Middle Ear Fluid Of Children Research Articles

Clinical Uses of Nanopore Sequencing in Recurrent Acute and Chronic Otitis Media

Background. Otitis Media (OM) is a widespread problem that has an incidence of 10.85% worldwide, 51% occurring under the age of 5. Emergence of drug resistant bacteria contribute to high incidence of recurrent acute otitis media (AOM) and chronic otitis media with effusion (COME). Despite this, the standard of care for treatment is empiric antibiotics. Molecular microbiology diagnostics may enable clinicians to provided pathogen directed antibiosis. Nanopore sequencing is a highly accurate method of identifying a broad range of pathogens when compared with multiplex PCR. We hypothesize that nanopore sequencing is an effective method for detection of pathogens in the middle ear and nasopharynx of patients with recurrent or chronic OM.
 Methods. Bacterial Culture, multiplex PCR (BioFire PNA panel), and nanopore sequencing (MinIon, Oxford Nanopore Technologies) were tested on 60 middle ear and nasopharynx samples. Chi-Square test was used to examined differences in bacterial identification among the methods.
 Results. Bacterial identification using nanopore proves to be more sensitive in identification of 16 of the 18 unique pathogens when compared with multiplex testing. Of the 5 most common OM bacterial pathogens (S. pneumoniae, M. catarrhalis, H. influenza, S. aureus, P. aeruginosa), nanopore identified 3 pathogens (S. pneumoniae, M. catarrhalis, P. aeruginosa) at higher levels, 1 at the same rate (H. influenza), and 1 did not receive any readings (S. aureus).
 Conclusion. This is the first study to utilize nanopore sequencing to assess pathogens in the middle ear fluid of children with OM. While nanopore genomic sequencing is still in the early stages of use, it has potential to comprehensively identify bacteria with more sensitivity when compared to the current clinical standards. The next step of data analyzation will include bacterial identification in nanopore that multiplex PCR and traditional bacterial cultures cannot test for.

Moraxella Catarrhalis Infrequently Cultured from Middle Ear Fluid of Children with Acute Otitis Media.

We compared the distribution of organisms in the nasopharynx and the middle ear fluid in 164 episodes of acute otitis media in children 6-35 months of age. In contrast to Streptococcus pneumoniae and Haemophilus influenzae , Moraxella catarrhalis is isolated from the middle ear in only 11% of episodes with nasopharyngeal colonization with this organism.

Pathogenesis of Streptococcus pneumoniae serotype 3 during natural colonization and infections among children and its IgG correlate of protection in a mouse model

Current licensed pneumococcal conjugate vaccines (PCVs) are effective against pneumococcal diseases caused by the serotypes contained in the PCvs However; several studies evaluating pneumococcal colonization and acute otitis-media (AOM) prevention in young children vaccinated with PCV13, observed less effectiveness against serotype-3. One possible reason for less effectiveness may be release of the capsular polysaccharide (CPS) of serotype-3 (CPS-3) as an immune evasion mechanism. Here we evaluated free CPS-3 levels released from 6 clinical isolates from young children compared to WU2 strain and to serotype-19A CPS (CPS-19A) released in vitro when interacting with nasopharyngeal, middle-ear and lung cell-lines. Clinical serotype-3 strains showed greater release of CPS than WU2 with the interaction to 2 cell-lines and all 6 clinical serotype-19A strains. We next evaluated CPS-3 vs CPS-19A levels in middle-ear fluid (MEF) and the nasopharynx (NP) of young children and found higher levels of CPS-3 compared to CPS-19A in MEF during AOM but not in NP secretions during colonization. With anti-CPS-3 IgG in MEF and NP secretions at time of health and onset of AOM, a significant negative correlation (r = -0.75, p < 0.05) between unbound anti-CPS-3 IgG levels and free- anti-CPS-3 in MEF were found, and a significant lower detection of unbound anti-CPS-3 IgG in NP at the time of health with serotype-3 SPN (p < 0.05) compared to irrelevant SPN serotypes were found. In a mouse model of AOM and pneumonia, we sought a correlate of protection against serotype-3 infection using human serum-derived anti-CPS-3 IgG. We conclude that serotype-3 clinical isolates from children release more capsule than WU2 strains or 19A strains during in vitro testing; release more capsule in the MEF of children during AOM than serotype 19A; unbound anti-CPS-3 IgG levels negatively correlate with free-anti-CPS-3; and a level of 2.8 µg/ml anti-CPS-3 antibody protects mice from AOM and pneumonia but not colonization.

Otitis Media Practice During the COVID-19 Pandemic

The global coronavirus disease-2019 (COVID-19) pandemic has changed the prevalence and management of many pediatric infectious diseases, including acute otitis media (AOM). Coronaviruses are a group of RNA viruses that cause respiratory tract infections in humans. Before the COVID-19 pandemic, coronavirus serotypes OC43, 229E, HKU1, and NL63 were infrequently detected in middle ear fluid (MEF) specimens and nasopharyngeal aspirates in children with AOM during the 1990s and 2000s and were associated with a mild course of the disease. At times when CoV was detected in OM cases, the overall viral load was relatively low. The new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen responsible for the eruption of the COVID-19 global pandemic. Following the pandemic declaration in many countries and by the World Health Organization in March 2020, preventive proactive measures were imposed to limit COVID-19. These included social distancing; lockdowns; closure of workplaces; kindergartens and schools; increased hygiene; use of antiseptics and alcohol-based gels; frequent temperature measurements and wearing masks. These measures were not the only ones taken, as hospitals and clinics tried to minimize treating non-urgent medical referrals such as OM, and elective surgical procedures were canceled, such as ventilating tube insertion (VTI). These changes and regulations altered the way OM is practiced during the COVID-19 pandemic. Advents in technology allowed a vast use of telemedicine technologies for OM, however, the accuracy of AOM diagnosis in those encounters was in doubt, and antibiotic prescription rates were still reported to be high. There was an overall decrease in AOM episodes and admissions rates and with high spontaneous resolution rates of MEF in children, and a reduction in VTI surgeries. Despite an initial fear regarding viral shedding during myringotomy, the procedure was shown to be safe. Special draping techniques for otologic surgery were suggested. Other aspects of OM practice included the presentation of adult patients with AOM who tested positive for SARS-2-CoV and its detection in MEF samples in living patients and in the mucosa of the middle ear and mastoid in post-mortem specimens.

Pneumococcal conjugate vaccines for preventing acute otitis media in children.

Pneumococcal conjugate vaccines for preventing acute otitis media in children.

Pneumococcal conjugate vaccines for preventing acute otitis media in children.

Pneumococcal conjugate vaccines for preventing acute otitis media in children.

Prediction of Fluid Characteristics in Pediatric Otitis Media with Effusion from the Tympanometry

Objective: To correlate the characteristics of type B tympanogram with the type of middle ear fluid in children suffering from persistent OME. Methodology: A prospective study was conducted on children with persistent OME for more than 3 months who failed to respond to medical treatment with type B tympanogram in one or both ears and were candidate for myringotomy. The middle ear fluid was aspirated and the viscosity of the fluid was evaluated objectively by measuring the diameter of the diffusion circle on a filter paper in mm. Results: Thirty eight children (70 ears) were enrolled in this study. The shape of type B tympanogram was either flat (67.14%) or peaked (32.86 %).Three fluid types were noticed in OME namely serous, mucoid and glue. The shape was highly correlated with the type of secretions. Compliance and gradient of type B tympanogram were negatively correlated with the viscosity of the secretions. Conclusion: Tympanometry is a beneficial clinical tool in prediction, diagnosis and management of OME. Characteristics of type B tympanogram were correlated with the viscosity of middle ear fluid. Detailed analysis of type B tympanogram can assist the clinicians in determining the indication of comprehensive treatment.

Pneumococcal vaccine impact on otitis media microbiology: A New Zealand cohort study before and after the introduction of PHiD-CV10 vaccine

We compared the microbiology of middle ear fluid (MEF) in two cohorts of children having ventilation tube (VT) insertion; the first in the era of 7-valent Streptococcus pneumoniae conjugate vaccine (PCV7) and the second following introduction of the ten-valent pneumococcal vaccine (PHiD-CV10). MethodsDuring 2011 (Phase 1) and again in 2014 (Phase 2) MEF and NP samples from 325 children and 319 children were taken at the time of VT insertion. A matched comparison group had NP swabs collected with 137 children (Phase 1) and 154 (Phase 2). Culture was performed on all NP and MEF samples with further molecular identification of Haemophilus species, serotyping of S. pneumoniae, and polymerase chain reaction (PCR) testing on all MEF samples. ResultsIn Phase 2 immunisation coverage with ⩾3 doses of PHiD-CV10 was 93%. The rate and ratios of culture and molecular detection of the 3 main otopathogens was unchanged between Phase 1 and Phase 2 in both MEF and NP. Haemophilus influenzae was cultured in one quarter and detected by PCR in 53% of MEF samples in both time periods. S. pneumoniae and Moraxella catarrhalis were cultured in up to 13% and detected by PCR in 27% and 40% respectively of MEF samples. H. influenzae was the most common organism isolated from NP samples (61%) in the children undergoing VT surgery whilst M. catarrhalis (49%) was the most common in the non-otitis prone group. 19A was the most prominent S. pneumoniae serotype in both MEF and NP samples in Phase 2. Of Haemophilus isolates, 95% were confirmed to be non-typeable H. influenzae (NTHi) over both time periods. ConclusionFollowing implementation of PHiD-CV10 in New Zealand, there has been no significant change in the 3 major otopathogens in NP or MEF in children with established ear disease. For these children non-typeable H. influenzae remains the dominant otopathogen detected.

Impact of the Seven-valent Pneumococcal Conjugate Vaccine on Acute Otitis Media in Japanese Children: Emergence of Serotype 15A Multidrug-resistant Streptococcus pneumoniae in Middle Ear Fluid Isolates.

Streptococcus pneumoniae is one of the most common bacteria causing acute otitis media (AOM). In Japan, a 7-valent pneumococcal conjugate vaccine (PCV7) was introduced for voluntary vaccination of children in 2010, and it became a recommended vaccination in April 2013. We surveyed the serotypes and antimicrobial susceptibility profile of S. pneumoniae isolates obtained from the middle ear fluid of Japanese children with AOM. Between April and September 2013, a total of 176 S. pneumoniae isolates were obtained from the middle ear fluid of children aged 0-3 years with AOM. Isolates were collected from various regions of Japan. Minimum inhibitory concentrations were measured by the broth microdilution method. Serotyping was performed by observing the Quellung reaction. Although 45.5% of the strains were susceptible to penicillin G, 42.6% were penicillin-intermediate strains and 11.9% were penicillin-resistant strains. Serotype 19A (27.3%), serotype 15A (14.2%) and serotype 3 (11.9%) showed a high frequency. Although PCV7 types only accounted for 4.5% of all strains, 44.9% were PCV13 types and 55.1% were non-PCV types. Serotype 15A strains were 100% nonsusceptible to penicillin G and all of these strains showed multidrug resistance. Serotype 15A was frequent in children up to 1 year old. After this research was completed, PCV7 was switched to a PCV13 that also contained serotype 3 and serotype 19A. We need to consider the possibility that serotype 15A, which is not included in PCV13, may increase and cause intractable AOM in the future.

Pepsinogen Assay as a Marker of Reflux in Children with Otitis Media with Effusion

Background: Otitis media with effusion remains the most common cause of deafness in childhood. Reflux of gastric contents into the nasopharynx and then into the middle ear cleft has been reported as a contributing factor in the development of OME. Objective: To investigate the relationship between gastro esophageal reflux diseases (GERD) and Otitis media with effusion (OME) by evaluation of the presence of gastric pepsinogen in middle ear fluid of children with OME. Materials and Methods: In this prospective study, middle ear effusions as well as blood samples were collected from 25 patients suffering from OME and undergoing bilateral myringotomy with ventilation tube insertion. Total pepsinogen concentrations of effusions and serum samples were measured with an enzyme-linked immuno sorbent assay (ELISA) using Human Pepsinogen I ELISA Kit. Results: 64% (16 cases) of the study sample (25 children) were found with pepsinogen in the middle ear effusions while 36% (9 cases) were not found to have pepsinogen in the middle ear. The concentrations of pepsinogen measured in the effusions were found to be higher than the levels found in the serum of the same patients as detected in each case separately. Conclusion: GERD should be considered one of the contributing factors in the etiopathogenesis of OME.

Etiological and demographic characteristics between unilateral and bilateral otitis media in Costa Rican children

Data from Latin America describing the potential differences among children with unilateral or bilateral otitis media are scarce and needed in order to implement appropriate prevention actions and specific antimicrobial recommendations. The present study analyzed the demographic and microbiologic characteristics of 1,427 Costa Rican children with unilateral and bilateral otitis media in whom a tympanocentesis was performed between 1992 and 2006 (median age: 23 months; range two to 150 months). There were no significant differences in the demographics, microbiology and antimicrobial susceptibility among middle ear fluid isolates obtained from both populations. The only significant finding was observed in children with Moraxella catarrhalis otitis media that was observed more frequently than other bacterial pathogen in the middle ear fluid of children with unilateral acute otitis media that were < 24 months of age and during the rainy season.

Cytokine, chemokine, and Toll-like receptor expression in middle ear fluids of children with acute otitis media.

Acute otitis media (AOM) is a common bacterial infection in childhood that causes an inflammatory response in the middle ear. Leukocytes produce different inflammatory molecules in vitro when stimulated with Gram-positive and Gram-negative bacteria. The major causes of AOM are Streptococcus pneumoniae, nontypeable Haemophilus influenza, and Moraxella catarrhalis. We sought to assess differences in cytokines, chemokines, and expression of Toll-like receptors (TLRs) at onset of AOM based on bacterial culture results. Middle ear fluid (MEF) from 66 children with AOM was studied. Innate immune genes, cytokines (interleukin [IL]-6, IL-8, IL-10, IL1-β; tumor necrosis factor-α), chemokines (CCL2, CCL3, CCL4, CCR5, CXCR3), and Toll-like receptors (TLR2, TLR4, TLR9) expression was measured using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) from MEF collected in vivo by tympanocentesis. Culture-positive MEF had higher levels of all cytokines and chemokines (9-300-fold) as compared to MEF that was culture negative. Polymerase chain reaction (PCR)-positive/culture-negative MEF for otopathogens showed significant differences (P < .01) in TLR2, TLR4, and TLR9 expression (6-31-fold), but cytokine and chemokine levels were similar compared to PCR-negative/culture-negative MEF. No significant differences were found in the cytokine/chemokine/TLR levels among the bacterial otopathogen species. However, higher levels of TLRs, and all the cytokine and chemokines were detected when more than one bacterial species was present compared to single otopathogens. Expression levels of proinflammatory cytokines/chemokines and TLRs are elevated in AOM children with a bacterial otopathogen, and are dependent on the number of bacterial species identified.

Detection of gastric pepsin in middle ear fluid of children with chronic otitis media with effusion

ObjectivesTo study the possible relationship between gastroesophageal reflux and chronic middle ear effusion by (i) evaluating the presence of pepsin/pepsinogen in middle ear fluid of children with chronic otitis media with effusion (OME) and to (ii) differentiate between pepsin from gastric juice and pepsin derived from plasma pepsinogen.Patients and methodsThis was a prospective study carried out in the Departments of Otorhinolaryngology and Clinical Pathology, Alexandria University. Middle ear effusions and blood samples were collected from 25 patients undergoing bilateral myringotomy with tube placement for chronic OME. These samples were prepared for pepsin assay. The total pepsin/pepsinogen concentrations of effusions and serum samples were measured with an enzyme-linked immunosorbent assay using human pepsin-specific antibody.ResultsPepsin was detected in 22 of 25 (88%) patients and in 42 of 50 (84%) of the ears, and it was detected in all serum samples. A total of seven of 25 (28%) patients and nine of 50 (18%) ears were found to have higher pepsin levels in their middle ear effusion samples than the cut-off value of serum. The average pepsin level in all effusion samples was 109.99 ng/ml, whereas it was 55.72 ng/ml in serum samples.ConclusionThe gastroesophageal reflux is one of the contributing factors in the etiopathogenesis of middle ear effusion as gastric pepsin reaches the middle ear through the nasopharynx and Eustachian tube to cause OME.

Incorporation of phosphorylcholine into the lipooligosaccharide of nontypeable Haemophilus influenzae does not correlate with the level of biofilm formation in vitro.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that causes otitis media in children and community-acquired pneumonia or exacerbations of chronic obstructive pulmonary disease in adults. A large variety of studies suggest that biofilm formation by NTHi may be an important step in the pathogenesis of this bacterium. The objective of this report was to determine the relationship between the presence of phosphorylcholine in the lipooligosaccharide of NTHi and the level of biofilm formation. The study was performed on 111 NTHi clinical isolates collected from oropharyngeal samples of healthy children, middle ear fluid of children with otitis media, and sputum samples of patients with chronic obstructive pulmonary disease or community-acquired pneumonia. NTHi clinical isolates presented a large variation in the level of biofilm formation in a static assay and phosphorylcholine content. Isolates collected from the oropharynx and middle ear fluid of children tended to have more phosphorylcholine and made denser biofilms than isolates collected from sputum samples of patients with chronic obstructive pulmonary disease or community-acquired pneumonia. No correlation was observed between biofilm formation and the presence of phosphorylcholine in the lipooligosaccharide for either planktonic or biofilm growth. This lack of correlation was confirmed by abrogating phosphorylcholine incorporation into lipooligosaccharide through licA gene deletion, which had strain-specific effects on biofilm formation. Altogether, we present strong evidence to conclude that there is no correlation between biofilm formation in a static assay and the presence of phosphorylcholine in lipooligosaccharide in a large collection of clinical NTHi isolates collected from different groups of patients.

Effect of combination of montelukast and levocetirizine on otitis media with effusion: a prospective, placebo-controlled trial

Conclusion: This study demonstrated that treatment with montelukast in combination with levocetirizine may have a beneficial effect on clinical improvement of middle ear fluid in children with otitis media with effusion (OME). Objective: To evaluate the effect of the combination of montelukast and levocetirizine on shortening the duration of effusion in successfully treated OME in children. Methods: This was a randomized, prospective, double-blind, placebo-controlled trial. The study group consisted of 120 children (age range 2–6 years), who were diagnosed with OME of at least 2 months duration. The children were randomized into one of four groups. The treatment packs containing montelukast 4 mg (tablet) and/or levocetirizine 2.5 mg/5 ml as the active drug or placebo were prepared, numbered, and distributed by the second author. Results: The amelioration of bilateral otoscopic sign scores in the combination therapy group was statistically significantly greater than in all the other groups (p < 0.05). Improvement in bilateral tympanometric findings in the combination therapy group and montelukast group was greater than in the other groups. However, this difference was not statistically significant (p > 0.05). When we compared the difference between otoscopic sign scores before and after treatment we found that it was statistically significant in every group (p > 0.05). However, the significant improvement in tympanometric findings occurred in all groups except the levocetirizine group (p > 0.05).

Microbiological characterization of Streptococcus pneumoniae and non-typeable Haemophilus influenzae isolates as primary causes of acute otitis media in Bulgarian children before the introduction of conjugate vaccines

BackgroundPneumococcal and Haemophilus influenzae type b (Hib) vaccines were introduced in our national immunisation program in April 2010. The aims of this retrospective, laboratory-based study were to determine the serotypes and antibiotic resistance of Streptococcus pneumoniae and H. influenzae isolates from middle ear fluid (MEF) collected before the introduction of immunization.MethodsS. pneumoniae (n = 128) and H. influenzae (n = 40) strains isolated from MEF of children with AOM between 1994 and 2011 were studied. MICs were determined by a microdilution assay. Serotyping of S. pneumoniae was done by Quellung method and PCR capsular typing was used for H. influenzae. Macrolide resistance genes were detected by PCR for erythromycin resistant S. pneumoniae (ERSP). DNA sequencing of ftsI gene was performed for ampicillin nonsusceptible H. influenzae.ResultsThe most common serotypes found among children with pneumococcal AOM were 19 F (20.3%), 6B (15.6%), and 19A (10.9%). The potential coverage rates by the PCV7, PCV10 and PCV13 of children aged < 5 years were 63.6%, 66.4% and 85.5%, respectively. Reduced susceptibility to oral penicillin was seen in 68.1%; resistance to erythromycin was 46.9%. We found erm(B) gene in 56.7% of the ERSP, mef(E) gene in 25%; 15% harbored both genes erm(B) + mef(E) and 3.3% had mutations of L4 ribosomal protein. Of the 40 H. influenzae isolates 97.5% were nontypeable. Nonsusceptibility to ampicillin occurred in 25%. Ampicillin resistance groups were: β-lactamase-positive ampicillin resistant (BLPAR) strains (10%), β-lactamase-negative ampicillin resistant (BLNAR) strains (12.5%) and β-lactamase-positive amoxicillin-clavulanate resistant (BLPACR) strains (2.5%). Among BLNAR and BLPACR most of the isolates (5/6) belonged to group II, defined by the Asn526Lys substitution.ConclusionsThe levels of antibiotic resistance among S. pneumoniae and H. influenzae causing severe AOM in children are high in our settings. The existence of multidrug-resistant S. pneumoniae serotype 19A is of particular concern. The rate of BLNAR and BLPACR strains among H. influenzae isolates was 15%.

925 Antibiotic Resistance and Pneumococcal Conjugated Vaccines Coverage of Streptococcus Pneumonia from Middle Ear Fluid of Children < 5 Years

AimsA prospective study was initiated in Brasov, Romania in 2009 to assess the antibiotic resistance pattern of Streptococcus pneumoniae (Pnc) isolated from middle ear fluid in children with acute otitis...

Antibody in Middle Ear Fluid of Children Originates Predominantly from Sera and Nasopharyngeal Secretions

The human middle ear is devoid of any immunocompetent cells in normal mucosa. We sought to determine the source of antibody present in the middle ear of children. Total IgG, IgA, and secretory IgA antibodies were determined by enzyme-linked immunosorbent assay from the nasopharyngeal, middle ear, and serum samples of children with acute otitis media. The two-dimensional gel electrophoresis pattern of the entire array of IgA antibodies in the nasal wash (NW) and middle ear fluid (MEF) was compared from the MEF and NW samples using isoelectric focusing and Western blotting. The total IgG and IgA antibodies in the MEF and NW samples of 137 children were compared. The ratio of IgG to IgA in the MEF was significantly different (P < 0.008) compared to NW because IgA levels were higher and IgG levels lower in NW. The IgG/IgA ratio of MEF resembled serum consistent with transudation to the MEF. Small amounts of secretory IgA were detected in MEF but the electrophoresis patterns of the entire array of IgA antibodies in the MEF and NW were virtually identical in each child evaluated; thus, IgA in MEF derived predominantly from serum and the nasopharynx by reflux via the Eustachian tube. The IgG/IgA antibody levels in the MEF and the same composition of IgA antibody in the MEF and NW identifies the predominant source of antibody in the MEF as a transudate of serum combined with nasal secretions refluxed from the nasopharynx in children.

Decline and Rise of the Antimicrobial Susceptibility of Streptococcus pneumoniae Isolated from Middle Ear Fluid in Children: Influence of Changes in Circulating Serotypes

Changes in the antimicrobial susceptibility of Streptococcus pneumoniae causing otitis media were studied in 916 isolates from children <5 years old between 1999 and 2010 in a region of northern Spain. The rate of antimicrobial resistance decreased between the period before the introduction of the heptavalent pneumococcal conjugate vaccine (from 1999 to 2001) and the period from 2005 to 2007. However, in 2008 to 2010, resistance rates increased again due to the spread of serotype 19A, especially the multidrug-resistant ST320 and ST276 clones.

Presence of Viral Nucleic Acids in the Middle Ear

Viruses play an important role in acute otitis media (AOM) pathogenesis, and live viruses may cause AOM in the absence of pathogenic bacteria. Detection of AOM pathogens generally relies on bacterial culture of middle ear fluid. When viral culture is used and live viruses are detected in the middle ear fluid of children with AOM, the viruses are generally accepted as AOM pathogens. Because viral culture is not sensitive and does not detect the comprehensive spectrum of respiratory viruses, polymerase chain reaction assays are commonly used to detect viral nucleic acids in the middle ear fluid. Although polymerase chain reaction assays have greatly increased the viral detection rate, new questions arise on the significance of viral nucleic acids detected in the middle ear because nucleic acids of multiple viruses are detected simultaneously, and nucleic acids of specific viruses are detected repeatedly and in a high proportion of asymptomatic children. This article first reviews the role of live viruses in AOM and presents the point-counterpoint arguments on whether viral nucleic acids in the middle ear represent an AOM pathogen or a bystander status. Although there is evidence to support both directions, helpful information for interpretation of the data and future research direction is outlined.