Short-course aminoglycosides as adjunctive empirical therapy in patients with Gram-negative bloodstream infection, a cohort study.

OBJECTIVE: Short-course aminoglycosides as adjunctive empirical therapy to ß-lactams in patients with a clinical suspicion of sepsis are used to broaden antibiotic susceptibility coverage and to enhance bacterial killing. We quantified the impact of this approach on 30-day mortality in a subset of sepsis patients with a Gram-negative bloodstream infection. METHODS: From a prospective cohort study conducted in seven hospitals in the Netherlands between June 2013 and November 2015, we selected all patients with Gram-negative bloodstream infection (GN-BSI). Short-course aminoglycoside therapy was defined as tobramycin, gentamicin or amikacin initiated within a 48-hour time window around blood-culture obtainment, and prescribed for a maximum of 2 days. The outcome of interest was 30-day all-cause mortality. Confounders were selected a priori for adjustment using a propensity score analysis with inverse probability weighting. RESULTS: A total of 626 individuals with GN-BSI who received ß-lactams were included; 156 (24.9%) also received aminoglycosides for a median of 1 day. Patients receiving aminoglycosides more often had septic shock (31/156, 19.9% versus 34/470, 7.2%) and had an eight-fold lower risk of inappropriate treatment (3/156, 1.9% versus 69/470, 14.7%). Thirty-day mortality was 17.3% (27/156) and 13.6% (64/470) for patients receiving and not receiving aminoglycosides, respectively; yielding crude and adjusted odds ratios for 30-day mortality for patients treated with aminoglycosides of 1.33 (95% CI 0.80-2.15) and 1.57 (0.84-2.93), respectively. CONCLUSIONS: Short-course adjunctive aminoglycoside treatment as part of empirical therapy with ß-lactam antibiotics in patients with GN-BSI did not result in improved outcomes, despite better antibiotic coverage of pathogens.

Development of diagnostic prediction tools for bacteraemia caused by third-generation cephalosporin-resistant enterobacteria in suspected bacterial infections: a nested case-control study.

OBJECTIVES: Current guidelines for the empirical antibiotic treatment predict the presence of third-generation cephalosporin-resistant enterobacterial bacteraemia (3GCR-E-Bac) in case of infection only poorly, thereby increasing unnecessary carbapenem use. We aimed to develop diagnostic scoring systems which can better predict the presence of 3GCR-E-Bac. METHODS: A retrospective nested case-control study was performed that included patients ≥18 years of age from eight Dutch hospitals in whom blood cultures were obtained and intravenous antibiotics were initiated. Each patient with 3GCR-E-Bac was matched to four control infection episodes within the same hospital, based on blood-culture date and onset location (community or hospital). Starting from 32 commonly described clinical risk factors at infection onset, selection strategies were used to derive scoring systems for the probability of community- and hospital-onset 3GCR-E-Bac. RESULTS: 3GCR-E-Bac occurred in 90 of 22 506 (0.4%) community-onset infections and in 82 of 8110 (1.0%) hospital-onset infections, and these cases were matched to 360 community-onset and 328 hospital-onset control episodes. The derived community-onset and hospital-onset scoring systems consisted of six and nine predictors, respectively. With selected score cut-offs, the models identified 3GCR-E-Bac with sensitivity equal to existing guidelines (community-onset: 54.3%; hospital-onset: 81.5%). However, they reduced the proportion of patients classified as at risk for 3GCR-E-Bac (i.e. eligible for empirical carbapenem therapy) with 40% (95%CI 21-56%) and 49% (95%CI 39-58%) in, respectively, community-onset and hospital-onset infections. CONCLUSIONS: These prediction scores for 3GCR-E-Bac, specifically geared towards the initiation of empirical antibiotic treatment, may improve the balance between inappropriate antibiotics and carbapenem overuse.

An Enzyme-Linked Immunosorbent Spot Assay Measuring Borrelia burgdorferi B31-Specific Interferon Gamma-Secreting T Cells Cannot Discriminate Active Lyme Neuroborreliosis from Past Lyme Borreliosis: a Prospective Study in the Netherlands.

Two-tier serology testing is most frequently used for the diagnosis of Lyme borreliosis (LB); however, a positive result is no proof of active disease. To establish a diagnosis of active LB, better diagnostics are needed. Tests investigating the cellular immune system are available, but studies evaluating the utility of these tests on well-defined patient populations are lacking. Therefore, we investigated the utility of an enzyme-linked immunosorbent spot (ELISpot) assay to diagnose active Lyme neuroborreliosis. Peripheral blood mononuclear cells (PBMCs) of various study groups were stimulated by using Borrelia burgdorferi strain B31 and various recombinant antigens, and subsequently, the number of Borrelia-specific interferon gamma (IFN-γ)-secreting T cells was measured. We included 33 active and 37 treated Lyme neuroborreliosis patients, 28 healthy individuals treated for an early manifestation of LB in the past, and 145 untreated healthy individuals. The median numbers of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs did not differ between active Lyme neuroborreliosis patients (6.0; interquartile range [IQR], 0.5 to 14.0), treated Lyme neuroborreliosis patients (4.5; IQR, 2.0 to 18.6), and treated healthy individuals (7.4; IQR, 2.3 to 14.9) (P = 1.000); however, the median number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs among untreated healthy individuals was lower (2.0; IQR, 0.5 to 3.9) (P ≤ 0.016). We conclude that the Borrelia ELISpot assay, measuring the number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs, correlates with exposure to the Borrelia bacterium but cannot be used for the diagnosis of active Lyme neuroborreliosis.

Pneumococcal conjugate vaccination response in patients after community-acquired pneumonia, differences in patients with S. pneumoniae versus other pathogens.

OBJECTIVES: The goal of this study is to investigate the immune response to the 13-valent pneumococcal conjugate vaccine (PCV13) in former pneumococcal CAP patients. We hypothesize that an impaired or suboptimal humoral immune response against (specific) pneumococcal serotypes might explain the vulnerability for pneumococcal disease. METHODS: Hospitalised adult CAP patients who participated in two trials (2004-2006 (n=201) and, 2007-2009 (n=304)) were screened. Patients eligible for inclusion had CAP caused by either S. pneumoniae (pneuCAP) or due to another well-defined pathogen (otherCAP). Serotype-specific pneumococcal antibody concentrations (total IgG and IgG2/IgG1) before and 3-4weeks after PCV13 administration were measured (Luminex) and compared between pneuCAP and otherCAP patients. RESULTS: We vaccinated 60 patients:i.e. 34 pneuCAP and 26 otherCAP patients. In the pneuCAP group, 74% of patients were categorized as good responders (≥9/13 serotypes with concentration≥1300ng/ml), versus 77% in the otherCAP group. Significantly fewer full responders (i.e. 13/13 serotypes with a concentration≥1300ng/mL) were identified in the pneuCAP group (15% vs 42% respectively, p=0.02). For serotype 1, total IgG and IgG2/IgG1 subset post-vaccination concentrations were significantly lower among pneuCAP patients. Our additional case-series showed that of 16 pneuCAP patients who were infected by a serotype included in PCV13 three patients did not respond against the serotype originally responsible for their CAP episode, including one former bacteraemic pneumococcal CAP patient who also failed to show a response against the serotype responsible for CAP during infection. Thirteen patients did respond to the previously infecting serotype following PCV13 including three patients who had bacteraemic pneumococcal pneumonia and did not show a response during infection against the serotype responsible for CAP. CONCLUSIONS: Our results confirm the immunogenic properties of PCV13 in former pneumococcal CAP patients including patients previously regarded as potential hyporesponders. A slightly diminished overall humoral response to polysaccharides characterizes the former pneumococcal CAP patients. ClinicalTrials.gov Identifier: NCT02141009.

Disagreement between the results from three commercial tests for the detection of Borrelia-specific serum antibodies in the Netherlands associated with antibiotic treatment for Lyme borreliosis: a retrospective study.

The diagnosis of Lyme borreliosis is challenging because of the often non-specific symptoms and persisting antibodies after infection. We investigated the diagnostic characteristics of two enzyme-linked immunosorbent assays (ELISAs) and an immunoblot for the detection of Borrelia-specific serum antibodies using different test strategies in individuals with and without antibiotic treatment for Lyme borreliosis. This retrospective study included healthy individuals, patients with active Lyme neuroborreliosis and patients treated for Lyme neuroborreliosis. Two ELISAs were compared: the C6 ELISA and the SERION ELISA. Equivocal and positive results were confirmed by immunoblot. We included 174 healthy individuals, of whom 27 (15.5%) were treated for Lyme borreliosis in the past, 36 patients were treated for Lyme neuroborreliosis and 27 patients had active Lyme neuroborreliosis. All the active Lyme neuroborreliosis patients were reactive in both ELISAs (100% sensitivity); less reactivity was seen in the other three groups (range 17.7% to 69.4%). The concordance between the ELISA results was high in active Lyme neuroborreliosis patients (26/27; 96.3%) and healthy individuals (131/147; 89.1%), but lower in treated healthy individuals (18/27; 66.7%) and treated Lyme neuroborreliosis patients (18/36; 50.0%) (p ≤ 0.005). This study showed that antibiotic treatment against Lyme borreliosis was strongly associated with discordant ELISA and test strategy results (odds ratio: 10.52; p < 0.001 and 9.98; p = 0.014, respectively) suggesting antibiotic treatment influences the pace at which the various antibodies directed to the different antigens used in both ELISAs wane. Among treated neuroborreliosis patients, the SERION ELISA stayed positive for a longer period after infection compared to the C6 ELISA. This should be taken into consideration when requesting and/or interpreting Lyme serology.

Randomized clinical trial of extended versus single-dose perioperative antibiotic prophylaxis for acute calculous cholecystitis.

BACKGROUND: Many patients who have surgery for acute cholecystitis receive postoperative antibiotic prophylaxis, with the intent to reduce infectious complications. There is, however, no evidence that extending antibiotics beyond a single perioperative dose is advantageous. This study aimed to determine the effect of extended antibiotic prophylaxis on infectious complications in patients with mild acute cholecystitis undergoing cholecystectomy. METHODS: For this randomized controlled non-inferiority trial, adult patients with mild acute calculous cholecystitis undergoing cholecystectomy at six major teaching hospitals in the Netherlands, between April 2012 and September 2014, were assessed for eligibility. Patients were randomized to either a single preoperative dose of cefazolin (2000 mg), or antibiotic prophylaxis for 3 days after surgery (intravenous cefuroxime 750 mg plus metronidazole 500 mg, three times daily), in addition to the single dose. The primary endpoint was rate of infectious complications within 30 days after operation. RESULTS: In the intention-to-treat analysis, three of 77 patients (4 per cent) in the extended antibiotic group and three of 73 (4 per cent) in the standard prophylaxis group developed postoperative infectious complications (absolute difference 0·2 (95 per cent c.i. -8·2 to 8·9) per cent). Based on a margin of 5 per cent, non-inferiority of standard prophylaxis compared with extended prophylaxis was not proven. Median length of hospital stay was 3 days in the extended antibiotic group and 1 day in the standard prophylaxis group. CONCLUSION: Standard single-dose antibiotic prophylaxis did not lead to an increase in postoperative infectious complications in patients with mild acute cholecystitis undergoing cholecystectomy. Registration number: NTR3089 (www.trialregister.nl).

Bacteraemic and non-bacteraemic/urinary antigen-positive pneumococcal community-acquired pneumonia compared.

The diagnosis of invasive pneumococcal pneumonia is based mainly on bacteraemia. Episodes without bacteraemia, but with a positive urinary antigen test (UAT), are considered non-invasive. We determined differences in outcome between patients with bacteraemic and non-bacteraemic/UAT-positive pneumococcal community-acquired pneumonia (CAP). Adult patients with clinical and radiological evidence of CAP with blood cultures and UAT tests performed at presentation in three Dutch laboratories between June 2008 and May 2010 were included. Clinical characteristics were retrospectively extracted from hospital records. Overall, 168 patients had non-bacteraemic/UAT-positive pneumococcal CAP and 123 had bacteraemic pneumococcal CAP. The day-30 mortality was 9% and 13% for non-bacteraemic/UAT-positive and bacteraemic pneumococcal CAP patients, respectively [risk difference -4%, 95% confidence interval (CI) -11% to +3%, p = 0.28]. In a multivariable logistic regression model, age ≥ 65 years, admission to the intensive care unit/coronary care unit (ICU/CCU) and presence of an immunocompromising condition were associated with day-30 mortality. A non-significant association with mortality was found for bacteraemia [odds ratio (OR) 2.21, 95% CI 0.94-5.21, p = 0.07). No such trend was found for UAT positivity. The median lengths of hospital stay were 8 [interquartile range (IQR) 5-14] and 10 (IQR 6-18) days for non-bacteraemic/UAT-positive and bacteraemic pneumococcal CAP patients, respectively (p = 0.05). As compared to non-bacteraemic/UAT-positive pneumococcal CAP, bacteraemic pneumococcal CAP has a stronger association with day-30 mortality.

Increased incidence of serotype-1 invasive pneumococcal disease in young female adults in The Netherlands.

Analysis of the Dutch national invasive pneumococcal disease (IPD) surveillance data by sex reveals an increase in the incidence of serotype-1 disease in young female adults in The Netherlands after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in the national immunization schedule. This has led to an overall increase in IPD in women aged 20-45 years, which was not observed in men of the same age. No other differences in serotype shifts possibly induced by the introduction of PCV7 were observed between the sexes in this age group. Serotype 1 is a naturally fluctuating serotype in Europe and it has been associated with disease in young healthy adults before. It remains uncertain whether or not there is an association between the observed increase in serotype-1 disease in young female adults and the implementation of PCV7 in The Netherlands.

Incidence of bactobilia increases over time after endoscopic sphincterotomy.

Patients with choledochocystolithiasis are usually treated by endoscopic retrograde cholangiography with endoscopic sphincterotomy (ES) followed by laparoscopic cholecystectomy (LC). LC after ES is more difficult than in uncomplicated gallstone disease, possibly due to bacterial colonization of the common bile duct. The goal of this study was to evaluate if bactobilia influences the peri- and postoperative outcomes. Data were obtained from a randomized trial on the timing of LC after ES. Ninety-six patients were randomized after ES to LC either within 72 h (early LC [ELC]) or in 6-8 weeks (delayed LC [DLC]). In 64 of 96 patients bile samples were obtained peroperatively. The overall prevalence of bactobilia was 62.5% [40/64; 50% of ELC patients (n = 13) vs. 71.1% in the DLC group (n = 27); p = 0.088]. Age and group (i.e. ELC/DLC) were independent and significant predictors for the presence of bactobilia. The presence of bactobilia did not influence operating time and difficulty or conversion rate. Patients with bactobilia developed more biliary events in the period between ES and LC (44 vs. 28%). After ES for choledochocystolithiasis, 62.5% of patients have bactobilia at the time of surgery. The prevalence of bactobilia increases with age and time. Patients with bactobilia tend to develop more biliary-related complications awaiting surgery.

Highly resistant gram-negative microorganisms: incidence density and occurrence of nosocomial transmission (TRIANGLe Study).

OBJECTIVES: The objectives of this study were to determine the incidence density and the occurrence of horizontal spread of highly resistant gram-negative rods (HR-GNRs) in Dutch hospitals. The factors that influence these outcome measures were also investigated. METHODS: All patients with HR-GNRs, as determined by sample testing, who were hospitalized in 1 of 18 hospitals during a 6-month period (April through October 2007) were included in this study. For all available isolates, the species was identified, susceptibility was determined (including the presence of extended-spectrum ß-lactamases [ESBLs]), and molecular typing was performed. On the basis of a combination of species identification, molecular typing, and epidemiological data, the occurrence of nosocomial transmission was determined. RESULTS: The mean incidence density of patients with HR-GNRs was 55 per 100,000 patient-days (cumulative incidence, 39 per 10,000 patients admitted). A facility being a university hospital was a statistically significant (P = .03) independent determinant of a higher incidence of patients with HR-GNRs. The majority of HR-GNR isolates were ESBL producers. The adjusted transmission index-the ratio between secondary and primary cases-in the participating hospitals ranged from 0.0 to 0.2. The overall adjusted transmission index of HR-GNRs was 0.07. No determinants for a higher transmission index were identified. DISCUSSION: The nosocomial transmission rate of HR-GNRs was relatively low in all hospitals where well-established transmission-based precautions were used. The incidence density of patients with HR-GNRs was higher in university hospitals, probably due to the patient population and the complexity of the care provided.

Impaired antibody response to conjugated meningococcal serogroup C vaccine in asplenic patients.

The purpose of this study was to determine the quantity and quality of antibodies against the meningococcal serogroup C (MenC) conjugated vaccine in asplenic patients. In 116 asplenic patients, antibody concentrations (IgG) were measured against meningococcal serogroup C before and after immunisation. Of MenC-specific IgG, both antibody avidity and subclasses of IgG1 and IgG2 were determined. The mean MenC IgG concentration rose from 0.16 µg/mL prior to vaccination to 3.69 µg/mL 3 weeks post-vaccination, with 67% of patients reaching the threshold of ≥ 2.0 µg/mL. The mean IgG concentration at 35 weeks post-vaccination was 3.10 µg/mL. IgG2 concentrations increased more than IgG1. Marginal avidity maturation was seen. Hypo-responders to the first MenC vaccine (IgG anti-MenC ≤ 2.0 µg/mL) were offered a booster dose. After revaccination, 59% reached the chosen IgG threshold. The IgG concentration rose from 0.29 to 1.12 µg/mL, with an increase in the IgG1/IgG2 ratio. Avidity indices remained below 33%. In asplenic patients, the quantity and quality of antibodies produced after one dose of conjugated MenC vaccination is lower than that observed in previous studies in healthy adults. Booster vaccination does, indeed, lead to a rise in IgG geometric mean concentrations (GMCs), but does not lead to higher avidity of antibodies.

[Invasive Lancefield group A streptococcal infections in the Netherlands]. / Invasieve infecties met streptokokken uit lancefield-groep A in Nederland.

After a steady decrease in morbidity and mortality resulting from severe group A streptococcal (GAS) infections, the 1980s witnessed a resurgence of invasive GAS disease. As a result a nationwide laboratory-based surveillance for invasive GAS infections was conducted at the National Institute of Public Health (RIVM) from 1994 to 2003. The estimated annual incidence ranged from 2.0 to 4.0 cases per 100,000 individuals per year. The case-fatality rate was 18% overall but varied substantially depending on the manifestation of the disease. GAS infections may be complicated by toxic shock-like syndrome (TSS) which is caused by bacterial exotoxins. Case fatality among TSS cases was 59%. The M-protein that extends from the cell membrane is used for sub-typing GAS in > 150 different M-types. Increased intrinsic virulence has been reported in Streptococcus pyogenes of certain M-types, notably M1 and M3. In the Netherlands these M-types have been independently associated with fatality. Over the last 50 years the genome of these M-types appears to have become enriched with phage-encoded virulence factors, possibly contributing to the altered epidemiology of invasive GAS disease. Despite this genetic plasticity, GAS have remained uniformly susceptible to penicillin. In-vitro studies have shown that the administration of immunoglobulin G can have a neutralising effect in cases ofTSS but clinical studies have failed to provide any statistical support for this.

[HIV should become a notifiable disease]. / Hiv moet meldingsplichtig worden.

In recent years, it has become evident that primary HIV infections are largely responsible for new cases. In order to identify the chains of transmission involved, HIV should become a notifiable disease in the Netherlands.

[The Dutch MRSA policy should be retained]. / Het Nederlandse MRSA-beleid is succesvol.

The Dutch methicillin-resistant Staphylococcus aureus (MRSA) 'search and destroy' policy is effective. MRSA should be banned from hospitals: MRSA infections are associated with increased mortality and costs. In addition, widespread use of vancomycin for treating MRSA infections encourages the spread and development of vancomycin-resistant micro-organisms.

Genes for the majority of group a streptococcal virulence factors and extracellular surface proteins do not confer an increased propensity to cause invasive disease.

BACKGROUND: The factors behind the reemergence of severe, invasive group A streptococcal (GAS) diseases are unclear, but it could be caused by altered genetic endowment in these organisms. However, data from previous studies assessing the association between single genetic factors and invasive disease are often conflicting, suggesting that other, as-yet unidentified factors are necessary for the development of this class of disease. METHODS: In this study, we used a targeted GAS virulence microarray containing 226 GAS genes to determine the virulence gene repertoires of 68 GAS isolates (42 associated with invasive disease and 28 associated with noninvasive disease) collected in a defined geographic location during a contiguous time period. We then employed 3 advanced machine learning methods (genetic algorithm neural network, support vector machines, and classification trees) to identify genes with an increased association with invasive disease. RESULTS: Virulence gene profiles of individual GAS isolates varied extensively among these geographically and temporally related strains. Using genetic algorithm neural network analysis, we identified 3 genes with a marginal overrepresentation in invasive disease isolates. Significantly, 2 of these genes, ssa and mf4, encoded superantigens but were only present in a restricted set of GAS M-types. The third gene, spa, was found in variable distributions in all M-types in the study. CONCLUSIONS: Our comprehensive analysis of GAS virulence profiles provides strong evidence for the incongruent relationships among any of the 226 genes represented on the array and the overall propensity of GAS to cause invasive disease, underscoring the pathogenic complexity of these diseases, as well as the importance of multiple bacteria and/or host factors.

Epidemiological considerations following long-term surveillance of invasive group A streptococcal disease in The Netherlands, 1992-2003.

A nationwide laboratory-based surveillance system for invasive group A streptococcal (GAS) infections was conducted in The Netherlands from March 1992 until December 2003. Until 1996, all isolates submitted were evaluated clinically and demographically. During this period there was a transition from passive to active surveillance for some of the participating laboratories, corresponding to a national coverage of 50%. During active surveillance, participating laboratories submitted twice as many isolates from invasive GAS disease, whereas the relative submission of isolates representing very severe manifestations (toxic shock-like syndrome, fatality) did not increase. From 1997 onwards, invasiveness was defined solely on the basis of source of isolation (without clinical evaluation). During the period of microbiological and clinical evaluation, microbiological evaluation alone was found to be specific (> 99%), but had limited sensitivity (66%). Estimation of the true rate of invasive GAS disease should be based on an active surveillance system with inclusion of both microbiological and clinical data.

Long-term surveillance of invasive group A streptococcal disease in The Netherlands, 1994-2003.

A nationwide laboratory-based surveillance study of invasive group A streptococcal (GAS) infections was conducted in The Netherlands from May 1994 until December 2003 (average population during this period was 15 729 704). Microbiologically invasive isolates were obtained from 1504 patients, with most (70%) isolates cultured from blood. There was a clear seasonal pattern in invasive streptococcal infections, with an estimated annual incidence that peaked in 1996 (4.0 cases/100 000 individuals/year) and was at its lowest in 1999 (2.0 cases/100 000 individuals/year). Twenty-eight different M-types were identified, of which the most frequent were M1 (339/1504, 23%), M3 (187/1504, 12%), M89 (174/1504, 12%), M28 (164/1504, 11%), M12 (109/1504, 7%) and M6 (55/1504, 4%). There was a high degree of variation in the relative annual contributions of the predominant M-types, but variations in M1 and M3 combined correlated with overall changes in the annual incidence. The contribution of the patient group aged > or = 56 years to all cases of invasive GAS disease increased during the study period, whereas that of the group aged 0-20 years decreased. A peak in the incidence of invasive GAS disease among the patient group aged 30-34 years did not vary during the study period, indicating that the high incidence of invasive GAS disease in this age group was age-specific rather than cohort-related.

Epidemiological features of invasive and noninvasive group A streptococcal disease in the Netherlands, 1992-1996.

A prospective, nationwide, laboratory-based surveillance of invasive group A streptococcal infections was conducted in the Netherlands from 1992 through 1996. Clinical and demographic data were obtained and all isolates were T/M typed. All noninvasive group A streptococcal isolates were registered from 1994 through 1996. A total of 880 patients with invasive streptococcal disease were identified. The annual incidence was found to be 2.2 per 100,000. Predominant M types were M1 (21%), M3 (11%), M6 (5%), M12 (5%), and M28 (8%). Particular age and M-type distributions were observed in different clinical entities. The case-fatality rate was 18% overall, but it reached 59% among cases of toxic shock-like syndrome. Older age, necrotizing fasciitis, sepsis without focus, pneumonia, infection with type M1 or M3 strains, and underlying cardiopulmonary disease were associated with fatality. A total of 10,105 patients with noninvasive group A streptococcal disease were registered. These patients differed significantly from patients with invasive disease with regard to age distribution and primary foci of infection.