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.

Innate and adaptive immune responses in subjects with CPA secondary to post-pulmonary tuberculosis lung abnormalities.

BACKGROUND: Post-tuberculosis lung abnormality (PTLA) is the most common risk factor for chronic pulmonary aspergillosis (CPA), and 14%-25% of the subjects with PTLA develop CPA. The pathogenesis and the host immune response in subjects with PTLA who develop CPA need to be better understood. METHODS: We prospectively compared the innate and adaptive immune responses mounted by patients of PTLA with or without CPA (controls). We studied the neutrophil oxidative burst (by dihydrorhodamine 123 test), classic (serum C3 and C4 levels) and alternative (mannose-binding lectin [MBL] protein levels) complement pathway, serum immunoglobulins (IgG, IgM and IgA), B and T lymphocytes and their subsets in subjects with PTLA with or without CPA. RESULTS: We included 111 subjects (58 CPA and 53 controls) in the current study. The mean ± SD age of the study population was 42.6 ± 15.7 years. The cases and controls were matched for age, gender distribution and body weight. Subjects with CPA had impaired neutrophil oxidative burst, lower memory T lymphocytes and impaired Th-1 immune response (lower Th-1 lymphocytes) than controls. We found no significant difference between the two groups in the serum complement levels, MBL levels, B-cell subsets and other T lymphocyte subsets. CONCLUSION: Subjects with CPA secondary to PTLA have impaired neutrophil oxidative burst and a lower Th-1 response than controls.

A novel indirect ELISA for serodiagnosis of mucormycosis using antigens from Rhizopus arrhizus.

BACKGROUND: Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection. OBJECTIVE: To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis. METHODS: We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 µg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample. RESULTS: Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity. CONCLUSION: The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.

Efficacy of generic forms of itraconazole capsule in treating subjects with chronic pulmonary aspergillosis.

BACKGROUND: Itraconazole capsules have variable and unpredictable bioavailability. OBJECTIVE: Whether the generic brands are as effective as the innovator itraconazole in treating subjects with chronic pulmonary aspergillosis (CPA) remains unclear. METHODS: In this retrospective study, we treated CPA subjects with 6-month itraconazole capsule and measured itraconazole levels at 2 weeks, 3 months and 6 months. Our primary outcome was to compare the proportion of subjects achieving therapeutic drug levels (≥0.5 mg/L) with the generic and the innovator itraconazole after 2 weeks. We performed a multivariate logistic regression analysis to ascertain whether trough itraconazole levels affected treatment outcomes. We categorised treatment response as favourable or unfavourable based on improvement (or worsening) in clinical symptoms, microbiology and imaging. We also performed morphometric analysis of different brands of itraconazole by video-dermoscopy. RESULTS: We included 193 (generic brands [n = 94] and innovator itraconazole [n = 99]) CPA subjects. A higher proportion of subjects achieved therapeutic levels at 2 weeks with the innovator than with the generic brands (72/99 [73%] vs. 27/94 [29%], p < .0001). The median trough level at 2 weeks was higher with the innovator than the generic brands (0.8 vs. 0 mg/L). The mean trough itraconazole levels achieved (average of three values measured over 6 months) independently predicted a favourable treatment response after adjusting for age, gender and CPA severity. On morphometric analysis, the generic brands had variable pellet numbers and sizes, and dummy pellets. CONCLUSION: At 2 weeks, a significantly higher proportion of CPA subjects achieved therapeutic drug levels with the innovator than the generic itraconazole. The mean serum itraconazole levels independently predicted a favourable treatment response in CPA.

Saksenaea vasiformis infection: Extensive abdominal wall necrotizing fasciitis with systematic review and analysis of 65 cases.

INTRODUCTION: Saksenaea vasiformis is a rarely reported Mucorales causing mucormycosis in both immunocompromised and immunocompetent individuals. Due to few reported cases, the clinical characteristics and optimal management strategy for this rare agent are not clearly described. METHODS: We systematically reviewed Medline, EmBase and CINHAL for studies on S. vasiformis infections reported until 1 January 2022 and 57 studies (63 patients) were retrieved. Additionally, one more case of extensive abdominal wall necrotizing fasciitis managed by our team was also included. The clinical and demographic characteristics and outcomes were extracted and analysed. RESULTS: Out of the 65 included cases, the majority were reported from India (26.6%). The most common risk factors for infection were accidental trauma wounds (31.3%), health-care-related wounds (14.1%) and animal/insect bites (12.5%). Most common clinical presentation was subcutaneous mucormycosis (60.9%) followed by rhino-orbito cerebral mucormycosis (14%), necrotizing fasciitis (10%), disseminated infection (9.3%), pulmonary mucormycosis (3.2%) and osteomyelitis (1.6%). Mortality was observed in 24 (37.5%) patients and health care related injuries were significantly associated with higher mortality (p = .001). The use of posaconazole (p = .019) and the use of surgical management (p = .032) was associated with significantly better survival. DISCUSSION: In this study, we describe the largest compendium of mucormycosis due to S. vasiformis, which can be useful in increasing awareness regarding this rare Mucorales and guiding patient management.

Serum glucose-regulated protein 78 (GRP78) levels in COVID-19-associated mucormycosis: results of a case-control study.

BACKGROUND: In experimental models, the expression of glucose-regulated protein 78 (GRP78) in endothelial cells played a role in the pathogenesis of mucormycosis. However, the role of GRP78 in COVID-19-associated mucormycosis (CAM) has not been studied. We hypothesized that serum GRP78 levels are elevated in subjects with CAM. OBJECTIVE: To compare the serum GRP78 levels in subjects with CAM and COVID-19 controls without mucormycosis. DESIGN AND SETTING: We performed a hospital-based, case-control study between 1 April 2021 and 31 May 2021. PARTICIPANTS: We enrolled 24 subjects each of CAM and COVID-19 subjects without mucormycosis. We also measured serum GRP78 levels in ten healthy controls. EXPOSURE: The primary exposure studied was serum GRP78 concentration, estimated using a commercially available ELISA kit in stored serum samples. RESULTS: We found the mean ± standard deviation (SD) serum GRP78 levels significantly higher (p = 0.0001) among the CAM (374.3 ± 127.3 pg/mL) than the COVID-19 (246.4 ± 67.0 pg/mL) controls. The proportion of subjects with an abnormal GRP78 level (> mean [184.8 pg/mL] plus two SD [23.2 pg/mL] of GRP78 from healthy participants) was 87.5% and 45.8% in the CAM group and COVID-19 controls, respectively. Serum GRP78 level was independently associated with CAM (odds ratio 1.011; 95% confidence interval [1.002-1.019]) after adjusting for diabetes mellitus and hypoxemia during acute COVID-19. CONCLUSION: Serum GRP78 levels were significantly higher in CAM than in COVID-19 controls. Further studies are required to the role of GRP78 in the pathogenesis of CAM.

Is there an association between zinc and COVID-19-associated mucormycosis? Results of an experimental and clinical study.

BACKGROUND: The enormous increase in COVID-19-associated mucormycosis (CAM) in India lacks an explanation. Zinc supplementation during COVID-19 management is speculated as a contributor to mucormycosis. We conducted an experimental and clinical study to explore the association of zinc and mucormycosis. METHODS: We inoculated pure isolates of Rhizopus arrhizus obtained from subjects with CAM on dichloran rose Bengal chloramphenicol (DRBC) agar enriched with (three different concentrations) and without zinc. At 24 h, we counted the viable colonies and measured the dry weight of colonies at 24, 48 and 72 h. We also compared the clinical features and serum zinc levels in 29 CAM cases and 28 COVID-19 subjects without mucormycosis (controls). RESULTS: We tested eight isolates of R arrhizus and noted a visible increase in growth in zinc-enriched media. A viable count percentage showed a significantly increased growth in four of the eight isolates in zinc-augmented DRBC agar. A time- and concentration-dependent increase in the mean fungal biomass with zinc was observed in all three isolates tested. We enrolled 29 cases of CAM and 28 controls. The mean serum zinc concentration was below the reference range in all the subjects and was not significantly different between the cases and controls. CONCLUSIONS: Half of the R arrhizus isolates grew better with zinc enrichment in vitro. However, our study does not conclusively support the hypothesis that zinc supplementation contributed to the pathogenesis of mucormycosis. More data, both in vitro and in vivo, may resolve the role of zinc in the pathogenesis of CAM.

Rapid and Simple Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantifications of Triazole Antifungals in Human Serum.

PURPOSE: To develop and validate a one-step, rapid and simple reversed-phase high-performance liquid chromatography (HPLC)-based protocol for the simultaneous measurement of voriconazole (VCZ), posaconazole (POSA), itraconazole (ITC) in serum/plasma. METHODS: Calibration standards (CS) and quality control samples were prepared in drug-free serum by spiking with the triazoles at different concentrations. HPLC was performed with C18 column, isocratic mobile phase after extraction with cold acetonitrile. The standardized method was tested in 2693 patients' serum/plasma samples. RESULTS: Linearity of CS for ITC, VCZ and POSA was proportional to the nominal concentration (correlation coefficient > 0.999). Limit of detection (mg/L) for ITC, VCZ and POSA was 0.25, 0.25 and 0.125, respectively. The lower limit of quantification (mg/L) for ITC, VCZ and POSA was 0.5, 0.5 and 0.25, respectively. Precision and accuracy were in acceptable range with 100% average percentage recovery. No interferences from endogenous substances and other antimicrobial compounds were noted. In clinical samples, the therapeutic range achieved for VCZ was 39.9%. Whereas, 61.1% and 44% of samples with ITC and POSA, respectively, were in the sub-therapeutic range. CONCLUSION: We developed a rapid and simple HPLC method to quantify common triazoles in a single chromatographic run allowing simultaneous measurement of different antifungals in a small volume of serum/plasma. Thus, therapeutic drug monitoring requests can be processed in one run without changing the protocol parameters, column or column conditioning thereby improving turnaround time.

Eumycetoma of the Foot due to Fusarium solani in a Person with Diabetes Mellitus: Report of a Case and Review of Literature.

Eumycetomas are chronic suppurative granulomas caused by fungi characterised by invasive tumefactive lesions, sinuses and discharging grains. Herein, we describe a case of pedal eumycetoma due to Fusarium solani sensu stricto in a person with diabetes mellitus. A 45-year-old gentleman presented with an insidious onset swelling over his right foot with nodules and discharging grains. He had received itraconazole and anti-tuberculous therapy elsewhere, without response. Re-evaluation included a biopsy which confirmed eumycetoma and newly diagnosed diabetes. Surgical excision followed by histopathological, microbiological and multigene sequencing analyses [translation elongation factor, calmodulin and internal transcribed spacer region of rDNA] of the mould on culture were performed. Histopathology revealed septate fungal hyphae amidst a dense inflammatory infiltrate (Splendore-Hoeppli) reaction. Oral voriconazole was started and good glycemic control attained. Tissue growth sequences showed > 99% similarity with Fusarium solani sensu stricto. Antifungal susceptibility testing showed lowest MIC to voriconazole (0.5 mg/L). The patient showed excellent response to combined therapeutic modality with a near-complete resolution in size of lesion and obliteration of sinuses following 4 months of therapy and is planned for prolonged voriconazole therapy till complete radiological resolution. Diabetes predisposes to fungal infections of foot but eumycetomas are uncommon. Combined surgery and antifungals can improve morbidity and avoid amputations.

Role of recombinant Aspergillus fumigatus antigens in diagnosing Aspergillus sensitisation among asthmatics.

BACKGROUND: The diagnosis of Aspergillus-sensitised asthma (ASA) and allergic bronchopulmonary aspergillosis (ABPA) is made using IgE against crude antigens of A fumigatus (cAsp). However, the IgE against cAsp has limitations due to cross-reactivity with other fungi. OBJECTIVE: To evaluate the utility of recombinant A fumigatus (rAsp) antigens in detecting ASA and their role in differentiating true from cross-sensitisation. METHODS: We performed IgE against rAsp (f 1, f 2, f 3, f 4 and f 6), cAsp and other fungal (Alternaria, Candida, Cladosporium, Malassezia and Trichophyton) antigens in subjects with A fumigatus-unsensitised asthma (Af-UA [n = 51]), ASA (n = 71) and ABPA (n = 123). The diagnoses were made using cAsp-IgE and compared using rAsp-IgE. Subjects with elevated cAsp-IgE, but negative rAsp f 1 and f 2, were presumed to lack true A fumigatus sensitisation. RESULTS: The prevalence of any rAsp antigen positivity (cut-off, 0.35 kUA/L) varied from 2%-22%, 32%-73% and 84%-98% for Af-UA, ASA and ABPA, respectively. The prevalence of sensitisation to other fungi ranged from 29%-65%, 59%-85% and 87%-95%, respectively, among subjects with Af-UA, ASA and ABPA. Nineteen subjects of ASA and one subject with ABPA were positive with cAsp-IgE but negative for rAsp f 1 and f 2 and were also cross-sensitised to at least one of the other fungi. Five subjects of Af-UA (cAsp-IgE negative) were rAsp f 1 or f 2 positive. CONCLUSIONS: Crude Aspergillus antigens may misclassify Aspergillus sensitisation among asthmatics. IgE against rAsp antigens (f 1 and f 2) potentially detect true Aspergillus sensitisation and could be used for this purpose.

Utility of Serum and Bronchoalveolar Lavage Fluid Galactomannan in Diagnosis of Chronic Pulmonary Aspergillosis.

The value of serum and bronchoalveolar lavage fluid galactomannan (BALF-GM) in diagnosing chronic pulmonary aspergillosis (CPA) remains unclear. Here, we study the diagnostic efficacy of GM in the diagnosis of CPA. We included consecutive treatment-naive subjects with CPA. For calculating the specificity of serum GM, we enrolled diseased controls (minimally symptomatic subjects previously treated for pulmonary tuberculosis, not meeting the criteria for CPA). To calculate the specificity of BALF-GM, subjects with pulmonary disorders other than CPA who underwent bronchoscopy were included. We determined the cutoff of serum and BALF-GM levels using receiver operating characteristic (ROC) curve analysis. We enrolled 243 consecutive treatment-naive subjects (53.5% males) of CPA with a mean (standard deviation) age of 43.6 (14.7) years. Forty-five (60% males; age, 46.7 [15.7] years) and 27 (59.3% males; age, 52.6 [12.8] years) subjects served as controls for serum and BALF-GM, respectively. The best cutoff value for serum and BALF-GM was 0.55 (area under the ROC curve [AUROC], 0.605; sensitivity, 38%; specificity, 87%) and 1.375 (AUROC, 0.836; sensitivity, 68%; specificity, 93%), respectively. At a cutoff value of 2.5, BALF-GM had a sensitivity and specificity of 50% and 100%, respectively. BALF-GM performs better than serum GM and may be helpful in the diagnosis of CPA in selected patients. More studies are required to confirm our findings.

Prevalence of sensitization to Aspergillus flavus in patients with allergic bronchopulmonary aspergillosis.

Aspergillus fumigatus is the most common Aspergillus species worldwide; however, A. flavus has also been shown to be prevalent in North India. Herein, we investigate the prevalence of sensitization to A. flavus in subjects with allergic bronchopulmonary aspergillosis (ABPA). We also evaluate the occurrence of allergic bronchopulmonary mycosis (ABPM) due to A. flavus. Treatment-naive subjects with ABPA underwent sputum culture; and, skin testing, fungal-specific immunoglobulin E (IgE) and serum precipitation tests for A. fumigatus and A. flavus. Sensitization to A. flavus was diagnosed if any immunological test for A. flavus was positive in subjects with ABPA. ABPM was labelled as probable if sputum cultures grew A. flavus and A. flavus-specific IgE was greater than A. fumigatus-specific IgE; and, possible if only A. flavus-specific IgE was greater than A. fumigatus-specific IgE. Fifty-three subjects with a mean (SD) age of 34.2 (12.8) years were included. Sensitization to A. flavus was seen in 51 (96.2%) subjects, with overlap occurring in 49 (92.5%), 21 (39.6%), and 12 (22.6%) instances on fungal-specific IgE, skin prick test and precipitins, respectively. Sputum culture was positive in 18 (33.9%; A. flavus [n = 12], A. fumigatus [n = 6]) subjects. ABPM due to A. flavus was diagnosed in 16 (30.2%) subjects (10 probable, 6 possible). They were more likely to have high-attenuation mucus and a trend towards higher occurrence of sinusitis, compared to ABPA. We found a high occurrence of sensitization to A. flavus in subjects with ABPA. Subjects with A. flavus-related ABPM had a higher likelihood of high-attenuation mucus and probability of sinusitis. More studies are required to confirm this observation.

Efficiency of A fumigatus-specific IgG and galactomannan testing in the diagnosis of simple aspergilloma.

BACKGROUND: An early diagnosis of chronic pulmonary aspergillosis (CPA) at the stage of simple aspergilloma (SA) remains a challenge in low- and middle-income countries, where imaging may not be routinely available. OBJECTIVE: We investigate the role of Aspergillus fumigatus-specific IgG in serum, and galactomannan (GM) in bronchoalveolar lavage fluid (BALF) and serum for the diagnosis of SA. METHODS: We included 46 consecutive treatment-naïve subjects with SA. The 81 controls were subjects of treated pulmonary tuberculosis with residual radiological abnormality and minimal symptoms; and subjects with pulmonary disorders other than CPA who underwent bronchoscopy. The diagnosis of SA was based on consistent clinical features along with radiological manifestations (cavity with fungal ball). RESULTS: Using receiver operating characteristic (ROC) curve analysis, the best cut-off value for A fumigatus-specific IgG was 27.3 mgA/L (AUROC, 0.839; sensitivity, 63.5%; specificity, 98.3%). The best cut-off value for serum and BALF-GM was 0.7 (AUROC, 0.636; sensitivity, 32%; specificity, 96.2%) and 2.5 (AUROC, 0.833; sensitivity, 63.7%; specificity, 97.1%), respectively. A combination of A fumigatus-specific IgG (>27 mgA/L) or serum GM (≥0.7) or BALF-GM (≥2.5) had a sensitivity and specificity of 82.6% and 96%, respectively. CONCLUSIONS: A combination of serological tests has the best sensitivity in diagnosing SA. More studies are needed to confirm our findings.

Diagnostic cut-off of Aspergillus fumigatus-specific IgG in the diagnosis of chronic pulmonary aspergillosis.

Aspergillus fumigatus-specific IgG is pivotal in making the diagnosis of chronic pulmonary aspergillosis (CPA). However, the cut-off value for A. fumigatus-specific IgG remains unknown. We included consecutive treatment-naïve subjects with chronic cavitary pulmonary aspergillosis (CCPA, cases). The controls were subjects with treated pulmonary tuberculosis, who had residual radiological abnormality and minimal symptoms. The diagnosis of CCPA was based on consistent clinicoradiological features along with demonstration of Aspergillus infection (growth of Aspergillus in sputum or bronchoalveolar lavage fluid [BALF] culture; serum or BALF galactomannan index >0.5 and >1, respectively). For determining the cut-off of A. fumigatus-specific IgG (Phadia), subjects were randomly classified as derivation (two-thirds) and validation (one-third) cohort. One hundred and thirty-seven cases and 50 controls were included. The best cut-off value for A. fumigatus-specific IgG (derivation cohort) was 27.3 mgA/L (AUROC, 0.976) at a sensitivity and specificity of 95.6% and 100%, respectively. Using a cut-off of 27 mgA/L, the sensitivity and specificity in the validation cohort was 91.3% and 100%, respectively. In contrast, the sensitivity of Aspergillus precipitins was only 25.5%. At a cut-off value of 27 mgA/L, A. fumigatus-specific IgG is a reliable test with high sensitivity and specificity in the diagnosis of CPA. More studies are required to confirm our findings.

Aspergillus sensitisation in bidi smokers with and without chronic obstructive lung disease.

Recent studies have described fungal sensitisation in patients with chronic obstructive pulmonary disease (COPD). However, no study has evaluated fungal sensitisation specifically in bidi smokers. Herein, we evaluate the prevalence of Aspergillus sensitisation in bidi smokers. Bidi smokers with and without COPD underwent chest radiography, spirometry, Aspergillus skin test, A. fumigatus precipitins, A. fumigatus-specific IgE and total IgE. Aspergillus sensitisation was defined as the presence of either immediate cutaneous hyperreactivity to Aspergillus antigen or raised A. fumigatus-specific IgE level >0.35 kUA/L. Bidis were obtained from a subset of cases and controls and cultured for the growth of any fungus. Two hundred subjects with COPD and 72 chronic bidi smokers without COPD were included in the study (258 men; mean age, 56.8 years). Aspergillus sensitisation was found to be significantly higher in bidi smokers without COPD (27.8%) compared to the COPD cases (16%). Age, COPD, lung function, severity of smoking and current smoking were not associated with Aspergillus sensitisation, on a multivariate logistic regression analysis. We found a high prevalence of Aspergillus sensitisation in bidi-smoking subjects. More studies are required to confirm the findings of our study.

Role of Aspergillus fumigatus-specific IgG in diagnosis and monitoring treatment response in allergic bronchopulmonary aspergillosis.

Few studies have evaluated the utility of Aspergillus fumigatus-specific IgG in allergic bronchopulmonary aspergillosis (ABPA). Herein, we evaluate the role of specific IgG in diagnosis and monitoring treatment response in ABPA. Forty-eight control subjects with A. fumigatus-associated asthma underwent A. fumigatus-specific IgG measurements at baseline, while specific IgG was assayed in 102 treatment-naïve subjects of ABPA at baseline, after eight weeks of glucocorticoid therapy, and during exacerbations. For determining the cut-off of A. fumigatus-specific IgG, we randomly classified two-thirds of the study subjects (cases and controls) as the derivation cohort, while the remaining one-thirds were labelled as the validation cohort. The best cut-off value of A. fumigatus-specific IgG in the derivation cohort was 26.9 mgA /L (sensitivity: 88%; specificity: 100%). Using this limit, the sensitivity and specificity of A. fumigatus-specific IgG in diagnosis of ABPA was 89% and 100%, respectively, in the validation cohort. In contrast, the sensitivity of Aspergillus precipitins was only 27.4%. Following treatment, the A. fumigatus-specific IgG increased in 38 (37.2%) subjects, while it decreased in three (23.1%) of the 13 subjects experiencing an exacerbation. The A. fumigatus-specific IgG was found to be an extremely useful test in the diagnosis and differential diagnosis of ABPA but is unreliable in monitoring treatment response in this disorder.

Basidiobolomycosis caused by a rare species - Basidiobolus meristosporus.

A five-year girl was referred to our centre with swelling over the right lower back. The child was evaluated to rule out chronic cutaneous tuberculosis, lymphoma and soft tissue tumor. Biopsy of the lesion on culture yielded Basidiobolus species. Whole genome sequencing of the isolate identified it as Basidiobolus meristosporus. Sequencing of fungi pathogenic to humans which cannot be differentiated by conventional methods of speciation becomes essential to assign pathogenicity, understand epidemiology and resolve the nuances in the ever-evolving taxonomical classification.

LDBio Aspergillus immunochromatographic test lateral flow assay for IgG/IgM antibody detection in chronic pulmonary aspergillosis: Single-centre evaluation and meta-analysis.

PURPOSE: Chronic pulmonary aspergillosis (CPA) is an infection of the lung usually caused by Aspergillus fumigatus in patients with pre-existing pulmonary diseases. Its diagnosis hinges on demonstrating IgG antibodies against A. fumigatus. Herein, we evaluated the performance of a newly introduced point of care test (POCT) kit, the LDBio Aspergillus IgG/IgM lateral flow assay (LFA) in India with the standard ImmunoCAP kit for diagnosing CPA. METHODS: A total of 60 serum samples (30 CPA cases and 30 controls) were evaluated by the Aspergillus immunochromatographic test (ICT) IgG/IgM LFA. Fluorescent-enzyme immunoassay was used to determine specific A. fumigatus-IgG concentrations (positive >27 mgA/L). Further, a systematic review and meta-analysis of studies (up to August 26, 2021) reporting the performance of LDBio ICT for the diagnosis of CPA was performed. RESULT: A sensitivity of 86.7%, specificity of 90%, negative predictive value of 87.1%, positive predictive value of 89.7%, negative likelihood ratio of 0.15, positive likelihood ratio of 8.67, and was observed for the LDBio IC. There was good agreement between LDBio ICT and ImmunoCAP (88.3%) with a Cohen's Kappa score of 0.77. Our systematic review identified four studies and the pooled sensitivity of 90%, specificity of 91%, area under the curve of 0.94 and diagnostic odds ratio of 57.2, for CPA diagnosis by LDBio ICT. CONCLUSION: Aspergillus LDBio ICT assay exhibits good sensitivity and can be used to screen CPA cases.

Efficacy of 12-months oral itraconazole versus 6-months oral itraconazole to prevent relapses of chronic pulmonary aspergillosis: an open-label, randomised controlled trial in India.

BACKGROUND: Chronic pulmonary aspergillosis has a 5-year mortality of 50-80% globally, and the optimal duration of treatment for chronic pulmonary aspergillosis remains unclear. We aimed to compare the effect of 6-months of oral itraconazole with 12-months of oral itraconazole on chronic pulmonary aspergillosis clinical outcomes. METHODS: In this single-centre, open-label, randomised controlled trial conducted in one chest clinic in Chandigarh, India, we screened consecutive patients with chronic pulmonary aspergillosis who were naive to antifungal treatment and randomised eligible patients, using block randomisation, to receive a starting dose of 400 mg/day of oral itraconazole for either 6 months or 12 months. There was no masking of participants or investigators. We excluded patients who were unable to provide informed consent; had an intake of any antifungal drugs for more than 3 weeks in the preceding 6 months; had active Mycobacterium tuberculosis or non-tuberculous mycobacterial pulmonary disease; and had allergic, subacute, or invasive forms of aspergillosis. The primary outcome was the proportion of patients having relapse 2 years after treatment initiation. We performed an intention-to-treat analysis for all outcomes. The study is registered with ClinicalTrials.gov, NCT03920527. FINDINGS: We recruited participants between July 1, 2019, and Aug 31, 2021. We screened 250 patients, of which 164 were included in the trial. 81 patients were randomised to the 6-month group and 83 patients were randomised to the 12-month group. The study population was 78 (48%) women and 86 (52%) men, and the mean age of participants was 44·3 (SD 13·3) years. The proportion of patients experiencing relapse was significantly lower in the 12-month group, 31 (38%) had a relapse in the 6-month group compared with 8 (10%) in the 12-month group, with an absolute risk reduction of 0.29 [95% CI 0·16-0·40]. The mean time to first relapse was 23 months in the 12-month group, which is significantly longer than the mean of 18 months in the 6-month group (p<0.0001). There were 16 deaths in total, eight in each group. Ten (12%) of 81 patients in the 6-months group and 18 (22%) of 83 patients in the 12-months group had adverse effects, with none requiring treatment modification. Nausea and anorexia were the most common adverse events in both groups. INTERPRETATION: Treatment of chronic pulmonary aspergillosis with 12 months of oral itraconazole was superior to 6 months of oral itraconazole in reducing relapses at 2 years. Itraconazole should be given for at least 12 months for treating chronic pulmonary aspergillosis. FUNDING: None. TRANSLATION: For the Hindi translation of the abstract see Supplementary Materials section.

Evaluation of environmental Mucorales contamination in and around the residence of COVID-19-associated mucormycosis patients.

Introduction: Recently, India witnessed an unprecedented surge of coronavirus disease 2019 (COVID-19)-associated mucormycosis (CAM) cases. In addition to patient management issues, environmental Mucorales contamination possibly contributed to the outbreak. A recent study evaluated environment contamination by Mucorales in the hospital setting. However, a considerable number of CAM patients were never admitted to a hospital before the development of the disease. The present study, therefore, planned to evaluate Mucorales contamination of patients' residences. Methods: The residential environment of 25 patients with CAM living in north India was surveyed. Air samples were collected from indoor and immediate outdoor vicinity of the patients' residence and cultured on Dichloran Rose-Bengal Chloramphenicol (DRBC) agar with benomyl for selective isolation of Mucorales. Surface swab samples were also collected from the air coolers fitted in those residences and cultured on DRBC agar. The isolates were identified by phenotypic and genotypic methods. Amplified fragment length polymorphism (AFLP) was employed to evaluate the genetic relatedness of the environmental and patients' clinical isolates. Results: The median spore count (mean ± SD, cfu/m3) of Mucorales in the air of patients' bedrooms was significantly higher than in the air in other rooms in those residences (3.55 versus 1.5, p = 0.003) or the air collected directly from the front of the air cooler (p < 0.0001). The Mucorales spore count in the environment did not correlate with either ventilation of the room or hygiene level of the patients' residences. Rhizopus arrhizus was isolated from the environment of all patients' residences (n = 25); other Mucorales species isolated were Cunninghamella bertholletiae (n = 14), Rhizopus microsporus (n = 6), Rhizopus delemar (n = 6), Syncephalastrum racemosum (n = 1), Lichtheimia corymbifera (n = 1), and Mucor racemosus (n = 1). Genetic relatedness was observed between 11 environmental isolates from the patients' bedrooms and respective clinical isolates from patients. Discussion: The study supported the view that the patients might have acquired Mucorales from the home environment during the post-COVID-19 convalescence period. Universal masking at home during patients' convalescence period and environmental decontamination could minimize exposure in those susceptible patients.