Rhizopus microsporus and Mucor racemosus coinfection following COVID-19 detected by metagenomics next-generation sequencing: A case of disseminated mucormycosis

Abstract

Mucormycosis is an invasive opportunistic fungal infection, which may be lethal and mostly affects patients with immunodeficiency or diabetes mellitus. Among Mucorales fungi, Rhizopus spp. is the most common cause of mucormycosis, followed by genera such as Mucor and Lichtheimia. Here we report a patient with severe COVID-19 infection who developed nasal pain, facial swelling, prominent black eschar on the nasal root. CT scan revealed pansinusitis along the maxillary, ethmoidal, and sphenoid sinuses. Mixed mold infection with Rhizopus microsporus and Mucor racemosus was detected by blood metagenomics next-generation sequencing (mNGS) and later nasal mucosa histological investigation confirmed mucormycosis. Severe COVID-19 infection led to the patient's thrombocytopenia and leukopenia. Later disseminated mucormycosis aggravated the infection and sepsis eventually resulted in death.
It is the first case report of mucormycosis in which R. microsporus and M. racemosus as the etiologic agents were found simultaneously in one patient. COVID-19 infection combined with disseminated mucormycosisis can be fatal and mNGS is a fast, sensitive and accurate diagnostic method for fungi detection.

Full text

Heliyon 10 (2024) e25840
Available online 3 February 2024
2405-8440/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Case report
Rhizopus microsporus and Mucor racemosus coinfection following
COVID-19 detected by metagenomics next-generation sequencing:
A case of disseminated mucormycosis
Lihan Hai
a
, Peihong Li
b
, Zheng Xiao
c
, Jinxia Zhou
b
, Bo Xiao
b
, Luo Zhou
b
,
d
,
*
a
Department of Neurology, Xing’an League People’s Hospital, Ulanhot, Inner Mongolia, China
b
Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, China
c
Department of Pathology, First Hospital of Changsha, Changsha, Hunan, China
d
National Clinical Medical Research Center for Geriatric Diseases (Xiangya Hospital), Central South University, Changsha, Hunan, China
ARTICLE INFO
Keywords:
Mucormycosis
mNGS
Rhizopus microsporus
Mucor racemosuss
Severe COVID-19
ABSTRACT
Mucormycosis is an invasive opportunistic fungal infection, which may be lethal and mostly af-
fects patients with immunodeciency or diabetes mellitus. Among Mucorales fungi, Rhizopus spp.
is the most common cause of mucormycosis, followed by genera such as Mucor and Lichtheimia.
Here we report a patient with severe COVID-19 infection who developed nasal pain, facial
swelling, prominent black eschar on the nasal root. CT scan revealed pansinusitis along the
maxillary, ethmoidal, and sphenoid sinuses. Mixed mold infection with Rhizopus microsporus and
Mucor racemosus was detected by blood metagenomics next-generation sequencing (mNGS) and
later nasal mucosa histological investigation conrmed mucormycosis. Severe COVID-19 infec-
tion led to the patient’s thrombocytopenia and leukopenia. Later disseminated mucormycosis
aggravated the infection and sepsis eventually resulted in death.
It is the rst case report of mucormycosis in which R. microsporus and M. racemosus as the
etiologic agents were found simultaneously in one patient. COVID-19 infection combined with
disseminated mucormycosisis can be fatal and mNGS is a fast, sensitive and accurate diagnostic
method for fungi detection.
1. Introduction
The global epidemic of coronavirus disease 2019 (COVID-19) continues to be a major health issue worldwide. The most common
symptoms are fever, dry cough, fatigue, and shortness of breath and sometimes in severe cases, the disease leads to acute respiratory
distress syndrome. According to the World Health Organization, disease severity is determined as mild, moderate, severe, or critical.
COVID-19 can lead to severe fungal co-infections in patients with underlying health conditions, and previous reports have documented
the occurrence of associated mucormycosis in severe cases.
Mucormycosis is an invasive opportunistic fungal infection, mostly affecting immunocompromised patients, such as those with
uncontrolled diabetes and long-term use of corticosteroids. There is an approximately 46% mortality rate in the patients infected [1].
Among Mucorales fungi, Rhizopus spp. is the most common cause of mucormycosis, followed by genera such as Mucor and
* Corresponding author. Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, China.
E-mail addresses: zhouluo33@163.com, zhouluo@csu.edu.cn (L. Zhou).
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https://doi.org/10.1016/j.heliyon.2024.e25840
Received 29 August 2023; Received in revised form 27 December 2023; Accepted 2 February 2024

Heliyon 10 (2024) e25840
2
Lichtheimia, accounting for 70–80% of all mucormycosis cases [2]. It is reported that in all those cases only 50% of fungal cultures
were positive [3]. Moreover, in many clinical reports the pathogens of mucormycosis cases were not identied to the genus or species
level.
Compared to fungal culture, mNGS is a relatively precise and fast method to obtain pathogenic results. Here we report a severe
COVID-19 patient diagnosed with mucormycosis conrmed by the metagenomics next-generation sequencing (mNGS) and histo-
pathological ndings. Based on the mNGS results, our case was positive for the presence of Rhizopus microsporus and Mucor
racemosus.
2. Case report
A 67-year-old male patient with uncontrolled diabetes who experienced COVID-19 infection at the end of December 2022 was
admitted to our hospital on January 1st，2023, with shortness of breath and hypoxia.
On admission, he had a temperature of 36.5 ◦C, heart rate of 116, respiratory rate of 26, blood pressure of 150/88 mmHg, and O
2
saturation of 90% in room air. The laboratory examinations showed: white blood cell count (10^9/L): 2, creatinine (umol/L): 178,
blood sugar (mmol/L): 11.6, hemoglobin A1C (%): 10.1, platelet count (10^9/L): 17, albumin (g/L):20.5, ferritin (ng/ml):＞2000,
indicating inammatory state with severe thrombocytopenia and leukopenia.
After admission, the patient was placed on Paxlovid against COVID-19. He wasn’t treated with steroids because of high blood sugar.
Other therapies also included granulocyte-macrophage colony-stimulating factor (GM-CSF) injection, use of gamma-globulin and
Albutein, etc. The patient gradually improved with platelets increasing, and without hypoxia and shortness of breath. However, the
patient suddenly progressed on January 11th when he complained the new symptom of nasal ache, along with dysnea and platelets
decreasing again (Table 1). Physical examination showed facial and nasal swelling. Uncontrolled diabetes combined with COVID-19
infection made it easier for the patient to develop secondary fungal infection, so when the condition had worsened abruptly, we had a
lot of microbiological workups done, including blood, lower respiratory tract sputum, nasal secretion and urine cultures. All came back
negative. The mNGS of blood was underwent On January 11th. Since highly suspected of fungi infection, though β-D-Glucan (G) and
Galactomannan (GM) tests of blood sample remained negative, the patient was treated with liposomal amphotericin B at a daily dosage
of 250mg on January 13th, later on which day the result of mNGS came back, indicating a total of 22 sequence reads of Rhizopus
microsporum and 19 reads of Mucor racemosus that accounted for 0.16% and 0.13% of all reads respectively (Supplementary
Material).
The next day black eschar on the patient’s nasal root was observed (Fig. 1, a). A noncontrast Computed tomography (CT) scan of the
head was then conducted which revealed pansinusitis along the maxillary, ethmoidal, and sphenoid sinuses (Fig. 1b and c). CT scan of
the lungs showed bilateral lung inltrations consistent with pulmonary mucormycosis (Fig. 1, d). Subsequently the diagnosis of
mucormycosis was further reconrmed by histopathological evaluation of tissue samples from nasal mucosa (Fig. 1e and f). Platelet,
cryoprecipitate and plasma transfusion were administered given his thrombocytopenia with hemorrhagic tendency. Continuous renal
replacement therapy (CRRT) was performed in view of acute kidney dysfunction accompanied by severe metabolic acidosis and sepsis.
Adequate management of the hyperglycemia and metabolic acidosis with intravenous uids and continuous insulin infusion was
initiated. Unfortunately, the patient rapidly aggravated with respiratory and circulatory failure. Norepinephrine, terlipressin and
Table 1
Laboratory results of the patient from admission (01.01.23), condition improved (09.01.23), suspected mucor diagnosis (11.01.23), and after
treatment (16.01.23).
Date 01.01.23 09.01.23 11.01.23 16.01.23 Normal range
Blood cell count
Red cell count 3.69 3.56 3.37 3.32 4.3–5.8 ×10^12/L
Hemoglobin 100 98 91 90 130–175g/L
White cell count 2.0 1.8 1.3 33.0 3.5–9.5 ×10^9/L
Platelets 17 81 28 9.0 125–350 ×10^9／L
inammatory markers
CRP 106 89.8 106 123.0 0–8mg／L
Ferritin ＞2000 – 1543.0 4144.0 30–400ng／mL
Procalcitonin 3.97 4.827 15.14 34.07 0–0.1ng／ml
Kidney function
Creatinine 178 336.0 355.9 410 41.0–111.0
μ
mol／L
BUN 10.5 24.33 29.48 38.22 3.60–9.50mmol／L
Arterial blood gas
PH – 7.34 7.32 7.12 7.350–7.450
PCO
2
– 25 18 30 35–45 mmHg
PaO
2
– 124 149 121 80–100 mmHg
Glucose – 16.2 14 22.3 3.9–5.8mmol/L
hemoglobin A1C 10.1 – – – 4–6%
BS 11.6 – – – 3.9–6.1mmol/L
albumin 20.5g 22.1 16.6 19.1 40.0–55.0g/L
Abbreviations: CRP, c-reactive protein; BUN, blood urea nitrogen; PH, pondus hydrogenii; PCO
2
, partial pressure of carbon dioxide; PaO
2
, arterial
partial pressure of oxygen; BS, blood sugar.
L. Hai et al.

Heliyon 10 (2024) e25840
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hydrocortisone were used but still unable to maintain stable blood pressure. The patient lost the chance to operation and showed no
response to all those treatments above. He was discharged from hospital on January 18th and nally deceased.
3. Discussion
The case emerged when Omicron variant was the primary pathogenic strain. Probably due to weakened virulence of Omicron
compared to Delta and the racial differences, in the mainland of China, the literature about COVID-19 associated mucomycosis is
scarce.
Our case is consistent with others indicating that diabetes mellitus is the most common underlying risk factor of mucormycosis [1,
2]. COVID-19 would further increase the risk. During the COVID-19 pandemic, patients are on immunosuppressive drugs, glucocor-
ticoids, thus are at a higher risk of mucormycosis [3]. Though our patient wasn’t treated with glucocorticoids, his diabetes and
hyperglycaemia brought about an inammatory state that could be potentiated by the activation of antiviral immunity to SARS-CoV2,
which favoured fungal infections.
The patient’s new symptom of nasal ache was subtle and may be easily neglected. We strongly suggest nasal ache may be an
important initial symptom indicating mucor infection, since it represents nasal cavity affected where mucor spores like to invade and
reside rstly.
A study showed that in the diagnosis of invasive fungal rhinosinusitis the sensitivities of the G and GM tests alone were 60.0% and
28.6%, respectively. When the G and GM tests were parallel combined, the sensitivity was 66.7% [4]. So negative results denitely
cannot rule out fungal infection. Therefore, we remained a high suspicion of fungal infection though results of G and GM tests in our
patient were negative. Histopathologically, mucorales hyphae typically have a variable width of 6–16
μ
m, and are nonseptate.
Obtaining a diagnosis of mucormycosis on histomorphological basis sometimes is challenging, because it is hard to distinguish un-
typical hyphae of mucorales from Aspergillus spp. It is strongly recommended to conrm the diagnosis of mucormycosis in tissue by
culture or by application of molecular identication technique [5]. In our case the mNGS provided the initial diagnosis accurately,
while the gold standard diagnostic histological conrmation of non-septate hyphae in nasal tissue was six days later when the patient
was terminally ill. Moreover, the mNGS provided the exact pathogens of Rhizopus microsporus and Mucor racemosus in species. It is
the rst case that these two pathogens were found simultaneously in a disseminatedly infected patient. The Rhizopus microspores and
Mucor racemosus related death rate is reported to be 64% and 52% respectively [6].
Besides bloodstream infection, CT scan conrmed both rhinosinusitis and pulmonary mucormycosis. Therefore disseminated
mucormycosis was diagnosed. The characteristic radiological nding of mucormycosis on CT of the lungs is the halo sign, a ring of
ground glass opacity surrounding a nodular inltrate. The noncontrast CT scan revealed mucosal thickening of the sinuses, indicating
an ongoing sinonasal inammatory process.
The concurrent infection with severe COVID-19 and mucor made timely diagnosis challenging. A large study showed that among
Fig. 1. (a) Black eschar on the nasal root. (b and c) Computed tomography (CT) scan of the head revealing much inammatory effusion along
bilateral maxillary, ethmoidal, and sphenoid sinuses. (d) CT of the lungs showing bilateral lung inltrations consistent with pulmonary mucor-
mycosis. (e and f) Haematoxylin and eosin staining of nasal mucosa showing presence of a large amount of fungal hyphae (red arrows)and spores
(black arrows). (For interpretation of the references to colour in this gure legend, the reader is referred to the Web version of this article.)
L. Hai et al.

Heliyon 10 (2024) e25840
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patients with severe COVID-19, 17% had second concurrent active condition, including second infections, and they were highly prone
to a delayed second diagnosis [7]. In our case, because of the concurrent infection situation, we actually didn’t know the exact time of
mucor infection. Nasal symptom may be a sign. We suggest it is vital to maintain a close surveillance of patients post severe COVID-19
infection.
Simultaneous infection is rare and fatal even when treated with antifungal drugs. Surgical debridement plays a vital role as an
adjunctive treatment in mucormycosis if the patient’s condition permits [5]. Early diagnosis and treatment are critical in order to
improve clinical outcomes. In our case, anti-fungal treatment was not initiated until two days after nasal symptom appeared. The
delayed treatment may be partially responsible for the poor outcome. Based on a study by Son HJ et al., neutropenia, thrombocy-
topenia, positivity of non-sterile culture, use of steroid and treatment without surgery contribute to a poor outcome in patients with
pulmonary mucornycosis [8]. Our case is consistent with the above conclusions. We hold that thrombocytopenia, neutropenia and
treatment without surgery in our case contributed to the poor prognosis. The worst complication of thrombocytopenia in a patient with
mucormycosis is intracranial hemorrhage [9], as a case report has indicated. Treatment is determined by the underlying cause of the
thrombocytopenia. In our case, getting the infection under control was the top priority. Another vital management was platelet
transfusion. However, we failed to elevate the level of platelet count. As shown in Table 1, the patient’s kidney function was getting
worse and worse. Moreover, inammatory markers, including serum levels of ferritin, procalcitonin, and C-reactive protein were
elevated in relation to high levels of circulating pro-inammatory cytokines and chemokines. Performing CRRT was essential to
improve the kidney function, get rid of the harmful circulating cytokines and correct acid-base imbalance. Mechanical ventilation
support and vasoactive drugs were needed to sustain patient’s vital signs at the late stage of the disease. In general, comprehensive
managements are needed for mucormycosis patients with complicated conditions.
Overall, the mNGS may be a fast, sensitive and accurate diagnostic method for early pathogen detection and point the way toward
treatment earlier. We highlight the need for high index of suspicion of mucormycosis after severe COVID-19 infection, performing
imaging earlier and initiating empiric anti-fungal treatment from the onset of presenting symptoms given the worse outcomes of this
disease.
4. Conclusion
COVID-19 associated mucormycosis is life-threatening especially for those with mixed mold infection. In view of the severity of this
disease, the promptly empiric anti-fungal treatment is suggested once mucormycosis is suspected. The mNGS may provide a fast,
sensitive and accurate diagnosis. Our case highlights the diagnostic value of the mNGS in fungal infection.
Consent
Patient consent for publication was obtained from the relative for the publication of all images, clinical data and other data
included in the manuscript.
Funding
This work was supported by the following funding: National Natural Science Foundation of China (Grant No. 81601139) and
Natural Science Foundation of Hunan Province (Grant No. 2017JJ3500).
Data availability statement
Data associated with our study has not been deposited into a publicly available repository. Since our paper is a case report. All data
supporting the ndings of this study are available within the paper and its Supplementary information.
Ethics declarations
All patient’s legal guardians provided informed consent for the publication of patient’s anonymised case details and images.
Approval by an ethics committee was not needed for this study because it is a case report that does not involve experiments.
CRediT authorship contribution statement
Lihan Hai: Writing – original draft, Writing – review & editing, Data curation, Formal analysis, Investigation. Peihong Li: Formal
analysis, Writing – original draft. Zheng Xiao: Formal analysis, Writing – original draft. Jinxia Zhou: Data curation, Formal analysis,
Writing – review & editing. Bo Xiao: Conceptualization, Funding acquisition, Writing – review & editing. Luo Zhou: Conceptuali-
zation, Funding acquisition, Writing – review & editing, Supervision, Writing – original draft.
Declaration of competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to
inuence the work reported in this paper.
L. Hai et al.

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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e25840.
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