20 Jan 2023 Episode 308: Invasive Group A Streptococcus (iGAS).

Episode 308: Invasive Group A Streptococcus (iGAS).

Dear colleagues,

After Ep 306 on pneumococcus (Group B Streptococcus) as a complication of Influenza, I received several mails, indicating that the bigger problem today in Europe is in fact Group A Streptococcus (GAS), also known as “pyogenes” and more particularly the “invasive” (iGAS) subvariants.

Quoting colleague Erika Vlieghe:

The press attention focuses even more on iGAS (invasive group A streptococci) than on pneumococci (although these can of course also be very virulent and relevant).

In particular, there is a lot to be said about the increase in iGAS with streptococcal septic shock, necrotizing fasciitis, etc.

Much morbidity and even mortality due to very pronounced virulence, need for extensive surgery, need to add clindamycin (anti-toxin effect), need IV IG.

In prevention, there is no vaccine available, and few concrete NPis, but there is an indication to give prophylactic antibiotics to household contacts.

The initial registration can be very vague and is therefore often missed.

Available data in Belgium are scarce, reporting obligations for these invasive infections are not well known

 

And colleague Eric Florence

… we've had a lot of trouble with strep infection, not so much S pneumoniae, but very serious co-infection with GAS, it's also been in the press.

Really striking many children and young adults admitted to ICU and eventually got to ECMO to bridge to healing (with a lot of seborrhea). In the children, of course, also a lot of RSV

 

… about the serotype shift (of Pneumococcus), that would not prevent us from continuing to vaccinate widely,

Indeed, carrier status of uncovered strains seems to be on the rise, but the contribution of these strains to invasive disease is not clear and current vaccine coverage for strains responsible for invasive disease remains reasonably good (I have not found any data to reduction was – ie an “escape” to non-vaccine serotype as a causative agent of invasive diseases)

 

Therefore, in this Episode, I will summarize some of the present-day knowledge on clinical and pathogenic aspects of Strep A, including the slow progress towards vaccination.  The information is not complete: I will limit discussion to the main syndromes and I will not touch on treatment. As you will see, I try to be didactic by providing lots of pictures and schemes. I’m not a specialist in this matter: please let me know if I’m incomplete or too superficial.    

See:

Par 1 Epidemiological notes on today’s surge

Ep 308-1: Press release of ECDC 12 Dec 2022:

• Several deaths associated with iGAS in children less than 10 years reported in UK, Ireland and France
• iGAS infections can present initially with non-specific symptoms (fever, general tiredness, loss of appetite) and children, in particular, can have rapid progression to severe disease with bacteraemia, pneumonia, or skin and bone infection (cellulitis, osteomyelitis, necrotising fasciitis).
• In children often in context of a respiratory viral infection (seasonal Influenza, Respiratory Syncytial Virus).
• Fortunately, there is no indication of a new strain or increased antibiotic resistance.
• Unfortunately, there is no specific vaccine

What to do:

• Preventive:
  • Vaccination against seasonal influenza and COVID-19 should be promoted
  • NPI: adequate hand hygiene (colonize skin) and respiratory hygiene, as well as good indoor ventilation (asymptomatic respiratory colonization and droplet transmission)
  • (Prophylactic antibiotics to household contacts (according to E Vlieghe)
• Therapeutic: in principle sensitive to penicillin and amoxycillin; with S pyogenes is very sensitive to penicillin with erythromycin or an oral cephalosporin as back-up  in patients with penicillin allergy

 

Ep 308-2: RIVM Dramatic rise of septic shock, puerperal fever/shock (= “maternal bed fever) and necrotizing fasciitis ( = typical syndromes by invasive GAS) in the Netherlands

 

With a striking number of young children

 

Ep 308-3: UK Health Security Agency notes both spectacular rise in scarlet fever and iGAS much earlier in the season than usually

1. Scarlet fever

 

 

This season (from 19 September to 8 January), there have been 37,068 notifications of scarlet fever.

In 2017-18 at the same time only 4,490 at the same point: scarlet started later withy final total 30,768 that season

2. iGAS:

 

 

1,539 iGAS cases across all age groups, compared to 2,967 across the whole high season in 2017 to 2018.

177 iGAS cases in children aged 1 to 4 compared to 194 cases in whole of the 2017 to 2018 season.

 

Par 2 Bacteriological background summary  

 

 

 

 

 

Par 2 Syndromes (based on Ep 308-4 and other sources)

 

1. Streptococcal Pharyngitis

 

 

Strarts with fever, malaise, soar throat.  Can be associated with abdominal pain and vomiting in children.

 

The pharynx is erythematous, and the tonsils have purulent exudates.

 

To be differentiated from many different viral infections (without purulent exudate) as well as from Mycoplasma species, Corynebacterium diphtheria: Streptococcl pharyngitis :  Cough, rhinorrhea, hoarseness, conjunctival irritation, and diarrhea are notably absent in streptococcal pharyngitis, and the presence of these symptoms should suggest a non-streptococcal etiology

 

Infectious complications include peritonsillar or retropharyngeal abscess, sinusitis, otitis media, cervical lymphadenitis, bacteremia, endocarditis, pneumonia, and meningitis

 

Auto-immune complication: acute rheumatic fever (see Par 4)

 

2. Scarlet Fever + Strawberry Tongue; also known as “scarlatina” (roodvonk in Dutch)

NHS:

• The first signs of scarlet fever can be flu-like symptoms, including a high temperature, a sore throat and swollen neckglands.
• A rash appears 12 to 48 hours later. The classic scarlet fever rash initially appears as tiny red papules on the chest and abdomen. Papules may then spread over the body. The rash resembles sunburn, feels like rough sandpaper, and lasts about 2 to 5 days.
• On white skin the rash looks pink or red. On brown and black skin it might be harder to see a change in colour, but youcan still feel the rash and see the raised bumps

 

 

 

Strawberry Tongue

 

 

This tongue is erythematous with prominent papillation

Scarlet fever can be confused with measles and rubella. However, in those diseases, upper respiratory tract infection is less common and confluent rash in measles can help to differentiate these diseases.

Complication: Scarlet fever results from and “erythrogenic or scarlatina toxins”, a group of related streptococcal pyrogenic exotoxins (SPEs), which have a role in severe invasive streptococcal infections, of which scarlet fever can be a symptom.

3. Skin and soft tissue infections.

 

Anatomical relationship between skin and skin structures and different S. pyogenes infections

 

3.1. Impetigo is a superficial skin infection that causes crusting or bullae.

 

 

 

In impetigo, clusters of vesicopustular or bullous lesions form, rupture, and develop a honey-colored crust, usually around the mouth.

To be differentiated from the bullous, blister-like impetigo caused by Staphylococcus aureus, usually in skin folds, like the armpit, groin, between the fingers or toes, beneath the breast and between the buttocks

3.2 .Erysipelas is a superficial cellulitis that also involves the lymphatics.

 

Erysipelas is characterized by shiny, raised, indurated, and tender plaque-like lesions with distinct margins.

3.3.Cellulitis involves the deeper layers of skin and may spread

 

 

Streptococcal Cellulitis with Accompanying Tissue Necrosis

 

3.4. Necrotizing fasciitis is a severe dermal (and sometimes muscle) infection that spreads along fascial planes: can proceed to  myositis and osteomyelitis.

 

 

 

 

4. The systemic and sometimes life-threatening complications  distinguished in two categories

 

1. 1. Par.3 Severe Streptococcal infections by invasive GAS: as a consequence of “streptococcal pyrogenic exotoxins” (Spe), including classical scarlatina toxins SpeA and SpeC, the cysteine proteinase SpeB, and others.  These are mostly present in M-serotypes 1 and 3, but also 11, 12 and 28.

Patients are usually otherwise healthy children or adults with skin and soft-tissue infections with invasive GAS strains.

The life-threatening syndromes include scarlet fever, bacteremia, pneumonia, necrotizing

fasciitis, myonecrosis and Streptococcal Toxic Shock Syndrome, including puerperal sepsis

 

1. 2. Delayed auto-immune complications: as a consequence of cross-reactivity of streptococcal antibodies against host tissue.

Par 4  Rheumatic fever (See Par 4) = an inflammatory disorder, occurs in < 3% of patients in the weeks after untreated GAS pharyngitis.

-  It has become much less common in developed countries but is still common in developing countries.

-  Diagnosis of a first episode is based on a combination of arthritis, carditis, chorea, specific cutaneous manifestations, and laboratory test results

One of the most important reasons for treating GABHS pharyngitis is to prevent rheumatic fever.

Par 5 Poststreptococcal acute glomerulonephritis (See Par 5)  = an acute nephritic syndrome following pharyngitis or skin infection due to a certain limited number of nephritogenic strains of GAS (eg, M protein serotypes 12 and 49).

- After a throat or skin infection with one of these strains, about 10 to 15% of patients develop acute glomerulonephritis.

- It is most common among children, occurring 1 to 3 weeks after infection.

 

Nearly all children, but somewhat fewer adults, recover without permanent renal damage.

Antibiotic treatment of GABHS infection has little effect on development of glomerulonephritis.

 

 

SUMMARY of Streptococcal infection in children (Ep 308-5 Theresa Leung HKMJ 2018)

• Mild Streptococcus pyogenes infections: pharyngitis (strep throat) and localised skin infections (impetigo).
• Deeper skin infection = erysipelas and cellulitis,  characterised by multiplication and lateral spread of.
• Invasion and fascial involvement can lead to necrotising fasciitis, a life-threatening condition.
• Scarlet fever is characterised by a sandpaper-like rash in children with fever, caused by a streptococcal toxin.
• Severe infections that lead to septicaemia or toxic shock syndrome are associated with high mortality.
• Autoimmune reactions cause characteristic syndromes such as rheumatic fever and nephritis.

 

 

Par 3: Invasive GAS (iGAS) Syndromes

3.1. Introduction

 

Ep 308-6 Metzgar Virulence 2011 vol 2 p. 402: Some association of M-serotypes with particular diseases entities.

 

 

However, these associations may vary with time: a particular serotype will become less “virulent”, after a while, because of “herd immunity” in the human population.

 

Portal of entry?

 

The vagina, pharynx, mucosa, and skin: can be: wounds (including surgery) or varicella infection:

 

Surgical procedures such as suction lipectomy, hysterectomy, vaginal delivery, toe correction,, reduction mammoplasty, hernia repair, bone pinning, and vasectomy have provided portals

 

Also use of non-steroidal anti-inflammatory drugs (NSAID) has been associated with invasive GAS infection.  

 

In many cases no obvious port of entry.

 

Note on varicella

• Multiple case reports on epiglottitis, necrotizing fasciitis, septic shock in children, associated with varicella

 

• Recent evidence from Canada and Israel on lower case load of iGAS after introduction of varicella vaccine

 

Ep 308-7: Laupland Pediatrics 2000

1.9 cases of invasive GAS disease per 100 000 children per year in Canada.

- Streptococcal toxic shock syndrome (STSS) occurred in 7% of cases and necrotizing fasciitis (NF) in 4%.

- Case-fatality rates were 56% for STSS, 10% for NF, and 4% overall.

The presence of chronic underlying illness other than asthma was associated with death (relative risk [RR]: 11.

15 % had preceding chickenpox infection, which increased acquisition of iGAS disease (RR: 58; 95% CI: 40–85).

Children with iGAS and recent chickenpox were more likely to have NF (RR: 6.3; 95% CI: 1.8–22.3).

 

Ep 308-8 : Julie Frère CID 2016: 70 % reduction of iGAS after introduction of varicella vaccine with changing microbiological (toxin) and clinical characteristics: less necrotizing fasciitis and less toxic shock

 

Ep 308-9: Ori Hasin J Ped Infect Dis Soc 2020

• Clear-cut overall decrease of iGAS in children after vaccination national program (NBP)

 

• No more iGAS following varicella infection and less toxic shock as a clinical characteristic

 

 

 

1. Invasive GAS are also seen in association with  severe respiratory infections e.g. Influenza

 

Ep 308-10:  Okamoto Microbiol Immunol 2018; 62: 141–149

 

 

(a) In intranasal infection with GAS alone, GAS binds to the alveolar epithelium surface proteins that are the ligands of GAS adhesion molecules. However, monocytes migrate into the infected site and differentiate into alveolar macrophages, which eliminate GAS by phagocytosis and digestion.

 

(b) In intranasal superinfection with influenza A virus and GAS,

• GAS adhesion/invasion to the epitheliums is enhanced: influenza virus hemagglutinin (HA) and GAS capsules (mainly M3 protein are important for adhesion and invasion in superinfections.
• Infection of macrophages with influenza virus results in suppression of phagocytosis and digestion, diminishing their ability to ingest bacteria.

Thus, GAS easily invades and proliferates in lung tissue and diffuses throughout the body through blood capillaries, leading to invasive GAS infections.

Note: Relation with COVID:

- Literature suggests that the lower prevalence of iGAS infections during the COVID pandemic was due to the non-pharmacological interventions taken.

- On the other hand, there is no evidence that COVID pneumonia would facilitate iGAS.

I will discuss Septic (and Puerperal) Shock in more detail.  For  other syndroms see Ep 308-23: Stevens DL Severe Group A Streptococcal Infections - Streptococcus pyogenes - NCBI Bookshelf 2016

3.2 Streptococcal Toxic Shock Syndrome (STSS)

3.2.1. Who is At Risk ? (Ep 308-11: Thompson Acad Forensic Pathol. 2018 Mar;8(1):136-149.)

Infection at any age, with more severe illnesses affecting the extremes of age (less than 2 and greater than 65)

 

 

 

3.2.2. Clinical presentation of Strep versus Staph shock (Ep 308-12 Wilkins J Infect 2017 Jun;74 Suppl 1:S147-S152)

 

 

 

Differential diagnosis from Staphylococcus aureus

 

 

 

 

 

 

 

3.2.3 Pathogenesis

 

1. Multiple pro-inflammatory virulence factors and their host interactions (Ep 308-13 Shira Wilde Frontiers in Cellular and Infection Microbiology 2021 | Volume 11 | Article 704099)

 

 

 

Multiple pathways of inducing of inflammation:

 

 

• The GAS protease SpeB is directly proinflammatory by activating pro-IL-1b, other host substrates, and inactivating anti-inflammatory GAS effectors.
• Superantigens (SAgn) forcibly bind T lymphocytes and APCs, leading to excessive T cell activation.  Activated T cells kill other immune cells and release a “cytokine storm” of IFN-g, TNF, and IL-6, hallmark of Septic Shock Syndrome.
• Pore-forming toxins SLO and streptolysin S (SLS) form large pores in host cells that can lead to the passive release DAMPs, and other cytosolic or organelle-associated proinflammatory compounds, or be detected by the inflammasome to further activate inflammatory cell death by pyroptosis.
• M protein proteolytically released from the GAS surface can similarly form complexes that induce pyroptosis in macrophages or hyper-degranulation by neutrophils.
• GAS has numerous TLR agonists that activate proinflammatory regulatory program

 

2. Strept superantigens (SAgn)= Streptococcal pyrogenic exotoxin (Spe A, C, G, H, I, J, K, L, M, Q, R); SmeZ; SSA.  

(Ep 308-14 Mark Reglinski Virulence 2014 5:1, 127-136

and Ep 308-15 Jacklyn Hurst PLoS Pathog 2021 17(12): e1010097)

 

Superantigens bind to α or β chain of MHC class II and to a set of specific βchains of T Cell Receptor 

 

 

 

 

Model of T cell activation by a conventional peptide antigen (Ag) and superantigen (SAg).

APC, antigen-presenting cell; MHC II, major histocompatibility class II molecule; TcR, T cell receptor; TNF-α, tumor necrosis factor alpha; IFN-γ, interferon gamma; IL, interleukin.

 

 

 

3. Role of host immunity: Ep 308-16: Yu-Yu Chuang Pediatr Drugs 2005; 7 (1): 11-25

 

The association between the lack of antibodies to Streptococcal super-antigens SPEs in healthy individuals and the development of invasive streptococcal disease has been established:

• Antibodies to the M protein confer protection against invasive infection by enhancing phagocytosis
• Low levels of protective anti-M1 and anti-superantigen neutralizing antibodies in plasma may contribute to host susceptibility to invasive streptococcal infection but do not modulate disease outcome.

 

 

 

Influence of HLA: Patients with a DRB1*1501/DQB1*0602 haplotype seem to have an attenuated inflammatory

cytokine response to the GAS superantigen, avoiding the “cytokine storml” and thus have protection against the development of severe systemic disease.

 

Some pathogenic differences between Staph and Strep Septic Shock

 

 

 

 

 

3.3. Invasive Streptococcal infections during pregnancy and “puerperal” fever (= “maternal bed fever” “kraambedkoorts”)

 

Ep 308-17: Gilbert Donders J Clin Med 2021: Port of entry = symptomatic vulvo-vaginitis??

 

 

 

These data clearly show that prevalence of GAS is much higher in women with symptoms of vaginitis.

• mostly in pre-pubertal girls and postmenopausal women: thin un-estrogenized vaginal epithelium = vulnerable to inflammation and infection.
• is rarely found in adult, premenopausal women.

 

 

 

COMPLICATIONS for fetus and neonate:

• Chorio-amnionitis,
• Preterm labor,
• Neonatal sepsis.

 

Ep 308-18: Kristin Harris Acta Obstet Gynecol Scand - 2023

 

A review of 1190 patients:

2. Most cases postpartum (91.9%), with 4.7% reported antepartum and 0.6% intrapartum.
3. Bacteremia in 49.0% of and endometritis in 45.9%.
4. Puerperal sepsis in 28.2% of cases and it progressed to streptococcal toxic shock syndrome in 1/3
5. Overall, the case fatality ratio was 2.0%,

 

 

 

 

Par 4 Acute Rheumatic Fever (ARF) complications:  (see Ep 308- )  

ARF leads to an illness that is characterized by various combinations of

• Arthritis = Joint pain and swelling,
• Rheumatic Heart Disease (RHD) Cardiac valvular regurgitation with the potential for secondary heart failure,
• Sydenham Chorea,
• Skin (erythema marginatum) and subcutaneous nodules
• and fever

 

 

 

 

The burden of Rheumatic Heart Disease is now mainly in ss-Africa and South-Asia

 

Pathogenesis = auto-immunity due to cross reactivity with M protein of Streptococcus

Following group A Streptococcus (GAS) adhesion to and invasion of the pharyngeal epithelium, GAS antigens activate both B and T cells. Molecular mimicry between GAS group A carbohydrate or serotype-specific M protein and the host heart, brain or joint tissues can lead to an autoimmune response, which causes the major manifestations of acute rheumatic fever (ARF). BCR, B cell receptor; TCR, T cell receptor        

 

1. Arthritis

 

 

2. Rheumatic heart disease (RHD) : The heart is affected by antibodies against the group A carbohydrate binding to the surface of the valve and upregulating vascular cell adhesion molecule 1 (VCAM1) on the surface of the valve endothelium. The upregulation of VCAM1 allows T cells expressing integrin α4β1 (also known as VLA4) to adhere to the endothelium and to extravasate into the valve.

The inner valve becomes infiltrated by T cells, primarily CD4+ T cells, and Aschoff bodies or granulomatous lesions form underneath the endocardium. Damage to the endothelium and infiltration of T cells into the valve causes malformation of the valve leading to regurgitation or stenosis of the valve.

 

 

 

3. Sydenham Chorea: Neurons in the basal ganglia are attacked by antibodies against the group A carbohydrate of Streptococcus spp. that react with the surface of the neuron. This reaction activates signaling through calcium/calmodulin-dependent protein kinase type II (CAMK2), which involves an increase in tyrosine hydroxylase in dopaminergic neurons. Receptors, such as the D1 and D2 dopamine receptors, and lysoganglioside might be autoantibody targets on the neuronal cell. This targeting could lead to altered cell signalling and increased levels of dopamine, in turn leading to abnormal movements and behaviours.

 

 

4. Skin manifestations: Erythema marginatum might be due to antibodies against group A carbohydrates cross-reacting with keratin and subcutaneous nodules might be caused by delayed hypersensitivity against group A streptococcal antigens.

 

 

 

 

 

 

There is a role of genetic polymorphism in various populations, involving different genes that have a role in immunity and inflammation (e.g. Tumor Necrosis Factor, Fc-immunoglobunlin receptor,  Toll-like receptor etc.) as well as the HLA class 2 (important for CD4 T cell activation).

  

 

Par 5 Acute post-streptococcal glomerulonephritis (ASPGN)

See Ep 308-20 : Anjala Satoskar Nat Rev Nephrol 2020

 

 

Epidemiology of APSGN (compared to Staphylococcus infection- associated glomerulonephritis or SAGN

6. The incidence of APSGN declined sharply in the Western world, whereas the number SAGN cases increased.
7. APSGN still occurs with high frequency among highly populated and economically disadvantaged communities around the world, where group A β- haemolytic streptococcal infections are common
8. APSGN remains the most common cause of acute GN among children, whereas SAGN has very different demographic features and mainly affects the elderly population.

 

 

 

Clinical and laboratory differentiation

 

 

 

Pathogenesis of Acute Post-Streptococal GN

 

 

a | = Immune-complex disease:  with leading antigens = Streptococcal pyrogenic protease exotoxin B (SPEB) and nephritis plasmin- binding protein (NAPlr)

→ Both interact with plasmin

→ activation of complement and the release of matrix metalloproteinases (MMPs) and collagenases,

→ immune cell infiltration and activation (including cytokine release),

→ changes in the glomerular basement membrane (GBM) and mesangial extracellular matrix (ECM).

These changes probably allow immune complexes to breach the filtration barrier and accumulate as subepithelial humps.

Fibrinogen is another acute phase reactant that binds to and is deposited with antigen–antibody complexes.

 

b | Light microscopy image of endocapillary proliferative GN, stained with haematoxylin & eosin (H&E), 400 × magnification.

c | Direct immunofluorescence staining for complement protein C3 shows lumpy- bumpy , coarsely granular staining in the mesangium and glomerular capillary loops, 400 × magnification.

d | Electron microscopy image of subepithelial ‘humps’; tissue stained with uranyl acetate, 30,000 × magnification

 

 

Par 6 Vaccine development

 

Ep 308-21: Elodie Pastural Vaccine 38 (2020) 1384–1392 provides a  nice overview on the challenges

 

Efforts to develop a Strep A vaccine ongoing for a century.

 

Surface-expressed M protein is a major Strep A protective antigen =  target for vaccine development.

 

Numerous experimental M protein-based vaccines, ranging from crude cell walls to highly purified M proteins, were evaluated between the 1930s and 1970s in trials involving thousands of participants

 

Halted when in the 1960s  when partially purified M3 protein vaccine showed increase rheumatic fever

 

In the 1980s:  type-specific amino-terminal regions of the M protein elicited bactericidal immune responses and could be separated from the potentially harmful cross-reactive epitopes

 

 

 

 

Characteristics of the complete M6 protein sequence. Blocks A, B, C, and D designate the location of the sequence repeat blocks. Pro/Gly denotes the proline- and glycine-rich region that is likely located in the peptidoglycan. The hydrophobic region is composed of a 19 hydrophobic amino acid region, adjacent to a 6 amino acid charged tail. The LPXTG motif is located N-terminal to the hydrophobic region, which places it outside of the cytoplasmic membrane. The cell-associated region would be imbedded from the inner side of the cytoplasmic membrane to the top of the surface carbohydrates (this begins at Ala-298). Pepsin identifies the position of the pepsin-sensitive site after amino acid 228. The helical rod region is essentially the coiled-coil segment of the M molecule.

 

 

 

 

Complex hybrid proteins that contained increasing numbers of amino-terminal M protein fragments of many serotypes were evaluated in preclinical and early clinical studies

 

StreptAvax, a 26-valent vaccine candidate was shown to be safe, well tolerated and immunogenic in phase I and II trial, but halted for commercial reasons.

 

Here a 30-valent N-terminal M protein, based on the strains in US and Europe  is tested.   

 

 

RESULT

1. Ep 308-22: James Dale Vaccine 2011 : rabbit experiments

(2.A.) Geometric mean antibody levels (Log2) in three rabbits after three 800 μg doses of the 30-valent GAS vaccine

 

(2.B.) Bactericidal antibodies evoked by 30-valent M protein-based GAS vaccine

 

 

Fig. 3. Bactericidal antibodies evoked by 30-valent vaccine against non-vaccine serotypes of GAS.

 

 

 

Conclusion: The vaccine induced bactericidal antibodies against all included serotypes and showed cross-reactivity against many nob-included serotypes.

2. Ep 308-21: Elodie Pastural: Phase 1 humans

 

 

The vaccine elicited significant increases in ELISA mean antibody levels to 24 of the 30 component M antigens. Vaccine-induced opsono-phagocytic killing activity was observed against selected M types of Strep A in vaccinated participants that seroconverted to specific M peptides.

 

The authors are encouraged, but the results do not seem spectacular to me….  It may still be a long way towards an effective Strep A vaccine?

 

Best wishes,

 

Guido