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Clinical data for cases 2 and 3 are present in Table 1.
On presentation, the dog was depressed, dehydrated, shivering, hypersalivating with blood stained saliva, and coughed spontaneously with haemorrhagic expectorate. The neck was slightly stretched and auscultation of thorax revealed increased vesicular sounds.
Thoracic radiographs showed moderately increased attenuation of the ventral part of the right middle lung lobe, moderately to severely increased attenuation of the ventrocaudal part of the right caudal lung lobe as well as air bronchograms (Figures 4 and 5). These changes are consistent with acute pneumonia. A faint soft-tissue opacity was seen in the lung fissures, interpreted as a possible low amount of free pleural fluid.
The dog was treated with intravenous (IV) ringer acetate 100 ml/kg/hr for eight hours, thereafter 50 ml/kg/hr, enrofloxacin (Baytril, vet. Bayer; 5 mg/kg bodyweight (BW) IV once daily), ampicillin (Pentrexyl, Bristol-Myers Squibb; 35 mg/kg BW IV three times daily) and buprenorfin (Temgesic, Schering-Plough; 0.02 mg/kg BW IV three times daily). All medications were given for four days. The dog was hospitalised in an oxygen cage. Simultaneously as the treatment was initiated an expectorate sample was sent for routine bacteriological cultivation. S. equi subsp. zooepidemicus was isolated in pure culture and directly demonstrated to belong to the Lancefield Group C of streptococci. Complete blood count with serum biochemistry analysis was normal except for a mild leucopenia (Table 2). The coagulation profile was normal.
On the second day of hospitalisation the dog showed substantial clinical improvement and was normothermic (38.1°C), less dyspneic, less tachypneic (respiration rate (RR) = 44/min), and had reduced salivation and coughing. There was no longer blood in the saliva nor epistaxis. The result from the bacteriological investigation of the expectorate demonstrated sensitivity against penicillin, tetracyclin, cefalexin, ampicillin, amoxicillin/clavulanate, enrofloxacin and linkomycin. The antibiotic regimen was switched to phenoxymethylpenicillin (Apocillin; Actavis) 660 mg per os (PO) three times daily for 14 days. No diagnostic tests for respiratory viruses were performed. Clinical signs gradually resolved over the next few days and the dog was sent home seven days after hospitalisation. Control radiographs before departure from the clinic revealed absence of air bronchograms, though a mild to moderate increased attenuation with an interstitial pattern was still present.
This animal was presented to NSVS one day after Case 1. The dog had been coughing for several days and gradually worsened with reduced appetite and depression developing on the day of presentation. On physical exam there was moderate dyspnoea with abdominal respiration and increased vesicular sounds, slight neck extension, blood stained saliva as well as fever (Table 1). Haematology showed moderate leucocytosis due to neutrophilia and monocytosis (Table 2). Radiography of the thorax showed the same changes as described for Case 2.
The dog was hospitalised and medically treated in the same way as for Case 2. Clinical progression was similar to Case 2, with normalisation of temperature (38.8°C), respiration rate (28 breaths/min), heart rate (100 beats/minute) and appetite on the second day of hospitalisation. No salivation or spontaneous coughing was observed unless whilst excited after visiting the exercise pen. Repeat thoracic radiographs on the seventh day of hospitalisation revealed air bronchograms in the right middle lung lobe, but reduced consolidations. The dog was sent home on phenoxymethylpenicillin (Apocillin; Actavis) 660 mg PO three times daily for another 14 days.
Follow-up hospital care of both dogs included radiographs of the thorax after one, three, five and eight weeks (Figures 6 and 7) together with complete blood counts (Table 2). After thoroughly scrutinising the last taken radiographs together with assessing their clinical condition they started a step-wise training program.
The radiographs taken during recovery revealed a very mild interstitial attenuation of the lung lobes that had been most severely affected and mild to faint visualisation of fissure lines which was interpreted as either mild amount of free fluid or mild fibrosis. These minor findings were gradually reduced, but faint fissure lines could still be seen after five weeks for Case 3 and after eight weeks for Case 2 (Figures 6 and 7).
The dogs were kept confined for one week after they were released from the hospital, and did run free on a large (2–3 acres) fenced yard for two weeks. Case 2 was in full training eight weeks post infection and Case 3 was slightly behind.
Early in January the following year both dogs participated in a sled race resulting in a time track record, and medal placements in various championships were achieved the following season.
Some cats with mucopurulent nasal discharge maintain normal appetite and attitude and experience spontaneous resolution of illness within 10 days without antimicrobial treatment. The Working Group recommends that antimicrobial treatment be considered within the 10‐day observation period only if fever, lethargy, or anorexia is present concurrently with mucopurulent nasal discharge.
If antimicrobial treatment is chosen for a cat with acute bacterial URI, the optimal duration of treatment is unknown and so this recommendation is based on experiences of the Working Group members that are clinicians. The Working Group recommends empirical administration of doxycycline (Tables 1 and 2) for 7–10 days to cats with suspected acute bacterial URI as the first‐line antimicrobial option.27, 28 The Working Group believes that doxycycline is a good first choice because it is well tolerated by cats; most B. bronchiseptica isolates from cats are susceptible to doxycycline in vitro (by unapproved standards for testing), despite resistance to other agents such as beta‐lactams and sulfonamides,29, 30, 31 and doxycycline is effective in vivo for the treatment of cats with C. felis infections,27, 32, 33, 34 and Mycoplasma spp. infections.35 Doxycycline is also effective for the treatment of a variety of chlamydial and mycoplasma infections in cats and other mammalian host species. It also has activity against many opportunistic bacterial pathogens that are components of the normal microbiota of the respiratory tract. Of the 17 reviewers, 16 (94%) agreed with this Working Group recommendation and 1 disagreed because there is no breakpoint data for this antimicrobial for B. bronchiseptica or other bacteria in cats and there are no pharmacokinetics, controlled clinical trials, susceptibility data, or pharmacodynamic data on which to base the recommendation.
Due to delayed esophageal transit time for capsules and tablets, cats are prone to drug‐induced esophagitis and resultant esophageal strictures.36, 37 Although any table or capsule could cause this problem, doxycycline hyclate tablets and clindamycin hydrochloride capsules have been reported most frequently to cause problems.38, 39, 40 Thus, tablets and capsules should be given coated with a lubricating substance, followed by water, administered in a pill treat, concurrently with at least 2 mL of a liquid, or followed by a small amount of food.37 Doxycycline formulated and approved for use in cats is available in some countries and should be used if available. The use of compounded suspensions of doxycycline should be avoided because marketing of such formulations is in violation with regulations in some countries, including the USA. In addition, compounded aqueous‐based formulations of doxycycline are associated with a variable loss of activity beyond 7 days.41 Minocycline pharmacokinetics are now available for cats and this tetracycline should be evaluated further for efficacy against infectious disease agents in cats.42
The Working Group considers amoxicillin to be an acceptable alternate first‐line option for the treatment of acute bacterial URI when C. felis and Mycoplasma are not highly suspected. This is based on evidence that cats administered amoxicillin for the treatment of suspected secondary bacterial infections in shelter cats with acute bacterial URI often have apparent clinical responses.20, 43 Cats administered amoxicillin and clavulanate potassium (amoxicillin–clavulanate) had apparent clinical responses in 1 study of shelter cats with acute bacterial URI and so this drug also could be considered as an alternative to doxycycline in regions where a high prevalence of beta‐lactamase‐producing organisms has been identified (eg, based on regional antibiograms).44
In 1 study of shelter cats with suspected bacterial URI, the injectable cephalosporin, cefovecin was inferior to doxycycline or amoxicillin–clavulanate.44 One limitation of this study was the lack of a negative control group.44 Thus, it is the opinion of the Working Group that more evidence is needed before cefovecin can be recommended for the treatment of bacterial URI in cats (Table 2).
The Working Group recommends that treatment of pyothorax include IV fluid administration and critically, drainage of pus after placement of chest tubes with intermittent or preferably continuous suction with or without lavage.96, 97, 98, 99, 100, 101, 102, 103 Surgical debridement might be required in some cases. Sixteen reviewers (94%) agreed, and 1 (6%) disagreed with this Working Group recommendation. The primary comment was that evidence supporting the definitive need for thoracic lavage was lacking. However, based on lack of data supporting its use, the Working Group does not recommend administration of antimicrobial drugs into the pleural space.
The Working Group recommends the combination of parenteral administration of enrofloxacin or marbofloxacin (when available in parenteral form) with a penicillin or clindamycin combined with therapeutic drainage of the pleural space with or without lavage for the initial treatment or canine and feline pyothorax pending the results of culture and antimicrobial susceptibility testing. Sixteen reviewers (94%) agreed and 1 (6%) disagreed with this Working Group recommendation. The primary comment was that pradofloxacin administered PO as a single drug could be effective if available.
Treatment with an antimicrobial drug with activity against anaerobes should be continued regardless of culture results because fastidious anaerobic bacteria could be present. If combination treatment was initiated and the bacterial isolates are susceptible to both drugs in the initial treatment regime, then either of the treatment drugs could be discontinued. If organisms are grown that are resistant to one of the drugs and clinical improvement is not noted, that antimicrobial agent should be discontinued. A second drug to which the isolate is susceptible should be substituted if the animal has not responded sufficiently. If organisms are grown that are resistant to both antimicrobials or clinical evidence of improvement is not evident, antimicrobial treatment should be changed to a drug to which the organisms are susceptible in vitro. Fifteen reviewers (88%) agreed, 1 was neutral (6%), and 1 (6%) disagreed with this Working Group recommendation. The dissenting reviewer stated that mixed culture results can be difficult to interpret and so if the animal's clinical condition improves on the first therapeutic regimen, changes should not be made.
Consultation with a specialist is recommended when multidrug‐resistant organisms are isolated. In all situations, the clinical condition must be considered when interpreting culture results, and continuing apparently effective treatment despite in vitro resistance is recommended because of the potential that the offending organism was not isolated.
Infectious causes of respiratory disease are common in dogs; canine distemper virus, adenovirus 2, parainfluenza, influenza, herpesvirus, pneumovirus, respiratory coronavirus, Bordetella bronchiseptica, various Mycoplasma spp., and Streptococcus equi var. zooepidemicus are documented causes.1 Molecular diagnostic assays to detect viral and bacterial pathogens are available for these agents. In the United States, modified live vaccines (MLVx) for intranasal (IN) administration are currently available for adenovirus 2, B. bronchiseptica, and parainfluenza. These vaccines do not induce sterilizing immunity, and vaccinated dogs can still develop clinical signs of disease if exposed to virulent strains of the organisms.2 It is currently unknown if IN administration of MLVx against these agents results in positive molecular diagnostic assay results in dogs without previous vaccination. If transient positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays to predict disease caused by these agents in dogs would be decreased.
The purpose of this study was to determine the impact of administration of a single IN dose of a commercially available MLVx adenovirus 2, B. bronchiseptica, and parainfluenza containing vaccine,1 included as part of a facility standard initial vaccination series with a parenteral administration of MLVx containing adenovirus 2, canine distemper virus, and parvovirus, on the results of a commercially available polymerase chain reaction (PCR) panel that amplifies the RNA or DNA of the agents.2
The study was completed with Institutional Animal Care and Use approval. Beagle puppies housed at a commercial breeding facility were used.3 The puppies were housed in a closed facility without contact with other dogs and staff members followed facility barrier precautions over the course of the study. A sterile cotton swab was gently rubbed at the entrance to the external nares, and a second swab gently rubbed against the mucosa of the oropharynx in nonsedated puppies. The swabs were stored separately at 4°C in sterile plastic tubes and stored until shipped by overnight express on cold packs for performance of the molecular assays.2
A total of 12 puppies were screened twice as described, 1 week apart, and all were negative for nucleic acids of the target organisms. Eight puppies were randomly selected for the study and housed in a separate room at the breeding facility for the duration of the study. The puppies were approximately 9 weeks of age when samples were collected on Day 0 before the SQ administration of a MLVx containing adenovirus 2, canine distemper virus, and parvovirus4 and the IN administration of a MLVx1 containing adenovirus 2, B. bronchiseptica, and parainfluenza following manufacturer's instructions (approximately ½ mL per nares). Nasal and pharyngeal swabs were then collected on days 1, 2, 3, 4, 5, 6, 7, 10, 14, 17, 21, 24, and 28 for molecular analysis.2
Sneezing or coughing which have been associated with IN MLVx administration was not noted over the course of the study. Adverse effects associated with the collection of the nasal and oropharyngeal swabs were not noted. At the time the study was performed, the PCR panel utilized also included primers for canine distemper virus RNA; and none of the samples collected over the course of the study were positive. In contrast, nucleic acids of adenovirus 2, B. bronchiseptica, and parainfluenza were amplified from both sampling sites, from all 8 puppies, on multiple days after vaccine administration (Table 1). Because adenovirus 2 was administered in both vaccine types, source of that virus cannot be determined. Increasing numbers of positive samples after vaccination suggest local replication of the vaccinal strains. Decreasing numbers of positive samples over time suggest immune responses inhibiting organism replication. However, quantitative PCR assays normalized to total DNA/RNA on the swab would be needed to confirm or deny these hypotheses. The PCR laboratory adheres to standard operating procedures including use of positive and negative controls thus erroneous results are unlikely.
Agents considered most common for kennel cough syndrome include canine distemper virus, adenovirus 2, parainfluenza, and B. bronchiseptica. However, emerging pathogens include influenza, herpesvirus, respiratory coronavirus, pantropic coronavirus, pneumovirus, and others.1 All of these agents, as well as S. equi var. zooepidemicus and Mycoplasma spp., have been identified as causes of canine infectious respiratory disease. Determination of the agent is important for targeting treatment, particularly for dogs who fail to respond to standard treatment recommendations.2 In animal shelter environments, agent identification is critical for outbreak control and individual case management.3
Bacterial and viral shedding postvaccine administration complicates diagnostic testing and treatment. This is especially problematic in shelter environments as dogs are routinely vaccinated on intake. Viral shedding after vaccination has been detected in cats,4 people,5 cattle,6 pigs,7 and dogs.8 A vaccine strain of B. bronchiseptica was detected via nasal culture up to 4 weeks after IN vaccination of 2–week‐old puppies.9
Commercially available respiratory PCR panels are a relatively cost and time effective diagnostic method for identifying multiple respiratory pathogens. However, amplification of nucleic acids may inherently lead to inaccurate clinical diagnosis because small amounts can be amplified from some animals even though the agent may not be present in sufficient quantity to cause disease. In this study, nucleic acids of all 3 organisms contained in the IN vaccine were amplified from both sites on multiple days via PCR, although no clinical signs of respiratory disease were observed. Thus, interpretation of PCR panel results for diagnoses should include consideration of recent vaccination status and clinical signs of disease. Use of quantitative PCR and wild‐type sequence differences may be able to differentiate between vaccine and pathogenic agent shedding and may be used diagnostically in the future.
Real‐time reverse transcriptase PCR has been used to amplify canine distemper virus RNA in blood, urine, and conjunctival swabs after administration of SQ MLVx.10 In this study, the PCR panel did not amplify distemper virus RNA from nasal or pharyngeal swabs. Further studies are needed to determine whether the negative result is because this strain of vaccine virus does not reach the nasal or pharyngeal tissues or was present at levels below the detectable limit of the assay used.
A 76-year-old Caucasian man who underwent laryngectomy 10 years earlier, presented with fever (38.9 °C; 102.0 °F), increased sputum production, and purulent conjunctivitis. These symptoms emerged gradually over a period of 48 hours. He noted increasing difficulty in coughing out his sputum that became brownish and viscous. He had been wearing a heat and moisture exchanger (HME) filter that covered his stoma and spoke through a tracheoesophageal voice prosthesis. The symptoms started a day after a very cold weather spell with temperatures of −7 to −1 °C (19–31 °F). He had to remove his HME on several occasions for extended periods of time to enable him to breathe when he walked outside his home.
His past medical history included hypopharyngeal squamous cell carcinoma which was treated with intensity-modulated radiotherapy (IMRT) 12 years earlier. A recurrence of the cancer 2 years later required laryngectomy. He had no signs of tumor recurrence since then. He also suffered from paroxysmal hypertension, diverticulitis, and migraines.
He was vaccinated with the current Influenza virus vaccine 3 month earlier. He had also received a pneumococcal polysaccharide vaccine (PPSV23) 2 years earlier.
He was in mild respiratory distress especially when coughing. He had coughing spells and expectorated green-brown dry and viscous sputum. A physical examination revealed bilateral purulent conjunctivitis and auscultation of his lungs revealed coarse rhonchi and no crepitations. No lymphadenopathy was noted. The results of the rest of the physical and neurological examinations were within normal limits. A chest X-ray was normal.
Sputum and conjunctival culture grew heavy growth of beta-lactamase-producing nontypeable Haemophilus influenzae (NTHi) that was susceptible to levofloxacin and amoxicillin- clavulanate. A FilmArray® Respiratory Panel 2 (RP2) polymerase chain reaction (PCR) system test did not detect 14 viruses (adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, human rhinovirus/enterovirus, human metapneumovirus, influenza A, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus) and four bacteria (Bordetella pertussis, Bordetella parapertussis, Chlamydophila pneumoniae, Mycoplasma pneumoniae).
He was treated with orally administered levofloxacin 500 mg/day, ciprofloxacin eye drops, acetaminophen, and guaifenesin. Humidification of his trachea and the airway was maintained by repeated insertions of 3–5 cc respiratory saline into the stoma at least once an hour and by breathing humidified air.
The main challenge was to maintain a patent airway as the mucus was very dry and viscous and tended to stick to the walls of his trachea and the stoma. The mucus had to be repeatedly expectorated by vigorous coughing and by manual removal from the upper part of his trachea and stoma.
He experienced repeated episodes of sustained elevated blood pressure (up to 210/110) and tachycardia (112/minute). This was managed by administration of clonidine 0.1 mg as needed (1–2/day).
His fever started to decline 48 hours after antimicrobial therapy was started. The conjunctivitis improved within 36 hours. The sputum production declined and became less viscous over time, but persisted for 5 days.
Antimicrobial therapy was discontinued after 7 days.
His condition improved and he had a complete recovery in 7 days. He was seen in the clinic every 2 months and showed no recurrence of his infection for the following 8 months. He received vaccination for H. influenzae B and Prevnar 13® (pneumococcal conjugate vaccine; PCV13) 4 weeks after his recovery.
Reflecting the clinical impact compared to other CARVs, the working group distinguishes the need of treatment for influenza A and B, RSV and HPIV, taking into account the higher risk for poor outcome in specific patient groups. The treatment of RSV and HPIV may involve the deferral of conditioning therapy, treatment with aerosolized ribavirin, or off-label use of systemic ribavirin, whereas no general recommendations for other CARVs can be made at this time (Table 5).
The corresponding modalities of RSV therapy and systemic ribavirin are summarized in Tables 6 and 7, respectively. The working group is cautious about the use of intravenous monoclonal antibody specific for the RSV-F protein, because existing data outside of single case reports do not support its beneficial effect and the cost is very high. Therefore, only very young (age <2 years) allogeneic HSCT patients with LRTID or at high risk for progression to RSV LRTID might be considered for treatment with intravenous monoclonal antibody specific for the RSV-F protein (eg, palivizumab 15 mg/kg body weight) (CIII; Supplementary Table 1), while this drug should not be considered in other patient groups.
Withholding treatment for RSV infection might be considered for selected stable leukemia and HSCT patients after careful evaluation of risk factors for morbidity and mortality and the possibility of appropriate follow-up visits considering, for example, remission of underlying disease, absence of immunosuppressive drug treatment, absence of the risk factors associated with LRTID, or mortality (CIII). Although some centers would treat patients with HPIV URTID and risk factors listed in Table 3, treatment of HPIV URTID is not generally recommended given the clinically undefined risk and benefit ratio (CIII).
Overall, the evidence is more limited for patients with autologous HSCT and/or hemato-oncological disease.
Infection control measures should be applied to patients undergoing autologous HSCT or chemotherapy for hemato-oncological diseases with CARV URTID or LRTID (BIII).
Deferral of conditioning/chemotherapy should be considered for patients with CARV-RTID scheduled for autologous HSCT or chemotherapy for hemato-oncological diseases (BIII). Treatment of CARV RTID other than influenza is not generally recommended for patients undergoing autologous HSCT or chemotherapy for hemato-oncological diseases (CIII).
In general, the APN receptor is used by alphacoronaviruses in a species-specific manner, that is, human APN is the cellular receptor for HCoV-229E, but not for the porcine coronaviruses, and conversely, porcine APN serves as a receptor for the porcine coronaviruses, but not for HCoV-229, FCoV, or CCoV [104, 105]. However, feline APN is a functional receptor for many alphacoronaviruses, including feline (FECV and FIPV), human (HCoV-229E), porcine (TGEV), and canine coronaviruses. Human, feline, and porcine APN show strong amino acid conservation and display about 78% identity. Yet, species-specific tropism is influenced by minor differences in certain regions of APN. Chimeras of mouse-feline APN were used by Tusell et al. to identify the three small, discontinuous regions in feline APN that are critical determinants for the host range of these coronaviruses. Amino acids (aa) 288 to 290 are essential for the entry of HCoV-229E, particularly the presence of an N-glycosylation sequon prevents virus infection. TGEV requires the region corresponding to aa 732 to 746 of feline APN, while FCoV and CCoV necessitate both aa 732 to 746 and aa 764 to 788 for entry. The entry of all of these viruses is blocked by the same monoclonal antibody directed against feline APN, suggesting that these three regions are closely link together in the three dimensional structure of feline APN. HCoV-229E, FCoV, TGEV, and CCoV probably evolved from the same ancestral alphacoronavirus, which may have infected cats using feline APN. The selection of mutations in the S protein may then have led to the appearance of viruses able to infect other host species by means of their cognate APN proteins, although all of them retained their capacity to use feline APN as a receptor in vitro.
The pre-publication history for this paper can be accessed here:
The study was approved by The First Affiliated Hospital of Guangzhou Medical University Ethics Committee for research on human beings, and all participants or their guardians gave signed informed consent for participation in the study.
Canine infectious respiratory disease complex (CIRDC) is a major cause of respiratory illness and morbidity. It is a complex condition for disease occurrence, involving multifactorial etiologies. Overcrowding, stress, age and other underlying factors result in interference with the dog's immune response and play a role in disease predisposition [2, 3, 4, 5, 6]. Infectious agents serve as a key factor in promoting the severity of a clinical symptom. The CIRD viruses (CIRDVs) that are commonly associated with CIRDC are canine parainfluenza (CPIV), canine distemper (CDV), canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CaHV-1), canine respiratory coronavirus (CRCoV), and canine influenza virus (CIV) [1, 7, 8, 9].
During the last decade, novel viruses, such as canine pneumovirus (CnPnV) [10, 11] and pantropic canine coronavirus, which induce respiratory problems in dogs, have also been discovered. In addition, canine bocavirus [13, 14], canine hepacivirus and canine circovirus have been detected in respiratory tract samples of dogs showing respiratory illness, but the pathogenic roles of those viruses are poorly determined. Although new viruses have emerged, the common CIRDVs are still important contributors to respiratory disease [17, 18]. Additionally, current core vaccines that are routinely used in dogs prevent some CIRDVs, such as CPIV, CDV, and CAdV-2, but not CaHV-1, CRCoV and CIV. This lack of a vaccine for CaHV-1, CRCoV, and CIV is likely to allow the spread of infection.
Hospital-associated infection (HAI) has recently been described as an “ever-present risk”. The current literature includes reported outbreaks of CPIV and CaHV-1 in animal healthcare facilities, where these nosocomial infections worsened any ongoing disease and enhanced the morbidity and mortality rates. In addition, infections among communities are documented and serve as a source for disease dissemination. Community-acquired infections (CAIs) are those that are acquired without exposure to hospitals or with limited regular exposure with a health care center or both. Currently, CAIs with CIRDVs are believed to have a concomitant effect in respiratory disease [1, 21, 22, 23, 24].
The identification of multiple-viral-induced CIRDC involves the simultaneous rapid nucleic acid-based detection, typically by the reverse transcription polymerase chain reaction (RT-PCR) for RNA viruses and PCR for DNA viruses. These detection methods have led to an increased and better realization of the prevalence of CIRDC worldwide [3, 25, 26]. However, whether the severity of the respiratory disease depends on the number of viral infections is equivocal. Although epidemiological evidence of CIRDC has been reported periodically, the classification among HAIs and CAIs has largely not been evaluated [7, 24].
A comprehensive understanding of the source of infection (HA or CA) and other associated risk factors may help guide the successful implementation of preventive strategies. To emphasize this point, this study focused on the associations between the incidence of all six common CIRDVs, source of infection and the possible related risk factors of the dog's age, sex and vaccine status. Moreover, the relationship between clinical severity and number of viral infections was also evaluated in respiratory-ill dogs during 2013–2016 in Thailand.
To balance costs and clinical benefit, screening all patients for CARVs is currently not indicated unless indicated in the context of an infection control investigation of nosocomial transmission and prevention, and thus laboratory testing should focus on symptomatic patients (Table 4). Taking into account the clinical impact of CARVs in HSCT and leukemia patients and the differences among centers in the technical and financial resources for comprehensive CARV diagnostics by multiplex NAT, the working group recommends prioritizing laboratory tests for specific CARVs such as influenza, RSV, and HPIV (Table 4).
For detection of antibodies against betacoronaviruses, 30 acute serum samples as well as the corresponding sera (obtained 2 to 3 weeks later) of the convalescent dogs were examined by an indirect immunofluorescence test. Madin‐Darby bovine kidney cells were disseminated on 96‐well microtitre plates (100 μL/well) and then incubated at 37°C in a humid 5% CO2‐atmosphere overnight. After washing the plates with phosphate‐buffered saline (PBS) solution, the adherent cells were infected with BCoV strain 15317/82 and incubated at 37°C for 48 hours. Subsequently cells were washed with PBS again and fixed with 100 mL of 96% ethanol.
The sera underwent twofold serial dilutions from 1:20 to 1:5120 with PBS and immunofluorescence test was performed as follows:
Ethanol was discharged, and the 96‐well microtitre plates were washed three times with PBS; 50 μL of the previously diluted sera per well were added and incubated at 37°C for 30 minutes. Thereafter, the plates were washed three times with PBS and 50 μL of 1:40 diluted fluorescein isothiocyanate (FITC)‐conjugate (anti‐dog IgG, Jackson) was added to each well. After incubation at 37°C for another 30 minutes and three washing cycles with PBS, 50 μL/well Eriochrome black T indicator (diluted 1:200 with PBS) was filled in each well of the 96‐well microtitre plate to reduce background fluorescence. Plates incubated for 5 minutes at room temperature before cells were washed three times with PBS once more. Finally, wells were filled with 50 μL/well of glycerine buffer solution to prevent the cells from drying.
For evaluation of the microtitre plates an inverse ultraviolet microscope was used. The highest dilution with a clear cytoplasmatic fluorescence was equivalent to the specific antibody titre of each serum sample. Samples that showed no fluorescence in dilution 1:20 were regarded as negative (no antibodies present). Each assay included a positive and a negative control serum.
Focussing on upper respiratory tract samples (nasal and tonsillar swabs), viral nucleic acids were detected in 31 of 214 diseased dogs (14.5%). Sixteen dogs tested positive for CRCoV (7.5%), 14 dogs for CPiV (6.5%) and one of these dogs additionally for CAV‐2‐specific nucleic acid (0.5%). One single dog tested positive for CDV‐specific nucleic acid (0.5%). In none of the obtained samples from the upper respiratory tract was CIV‐specific nucleic acid detected. Of those 31 positive dogs, 21 were privately owned (group A), and 10 kept in shelters (group B). They consisted of five puppies, 12 adolescent dogs and 14 adult dogs. Twenty‐seven of the 31 positive dogs (87.1%) showed acute onset of signs, three suffered from chronic disease (9.7%) and for one diseased dog this information was not available (Table 2).
Furthermore, upper respiratory tract samples from two dogs (4.0%) of the clinically healthy control group C tested positive for CRCoV‐specific nucleic acid (Table 2).
Nine dogs from group A (5.2%) and seven dogs out of group B (17.0%) tested positive for CRCoV in either nasal, tonsillar or both samples at one time. One of these dogs belonged to the subgroup of puppies; nine dogs were from the adolescent subgroup and six animals from the subgroup of adult dogs. With one exception, all these animals showed acute onset of CIRD (93.7%).
Fourteen diseased dogs (6.5%) tested positive for CPiV. From those, 11 belonged to group A and three to group B. They all harboured CPiV‐specific nucleic acid in sample material from the nose and one dog concurrently from the tonsils. Four of these dogs were classified as puppies; three dogs were from the adolescent subgroup and seven dogs were adults. Twelve out of these 14 animals showed acute onset of clinical signs (85.7%), one dog was chronically ill, and for another dog this information was not available. Seven dogs (50.0%) were regularly vaccinated‐including against CPiV.
In one of these 14 CPiV‐positive dogs, CAV‐specific nucleic acid was detected concurrently. This dog was privately owned (group A) and tested positive for CAV in both nasal and tonsillar swabs and CAV‐2 strain (Toronto) was confirmed by DNA sequencing. Belonging to the subgroup of adults this dog had been irregularly vaccinated and received its latest vaccine 45 days before sample collection. It presented with a several week history of clinical signs including severe coughing, nasal and ocular discharge, dyspnoea and fever. Apart from that case, in no other dog was viral nucleic acid of two or more different viruses detected. In addition, no other proband of the study tested positive for CAV.
One dog from group A tested positive for CDV‐specific nucleic acid in a sample retrieved from the tonsils. RNA sequencing enabled the identification of a CDV vaccine strain (Onderstepoort). The dog was an adult and presented with chronic respiratory disease but no other signs consistent with CDV infection. The vaccination status of this dog was unknown.
Additional information regarding all PCR‐positive dogs is summarised in Table 3.
All BALF samples collected from 31 chronically ill dogs revealed negative PCR results.
Patient characteristics stratified by age group are presented in Table 1.
Children under two years of age were admitted to the hospital significantly earlier after the onset of symptoms than older children, (p = 0.01, two-sample t-test). 61% of the children were discharged from hospital on the day of admission.
The most common respiratory manifestation was a cough (100%) together with an age-depending degree of wheezing (Table 1). At admission to the hospital, 92% of infants and children were diagnosed with an episode of troublesome lung symptoms lasting at least three days. Significantly more young children (less than three years of age) had objective wheezing and cough (asthma-like symptoms) than older children (p = 0.01, Z-score test). Previous and clinically significant chronic disease was diagnosed in 10% of the children. Of these 4/13 (31%) had previously diagnosed and current asthma, and had a severe exacerbation during the M. pneumoniae infection.
The majority of the patients (84%) had a chest x-ray taken, and 96% of these had positive radiological findings (Table 1). Among infants and young children, exclusive hilar adenopathy was more frequent, while older children usually had significant peripheral infiltration on the chest X-ray (Table 1). Children with atelectasis had a significantly longer duration of hospital stay; more than three days (35% versus 25%; p = 0.05). The rate of pulmonary complications was the same for children with CRP over 50 mg/l (POCT(Point of Care Testing), part of an adult definition of significant pneumonia) as below 50 mg/l (18% versus 19%, p > 0.05). No pulmonary complications were reported in the under two age-group. The number of severe manifestations of pneumonia was equal in the age-groups; 2–6 years and 7–15 years (Tabel 1). Overall 20% of the children had an increased CRP level of more than 50 mg/l, and they were all older than three years of age.
A total of 120 children received antibiotic treatment. The majority were treated with clarithromycin according to the local Guideline. Sixty-four patients, or 46%, were treated upon suspicion. Out of these, 53% had received other antibiotics (beta-lactam) prior to M. pneumoniae testing. Fifty-six patients (42%) were started on treatment upon receiving the positive test result. Only six children were treated with macrolide antibiotics twice due to suspicion of recurrence or treatment failure. One sample was tested for macrolide resistance and found negative.
The most common extra-pulmonary manifestation was nausea, with or without vomiting, reported by a third of all children. 23% of all children had some type of rash, and 9% had hives. In infants, there were skin manifestations in 33% of the cases. Two children developed Steven Johnson syndrome (SJS) with mucosal symptoms arising prior to or at the same time as the antibiotic treatment was started.
A total of 37 children had simultaneously been tested with sputum-culture for other bacterial pathogens. In 41% of these, a co-infection was diagnosed. The most common bacteria were Moraxella catarrhalis, Haemophilus influenzae and S. pneumonia. Due to methodological setup, all children were tested for Chlamydophila pneumoniae none were found positive.
Only two children were tested for viral infections during the clinical setup. The post-hoc analyses of 49 oropharyngeal-swabs showed that 27% of these had a single or mixed viral co-infection (RSV (1 child), influenza A (2), human metapneumovirus (1), rhinovirus (2), coronavirus (3), bocavirus (2) and adenovirus (5)). Four children were PCR positive for two viruses as well as for M. pneumoniae. Table 2 shows, in a similar matter as Table 1, the clinical characteristics of children with, without and of unknown viral infection. The data suggest that significantly more children with mixed infection of M. pneumoniae and a respiratory virus had rhinorrhoea (p = 0.02), and were wheezing (tendency, p = 0.07), compared to those who were only positive for M. pneumoniae. We could not identify other differences between the two groups, including no radiological discrepancies.
Acute respiratory infections are highly prevalent in the population, with the common cold, flu-like syndromes, tracheobronchitis, sinusitis, laryngitis and pneumonias being particularly important. When the aetiology is presumably bacterial, treatment is based on the use of antibiotics and medication for symptomatic relief. However, the most common clinical entities, the common cold and the flu-like syndrome, have a viral aetiology, for which symptomatic treatment remains, in most cases, the standard recommendation.
The common cold is the most frequently encountered disease in medical practice. It affects most adults, on average two to four times a year, and accounts for up to 40% of work absences among the economically active population in the United States.
This syndrome affects the upper airways, sometimes in association with low-grade fever and systemic symptoms, and usually presents with at least two of the following symptoms: cough, dysphonia, throat discomfort, sore throat, nasal congestion, rhinorrhoea, sneezing, headaches, myalgia and fever. Symptoms usually peak at 2 to 3 days and have a mean duration of 7 to 10 days. This definition has been prospectively validated in other studies and is that most frequently used in clinical studies of patients with the common cold. Although most cases are caused by the rhinovirus, other agents may be involved, such as the respiratory syncytial virus, adenovirus, coronavirus, and influenza and parainfluenza viruses.
Flu-like syndrome is characterized by sudden onset of fever, headache, cough, sore throat, myalgia, nasal congestion, weakness and loss of appetite. Complications, such as pneumonia, otitis and sinusitis, may occur.
Some of the medications studied for the treatment of the common cold are antihistamines, anticholinergics, alpha-adrenergic agonists, membrane stabilizers, nonsteroidal anti-inflammatory drugs, vitamin C, glucocorticoids, zinc, herbal medications and alpha-interferon. Clinical trials of high-dose vitamin C have not found any benefits in the treatment of the common cold. Recent studies of Echinacea purpurea, a herbal medicine, did not find any clinical benefits for patients with the common cold. The results of meta-analyses about the efficacy of zinc are contradictory, and there is weak evidence of its benefit in well-designed studies.
The symptomatic treatment of the common cold has been evaluated in Cochrane meta-analyses. The first included 32 studies with a total of 8930 patients and investigated the administration of antihistamines in the common cold. Results showed that monotherapy did not improve symptoms in either children or adults. The combined use of antihistamines and decongestants may alleviate symptoms in adults, but results are heterogeneous. Another meta-analysis investigated the use of nasal decongestants in the common cold in 286 adults, and found no benefit for the relief of nasal congestion. Another recent meta-analysis suggests that a triple combination of antihistamine, decongestant and analgesic provided some general benefit in adults and older children.
Most cases of influenza require the use of drugs for symptomatic relief. In clinical practice, treatment directed to the aetiological agent is not routine, but became considerably more frequent after the H1N1 pandemic in 2009.
Therefore, patients continue to use medications that produce symptomatic relief because of the epidemiological relevance and the intensity of symptoms of flu-like syndromes and the common cold.
This study evaluated the efficacy and safety of a fixed-dose combination of paracetamol, chlorphenamine and phenylephrine in the symptomatic treatment of the common cold and flu-like syndromes by analysing symptom score reductions during and after treatment, duration of symptoms, return to usual activities and adverse events in both groups.
No other randomized clinical trial (RCT) evaluated the safety and efficacy of this specific fixed-dose combination for the common cold in adults. This was the first RCT conducted in Brazil designed to also assess adverse cardiovascular effects of phenylephrine.
Three different CNV strains with maximum sequence difference were selected for VLP production. The sequences of the three CNV-VP1 genes were obtained from GenBank (table 1) and restriction enzymes sites designed (5′ NotI for all strains, 3′ BbsI for CNV strains 170, C33 and 3′ BsaI for CNV HK) to enable later ligation into the baculovirus transfer vector pTriex1.1. Sequences were synthesized by BioBasic Inc. in the vector pUC57. CNV-VP1 sequences were digested from pUC57 and re-ligated into pTriex1.1 that had been digested with NcoI and NotI (NEB). The correct sequence for all three CNV-VP1 inserts was confirmed by sequencing.
Recombinant baculoviruses were generated using the flash BAC baculovirus expression system as per the manufacturers instructions (Oxford Expression Technologies). Stock viruses were generated and titrated in Sf9 cells and stored in the dark at 4°C. Protein expression was performed in Hi5 insect cells (Invitrogen). Briefly, 1×107 Hi5 insect cells were seeded into 10×T150 flasks then infected with recombinant baculovirus at a multiplicity of infection of 5 pfu/cell. Infections were allowed to proceed for 6 days prior to protein harvest and VLP purification. VLP purification was performed essential as described. VLP was released from infected Hi5 cells by freeze-thaw, followed by clarification to remove cellular debris (6000×g, 30 minutes) then baculovirus removal (14,000×g for 30 mins). VLPs were partially purified through a 30% w/v sucrose cushion in TNC buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 10 mM CaCl2) containing the protease inhibitor leupeptin for 150,000×g for 2 hrs. The pelleted VLP was resuspended in TNC and further purified by isopynic centrifugation in caesium chloride (150,000×g, 18 hrs). The resultant VLP bands were collected by puncture and the solution containing VLPs was dialysed against PBS prior to quantification by BCA protein assay (Thermo Scientific) and storage at −80°C.
Table 2 summarizes the common applications of aerosol therapy in critical care.
Aerosolized bronchodilators and corticosteroids have been effectively utilized in critical care. Aerosolized antibiotics are quickly gaining more data to support their position in the critical care armamentarium. With improvements in drug formulation and delivery devices, more is now known about the optimal conditions required for effective aerosolized therapy as summarized in Fig. 2.
Despite these developments there are concerns that best evidence for administration is not being applied, particularly for aerosolized antibiotic therapy [118, 119]. Clinical and experimental study data for aminoglycosides and colistin are perhaps most numerous for antibiotics in critical care. Aminoglycosides are concentration-dependent antibiotics whereby the bactericidal effect is best described by the Cmax/MIC ratio. Studies have shown that intravenous aminoglycosides penetrate poorly into the epithelial lining fluid [48, 120]. In an Escherichia coli inoculation pneumonia model, aerosolized amikacin was seen to achieve significant lung concentrations. Figure 4 is an illustration of this phenomenon. On the other hand, with repeated administration, there was no accumulation effect and hence no toxicity concerns with aerosolized amikacin. In experimental studies, the serum concentration of amikacin was higher when aerosolized amikacin was used in a pneumonia model compared with that of healthy lungs. Moreover, a combination of intravenous and aerosolized aminoglycosides has not been shown to increase cure rates compared with that of aerosolized antibiotic alone. Thus, for the treatment of ventilator-associated pneumonia, aerosol therapy alone may be adequate without the need for intravenous therapy, decreasing the risk of systemic toxicity.
Colistin, also a concentration-dependent antibiotic, is another antibiotic used widely in aerosolized form. Colistin aerosolization is not approved by the FDA and is not licensed for human use in China. Like aminoglycosides, colistin has poor lung penetration when given intravenously. Experimental studies have shown that a rapid and high bactericidal effect can be achieved with aerosolized colistin. Figure 5 illustrates this phenomenon. As demonstrated by Lu et al., with low serum concentrations resulting from aerosolized colistin in an inoculation pneumonia model, the risk of toxicity is minimal. In a prospective observational study, Lu et al. demonstrated similar clinical cure for patients with VAP where susceptible P. aeruginosa or A. baumannii were treated with only intravenous colistin and MDR strains were treated with nebulized colistin. Combined intravenous aminoglycoside and aerosolized colistin has not been shown to be superior to aerosolized colistin alone although implemented worldwide. The benefit from the use of aerosolized colistin instead of systemic colistin is to avoid nephrotoxicity, and this was further confirmed in one randomized clinical trial.
Acute respiratory infections (ARI) result in the death of an estimated 4 to 5 million children each year in developing countries–. Most of these deaths are among children with pneumonia. Respiratory tract infection etiology is complex and diverse. In developed counties, the major causes of ARI in children and adults are influenza A and B viruses (infA, infB), parainfluenza virus type 1 (PIV1), PIV2, PIV3, respiratory syncytial virus (RSV), adenovirus (ADV) and rhinovirus–. However, there is a lack of data on the characteristics of ARI in developing nations. Over the past decade, a number of new pathogens have been reported, including human metapneumovirus (HMPV) and human bocaviurs (HBoV), thus increasing the urgency for the study of epidemiology of respiratory tract pathogen infections in developing countries.
Over the past two decades, virus isolation and serology have been the mainstay of clinical laboratory diagnosis for respiratory virus infections. The introduction of molecular-based detection methods has made diagnosis quicker and cheaper and increased the ability to detect more than one virus simultaneously. In this work, the epidemiological features of 17 respiratory pathogens in children with ARI were studied in Guangzhou, southern China. This study should serve as a valuable resource for information on ARI and provide useful data for future research and development of vaccines.
Aerosol therapy provides effective drug delivery in the critically ill patient. Careful consideration of the various elements that affect pharmacological effect of aerosolized therapies is essential to derive optimal therapeutic benefit. Effective drug delivery alone does not ensure successful aerosol drug therapy. It is crucial that the drug in its aerosolized form should have efficacy in the specific disease condition to derive clinical benefit.
Good quality data and clinical experience support use of bronchodilators such as salbutamol, anti-infectives such as tobramycin, aztreonam and colistin, and anti-inflammatory agents such as budesonide. Although with application of principles it is possible to provide aerosol drug delivery, the effectiveness of the therapy in disease conditions is yet to be proven. This is because there is a scarcity of high-quality trial-based data in this area to quantify how effective these agents are in the critically ill patient.
Given the challenges of effective treatment of the critically ill patient, it is necessary to optimize as many factors as possible for effective drug delivery. Hence, it is important that guidelines for aerosol therapy are developed. It is envisaged that as the technologies become mature through rigorous evaluation, a diverse range of aerosol therapies with unique advantages (i.e. controlled release/sustained release or direct targeting) and or for specific indications may be possible.
Canine infectious respiratory disease (CIRD), also known as “Kennel cough”, is an endemic syndrome with multiple viral and bacterial pathogens being involved in disease causation. CIRD is most common when dogs are kept in large groups with continuous intake of new animals, particularly in kennels, but also occurs in singly housed pets. Clusters of infection have also been documented in veterinary hospitals. Common clinical signs include nasal discharge, coughing, respiratory distress, fever, lethargy and lower respiratory tract infections [1, 3–5]. The clinical signs caused by the different pathogens associated with this syndrome are similar, which makes differential diagnosis challenging. Vaccination plays an important role in managing CIRD, and as such, several mono and multivalent vaccines are available; however, despite the widespread use of vaccines to prevent CIRD, clinical disease is still common in vaccinated dogs [2, 6]. Vaccines are commercially available for some, but not all pathogens, which may explain the occasional lack of protection.
The complex multifactorial etiology of this disease involves the traditional CIRD viral and bacterial agents, canine parainfluenza virus (CPIV), canine adenovirus (CAV), canine distemper virus (CDV), canine herpesvirus (CHV), and Bordetella bronchiseptica. New or emerging microorganisms associated with CIRD include canine influenza virus (CIV), canine respiratory coronavirus (CRCov), Mycoplasma cynos and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus). Other novel canine respiratory agents include canine pneumovirus, canine bocavirus, canine hepacivirus [17, 18] and canine picornavirus. There is debate on whether these are truly new emerging pathogens or pre-existing pathogens that are now easier to detect due to the advent of sophisticated molecular diagnostic tools and more frequent diagnostic testing. In recent years, the role of other bacterial agents such as Mycoplasma canis has been questioned [13, 20]. It is unknown whether certain Mycoplasma species such as M. canis act as a commensal, primary or secondary agent.
The detection of co-infections of CIRD pathogens in a single dog has been previously documented [2, 12, 20]. It is most likely that a single pathogen alters the protective defense mechanisms of the respiratory tract, thereby allowing additional pathogens to infect the respiratory tissues. The presence of co-infections may increase disease severity compared with single pathogen infections [2, 5, 20]; however, the prevalence and role of co-infections in CIRD causation remain unclear.
Previous epidemiologic studies of CIRD pathogens in the United States have focused on asymptomatic dogs or on specific pathogens implicated in clinical cases [11, 22, 23]; therefore, a comprehensive etiologic and epidemiologic study involving multiple CIRD agents in a diverse population of dogs has not yet been reported. Understanding disease prevalence facilitates the improvement or establishment of new vaccination programs and alternative treatments. To aid in addressing this question, we conducted a disease surveillance study using molecular methods to detect nine pathogens currently known to be involved in CIRD using samples from symptomatic and asymptomatic dogs that were received at a veterinary diagnostic laboratory. The aim was to attain information regarding pathogen occurrence according to age, seasonality, sex, clinical signs, and vaccination history. This study also aimed to evaluate the role of co-infections in disease severity, and to develop a novel probe-based multiplex real-time PCR assay to simultaneously detect and differentiate M. cynos and M. canis.
We present data from a large cohort of children with M. pneumoniae infection. All children enrolled were referred from the primary healthcare system to hospitalisation due to character and severity of symptoms. The majority of M. pneumoniae PCR test-positive children had LRTI, which we confirmed by a high rate of radiological findings (94%).
We found a higher rate of positive samples in the later wave of the epidemic in 2010 and 2011. School-aged children were more often M. pneumoniae positive (65%) than younger children, but even amongst the 2 to 6-year-old children 30% were M. pneumoniae positive substantiating our initial suspicion that M. pneumoniae also affects small children. Even a small number of infants; 6 out of 276 were diagnosed.
This study was conducted as a retrospective chart study. The doctor on call decided whom to test for M. pneumoniae. Children with M. pneumoniae infection might have been under-diagnosed if they had minor respiratory symptoms especially during the first wave of the epidemic period. Due to commonly held concepts of CAP epidemiology, originally based on a long-term study conducted in primary care from 1963–1975, we expect infants and young children to be under-diagnosed due to selection-bias.
Even very young children can become ill from M. pneumoniae even though it is less common. The differential diagnosis of respiratory viral infections and exacerbation of asthma-like symptoms must be considered. The clinical presentation with a cough, wheezing, low-grade-fever, CRP below 50 mg/L and rhonchi on auscultation in 33% of the youngest children can also be considered as a childhood asthma-like exacerbation, primarily due to viral infection in pre-school children. Indeed, we also had a minor degree of mixed viral co-infections discovered in our post-hoc analysis. It can only be speculated in what pathogen was the primary cause of disease in these cases. Due to our post-hoc findings, we would advise that small children with wheezing and rhinorrhoea should be tested for both M. pneumoniae and respiratory viral infections simultaneously. During the Norwegian M. pneumoniae epidemic, Inchly et al. described a similar relative number of viral co-infections.
In a Dutch childhood study of carriage of M. pneumoniae in the upper respiratory tract (URT), season and year of enrolment affected the prevalence of asymptomatic carries ranging from 3% to 52%. In our study, some of the children discharged from the ward on the same day as admitted to the hospital could have been carriers of M. pneumoniae. However, several of these children were treated with first-line antibiotics, prior to admission, and referred to our department because of insufficient response to beta-lactam antibiotic management.
Kroppi et al. found that 50% of children with LRTI caused by M. pneumoniae were co-infected with primarily S. pneumoniae or Chlamydiae spp. Only a small part of this cohort was tested for bacterial co-infection, but we did not regard this as a major problem. Again, it is noteworthy that 59% of the children had been treated with a beta-lactam antibiotic before examination for M. pneumoniae without improvement of the infection.
In parts of the same epidemic period in Denmark (2010–2011), Stockholm et al. identified an effect of azithromycin (a macrolide antibiotic) on episode duration of asthma-like symptoms in young children. No investigations for M. pneumoniae were done, and exclusion criteria of respiratory rate over 50/min, temperature over 39°C and CRP over 50 mg/L would not exclude all children with a possible M. pneumoniae infection [19–21]. Two recently published Norwegian studies described the discrepancy of the incidence of clinical symptomatic M. pneumoniae infections in preschool children between epidemic and endemic periods. Randomised Controlled Trials concerning the efficacy of macrolides on asthma-like symptoms should be conducted in endemic periods or better controlled for M. pneumoniae infections, especially in young children born after an epidemic period. The anti-inflammatory effect of macrolides still has to be further addressed.
10% of this cohort was affected by chronic illness, mainly respiratory severe illness. Severe asthma exacerbations were diagnosed in the current asthmatics.
Older children tended to be seen later after onset of symptoms and were accountable for the longer hospitalisations. This might indicate that older children had more severe infections, or that the delay in admission to the hospital resulted in more severe disease and thereby a prolonged period of rehabilitation. Despite that, we also identified severe pneumonia based on the radiological findings (atelectasis, pleural effusions) in the 2-6-year-olds. If adjusted for population size these preschool children had an increased risk of developing severe pneumonia compared with school children during this epidemic. Inchley et al. showed the same pattern even if their definition of severe pneumonia differed. Treating the infections earlier might reduce severe morbidity and length of hospital stays.
Treatment of M. pneumoniae infections with macrolide antibiotics is controversial since a Cochrane review, concluded that there is insufficient evidence to draw any specific conclusions about the efficacy of antibiotics in M. pneumoniae infections in children. The efficacy of antibiotic treatment should be discussed in light of a correct diagnostic test. Asymptomatic carriers of M. pneumoniae have to be differentiated from children suffering from symptomatic infections, LRTI, caused by M. pneumoniae. Gardiner et al. underline the need for RCT on this topic. In Denmark, SSI still recommends treatment of M. pneumoniae positive LRTI in children. Macrolide resistance is a growing problem worldwide. In Denmark, the occurrence is estimated to be 2%. No macrolide resistance was identified in our childhood cohort.
We found radiological changes in 94% of the chest x-rays taken in this study. The radiological findings were quite diverse, but notably, over 80% of children older than two years had a lobar infiltration while the younger children had significantly more subtle findings. This was in accordance with an Italian prospective childhood study.
Even in older children, symptoms could not be distinguished from CAP caused by other pathogens. Radiological findings in M. pneumoniae pneumonia were not distinguishable from CAP in general.
Almost 25% of all children had some kind of rash (erythema/hives) during the illness, and 33% had gastrointestinal symptoms like nausea and or vomiting. Severe extra-pulmonary manifestations accompanying respiratory infections caused by M. pneumoniae are expected to occur. Two children in our cohort were diagnosed with SJS, which is a known complication of M. pneumoniae. Outbreaks of M. pneumoniae–associated SJS in children has recently been reported. We did not see any children with neurological symptoms in this cohort which would be expected.
Laryngectomees are at a higher risk of developing lower respiratory tract infections especially in the winter and when not wearing an HME. Maintaining the patency of the airway is of utmost importance as the mucus can be very dry and viscous and can stick to the walls of the trachea and the stoma. The risk of acquiring these infections can be reduced by: getting vaccinated for respiratory pathogens that include Streptococcus pneumoniae, H. influenzae, and the influenza viruses; washing hands before any stoma care; wearing an HME at all times; maintaining adequate respiratory tract humidification; and avoiding hypothermia or inhaling cold air.
Ninety-six-well polystyrene microtiter plates (Nunc maxisorb, Fisher Scientific) were coated overnight at 4°C with 75 ng of pooled CNV VLPs consisting of 25 ng of each strain; 170, C33 and HK in 0.05 M carbonate/bicarbonate buffer (pH 9.6). Plates were washed three times with 0.05% Tween 20 in phosphate buffered saline (PBS-T) before blocking in 5% skimmed milk-PBS-T for 1 h at 37°C and then three PBS-T washes. Plates were then incubated for 3 h at 37°C with 1∶50 dilution of each serum sample in duplicate in 5% skimmed milk-PBS-T. Pooled human sera (Sigma Aldrich), diluted 1∶400, and 100 ng pooled GII human norovirus VLPs were used as a positive control until a canine positive control was identified. After three washes with PBS-T, 50 µl of horseradish peroxidase (HRP)-conjugated anti-dog IgG antibody (Sigma Aldrich) diluted 1∶5000 in 5% milk PBS–T, was added to each well and incubated at 37°C for 1 h. The plates were washed four times with PBS-T and bound antibody detected with 50 µl tetramethylbenidine (TMB, Sigma Aldrich) followed by incubation at room temperature for 10 min. The reaction was stopped with 1 N H2SO4 and the optical density (OD) was read at 450 nm (Spectromax M2 plate reader, Molecular Devices).
To eliminate the possibility that non-specific components of the VLP preparation were identified by the canine sera, an antigenically distinct vesivirus 2117 VLP was included in the assay. The OD450 of a selection of serum samples incubated on either carbonate/bicarbonate buffer coated wells or vesivirus 2117 coated wells was highly comparable. This confirmed that no non-specific reactivity relating to the VLP preparation was occurring. The background signal for each sample was hence determined by measuring the OD450 of serum samples incubated with carbonate/bicarbonate buffer alone. Background signal was then subtracted from the OD450 of VLP coated wells to generate the corrected OD450 value. A threshold value was established as the mean of the OD450 of all buffer coated cells plus 3 standard deviations. A serum sample was considered positive when the corrected OD450 was higher than the threshold. Any serum samples showing a positive response to pooled CNV VLPs were subjected to further testing with individual CNV VLPs. Plates were coated with 25 ng of individual VLPs in carbonate/bicarbonate buffer and the protocol then repeated as above.
Evaluation of serological cross reactivity between different norovirus strains was achieved using VLP competition assays. Plates were coated with 25 ng/well of VLP overnight at 4°C. CNV positive canine sera was incubated with a range of concentrations of each of the either human norovirus VLPs, or individual CNV VLPs (0.5, 1, 2 and 4 µg/ml) for 1 h at 37°C. Vesivirus 2117 VLP was incubated with the canine sera as a negative control. After the incubation period, 50 µl of each serum-VLP combination was added to the previously VLP coated plates. The remainder of the ELISA protocol was followed as detailed above.