Search

SUMMARY We investigated the positive predictive value (PPV) of a solitary positive immunoglobulin M (IgM) phase II response for the serodiagnosis of acute Q fever detected with either an indirect immunofluorescence assay (IFA) or an enzyme-linked immunosorbent assay (ELISA). Initial and follow-up sera from patients suspected of acute Q fever were included if initially only IgM phase II tested positive with IFA in 2008 (n=92), or ELISA in 2009 (n=85). A seroconversion for Q fever was defined as an initial sample being IgG phase II negative but positive in the follow-up sample. The PPV of an initial isolated IgM phase II result detected by IFA or ELISA was 65% and 51%, respectively, and therefore appeared not to adequately predict acute Q fever. For this reason it cannot be used as a diagnostic criterion nor should it be included in public health notification without confirmation with other markers or a follow-up serum sample. [ABSTRACT FROM AUTHOR]

BackgroundIn the Netherlands, obstacle, mud and survival runs are increasingly popular. Although outbreaks of gastroenteritis have been reported following these events, associated health risks have not been systematically assessed.AimTo investigate the incidence of acute gastrointestinal infections (AGI), skin infections (SI) and respiratory infections (RI) among obstacle run participants, as well as risk factors.MethodsBetween April and October 2017, we conducted a retrospective cohort study among 2,900 participants of 17 obstacle runs in the Netherlands. Demographic, symptomatic and behavioural data were collected from participants via an online questionnaire 1 week after participation in an obstacle run. Stool specimens were obtained from respondents for microbiological tests. Adjusted relative risks (aRR) and 95% confidence intervals (CI) using multilevel binomial regression analysis were calculated.ResultsOf 2,646 respondents (median age: 33 years; 53% male), 76 had AGI after the obstacle run; ingesting mud was associated with AGI (aRR: 1.7; 95% CI: 1.2-4.9) and 38 respondents had AGI during or in the week before the obstacle run. Overall, 103 respondents reported SI and 163 RI. Rinsing off in a hot tub was associated with SI (aRR: 2.2; 95% CI: 1.7-2.8). Of 111 stool specimens, 13 tested positive for six different pathogens. No clusters were found.ConclusionThe reported incidence of AGI, SI and RI was low. Risk of these infections could be decreased by informing participants on preventive measures, e.g. showering vs rinsing in the hot tub, avoiding ingesting mud and not participating with symptoms of AGI.

This case study discusses how we diagnosed and treated a patient with a late haematogenous bilateral periprosthetic joint infection (PJI) after total knee arthroplasties caused by Ureaplasma urealyticum. This has never been reported before. We will discuss how we used a PET-CT, synovial fluid cell count, and synovial fluid analysis by 16S rRNA gene sequencing to diagnose this PJI. We will also discuss how we treated this patient to obtain full recovery.

Background: Serological follow-up of acute Q-fever patients is important for detection of chronic infection but there is no consensus on its frequency and duration. The 2007-2009 Q-fever epidemic in the Netherlands allowed for long-term follow-up of a large cohort of acute Q-fever patients. The aim of this study was to validate the current follow-up strategy targeted to identify patients with chronic Q-fever., Methods: A cohort of adult acute Q-fever patients, diagnosed between 2007 and 2009, for whom a twelve-month follow-up sample was available, was invited to complete a questionnaire and provide a blood sample, four years after the acute episode. Antibody profiles, determined by immunofluorescence assay in serum, were investigated with a special focus on high titres of IgG antibodies against phase I of Coxiella burnetii, as these are considered indicative for possible chronic Q-fever., Results: Of the invited 1,907 patients fulfilling inclusion criteria, 1,289 (67.6%) were included in the analysis. At any time during the four-year follow-up period, 58 (4.5%) patients were classified as possible, probable, or proven chronic Q-fever according to the Dutch Q-fever Consensus Group criteria (which uses IgG phase I ≥1:1,024 to as serologic criterion for chronic Q-fever). Fifty-two (89.7%) of these were identified within the first year after the acute episode. Of the six patients that were detected for the first time at four-year follow-up, five had an IgG phase I titre of 1:512 at twelve months., Conclusions: A twelve-month follow-up check after acute Q-fever is recommended as it adequately detects chronic Q-fever in patients without known risk factors. Additional serological and clinical follow-up is recommended for patients with IgG phase I ≥1:512, as they showed the highest risk to progress to chronic Q-fever.

Knowledge of Q fever has increased over the last decades, but research has mainly focused on adults. Data in children are scarce, and current knowledge is mostly based on case reports. The aim of this study was to determine predictors for acute Q fever in children in the general population. We retrospectively studied all children tested for Coxiella burnetii by serology and/or PCR upon request of their general practitioner in the regional laboratory for Medical Microbiology of the Jeroen Bosch during the Q fever outbreak in the Netherlands between 2007 and 2011. A total of 1061 patients was analyzed. Influenza-like illness and respiratory tract infection were the most common presentations of acute Q fever, mimicking other common childhood illnesses. None of the reported symptoms was significantly related to a positive test outcome and therefore presenting signs or symptoms have no predictive value in diagnosing Q-fever in children. Only diagnostic tests are reliable. As the infection generally follows a mild and uncomplicated course, we question if the difficulty of recognizing pediatric Q fever is a problem worth solving.

Between 2007 and 2010, the Netherlands experienced one of the largest outbreaks of Q fever. Since asymptomatic Coxiella burnetii infection has been associated with maternal and obstetric complications, evidence about the effectiveness of routine screening during pregnancy in outbreak areas is needed. We performed a clustered randomised controlled trial during the Dutch outbreak, in which 55 midwife centres were randomised to recruit pregnant women for an intervention or control strategy. In both groups a serum sample was taken between 20 and 32 weeks of gestation. In the intervention group (n=536), the samples were analysed immediately by indirect immunofluorescence assay for the presence of IgM and IgG (phase I/II) and treatment was given during pregnancy in case of an acute or chronic infection. In the control group (n=693), sera were frozen for analysis after delivery. In both groups 15% were seropositive. In the intervention group 2.2% of the women were seropositive and had an obstetric complication, compared with 1.4% in the control group (Odds ratio: 1.54 (95% confidence interval 0.60-3.96)). During a large Q fever outbreak, routine C. burnetii screening starting at 20 weeks of gestation was not associated with a relevant reduction in obstetric complications and should therefore not be recommended.

Large outbreaks of Q fever in The Netherlands have provided a unique opportunity for studying longitudinal serum antibody responses in patients. Results are presented of a cohort of 344 patients with acute symptoms of Q fever with three or more serum samples per patient. In all these serum samples IgM and IgG against phase 1 and 2 Coxiella burnetii were measured by an immunofluorescence assay. A mathematical model of the dynamic interaction of serum antibodies and pathogens was used in a mixed model framework to quantitatively analyse responses to C. burnetii infection. Responses show strong heterogeneity, with individual serum antibody responses widely different in magnitude and shape. Features of the response, peak titre and decay rate, are used to characterize the diversity of the observed responses. Binary mixture analysis of IgG peak levels (phases 1 and 2) reveals a class of patients with high IgG peak titres that decay slowly and may represent potential chronic cases. When combining the results of mixture analysis into an odds score, it is concluded that not only high IgG phase 1 may be predictive for chronic Q fever, but also that high IgG phase 2 may aid in detecting such putative chronic cases.

Little is known about the effect of timing of antibiotic treatment on development of IgG antibodies following acute Q fever. We studied IgG antibody responses in symptomatic patients diagnosed either before or during development of the serologic response to Coxiella burnetii. Between 15 and 31 May 2009, 186 patients presented with acute Q fever, of which 181 were included in this retrospective study: 91 early-diagnosed (ED) acute Q fever patients, defined as negative IgM phase II enzyme-linked immunosorbent assay (ELISA) and positive PCR, and 90 late-diagnosed (LD) acute Q fever patients, defined as positive/dubious IgM phase II ELISA and positive immunofluorescence assay (IFA). Follow-up serology at 3, 6, and 12 months was performed using IFA (IgG phase I and II). High IgG antibody titers were defined as IgG phase II titers of ≥1:1,024 together with IgG phase I titers of ≥1:256. At 12 months, 28.6% of ED patients and 19.5% of LD patients had high IgG antibody titers (P = 0.17). No statistically significant differences were found in frequencies of IgG phase I and IgG phase II antibody titers at all follow-up appointments for adequately and inadequately treated patients overall, as well as for ED and LD patients analyzed separately. Additionally, no significant difference was found in frequencies of high antibody titers and between early (treatment started within 7 days after seeking medical attention) and late timing of treatment. This study indicates that early diagnosis and antibiotic treatment of acute Q fever do not prohibit development of the IgG antibody response.

A commercially available enzyme-linked immunosorbent assay (ELISA) detecting Coxiella burnetii phase II-specific IgM for the diagnosis of acute Q fever was compared with indirect immunofluorescent antibody assay (IFA). IFA is the current reference method for the detection of antibodies against C. burnetii, but has disadvantages because the judgment of fluorescence is subjective and tiring, and the test is expensive and automation is not possible. To examine whether phase II IgM ELISA could be used as a screening assay for acute Q fever, we compared the sensitivity and specificity of IFA and ELISA. The sensitivity of the IFA and ELISA tests were 100 and 85.7%, respectively, with a specificity of 95.3 and 97.6%, respectively. Because of the high sensitivity and specificity of the ELISA in combination with the practical disadvantages of the IFA, we introduced a new algorithm to screen samples of patients with symptoms of acute Q fever infection.

In Europe the incidence of human Q fever has dramatically increased over the previous years. Untreated infections with Coxiella burnetii, the causal agent of Q fever, have been associated with both obstetric and maternal complications. The majority of pregnant women with a C. burnetii infection remain asymptomatic, hence screening could be of value to prevent unwanted outcomes in this high-risk group. We applied the updated Wilson and Jungner criteria to review the evidence for routine screening for C. burnetii infection during pregnancy. Since much uncertainty remains about the incidence, clinical consequences, diagnostics and treatment of C. burnetii infection during pregnancy, routine screening for C. burnetii infection during pregnancy should not be recommended. Rigorous studies to assess the effectiveness of C. burnetii screening are warranted.

In 2005, Q fever was diagnosed on two dairy goat farms and 2 years later it emerged in the human population in the south of the Netherlands. From 2007 to 2010, more than 4,000 human cases were notified with an annual seasonal peak. The outbreaks in humans were mainly restricted to the south of the country in an area with intensive dairy goat farming. In the most affected areas, up to 15% of the population may have been infected. The epidemic resulted in a serious burden of disease, with a hospitalisation rate of 20% of notified cases and is expected to result in more cases of chronic Q fever among risk groups in the coming years. The most important risk factor for human Q fever is living close (<5 km) to an infected dairy goat farm. Occupational exposure plays a much smaller role. In 2009 several veterinary control measures were implemented including mandatory vaccination of dairy goats and dairy sheep, improved hygiene measures, and culling of pregnant animals on infected farms. The introduction of these drastic veterinary measures has probably ended the Q fever outbreak, for which the Netherlands was ill-prepared.

During 2007-2009, we tested serum samples from 2,004 pregnant women living in an area of high Q fever incidence in the Netherlands. Results confirmed that presence of antibodies against Coxiella burnetii is related to proximity to infected dairy goat farms. Pregnant women and patients with certain cardiovascular conditions should avoid these farms.

Background: Sclerotherapy with foam is becoming increasingly popular for the treatment of varicose veins. There is no consensus on the necessity of sterile air or other gases to produce foam., Objectives: To evaluate the potential risk of bacterial inoculation of polidocanol (POL) foam using room air and the antimicrobial properties of polidocanol., Materials and Methods: The amount of airborne microorganisms was quantitatively measured. Four bacterial strains were tested for susceptibility to polidocanol: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Streptococcus pyogenes., Results: Air measurements varied as a result of air movement and the number of people in the room. Although the risk of introducing one colony-formin unit can be calculated as less than 1 in 330, the clinical relevance is still to be determined. No inhibition of bacterial growth was achieved with POL in of any of the tested strains., Conclusions: Foam sclerotherapy with POL prepared in a standard treatment room is a safe procedure without the risk of introducing a severe bacterial complication. The use of sterile air, nitrogen, or carbon dioxide is unnecessary and will make foam sclerotherapy with POL more elaborate and more expensive to use., (© 2011 by the American Society for Dermatologic Surgery, Inc.)

Background: Recent outbreaks in the Netherlands allowed for laboratory follow-up of a large series of patients with acute Q fever and for evaluation of test algorithms to detect chronic Q fever, a condition with considerable morbidity and mortality., Methods: For 686 patients with acute Q fever, IgG antibodies to Coxiella burnetii were determined using an immunofluorescence assay at 3, 6, and 12 months of follow-up. Polymerase chain reaction (PCR) was performed after 12 months and on earlier serum samples with an IgG phase I antibody titer ≥ 1:1024., Results: In 43% of patients, the IgG phase II antibody titers remained high (≥ 1:1024) at 3, 6, and 12 months of follow-up. Three months after acute Q fever, 14% of the patients had an IgG phase I titer ≥ 1:1024, which became negative later in 81%. IgG phase I antibody titers were rarely higher than phase II titers. Eleven cases of chronic Q fever were identified on the basis of serological profile, PCR results, and clinical presentation. Six of these patients were known to have clinical risk factors at the time of acute Q fever. In a comparison of various serological algorithms, IgG phase I titer ≥ 1:1024 at 6 months had the most favorable sensitivity and positive predictive value for the detection of chronic Q fever., Conclusions: The wide variation of serological and PCR results during the follow-up of acute Q fever implies that the diagnosis of chronic Q fever, necessitating long-term antibiotic treatment, must be based primarily on clinical grounds. Different serological follow-up strategies are needed for patients with and without known risk factors for chronic Q fever.

In the peak of the 2009 Q fever outbreak in the Netherlands, we introduced a diagnostic algorithm for acute Q fever with an enzyme-linked immunosorbent assay for immunoglobulin M antibodies to Coxiella burnetii phase II antigens (MII screen) as an initial step. Subsequently, an immunofluorescence assay or PCR was performed depending on the MII screen outcome, date of onset of disease, and inpatient or outpatient setting. The impact of MII screen on the number of immunofluorescence assays performed and the contribution of PCR to diagnosis were retrospectively evaluated in 825 patients referred in a 17-day period. Acute Q fever was diagnosed in 256 patients. The introduction of MII screen reduced the number of immunofluorescence assays performed by more than 80%. In 103 patients, PCR analysis contributed to the diagnosis of acute Q fever. Q fever diagnostics were hampered by the fact that for a high number of patients the date of onset of disease was not provided and the requested follow-up serum samples were not received.

Background: Q fever has become a major public health problem in The Netherlands. Infection with Coxiella burnetii (Q fever) during pregnancy has resulted in adverse pregnancy outcome in the majority of reported cases. Therefore, we aimed to quantify this risk by examining the earliest periods corresponding to the epidemic in The Netherlands., Methods: Serum samples that had been collected from the area of highest incidence by an existing national prenatal screening programme and data from the Netherlands Perinatal Registry (PRN) on diagnosis and outcome were used. We performed indirect immunofluorescence assay to detect the presence of IgM and IgG antibodies against C. burnetii in the samples. The serological results were analyzed to determine statistical association with recorded pregnancy outcome., Results: Evaluation of serological results for 1174 women in the PRN indicated that the presence of IgM and IgG antibodies against phase II of C. burnetii was not significantly associated with preterm delivery, low birth weight, or several other outcome measures., Conclusion: The present population-based study showed no evidence of adverse pregnancy outcome among women who had antibodies to C. burnetii during early pregnancy.

A 42-year-old woman visited the pulmonologist for follow-up after a pneumonia. In retrospect the pneumonia appeared to be a manifestation of an acute Q fever infection. A few weeks later the patient was found to be unexpectedly pregnant. At the normal serological follow-up six months after the primary infection chronic Q fever infection was diagnosed. Doxycycline and hydroxychloroquine are contraindicated in pregnancy and the patient was found to be allergic to co-trimoxazole. Therefore treatment with erythromycin was chosen on empirical grounds. The patient had many symptoms during pregnancy. After 38 weeks and 2 days amenorrhea labour was induced on maternal indication. Finally a healthy boy of 3850 grams was born by caesarean section. In view of the increased risk of chronic Q fever infection during pregnancy we advise intensified serological monitoring of patients with acute Q fever who subsequently become pregnant.

Background: In The Netherlands the largest human Q fever outbreak ever reported in the literature is currently ongoing with more than 2300 notified cases in 2009. Pregnant women are particularly at risk as Q fever during pregnancy may cause maternal and obstetric complications. Since the majority of infected pregnant women are asymptomatic, a screening strategy might be of great value to reduce Q fever related complications. We designed a trial to assess the (cost-)effectiveness of a screening program for Q fever in pregnant women living in risks areas in The Netherlands., Methods/design: We will conduct a clustered randomized controlled trial in which primary care midwife centres in Q fever risk areas are randomized to recruit pregnant women for either the control group or the intervention group. In both groups a blood sample is taken around 20 weeks postmenstrual age. In the intervention group, this sample is immediately analyzed by indirect immunofluorescence assay for detection of IgG and IgM antibodies using a sensitive cut-off level of 1:32. In case of an active Q fever infection, antibiotic treatment is recommended and serological follow up is performed. In the control group, serum is frozen for analysis after delivery. The primary endpoint is a maternal (chronic Q fever or reactivation) or obstetric complication (low birth weight, preterm delivery or fetal death) in Q fever positive women. Secondary aims pertain to the course of infection in pregnant women, diagnostic accuracy of laboratory tests used for screening, histo-pathological abnormalities of the placenta of Q fever positive women, side effects of therapy, and costs. The analysis will be according to the intention-to-screen principle, and cost-effectiveness analysis will be performed by comparing the direct and indirect costs between the intervention and control group., Discussion: With this study we aim to provide insight into the balance of risks of undetected and detected Q fever during pregnancy., Trial Registration: ClinicalTrials.gov, protocol record NL30340.042.09.

The world's largest Q fever outbreak is ongoing in The Netherlands with around 3,000 confirmed cases since the first half of 2007. Increased awareness has resulted in early referral of patients for diagnostics. An important drawback to serological diagnosis of acute Q fever is the lag phase in antibody response. Therefore, we evaluated the performance of a real-time PCR for detection of Coxiella burnetii DNA using serum samples from patients with acute Q fever. PCR, targeting IS1111, was retrospectively performed on acute-phase and follow-up convalescent-phase serum samples from 65 patients with acute Q fever as diagnosed by immunofluorescence assay. The results obtained by PCR were related to disease stage as defined by subsequent appearance of phase II IgM, phase II IgG, phase I IgM, and phase I IgG (IgM-II, IgG-II, IgM-I, and IgG-I, respectively) antibodies and time since onset of disease. In addition, we analyzed seronegative acute-phase serum samples from patients with inconclusive Q fever serology, because no convalescent-phase serum samples were available. PCR was scored positive in 49/50 (98%) seronegative sera, 9/10 (90%) sera with isolated IgM-II antibodies, 3/13 (23%) sera with IgM-II/IgG-II antibodies, 2/41 (5%) sera with IgM-II/IgG-II/IgM-I antibodies, 0/15 (0%) sera with IgM-II/IgG-II/IgM-I/IgG-I antibodies, and 0/1 (0%) serum sample with IgM-II/IgG-II/IgG-I antibodies. The latest time point after onset of disease in which C. burnetii DNA could be detected was at day 17. In patients with inconclusive Q fever serology, PCR was positive in 5/50 (10%) cases. We conclude that real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever. C. burnetii DNA becomes undetectable in serum as the serological response develops.

Background: Query-fever (Q-fever) is a zoonotic infection caused by the intracellular Gram-negative coccobacillus Coxiella burnetii. A large ongoing outbreak of Q-fever has been reported in the Netherlands. We studied various markers of infection in inpatients (hospitalised) and outpatients (treated by a general physician) with acute Q-fever in relation to disease severity., Methods: Leukocyte counts, C-reactive protein (CRP) and procalcitonin (PCT) concentrations were measured in 25 inpatients and 40 outpatients upon presentation with acute Q-fever. Chest X-rays, if available, were analysed and confusion, urea, respiratory rate, blood pressure-age 65 (CURB-65) scores, indicating severity of pneumonia, were calculated., Results: CRP was the only marker that significantly differentiated between inpatients and outpatients. It was increased in all patients from both groups. Leukocyte counts and PCT concentrations did not differ between inpatients and outpatients. Overall, only 13/65 patients had an increased leukocyte count and only 11/65 patients presented with PCT concentrations indicative of possible bacterial respiratory tract infection. Infiltrative changes on the chest X-ray were observed in the majority of patients. CURB-65 score was 0+/-1 (mean+/-SD)., Conclusions: Acute Q-fever, a relatively mild pneumonia with low CURB-65 scores, specifically induces a response in CRP, while PCT concentrations and leukocytes are within the normal range or increased only marginally.

In The Netherlands it has been shown that people in contact with pigs have a higher risk of meticillin-resistant Staphylococcus aureus (MRSA) carriage than the general population. Isolates of closely related spa types, corresponding to multilocus sequence type (MLST) ST398, were found in pig farmers, pig veterinarians and pigs. The objective of this study was to investigate whether contact with pigs and veal calves or other livestock is a risk factor for MRSA carriage in Dutch healthcare workers (HCWs). HCWs at four general hospitals and one university hospital were asked to fill in questionnaires covering contact with animals and to take MRSA cultures of their throat and nares. Cultures of HCWs in contact with livestock were processed with samples from HCWs with no contact with livestock as controls. Seventy-seven of 1721 HCWs (4.4%) reported direct or indirect contact with pigs and/or veal calves and 145 reported contact with other livestock animals. The MRSA carriage rate in the group in contact with pigs and veal calves was 1.7% and in the control group was 0.15%. No carriers were found among HCWs in contact with other livestock. An estimated 3% of hospital staff working in Dutch hospitals serving rural populations belong to a high risk group for MRSA carriage according to the Dutch guidelines. Although MRSA carriage in HCWs in contact with livestock is 10-fold higher than in other HCWs, the difference is not statistically significant.

Objective: To determine the antibiotic sensitivity of methicillin-resistant Staphylococcus aureus (MRSA) isolated from persons in contact with pigs., Design: Retrospective., Method: The pig-related MRSA collection, built up between January 1st 2003 and November 30th 2006 in the Regional Laboratory for Medical Microbiology and Infection Prevention (RLMMI) of the Jeroen Bosch Hospital, Den Bosch, The Netherlands, was tested for sensitivity to a large number ofantibiotics., Results: A total of 65 isolates were obtained from 53 patients. All (100%) of the pig-related MRSA isolates were sensitive to vancomycin, teicoplanin, nitrofurantoin, rifampicin, linezolid, and quinupristin-dalfopristin. Variable sensitivity was found for erythromycin (40%), clindamycin (48%), cotrimoxazole (48%), aminoglycosides (92%), tetracycline (6%), and quinolones (94%)., Conclusion: In view of the sensitivities found, clindamycin does not seem suitable for the empirical therapy ofpig-related MRSA-infections. In case of severe infection, therapy should be started either with an intravenous glycopeptide or with oral ciprofloxacin, possibly combined with rifampicin or linezolid.

Background: Previous studies on the molecular epidemiology of hepatitis A virus (HAV) in Amsterdam, The Netherlands, show that subgenotype 1A is mainly seen among homosexual men practising anonymous oral-anal sex in saunas and darkrooms, while subgenotype 1B is usually detected among children originating from Morocco, and subgenotype 3A is mostly found among travellers to Pakistan., Objective: We studied the genotype distribution in a more rural area of The Netherlands, Noord-Brabant, and compared it with Amsterdam., Study Design: We collected blood and feces samples from 34 HAV IgM(+) individuals who were reported from August 2001-March 2003 at the Municipal Health Service (MHS) Heart for Brabant (Brabant). We also collected feces samples from nine household contacts of whom the HAV IgM status was not known. HAV RNA was isolated and subsequently amplified by reverse transcriptase polymerase chain reaction (RT-PCR) at the VP1-P2a and the VP3-VP1 region, sequenced and analysed., Results and Conclusions: In most cases, relations between risk groups and HAV subgenotypes in Noord-Brabant were similar to those in Amsterdam. Next to genotypes 1 and 3 we also detected a genotype 2/7 strain in a Noord-Brabant case. Also, in contrast to the Amsterdam study, sporadic transmission occurred among various risk groups. Children involved in a school-related outbreak were infected with strains identical to one that was previously isolated from a man who has sex with men (MSM). Also, Dutch patients having no epidemiological link with Turkish or Moroccan children harboured strains imported from high-endemic countries. Furthermore, we report a special case in which HAV may be causally involved in meningitis. The results of this study show that the molecular epidemiology of HAV in The Netherlands can be more complicated than previously anticipated and that HAV phylogenetic studies can provide important information for the design of appropriate public health measures.

After five patients were diagnosed with nosocomial invasive aspergillosis caused by Aspergillus fumigatus and A. flavus, a 14-month surveillance program for pathogenic and nonpathogenic fungal conidia in the air within and outside the University Hospital in Rotterdam (The Netherlands) was begun. A. fumigatus isolates obtained from the Department of Hematology were studied for genetic relatedness by randomly amplified polymorphic DNA (RAPD) analysis. This was repeated with A. fumigatus isolates contaminating culture media in the microbiology laboratory. The density of the conidia of nonpathogenic fungi in the outside air showed a seasonal variation: higher densities were measured during the summer, while lower densities were determined during the fall and winter. Hardly any variation was found in the numbers of Aspergillus conidia. We found decreasing numbers of conidia when comparing air from outside the hospital to that inside the hospital and when comparing open areas within the hospital to the closed department of hematology. The increase in the number of patients with invasive aspergillosis could not be explained by an increase in the number of Aspergillus conidia in the outside air. The short-term presence of A. flavus can only be explained by the presence of a point source, which was probably patient related. Genotyping A. fumigatus isolates from the department of hematology showed that clonally related isolates were persistently present for more than 1 year. Clinical isolates of A. fumigatus obtained during the outbreak period were different from these persistent clones. A. fumigatus isolates contaminating culture media were all genotypically identical, indicating a causative point source. Knowledge of the epidemiology of Aspergillus species is necessary for the development of strategies to prevent invasive aspergillosis. RAPD fingerprinting of Aspergillus isolates can help to determine the cause of an outbreak of invasive aspergillosis.

It has been suggested that a better outcome of neutropenia-associated invasive fungal infections can be achieved when high doses of lipid formulations of amphotericin B are used. We now report a randomized multicentre study comparing liposomal amphotericin B (AmBisome, 5 mg/kg/d) to amphotericin B deoxycholate (AmB, 1 mg/kg/d) in the treatment of these infections. Of 106 possible patients, 66 were enrolled and analysed for efficacy: nine had documented fungaemia, 17 had other invasive mould infections and 40 had suspected pulmonary aspergillosis. After completion of the course medication, in the AmBisome group (n = 32) 14 patients had achieved complete response, seven a partial response and 11 were failures as compared to 6, 13 and 15 patients (n = 34) treated with AmB (P=0.09); P=0.03 for complete responders. A favourable trend for AmBisome was found at day 14, in patients with documented infections and in patients with pulmonary aspergillosis (P=0.05 and P=0.096 respectively). Mortality rates were lower in patients treated with AmBisome (adjusted for malignancy status, P=0.03). More patients on AmB had a >100% increase of their baseline serum creatinine (P<0.001). The results indicate that, in neutropenic patients with documented or suspected invasive fungal infections AmBisome 5 mg/kg/d was superior to AmB 1 mg/kg/d with respect to efficacy and safety.

Objective: Amphotericin B deoxycholate initial therapy and fluconazole maintenance therapy is the treatment of choice for AIDS-associated cryptococcal meningitis. However, the administration of amphotericin B is associated with considerable toxicity. A potential strategy for reducing the toxicity and increasing the therapeutic index of amphotericin B is the use of lipid formulations of this drug., Design and Methods: HIV-infected patients with cryptococcal meningitis were randomized to treatment with either liposomal amphotericin B (AmBisome) 4 mg/kg daily or standard amphotericin B 0.7 mg/kg daily for 3 weeks, each followed by fluconazole 400 mg daily for 7 weeks. During the first 3 weeks, clinical efficacy was assessed daily. Mycological response was primarily evaluated by cerebrospinal fluid (CSF) cultures at days 7, 14, 21 and 70., Results: Of the 28 evaluable patients, 15 were assigned to receive AmBisome and 13 to receive amphotericin B. Baseline characteristics were comparable. The time to and the rate of clinical response were the same in both arms. AmBisome therapy resulted in a CSF culture conversion within 7 days in six out of 15 patients versus one out of 12 amphotericin B-treated patients (P = 0.09), within 14 days in 10 out of 15 AmBisome patients versus one out of nine amphotericin B patients (P = 0.01), and within 21 days in 11 out of 15 AmBisome patients versus three out of eight amphotericin B patients (P = 0.19). When Kaplan-Meier estimates were used to compare time to CSF culture conversion, AmBisome was more effective (P < 0.05; median time between 7 and 14 days for AmBisome versus > 21 days for amphotericin B). AmBisome was significantly less nephrotoxic., Conclusions: A 3-week course of 4 mg/kg AmBisome resulted in a significantly earlier CSF culture conversion than 0.7 mg/kg amphotericin B, had equal clinical efficacy and was significantly less nephrotoxic when used for the treatment of primary episodes of AIDS-associated cryptococcal meningitis.

Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, serotype, and killer toxin sensitivity patterns of a wide range of saprobic, clinical, and veterinary isolates of both varieties of Cryptococcus neoformans were examined. C. neoformans var. neoformans and C. neoformans var. gattii differed in chromosomal makeup, RAPD patterns, and killer sensitivity patterns. These results suggest that there are two separate species rather than two varieties. No clear genetic or phenotypic differences were observed among the clinical, saprobic, and veterinary isolates within each taxon. The serotypes differed substantially in their RAPD characteristics. Geographical clustering was observed among the isolates of C. neoformans var. gattii, but not among the isolates of C. neoformans var. neoformans. The isolates of each taxon that originated from restricted geographical areas often had identical or similar karyotypes and RAPD patterns, suggesting that clonal reproduction had occurred. The combination of PFGE and RAPD analysis allowed us to distinguish almost all isolates. This combination of techniques is recommended for further research on epidemiological, ecological, and population issues.

Despite its considerable toxicity, amphotericin B (AmB) remains the 'golden standard' in the treatment of many systemic fungal infections. To reduce this toxicity, with the aim of increasing its therapeutic index, AmB can be encapsulated into liposomes or bound to lipid carriers. Following promising clinical results with investigational formulations, three industrial compounds are available at this moment: Abelcet (Amphotericin B Lipid Complex, ABLC), Amphocil (Amphotericin B Colloidal Dispersion) and AmBisome. These three formulations differ significantly in composition and pharmacokinetics. All three compounds share a considerable reduction of nephrotoxicity, but the number of acute reactions differ among these compounds, Amphocil showing the highest and AmBisome the lowest rate. Increased therapeutic indexes for all three formulations were shown only in some of the animal models for several fungal infections. Four recent clinical trials comparing these formulations with AmB demonstrated their clinical efficacy but failed to clearly show an increased therapeutic index. Therefore these compounds can be recommended in cases of intolerance to or failure on AmB therapy. The optimal therapeutic dosages have not been established, but dosages as low as 1 mg/kg should probably be avoided in the initial treatment of fulminant fungal infections, since efficacy may be inferior to equal dosages of conventional AmB.

The efficacy of AmBisome, a liposomal formulation of amphotericin B, was compared with that of Fungizone (amphotericin B desoxycholate), in a rat model of unilateral, pulmonary aspergillosis. Repeated administration of cyclophosphamide resulted in persistent, severe granulocytopenia. The left lung was inoculated with a conidial suspension of Aspergillus fumigatus, thus establishing an unilateral infection. Antifungal treatment was started 40 h after fungal inoculation, at which time mycelial disease was confirmed by histological examination. Both Fungizone 1 mg/kg and AmBisome 10 mg/kg resulted in increased survival in terms of delayed as well as reduced mortality. Quantitative cultures of lung tissue showed that only AmBisome 10 mg/kg resulted in reduction of the number of fungal cfus in the inoculated left lung. Compared with Fungizone, both AmBisome 1 mg/kg/day and AmBisome 10 mg/kg/day significantly prevented dissemination from the infected left lung to the right lung. In addition, both AmBisome regimens reduced hepatosplenic dissemination, and the 10 m/kg dosage fully prevented this complication. In conclusion, when compared with Fungizone, in this model AmBisome is more effective in reducing dissemination of unilateral, pulmonary aspergillosis, even when given in relatively low dosage. Such low dosages may have a place in prophylactic settings.