Role of interferon gamma in SARS-CoV-2-positive patients with parasitic infections

Background By 27 June 2020, almost half a million people had died due to COVID-19 infections. The susceptibility and severity of infection vary significantly across nations. The contribution of chronic viral and parasitic infections to immune homeostasis remains a concern. By investigating the role of interferon (IFN)-γ, we conducted this study to understand the connection between the decrease in numbers and severity of COVID-19 cases within parasitic endemic regions. Our research included 375 patients referred to hospitals for diagnosis of COVID-19 infection. Patients were subjected to full investigations, in particular severe acute respiratory syndrome coronavirus-2 nucleic acid and Toxoplasma IgM and IgG antibody detection, stool examination, and quantitative IFN-γ measurement. Results The majority of the studied cases had chest manifestation either alone (54.7%) or in association with gastrointestinal (GIT) manifestations (19.7%), whereas 25.6% had GIT symptoms. We reported parasitic infections in 72.8% of mild COVID-19 cases and 20.7% of severe cases. Toxoplasma gondii, Cryptosporidium, Blastocyst, and Giardia were the most common parasitic infections among the COVID-19 cases studied. Conclusion The remarkable adaptation of human immune response to COVID-19 infection by parasitic infections with high levels of IFN-γ was observed in moderate cases compared with low levels in extreme cases. The potential therapeutic efforts aimed at the role of parasitic infection in immune system modulation are needed if this hypothesis is confirmed.


Introduction
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an intercontinental pandemic triggered by the universal human-to-human transmission of the virus [1]. Meaningful variations are observed in the way COVID-19 spreads across different regions on various continents [2].
Malaria-endemic countries have reported a low number of COVID-19 cases [3]. The populations in these areas have natural protection against malaria infection by producing a combination of Th1 and Th2 responses. In several coinfections, malaria-induced immunosuppression also severely inhibits immune responses to other infections, such as Salmonella spp. [4]. By inhibiting the activity of CD8+ T-cell migration, Plasmodium infection protects against the Chikungunya virus [5].
Toxoplasma gondii is an intracellular parasite that generates dense granuleprotein-7 (GRA-7) into the host cell, which inhibits viral replication [6]. Based on the antagonistic phenomenon in which certain pathogens can

Open Access
Gut Pathogens *Correspondence: enas_refae1983@yahoo.com 1 Medical Parasitology Department, Faculty of Medicine, Zagazig University, El Kawmia Square, Zagazig 44511, Sharkia Governorate, Egypt Full list of author information is available at the end of the article inhibit another infection by limiting its invasion, survival, and reproduction, various parasites have weakened the severity of viral infections [7]. In addition, the concurrent infection of Giardia and Rotavirus reduces the severity of the related viral infection [8].
The most frequent symptoms of patients infected with SARS-CoV-2 upon admission are history of fever, shortness of breath, cough, fatigue/malaise, and confusion, according to the Center for Disease Control and Prevention [9]. The pathogenesis of human infection with SARS-CoV-2 manifests as mild symptoms to extreme respiratory failure. The virus replicates and migrates across the airways upon binding to epithelial cells of the respiratory tract. The rapid replication of SARS-CoV-2 in the lungs elicits an effective immune response. After the relief of clinical symptoms and negative reversetranscriptase polymerase chain reaction (RT-PCR) assay findings, this viral infection displays an inflammatory response resulting in lung fibrosis [10].
Cytokine storm syndrome, which is considered the primary cause of death in patients, induces acute respiratory distress syndrome and respiratory failure. The serum of hospitalized patients with severe COVID-19 contain elevated levels of tumor necrosis factor, interleukin (IL)-2, IL-10, IL-7, granulocyte colony-stimulating factor, C-X-C motif chemokine ligand 10, and macrophage inflammatory protein 1α [11]. Interferons (IFNs) are well-known cytokines for their antiviral effects; they play a critical role in viral multiplication drop off [12]. The expression of IFN-γ receptors is limited to epithelial cell-enriched tissues, such as the lungs, skin, gastrointestinal tract (GIT), and respiratory tract. This receptor allocation pattern renders IFN-γ with the capability to target specific tissues [13]. The IFN-γ levels are negatively related to the increase in the amount of COVID-19 fibrosis at discharge. These results indicate the early interference of IFN-γ antiviral infection and inhibition of fibrosis for improved recovery [14].
We conducted this study to realize the relationship between the decrease in numbers and severity of COVID-19 cases within parasitic endemic areas by investigating the role of IFN-γ.

Results
This research included 375 patients diagnosed with COVID-19 positive (+ ve) via PCR tests. The patients were aged18-69 years old, with a mean age of 44.72 years, and of which 65.6% were male, and 34.4% were female. Employers (24.8%) and specialists (18.4%) were the most frequent occupations among the studied population, as shown in Table 1. In terms of comorbidity, 40% of patients had other systemic disorders, with chronic lung disease (19.7%) being the most common.
The majority of cases examined had chest manifestations either alone (54.7%) or in association with GIT manifestations (19.7%), whereas only GIT symptoms were present in 25.6%. In 92.3% of the studied cases, the severity of COVID-19 was mild, and a severe condition was noted in 7.7% (required intensive care unit (ICU) admission) ( Table 1).
The prevalence of parasitic infections among the studied COVID-19 cases was 68.8%. The most commonly found parasites were Toxoplasma gondii (22.4%), Cryptosporidium (19.7%), Blastocyst (17.6%), Giardia (9.1%), and Cryptosporidium + Blastocyst + Entamoeba histolytica combined infection (6.1%) (Fig. 1). Table 2 reveals the relationship between the severity of COVID-19 infection and symptoms, where all confirmed severe cases showed chest symptoms only with a highly statistically significant difference (P < 0.001) compared with the mild cases. The mild cases showed chest symptoms (50.9%), GIT symptoms (27.7%), and mixed chest and GIT symptoms (21.4%) with a highly statistically significant difference (P < 0.001). Reviewing the relation between parasitic infected cases and the severity of this viral infection, 72.8% and 20.7% of mild and severe cases had parasitic infection, respectively (Table 3). Additionally, 91.7% of cases with diabetes mellitus had parasitic infections, and 75% of hypertensive cases and 100% of immune-compromised patients had parasitic infections with a statistically significant difference (P < 0.05). A highly statistically significant increase was observed in the frequency of infections among cases with GIT symptoms (78.1%) and combined GIT and chest symptoms (83.8%) compared with cases with chest symptoms only (59%), indicating a high statistically significant difference (P < 0.001).
The parasitic infection modulated the human immune response to COVID-19 infection in mild cases with high levels of IFN (50.17 ± 8.24) compared with those with low levels of IFN (9.17 ± 1.60) in severe cases (Table 4).
In our research, the path of the disease was mild given that spontaneous resolution occurred in Toxoplasma gondii-infected patients. These results can be explained  by the production of GRA-7, which displays immune stimulation and a broad spectrum of antiviral activities via type I IFNs signaling [6]. Another clarification was obtained by Degrandi et al. [15], who announced that Toxoplasma-infected mice exhibited up-regulated immune responsive gene 1 in their lungs. This gene is Table 3 Comparison between parasitic infected cases and free cases in terms of demographic data, comorbidity, SARS-CoV-2 manifestation, and severity SD standard deviation, t independent t test, χ2 chi-square test, NS non-significant, NS non-significant (P > 0.05) * significant (P < 0.05) ** highly significant (P < 0.01)  an IFN-stimulated gene that mediates antiviral effects against RNA viruses [16]. Moreover, the apicoplast proteins of protozoa have immunogenic potential [17]. Priming with viable C. parvum oocysts provides a protective immunity at an extremely low dose. This condition restores the Th1-partner mucosal (CD8+ T-cells) and their cytokine IFN-γ effectors that otherwise decrease with ongoing protein malnutrition [18]. Nevertheless, after respiratory syncytial virus, Heligmosomoide spolygyrus considerably reduces pulmonary complications [19]. In our study, we obtained high levels of IFN-γ (50.13 pg/ml) in mild cases compared with the low levels (0.7385 pg/ml) in severe cases of COVID-19 with parasitic infections. Chen et al. [10], who reported that IFN-γ was lower in severe COVID-19 patients compared with those with a moderate form of the disease, approved our findings. Bajwa et al. [20] also noted the antiviral activity of IFN-γ by exerting cellular effects at various levels. IFN-γ interacts with the viral receptor, resulting in the consequent reduction of several virus replication downregulating genes and gene products [21]. Li and De Clercq [22] showed that IFNs are potential drug choices for SARS-CoV-2 infection.

Characteristics
Our main finding supports the protective role of parasitic infections against infection with COVID-19. Compared with parasite-free patients, the general improvement in the overall health status of COVID-19 +ve patients was demonstrated by a rapid exit without any respiratory complications. Our findings are endorsed by Cohen et al. [23], who stated that CD4+ and CD8+ T-cells play imperative synergistic roles given that both secrete IFN-γ during T. gondii infection. The IFN-γ produced by CD4+ cells is required to prime macrophages, inducing powerful antimicrobial responses. IFN-γ regulates the expression of inducible nitric oxide synthase, which is eventually required for parasite control [24]. Additionally, treatment with soluble T. gondii antigen 24 h post-P.berghei infection resulted in a rapid increase in serum IL-12 and IFN-γ levels [25]. The development of immunity in Cryptosporidium-infected humans and murines is associated with the elevated expression of IFN-γ Th1 cytokines [26]. In addition, the immunomodulation caused by malaria is effective against extreme manifestations of certain respiratory viruses. Hospitalized children diagnosed with influenza and malaria in Kenya are less likely to suffer respiratory distress than those with influenza alone [27].

Conclusion
Among the parasite-infected cases, the COVID-19 disease sequence was moderate. Cryptosporidium, T. gondii, Blastocyst, and Giardia were parasitic infections among the studied COVID-19 infected cases. A high percentage of moderate COVID cases accompanied with parasitic infections was observed in our results, and an increased number of serious cases were parasite free. The +ve role of parasitic infections in modulating the human immune response to COVID-19 infection was evident in the high level of IFN γ in mild cases. A new prospect for the development of novel successful protection against COVID-19 infection has been provided by the unclear link between parasitic and viral infections. Testing the safety and efficacy of parasite-derived antigens in more patients with SARS-CoV-2 infection may be worthwhile. Our future research will focus on the cytokine storm concerning parasitic infections triggering the immunocompromised population.

Population study
This cross-sectional analysis was conducted on 375 patients who were referred to Zagazig University Hospital and Al-Ahrar Hospital in Sharkia Province, Egypt for diagnosis of COVID-19 infection between 15 July and 10 October 2020. The requirements for diagnosis of the suspected cases of infection were sent to the Egyptian Ministry of Health Protocol for Diagnosing and Treating Cases of COVID-19. The reported patient had a strong history of epidemic exposure and had at least two of the following clinical manifestations: fever, respiratory symptoms, viral pneumonia imaging, and normal or decreased number of white blood cells and lymphocytes at the early stage of the disease. Complete blood count, SARS-CoV-2 nucleic acid detection, lung computed tomography analysis, specific Toxoplasma IgM and IgG antibody detection, and stool examination were conducted onall patients involved. No exclusion criteria were applied for the patients. The cases examined were subjected to medical history and clinical examination and then divided into those with mild disease with fever, fatigue, dry cough, and ground-glass opacities of pneumonia. The total number of white blood cells in the early stage of disease was normal or decreased, the lymphocyte count decreased, and severe disease occurred with respiratory distress (respiration rate ≥ 30 times/min), oxygen saturation (≤ 93% in the resting state), PaO 2 /FiO 2 ≤ 300 mm Hg. Upon admission, blood samples were obtained from the patients. Nine days after symptoms appeared, the medication regimen was started. Mild cases appeared after a week. On the other hand, in severe cases, oral intubation was conducted in ICU during treatment.

Diagnosis of toxoplasmosis
Venous blood (approximately 5 ml) was collected from each patient. The blood samples were labeled and refrigerated by shipment to the laboratory at 4 °C. The blood samples were then centrifuged in 1.5 ml centrifuge tubes to obtain the serum. The samples were held at − 80 °C before use. The electrochemiluminescence immunoassay (Cobas E411 immunoassay analyzers, Mannheim, Germany) was used to detect anti-Toxoplasma IgG and IgM antibodies. Master curve minimum and maximum limits specified by 0.13-650 IU/ml were detected for reference measurement.

Stool examination
Fresh stool samples from patients with manifestations of COVID-19 were collected. Direct smear, formol-ether concentration technique [28], and modified Ziehl-Neelsen stain [29] were performed. On the basis of GIT manifestation of most patients, parasitological evaluation was requested.

IFN-γ measurement
The human IFN γ enzyme-linked immunosorbent assay kit (NOVA, No. 18, Keyuan Road, DaXing Industry Region, Beijing, China) was used to quantitatively calculate IFN-γ in the serum of the studied population, following the manufacturer's instructions. Optical density was estimated at 450 nm, the standard curve was built, and the mean absorbance concentration of the samples from the standard curve was measured.

Statistical analysis
The data were computerized and statistically analyzed using Statistical Package for the Social Sciences, version 25.0. Qualitative data were expressed as the number and percentage, and quantitative data were expressed as the mean, standard deviation, and range. Chi-square (χ 2 ) and independent t-tests were used in comparing data. The p-value was considered significant if < 0.05 and highly significant if < 0.01.