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Background: Allograft bone used in joint replacement surgery can additionally serve as a carrier for antibiotics and serve as a prophylaxis against infections. However, in vitro dose-response curves for bone chips impregnated with different kinds of antibiotics are not available. In addition, while it would be desirable to add the antibiotics to allograft bone chips before these are stored in a bone bank, the effects of different storage temperatures on antibiotics are unknown.Methods: Five different antibiotics (cefazolin, clindamycin, linezolid, oxacillin, vancomycin) were stored, both as pills and as solutions, at -80 degrees C, -20 degrees C, 4 degrees C, 20 degrees C and 37 degrees C; in addition, bone chips impregnated with cefazolin and vancomycin were stored at -80 degrees C and -20 degrees C. After 1 month, 6 months and 1 year, the activity of the antibiotics against Staphylococcus epidermidis was measured using an inoculated agar. The diameter of the S. epidermidis-free zone was taken as a measure of antibiotic activity. In a separate experiment, in vitro dose-response curves were established for bone chips impregnated with cefazolin and vancomycin solutions at five different concentrations. Finally, the maximum absorbed amounts of cefazolin and vancomycin were established by impregnating 1 g of bone chips with 5 ml of antibiotic solution.Results: A decrease of the S. epidermidis-free zone was seen with oxacillin and cefazolin solutions stored at 37 degrees C for 1 month, with vancomycin stored at 37 degrees C for 6 months and with cefazolin and oxacillin solutions stored at 20 degrees C for 6 months. The activity of the other antibiotic solutions, pills and impregnated bone chips was not affected by storage. The in vitro dose-response curves show that the free-zone diameter increases logarithmically with antibiotic concentration. The absorbed antibiotic amount of one gram bone chips was determined.Conclusions: Storage of antibiotics in frozen form or storage of antibiotic pills at temperatures up to 37 degrees C for 12 months does not affect their activity. However, storage of antibiotic solutions at temperatures above 20 degrees C does affect the activity of some of the antibiotics investigated. The in vitro dose-response curve can be used to determine the optimal concentration(s) for local application. It provides the opportunity to determine the antibiotic content of bone chips, and thus the amount of antibiotics available locally after application. [ABSTRACT FROM AUTHOR]

An electronic surveillance network for monitoring antibiotic resistance in The Netherlands has been in operation since 1989. Seven public health laboratories participate and the system covers about 25% of all bacteriological determinations in The Netherlands. This paper reports the results of staphylococci isolated in the period 1989-1995. About 0.3% of the Staphylococcus aureus isolates in the study period were resistant to methicillin. This low percentage may be due to the restrictive use of antibiotics and to strict isolation measures aimed at eradicating methicillin-resistant S. aureus. Low frequencies of resistance among methicillin-resistant S. aureus were found for vancomycin (0%), chloramphenicol (11%), cotrimoxazole (11%), mupirocin (3% low-level resistance) and fusidic acid (7%). Twenty-one percent of the coagulase-negative staphylococci were resistant to methicillin. Low frequencies of resistance among these methicillin-resistant coagulase-negative staphylococci were those to vancomycin (0.4%), nitrofurantoin (2%), doxycycline (20%) and amikacin (20%). Coagulase-negative staphylococci from cerebrospinal fluid, blood and skin were less often resistant to quinolones than isolates from respiratory tract, faeces and urine. A significant increase in resistance of coagulase-negative staphylococci to methicillin, erythromycin, gentamicin and ciprofloxacin was observed in the investigated period but the resistance to doxycycline and co-trimoxazole decreased in the last few years. To confirm the determination of methicillin resistance and coagulase production, a PCR method was developed which detects both the mecA and the coagulase gene. The results of the PCR method correlated well with the methicillin MIC as determined by an agar-dilution method. [ABSTRACT FROM PUBLISHER]

Background: The aim of this study was to investigate the effect of door opening rates on air quality in the operation room during hip revision surgery by measuring the number of colony forming units per cubic meter (CFU/m(3))., Methods: During 70 hip revision operations the number of CFU/m(3) was measured at four time points. Factors that may influence air quality were recorded, including the number of persons present, duration of surgery, and door opening rates. The measured CFU/m(3) was dichotomized as either acceptable (≤20 CFU/m(3)) or not acceptable (>20 CFU/m(3)). To determine whether door openings were associated with CFU/m(3) values, we used generalized linear mixed models to model the dichotomized repeatedly measured CFU/m(3) values., Results: The median number of door openings per operation was eight (range, 0-72), the median duration of surgery was 145 min (range, 60-285), and the median number of persons present during surgery was eight (range, 5-10). Adjusted for number of persons in the operation room and duration of surgery, the number of door openings per operation was associated (odds ratio [OR] 1.05 [95% confidence interval {CI} 1.02-1.09]; p = 0.003) with an unacceptable number of CFU/m(3)., Conclusions: In conclusion, a substantial association between number of door openings and an unacceptable number (>20) of CFU/m(3) was found. Adjusted for number of persons in the operation room and duration of surgery, every door opening increased the odds of unacceptable CFU/m(3) values by 5%. Number of persons present during surgery and duration of surgery were not related to CFU/m(3).

With bone impaction grafting, cancellous bone chips made from allograft femoral heads are impacted in a bone defect, which introduces an additional source of infection. The potential benefit of the use of pre-processed bone chips was investigated by comparing the bacterial contamination of bone chips prepared intraoperatively with the bacterial contamination of pre-processed bone chips at different stages in the surgical procedure. To investigate baseline contamination of the bone grafts, specimens were collected during 88 procedures before actual use or preparation of the bone chips: in 44 procedures intraoperatively prepared chips were used (Group A) and in the other 44 procedures pre-processed bone chips were used (Group B). In 64 of these procedures (32 using locally prepared bone chips and 32 using pre-processed bone chips) specimens were also collected later in the procedure to investigate contamination after use and preparation of the bone chips. In total, 8 procedures had one or more positive specimen(s) (12.5 %). Contamination rates were not significantly different between bone chips prepared at the operating theatre and pre-processed bone chips. In conclusion, there was no difference in bacterial contamination between bone chips prepared from whole femoral heads in the operating room and pre-processed bone chips, and therefore, both types of bone allografts are comparable with respect to risk of infection.

Owing to multidrug resistance, quinolones and third-generation cephalosporins are currently used as key antibiotics to combat Salmonella organisms. Therapy failure due to reduced ciprofloxacin susceptibility has been reported in endemic areas, but also in imported disease. Different bacterial resistance mechanisms may result in reduced ciprofloxacin susceptibility. In this study, the presence and expression of different resistance mechanisms resulting in reduced minimum inhibitory concentrations (MICs) for ciprofloxacin were evaluated in 23 blood-culture-derived Salmonella enterica serotypes Typhi and Paratyphi A organisms from ill-returned travellers to Asia. The presence of mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene as well as an activated efflux pump and plasmid-mediated quinolone resistance genes was determined. Resistance selection during therapy and the clonal relatedness of all isolates were established. Efflux pump inhibition did not appear to affect the MICs of ciprofloxacin and activity of the efflux pump appeared to be specific for nalidixic acid. Repeated exposure of the isolates to ciprofloxacin did not result in a significant increase in the MICs for ciprofloxacin. Repetitive sequence-based polymerase chain reaction (rep-PCR) profiles identified five different genotypes, but no correlation with resistance was observed. However, a significant relation was found with geographic region; reduced ciprofloxacin susceptibility was only found in travellers returning from India and Pakistan. All isolates with reduced ciprofloxacin susceptibility had a mutation at position 83 in the QRDR region of the gyrA gene. Plasmid-mediated quinolone resistance was not found. These findings confirm that the reduced ciprofloxacin MIC in S. Typhi and S. Paratyphi A is solely due to an amino acid substitution in the QRDR 'cluster' of the gyrA gene., (Copyright © 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Background: Most clinicians in developed, non-malaria endemic countries have limited or no experience in making clinical assessments of malaria disease severity and subsequent decisions regarding the need for parenteral therapy or high-level monitoring in febrile patients with imported malaria. In the present study, the diagnostic accuracy of plasma soluble Triggering Receptor Expressed on Myeloid cells 1 (TREM-1), neopterin and procalcitonin levels as biomarkers for severe Plasmodium falciparum disease was evaluated in 104 travellers with imported malaria (26 patients with non-P. falciparum malaria, 64 patients with uncomplicated P. falciparum malaria and 14 patients with severe P. falciparum malaria)., Methods: TREM-1, neopterin and procalcitonin were determined in serum using commercially available ELISA or EIA tests. The diagnostic performance of these biomarkers for severe disease was compared with plasma lactate, a well-validated parameter for disease severity in patients with malaria, as reference. Severe malaria was defined according to the modified WHO criteria., Results: No significant differences in TREM-1 levels were detected between the different patient groups. Patients with severe P. falciparum malaria had significantly higher neopterin and procalcitonin levels on admission when compared to patients with uncomplicated P. falciparum malaria or non-P. falciparum malaria. Receiver Operating Characteristic (ROC) curve analysis showed that neopterin had the highest Area-Under-the-ROC curve (AUROC 0.85) compared with plasma lactate (AUROC 0.80) and procalcitonin (AUROC 0.78). At a cut-off point of 10.0 ng/ml, neopterin had a positive and negative predictive value of 0.38 and 0.98 whereas procalcitonin, at a cut-off point of 0.9 ng/ml, had a positive and negative predictive value of 0.30 and 1.00., Conclusion: Although the diagnostic value of neopterin and procalcitonin is limited, the high negative predictive value of both neopterin and procalcitonin may be helpful for a rapid exclusion of severe malaria disease on admission. This may be a valuable tool for physicians only occasionally dealing with ill-returned travellers from malaria-endemic regions and who need to decide on subsequent oral anti-malarial treatment or timely referral to a specialized centre for high-level monitoring and intensified parenteral treatment.

To assess the role of non-tuberculous mycobacteria (NTM) as a cause of tuberculosis-like diseases in Zambia, 167 chronically ill patients, hospitalized in three rural hospitals in Katete, Sesheke and Chilonga, were examined by microscopy and liquid culture for the presence of NTM. The percentages of patients with a positive culture for Mycobacterium tuberculosis complex were similar in the three geographical locations (19-25%). In contrast, the percentage of NTM ranged from 78% in Katete and 65% in Sesheke to 21% in Chilonga. Furthermore, the distribution of NTM species was different at the three geographical sites. In seven patients, true NTM-associated disease was suspected: five with Mycobacterium lentiflavum and two with Mycobacterium intracellulare. Analysis of possible risk factors indicated that the OR for NTM culture-positive sputum was significantly higher for patients living in Katete and Sesheke. Female gender and chest X-ray appearances of tuberculosis were independently associated with NTM culture-positive sputum. NTM colonization and disease in hospitalized, chronically ill patients in rural Zambia appear to be common.

Objective: Microbiological examination of donated human cardiac tissue is a necessary procedure for Heart Valve Banks to determine the biological safety of preserved allografts. Test protocols must be validated to prevent false-negative outcomes that pose a risk of infection to recipients of the tissue. The Heart Valve Bank in Rotterdam evaluated a validated, alternative entry test for donated tissues to compare the performance of its standard microbiological examinations., Methods: Samples of explanted heart transport medium from 275 donors were examined for the presence of microorganisms using blood culture flasks (standard test) and fluid thioglycolate medium (alternative test). Results were compared with the outcome of microbiological assessment of subvalvular myocardial fragments and the cryoprotective medium that were collected before and after treatment of the grafts with antibiotics, respectively., Results: Microorganisms, mainly skin flora, were detected in transport medium of 177 hearts (64%). The alternative validated culture method detected a growth in 80 transport medium samples that was not identified by the standard method. Microorganisms were only identified in the cultivated cardiac tissue fragments from 56 donors (20%). After antibiotic treatment of the tissue, microorganisms could still be encountered in cryoprotective medium samples from 55 donors (20%). Most of the contaminants in these final samples were identified as Propionibacterium species and Corynebacterium species and had already been detected in the transport medium by the alternative validated culture method., Conclusions: The use of blood culture flasks for microbiological assessment of non-blood liquid media and the cultivation of myocardial tissue fragments may hamper detection of certain microorganisms and therefore provide less complete information about microbiological safety. Heart Valve Banks may want to review their microbiological examination and decontamination procedures regarding the ability to detect and eliminate anaerobic skin flora, respectively., (Copyright 2009 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.)

Two Indian migrant workers suffering from fever and malaise were admitted to the hospital directly after arrival in the Netherlands. The first patient was 25-year-old man who had fever and rigors on admission. The patient was treated for presumptive typhoid fever with ciprofloxacin. Cefotaxime was added the following day because of the possibility of a nalidixic-acid resistant strain of S. typhi. The clinical course was complicated by a small bowel perforation on the third day of the disease. Blood cultures grew a nalidixic acid resistant strain of Salmonella enterica serovar typhi. The patient recovered completely. The second patient, a 22-year-old man, suffered from fever, malaise and hearing loss. A sensorineural hearing loss with vestibular dysfunction was diagnosed. Cultures of blood and bone marrow aspirate showed a nalidixic acid resistant strain of S. typhi. Treatment with ciprofloxacin and ceftazidime improved the hearing loss significantly. The clinical features of typhoid fever are heterogeneous and rare complications may occur. The emergence of multidrug and nalidixic acid resistance may complicate further the treatment of this serious systemic infection.

A novel olfactory method for bacterial species identification using an electronic nose device called the MonoNose was developed. Differential speciation of micro-organisms present in primary cultures of clinical samples could be performed by real-time identification of volatile organic compounds (VOCs) produced during microbial replication. Kinetic measurements show that the dynamic changes in headspace gas composition are orders of magnitude larger than the static differences at the end of fermentation. Eleven different, clinically relevant bacterial species were included in this study. For each of the species, two to eight different strains were used to take intra-species biodiversity into account. A total of 52 different strains were measured in an incubator at 37 degrees C. The results show that the diagnostic specificities varied from 100% for Clostridium difficile to 67% for Enterobacter cloacae with an overall average of 87%. Pathogen identification with a MonoNose can be achieved within 6-8 h of inoculation of the culture broths. The diagnostic specificity can be improved by broth modification to improve the VOC production of the pathogens involved.

Clinical relevance of nontuberculous mycobacteria (NTM) isolated from 180 chronically ill patients and 385 healthy controls in Zambia was evaluated to examine the contribution of these isolates to tuberculosis (TB)-like disease. The proportion of NTM-positive sputum samples was significantly higher in the patient group than in controls; 11% and 6%, respectively (p<0.05). NTM-associated lung disease was diagnosed for 1 patient, and a probable diagnosis was made for 3 patients. NTM-positive patients and controls were more likely to report vomiting and diarrhea and were more frequently underweight than the NTM-negative patients and controls. Chest radiographs of NTM-positive patients showed deviations consistent with TB more frequently than those of controls. The most frequently isolated NTM was Mycobacterium avium complex. Multiple, not previously identified mycobacteria (55 of 171 NTM) were isolated from both groups. NTM probably play an important role in the etiology of TB-like diseases in Zambia.

Fever in a returned traveller may be the manifestation of a self-limiting, trivial infection but it can also presage an infection that can be rapidly progressive and lethal. We studied the diagnostic accuracy of procalcitonin (PCT) as a biomarker for a bacterial cause of fever in a cohort of 157 consecutive travellers with fever after a stay in the (sub)tropics. Elevated procalcitonin levels were observed not only in about 50% of travellers with proven bacterial infection, but also in a significant proportion of travellers with a likely infection. Using a cutoff point of 0.5 ng/mL, procalcitonin had a sensitivity of 0.52 and a specificity of 0.76 for a bacterial cause of fever on admission. Interestingly, only 1 out of 16 patients with a proven viral infection had a marginally elevated PCT concentration on admission, suggesting that an increased PCT level likely excludes a viral infection as the cause of fever. However, the diagnostic accuracy of this semiquantitative procalcitonin test for a bacterial cause of fever on admission is too poor to advocate its use in the initial clinical evaluation of fever in a setting of ill-returned travellers.

Polymorphism in various genes that may influence susceptibility to tuberculosis (TB) was examined in 46 TB patients and 119 healthy tuberculin-positive controls in Zambia. The odds of having TB was 2.8-fold higher in carriers of the -2518 AG single-nucleotide polymorphism in the promoter region of the CC-chemokine ligand 2 than in those carrying the homozygous genotype AA (95%CI 1.3-5.5).

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.

A 26-year-old woman presented with a 2-day history of fever peaking to 39 degrees C and cold shivers that developed after a 2-weeks trip to Guatemala and Belize. Prior to the fever the patient had felt symptoms of a common cold and general malaise. Moreover, she complained of generalised myalgia and nausea. She had taken paludrine as a prophylactic against malaria. Borrelia spirochaetes, the pathogen of relapsing fever, were detected in a thick blood smear preparation. On the basis of the anamnesis, geography and specific exposure, the patient had a form of relapsing fever that is transmitted by ticks and not by lice: tick-borne relapsing fever. She was treated with doxycycline, 100 mg b.i.d. for 7 days. She could be discharged home in good condition after 2 days.

The isolation of nontuberculous mycobacteria (NTM) raises the question of their clinical significance, especially in an African setting. We found a high percentage of NTM isolated from various specimens, including ones that are normally sterile, among 213 patients in Zambia. Because tuberculosis can affect all parts of the body, we decided to include patients who had signs and symptoms in any part of the body for more than 2 weeks. Most patients had tractus respiratorius (80%) and tractus digestivus (10%) symptoms. During three consecutive days, sputum was collected and two separate sputum specimens were cultured for mycobacteria. Depending on the clinical picture, pleural effusion, ascites, abscess material, or enlarged lymph nodes were also cultured for mycobacteria. A specimen from one sterile body site was collected from 25 patients (60% human immunodeficiency virus [HIV] positive). NTM were isolated from 8 of these 25 specimens. Mycobacterium lentiflavum was isolated from four patients, and Mycobacterium goodii was isolated from one patient. In order to exclude the possibility of laboratory cross-contamination, a novel amplified fragment length polymorphism DNA typing method for M. lentiflavum was developed. Genetic variation was detected, rendering the likelihood of laboratory contamination unlikely. Clinically relevant infection due to NTM occurs in both HIV-positive and HIV-negative patients in Zambia, and their clinical impact seems to be underestimated. This is the first report of M. lentiflavum and M. goodii infections in Africa.

Background: Rapid immunochromatographic dipstick assays are used increasingly in many tropical and Western countries as a tool to diagnose Plasmodium falciparum malaria. However, these tests do not provide any information about the severity of the infection. We evaluated the usefulness of plasma lactate as a parameter for disease severity on admission in imported P. falciparum malaria., Methods: In a cohort of 61 nonimmune travelers with imported P. falciparum malaria, plasma lactate levels on admission were related to the severity of the infection. Results from 12 of 61 patients fulfilled the criteria of severe malaria., Results: Logistic regression analysis showed that a plasma lactate level above the upper range of normal was associated with an odds ratio of 31 (95% CI 6-164) for severe malaria. As a continuous variable, a 1 mmol/L increase in plasma lactate level was associated with an odds ratio of 12 (95% CI 3-50) for severe malaria. The sensitivity of an increased plasma lactate level on admission for severe malaria was 67% with a specificity of 94%., Conclusions: A timely determination of plasma lactate on admission may be helpful in the assessment of disease severity in travelers with imported P. falciparum malaria. An increased plasma lactate level should raise suspicion of a severe P. falciparum malaria infection, in particular when concomitant infections are not considered likely.

We compared the NaOH-N-acetyl cysteine (NaOH-NALC) and the sulfuric acid decontamination procedure in the detection of mycobacteria using the Mycobacteria Growth Indicator Tube (MGIT). In total 219 sputum specimens were collected from 142 Zambian patients and subjected to mycobacterial culture. One half of the specimen was decontaminated with NaOH-NALC and the other half was decontaminated with sulfuric acid. From the 438 samples a total of 261 (60%) cultures yielded growth of mycobacteria, consisting of 22 different species. The sulfuric acid method was more successful than the NaOH-NALC method in recovering mycobacteria in MGITs (146 versus 115 respectively, p = 0.001). Of the 146 positive mycobacterial cultures recovered after sulfuric acid decontamination 28 were Mycobacterium tuberculosis, 84 nontuberculous mycobacteria (NTM) and 34 acid fast bacterial isolates which could not be identified to the species level. The 115 mycobacteria recovered by the NaOH-NALC method consisted of 34 M. tuberculosis strains, 55 NTM and 26 acid fast bacteria that could not be identified. The most frequently isolated NTM were Mycobacterium lentiflavum and Mycobacterium intracellulare. Comparing the two decontamination methods the recovery of NTM in the sulfuric acid group was significant higher than in the NaOH-NALC group (p = 0.001). In contrast, no significant difference was found for the recovery of M. tuberculosis. These results show that the decontamination method used affects the recovery of nontuberculous mycobacteria in particular.

Helicobacter cinaedi was initially recovered from homosexual men infected with human immunodeficiency virus. Later, it was also isolated from patients with chronic alcoholism, and anecdotally from non-immunocompromized men, women, and children. We describe a case of H. cinaedi-associated bacteraemia and erysipelas in a homosexual but immunocompetent male and the diagnostic problems in identifying the causative organism.

Objective: To determine prognostic factors and the significance of (non-invasive) microbiological tests for the clinical course of patients admitted to one general hospital with community-acquired pneumonia (CAP)., Design: Prospective observational study., Method: Patients admitted to one location of a general hospital with symptoms of CAP during the period January 1998-December 1999 were included. Data from the anamnesis, physical examination and laboratory tests were recorded and samples were taken for diagnosis of the possible pathogens. Cultures were made of sputum and blood, serum and sputum were examined for infection with viral and atypical microorganisms, and the urine was screened for Streptococcus pneumoniae and Legionella antigens., Results: Of the 157 patients studied, 28 (18%) died as a direct result of CAP. In a stepwise multivariate analysis, age > or = 65 years, increased serum-creatinine levels and hypercapnia were independent predictors of mortality. Streptococcus pneumoniae and Haemophilus influenzae were detected 53 and 19 times, respectively, and were the bacterial pathogens most frequently found. Among the nonbacterial pathogens, Mycoplasma pneumoniae was found 37 times and serologic tests for influenza A or B virus were positive in 34 cases. In 62 patients (39%), extensive microbiological tests revealed signs of a combined infection and in 20 patients (13%) no microorganism could be detected. An elevated serum-procalcitonin level was associated with bacterial infections. As a consequence of their retrospective nature, the results of the extensive serological tests did not contribute to reaching a diagnosis in daily clinical practice. In comparison with the literature, there was a markedly high number of subclinical or atypical infections with Legionella (8%) and Bordetella (18%) and a high incidence of viral and atypical microorganisms as the cause of CAP.

We analyzed the bacterial contamination rate of femoral head allografts from living donors and determined the true bacterial load with cultures from the grafts in their entirety in a specially prepared medium. During 4 years we took swab cultures from 2,679 grafts of which 2,414 (90%) were negative. In a period of 12 months, grafts rejected for reasons other than infectious disease were cultured in their entirety to determine the true bacterial load. Of the 106 grafts included, 15 were contaminated. Microorganisms were isolated from 10 of the 91 initially swab culture-negative grafts (9%) and from only 5 of the 15 swab culture-positive grafts, most of which were normal skin contaminants. Swab culture negative grafts apparently may still be contaminated. It seems wise to subject all femoral head allografts from living donors to antibacterial processing.

This study assesses the value of blood cultures in combination with swab culturing techniques in association with bone banking procedures. The results of blood and swab cultures of two postmortem bone donors were compared with procured grafts, cultured in their entirety. In one donor, who died of drowning, three of the 12 entire graft cultures were positive with the same microorganism of high pathogenicity as the blood culture, whilst the swab culture of only one graft was positive. In the second donor, who died from myocardial infarction, four entire cultures were positive with the same organism of high pathogenicity as the blood culture, whilst the swab cultures of three grafts were positive. In both donors identical organisms were cultured from the myocardium of the pulmonary or aortic heart valve. The results confirm the limited sensitivity of swab culturing techniques. Especially micro-organisms inside a graft, disseminated through the bloodstream, can remain unnoticed. Blood cultures seem to provide additional information on micro-organisms that have spread haematogenously pre-mortem and may even provide information on the source from which they have spread., (Copyright 2001 The Hospital Infection Society.)

In continuous surveillance of routine samples from five Dutch laboratories, we studied resistance to the antibiotics most commonly prescribed for urinary tract infections (UTI) in The Netherlands, namely norfloxacin, amoxycillin, trimethoprim and nitrofurantoin, from 1989 to 1998 in >90000 Escherichia coli isolates. Resistance to norfloxacin increased from 1.3% in 1989 to 5.8% in 1998. Multiresistance, defined as resistance to norfloxacin and at least two of the other three antibiotics, increased from 0.5% in 1989 to 4. 0% in 1998. Multivariate analysis of the norfloxacin resistance demonstrated that this yearly increase (the odds ratio was 1.0 in 1989, 1.6 in 1992, 2.9 in 1995 and 6.1 in 1998) was independent of other determinants of resistance to norfloxacin, such as age, gender and origin of the isolate. Analysis of strata, classified by year, age and gender, demonstrated an association between prescription of fluoroquinolones (defined daily doses per case of UTI) and resistance to norfloxacin in E. coli (P < 0.001). There was no significant association with the prescription of nitrofuran derivatives (nitrofurantoin) and trimethoprim with or without sulphamethoxazole. The yearly increase of resistance to fluoroquinolones in E. coli from UTI may stem from increased prescription of fluoroquinolones for UTI. Resistance of E. coli to these agents is likely to increase further as fluoroquinolone use increases in future.

The results of blood cultures taken from cadaveric bone donors were compared with bone marrow and also swab cultures of the procured grafts. In eight of the 95 donors evaluated, pathogenic micro-organisms were detected in the blood sample. In two, identical micro-organisms were cultured from the blood and bone marrow sample whilst the swab cultures were negative. Considering the low sensitivity of the swab culture, the organisms detected in the blood culture were likely to have spread haematogenously and considered to be present in the explanted grafts. Bacteriological screening of bone donors is extremely important since the transmission of micro-organisms via an allograft can lead to serious complications in the recipient. Positive blood cultures provide important information on the presence of pathogenic micro-organisms in grafts obtained from cadaveric bone donors and are therefore essential in deciding whether a graft is bacteriologically acceptable for transplantation.

Unlabelled: The intraperitoneal insufflation of carbon dioxide during laparoscopic procedures is associated with an increase in mean arterial pressure and systemic vascular resistance. To evaluate the time course of the circulatory changes related to intraabdominal pressure variation, we used transesophageal echocardiography in 10 patients anesthetized for laparoscopic cholecystectomy. Left ventricular dimensions, fractional area shortening (FAS), and left ventricular afterload assessed by the left ventricular end-systolic wall stress (LVESWS) were measured from echocardiographic data before insufflation, during pneumoperitoneum, and during exsufflation. Three minutes after the onset of pneumoperitoneum, we observed a 25.7% +/- 10.5% (mean +/- SD) increase in mean arterial pressure, a 49.1% +/- 14.4% increase in LVESWS, and a 17.0% +/- 16.2% decrease in FAS. All measured variables returned to preinsufflation values after 30 min of pneumoperitoneum and thereafter were no longer significantly affected by postural changes (10 degrees head-up position) or pneumoperitoneum exsufflation. We conclude that the circulatory changes associated with pneumoperitoneum are transient and are probably mediated by factors other than intraabdominal pressure variations., Implications: Insufflation of gas into the abdominal cavity results in hemodynamic changes during laparoscopic procedures. We found that echocardiographic and blood pressure effects associated with gas insufflation were transient, indicating mediation by factors besides intraabdominal pressure.

We analysed the bacterial contamination of 1999 bone allografts retrieved from 200 cadaver donors under sterile operating conditions. The effect of various factors on the relative risk of contamination was estimated using a multiple logistic regression model. Organisms of low pathogenicity were cultured from 50% of the grafts and of high pathogenicity from 3%. The risk of contamination with low pathogenic organisms (mainly skin commensals) increased by a factor of 1.6 for each member added to the procurement team. The risk of contamination with high pathogenic organisms (mainly contaminants from the gastrointestinal tract) was 3.4 times higher in donors with a traumatic cause of death and 5.2 times higher in those with a positive blood culture. Preceding organ procurement did not significantly influence the risk of contamination. Rinsing the graft with an antibiotic solution was not an effective decontamination method. The major source of contamination is exogenous and is strongly influenced by the procurement team. Contamination from endogenous sources can be controlled by donor selection. We discuss methods that can be used to decrease contamination and the rate of discarding of bone allografts.

There is a high prevalence of Ebola antibodies found in the Kenya population, related to geographical area and season, although the clinical disease was never found and the virus was not isolated. A field study was carried out in 7 hospitals in western Kenya, 1986 -1987 (including surveillance studies in suspect areas), to intensify collection and transport of samples, testing facilities, patient observation with record keeping and follow-up. This study involved 1109 admitted patients with fever and/or bleeding, 155 contacts of haemorrahagic fever antibody (Hfab) patients, and 916 people in suspect areas. Respectively 160,44 and 80 persons were found Hfab positive mainly to Ebola, using an indirect immunofluorescent assay. From 676 viral cultures no virus was isolated. A relationship between antibody titres and ecological factors, social habitat, age, sex or season was not found. The non-specificity of IF testing was demonstrated by: 1) the disagreement between the results of two reference laboratories; 2) the unpredictability of the titre conversation course; and 3) by proving a significant cross-reactivity with Borrelia burgdorferii antibodies, Plasmodium falcparum antibodies and Salmonella typhi antibodies. Renewed testing in 1995 of 90 positive sera (with low titres) showed 19 sera to be positive by Elisa (2 in Zaire, 1 in Sudan, 9 in Reston and 7 in Cote d'Ivoire) from which 4 were confirmed by IFI 2 in Reston and 2 in Cote d'Ivoire. These findings are more proof that non-human virulent strains of Filoviridae, especially Ebola virus, are around in Kenya.

In two studies on the causative agents of bacteraemia in Malawi and Kenya, 33 Salmonella strains were isolated. Fourteen strains of Salmonella typhimurium and Salmonella enteritidis were found to exhibit resistance to amoxicillin, amoxicillin/clavulanic acid and cotrimoxazole as well as decreased susceptibility to a range of aminoglycosides. The resistant strains were studied to establish their resistance mechanisms. Beta-lactamase co-focusing with TEM-1 was present in 12 strains. In two strains, both S. typhimurium from Kenya, an OXA-1 beta-lactamase was detected. The aminoglycoside-modifying enzyme ANT(2") was found in 10 strains. The presence of the encoding genes was confirmed by PCR. For comparison, susceptibility records of 73 Salmonella strains isolated during the past 14 years in our hospital were studied retrospectively. Only one of these strains was resistant to amoxicillin. This resistance was acquired during therapy.

Detailed standardized annual reports are analysed for 17 rural hospitals in four African countries, with admission figures of 1.2 million patients (excluding deliveries) and more than 67,000 deaths over a period of 16 years. The countries involved are Zambia, Tanzania, Kenya and Ghana. Figures on admission, causes of death and clinical case fatality rates are presented per country and per 4-year calendar period for the most important infectious diseases. The number of admissions increased substantially in 3 of the 4 countries (50-77%) between 1976 and 1990, but admission rates (per 1,000 population per year) by much less (6-25%), indicating that a large part of the increase in the volume of services was due to population growth. The number of infectious disease admissions in Ghana, however, decreased by 12% and even more in terms of admission rates (by 42%). About 75% of all admissions in children below 15 years of age were due to infections and infection related diseases; for adults this figure was 31%. Malaria is the single most important infectious disease both in terms of admissions and as a cause of death; it has increased substantially in three of the four countries. Bacterial infections, in particular pneumonia, gastroenteritis, meningitis and tuberculosis, are also important diseases in terms of admissions and deaths. On the whole they have remained at more or less the same level in 1975-1990 in terms of both admissions and deaths. Immunizable diseases and measles, once important as causes of admissions and deaths, have declined in all countries. Case fatality rates vary substantially by type of disease. They are very high for tetanus (36.7-68.8%) and meningitis (14.7-43%), and low for malaria (0.6-4.6%). However, they vary considerably in the four countries included in the study and are usually lower in children than in adults. A need for detailed studies with good "standardized" hospital records is emphasized. Representative data are needed from all hospitals in a given catchment area, with defined diagnoses for diseases and details regarding age and sex. This kind of information is highly desirable for planning and operation of curative and preventive medical care in developing countries.

In three studies, in Ghana and Kenya, blood from 639 patients admitted with fever was cultured. Standard treatments were antimalarials (54-100%) and antibiotics (39-90%). According to the criteria in use, however, only 10-31% had malaria alone; of those who received antibiotics, 66% were diagnosed with malaria, gastrointestinal infections, post-operative recuperations, circulatory problems, central nervous system disorders or FUO, and did not need antibiotics at the first encounter. For those with wounds and abscesses (8%), generalised antibiotic treatment can also be questioned. Bacteraemia was found in 71 (11.3%) patients; in the HIV patients, however, 5 (23%) of 22 had bacteraemia. This is a minimum incidence, since culture techniques were not optimal for the isolation of fastidious microorganisms. The most prevalent organisms isolated were Salmonella, Klebsiella/Enterobacter and S. aureus. Resistance (intrinsic and extrinsic) in the Gram- bacteria was high: 31-100% were resistant to amoxycillin, 0-80% to cotrimoxazole, 15-95% to chloramphenicol and 9-15% to gentamicin. The need for cultures and sensitivity tests for patients with prolonged or undiagnosed fever is stressed. Specific treatment should be given only when infections, whether malarial or bacterial, have been positively diagnosed.

Endocarditis due to a bacterium of CDC coryneform group ANF-3 developed in the native aortic valve of a patient without predisposing factors other than valvular calcification. A review of the literature indicates that corynebacteria are an increasingly important cause of endocarditis. Problems in identification and treatment remain. Techniques for the culture and quick identification of these organisms and effective regimens for treatment of the infections they cause are needed.

A study of 256 annual reports from 17 rural tropical hospitals in 4 African countries over a period of 16 years showed an absolute increase in the number of patients admitted with infectious diseases. Admissions were highest for malaria, followed by pneumonia and gastroenteritis. Admissions for immunisable diseases are decreasing in all countries. Fever remains the most important indicator of infectious diseases. Analysis of fever patients in rural tropical hospitals relies on knowledge of the epidemiology of diseases, plus expertise in physical examination. In this study, a detailed analysis of 900 fever patients indicated that 4% showed no infection, 21% of infections could be diagnosed by physical examination, 35% were diagnosed with the help of additional laboratory tests and 40% of patients were diagnosed as FUO (fever of unknown origin). 17% of FUO patients had a short, self limiting fever, but the remaining 23% were severely ill, suggesting bacterial sepsis, as was indicated by earlier studies. Undiagnosed fevers with resulting over-treatment and high resistance are costly and dangerous. These effects stress the need for better and more laboratory facilities, including possibilities for bacterial cultures. At present, patients are generally over-treated with antimalarials and antibiotics, since further diagnostic facilities are not available. Resistance is high for antimalatials ( Malaria) and for Amoxycillin, Cotrimoxazole and Gentamicin (Gram-bacteria from urine and blood).

In a 84-year-old woman extraintestinal infection by non-OI Vibrio cholerae was diagnosed. She had septicaemia with cholangitis and cholecysto- and choledocholithiasis. Until now 26 patients with non-OI V. cholerae septicaemia have been reported. Most had an underlying disease, usually a chronic liver disease or haematological malignancy. These disorders were not present in our patient. She was treated with co-trimoxazole and afterwards she underwent a cholecystectomy and common bile duct exploration. At the time of operation no non-OI V. cholerae could be isolated from the gallbladder or the bile from the common bile duct. A possible cause of the infection was a herring which the patient had eaten six weeks before hospital admission.

A retrospective analysis was done on the diagnosis of typhoid fever based on clinical symptoms and available laboratory data over the last 16 years from rural areas of four African countries. This analysis concentrated on the reliability of diagnosis without cultures which cannot be performed in most rural hospitals due to lack of the necessary expertise and equipment. The analysis showed the problem to be increasing perhaps because of interaction of salmonella infection with human immunodeficiency virus (HIV), malnutrition and other infections together with neglected sanitary facilities and lack of clean water. The use of certain cardinal clinical symptoms combined with available laboratory tests were shown to enhance the diagnosis of typhoid fever, especially in vulnerable persons. In conclusion the paper suggests that using the approach followed to obtain this data in rural tropical areas one can confidently make a diagnosis of typhoid fever.

Seventy-five fibular specimens were obtained from postmortem donors using aseptic surgical techniques. All specimens were swabbed following the same technique as routinely used for retrieved musculoskeletal grafts. After swabbing, the specimens were placed in BHI-culture medium. Three different protocols were subsequently followed: (1) culture of the entire bone specimen in BHI-culture medium, (2) culture of the swab incubated on blood agar and chocolate plates, and (3) culture of the swab in BHI-culture medium. A control group included 20 sterilized bone specimens that were cultured entirely in BHI-culture medium according to Protocol 1. The sensitivity and negative predictive value were found to be 10% and 9% in Protocol 2 and 13% in Protocol 3. These findings imply that swab cultures are inadequate to detect bacterial contamination of musculoskeletal allografts in all cases. However, the instances of infection after transplantation of allografts bacteriologically screened according to Protocols 1 and 2 do not exceed those reported in other similar series. This suggests an acceptable bioburden.

The cytotoxicity of commonly used antimicrobial and antiseptic agents to cultured human keratinocytes was investigated, combining a morphologic assay with a quantitative neutral red (NR) spectrophotometric assay. Main outcome criteria of response were the initial cytotoxicity (NR 90) and midpoint cytotoxicity (NR 50) and the highest tolerated dose (HTD), a concentration causing the first observed morphologic alterations. These values were compared with commonly administered clinical doses or calculated doses. Thirty-five agents were evaluated; five agents were also subjected to an assay for cell morphologic alterations, and HTD and percentage of cells taking up trypan blue and annihilation dose were assessed. These studies show that in clinical concentrations many, but not all, agents exert profound cytotoxic effects. The results suggest that under certain conditions cultured epithelial grafts may be exposed to clinical concentrations of neomycin, clindamycin, framycetin, erythromycin, gentamicin, and 0.1% solutions of povidone-iodine. Tetrachlorodecaoxygen anion complex (TCDO) should be further evaluated as a likely effective cleansing agent before culture grafting.

An in vitro study was performed to evaluate the effect of Staphylococcus aureus and Staphylococcus epidermidis on the integrity of a hydroxylapatite coating. The coating plasma-sprayed on poly(L-lactide), showed dissolution during a 24 h incubation period. This was indicated by an increase in pH and calcium release in the buffer solution. After 4 h of incubation, calcium levels decreased due to the precipitation of calcium phosphate complexes on the coating. The bacteria digested or dissolved the coating, creating irregularly shaped holes. Although the integrity of the hydroxylapatite coating was focally damaged within 2-4 h of incubation with staphylococci, the extent of the damage was only marginal. Due to the formation of a layer of CaP precipitates though, bacteria could not be counted accurately after 4-8 h of incubation. This model could reveal part of the failure mechanism of infected hydroxylapatite coated implants.

Bacterial adherence on to several materials with a potential application in reconstructive surgery was studied. Polymer (poly(L-lactide)), composite (hydroxyapatite/poly(L-lactide)) and metal (316L stainless steel) were evaluated both as smooth and sandblasted specimens. All materials were incubated in phosphate-buffered saline, challenged with Staphylococcus aureus or S. epidermidis and evaluated for up to 24 h. S. aureus showed a preference for the metal and composite tested over the polymer used. For S. epidermidis no preference was found for one of the investigated materials. The influence of surface roughness on bacterial growth was demonstrated by increased colonization on the sandblasted specimens.

The occurrence of high-level aminoglycoside resistance (HLAmR) was determined for 73 enterococci and 54 group A streptococci by the high-load disc method, tube macrodilution and the polymerase chain reaction (PCR). The PCR method revealed the presence of genes coding for aminoglycoside-3'-O-phosphoryltransferase-III (APH(3')-III), aminoglycoside-6'-N-acetyltransferase/2''-O-phosphoryltransferase (AAC(6')/APH(2'')), or both, in 20.6%, 9.6% and 4.1% of the enterococci, respectively. The prevalence of HLAmR to at least one aminoglycoside among local enterococci was 37% (27/73). Only one of 54 Streptococcus pyogenes isolates produced APH(3')-III and exhibited high-level resistance to kanamycin and streptomycin. In general, the three methods yielded comparable results, with only three discrepancies among the 127 isolates examined. High-load disc screening and tube macrodilution proved to be practical, reliable and reproducible, and thus suitable for routine screening. Of 20 Enterococcus faecalis strains tested, all were penicillin-tolerant. Only one of seven penicillin-tolerant S. pyogenes strains was HLAmR. No association between the two forms of resistance was found.

Of 90 isolates of Neisseria gonorrhoeae in a rural area in Kenya, 44 (48.9%) produced beta-lactamase (penicillinase). Testing for susceptibility of 35 penicillinase producing N. gonorrhoeae (PPNG) strains to four antibiotics yielded the following results: 16 (45.7%) showed a decreased susceptibility to tetracycline; six (17.1%) showed resistance, probably plasmid mediated; 10 (28.6%) had intermediate susceptibility to gentamicin; one (2.9%) was resistant; and two (5.7%) isolates were resistant to cefotaxime. 16 (57.1%) of 28 non PPNG strains showed a decreased susceptibility to penicillin; 10 (35.7%) were resistant. Nine (32.1%) of 28 non-PPNG isolates showed intermediate susceptibility to tetracycline; one (3.6%) was resistant. Eight of non PPNG isolates (28.6%) showed decreased susceptibility to gentamicin. These results imply that penicillin and tetracycline should be abandoned as primary therapy. For the time being, thiamphenicol and spectinomycin seem to be good alternatives. The observation of cefotaxime resistance in N. gonorrhoeae in a rural area implies a warning concerning future possibilities for use of third generation cephalosporins.

No microbial data are available for rural tropical areas. Yet most people in Africa live in rural areas where the burdens of infectious diseases remain the primary cause for morbidity and mortality. A pilot study was done by culturing midstream urine from patient with dysuria (100 with Gram-negative fermentative rods), blood from patients with FUO (55/307 positive) and swabs from patients with infected wounds (29 with Staphylococcus aureus). Bacteria were identified and susceptibility tests were performed according to protocols. Klebsiella was the most common isolate from urine (48%). The overall resistance in urine isolates for ampicillin was 76% and for cotrimoxazole, 60%. The main isolates from blood were Klebsiella Enterobacter sp (21/55) and Salmonella (14/55) in patients from agricultural areas, and Staphylococcus aureus (12/55) in patients from desert areas. The Staphylococcus aureus from wounds were all penicillin resistant and 9/29 were cloxacillin resistant. Influence of the environment, underlying pathology and previous use of antibiotics give unexpected predominance of Klebsiella and high resistance in all isolates. Large scale surveillance studies are needed so that prescription of antibiotics can be based on locally obtained data.

A patient is described with an infection with Aeromonas hydrophila after the use of medical leeches to improve the circulation of a congested region of surgery. The use of medical leeches and the treatment of a leech-related infection are discussed.

The behavioural objectives model of curriculum design was introduced to the Medical Auxiliary Training Sector in Tanzania during the 1970s while problems of expansion, novelty and economic stress were abundant. Experience in a relatively privileged school, Rural Medical Training Centre, Sengerema, shows that the model did not result in more practical teaching time, due mainly to various constraints. The students did, however, appreciate the 'sharing of objectives' at the onset of a teaching block as a great help for their (theoretical) learning. Due to unfamiliarity of teachers with new subject matter related to primary health care, the model has failed thus far to result in a relevant practical course in public health promotion. A more problem-oriented approach to curriculum development is recommended.

In 226 women requesting chorionic villus sampling (CVS), routine cervical cultures were obtained before the procedure. Transcervical CVS was performed irrespective of the test results. The prevalence of potential pathogens in cervical cultures at our institution is low. Beta haemolytic Streptococcus was cultured in 3 per cent of the women. No pathogenic microorganisms were isolated in 64 per cent of the women. There was no relationship between culture results and the outcome of pregnancy. These observations suggest that adequate antiseptic cleansing of the genital tract is a suitable approach and there is no need to routinely perform cultures before CVS.