Heparan Sulphate Binding, Disease Development and Treatment Options

Nephronin Specifically Binds to Heparin / Heparan Sulphate

Nephronin was found to be highly negatively charged based on its tight binding to anion exchange columns (Q-column) at neutral pH. Cellulose acetate electrophoresis was used to demonstrate specific binding of Nephronin to Heparin and Heparan sulphate (Figure 1).

Figure 1: A one dimensional cellulose acetate electrophoresis, that is often used in the characterisation of glycosaminoglycans (GAGs) was used for the detection of Nephronin binding and inhibition of migration of each specific product [12].

1. Heparin.

2. Heparin + Nephronin.

3. Heparan sulphate.

4. Heparan sulphate + Nephronin.

5. Chondroitin sulphate.

6. Chondroitin Sulphate + Nephronin.

7. Dermatan Sulphate.

8. Dermatan Sulphate + Nephronin.

The Major Biological Activities Associated with this Compound

a) Nephronin is a potent inhibitor of endotoxin and endotoxin induced cytokine production. Endotoxin is the major factor in the onset of sepsis, septic shock and related diseases such as Meningitis. Currently there is no effective treatment for inhibition of stimulation and production of these cytokines. The anti-inflammatory activity of Nephronin is of paramount importance in inflammatory disease conditions.

b) They bind to receptors of heparan sulphate and in turn impede development of diseases, where heparan sulphate plays a major role in the pathogenesis of disease development. Many viruses and bacteria use heparan sulphate as anchorage in their mechanism of infection, as well as metastasising tumourogenic cells utilising heparanase. I have been able to demonstrate inhibition of Herpes virus infection of human keratinocytes by separate preparation of Nephronin.

Anti-Inflammatory Activity of Nephronin

To date, huge amounts of money have been spent in developing new therapeutic agents, including antibodies against endotoxin and some immune mediators such as tumour necrosis factor (TNF) and various interleukins, their receptors and synthetically produced antagonists of endotoxin for the treatment of various diseases, in particular autoimmune conditions such as Psoriasis, Eczema, Rheumatoid Arthritis etc. as well as infectious diseases leading to sepsis and septic shock. Unfortunately each of these agents has their own particular side-effects and they become less effective with prolonged use. In the case of septic shock, which is caused by the cytokine cascade (cytokine storm) that can often be fatal, demonstrates the pressing need to develop effective, bioavailable therapeutics. A review article entitled “Treatment of Sepsis” by Baumgartner & Calandra, [13] confirms this conclusion. Endotoxin is a structural component of Gram-negative bacterial cell wall and it is also the major contributing factor in sepsis. We have demonstrated in laboratory tests, that Nephronin inhibits endotoxin induced cytokine production. We are currently exploring options in developing suitable products as anti-inflammatory for various conditions.

Treatment of Autoimmune Conditions, Psoriasis and Eczema

Psoriasis

Psoriasis is a chronic inflammatory skin disease characterised by thick, raised lesions. Psoriasis presents in a number of ways, each recognised as a specific form of the condition, regardless psoriasis tends to be unsightly and almost invariably is uncomfortable for the sufferer. There is no cure for this condition and patients need to be treated on long-term therapies. There are a number of therapeutic products for the treatment and maintenance of Psoriasis, including Steroidal products for the milder types, Biologics and other nonsteroidal anti-inflammatory drugs for the more severe and systemic cases. However, all these treatments have side effects and also seem to lose efficacy with prolonged use (Figure 2).

Figure 2: The data shown above represents a Dose-dependent inhibition of LPS-induced TNF- and IL-1, two major mediators in sepsis and septic shock by four different preparations of Nephronin. It is noteworthy that when these tests were carried out, it was difficult to accurately measure the concentration of each preparation, due to contaminating salts, however the concentration of each preparation is ng/ml. Therefore when 20ul was used, the amount of active constituent (Nephronin) was estimated to be approximately 20ng. The indicated amount of each preparation was added to chambers of 24 well culture plates. 1ml of PBMC at 1.5x106 cells /ml containing 1ng/ml LPS was then added to each well. LPS was added to the cell suspension a few minutes prior to dispensing the cells into the wells of the tissue culture plates. The cells were harvested at 24 and 48 post-stimulation and the concentration of the cytokines measured by sandwich ELISA. Similar results were also obtained at 24h and 72h post-stimulation

a) Dose-dependent inhibition of LPS induced TNF-, 24h post-stimulation.

b) Dose-dependent inhibition of LPS-induced IL-1, 24h post-stimulation.

Eczema

Atopic Eczema (Atopic Dermatitis AD) is one of the most widespread autoimmune/inflammatory skin disorders with no known cure. Global rates in children have been estimated to be 10-15%, dropping to 1-3% in the adult population who suffer from persistent AD symptoms. The disease is characterized by the presence of pruritic, erythemic commonly oedemic and vesiculated patches known as AD lesions, which are inflamed and are areas of excessive skin production. There is also a significant physiological aspect to AD and many patients suffer from severe itch, or pruritus, which may cause significant distress and disturb sleep. Patients may also experience embarrassment from the presence of the skin lesions, which can affect their sociability and personal relationships. There may be a significant effect on patient’s quality of life and incidence of depression. AD is characterized by marked intercellular oedema within the epidermis (spongiosis), which leads to formation of intra epidermal vesicles. Lymphocytic infiltration into the epidermis is common, as well as the presence of histiocytes and occasionally neutrophils and eosinophils. AD is considered a chronic and pruritic inflammatory skin disorder and is commonly associated with other allergic conditions. Genetic factors have been linked to AD pathogenesis with various genes identified, that may have roles in disease onset and progression. The immune system is also largely involved; the recent understanding is that there exists an imbalance in the Th1 and Th2 immune pathways, with a shift to the Th2 arm largely driving the increased allergic sensitisation in this disease. Diet and environmental triggers are also an important factor in the development and flaring of AD and if identified and controlled, can significantly contribute to symptomatic relief. Widely used therapies for AD include moisturisers and irritant avoidance. Other topical treatments include topical steroids and pimecrolimun as they interfere with the inflammatory pathways. Systemic treatments are also used in the case that topical therapies do not sufficiently control AD symptoms, such as biologics, cyclosporine, methotrexate and azathioprine. Many new areas of AD research are now concentrated on targeting the skin barrier, to address the disruption and breakdown of the barrier, which is an important component of the pathogenesis of AD. Immune mediators are also a large target in the development of new treatments of AD, with many anti-inflammatory compounds being developed and trialled.

There is a strong preference to reduce systemic exposure to drugs, particularly those that have side effects and a strong preference and rationale for using naturally sourced products. Despite the availability of numerous topical and systemic treatment options for atopic dermatitis and psoriasis, all are lacking with respect to participant satisfaction and durability of treatment. Many of the systemic therapies for atopic dermatitis and psoriasis have significant side effects. Most current topical therapies and many treatments under development are based on modifications of a steroid structure, on inhibiting inflammatory pathways by inhibition of calcineurin, phosphodiesterase-4 (PDE4) and other pro-inflammatory molecules or removing certain immune modulators - as in the case of biologics. This supports the development of novel immunomodulatory treatments, which are safe to use and preferably naturally sourced, for the long-term management of atopic dermatitis and psoriasis.

Characterisation of Nephronin

In an effort to elucidate the structure and nature of Nephronin molecule, I became aware that ingredients pertaining to the composition of Nephronin, when added to a biological system show the same biological activity as the isolated Nephronin from the urine of children with SRNS. Based on this finding, I have developed a number of topical and one oral product, that contain naturally sourced ingredients. These ingredients are well tolerated and there has been no report of any side effects associated with them. These products are under the brand name of ENDOR and are with the Australian Therapeutic Goods Administration (TGA) as over the counter products and are commercially available for the treatment of mild – moderate psoriasis and Eczema. ENDORTM Dermatitis Care Capsules contain multivitamins and other natural antiinflammatory ingredients, such as curcumin. There are also two topical products for use on Psoriasis and Eczema: ENDOR cream for mild-moderate conditions and ENDOR 3.5 cream for the more severe conditions. We are currently conducting a clinical trial using ENDORTM Dermatitis Care capsules and ENDORTM 3.5 cream for the treatment of moderate – severe Psoriasis and Eczema, at the St George Dermatology and Skin Cancer Centre, Sydney Australia.

Heparan Sulphate Binding and Potential Inhibition of Viral Infection

Many pathogens utilise heparan sulphate as their binding to the cell surface for entry into the cell. Herpes virus has a prerequisite first step binding to the cell surface heparan sulphate prior to entry into the cell (Table 1).

Table 1: Column 2 shows the effect of different preparations of Nephronin for the inhibition of angiogenesis. Column 3 and 4, headed as APTT activity and PT-Fibrinogen activity show the data for coagulation studies as compared with anti-coagulation of heparin. Column 5, title Mouse Toxicity, shows the data for toxicity studies when preparations of Nephronin dissolved in sterile water were injected IP into mouse.

Successful Inhibition of Hsv-1 Caused Destruction of Epidermal Cells After Axonal Transmission in Human In Vitro Model

Following Experimental Study Was Conducted by Dr Zorka Mikloska, Phd

Background: Herpes Simplex Virus (HSV) infection is one of the commonest infections of mankind and results in asymptomatic shedding of virus in urogenital skin or mucosa, mild oral recurrences (cold sores) or painful episodes of genital herpes. Genital herpes is the most important sexually transmitted disease of the Western world (after AIDS) and can lead to neonatal death and other complications. HSV establishes latency (stays inactive) after infection of the spinal cord and can be transmitted to the skin (or mucosa) via axons. The transmission of virus from axons to epidermal cells is a potential site for immune control, possibly via cytokines, chemokines and/or neutralizing antibodies [14].

Figure 3: The data above shows the effectiveness of Nephronin in the inhibition of herpes virus in the infection of human skin cells. The data demonstrating the axonal inhibition of herpes virus by Nephronin is for the first time pointing to a possible cure for this disease.

Technique: In order to study the events after reactivation of HSV-1 in the neurons of Dorsal Root Ganglia (DRG), a neuronepidermal cell in vitro model consisting of autologous human foetal DRG and skin in 2 separate chambers was developed in our laboratory [15]. This allowed us to examine the events involving HSV infection of skin cells after axonal transmission, which highly resembles what actually happens in recurrent HSV infection. We previously have shown that neutralising antibodies applied to the epidermal cells of the model (or directly) at the different times after HSV-1 infection of DRG could inhibit viral destruction of these cells. In similar fashion Nephronin (in various concentrations of 5-40ml/ ml) was applied to epidermal cells in outer chamber of the model at times 24 and 12 hours before HSV-1 infection of DRG neurons in inner chamber, at the time of infection and 12, 18, 26 and 32 hours after HSV-1 infection. Epidermal cells were stained for HSV- 1 antigen 48 hours after infection and HSV-1- cytolytic plaques counted in each culture. The difference in the number of plaques between viral control (no Nephronin) and Nephronin treated cultures was calculated and expressed as %Reduction in the number of HSV-1 plaques (for detailed explanation of methods and techniques see J Virol 73:5934-44,1999) [16]. This was compared with direct HSV-1 infection of epidermal cells grown from the same tissue (Figure 3).

Results: The concentration of Nephronin that provides optimal inhibiting effect on HSV plaques and is not toxic in cell culture was tested in 2 cell lines and human foetal epidermal cell cultures and found to be 5ml/ml.Nephronin was added before, at the time of infection and after infection of epidermal cells grown in tissue culture dish. The best result was when the compound was added together with virus. In 3 experiments (with 6 replicates in each experimental group) involving our human foetal model maximal reduction in the number (and size) of HSV-1 plaques in epidermal cells was found at times 0 and 12 hours after infection of DRG and was in range of 55-67%. In directly infected epidermal cells, the reduction in number of plaques in epidermal cells was almost 100% up to 72 hours after infection.

Conclusion:

Simultaneous addition of Nephronin and HSV-1 to human foetal epidermal cells reduced the number of specific cytolytic plaques in these cells by almost 100% (as compared with viral control). Nephronin was also very efficient in our neuron-epidermal cell model. The ability of this compound to greatly reduce infection of epidermal cells after axonal transmission indicates its potential use as a topical ointment, e.g. in cream, lotion etc.

The Data Presented Below are the Results from the Above Studies: Nephronin totally inhibits infection of keratinocytes and fibroblasts by herpes virus. Our data suggests that Nephronin binds to HSV and prevents it from binding to heparan sulphate on the cells, thereby inhibiting infection by preventing the prerequisite step in HSV infection. The other aspect of HSV infection is that it can remain dormant in the nervous system with frequent episodes of infection, depending on the general health of the patient. Our studies have demonstrated that Nephronin potently inhibits axonal transmission of herpes virus, preventing the virus’s ability to reinfect the genitalia. We believe that for the first time, Nephronin offers the opportunity to develop a therapeutic drug to cure this debilitating disease. It is anticipated that a vaginal cream for the treatment and the prevention of the spread of HSV will be developed in the first instance. This will be followed by an oral medication for the inhibition of the axonal transmission of the virus, leading to a cure. Anecdotal data from people using the ENDOR Dermatitis Care capsules and the topical creams has shown effective treatment of herpes virus.

COVID19: The recent pandemic caused by Coronavirus Disease 2019 (COVID-19) poses a serious threat to global public health and the world economy. Although COVID19 infection and threat has been reduced considerably in many developed countries, it remains a threat, particularly if new mutations lead to new, more pathogenic strain(s) that develop resistance to the current vaccines and other therapies.Similar to herpes virus, the Receptor-Binding Domain (RBD) on epithelial cells, particularly of the respiratory tract, plays an important role in the adhesion of the coronavirus to the host cell surface. The RBD on the epithelial cell surface for COVID-19 has been identified as Heparan Sulphate (HS) [17]. The coronavirus spike protein, which plays the most important role in viral attachment, fusion and entry to the host cell has a pre-requisite heparan sulphate binding step, prior to binding to Angiotensin- Converting Enzyme 2 (ACE2) receptor and entry into the cell [18].

It has been hypothesized that compounds with HS binding ability may be potential treatment agents for patients with COVID19. There is some evidence that use of anticoagulant heparin treatment is associated with decreased mortality in severe coronavirus disease [19]. Development of the ENDOR dermatitis care capsules for the treatment of Psoriasis and eczema and anecdotal observation for the treatment of herpes infection, has provided sufficient anecdotal evidence in patients infected with COVID19, for using these capsules alongside recommended treatment in patients hospitalised with COVID19 infection. Clinical trial in collaboration with Tehran University and the Department of Virology at Imam Khomeini Hospital in Iran is currently underway.

Oncology Indication: Immune surveillance results in the destruction of cells, that grow outside their normal physiological geography. Tumour cells can evade immune recognition by being able to mimic surrounding cells, including the cell surface carbohydrates and receptors. Some carbohydrates, including heparan sulphate are required for anchorage and development to tumour mass. Furthermore, cancer cells have been shown to have considerably higher levels of heparanase, an enzyme that enables cancer cells to separate from their point of origin and spread. This process is called metastasis. Tests have shown that Nephronin very strongly inhibits angiogenesis in a rate Aorta model and heparanase in an enzymatic model. The therapeutical potential for Nephronin is huge, particularly in oncology and metastasis. Earlier studies using BAF-32 cells, that are deficient in cell surface Heparan Sulphate, were used to demonstrate the interaction of Nephronin with receptors of heparan sulphate. Other studies including inhibition of Herpes Simplex virus (HSV) infection of human fibroblasts and keratinocytes, were used to indicate that Nephronin is much more potent against different / abnormal or foreign receptors of heparan sulphate.

Normal and Tumourogenic Cell Lines: The importance of heparan sulphate in cell-cell interaction is becoming clear [20]. Furthermore, recent evidence suggests that tumourogenic cell surfaces might be covered by heparan sulphate, that is different from that found on the surface of normal cells. To test the hypothesis that Nephronin might distinguish between the heparan sulphate covering the surface of normal and cancer cells, a normal fibroblast cell line (BFT-3B) and a cancer cell line (HT1080) were used. The cells were grown to single layer confluence and up to 40ng/ ml of Nephronin was added to tissue culture media. In the case of carcinogenic (HT1080) cell line, the cells contracted soon after the addition of Nephronin and at higher concentrations of Nephronin within 24 hours after the addition of test samples large vacuoles within the cells could be seen. After 48h over 90% of the cells were found to be dead. In the case of normal (BFT-3B) cell line, addition of Nephronin did not seem to have any deleterious effect on the cells. To study further the effect of Nephronin on other cell types, normal mouse muscle cell line (C2C12) and mouse neuron tumourogenic cell (B35) lines were used. Increasing amounts of Nephronin were added to the cells (up to 4ng/ml) and similar results to those above were found. The fact that preparations of Nephronin were toxic to B35 and HT1080 but not C2C12 and BFT-3B suggests the interaction of Nephronin with cell surface molecules is specific to cancer cell lines.

Inhibition of Heparanase: The data shows the dose dependent inhibition of heparanase activity. Given the fact that heparanase activity is shown to be of particular importance in tumour development and metastasis, the ability of Nephronin to inhibit heparanase activity might suggest that Nephronin could play an important role in the inhibition of metastasis and inhibition of tumourogenic cells to solid tumours. Dose dependent inhibition of human platelet heparanase activity by four preparations of Nephronin. Samples 1-5 refers to separate preparations of Nephronin. Preparation 6 is a control preparation. Blank (Blk) refers to a control for the enzyme reaction. At the time of study it was difficult to quantitate the active component of the preparations accurately. However it was estimated to be at 1 ng of Nephronin per 1ml of sample (Figure 4).

Figure 4.

Angiogenesis: The anti-angiogenesis activities of preparations of Nephronin were assessed in an assay based on quantitative measurement of new vessel growth in rat aortic rings cultured in a minimally enriched medium [21-23]; the table below demonstrates the percent inhibition achieved by the chosen preparations of Nephronin. Because of the functional nature of Nephronin, their ability to inhibit coagulation was studied using Anti Partial Thromboplastin Time (APTT) and (Prothrombin Time) PT-fibrinogen assay systems were used. In each case a Coulter ACL3000 Blood Analyser was used and the data presented as described below:

For APTT: In this test the expected values for human plasma are quoted as being in the following range: - Coagulation Time 24.3 - 35.0 seconds with Coagulation Ratio 0.82 – 1.18 – these are for calibration plasma (IL 8469210).

PT-Fib: In this test the expected values for human plasma are quoted as being in the following range: - PT Coagulation Time 9.0 – 12.6 seconds with Coagulation Ratio 0.81 – 1.13, Fibrinogen 168 –392 mg/L and R= 0.92 – these are for calibration plasma (IL 8469210) [Fibrinogen values were not tabled because they relate to the constant use of calibrated plasma rather than changing samples]

The assays were performed in triplicates and the data presented is averaging of the results.

Protocol for Mouse Toxicity Tests

All tests were carried out on 6-8week old male white mice, strain BALB/c, weighing 20 to 25g, supplied by the Animal Breeding Unit of the University of N.S.W. or CSIRO Molecular Science Animal House, North Ryde, Sydney Australia. The mice were obtained at least one day prior to the test day and acclimatised in the test room (Temp 25oC), where they were held, five to a box and given commercial rat pellets and water ad lib. On the day of the test, the compound to be tested was weighed out and dissolved in sterile water to give a stock solution which when injected at the rate of 100 μl per mouse gave a dose of 50mg/Kg (i e 1mg/mouse). 100 μl of the test solution was given by intraperitoneal injection to each test mouse. Five mice were dosed at 50mg/Kg along with five control mice for each series of tests. On the day of testing, mice were initially observed at half-hourly intervals for the first three hours and symptoms recorded. Further readings were taken for the next seven days after which the surviving mice were disposed of by overanaesthetisation with carbon dioxide.

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Soil Weed Seed Bank Dynamics After Two-Year of On-Farm Trials Under Conservation Agriculture in Bangladesh

Introduction

While Conservation Agriculture (CA) has been recognized as an excellent method for boosting crop yields sustainably, weed management is often regarded as one of the most difficult aspects [1]. Due to the reduced tillage operation, the composition and dynamics of the weed seed bank in CA will vary as compared to conventional tillage. The soil weed seed bank is a repository for weed seeds that influences the species composition. The seed bank is the primary source of weeds, represents a critical stage in the weed life cycle, and the weed population is inextricably linked to its seed bank. Knowledge of the size and composition of the soil weed seed bank is critical for forecasting future weed infestations and management techniques, weed seed production after the cropping season, calculation of crop-weed competition and crop yield loss, as well as agricultural economics [2]. There are relatively few research that investigate the influence of CA principles on the dynamics of weed seed banks. These sorts of research are necessary to include weed management into cost-benefit assessments of CA adoption. In light of this, long-term CA studies were done to determine the influence of CA principles on the dynamics of weed seed banks.

Materials and Methods

We conducted green-house experiments at the Department of Agronomy, Bangladesh Agricultural University in Mymensingh, Bangladesh. Soil was taken from the site of long-term CA experiments in the Durbacahra village of Gouripur upazila in Bangladesh’s Mymensingh district. Under the Summer Rice– Mustard–Winter Rice cropping system, crops were cultivated using conventional tillage (CT) and strip tillage (ST), with 50% standing residues from previous harvests retained compared to no-residue. CT was performed using a two-wheeler tractor, whereas ST was performed using a Versatile Multi-Crop Planter machine [3]. Prior to the trials, five soil samples from each plot were obtained at depths of 0–5 cm, 5–10 cm, and 10–15 cm in a “W” pattern [4]. One kilogram of dirt from each plot was deposited in a 32 cm diameter plastic dish. To ensure adequate weed germination, the samples were maintained wet. During a one-year period, emerging seedlings were recognized, numbered, and destroyed at 30-day intervals. To help identification, seedlings of dubious identity were transplanted to another plate and nurtured to maturity. Following the removal of each batch of seedlings, the soils were carefully mixed and rewetted to allow for further emergence. This procedure was done a total of 12 times. The seedlings that were counted were translated to numbers per m2. Following the conclusion of the two-year field experiments in Gouripur, soil samples were gathered again using a similar approach, and the same experimental procedures were used in the green house.

Results and Discussion

Effect of CT, ST, No-Residue and 50% Residue Mulching on the Weed Abundance (Number Per M2): Effect of ST and retention of 50% crop residues was significant on the number of weed species. Before setting the long-term CA trials in 2013, there was no significant difference in the weed species for CT and ST. During this time, there was 59 species in CT and 62 species in ST indicating the homogeneity of weed seed bank in the field. After 6 field trials of two-year study, there was 20% higher weed species in CT (71 species) but 7% less species in ST (58 species) (Figure 1). Retention of 50% crop residue in the field caused to decrease the weed species by 9% after 2-year study compared to the before study (Figure 2).

Figure 1: Effect of CT and ST on the weed abundance (number per m2) after 2-year field trials of Conservation Agriculture (CT: Conventional Tillage, ST: Strip Tillage).

Figure 2: Effect of no-residue and 50% residue mulching on the weed abundance (number per m2) after 2-year filed trials of Conservation Agriculture.

Table 1: Effect of tillages and residues on weed density (number per m2) at different soil depths.

Note: CT: Conventional Tillage, ST: Strip Tillage, (+): increase (-): decrease.

Effect of CT, ST, No-Residue and 50% Residue Mulching on the Weed Abundance (Number Per M2) At Different Soil Depths: The highest weed density was recorded from 0–5 cm depth followed by 5–10 and 10–15 cm depth both in CT and ST during both of before and after field trials (Table 1). After 2-year crop cultivation CT increased the weed density by 13%. Data recorded from after 2-year study also reveals that, heavy pulverization in CT caused to increase density by 11% at 0–5 cm depth and 25% at 5–10 cm depth but decreases by 53% at 10–15 cm depth which might be attributed from continuous upward movement of weed seeds to the upper layer of soil. After 2-year cropping, ST reduced the seed bank size by 22%. It was also found that, ST reduced the weed seeds by 24% at 0–5 cm and 57% at 10–15 cm depth but increased by 25% at 5–10 cm depth. Minimal soil disturbance may cause to emerge the weeds from upper most layer leading to reduce seed bank size and deposition of seed to the middle layer leading to enrich seed bank. Soil compactness at the lowest layer may cause to increase seed dormancy and mortality and reduce seed bank size [5]. Two-year cropping with the retention of 50% residue reduced the seed bank by 11% and there was a decreasing trend in seed composition from upper to lower soil layer. Residue may cause to hinders the optimal atmosphere for weeds and favors the weeds seed predation by soil fauna and reduce seed bank status [6].

Conclusion

Results of the present on-farms study claimed that two years’ continuous practice Conservation Agriculture based on Strip Tillage with the retention of 50% anchored residues of previous crop may lead to reduce soil weed seed bank status. It is recommended to conduct long-term trials across the Agro-ecological Zones of Bangladesh to validate the results.

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A Brief Summary of the Global Status of the 2022 Monkeypox Outbreak in Non-Endemic Countries

Introduction

Since the second week of May 2022, a sudden outbreak of Human monkeypox cases has been observed in as many as 21 non-endemic countries [1-5]. The concerning aspect of this 2022 outbreak of Human monkeypox is that many of the diagnosed cases didn’t exhibit any direct travel history to any endemic country or region [6]. Monkeypox ICD-10 B04 [7,8] is an infection that is primarily seen in Central and West African countries that have tropical forests populated with animals which can act as hosts for this virus [3,6,9-12]. Human monkeypox refers to a virus-based zoonosis that is caused by the monkeypox virus. The monkeypox virus is a member of the Orthopoxvirus family that also includes the variola virus responsible for causing smallpox. Since it’s discovery, there have been found to be two genetically separate strains of the monkeypox virus namely the Congo Basin (Central African) strain and the West African strain. In the past, Human monkeypox infections caused by the West African strain have shown to be of low severity in comparison to the infections caused by the Congo Basin strain [1,2,11,12].

Facts known about the Ongoing Human Monkeypox Virus Infection

Human monkeypox is a viral infection which has been transmitted to human beings from animals (mainly monkeys) and the human version spreads through direct or close contact with an infected individual [4-8]. Thus, it is safe to say that human monkeypox is generally transmitted by both direct and indirect contact with the bodily fluids of an infected person like their blood, bodily fluids, their skin or mucosal lesions and their clothes or belongings that have some degree of infected bodily fluids on them [1,4,5,7,8,12]. Secondary or person-to-person spread may take place also in people who touch the infected respiratory secretions, the skin lesions or contaminated belongings of the infected individual [8,10]. In previous outbreaks, the main route of transmission was found to be close contact with infected respiratory droplets. Human monkeypox infections can also be transmitted through inoculation or via the transplacental route from mother to foetus (this is known as congenital monkeypox). Even though most of the initial cases of Human monkeypox have been diagnosed in sexual health care clinics amongst men who have sex with other men, the most probable route of infection was the close contact with skin lesions of the infected person [4,5,7-9,12].

The main symptoms of Human monkeypox infection are fever (>38.5℃), headache, joint and muscle pains with subsequent presentation of a skin rash that is generally localized to the patient’s face, palm section of the hands and the soles of their feet. Other symptoms include lymphadenopathy (this refers to the presence of swollen lymph nodes), profound myalgia (this means having severe muscle and body pains), pain in the back and presenting with asthenia (serious degree of weakness). The incubation period is usually 6 to 16 days but has been reported as ranging from 5 to 21 days [5-7]. The infected individuals remain contagious from the onset of the above mentioned symptoms till the skin lesions are completely cured [7]. A confirmed case of Human monkeypox is one in which the patient presents with symptoms and probable cause (direct contact with an infected person or a travel history to endemic regions) followed by laboratory confirmation of having the monkeypox virus (done using molecular protocols like the realtime PCR test as well as genetic sequencing where possible) [11,12].

Figure 1: The appearance and progression of the different types of skin lesions in the second stage of the Human monkeypox infection (Source: 1).

The Human monkeypox infection in a patient has two clear stages. The first stage is the period of incubation and invasion (generally lasts between 5-21 days). This first stage is the one in which the patient presents with fever, serious headaches, lymphadenopathy, acute pain within the lower back as well as serious asthenia. The second stage is the skin rash period (this occurs within three days after presenting with fever) and is characterized by the different phases of rash appearance. It usually affects the face first and then spreads to the rest of the body (see Figure 1 below) [1]. The most affected areas are the face (in 96% of cases), the palms of the hands and the soles of the feet (in 75% of cases). The progression of skin rashes from maculopapular (the flat-based type of lesions) to vesicles (the fluid-filled blister type of lesions), pustules, and the formation of crusts in the end takes place within a 10-12 day period. The drying and falling of the scabs may occur or continue for almost 21 days.

Status of the Present Ongoing Human Monkeypox Outbreak on a Global Scale

Human monkeypox which spreads via direct person-to-person contact has always been low, but the appearance of the present outbreaks in as many as 24 non-endemic nations poses a definite risk to the general public (see Table 1 below) [8,11,12].

Table 1: Status Report of the present Human monkeypox outbreak as of 30th May 2022 (Sources of data: 8, 11-15).

Potential Mitigation of Transmission, the Guidelines for Diagnosis and Existing Treatment Options

Monkeypox symptoms usually get resolved in the patient with only supporting management and treatment. Still, in the present outbreak, it is of vital importance to contain the outbreak by limiting contact with patients, contact tracing, swift information of contact status to the contacts and isolation of such contacts. The risk summary in the present 2022 Human monkeypox virus for various populations was carried out by the EDEC and is presented in Table 2 below [12]. Patients must be kept isolated till the skin rashes are totally cured and must limit or totally abstain from contact with immune-suppressed individuals (at high risk of contracting the Human monkeypox infection) persons and pets. There is also need to avoid sexual activity and close physical proximity until the skin lesions are healed. Most patients can be cared for at home with supportive care. Those who come in close contacts of Human monkeypox cases need to carry out self-monitoring for any presentation of the disease symptoms up to 21 days from the last exposure to the patient.

Table 2: Summary of risk assessment for contracting the Human monkeypox virus in various population groups as published by the EDEC [12].

Health care staff and certain populations are more susceptible to getting infected. Health care staff needs to wear appropriate PPE (personal protection equipment) like gloves, water-resistant gown, FFP2 respirator when in touch with suspected cases or providing care to diagnosed patients. Laboratory personnel that test for the Human monkeypox virus must take mandatory precautions to mitigate occupational exposure [12]. A summary of treatment and management guidance for Human monkeypox cases has been published by the EDEC and is shown in Table 3 below. This will be useful as a reference point for health care professionals [12].

Table 3: Summary of treatment and management strategy for those in close contact with a Human monkeypox patient as published by the EDEC [12].

The strict compliance with sanitary hygiene protocols (regularly disinfecting hands after contact with the patient) by care givers and all in contact with the patients is mandatory. The surveillance, case assessment, contact tracing, follow-up, laboratory diagnosis, assessment and management of the patients should follow the WHO guidelines [10,11] or the EDEC guidelines [12] as per the country’s health care policies [5-7,11,12].

The WHO, the EDEC and USA’s CDC (The Centre for Disease Control and Prevention) are responding to this event as a high priority to avoid further spread [6-15]. The WHO has also recommended safe sex as many of the present cases on a global scale seem to have been diagnosed in men who have sex with other men. Infected people and those in contact with them have to abstain from contact with pet or domestic animals to prevent the jumping of the monkeypox virus infection in these susceptible populations [5-7,11,12]. At present, there are no validated as well as licensed treatment protocols for human monkeypox. However, two orally used medicines namely brincidofovir and tecovirimat have been given approval by the FDA (Food and Drug Administration), USA for the treatment of smallpox as emergency strategies in the event of any future bioterrorism event. None of these two medications have been assessed properly in human clinical trials for effectiveness against other members of the Orthopoxvirus family including the Human monkeypox virus. There has been some compassionate usage of tecovirimat in the Central African Republic, where Human monkeypox outbreaks are common [1].

In terms of prevention through vaccination, there are a few vaccines available for preventing smallpox which give a certain degree of protection against the Human monkeypox virus. One of the more recent vaccines that was made for smallpox namely MVABN (also called Imvamune or Imvanex or Jynneos) has been given approval in the year 2019 for usage in prevention of the spread of the Human monkeypox infection. However the drawback is that this vaccine isn’t freely accessible globally at present. The WHO is presently working towards better availability by collaborating with the makers. It is of importance to know that individuals who are in the above 40 age group were vaccinated against smallpox as it had not been eradicated and this provides them some measure of protection against the Human monkeypox virus. The issue that is worrisome at present is that these original smallpox vaccines were withdrawn from public circulation after smallpox was eradicated. As such, people in the below 40 population were most probably not given smallpox vaccination after the year 1980. In some countries, some of the highly at risk health care staff might be given a more recent smallpox vaccine [15].

Conclusion

The UN and WHO are of the opinion that the present global outbreak of Human monkeypox in non-endemic countries is still a controllable public health situation. However, there is need for every country to carry out appropriate surveillance, reporting of cases, contact tracing, adequate management as well as the compliance with the prevention, management and treatment guidelines provided by WHO [10,11,15] as well as the EDEC guidelines [12] or the CDC [13,14], depending on the country’s health care policies. The general public can consult the WHO factsheet [15] and the EDEC or CDC websites for general awareness and information on the Human monkeypox infection.

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