Nasopharyngeal Wash with Normal Saline Decreases SARS-CoV-2 Viral Load: A Randomized Pilot Controlled Trial.

Background: Although great progress has been made over the past 2 years in the scientific understanding of the biology, epidemiology, and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), case morbidity and fatality rates remain a great concern and continue to challenge the healthcare resources worldwide as novel variants emerge. There is therefore an urgent need for affordable and readily available strategies to reduce viral transmission. Previous studies in non-COVID-19 patients have demonstrated that administration of low-salt (isotonic but 0.0375% Na) and isotonic saline (0.9% Na) solutions has been associated with an immediate, significant reduction in the microbial antigens and a related decline of microbial burden. The aim of the present study was to determine the effect of nasal washes with normal saline 0.9% on nasopharyngeal viral load and outcome in hospitalized patients with COVID-19 pneumonia. Methods: We performed a prospective, randomized, pilot, controlled trial in 50 patients with confirmed COVID-19 disease. Patients were randomized into two groups, the normal saline group (received normal saline 0.9% solution for nasopharyngeal wash) and the control group (no treatment). In the normal saline group, nasopharyngeal wash was performed every 4 hours for a 16-hour period. Twenty-four hours after the baseline nasopharyngeal swab (and 8 hours after the last wash in the normal saline group), a second nasopharyngeal swab was collected for the semiquantitative estimation of the SARS-CoV-2 viral load as determined by cycle threshold (Ct) values. Results: In the normal saline group, mean N gene Ct values increased significantly 24 hours after the baseline measurement [ΔCtday2-day1 = 1.87 ± 3.11 cycles, p = 0.007 (95% CI: 0.55 to 3.18)], indicating a decline in SARS-CoV-2 nasopharyngeal viral load by 8.9%. A significant decrease in mean N gene Ct values was observed in the control group, indicating an increase in viral load [ΔCtday2-day1 = -2.12 ± 2.66, p < 0.001 (95% CI: -3.20 to -1.05)] by 9.7%. The difference between the two groups 24 hours after admission and nasopharyngeal wash was 3.09 cycles (p = 0.005, 95% CI: 0.97 to 5.20). Conclusion: Nasal washes with normal saline effectively decreased the viral load during hospitalization and at follow-up.

Knowledge, behaviours and attitudes for human papillomavirus (HPV) prevention among educators and health professionals in Greece.

OBJECTIVE: Globally, human papillomavirus (HPV) is one of the most common sexually transmitted diseases in both men and women. The objective of this study was to investigate the knowledge, attitudes, and behaviour of educators and health professionals regarding the prevention of HPV infection in Greece. The analysis of the results shows that most of the participants (44.7%) had good knowledge about the human papillomavirus, regardless of occupation. PATIENTS AND METHODS: A cross-sectional survey was carried out with seven hundred and fifty participants. The participants completed a thirty-one-item questionnaire regarding their knowledge, attitude and behaviour towards the prevention of HPV. RESULTS: Knowledge scores were calculated with a grading system which was designed for accurate knowledge assessment. The Score was adjusted on a scale from 0 to 100 and was divided into five levels, from Excellent to Very Bad level of total knowledge. Of all the participants, 26.4% had excellent knowledge, 44.7% had good, 23.6% had moderate, 4.7% had bad, and 4.7% had a very bad level of total knowledge. Specifically, physicians of different specialities had 80.32%, and educators 65,31 % excellent level of knowledge, respectively. Also, participants had a high degree of awareness regarding prevention and adherence to the annual Papanikolaou test (65.8%) and gynaecological exam. Despite the positive response (94.1%) in terms of vaccination of boys, and girls, as well as information on HPV vaccination, many participants themselves were not vaccinated (74.8%), nor were their children (19.7%). CONCLUSIONS: The present study showed good level of knowledge for HPV prevention regardless of occupation and is characterized by a high degree of awareness of the usefulness of prevention in adhering to the annual Pap smear test and gynaecological examination. The study shows the need for more information workshops for healthcare professionals, because physicians and midwives had high levels of knowledge, but not excellent as expected and required due to health-related profession.

Knowledge, behaviours and attitudes regarding HPV infection and its prevention in female students in West Greece.

OBJECTIVE: Infection with several types of human papilloma viruses (HPV) has been correlated with the development of cervical cancer. Apart from other preventive strategies, two prophylactic vaccines have been added recently to the HPV prevention arsenal. The objectives of this study were to assess HPV vaccination coverage rates and to evaluate the level of knowledge regarding cervical cancer, HPV and Papanicolaou test among female students in a Greek city. PATIENTS AND METHODS: A cross-sectional study was carried out among five hundred female students of the Technological Educational Institute (TEI) of Patras, Greece. They completed an eighteen-item self-administrated questionnaire regarding their knowledge related to cervical cancer. RESULTS: Only 31.7% of the students had a high level (> 66%) of total knowledge. The majority (70.4%) had not been vaccinated against HPV. Students who achieved low and moderate total knowledge scores were less likely to be vaccinated against HPV. CONCLUSIONS: Implementing strategies for improving young females' knowledge on the different aspects of the natural course of HPV infection and increasing HPV vaccination coverage rates seem to be crucial.

Female students receiving post-secondary education in Greece: the results of a collaborative human papillomavirus knowledge survey.

OBJECTIVES: Contrary to the optimistic forecasts, existing until 2008 and despite the incorporation of the vaccine into the Greek National Immunization Program, six years later, the percentage of HPV vaccination coverage in Greece remains disappointingly low. The aim of this extended study was to investigate the knowledge, behaviour and attitude of a representative sample of the initial target group; young female students of Greek higher education institutions to Pap cervical screening, biology of HPV infection and principles of HPV vaccination. STUDY DESIGN: Cross-sectional study. METHODS: One thousand two hundred ten (1210) questionnaires were completed by young female students aged 17-24 years. The survey questionnaire sought data relating to sociodemographic characteristics, health behaviour and knowledge about HPV, as well as vaccination status. RESULTS: 79.6% of the sample reported at least one annual gynaecologic examination and 92.6% were familiar with the rationale of cervical screening; however only 52.9% had undergone a Pap smear. 69.7% reported adequate knowledge about HPV and 89.3% were aware of the possible course of HPV infection. Despite most (95.9%) were aware of vaccine availability, vaccinated students represented only 33.1%. According to the multivariate analysis, vaccination status was associated with university studies (OR 1.96; 95% CI: 1.19-3.20), parental area of expertise (OR 2.77; 95% CI: 1.18-6.53, OR 2.03; 95% CI: 1.05-3.94), and adequate knowledge of the reasons for which women should undergo regular cervical screening (OR 4.23; 85% CI: 1.55-11.55). Fear of side-effects and equivocal information were the main reasons of non-vaccination (52.2% and 33.1% respectively). Finally, the majority of unvaccinated individuals showed a positive attitude towards prospective HPV vaccination, providing they received well-documented advising. CONCLUSIONS: Young women attending Greek higher education exhibit a good level of knowledge about HPV and its correlation with cervical cancer. These data highlight the need for further sensitization of the general population.

Epidemiological and immunological profile of muscle-specific kinase myasthenia gravis in Greece.

OBJECTIVES: The purposes of this study were to determine the epidemiological characteristics of muscle-specific kinase-myasthenia gravis (MuSK-MG) in Greece and the IgG subclass of the anti-MuSK antibodies. METHODS: This population-based study was performed on MuSK-MG patients in Greece between 1 January 1986 and 30 June 2006. Epidemiological and clinical data for 33 patients were collected. In addition, the distribution of anti-MuSK IgG autoantibody subclasses in the sera of 14 patients was determined by immunoprecipitation. RESULTS: The average annual incidence was 0.32 patients/million population/year. On 1st July 2006, there were 33 prevalent cases, giving a point prevalence rate of 2.92/million (women 4.56 and men 1.25). In females, onset of MuSK-MG occurred after the age of 30, whilst, in males, the disease appears in any decade. The female:male incidence ratio was 3.33:1, whilst the prevalence ratio was 3.65:1. Most patients presented with involvement of the facial and bulbar muscles. Amongst about 800 MG patients seropositive for antibodies against either the AChR or MuSK, one patient was found to be seropositive for anti-MuSK antibodies and ambiguous for anti-acetylcholine receptor (anti-AChR) antibodies. The vast majority of anti-MuSK antibodies were IgG4, whilst total IgG4 levels in these patients were similar to those in two healthy controls. CONCLUSIONS: The incidence and prevalence of MuSK-MG in Greece are amongst the highest reported previously for other countries. MuSK-MG in Greece affects both sexes, but mainly females. The main epidemiological indices were calculated. The vast majority of anti-MuSK antibodies were IgG4.

Epidemiology of seropositive myasthenia gravis in Greece.

OBJECTIVES: To study the epidemiological characteristics of myasthenia gravis in Greece. METHODS: A population based study was carried out of seropositive myasthenia gravis in Greece for the period from 1 January 1983 to 30 June 1997; 843 patients were studied. RESULTS: The average annual incidence for the period 1992-7, for which the database is complete, was 7.40/million population/year (women 7.14; men 7.66). On 1 July 1997, there were 740 prevalent cases. The point prevalence rate was 70.63/million (women 81.58; men 59.39). The average overall annual mortality rate in the patients was 0.67/million population (women 0.53; men 0.82), and the mortality rate attributed to myasthenia gravis was 0.43/million population (women 0.41; men 0.45). The average age at onset was 46.50 years (women 40.16; men 54.46), and the mean age of the prevalent patients was 52.58 (women 47.65; men 59.48). The women:men incidence ratio was 1:1.04, and the prevalence ratio was 1.41:1. It is predicted that the prevalence and women: men prevalence ratio would increase if the patient list included all patients with a date of onset before 1983. CONCLUSIONS: The largest epidemiological study ever performed on myasthenia gravis is presented. The most important epidemiological indexes are provided.

Crystal structure of Fab198, an efficient protector of the acetylcholine receptor against myasthenogenic antibodies.

The crystal structure of the Fab fragment of the rat monoclonal antibody 198, with protective activity for the main immunogenic region of the human muscle acetylcholine receptor against the destructive action of myasthenic antibodies, has been determined and refined to 2.8 A resolution by X-ray crystallographic methods. The mouse anti-lysozyme Fab D1.3 was used as a search model in molecular replacement with the AMORE software. The complementarity determining regions (CDR)-L2, CDR-H1 and CDR-H2 belong to canonical groups. Loops CDR-L3, CDR-H2 and CDR-H3, which seem to make a major contribution to binding, were analyzed and residues of potential importance for antigen-binding are examined. The antigen-binding site was found to be a long crescent-shaped crevice. The structure should serve as a model in the rational design of very high affinity humanized mutants of Fab198, appropriate for therapeutic approaches in the model autoimmune disease myasthenia gravis.

Treatment of passively transferred experimental autoimmune myasthenia gravis using papain.

Antibody-mediated acetylcholine receptor (AChR) loss at the neuromuscular junction, the main cause of the symptoms of myasthenia gravis, is induced by bivalent or multivalent antibodies. Passive transfer of experimental autoimmune myasthenia gravis (EAMG) can be induced very efficiently in rats by administration of intact MoAbs directed against the main immunogenic region (MIR) of the AChR, but not by their monovalent Fab fragments. We tested whether papain, which has been used therapeutically in autoimmune and other diseases, is capable of preventing EAMG by in vivo cleavage of the circulating anti-AChR antibodies into Fab fragments. EAMG was induced in 4-week-old female Lewis rats by i.p. injection of anti-MIR mAb35. A total of 0.75 mg of papain was given as one or three injections 3-7 h after MoAb injection. The mAb35 + papain-treated animals developed mild weakness during the first 30 h and subsequently recovered, while all animals that received only mAb35 developed severe myasthenic symptoms and died within 24-30 h. Animals treated only with papain showed no apparent side effects for up to 2 months. Serum anti-AChR levels in mAb35 + papain-treated rats decreased within a few hours, whereas in non-papain-treated rats they remained high for at least 30 h. Muscle AChR in mAb35 + papain-treated animals was partially protected from antibody-mediated degradation. These results show that treatment of rats with papain can prevent passively transferred EAMG without any apparent harm to the animals, and suggest a potential therapeutic use for proteolytic enzymes in myasthenia gravis.

Prevention of passively transferred experimental autoimmune myasthenia gravis by Fab fragments of monoclonal antibodies directed against the main immunogenic region of the acetylcholine receptor.

The muscle acetylcholine receptor loss, responsible for the clinical symptoms of myasthenia gravis, is due mainly to mechanisms dependent on the bivalent character of the anti-receptor antibodies. In cell culture, univalent Fab fragments of monoclonal antibodies (mAbs) directed against the main immunogenic region (MIR) of the acetylcholine receptor are able to protect the receptor against the action of the intact antibodies. To investigate the potential therapeutic use of this approach, we examined the ability of the Fab fragment of anti-MIR mAb195 (Fab195) to protect the receptor in vivo against two anti-MIR mAbs. Because of the rapid clearance of Fab fragments from the circulation, Lewis rats were treated repeatedly with Fab195. The Fab fragment significantly protected muscle receptors against antibody-mediated loss and was very efficient in providing protection against clinical symptoms when its administration was commenced before, simultaneously with, or 2 h after, mAb injection. Twenty-four hours after mAb injection, the protected rats only showed mild myasthenic symptoms, whereas those which only received intact antibodies were moribund or dead. These results suggest that, once modified to ensure their low immunogenicity and a long half-life, anti-MIR Fab fragments might be useful in the specific immunotherapy of myasthenia gravis.

Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor.

The single chain Fv fragment of mAb198 (scFv198) directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR), can efficiently protect the AChR in muscle cell cultures against the destructive activity of human myasthenic autoantibodies. Humanization of the scFv198 antibody fragment should prove useful for therapeutic application by reducing its immunogenicity. Framework sequences from human immunoglobulins homologous to the rat scFv198 sequences were selected and a totally synthetic humanized scFv198 antibody fragment was constructed in vitro. Humanized VH and VL domains were synthesized using two overlapping sets of 225 bases long oligonucleotides overlap extension and polymerase chain reaction (PCR), then assembled into a full-length gene by overlap extension of single-stranded DNA (ssDNA) fragments and PCR. The initial humanized antibody fragment had a very low affinity for the AChR. Molecular modeling was then performed and four residues from the framework regions (FR) of the humanized VH domain were selected to be replaced by the corresponding amino acid from the rat sequence. Three mutants were constructed by overlap extension, using PCR. The humanized variant containing replacements at VH residues 27, 29, 30 and 71 showed very good recovery of AChR binding activity; its binding affinities for Torpedo or human AChR (K(D): 8.5 or 323 nM, respectively) being only four times lower than those of the parental scFv198 (K(D): 2 or 80 nM, respectively). This variant was able to protect the human AChR against the binding of anti-MIR mAb and anti-alpha autoantibodies from a myasthenic patient. It was also able to protect AChR against antigenic modulation induced by the anti-MIR mAb198.

Accumulation of acetylcholine receptors is a necessary condition for normal accumulation of acetylcholinesterase during in vitro neuromuscular synaptogenesis.

To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes cultured in the presence of spinal cord cells from embryonic rat, numerous AChE clusters appeared. Such clusters are always co-localized with AChR clusters, but the reverse is not true: the number of AChR clusters largely exceeds that of AChE clusters. Very few AChE clusters formed when such co-cultures were treated with monoclonal antibodies (mAbs) against the main immunogenic region (MIR) of the AChR, which provoke internalization and degradation of the AChRs of the muscular membrane. The total levels of AChE and proportions of molecular forms were unaffected. We also used non-innervated myotubes in which addition of agrin, a protein normally synthesized by motoneurons, transported to nerve terminals and inserted into the synaptic basal lamina, induces the formation of small clusters of AChE. When added to rat myotubes devoid of membrane AChR, agrin-induced AChE clusters did not form. Finally, we analysed the capacity of the variant of the C2 mouse muscle cell line deficient in AChR (1R-) to form clusters of AChE in co-cultures with spinal cord cells from rat: no formation of AChE clusters could be observed. In all these different systems of cultures, the conditions which prevented clustering of AChR (anti-AChR antibodies, deficiency of the variant C2 cell line) also suppressed AChE clustering. We concluded that clustering of AChR is a prerequisite for clustering of AChE, so that NMJ formation implies the sequential accumulation of these two components.