Children with Kawasaki disease or Kawasaki-like syndrome (MIS-C/PIMS) at the time of COVID-19: are they all the same? Case series and literature review.

Since the coronavirus disease 2019 (COVID-19) outbreak started, children have been considered marginally involved compared to adults, with a quite significant percentage of asymptomatic carriers. Very recently, an overwhelming inflammatory activation, which shares clinical similarities with Kawasaki disease (KD), has been described in children exposed to COVID-19. We report three KD-like cases that occurred during the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a highly affected area of Northern Italy. The clinical presentation was characterized by the presence of unremitting fever, diarrhea and elevated inflammatory markers. Case #1 and Case #2 occurred one week apart and shared other clinical features: laboratory tests confirmed COVID-19 exposure and high inflammatory activation with myocardial involvement. Case #3 followed a more typical pattern for KD. Interestingly, this patient showed lower levels of procalcitonin, C-reactive protein, D-dimers, and ferritin compared to the other two cases, whereas platelet count was higher. We hypothesize that SARS-CoV-2 might act in children as a trigger, either inducing a classical KD phenotype or causing a systemic inflammatory response leading to a severe KD-like phenotype, eventually characterized by myocardial impairment. We think that bringing these cases and their differences to the attention of the rheumatology community during the COVID-19 pandemic will be beneficial in order to highlight the importance of early diagnosis and to increase awareness of this new phenomenon.

Isolation and partial characterization of biologically active Fc receptor of chicken red cells.

It has previously been demonstrated that chicken red cells have a receptor with the capacity to bind aggregated IgG, IgM 7 S or antigen-complex IgG. This receptor was isolated from Nonidet P-40 soluble extracts of chicken red cells by immunoadsorption with either immobilized aggregated IgG or monomeric IgM (IgM 7 S) and further gel filtration through a Sephacryl S-300 column. The Fc binding material was characterized as a glycoprotein with a molecular weight of 30,000 which retained its Fc receptor activity after the isolation procedure. This was demonstrated by its capacity to inhibit the binding of 125I-IgM 7 S or 125I-labelled aggregated IgG to chicken red cells. After Bacillus cereus phospholipase C treatment the Fc receptor activity remained unchanged, but the molecular weight (15,000) did not, suggesting that the phospholipids cleaved by this treatment were not essential for the interactions of the receptor with specific ligands. However, this Fc-binding component was shown to have a molecular weight of 13,000 and a diminished Fc receptor activity after reduction with dithiothreitol, suggesting the presence of at least one disulphide bridge, necessary to maintain the total ligand-binding activity.

Trichinosis.

Trichinosis is a worldwide zoonotic disease closely related to cultural and dietary habits caused by a nematode Trichinella spp. Human infection is acquired through ingestion of undercooked meat containing infective encysted larvae. There are two cycles of transmission, one domestic and the other wild. A complete life cycle develops in a single host harboring adult worms in the small intestine, from which newborn larvae migrate and finally encyst in striated muscle. Traumatic and immunological alterations are responsible for the main clinical features, including diarrhea, febrile syndrome, myalgias, oculopalpebral signs and eosinophilia. Cardiovascular, lung and CNS involvement characterize severe trichinosis. CNS inflammatory infiltration and damage may result from larval migration and vascular obstruction, or from the effect of toxic parasite antigens, or eosinophil infiltration. Humoral and cellular immune host response are relevant both to protect against re-infection and for immunodiagnosis. DNA probes and PCR technology may help to identify Trichinella spp. Muscle biopsy may disclose T spiralis larvae coiled within a muscle fibre host nurse cell surrounded by a capsule. Inflammatory infiltration includes monocytes, plasma cells, eosinophils and T lymphocytes mainly of the suppressor/cytotoxic phenotype. Histological appearance and histochemical profile of the host nurse cell differ from that of striated muscle fibre and are partly indicative of regeneration. Our own histological and histochemical findings in experimental studies of infected mouse muscle support the concept that changes induced by the larva encysting within a single host skeletal muscle fibre which becomes a nurse cell are unique of Trichinella infection. Interestingly, no dystrophin could be detected within the host nurse cell-capsule interface. It has been advanced that larva-induced host muscle fibre changes may be regulated at muscle gene transcription level whilst host regulatory pathways governed by cell cycle phase may also contribute to larval development.

Echinococcosis.

Echinococcosis is a human disease caused by the larval form of Taenia echinococcus, which lives in the gut of the dog, wild canides and other carnivorous animals which represent the definitive hosts and involves as intermediate hosts both domestic and wild animals. Humans become accidental intermediate hosts by ingesting Taenia eggs. The main species pathogenic for man are E granulosus causing cystic echinococcosis with worldwide distribution and endemic in sheep and cattle breeding countries, and E multilocularis causing alveolar echinococcosis, with preferential distribution in the northern hemisphere. After ingestion of contaminated food, hexacanth embryos migrate by the portal system to liver and later lung, brain and other tissues. Symptoms are related to both cyst location and size. E granulosus infection of the central nervous system (CNS) may be primary or secondary and has been estimated to be low (2%). Sharply demarcated, spherical and intraparenchymal, cysts may reach a large size causing neurological symptoms. Spilling of cyst fluid due to trauma or surgery may trigger anaphylaxis as well as disseminated infection. Host reaction is minimal in the brain but a foreign giant cell reaction may develop. E multilocularis develops within the liver as a rapid invasive pseudomalignant growth and may metastasize to the CNS, where estimated incidence reaches 5%. Hydatid antigens induce an immune reaction in the host which is helpful for the diagnosis. DNA probes and PCR may be applied to differentiate between Echinococcus spp. Although the host develops an immunological protection from reinfection, the parasite evades host immune attack. A wide range of evasion mechanisms have been advanced, including a barrier for host cells due to hydatid cyst laminated cuticle, polyclonal activation of lymphocytes by parasite soluble antigens, and depression of host cell immune responses. Chronic stimulation of the host by cyst fluid antigens leads to increased specific IgG4 production, which might act as blocking antibodies against anaphlaxis suggestive of host response immunomodulation.

Diagnosis of porcine trichinellosis: parasitological and immunoserological tests in pigs from endemic areas of Argentina.

In order to compare the reliability of serological and parasitological techniques for the diagnosis of porcine trichinellosis from endemic areas in Argentina, 116 pigs were studied: 61 animals from two separate outbreaks and 55 from a small abattoir. Direct diagnostic techniques included trichinoscopy and the artificial digestion method. Indirect diagnostic tests used in this study were the enzyme-linked immunosorbent assay (ELISA), employing the excretory-secretory products of muscle larvae (ML) as antigen, and the indirect immunofluorescence assay using as antigen ML in suspension (IIF-susp), cryostat sections of infected rat muscle or of free ML (IIF-slide). The percentage of parasitologically positive pigs was invariably lower than that of serologically positive animals (IIF-slide), even when digestion studies were carried out individually with a greater amount of muscle sample than required by current regulations. Close correlation was found between IIF using as antigen tissue sections and IIF using free ML sections, while IIF-susp proved unsuitable for diagnosis since this assay presented a high percentage of false negative results (20%). The IIF-slide technique proved positive in all parasitologically positive animals. ELISA rendered a lower percentage of positive reactions than IIF-slide, especially when worm burden was low. Since most parasitologically positive animals rendered at least two positive serological tests (two variations of IIF or IIF plus ELISA), those negative by digestion and positive by two serological methods were strongly suspected of having trichinellosis. Upon studying swine from a abattoir it was found that 9% of the pigs were positive when assayed by two serological techniques, but Trichinella spiralis infection could not be parasitologically confirmed. To sum up, serological methods may be used for screening all pigs and positive findings should be tested by the digestion method by analysing a greater quantity of pork than that required by current regulations, above all in areas with reported clinical trichinellosis in humans, to ensure that the pork is safe for human consumption.

Immunoelectrotransfer blot assay in acute and chronic human trichinellosis.

An immunoelectrotransfer blot assay (IETB) using excretory secretory products of muscle larvae of Trichinella spiralis (ML-ESP) and the avidin biotin system was developed in order to characterize reactivity against ML-ESP in sera from patients with acute and chronic trichinellosis. A complete pattern of up to 13 bands was developed by sera from individuals with trichinellosis where doublets, triplets, or single bands were shown to have molecular weights of roughly 66, 55, 45, 36, 29, 24, and 14 kDa. The bands at approximately 55, 36, 29, and 14 kDa proved specific for T. spiralis. The band at approximately 55 kDa was present in all trichinellosis sera, whereas the approximately 14-kDa band was present in only a small percentage of sera. The development of approximately 36- and 29-kDa bands suggests a modulation of the reactivity against ML-ESP over time. IETB proved more sensitive for the population of chronic trichinellosis under study than a conventional diagnostic enzyme-linked immunosorbent assay, allowing negative or borderline serum samples to be determined. Thus, this technique, when applied for human trichinellosis surveillance, should provide a useful tool in endemic areas.

Diagnosis of human trichinellosis: pitfalls in the use of a unique immunoserological technique.

Serum samples belonging to three outbreaks in Argentina (47 patients) taken at different times post-ingestion were analysed employing IIF and ELISA simultaneously. Results show that: a) the number of patients diagnosed by a unique technique, especially by ELISA (31 patients), was lower than the one obtained by the simultaneous use of both assays (38 patients); b) four patients out of the seven diagnosed by a unique technique were negative by the other assay over the period of time evaluated. Therefore, it can be concluded that the use of a sole immunoserological technique can not only lead to the delay in the detection but also to the misdiagnosis of this parasitic infection.

[Serological, clinical, and epidemiological aspects of an outbreak of trichinosis in Azul, province of Buenos Aires]. / Aspectos serológicos, clínicos y epidemiológicos de un brote de triquinosis en Azul, Provincia de Buenos Aires.

An outbreak of human trichinellosis that took place in Southern Buenos Aires Province, an endemic area for this zoonosis, was studied. Eighteen individuals were infected by eating swine sausages lacking veterinary control. Clinical symptoms, as well as serological studies related to antibody production and cytotoxic capacity of the sera are described. Eight interdicted pigs and their products were analysed. In order to determine the transmission network of this zoonosis, wild rats were hunted in the farm where the swine were bred. The clinical symptoms of the infected individuals were: myalgia, edema, fever and cephalea. Sixty-one percent of the patients showed antibodies against muscle larvae 15-20 days after ingestion of the infected meat. Five out of eighteen patients showed anti-newborn larvae antibodies 40-60 days postinfection. The sausage burden was 5.3 muscle larvae/g. Two out of eight swine were diagnosed employing serological techniques, while parasitic techniques detected only one. The rat is an important reservoir in the domestic cycle of this zoonosis.

Description of an outbreak of human trichinellosis in an area of Argentina historically regarded as Trichinella-free: the importance of surveillance studies.

Trichinellosis is an important food-borne zoonosis which is not treated as a major public health concern in Argentina. After more than 20 years without reports of infection in an area regarded as Trichinella-free, research studies reported that infection occurred in humans, pigs and game animals, including the recent outbreak of human trichinellosis revealed here. The outbreak, affecting 64 individuals, occurred in July 2010 in the province of Entre Ríos. Epidemiological studies, clinical observations, laboratory analyses and immunoserological specific assays (indirect immunofluorescence, IIF, and ELISA) were performed. Food samples were analyzed by artificial digestion, and Trichinella larvae isolates were identified to the species level by multiplex PCR. The main source of infection, commercially available food, had a parasite load of 1.1 muscle larvae per gram. Larvae were identified as Trichinella spiralis. Patients presented predominantly with oedema, fever and myalgia; and laboratory findings and/or immunoserological tests were positive for trichinellosis. Individuals received outpatient treatment. No deaths or secondary sequelae were recorded. Results suggest that the presence of T. spiralis infection should be suspected in all endemic areas, especially where animal husbandry and official food safety controls are not properly conducted. The lack of the cases reported ought not to be taken as a proof of parasite absence. We highlight the importance of the urgent need to implement interdisciplinary and inter-institutional programs aimed to control infection transmission, to guarantee food safety and to conduct epidemiological surveillance studies.

Detection of coproantibodies and faecal immune complexes in human trichinellosis.

Trichinella spiralis is the nematode causative agent of trichinellosis, an intestinal and tissular parasitosis. Even though an early diagnosis during the intestinal phase is essential to limit the infection in humans, to date, there are no available tests to achieve this goal. Based on the immune response generated by the host's intestinal mucosa, the aim of this work was to develop ELISAs to assess the presence of coproantigens (CAgs), coproantibodies (CAbs) and faecal immune complexes in stool samples of 18 individuals belonging to different outbreaks that have arisen in Argentina. By the methodologies developed in this work it was found that anti-muscle larva excretory-secretory products (ML-ESP) CAbs were detected in 89% of the samples analysed regardless of the time p.i. Anti-ML-ESP IgA, IgG, IgE and IgM were detected in 56%, 56%, 28% and 22% of the individuals respectively. Those samples negative for anti-ML-ESP total immunoglobulins proved positive for anti-adult worm-ESP CAbs. No CAgs were detected in any of the samples. The results obtained in this work indicate that the intestinal immune response in human trichinellosis is featured by all the isotypes of specific immunoglobulins. Furthermore, the detection of antibodies in stool samples, in either the free or complexed form, could be applied to confirm early human trichinellosis.

Early pulmonary response in rats infected with Trichinella spiralis.

The migratory stage of Trichinella spiralis, the newborn larva, travels along the pulmonary microvascular system on its way to the striated muscle cells. In the present study, an important inflammatory reaction was observed on days 5 and 14 post-infection (p.i.) in the lungs of infected rats. This inflammation was characterized by a Th2 cell phenotype of hyperplastic bronchus-associated lymphoid tissue and by goblet cell hyperplasia. Among the inflammatory cells were eosinophils and mast cells scattered over the pulmonary parenchyma. On day 5 p.i. the number of IgE(+), CD4(+) and CD5(+) cells in the bronchus-associated lymphoid tissue were increased and IgE-secreting lung cells were also detected. At the end of the migratory phase of the infection (day 14 p.i.), only IgE(+) cells were detected in high numbers and in the bronchoalveolar lavage fluid, an increment in the total IgE levels as well as the presence of IgE and IgA anti-larvae surface were also detected. In cytotoxicity assays, cells from the bronchoalveolar lavage had considerable biological activity since they were able to kill the larvae even in the absence of specific antibodies. These results show that the lung is an organ involved in the immune response developed early during a T. spiralis infection and suggest its importance in the protection of the host.

In vitro and in vivo effects of progesterone on Trichinella spiralis newborn larvae.

We have previously demonstrated that during pregnancy there exists an increased parasiticide activity against Trichinella spiralis newborn larvae (NBL) in infected rats. In this work we analysed the contribution of peritoneal cells from noninfected pregnant rats to the mortality of the NBL in cytotoxicity assays, and evaluated the role of progesterone in this effector mechanism. Our findings suggest that progesterone can induce activation of effector peritoneal cells to destroy the NBL in a rapid and antibody-independent manner. The administration of progesterone to ovariectomized rats also led to a significant decrease in the parasite load of the animals, thus demonstrating that progesterone induces the increase of the parasiticide activity of the leukocytes involved in the mechanisms of NBL death.

Immune killing of newborn Trichinella larvae by human leucocytes.

The capacity of human leucocytes from normal donors to kill the newborn larvae of the nematode Trichinella spiralis in vitro, in the presence of serum from infected individuals, was studied using newborn larvae (NBL) less than 2 h of age or NBL that had been maintained in culture at 37 degrees C for 20 h. Neutrophils and monocytes attached to newborn Trichinella larvae and killed them, regardless of their age. When eosinophils were used, 20 h old NBL were killed whereas 2 h old NBL were not. Complement was essential in the cytotoxic effect of leucocytes. These results indicate that host defence against T. spiralis in humans may be a complex mechanism in which different cell types can be involved. They also show that the age of maturation of the NBL is of paramount importance in the antibody-dependent cellular cytotoxicity reaction.

Immunoparasitological parameters of the intestinal phase of trichinellosis in rats.

Enzyme-linked immunosorbent assays (ELISAs) were developed in order to detect coproantigens (CAgs), coproantibodies (CAbs) and faecal immune complexes (FIC) in rats experimentally infected with Trichinella spiralis. The usefulness of these assays was compared to that of a conventional ELISA for detection of serum antibodies (Abs) to muscle larvae excretory-secretory products (ML-ESP). The ELISA for CAgs was the first parameter to give a positive result but the detection was limited only to day 2 p.i. CAbs against ML-ESP and adult worm excretory-secretory products (AW-ESP) was first positive on day 4 p.i. Anti-ML-ESP remained positive until day 12 p.i. while CAbs against AW-ESP remained positive throughout the study period. Specific IgE and IgA were found. FIC were detected between days 2 and 8 p.i. Serum Abs began to appear on day 10 p.i. Therefore, the ELISA for CAbs was a suitable assay for the detection of the enteral and early phases of the infection.

Immune cytotoxic activity of human eosinophils against Trichinella spiralis newborn larvae.

The ability of human eosinophils to kill the newborn larvae (NBL) of Trichinella spiralis of different maturation status, in the presence of antibody, was studied. A cytotoxic in vitro test was performed using NBL less than 2 h of age (NBL2) or NBL maintained in culture at 37 degrees C for 20 h (NBL20), peripheral blood eosinophils, anti-Trichinella serum and human fresh serum as source of complement. Under these experimental conditions eosinophils from normal individuals attached to NBL2 as well as to NBL20 but only the latter were killed. On the other hand, eosinophils from volunteers with eosinophilia killed NBL regardless of larval age. Neither adherence nor significant mortality was observed in the absence of immune serum. These results indicate that NBL maturation and eosinophil activation status are crucial for antibody-dependent cellular cytotoxic reaction (ADCC).

Blocking anti-Trichinella spiralis antibodies in chronically infected rats.

Specific anti-newborn larva antibodies present in the serum of rats chronically infected with Trichinella spiralis were incapable of inducing killing of newborn larvae by activation of normal peritoneal cells. These late antibodies blocked the cytotoxic reaction induced by early antibodies produced a few weeks after infection. Passive transference of late serum to normal mice failed to induce protective immunity against infection by newborn T. spiralis larvae. When late immunoserum was fractionated by gel filtration, blocking activity was found only in the fraction containing IgG subclasses. By indirect immunofluorescence assay and cytotoxic reaction it was shown that blocking antibodies were specific for newborn larvae and could not be adsorbed with muscle larvae. It is concluded that the synthesis of anti-newborn larva antibodies is modulated in the course of a chronic infection: early antibodies developing shortly after infection are cytotoxic, whereas blocking antibodies predominate in the late population. Furthermore, the results suggest that during a chronic infection, resistance to reinfection may be modified.

Cytotoxicity-blocking antibodies in human chronic trichinellosis.

Antisurface newborn larva (NBL) antibodies (Abs) were found in sera from individuals chronically infected with Trichinella spiralis. These Abs were incapable of inducing NBL death by activation of normal human leukocytes of peripheral blood as determined by in vitro assays of antibody-dependent cell cytotoxicity (ADCC). Besides, such sera blocked the cytotoxic reaction mediated by Abs produced a few weeks after infection. The blocking activity could not be attributed to any particular isotype by the indirect immunofluorescence technique. Purified antisurface NBL Abs obtained from sera from chronically infected patients recognized antigens of muscle-larva excretory-secretory products (ML-ESP) in an enzyme-linked immunosorbent assay (ELISA) and an immunoelectrotransfer blot assay. Likewise, as did chronic sera, a monoclonal Ab raised against ML-ESP blocked NBL death in ADCC assays. These results suggest that during the course of an infection by T. spiralis, Abs related to ML-ESP provide an immunoevasive mechanism for avoidance by NBL of an important anti-NBL host effector mechanism.

Study of immune defence against Trichinella spiralis in BCG treated mice.

In vivo and in vitro studies were performed to determine the effect of BCG- activated macrophages on 2-hour-old newborn larvae (NBL2) of Trichinella spiralis. In vitro studies were done by the technique of antibody-dependent cellular cytotoxicity (ADCC) using peritoneal cells of BALB/c mice which had been treated with BCG 4-6 days before. No significant differences were found between the percentage of mortality caused by cells from treated animals and that corresponding to the untreated ones (control mice). The same result was obtained when only glass-adherent cell populations only were used. Intravenous administration of NBL2 and anti-T. spiralis antibodies to BCG-treated mice resulted in the same percentage of infectivity as that corresponding to control animals. These results indicate that an increase in the activity of macrophages induced by BCG neither alters the cytotoxic activity of the effector cells nor plays a direct role in the destruction of NBL2 of T. spiralis.

Antibody-dependent in-vitro cytotoxicity of newborn Trichinella spiralis larvae: nature of the cells involved.

The cytotoxic reaction of normal peritoneal mouse cells, containing less than 3% eosinophils, against newborn Trichinella spiralis larvae, in the presence of antibody, was studied using newborn worms less than 2 h of age, or newborn worms that had been maintained in culture at 37 degrees C for 20 h. Newborn worms 2 h old were killed, whereas 20 h newborn worms were not. The cells that initially adhered to the larvae were examined by electron microscopy. Only eosinophils adhered to 2 h newborn worms and only macrophages to 20 h ones. The attached eosinophils degranulated and died after a few hours. The macrophages that adhered to, but did not kill the 20 h newborn worms were morphologically in good state and no lysis of the larvae was observed. These results suggest that different antibody classes are involved in eosinophil and macrophage adherence, and strongly support the hypothesis that eosinophils mediate larval destruction. They also show that rapid changes are taking place after birth in the structure of the larval cuticle and that the age of Trichinella newborn worms is a major factor in the antibody-dependent cellular cytotoxicity reaction.