral disease research laboratories (VRDLs) and other ICMR designated laboratories for genome characterisation, cluster identification and identification of novel agents.

Evaluation of kit-based loop-mediated isothermal amplification (TB-LAMP) assay in the diagnosis of tubercular lymphadenitis.

Background: The rapid and accurate diagnosis of tubercular lymphadenitis remains a challenging task today. The World Health Organization (WHO) endorsed the LoopAMP MTBC kit (TB-LAMP) as a replacement for sputum smear microscopy in the diagnosis of pulmonary tuberculosis (PTB). However, no prospective diagnostic accuracy study of TB-LAMP for tubercular lymphadenitis in adults has been performed yet. The current study evaluated the diagnostic performance of TB-LAMP in tubercular lymphadenitis (LNTB). Methods: In a prospective observational study conducted at a tertiary care hospital in India, 90 subjects (age >18 years) suspected of LNTB were recruited consecutively and followed up for 6 months between January 2019 and December 2020. Samples were processed for microscopy, culture, GeneXpert, histopathology and TB-LAMP. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of TB-LAMP against the composite reference standard (CRS) and culture were determined. Results: TB-LAMP showed a sensitivity of 83.78 % (95 % CI, 73.76-90.47) and a specificity of 81.25 % (95 % CI, 56.99-93.41), respectively, against the CRS. The PPV and NPV were 95.38 % (95 % CI, 87.29-98.42) and 52.00 % (95 % CI, 33.50-69.97), respectively. TB-LAMP showed a sensitivity of 88.89 % (95 % CI, 71.94-96.15) and a specificity of 36.17 % (95 % CI, 23.97-50.46), respectively, against culture. The PPV and NPV were 44.44 % (95 % CI, 32-57.62) and 85 % (95 % CI, 63.96-94.76), respectively. Conclusion: TB-LAMP can be used instead of conventional microscopy for the diagnosis of TB in lymph node specimens at primary healthcare centres. It provides rapid and cost-effective diagnosis of LNTB in resource-limited settings due to good sensitivity and NPV.

Subphenotypes of SARS-CoV-2-Associated ARDS Overlap Each Other: A Retrospective Analysis.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus-associated pneumonia and acute respiratory distress syndrome (ARDS) were often associated with hyperinflammation and elevation of several serum inflammatory markers but usually less than what is observed in non-coronavirus disease (COVID) ARDS. Elevated inflammatory markers such as C-reactive protein, interleukin (IL)-6, etc., are associated with severe infection. This study identified subphenotypes of COVID-19 ARDS patients by latent profile analysis in a cohort of Indian patients. Methods Data of n = 233 adult Indian patients with laboratory-confirmed SARS-CoV-2 infection admitted to a tertiary care teaching hospital were analyzed in this retrospective study. Only patients with acute respiratory failure (defined by partial pressure of oxygen/fraction of inspired oxygen ratio < 200 mm Hg) and chest X-ray showing bilateral infiltrates were included. Results The patients' mean (standard deviation) age was 53.3 (14.9) years, and 62% were male. A two subphenotypic model was formulated based on the lowest Bayesian information criterion. Neutrophil-to-lymphocyte ratio and serum IL-6 were latent variables in that model (entropy 0.91). The second phenotype (hyperinflammatory) had lower platelet count ( p = 0.02), higher serum creatinine ( p = 0.004), higher C-reactive protein ( p = 0.001), higher ferritin ( p < 0.001), and serum lactate dehydrogenase ( p = 0.009). Age-adjusted hospital mortality ( p = 0.007), duration of hospital stay ( p < 0.001), and duration of intensive care unit stay ( p < 0.001) were significantly higher in the second subphenotype. Conclusion Two distinct but overlapping subphenotypes were identified in SARS-CoV-2-associated respiratory failure. Hyperinflammatory subphenotype was associated with significantly poor short-term outcomes.

Baroreflex sensitivity is impaired in survivors of mild COVID-19 at 3-6 months of clinical recovery; association with carotid artery stiffness.

The association between the stiffening of barosensitive regions of central arteries and the derangements in baroreflex functions remains unexplored in COVID-19 survivors. Fifty-seven survivors of mild COVID-19 (defined as presence of upper respiratory tract symptoms and/or fever without shortness of breath or hypoxia; SpO2 > 93%), with an age range of 22-66 years (27 females) participated at 3-6 months of recovering from the acute phase of RT-PCR positive COVID-19. Healthy volunteers whose baroreflex sensitivity (BRS) and arterial stiffness data were acquired prior to the onset of the pandemic constituted the control group. BRS was found to be significantly lower in the COVID survivor group for the systolic blood pressure-based sequences (BRSSBP ) [9.78 (7.16-17.74) ms/mmHg vs 16.5 (11.25-23.78) ms/mmHg; p = 0.0253]. The COVID survivor group showed significantly higher carotid ß stiffness index [7.16 (5.75-8.18) vs 5.64 (4.34-6.96); (p = 0.0004)], and pulse wave velocity ß (PWVß ) [5.67 (4.96-6.32) m/s vs 5.12 (4.37-5.41) m/s; p = 0.0002]. BRS quantified by both the sequence and spectral methods showed an inverse correlation with PWVß in the male survivors. Impairment of BRS in the male survivors of mild COVID-19 at 3-6 months of clinical recovery shows association with carotid artery stiffness.

Transfusion trigger in the critically ill with sepsis or septic shock: A prospective study.

Background & objectives: Current practice around transfusion trigger in critically ill sepsis patients is not clear. Moreover, any association of haemoglobin trigger and other transfusion parameters such as age of red blood cells (RBCs) at transfusion and number of units of RBCs transfused with mortality and other adverse outcomes need further assessment. Methods: In this prospective study, patients aged 18-70 yr and admitted to intensive care with a diagnosis of sepsis were included (n=108). Baseline demographic, clinical and laboratory parameters were noted and various transfusion data, i.e., haemoglobin trigger, number of units of RBCs and the age of RBCs were recorded. Following outcome data were collected: 28 and 90 day mortality, duration of mechanical ventilation, vasopressor therapy, intensive care unit (ICU) and hospital stay and requirement of renal replacement therapy. Results: Of the total 108 participants, 78 (72.2%) survived till 28 days and 66 (61.1%) survived till 90 days. Transfusion trigger was 6.9 (6.7-7.1) g/dl [median (interquartile range)]. On multivariable logistic regression analysis, acute physiology and chronic health evaluation (APACHE) II [adjusted odds ratio (aOR) (95% confidence interval {CI}): 0.86 (0.78, 0.96); P=0.005], cumulative fluid balance (CFB) [aOR (95% CI): 0.99 (0.99, 0.99); P=0.005] and admission platelet count [aOR (95% CI): 1.69 (1.01, 2.84); P=0.043] were the predictors of 28 day mortality [model area under the receiver operating characteristics (AUROC) 0.81]. APACHE II [aOR (95% CI): 0.88 (0.81, 0.97); P=0.013], CFB [a OR (95% CI): 0.99977 (0.99962, 0.99993); P=0.044] and transfusion trigger [aOR (95% CI): 3 (1.07, 8.34); P=0.035] were the predictors of 90 day mortality (model AUROC: 0.82). Interpretation & conclusions: In sepsis, patients admitted to the ICU, current practice suggests transfusion trigger is below 7 g/dl and it does not affect any adverse outcome including 28 day mortality.

Functional and transcriptional heterogeneity within the massively expanding HLADR+CD38+ CD8 T cell population in acute febrile dengue patients.

IMPORTANCE: CD8 T cells play a crucial role in protecting against intracellular pathogens such as viruses by eliminating infected cells and releasing anti-viral cytokines such as interferon gamma (IFNγ). Consequently, there is significant interest in comprehensively characterizing CD8 T cell responses in acute dengue febrile patients. Previous studies, including our own, have demonstrated that a discrete population of CD8 T cells with HLADR+ CD38+ phenotype undergoes massive expansion during the acute febrile phase of natural dengue virus infection. Although about a third of these massively expanding HLADR+ CD38+ CD8 T cells were also CD69high when examined ex vivo, only a small fraction of them produced IFNγ upon in vitro peptide stimulation. Therefore, to better understand such functional diversity of CD8 T cells responding to dengue virus infection, it is important to know the cytokines/chemokines expressed by these peptide-stimulated HLADR+CD38+ CD8 T cells and the transcriptional profiles that distinguish the CD69+IFNγ+, CD69+IFNγ-, and CD69-IFNγ- subsets.

Utility of in-house and commercial PCR assay in diagnosis of Covid-19 associated mucormycoss in an emergency setting in a tertiary care center.

Introduction. Invasive mucormycosis (IM) is a potentially fatal infection caused by fungi of the order Mucorales. Histopathology, culture, and radiology are the mainstays of diagnosis, but they are not sufficiently sensitive, resulting in delayed diagnosis and intervention. Recent studies have shown that PCR-based techniques can be a promising way to diagnose IM.Hypothesis/Gap Statement. Early diagnosis of fungal infections using molecular diagnostic techniques can improve patient outcomes, especially in invasive mucormycosis.Aim. The aim of this study was to evaluate the utility of our in-house mould-specific real time PCR assay (qPCR) in comparison with the commercially available real time PCR (MucorGenius PCR), for the early diagnosis of mucormycosis in tissue samples from patients with suspicion of invasive mucormycosis (IM). This in-house assay can detect and distinguish three clinically relevant mould species, e.g. Aspergillus spp., Mucorales and Fusarium spp. in a single reaction with only one pair of primers, without the need for sequencing.Methodology. We enrolled 313 tissue samples from 193 patients with suspected IM in this prospective study. All cases were classified using EORTC/MSGERC guidelines. All samples were tested using traditional methods, in-house qPCR, and MucorGenius PCR.Results. Using direct microscopy as a gold standard, the overall sensitivity and specificity of in-house qPCR for detection of IM was 92.46% and 80% respectively, while that of the MucorGenius PCR was 66.67% and 90% respectively. However, co-infection of IM and IA adversely affected the performance of MucorGenius PCR in detection of IM.The in-house PCR detected Aspergillus spp. in 14 cases and Fusarium spp. in 4 cases which showed clinical and radiological features of fungal sinusitis. The in-house qPCR also performed better in detecting possible cases of IM. This aids early diagnosis and appropriate treatment to improve patient outcomes.Conclusion. Because the in-house PCR is not only sensitive and specific, but also entirely based on SYBR Green for detection of targets, it is less expensive than probe-based assays and can be used on a regular basis for the diagnosis of IM in resource-constrained settings. It can be used to distinguish between mucormycosis and fungal sinusitis caused by Aspergillus and Fusarium in high-risk patients, as well as to accurately detect Mucorales in fungal co-infection cases.

Th17 overexpression in severe COVID-19: a prospective observational study.

INTRODUCTION: The objective of the study was to determine T-cell subtypes, Natural Killer cell activity and cytokines in COVID-19 patients with mild to moderate disease and compare them between patients who had recovered and those who had progressed to severe disease. METHODS: Peripheral blood samples of COVID-19 patients were collected at the time of hospital admission and after one week. These samples were analysed for interleukins (IL-6, IL-17a) using chemiluminescence ELISA. The T-cell subsets (T naïve, T regulatory, Th17, Th1, Th2, CD8+ T cells] were studied using flow cytometry. Mild, moderate and severe COVID-19 are defined as per CDC guidelines. RESULTS: Nineteen COVID-19-positive patients were enrolled between June 2020 to December 2021. Nine had mild COVID-19 and 10 had moderate COVID-19 at recruitment. All mild cases recovered without progression to severe disease, while five patients from the moderate group progressed to severe disease. Overall, there is a decrease in lymphocyte count in patients with moderate-severe disease, but the ratio of Th17 [5.91 (2.69-12.01)] was higher compared to Th1 [1.12 (0.27-3.13)] and Th2[2.34 (2-3.5)]. The high baseline level of IL-6 observed in patients with moderate disease leads to the proliferation of more Th17 type of CD4+ T-cells(p=0.002) and suppression of Treg cells. A higher Th17 subset leads to neutrophilic inflammation in patients with severe COVID-19. CONCLUSION: Interpretation conclusions: Higher baseline IL-6 leads to depletion of regulatory T-cells, Th1 Th2 CD4 cells. IL-6 leads to the proliferation of Th17 type of CD4+ subsets in moderate COVID-19. Higher Th17 cells in moderate COVID-19 patients lead to the production of IL-17a, which may result in intense neutrophilic inflammatory response and cytokine storm.