Evaluating the Use of Blood based Real Time Polymerase Chain Reaction Testing for Early Detection and Monitoring of Mucormycosis.

Mucormycosis is a rare invasive fungal infection with exceedingly high mortality and few therapeutic options. The disease is caused by Mucorales, which is a large group of species within the order of zygomycetes1,2.

Amidst the catastrophe of the second wave of the Coronavirus, the challenge of black fungus is constantly increasing. Many cases of this rare fungal infection have been reported in different parts of the country. Deaths have also been recorded in many places. In Maharashtra alone, 90 people have lost their lives due to black fungus. Every day new cases are coming up in other states including Delhi, Rajasthan, Madhya Pradesh. Rajasthan has even declared black fungus as an epidemic1.

Which patients have the highest risk?

·       Patients with uncontrolled diabetes, diabetic ketoacidosis, and diabetics on steroids or tocilizumab

·       Patients on immune-suppressants or anti-cancer treatment, and patients with chronic debilitating illness

·       Patients on high dose steroids for longer duration or tocilizumab and clinical evidence of thromboembolic episodes like abnormal D’Dimer test.

·       Severe Covid cases

·       Patients on oxygen support – nasal springs, by mask, or on a ventilator.

Recently, molecular techniques have offered an additional opportunity to improve the diagnostic sensitivity3. PCR assays using either primers for specific Mucorales genera, as well as pan-fungal assays targeting ribosomal targets followed by ITS sequencing, have successfully been used to identify Mucorales in different matrices, such as bronchoalveolar lavage fluid or tissue specimens3,4. Additionally, two different assays for detecting circulating Mucorales DNA in peripheral blood have been developed4,5. The assay by Millon et al. uses three different PCR reactions for the detection of Mucor/Rhizopus, Lichtheimia, and Rhizomucor, respectively5. On the other hand, the assay by Springer et al. uses a more generic approach and targets specific fragments of the 18S and 28S genes4.

Early diagnosis of mucormycosis is of utmost importance, since it may improve outcome. In clinical practice, laboratory diagnosis of mucormycosis includes histopathology, direct examination. As such, the current diagnosis of mucormycosis continues to rely on traditional microbiologic techniques such as fungal culture, direct microscopy (using optical brighteners), and histology, all of which have a notoriously low sensitivity3,5,6. Recently, molecular techniques have offered an additional opportunity to improve the diagnostic of mucormycosis 3,4. Methods on detection of Mucorales DNA in blood have shown promising results for earlier and rapid diagnosis and could be used as screening tests in high-risk patients3,5,6. This makes blood based PCR tests attractive as screening assays, as well as a diagnostic test in cases in which more invasive sampling is not feasible.

Purpose of the study:

·       To diagnose early which leads to prompt treatment, to reduce morbidity and mortality, to improve survival and to reduce surgical intervention.

·       Advantage of blood based test- detection 3-68 days earlier that conventional methods

·       To evaluate the sensitivity of qPCR/ Multiplex PCR assay for the detection of circulating Mucorales DNA in patients with invasive mucormycosis.