Validation of Fourier Transform Infrared Spectroscopy for Serotyping of Streptococcus pneumoniae.

Fourier transform infrared (FT-IR) spectroscopy (IR Biotyper; Bruker) allows highly discriminatory fingerprinting of closely related bacterial strains. In this study, FT-IR spectroscopy-based capsular typing of Streptococcus pneumoniae was validated as a rapid, cost-effective, and medium-throughput alternative to the classical phenotypic techniques. A training set of 233 strains was defined, comprising 34 different serotypes and including all 24 vaccine types (VTs) and 10 non-vaccine types (NVTs). The acquired spectra were used to (i) create a dendrogram where strains clustered together according to their serotypes and (ii) train an artificial neural network (ANN) model to predict unknown pneumococcal serotypes. During validation using 153 additional strains, we reached 98.0% accuracy for determining serotypes represented in the training set. Next, the performance of the IR Biotyper was assessed using 124 strains representing 59 non-training set serotypes. In this setting, 42 of 59 serotypes (71.1%) could be accurately categorized as being non-training set serotypes. Furthermore, it was observed that comparability of spectra was affected by the source of the Columbia medium used to grow the pneumococci and that this complicated the robustness and standardization potential of FT-IR spectroscopy. A rigorous laboratory workflow in combination with specific ANN models that account for environmental noise parameters can be applied to overcome this issue in the near future. The IR Biotyper has the potential to be used as a fast, cost-effective, and accurate phenotypic serotyping tool for S. pneumoniae.

Molecular characterization and epidemiology of Streptococcus pneumoniae serotype 8 in Denmark.

BACKGROUND: Streptococcus pneumoniae serotype 8 incidence has increased in Denmark after the introduction of pneumococcal conjugated vaccines (PCV). The mechanism behind the serotype 8 replacement is not well understood. In this study, we aimed to present epidemiological data on invasive pneumococcal disease (IPD) and molecular characterization of 96 serotype 8 clinical isolates. METHODS: IPD data from 1999 to 2019 were used to calculate the incidence and age distribution. Whole-genome sequencing (WGS) analysis was performed on 96 isolates (6.8% of the total serotype 8 IPD isolates in the period) to characterize the isolates with respect to pneumococcal lineage traits, a range of genes with potential species discrimination, presence of colonization and virulence factors, and molecular resistance pattern. RESULTS: The serotype 8 IPD incidence increased significantly (P < 0.05) for the age groups above 15 years after the introduction of PCV13, primarily affecting the elderly (65+). All isolates were phenotypically susceptible to penicillin, erythromycin and clindamycin. Molecular characterization revealed seven different MLST profiles with ST53 as the most prevalent lineage (87.5%) among the analyzed serotype 8 isolates. The genes covering the cell-surface proteins: lytA, rspB, pspA, psaA & Xisco and the pneumococcal toxin pneumolysin = ply were present in all isolates, while genes for the membrane transporter proteins: piaA/piaB/piaC; the capsular genes: cpsA (wzg) & psrP; the metallo-binding proteins zmpB & zmpC; and the neuroamidase proteins: nanA/nanB were variably present. Surprisingly, the putative transcriptional regulator gene SP2020 was not present in all isolates (98%). Susceptibility to penicillin, erythromycin and clindamycin was molecularly confirmed. CONCLUSION: The observed serotype 8 replacement was not significantly reflected with a change in the MLST profile or changes in antibiotic resistance- or virulence determinants.

Epidemiological and molecular characterization of Streptococcus pneumoniae carriage strains in pre-school children in Arkhangelsk, northern European Russia, prior to the introduction of conjugate pneumococcal vaccines.

BACKGROUND: The 13-valent Pneumococcal Conjugate Vaccine (PCV-13) was introduced in the National Immunization Programme (NIP) schedule in Russia in March 2014. Previously, the 7-valent Pneumococcal Conjugate Vaccine (PCV-7) was marketed in Russia in 2009 but has never been offered for mass vaccination. A carriage study was performed among children in Arkhangelsk in 2006. The objective was to determine the prevalence of carriage, serotype distribution, antimicrobial susceptibility and the molecular structure of Streptococcus pneumoniae strains before marketing and introduction of PCV-13. METHODS: A cross-sectional study was conducted on a cluster-randomized sample of children and a self-administrated questionnaire for parents/guardians.  Nasopharyngeal samples were collected from 438 children younger than 7 years attending nurseries and kindergartens in the Arkhangelsk region, Russia. Detailed demographic data, as well as information about the child's health, traveling, exposure to antimicrobials within the last 3 months and anthropometric measurements were collected for all study subjects. Variables extracted from the questionnaire were analysed using statistic regression models to estimate the risk of carriage. All pneumococcal  isolates were examined with susceptibility testing, serotyping and multilocus sequence typing. RESULTS: The overall prevalence of asymptomatic carriage was high and peaking at 36 months with a rate of 57%. PCV-13 covered 67.3% of the detected strains. High rates of non-susceptibility to penicillin, macrolides and multidrug resistance were associated with specific vaccine serotypes, pandemic clones, and local sequence types. Nine percent of isolates represented three globally disseminated disease-associated pandemic clones; penicillin- and macrolide-resistant clones NorwayNT-42 and Poland6B-20, as well as penicillin- and macrolide-susceptible clone Netherlands3-31. A high level of antimicrobial consumption was noted by the study. According to the parent's reports, 89.5% of the children used at least one antimicrobial regime since birth. None of the hypothesised predictors of S. pneumoniae carriage were statistically significant in univariable and multivariable logistic models. CONCLUSIONS: The study identified a high coverage of the PCV-13-vaccine, but serotype replacement and expansion of globally disseminated disease-associated clones with non-vaccine serotypes may be expected. Further surveillance of antimicrobial resistance and serotype distribution is therefore required.

Molecular characterization and epidemiology of Streptococcus pneumoniae serotype 24F in Denmark.

Since 2012, have we in Denmark observed an increase of invasive pneumococcal infections (IPD) due to Streptococcus pneumoniae serotype 24F. We here present epidemiological data on 24F IPD cases, and characterization of 48 24F clinical isolates based on clonal relationship, antimicrobial resistance (AMR) determinants and virulence factors. IPD surveillance data from (1999-2016) were used to calculate the incidence and age-distribution of serotype 24F IPD and the effect of pneumococcal conjugated vaccines (PCV). Characterization of forty-eight 24F isolates (14.7% of all 24F isolates from the period) was based on whole-genome sequencing analysis (WGS). The IPD cases of serotype 24F showed a significant increase (p < 0.05) for all age groups after the PCV-13 introduction in 2010. The majority of tested 24F isolates consisted of two MLST types, i.e. the ST72 and the ST162. Serotype 24F IPD increased in Denmark after the PCV-13 introduction in parallel with an increase of the ST162 clone. The genotypic penicillin binding protein (PBP) profile agreed with the phenotypical penicillin susceptibility. The virulence genes lytA, ply, piaA, piaB, piaC, rspB and the cpsA/wzg were detected in all 24F isolates, while the pspA and zmpC genes were absent.

Measurements of AMPs in stratum corneum of atopic dermatitis and healthy skin-tape stripping technique.

Decreased levels of antimicrobial peptides (AMPs) in atopic dermatitis (AD) have previously been reported and have been linked to the increased susceptibility to skin infections found in AD patients. This study intents to identify AMPs: hBD-2, hBD-3, RNase7, psoriasin and LL-37 in AD patients and healthy controls, and determine concentrations in consecutive depths of the outer most skin layers. Tape stripping was used on lesional and non-lesional skin. From each skin site, 35 consecutive tape strips were collected and pooled in groups of 5. Commercially available ELISA kits were used to determine AMP concentration in stratum corneum samples. hBD-2, hBD-3, RNase7 and psoriasin were identified in stratum corneum samples. hBD-3-level was markedly higher in AD non-lesional skin compared to healthy controls, and a similar trend was observed for RNase7. Most AMPs were distributed evenly through 35 tape strips, implying a homogeneous distribution of antimicrobial defense in the outer most skin layers. The findings indicate that AD patients may not suffer from a general baseline deficiency in AMPs, and that the innate immune defense is present throughout the stratum corneum, both insights of importance for understanding the role of AMPs in AD.

The capacity of diagnostic laboratories in Kenya for detecting infectious diseases.

BACKGROUND: The aim of this study is to present data of the diagnostic capacity of Kenyan laboratories to diagnose a number of human pathogens. The study is based on the data obtained from a biosecurity survey conducted in Kenya in 2014/2015 and data from the Statistical Abstract of Kenya for 2015. The biosecurity survey has previously been published; however, the survey also included information on laboratory capacity to handle a number of pathogens, which have not been published. METHODS: Data were retrieved from the survey on 86 laboratory facilities. The data include information from relevant categories such as training laboratories, human diagnostic laboratories, veterinary diagnostic laboratories, and research laboratories. RESULTS: The disease incidence in Kenya ranges widely from malaria and diarrhea with an incidence rate of around 10.000 per year to diseases such as cholera and yellow fever with an incidence rate of 1 per year or less for all age groups. The data showed that diseases with the highest number of diagnostic facilities were mainly malaria-, HIV-, tuberculosis-, and diarrhea-related infectious diseases. CONCLUSION: The study generally shows that the laboratory facilities have the capacity of detecting the infectious diseases with the highest incidence rates. Furthermore, it seems that the number of facilities able to detect a particular disease is related to the incidence rate of the disease.

Nasopharyngeal bacterial carriage in young children in Greenland: a population at high risk of respiratory infections.

The incidence of childhood respiratory infections in Greenland is among the highest globally. We performed a population-based study of 352 Greenlandic children aged 0-6 years aiming to describe rates and risk factors for carriage of four key bacteria associated with respiratory infections, their antimicrobial susceptibility and inter-bacterial associations. Nasopharyngeal swabs were tested for Streptococcus pneumoniae grouped by serotypes included (VT) or not included (NVT) in the 13-valent pneumococcal conjugate vaccine, non-typable Haemophilus influenzae (NTHi), Staphylococcus aureus and Moraxella catarrhalis. S. pneumoniae was detected from age 2 weeks with a peak carriage rate of 60% in 2-year-olds. Young age and having siblings attending a daycare institution were associated with pneumococcal carriage. Overall co-colonization with ⩾2 of the studied bacteria was 52%. NTHi showed a positive association with NVT pneumococci and M. catarrhalis, respectively, M. catarrhalis was positively associated with S. pneumoniae, particular VT pneumococci, whereas S. aureus were negatively associated with NTHi and M. catarrhalis. Nasopharyngeal bacterial carriage was present unusually early in life and with frequent co-colonization. Domestic crowding increased odds of carriage. Due to important bacterial associations we suggest future surveillance of pneumococcal conjugate vaccine's impact on carriage in Greenland to also include other pathogens.

Tape Stripping Technique for Stratum Corneum Protein Analysis.

The aim of this study was to investigate the amount of protein in stratum corneum in atopic dermatitis (AD) patients and healthy controls, using tape stripping technique. Furthermore, to compare two different methods for protein assessment. Tape stripping was performed in AD patients and healthy controls to collect stratum corneum samples and subsequently analysed with two different methods: Squame Scan, which gives an estimate of total protein (soluble and insoluble) and Micro BCA protein determination kit which measures soluble protein. Significant differences in cumulative protein content between AD lesional, AD non-lesional and healthy control skin was found using the Squame Scan as well as the Micro BCA protein determination kit. AD patients had significantly lower amount of protein, both total protein and soluble protein compared to healthy controls. Furthermore, soluble protein formed 82% of total protein in AD lesional skin, compared to 17-24% for AD non-lesional skin and healthy control. A decreasing amount of total protein with increasing stratum corneum depth was found for all skin types. Significant differences in stratum corneum protein content between AD lesional, AD non-lesional and healthy control skin were revealed, independent of method used.

In vivo expression of antimicrobial peptides in atopic dermatitis.

The aim of this review is to present findings on expression of antimicrobial peptides (AMPs) in atopic dermatitis (AD) skin, focusing only on in vivo studies, and to discuss differences in results obtained using various skin sampling techniques and different methodology for analysis of AMPs. The review also includes a discussion of the effect of frequently used treatments on AMP expression. Many studies have shown a reduced level of AMPs in lesional AD skin when compared to psoriatic skin, explaining the high frequency of AD-related infections. Interestingly, however, non-lesional AD skin has shown the same upregulation of AMPs after barrier disruption as non-lesional psoriatic skin. Various methods have been used to analyse AMP expression in the skin, and when comparing these methods, differences are revealed in AMP expression depending on the method used for sampling and analysis. Comparisons indicate that analyses of mRNA levels of AMPs may find greater differences in expression than analyses of protein levels. Few studies evaluate the effect of topical treatments on the expression of AMPs, and these indicate an inhibition of AMP expression, particularly after use of corticosteroids. AMPs are important components of the skin as a defense against infections, and despite much research, the clinical importance of the effect of common treatments, including systemic treatments for AD and the interplay between AMPs and the skin microbiome, is still largely unknown.

Human ß-defensin-2 as a marker for disease severity and skin barrier properties in atopic dermatitis.

BACKGROUND: Skin infections related to disrupted antimicrobial defence are a common problem in atopic dermatitis (AD). Altered levels of antimicrobial peptides, including human ß-defensin (hBD)-2, have been reported in AD skin, and a link to impaired barrier function has been suggested. OBJECTIVES: To study hBD-2 in relation to skin barrier function in patients with AD and controls, and to study hBD-2 in relation to disease severity. METHODS: Twenty-five patients with AD and 11 controls were enrolled. hBD-2 peptide concentration was determined in stratum corneum samples collected by a minimally invasive tape-stripping method. Disease severity was assessed by SCORing Atopic Dermatitis (SCORAD), and skin barrier function was evaluated by measurement of transepidermal water loss (TEWL) and skin pH. Patients with AD were characterized according to filaggrin mutations. RESULTS: hBD-2 concentrations in the stratum corneum were found to differ between lesional and nonlesional AD skin and controls, with the highest values in lesional skin (P < 0·001). SCORAD and TEWL were significantly increased in participants with measureable hBD-2 (P < 0·018 and P < 0·007, respectively). Significant correlations between hBD-2 in lesional skin, and TEWL and SCORAD were observed (R = 0·55 and R = 0·44, respectively). No correlations with skin pH were found. hBD-2 was not found to relate to filaggrin mutations. CONCLUSIONS: A significant correlation was found between hBD-2, disturbed skin barrier function and disease severity. The minimally invasive skin sample technique enables evaluation of the stratum corneum and its proteins over time and provides the possibility of relating these findings to treatment, infections and physiological variations.

Characterisation of invasive group B streptococci from adults in Denmark 1999 to 2004.

The aim of this study was to characterise the group B streptococci (GBS) isolates causing severe invasive infections in patients >15 years of age in Denmark from 1999 to 2004. A total of 411 invasive GBS isolates were phenotypically characterised by the capsular polysaccharide (CPS) serotype and protein Calpha, Cbeta and R4. The incidence of invasive GBS disease ranged from 2.2 to 3.2 per 100,000 adults in the study period, being highest among adults over 65 years of age. Diabetes was observed in 15% of the cases, 12% had alcohol abuse and 7% had cancer. Of all isolates, 77% were CPS serotypes Ia, Ib, III or V. The surface proteins Calpha or R4 were detected as the only protein in 57% of the GBS isolates. Cbeta was detected in 12% of the isolates, but always in combination with either Calpha or both Calpha and R4. The incidence of invasive GBS infections continued to increase in Denmark from 1999 to 2004. In that period, the overall case fatality was 14%. The most prevalent CPS serotypes were serotypes III, Ia, V and Ib. The most prevalent surface protein was R4 when testing for R4, Calpha and Cbeta. There was no clear relation between the GBS phenotype and infections with fatal outcome.

Developing an ELISA using serotype-specific antigens from selected pneumococcal strains of own selection.

Streptococcus pneumoniae (pneumococcus) is a significant cause of morbidity and mortality in infants and elderly people. Pneumococcal strains possess a polysaccharide capsule, and 90 different serotypes have been identified. The pneumococcal ELISA suggested by World Health Organization (WHO) guidelines is based on capsular serotypes included in the 23-valent polysaccharide vaccine (Pneumovax((R))). Pneumococcal antigens were developed and applied in an ELISA, which elicited a low baseline cross-reaction with the common cell wall polysaccharide (C-Ps). The aim of this study was to develop an ELISA using type-specific antigens from S. pneumoniae strains with serotypes of own selection in order to quantify type-specific pneumococcal antibodies in human serum.

The effect of broth media on pneumococcal growth and the latex serotyping result.

The aim was to test Todd-Hewitt broths (TH-broths) for the ability to propagate pneumococci and thereafter to evaluate the serotyping result obtained by the Pneumotest-Latex kit (SSI). TH-broths from four different producers (Oxoid, Sigma, Difco, and SSI) were tested and compared separately and with Serum broth (SSI). Twenty-three pneumococcal strains (different serotypes) were inoculated into the broths with start inoculums of 10(1), 10(3), and 10(6) CFU/ml. After incubation, overnight viable bacterial counts and visible growth were recorded. All pneumococci were serotyped with the Pneumotest-Latex kit. After incubation, the bacterial counts in all TH-broths were within the range of log 4.65-log 7.76 CFU/ml, while Serum broth showed an average growth ranging from log 8.05-log 8.90 CFU/ml. Comparing the growth of the four TH-broths showed no significant differences. In general, Serum broth had a more pronounced visual growth than each of the four TH-broths. Serotyping with Serum broth showed in general positive and correct latex typing results for all serotypes and initial inoculum, while the outcome of the TH-broths showed some false negative results depending on inoculum and serotype. Overall the Serum broth was found to be superior to the four TH-broths tested both with regard to CFU/ml and when used with the Pneumotest-Latex kit. However, if the Pneumotest-latex kit is only used on broths with visible growth as stated in the instruction manual, then the differences between the performances of the broths from the different producers was not significant and all broths could be used for Pneumotest-Latex typing.

Simple, rapid latex agglutination test for serotyping of pneumococci (Pneumotest-Latex).

The "gold standard" for epidemiological typing of Streptococcus pneumoniae (pneumococcus) is the capsular reaction test (Neufeld test) with antisera against the 90 pneumococcal polysaccharide capsules, i.e., serotyping. The method is labor intensive and requires a certain level of experience to be performed satisfactory, and thus it has been restricted for use in specialized reference or research laboratories. Surveillance of the serotype distribution of pneumococci that cause infections is important to secure an optimal composition of pneumococcal vaccines and to monitor antibiotic resistance in pneumococci. At Statens Serum Institut, a simple latex agglutination test for serotyping of pneumococci has been developed. The Pneumotest-Latex kit consists of 14 different pooled pneumococcus antisera (pools A to I and pools P to T) applied to latex particles. In a blind test of 352 isolates (with all 90 serotypes represented), 336 (95.5%) were typed or grouped correctly by the Pneumotest-Latex; in addition, 2 (7%) of 30 strains regarded as nontypeable or rough strains were serotyped, and the serotypes of these two isolates were confirmed by the capsular reaction test with type-specific antisera. The Pneumotest-Latex seems to be a sensitive method for serotyping or grouping of the majority of pneumococcal strains. By use of this ready-to-perform latex agglutination kit (Pneumotest-Latex), serotyping of pneumococci can gain more ground as a tool in prevention of pneumococcal diseases.

Latex assay for serotyping of group B Streptococcus isolates.

We developed a group B streptococcus (GBS) latex serotyping kit that reduces the numbers of GBS nontypeable isolates by nearly 50%. A total of 232 isolates were tested, and 203 isolates were serotyped by the GBS latex test, while the capillary precipitation test serotyped 184 isolates.

Alternative migration routes of Ascaris suum in the pig.

Experiments were conducted to investigate possible alternative routes of extraintestinal migration of Ascaris suum larvae in the pig. Pigs were infected with A. suum via injection of newly hatched larvae into cecal veins (i.v.), into cecal lymph nodes (LN), or intraperitoneally (i.p.), and control animals were inoculated orally with infective eggs (p.o.). Two pigs per inoculation route were necropsied on days 1, 4, and 13 postinoculation. The numbers of liver lesions and the percentage of larvae recovered was considerably greater in pigs inoculated i.v. or p.o. on each necropsy day. However, irrespective of inoculation route, at least a proportion of larvae passed through the livers and were able to complete migration to the small intestine by day 13. The results indicate that larval penetration of the intestinal wall is not necessary for liver-lung migration and that passage through the liver may be favorable for migrating A. suum larvae, although a delayed arrival in the small intestine cannot be ruled out for larvae following alternative routes.

Comparison of the IFAT and Iscom-ELISA response in bovine foetuses with Neospora caninum infection.

The study was carried out to evaluate the efficacy of foetal serology in the diagnosis of Neospora-associated bovine abortions. Fluids from 14 foetuses of cows with confirmed neosporosis (Group A), seven foetuses with confirmed bovine viral diarrhoea virus (BVD infection) (Group B) and 11 aborted foetuses without demonstrable infection (Group C) were examined. The age of the foetuses ranged from 4.5 months to 9 months. Albumin concentration (measured by Rocket Immunoelectrophoresis) was not significantly different in Group A compared with that in both Groups B and C, while that in Group B was significantly lower than in Group C. Levels of total IgG ranged from 0.01 to 1.78 (mg IgG) ml(-1) measured by single radial immunodiffusion technique. A measurable level of total IgG was found in all foetuses from Groups A and B, with no significant difference between levels in the two groups. Only one foetus in Group C had a detectable level of IgG. All foetuses in Group A had a specific IgG response (titre> or = 20) against Neospora caninum using the IFAT, while no positive responses in IFAT were found in Groups B and C. Measurement of specific IgG1 and IgG2 by Iscom-ELISA showed one and three false-negative results, respectively, in Group A. The IgG1 and IgG2 response in Group A was correlated according to the Spearman test (r = 0.66). Increasing age of the foetuses correlated significantly with the foetal IgG concentration, the specific IgG and IgG1 + IgG2. On the basis of the results obtained, it was concluded that the IFAT with a cut-off titre of 1:20, was a specific method for diagnosis of neosporosis in foetuses older than 4.5 months. The Iscom-ELISA also showed promising results as a method for screening specific antibodies against N. caninum in foetal fluid.

Early Ascaris suum migration in mice as a model for pigs.

A study was made of the early migratory pattern of Ascaris suum in mice. Mice were each inoculated orally with a single dose of 2,500 infective eggs and then killed at 2, 4, 6, 8, 10, and 12 hr postinoculation (PI). At necropsy, it was observed that the larvae had penetrated the mouse cecum and colon and had reached the liver by 4 hr PI. This migratory behavior closely mimics what is observed in the pig and suggests that the mouse may serve as an experimental model for intestinal immunity in the early phase of A. suum infection.

Experimental Ascaris suum infection in the pig: worm population kinetics following single inoculations with three doses of infective eggs.

To study population kinetics during primary Ascaris suum infections, 3 groups of 52 pigs each were inoculated with 100, 1000, or 10,000 infective eggs. In all groups, the majority of larvae was found in the liver on day 3 post inoculation (p.i.) and in the lungs on day 7 p.i. Liver white spots, caused by migrating larvae, were most numerous at day 7 p.i., whereafter they gradually healed, and only low numbers of granulation-tissue type white spots and lymphonodular white spots persisted at days 21-56 p.i. Independent of dose level, 47-58% of the inoculated eggs were recovered as larvae in the small intestine on day 10 p.i., but most larvae were eliminated at days 17-21 p.i. This elimination started earlier and removed a higher percentage of the worms with increasing inoculation dose, resulting in small strongly aggregated worm populations by day 28 p.i. (k of the negative binomial distribution was low: 0.2-0.4) without significant differences between groups. Thus, overdispersion, which is a characteristic of both porcine and human ascarosis, is found here under experimental conditions where aggregation factors like host behaviour, transmission rate, host status etc have been partly or totally controlled.