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.

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.

Efficacy of LD Bio Aspergillus ICT Lateral Flow Assay for Serodiagnosis of Chronic Pulmonary Aspergillosis.

Background: The diagnosis of CPA relies on the detection of the IgG Aspergillus antibody, which is not freely available, especially in resource-poor settings. Point-of-care tests like LDBio Aspergillus ICT lateral flow assay, evaluated in only a few studies, have shown promising results for the diagnosis of CPA. However, no study has compared the diagnostic performances of LDBio LFA in setting of tuberculosis endemic countries and have compared it with that of IgG Aspergillus. Objectives: This study aimed to evaluate the diagnostic performances of LDBio LFA in CPA and compare it with existing the diagnostic algorithm utilising ImmunoCAP IgG Aspergillus. Methods: Serial patients presenting with respiratory symptoms (cough, haemoptysis, fever, etc.) for >4 weeks were screened for eligibility. Relevant investigations, including direct microscopy and culture of respiratory secretions, IgG Aspergillus, chest imaging, etc., were done according to existing algorithm. Serums of all patients were tested by LDBio LFA and IgG Aspergillus (ImmunoCAP Asp IgG) and their diagnostic performances were compared. Results: A total of 174 patients were included in the study with ~66.7% patients having past history of tuberculosis. A diagnosis of CPA was made in 74 (42.5%) of patients. The estimated sensitivity and specificity of LDBio LFA was 67.6% (95% CI: 55.7−78%) and 81% (95% CI: 71.9−88.2%), respectively, which increased to 73.3% (95% CI: 60.3−83.9%) and 83.9% (95% CI: 71.7−92.4%), respectively, in patients with a past history of tuberculosis. The sensitivity and specificity of IgG Aspergillus was 82.4% (95% CI: 71.8−90.3%) and 82% (95% CI: 73.1−89%); 86.7% (95% CI: 75.4−94.1%) and 80.4% (95% CI: 67.6−89.8%), in the whole group and those with past history of tuberculosis, respectively. Conclusions: LDBio LFA is a point-of-care test with reasonable sensitivity and specificity. However, further tests may have to be done to rule-in or rule-out the diagnosis of CPA in the appropriate setting.

Invasive pulmonary aspergillosis infection in severely ill COPD patients in pulmonary ward and ICU.

PURPOSE: This study was planned to determine the trends and susceptibility pattern of invasive pulmonary aspergillosis (IPA) in severely ill chronic obstructive pulmonary disease (COPD) patients admitted in pulmonary ward and ICU of our tertiary care centre. METHODS: Fifty COPD patients suspected of IPA from pulmonary ward and ICU from April 2017 to September 2018 were investigated. Samples were processed by standard methods, culture positive isolates were confirmed by MALDI-TOF MS and antifungal susceptibility testing was performed by microbroth dilution method. RESULTS: Twenty-two critically ill COPD patients were microbiologically positive for IA infection, of which 13 were classified as putative invasive aspergillosis. The most common comorbid illness associated was diabetes. A. flavus and A. fumigatus were the commonest species isolated. The minimum inhibitory concentration of the antifungals was low. Morbidity due to IPA in COPD patients was very high. CONCLUSIONS: Prevalence of IPA in the pulmonary ward and ICU was found to be 9.6%. MALDI-TOF seems to be a promising tool for aiding rapid identification especially for slow growing and non-sporulating fungi. Heightened awareness and suspicion for pulmonary mould infections along with early diagnosis can substantially alter the patient prognosis.

Emerging Aspergillus lentulus infections in India.

Here we report first two cases of invasive pulmonary aspergillosis caused by Aspergillus lentulus from India, in non-neutropenic, critically ill patients with chronic obstructive pulmonary disease (COPD).

Development of a Sensitive and Specific Novel qPCR Assay for Simultaneous Detection and Differentiation of Mucormycosis and Aspergillosis by Melting Curve Analysis.

Molecular diagnostic assays can expedite the diagnosis of fungal infections, and subsequently help in early interventions and appropriate management of patients. The aim of this study was to develop a single set of primers for a real-time quantitative polymerase chain reaction (qPCR) assay to detect and identify commonly reported, clinically relevant molds i.e., Aspergillus spp, Mucorales and Fusarium spp., up to genus level by melting curve analysis. This assay was evaluated in whole blood from patients with suspected invasive aspergillosis (IA), and in tissue biopsy, bronchoalveolar lavage (BAL) fluid and other site-specific samples from patients with suspected invasive mucormycosis (IM). The limit of detection (LoD) was determined as 10 copies/µl for all three molds. The mean coefficient of variation (CV) across all sets of intra- and inter-assay data was 0.63% (ranging from 0.42 to 1.56%), showing high reproducibility of the assay. Sensitivity and specificity of the assay were 93.3 and 97.1% respectively for diagnosis of IA, and 99.29 and 83.84% respectively for diagnosis of IM. Fusarium was not detected in any of the clinical samples included and the few laboratory confirmed cases of fusariosis did not meet the inclusion criteria of the study. Hence no ROC curve or cutoff value could be generated for the same. This newly developed qPCR assay therefore appears to be a promising tool in detection of IA and IM.

Epidemiology and Antifungal Susceptibility Patterns of Invasive Fungal Infections (IFIs) in India: A Prospective Observational Study.

The epidemiology of invasive fungal infections (IFI) is ever evolving. The aim of the present study was to analyze the clinical, microbiological, susceptibility, and outcome data of IFI in Indian patients to identify determinants of infection and 30-day mortality. Proven and probable/putative IFI (defined according to modified European Organization for Research and Treatment of Cancer/Mycoses Study Group and AspICU criteria) from April 2017 to December 2018 were evaluated in a prospective observational study. All recruited patients were antifungal naïve (n = 3300). There were 253 episodes of IFI (7.6%) with 134 (52.9%) proven and 119 (47%) probable/putative infections. There were four major clusters of infection: invasive candidiasis (IC) (n = 53, 20.9%), cryptococcosis (n = 34, 13.4%), invasive aspergillosis (IA) (n = 103, 40.7%), and mucormycosis (n = 62, 24.5%). The significant risk factors were high particulate efficiency air (HEPA) room admission, ICU admission, prolonged exposure to corticosteroids, diabetes mellitus, chronic liver disease (CLD), acquired immunodeficiency syndrome (AIDS), coronary arterial disease (CAD), trauma, and multiorgan involvement (p < 0.5; odds ratio: >1). The all-cause 30-day mortality was 43.4% (n = 110). It varied by fungal group: 52.8% (28/53) in IC, 58.8% (20/34) in cryptococcosis, 39.8% (41/103) in IA, and 33.9% (21/62) in mucormycosis. HEPA room, ICU admission for IC; HEPA rooms, diabetes mellitus for cryptococcosis; hematological malignancies, chronic kidney disease (CKD), sepsis, galactomannan antigen index value ≥1 for IA and nodules; and ground glass opacities on radiology for mucormycosis were significant predictors of death (odds ratio >1). High minimum inhibitory concentration (MIC) values for azoles were observed in C. albicans, C. parapsilosis, C. glabrata, A. fumigatus, A. flavus, R. arrhizus, R. microsporus, and M. circinelloides. For echinocandin, high MIC values were seen in C. tropicalis, C. guillermondii, C. glabrata, and A. fumigatus. This study highlights the shift in epidemiology and also raises concern of high MICs to azoles among our isolates. It warrants regular surveillance, which can provide the local clinically correlated microbiological data to clinicians and which might aid in guiding patient treatment.

Two cases of Osteoarticular Mucor menace: A diagnostic and management conundrum.

Mucormycosis is an uncommon aggressive fungal infection usually seen in immunocompromised hosts or patients with burns and trauma. The common presentations include rhino-orbital-cerebral and pulmonary involvement. Osteoarticular involvement is a rare presentation of this disease. We present two cases of osteoarticular mucormycosis of pelvis and long bones of the lower limb, one in a patient with burn injury and other one in a patient with chronic granulomatous disease, hitherto a rarely reported association. Delayed diagnosis in a setting where tuberculosis is a common cause of chronic osteomyelitis, challenges in medical and surgical management of these patients are discussed in this report.