The Mystery of Mucormycosis in COVID-19: A Multifactorial Menace or an Enigmatic Delta Variant Associated Phenomenon? An Exploratory Study from a Tertiary Care Centre in North India with a Brief Literature Review.

Background: There was a global surge in cases of mucormycosis in COVID-19 patients during the second wave of the pandemic in 2021, reported especially from India. Various predisposing factors such as diabetes mellitus, rampant use of corticosteroids, and COVID-19 per se may be responsible for this spike. Some public health experts have postulated that the epidemiological link between the Delta variant of SARS-CoV-2 and mucormycosis should be explored. Material and
Methods: A retrospective exploratory study was conducted, in which data of 15 laboratory-confirmed cases of COVID-19 with mucormycosis and/or aspergillosis co-infections were collected after obtaining approval from the institute's ethics committee. These patients were admitted to the Mucor wards of our hospital. The positive COVID-19 status of these patients was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). The residual SARS-CoV-2 RNA containing elutes of these patients were stored at -80°C in deep freezers and subjected to whole-genome sequencing in June 2021 at the National Centre for Disease Control (NCDC), New Delhi, India as part of the Indian SARS-CoV-2 Genomic Consortia (INSACOG) program. Concomitant fungal infections in these patients were diagnosed by KOH wet mount and fungal culture as per standard guidelines. Descriptive statistics in the form of percentages and median were used to report the findings.
Results: Periorbital swelling and ocular pain (14/15; 93.33%), followed by facial swelling (11/15; 73.33%) and nasal obstruction (9/15; 60%), were the most common clinical features observed in these patients. Rhizopus arrhizus was the most common causative fungal agent (12/15; 80%). The majority of the patients (9/13; 69.23%) were infected with the Delta variant of SARS-CoV-2.
Conclusion: COVID-associated mucormycosis seems to be multifactorial in origin. Although there may be a possible association between mucormycosis and the Delta variant, more studies should be conducted to explore this seemingly reasonable proposition. Copyright:

INTRODUCTION

Coronavirus disease 2019 (COVID-19), which wreaked havoc globally, has been associated with various opportunistic bacterial and fungal infections.[1] Both Aspergillus and Candida species have been implicated as the main fungal pathogens causing co-infections in COVID-19 patients.[2] There was a global surge in cases of mucormycosis in COVID-19 patients during the second wave of the pandemic in 2021, reported especially from India.[3] Mucormycosis is a fatal angioinvasive infection caused by mould fungi of the order Mucorales, class Zygomycetes, which include Rhizopus, Mucor, Rhizomucor, Cunninghamella, and Absidia spp. Nearly 60% of mucormycosis cases in humans, of which 90% are of the Rhino-orbital-cerebral (ROCM) form, are caused by Rhizopus arrhizus (R. oryzae). The mode of contamination occurs through the inhalation of fungal spores.[3]

Few eminent Indian medical mycologists seem to have a different view on the seemingly enigmatic issue of mucormycosis and COVID-19 coinfection. Despite the well-known facts that diabetes mellitus (DM) and overuse of corticosteroids trigger fungal infections, they strongly believe that there are some missing links that need to be addressed.[4] Recent studies have revealed that the prevalence of mucormycosis in India is nearly 80 times higher (0.14 per 1000) as compared to developed countries, with diabetes mellitus being the most common risk factor.[567] However, the sudden rise in mucormycosis cases among COVID-19 patients during the second wave was unprecedented.[4] Mahalaxmi et al.[8] hypothesized that excessive use of high-flow industrial oxygen could have led to this menace. COVID-19-infected individuals were given oxygen therapy, and contamination in these devices may have served as points of mucormycosis infection. Inhalation of Mucor spores by immunocompromised patients, especially those with uncontrolled diabetes mellitus, would have led to the colonization of the fungus, followed by an invasion of the host and development of mucormycosis. To the best of our knowledge, there is no concrete evidence to date addressing the role of high-flow industrial oxygen leading to a surge in mucormycosis cases. An unpublished report from the Center of Advanced Research in Medical Mycology at PGIMER Chandigarh also ruled out the use of industrial oxygen as a cause behind the surge in cases. Mucor, Rhizopus, or similar species were not isolated, either in humidifiers for oxygen administration or in the oxygen supply pipelines.[4] Some public health experts have postulated that epidemiologically, the link between the Delta variant of SARS-CoV-2 (B.1.617.2) and mucormycosis has not been studied and should be explored further.[4] This variant is known to be highly transmissible and evades the host immune system.[9] Keeping the aforementioned hypothesis in mind, we conducted a preliminary study at a tertiary care teaching institute of National Importance located in Rishikesh, Uttarakhand, India.

MATERIAL AND METHODS

Study design and inclusion criteria: A retrospective exploratory study was conducted in which data of 15 laboratory-confirmed cases of COVID-19 with mucormycosis and/or aspergillosis coinfections were collected after obtaining approval from the institute's ethics committee vide letter number AIIMS/IEC/21/513 dated 02/09/2021. These patients were admitted to the Mucor wards of our hospital between May 16 and June 1, 2021.

Molecular testing for COVID-19: Positive COVID-19 status of these patients was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). Briefly, viral RNA was extracted from a 200-μL sample by using Kingfisher Flex system 96-well RNA extractor and MagMax Viral RNA extraction kit (Thermo Fisher Scientific, Waltham, MA USA 02451). Extracted RNA was subjected to SARS-CoV-2 testing by real-time RT-PCR by using Taqpath Covid-19 combo kit and Quantstudio-7 real-time Thermocycler (Thermo Fisher Scientific, Waltham, MA USA 02451).

Whole-genome sequencing: The residual SARS-CoV-2 RNA containing elutes of these patients were stored at −80°C in deep freezers and subjected to whole-genome sequencing in June 2021 at the National Centre for Disease Control (NCDC), New Delhi, India as part of the Indian SARS-CoV-2 Genomic Consortia (INSACOG) program. RNA library was prepared by Illumina Covidseq protocol (Illumina Inc, San Diego, CA, USA). Complementary DNA (cDNA) was prepared by annealing random hexamers to extracted RNA, which was obtained by the method described with PCR earlier. Initial strands of cDNA were synthesized by reverse transcriptase. Two separate pools of PCR plates (with two pools of primer, i.e., pool 1 and pool 2, covering the entire genome of SARS-CoV-2) were used to amplify synthesized cDNA. Tagmentation and post-tagmentation clean up (bead-based) processes were carried out next. The amplicon was further amplified now as per the manufacturer's instruction (Covidseq reference guide, Illumina Inc, San Diego, CA, USA). Pre-paired 10 base pair indexes (Set 1, 2, 3, 4 adapters) were added as a result of sequence cluster generation. For each of 96 wells, one Covidseq positive control (CPC) and one negative template control (NTC) were added. Libraries formed in batches of 96 samples per plate were pooled into one 1.7-mL tube. A magnetic bead-based clean-up process was utilized for library purification. Libraries were next quantified using a quantification kit (Kapa Bio systems, Roche Diagnostics Corporation, Indianapolis, IN, USA). In the 384 sample set, 25 μL of each normalized pool with index adapter set 1, 2, 3, 4 were combined in a new micro-centrifuge tube respectively. Libraries were denatured diluted and loaded to the machine at a concentration of 1.4 pM onto the NextSeq 500/550 system using NextSeq 500/550 High Output Kit v2.5 (75 Cycles) (Illumina Inc., San Diego, CA, USA). The reference mapping method was used to analyze the files as per CLC genomics workbench version 20.0 (CLC, QIAGEN, Aarhus, Denmark). Wuhan Hu-1 isolate (Accession Number: NC_045512.2) was used as the reference sequence.

Concomitant fungal infections in these patients were diagnosed by KOH wet mount and fungal culture as per standard guidelines.[10] Descriptive statistics in the form of percentages and median were used to report the findings.

RESULTS

Baseline patient-related information: 80% of the patients were males with median age (IQR) being 55 (40–61) years. The median cumulative duration of hospital stay (IQR) of the patients was 45 (8–64) days. Three patients were admitted more than once. While nine patients were normal at the time of discharge, six patients expired.

Clinical features and predisposing factors: Periorbital swelling and ocular pain (14/15; 93.33%), followed by facial swelling (11/15; 73.33%) and nasal obstruction (9/15; 60%), were the most common clinical features observed in these patients. All 15 patients had diabetes mellitus. None of the patients had diabetic ketoacidosis or any other underlying comorbidities.

Treatment and vaccination history of the patients: Steroids were administered to only six patients. None of them received immunomodulatory drugs such as tocilizumab or remdesivir. Ten patients were on high-flow oxygen due to severe hypoxia. Surgical intervention coupled with administration of antifungal drugs such as amphotericin B was resorted to for the treatment of patients. All patients were unvaccinated.

Laboratory profile of patients: Parameters such as HbA1c, blood urea, serum creatinine, serum ferritin, lymphopenia, and neutrophil to lymphocyte ratio were deranged in 15 (100%), 9 (60%), 5 (33.33%), 7 (46.7%), 10 (66.7%), and 5 (33.33%) patients, respectively. While Rhizopus arrhizus was found to be the causative fungal agent in 12 patients, two patients had a dual infection with Rhizopus arrhizus and Aspergillus flavus. Growth of Mucor/Rhizopus spp. could not be obtained in culture from one patient's sample, the microscopic findings of which revealed the presence of broad aseptate hyphae. Whole-genome sequencing could be performed on 13 out of 15 samples. The majority of the patients (9/13; 69.23%) were infected with the Delta variant of SARS-CoV-2 (B.1.617.2).

A summary of the clinical and laboratory profiles of all 15 patients has been summarized in Supplementary Table 1. Figures 1 and 2 depict direct microscopic findings in KOH wet mount and Gram stain, respectively, of debrided tissue samples obtained from patients, suggestive of Mucorales spp. (broad aseptate hyphae).

SUPPLEMENTARY TABLE 1

SUMMARY OF CLINICAL AND LABORATORY PROFILE OF PATIENTS

(a) CLINICAL PROFILE OF PATIENTS
Patient No.	Age (years); Gender	Clinical features								Underlying co-morbid conditions			Other relevant details			Treatment details			Cumulative duration of hospital stay; Patient outcome
		Fever	Headache	Malaise	Facial swelling	Periorbital swelling	Ocular pain	Nasal obstruction	Nasal discharge	Diabetes Mellitus	DKA	HTN/COPD/ CAD/CKD/CLD	Addiction (if any)	COVID-19 vaccination status	Travel history	Systemic steroids	Remdesivir &/or Toclizumab	High flow oxygen
		1.	62; Female	No	No	No	No	No	Yes	Yes	Yes	Yes	No	No	No	No	No	Yes		No	Yes	8 days; Death
2.	59; Male	No	No	Yes	Yes	Yes	Yes	Yes	No	Yes	No	No	No	No	No	No	No	Yes	8 days; Death
3.	28; Male	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	No	No	Smoking	No	No	Yes	No	Yes	59 days; Normal discharge
4.	62; Male	No	Yes	No	Yes	Yes	No	Yes	Yes	Yes	No	No	Smoking	No	No	Yes	No	Yes	55 days; Normal discharge
5.	43; Male	No	No	Yes	Yes	Yes	Yes	Yes	No	Yes	No	No	No	No	No	No	No	No	72 days; Normal discharge
6.	40; Male	No	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	No	No	Smoking	No	No	No	No	No	61 days; Normal discharge
7.	61; Male	No	No	No	Yes	Yes	Yes	No	No	Yes	No	No	No	No	No	No	No	No	64 days; Normal discharge
8.	50; Male	No	No	No	Yes	Yes	Yes	Yes	No	Yes	No	No	No	No	No	Yes	No	Yes	22 days; Normal discharge
9.	61; Male	No	No	No	Yes	Yes	Yes	No	No	Yes	No	No	No	No	No	No	No	Yes	64 days; Death
10.	50; Male	No	No	Yes	No	Yes	Yes	No	No	Yes	No	No	No	No	No	No	No	No	0 day; LAMA
11.	65; Male	No	No	No	No	Yes	Yes	No	No	Yes	No	No	No	No	No	No	No	Yes	3 days; Death
12.	58; Female	Yes	No	Yes	Yes	Yes	Yes	Yes	Yes	Yes	No	No	No	No	No	Yes	No	Yes	45 days; Death
13.	55; Male	No	No	No	Yes	Yes	Yes	No	No	Yes	No	No	No	No	No	No	No	Yes	45 days; Death
14.	29; Female	Yes	Yes	Yes	Yes	Yes	Yes	No	No	Yes	No	No	No	No	No	Yes	No	Yes	71 days; Normal discharge
15.	39; Male	No	Yes	No	No	Yes	Yes	Yes	Yes	Yes	No	No	Smoking	No	No	No	No	No	40 days; Normal discharge
(b) LABORATORY PROFILE OF PATIENTS
Patient No.	Biochemical parameters								Haematological parameters			Microbiological parameters
	RBS at admission (mg/dl)	HbA1c (%)	Blood urea (mg/dl)	Serum creatinine (mg/dl)	Total bilirubin (mg/dl)	SGOT (U/L)	SGPT (U/L)	Serum ferritin (ng/ml)	TLC (per mm3)	DLC N(%); L(%); NLR	Platelets (per mm3)	Microscopy (KOH wet mount)		Fungal culture		COVID-19 status (Real time RT PCR)		SARS-CoV-2 variants
																		PANGO lineage	WHO label
1.	170	10.2	131	1.2	0.81	19	23	46	22,300	95; 2; 48	2,60,000	Aseptate/pauciseptate & septate hyaline hyphae		Rhizopus arrhizus & Aspergillus flavus		Positive		Not done	Not applicable
2.	285	13.4	77	3.66	0.98	179	49.9	Not done	10,270	65; 21; 3	2,37,000	Aseptate/pauciseptate hyaline hyphae		Rhizopus arrhizus		Positive		B.1.617.2	Delta
3.	145	9.3	17.5	0.70	0.38	16	18	453	16,000	77; 13; 6	3,63,000	Aseptate/pauciseptate hyaline hyphae		Rhizopus arrhizus		Positive		B.1.617.2	Delta
4.	240	9.8	42	0.73	0.59	14	25	289	14,690	63; 26; 2	1,24,000	Aseptate/pauciseptate hyaline hyphae		Aspergillus niger		Positive		B.1.617.2	Delta
5.	210	11.6	63.2	1.28	0.26	36.6	44.4	776.89	23,890	80; 15; 5	4,84,000	Aseptate/pauciseptate hyaline hyphae		Rhizopus arrhizus		Positive		B.1.617.2	Delta
6.	420	8.6	31	1.60	0.60	28	34	32	8785	75; 14; 5	2,32,000	Aseptate/pauciseptate hyaline hyphae		Rhizopus arrhizus		Positive		B.1.617.2	Delta
7.	187	9.1	56	0.90	0.31	47	36	66	11,230	45; 20; 2	2,39,000	Aseptate/pauciseptate hyaline hyphae		Rhizopus arrhizus		Positive		B.1.617.2	Delta
8.	167	8.6	28.8	1.18	0.30	21	44	242	13,310	72; 17; 4	3,27,000	Aseptate/pauciseptate hyaline hyphae		Rhizopus arrhizus		Positive		B.1.617.2	Delta
9.	220	11.5	26	0.90	0.67	15	14	674	9277	55; 12; 5	2,42,000	Aseptate/pauciseptate hyaline hyphae		Rhizopus arrhizus		Positive		Not done	Not applicable
10.	351	14	146	4.99	0.32	36.3	24.7	4872	25,150	85; 7; 12	3,89,000	Aseptate/pauciseptate hyaline hyphae		Rhizopus arrhizus		Positive		B.1.617.2	Delta
11.	179	9.8	196	9.19	0.51	43.4	61.8	2507	28,810	92; 4; 23	3,99,000	Aseptate/pauciseptate hyaline hyphae		Rhizopus arrhizus		Positive		B.1.617.2	Delta
12.	322	15.6	125.6	2.54	0.80	26	28	522	9813	88; 5; 18	1,86,000	No fungal elements seen		Rhizopus arrhizus		Positive		B.1.575	Not assigned
13.	235	11.3	68.2	0.81	0.81	30.4	24.8	4729	11,190	56; 17; 3	3,17,000	Aseptate/pauciseptate & septate hyaline hyphae		Rhizopus arrhizus & Aspergillus flavus		Positive		B.1.1.7	Alpha
14.	134	7.3	9.8	0.46	0.62	29	28	342	6516	54; 33; 2	1,59,000	Aseptate/pauciseptate hyaline hyphae		Rhizomucor spp.		Positive		B.1.1.7	Alpha
15.	256	7.5	23	0.76	0.41	31	54	154	13,980	59; 24; 2	2,04,000	Aseptate/pauciseptate hyaline hyphae		Rhizomucor spp.		Positive		B.1.1.7	Alpha

Figure 1

Broad aseptate hyphae suggestive of Mucorales spp. in KOH wet mount

Figure 2

Gram-stained smear showing broad aseptate hyphae suggestive of Mucorales spp

DISCUSSION

A plethora of predisposing factors, particularly an unholy intersection of the trinity of diabetes mellitus, rampant use of corticosteroid, and COVID-19, may be responsible for the spike in mucormycosis cases in India. The presence of diabetes mellitus with or without diabetic ketoacidosis (DKA) increases the risk of contracting mucormycosis.[3] Diabetes mellitus is often associated with increased severity of COVID-19 infection due to the following reasons: (i) Autonomic dysfunction in DM leads to dysregulation of the inflammatory reflex that is responsible for mitigating macrophage activation; (ii) Structural and functional alterations in the lungs such as thickening of the pulmonary basal laminae and alveolar epithelium, restrictive spirometry pattern, decreased respiratory muscle endurance, and dysregulated control of ventilation and bronchomotor tone due to autonomic neuropathy are commonly observed in diabetic patients; (iii) Evidence suggests that DM is a pro-thrombotic state in which hyperglycemia coupled with inflammation directly contribute to abnormal platelet activation and increased concentration of fibrinogen and other coagulation factors.[11]

The use of systemic steroids is often associated with uncontrolled hyperglycemia and precipitation of DKA, thereby leading to low pH, which promotes germination of fungal spores. Steroid use causes immune suppression by causing reduced phagocytic activity of white blood cells and impairment of bronchoalveolar macrophages migration, ingestion, and phagolysosome fusion.[3]

COVID-19 per se is known to cause damage to pancreatic beta cells, thereby leading to reduced insulin levels and a hyperglycemic state. This infection also causes endothelialitis, endothelial damage, thrombosis, lymphopenia, and reduction in CD4+ and CD8+ T-cell levels. Increased free iron provides ideal conditions for Mucorales to thrive in the human body. This state can occur as a result of hyperglycemia, which causes glycosylation of transferrin and ferritin, leading to decreased iron-binding capacity. Also, cytokine storm (especially raised IL-6) observed in COVID-19 patients increases free iron by increasing ferritin levels due to increased synthesis and decreased iron transport. High glucose, low pH, free iron, and ketones can lead to enhanced expression of glucose-regulator protein 78 (GRP-78) of endothelium cells and fungal ligand spore coating homolog (CotH) protein, enabling angio-invasion, hematogenous dissemination, and tissue necrosis observed in mucormycosis.[412]

Although industrial oxygen may be regarded as a contributing factor for COVID-associated mucormycosis (CAM) during the second wave, a causal association has not been conclusively demonstrated. Researchers around the world have not been able to clearly explain the following findings to date: (a) Only some COVID-19 patients on oxygen therapy developed mucormycosis as a complication; (b) Cases were reported even after cessation of its use; (c) There were many patients with CAM for whom external oxygen was never used for the treatment of COVID-19.[13]

India witnessed a double blow in the form of oxygen crisis and mucormycosis epidemic during the second wave of COVID-19. Many patients, especially those with relatively minor symptoms, were treated at home without any oxygen because of a shortage of beds in the hospitals. A large majority of people suffered from protracted hypoxia throughout illness and recovery. A plausible explanation for the sudden spike in mucormycosis cases in India could be COVID-19-associated hypoxia, which stimulates the endocytosis mechanism in some Mucorales species. This in turn leads to a shift in the fungal metabolic activity from the utilization of carbohydrates to fatty acids such as extracellular lipids during infection. It is possible that the combination of severe hyperglycemia and prolonged hypoxia may have created the perfect conditions leading to a sudden rise in the incidence of mucormycosis.[14]

Recent evidence suggests the presence of high levels of autoantibodies against immunomodulatory proteins such as cytokines, chemokines, complement proteins, and cell-surface proteins in COVID-19 patients.[14] Wang et al.[15] in their study demonstrated that immune-targeting autoantibodies against type I IFNs, components of the IL-18 pathway, IL-1β, IL-21, and GM-CSF increased the disease severity in a mouse model of COVID-19. These findings suggest that SARS-CoV-2 can cause immune dysfunction by weakening the anti-viral effects exerted by cytokines that comprise the human exoproteome. Demonstration of such autoantibodies in patients infected with the Delta variant of SARS-CoV-2 (B.1.617.2) is the need of the hour. To establish any possible association of the Delta variant with Mucormycosis, genetic surveillance of SARS-CoV-2 along with anti-immunomodulatory protein antibody monitoring should be routinely carried out in countries with a high burden of CAM.

A noteworthy point regarding CAM is that most cases were reported from India. This could be due to a combination of environmental, geographic, and genetic factors (indicated by the high pre-pandemic frequency of mucormycosis in India) that may have predisposed immunologically dysregulated individuals to mucormycosis. However, CAM has also been reported from Brazil, Chile, Honduras, Mexico, Paraguay, the United States, Uruguay, Italy, and the United Kingdom.[14]

In a study conducted by Arora et al.,[16] CAM cases were diagnosed and reported during April–June 2021. During this time, the Delta variant of SARS-CoV-2 dominated the samples sequenced by INSACOG, constituting 58%, 88%, and 86% of isolates in April, May, and June, respectively. The authors of this study observed that most patients were male (73.8%), and had diabetes (80.0%). Only 3.1% had been taking long-term corticosteroids. None of the patients had any underlying comorbidities such as HIV, cancer, or a history of stem cell or solid organ transplant. The median age of patients was 56 years, and the median hemoglobin A1c level was 7.80%. COVID-19 was treated primarily with corticosteroids, remdesivir, or both. Favipiravir, doxycycline, azithromycin, ivermectin, and zinc were also commonly used. Tocilizumab was not administered to any patient. Most patients presented with fungal infections in the sinuses or eyes. Amphotericin B, posaconazole, and surgery were the most common antifungal treatments. Among 53 patients with available follow-up data at 42 days, 37 (69.8%) had recovered either partially or completely and six (11.3%) had died. The authors of this study also noted that diabetes, steroid use, and inadvertent administration of antibiotics were major risk factors for CAM. Most findings of the aforementioned study are in concordance with those of our study and point toward the fact that CAM is a multi-factorial disease with a possible role of the highly virulent Delta variant of SARS-CoV-2. In the present study, dual infection with Rhizopus and Aspergillus spp. was observed in two patients. Similar observations were made by Garg et al.[17] in their study.

Another intriguing observation of the present study was that all CAM patients were unvaccinated. Arakeri et al.[13] in their study also noted that a vast majority of mucormycosis cases were seen in unvaccinated people. At this juncture, one can only speculate that vaccination could have possibly prevented or decreased the severity of adverse effects causing immune dysregulation and mucormycosis. We will have to wait until more evidence emerges in this regard.

Several mutations have been observed in the different variants of concern (VOCs) that have led to increased transmissibility, high rates of disease severity and hospitalization, and possibly reduced neutralization by monoclonal antibody treatments and vaccine sera.[18] To the best of our knowledge, there is no data specifically pointing toward an association between these mutations and secondary infections such as mucormycosis in COVID-19-afflicted patients. Although a few reports of secondary infections in hospitalized COVID-19 patients were published during the pandemic,[192021] cases of mucormycosis were specifically observed during the second wave, when the Delta variant was the predominant strain across the globe. Therefore, it is imperative that the genetic basis of this apparent “epidemiological link” should be further explored.

The major lacunae of this study included small sample size, non-structured sampling, and inappropriate study design lacking a comparator group for establishing a causative association between the Delta variant of SARS-CoV-2 and mucormycosis.

CONCLUSION

CAM seems to be multifactorial in origin. Although there may be a possible association between mucormycosis and the Delta variant, more studies should be conducted to explore this seemingly reasonable proposition. At this juncture, we would like to surmise the findings of our study with the words of Claude Bernard, who said “Science admits no exceptions; otherwise there would be no determinism in science, or rather there would be no science.” We urge the scientific community to try and further explore this emerging public health threat. We believe that there are gaps in our current understanding of these diseases, and there are some missing links that need to be discovered.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.