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

Pandey, M. B.; Xess, Immaculata; Sachdev, Janya; Yadav, Usha; Singh, Gagandeep; Pradhan, Dibyabhaba; Xess, Ashit Bhusan; Rana, Bhaskar; Dar, Lalit; Bakhshi, Sameer; Seth, Rachna; Mahapatra, Manoranjan; Jyotsna, Viveka P; Jain, Arun K.; Kumar, Rakesh; Agarwal, Reshu; Mani, Prashant

doi:10.3389/ffunb.2021.800898

Cited by 8 publications

(5 citation statements)

References 37 publications

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“…The SYBR Green-based PCR demonstrated higher sensitivity and specificity compared to duplex PCR, as well. The sensitivity and PPV of the specific SYBR Green-based PCR for the direct detection of mucormycosis from clinical samples were mostly concordant with the results of previous studies on the detection of Mucorales using multiplex real-time PCR in tissue specimens (31). As evidenced in our results, with the specific primers, the probability of misleading or unintended amplification concerning the usage of pan-fungal primers in tissue specimens particularly those with mixed infections or those collected from unsterile sites will be aborted (32).…”

Section: Discussionsupporting

confidence: 87%

Molecular assays versus mycological methods for diagnosis of rhino orbital mucormycosis: analysis of 120 fresh clinical specimens from COVID-19 patients

Soltani,

Erami,

Ahmadikia

et al. 2024

Preprint

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Mucormycosis, a fungal emergency, poses a serious threat to both COVID-19 and non-COVID-19 individuals due to its invasive nature, rapid progression, and high rates of morbidity and mortality which highlights the crucial need for its timely detection and management. Here, we investigated the utility of Mucorales-specific real-time PCR (rt-PCR) assays for the detection of mucormycosis from clinical specimens and compared with conventional methods and duplex PCR. Methods: Both SYBR Green and TaqMan rt-PCR methodologies were optimized using Mucorales-specific oligonucleotides to amplify the conserved 18S rDNA targets. DNAs extracted from 120 rhino sinus specimens, which all were collected from COVID-19 patients upon suspicion of invasive fungal infections, were used for molecular diagnosis. The results of both rt PCR assays were compared with the result of direct microscopy, culture, and duplex Mucorales-specific PCR assay. Results: SYBR Green rt-PCR detected Mucorales in 51 out of 120 (91.67% of K0H-positive samples), yielding a unique Tm pattern (80.24 ± 0.70°C), whereas TaqMan-probe PCR and culture methods detected it in 34 (73.84%) and 24 samples, respectively. The SYBR Green-based PCR was also more sensitive/specific than the duplex PCR technique. The lower sensitivity in probe-based PCR can be influenced by various factors such as probe degeneracy, which can lead to false-negative results. Conclusion: SYBR Green-based PCR showed superiority over duplex PCR, culture, and TaqMan-probe PCR in terms of cost-effectiveness, rapidness, and sensitivity for the diagnosis of mucormycosis. As there is no serological test, SYBR Green-based PCR can be used as an affirmatory test to rule in or rule out mucormycosis, particularly in cases with atypical hyphae or septate and non-septate hyphae suggestive of mixed infections in direct examination but negative culture.

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Section: Discussionsupporting

confidence: 87%

Molecular assays versus mycological methods for diagnosis of rhino orbital mucormycosis: analysis of 120 fresh clinical specimens from COVID-19 patients

Soltani,

Erami,

Ahmadikia

et al. 2024

Preprint

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“…MucorGenius (PathoNostics; ADT India) is a fast RT-qPCR test kit that detects fungal nucleic acid sequences to help in early identification despite low loads. It is a pan-Mucorale test, as it can detect five different species of fungus that can aid in the early and prompt detection of mucormycosis (30).…”

Section: Advanced Diagnostic Techniquesmentioning

confidence: 99%

Mucormycosis diagnosis revisited: Current and emerging diagnostic methodologies for the invasive fungal infection (Review)

et al. 2022

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Mucormycosis, which is a life threatening condition, is one of the side effects experienced by post-COVID-19 patients. Early identification and timely treatment are essential to stop the dissemination of the disease, since invasive mucormycosis has a very high fatality rate and significant disease dispersion. Conventional diagnostic techniques, including clinical diagnosis, serology, histopathology and radiology, have limitations in diagnosing the disease at an early stage. This warrants the need for advanced diagnostic tools such as nucleic acid diagnostics, advanced serological tests (ELISpot), PCR (pan-Mucorale test) and multiplex PCR. These techniques have been introduced to identify this invasive fungal infection at an incipient stage, thereby helping clinicians to prevent adverse outcomes. The use of biosensors and micro-needle based diagnostic methodologies will pave the way for devising more point-of-care tests that can be employed for the detection of mucormycosis at an incipient stage. The present review discusses the current techniques available and their drawbacks, and the usefulness of advanced diagnostic tools. Furthermore, the possibility of using future diagnostic methods for the diagnosis of mucormycosis is highlighted.

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“…Our previous study showed higher sensitivity and specificity (99.29% and 83.84% respectively) in proven/probable cases of IM using the inhouse qPCR in tissue samples from ROCM cases [33]. This may be because the sample was severely inadequate for some cases in this study, owing to emergency collection settings which were not ideal.…”

Section: Discussionmentioning

confidence: 83%

“…The first real-time PCR assay was performed using our in-house qPCR protocol as described previously [33]. The target sequence was amplified by primers M1F (5′-ACCT TGRC CGRR AGGT CYGGGT −3′) and M1R (5′-CMSKRRRKGRYYGTTGCC −3′) and simultaneous melting curve analysis was performed for genus-level differentiation of the three target fungi.…”

Section: Detection and Identification Of Fungal Dna By Two Real-time ...mentioning

confidence: 99%

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

Pandey,

Sachdev,

Yadav

et al. 2023

Journal of Medical Microbiology

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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.

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

Cited by 8 publications

References 37 publications

Molecular assays versus mycological methods for diagnosis of rhino orbital mucormycosis: analysis of 120 fresh clinical specimens from COVID-19 patients

Molecular assays versus mycological methods for diagnosis of rhino orbital mucormycosis: analysis of 120 fresh clinical specimens from COVID-19 patients

Mucormycosis diagnosis revisited: Current and emerging diagnostic methodologies for the invasive fungal infection (Review)

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

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