Comparison of Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines for the interpretation of antibiotic susceptibility at a University teaching hospital in Nairobi, Kenya: a cross-sectional study Annals of Clinical Microbiology and Antimicrobials
2016

Ali Kassim firstname.lastname@example.com
Department of Pathology
Aga Khan University Hospital
P.O. Box 3027000100NairobiKenya

Geoffrey Omuse
Department of Pathology
Aga Khan University Hospital
P.O. Box 3027000100NairobiKenya

Zul Premji
Department of Pathology
Aga Khan University Hospital
P.O. Box 3027000100NairobiKenya

Gunturu Revathi
Department of Pathology
Aga Khan University Hospital
P.O. Box 3027000100NairobiKenya

Comparison of Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines for the interpretation of antibiotic susceptibility at a University teaching hospital in Nairobi, Kenya: a cross-sectional study Annals of Clinical Microbiology and Antimicrobials

Ann Clin Microbiol Antimicrob
1521201610.1186/s12941-016-0135-3RESEARCH This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. *Correspondence: Page 2 of 7 Kassim et al. Ann Clin Microbiol Antimicrob (2016) 15:21
Background: The Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines are the most popular breakpoint guidelines used in antimicrobial susceptibility testing worldwide. The EUCAST guidelines are freely available to users while CLSI is available for non-members as a package of three documents for US $500 annually. This is prohibitive for clinical microbiology laboratories in resource poor settings. We set out to compare antibiotic susceptibility determined by the two guidelines to determine whether adoption of EUCAST guidelines would significantly affect our susceptibility patterns.Methods: We reviewed minimum inhibitory concentrations (MIC) of various antibiotics routinely reported forEscherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) isolates from an automated microbiology identification system (VITEK-2) at the Aga Khan University Hospital Nairobi's Pathology department. These MICs were then analyzed using both CLSI 2015 and EUCAST 2015 guidelines and classified as resistant, intermediate or susceptible. We compared the susceptibility and agreement between the CLSI and EUCAST categorizations.Results: Susceptibility data from a total of 5165 E. coli, 1103 S. aureus and 532 P. aeruginosa isolates were included. The concordance rates of the two guidelines for E. coli, S. aureus and P. aeruginosa ranged from 78.2 to 100 %, 94.6 to 100 % and 89.1 to 95.5 % respectively. The kappa statistics for E. coli MICs revealed perfect agreement between CLSI and EUCAST for cefotaxime, ceftriaxone and trimethoprim-sulfamethoxazole, almost perfect agreement for ampicillin, ciprofloxacin, cefuroxime, gentamicin and ceftazidime, substantial agreement for meropenem, moderate agreement for cefepime and amoxicillin-clavulanate, fair agreement for nitrofurantoin and poor agreement for amikacin. For S. aureus the kappa statistics revealed perfect agreement for penicillin, trimethoprim-sulfamethoxazole, levofloxacin, oxacillin, linezolid and vancomycin, almost perfect agreement for clindamycin, erythromycin and tetracycline and moderate agreement for gentamicin. For P. aeruginosa the kappa analysis revealed moderate to almost perfect agreement for all the anti-pseudomonal antibiotics.Conclusion:The results show comparable antibiotic susceptibility patterns between CLSI and EUCAST breakpoints. Given that EUCAST guidelines are freely available, it makes it easier for laboratories in resource poor settings to have an updated and readily available reference for interpreting antibiotic susceptibilities.

Background

Over the last two decades, there has been emergence and spread of antibiotic resistance in many bacterial clinical pathogens [1]. Categorization of minimum inhibitory concentrations (MIC) of various antibiotics in antimicrobial susceptibility testing (AST) depends on breakpoints set by various international agencies. These breakpoints affect clinical decision making by determining whether an antibiotic is reported as susceptible or not. One of the most popular guidelines used worldwide is from the Clinical and Laboratory Standards Institute (CLSI) whose interpretive cut offs for antibiotics is based on MIC distributions, pharmacokinetic-pharmacodynamic (PK-PD) properties and the mechanisms of resistance [1]. The adoption of antibiotic susceptibility guidelines in the US is regulated by the Food and Drug Administration (FDA). In 1997 various national agencies in Europe came together to form the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that has since harmonized antibiotic interpretive breakpoints throughout Europe and most European countries have since switched from CLSI and their local guidelines to EUCAST guidelines [2,3]. EUCAST bases its clinical breakpoints on epidemiological MIC cut-offs (ECOFFS) and PK-PD properties. All documents on MIC distributions and ECOFFS are freely available on the EUCAST website [4]. Polsfuss et al. compared EUCAST with CLSI in screening for extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae isolates. They found no significant difference in the sensitivity of the two guidelines in the detection of ESBL-producing isolates [5]. A more recent study by Hombach et al. demonstrated significant differences in the susceptibility rates for drugs including cefepime, ceftazidime and cefotaxime in the detection of ESBLs between the CLSI 2013 and EUCAST 2013 AST guidelines. This study recommended adjustments to the clinical breakpoints to further harmonize the two guidelines [6]. Since then 2014 and 2015 guidelines for both systems have been released in an effort to harmonize the clinical breakpoints [7,8].

CLSI guidelines have a number of disadvantages. First, it is based on annual subscriptions of US $350 for members and a cost of US $500 to non-members annually and this may be a problem for microbiology laboratories in resource poor settings. Secondly, details on the decision making process are not accessible to the public. Thirdly, the FDA has a major influence in determining official clinical breakpoints before they are adopted by CLSI and this raises major concerns on the influence of pharmaceutical industries in establishing the guidelines. CLSI has a voting committee comprising representatives of both the profession and the industry and hence the industry plays a role in the decision making process. As for EUCAST, the industry only plays a consultative role and is not allowed to finance or participate in decision making [3]. EUCAST encourages the formation of National Antimicrobial Susceptibility Testing Committees (NAC) that can send a representative to sit in its General Committee (GC) thus fostering inclusivity in the decision making process [3]. Finally, antibiotics that are not registered in the US may not be included in the CLSI guidelines.

For resource poor settings like Africa, there is need for guidelines that are accessible and affordable while still maintaining quality of reported susceptibility. Given that antibiotic susceptibility is quite variable across different settings, we set out to compare the susceptibility rates of selected antibiotics based on CLSI 2015 and EUCAST 2015 AST guidelines and the level of agreement between the two guidelines.

Methods

The study was carried out at the Aga Khan University Hospital, Nairobi's (AKUHN) Department of Pathology. Ethical exemption was granted by the AKUHN's research and ethics committee (Ref 2015/REC-44) since this type of study is low risk and classified as a clinical audit. This was a retrospective study reviewing the MICs of various antimicrobials on one commonly isolated gram positive organism, Staphylococcus aureus (S. aureus), one fermenting gram negative organism, Escherichia coli (E. coli) and one non-fermenting gram negative organism, Pseudomonas aeruginosa (P. aeruginosa). MIC data for E. coli, P. aeruginosa and S. aureus isolates was collected from two Vitek 2 (version 4.01, bioMerieux, Marcyl'Etoile, France) automated microbiology systems for the period January 2012 to December 2014. The Vitek 2 AST-P580 and AST-GN26/AST-GN83 cards were used for antibiotic susceptibility for S. aureus and E. coli/P. aeruginosa respectively. Ceftriaxone MICs were only available for the year 2014 in the AST-GN83 cards for a total of 1673 E. coli isolates. The data was summarized using Microsoft Excel 2013 and imported into IBM (International Business Machines, Corporation; Armonk, New York, United States of America) SPSS (Statistical Package for the Social Sciences) Version 22.0 that was used for analysis. The MICs were then analyzed using both the CLSI 2015 and EUCAST 2015 guidelines to categorize them as either susceptible, intermediate or resistant [7,8]. The concordance rate between the two guidelines in percentage was compared. The susceptibility rates for the various antimicrobial agents were also calculated in percentages for each organism. Analysis of the extent of agreement between CLSI 2015 and EUCAST 2015 for the various antimicrobials was carried out using Cohen's kappa statistics and graded from perfect agreement to poor agreement [9]. Cohen's Kappa statistics determines the proportion of agreement over and above chance between two independent observations. This ranges from −1 to 1 and a p value less than 0.05 means that the agreement reported is significantly different from 0 and is not due to chance. For all inferential statistics, a p value less than 0.05 was considered statistically significant.

Results

A total of 5165 E. coli, 1103 S. aureus and 532 P. aeruginosa non-duplicate isolates were included in the analysis. Of the 5165 E. coli MICs analyzed, there was comparable susceptibility to most antibiotics including ampicillin, amoxicillin-clavulanate, cefuroxime, cefotaxime, ceftriaxone, trimethoprim-sulfamethoxazole, ciprofloxacin, gentamicin and meropenem between the two guidelines. The concordance between the two guidelines ranged from 78.2 to 100 %. Table 1 summarizes the susceptibilities, concordance and kappa statistics between the two guidelines for E. coli.

The E. coli susceptibility patterns achieved after analysis using EUCAST 2015 and CLSI 2015 guidelines are similar except for amoxicillin-clavulanate, amikacin and nitrofurantoin. Analysis year by year did not show any difference in the trends and overall susceptibilities. The kappa analysis revealed perfect agreement for ceftriaxone, cefotaxime and trimethoprim-sulfamethoxazole with kappa (κ) of 1 (p < 0.000). An almost perfect agreement was noted with ampicillin κ = 0.985 (95 % CI 0.979, 0.991), p < 0.000, ciprofloxacin κ = 0.969 (95 % CI 0.963, 0.975), p < 0.000, cefuroxime κ = 0.924 (95 % CI 0.914, 0.934), p < 0.000, ceftazidime κ = 0.859 (95 % CI 0.843, 0.85), p < 0.000 and gentamicin κ = 0.979 (95 % CI 0.973, 0.985), p < 0.000. Substantial agreement was noted with meropenem κ = 0.724 (95 % CI 0.573, 0.875), p < 0.000 while moderate agreement was noted with amoxicillinclavulanate κ = 0.581 (95 % CI 0.567, 0.595), p < 0.000 and cefepime κ = 0.600 (95 % CI 0.578, 0.622), p < 0.000. Fair agreement was seen with nitrofurantoin κ = 0.351 (95 % CI 0.314, 0.388), p < 0.000 while poor agreement was noted with amikacin κ = 0.112 (95 % CI 0.079, 0.145), p < 0.000.

Of the 1103 S. aureus MICs analyzed, susceptibility to penicillin, oxacillin, levofloxacin, linezolid, trimethoprim-sulfamethoxazole, vancomycin, clindamycin, erythromycin and tetracycline was comparable between the two guidelines. The susceptibilities, concordance and kappa statistics between the two guidelines are shown in Table 2. For S. aureus the susceptibilities are generally very similar between the two guidelines.

Year by year analysis did not show any differences in trends and susceptibility patterns. The kappa analysis for CLSI 2015 and EUCAST 2015 guidelines revealed perfect agreement for levofloxacin, linezolid, vancomycin, oxacillin, penicillin and trimethoprim-sulfamethoxazole with a kappa statistic of 1 (p < 0.000). An almost perfect agreement was noted with clindamycin κ = 0.904 (95 % CI 0.826, 0.982), p < 0.000, erythromycin κ = 0.978 (95 % CI 0.960, 0.996), p < 0.000 and tetracycline κ = 0.962 (95 % CI 0.940, 0.984), p < 0.000. Moderate agreement was noted with gentamicin κ = 0.537 (95 % CI 0.441, 0.633), p < 0.000.

For the 532 P. aeruginosa analyzed there were similar susceptibility patterns noted between CLSI 2015 and EUCAST 2015. Antibiotics analyzed included amikacin, ceftazidime, ciprofloxacin, cefepime, gentamicin, meropenem and piperacillin-tazobactum. The susceptibilities, concordance and kappa statistics between the two guidelines are shown in Table 3.

Year by year analysis did not show any differences in trends and susceptibility patterns. The kappa analysis for CLSI 2015 and EUCAST 2015 guidelines revealed moderate to almost perfect agreement for all the anti-pseudomonal antibiotics. P values for the kappa statistics for all the antibiotics were statistically significant at p < 0.000. Overall, the antibiotic susceptibility patterns were quite similar regardless of whether CLSI or EUCAST 2015 guidelines were used.

Discussion

The morbidity and mortality associated with communicable diseases including bacterial infections is quite significant in developing countries [10]. Antibiotics play a critical role in treating such infections especially when instituted in a timely fashion more so when the bacteria  is susceptible to the antibiotic given. The determination of accurate antibiotic susceptibility is therefore an important cog in the clinical care of bacterial infections especially in organisms that possess acquired resistance mechanisms and careful consideration should be given when deciding how to interpret phenotypic susceptibility data [11]. In Kenya, many laboratories have adopted CLSI guidelines as a basis for interpreting their susceptibility data despite the fact that it costs between US $300-$500. These guidelines are updated annually and therefore require laboratories to keep on purchasing them at a cost that is prohibitive to most public laboratories. Failure to stay updated may result in misinterpretation of susceptibility. For example in 2013, CLSI abandoned the use of oxacillin disc diffusion in determining whether a S. aureus isolate is methicillin resistant in favour of cefoxitin because it was more accurate in determining the presence of a mecA mediated mechanism of resistance [12]. In 2012, it adopted a new disc diffusion susceptibility cut off for ciprofloxacin of 31 mm up from 21 mm for non-typhoidal Salmonella (NTS) isolated from invasive specimens and all Salmonella Typhi isolated from both invasive and non-invasive specimens. The MIC for the same was reduced from 1 to 0.06 μg/mL resulting in many Salmonella spp. previously reported as susceptible to fall into the intermediate category, and many that were intermediate were now categorized as resistant [13]. In 2013, CLSI recommended that these cut offs should apply to all Salmonella spp. including NTS from non-invasive specimens [12,14]. In the same year new levofloxacin and ofloxacin breakpoints were introduced for Salmonella spp. including Salmonella Typhi. These changes were motivated by an increased risk of treatment failure in patients with decreased ciprofloxacin susceptible Salmonella spp [15]. In 2012 again, CLSI reduced meropenem and piperacillin-tazobactum MIC breakpoints for P. aeruginosa from ≤4 to ≤2 ug/mL and ≤64 ug/mL to ≤16 ug/mL respectively [13]. These examples highlight the importance of remaining up-todate and emphasizes the need for an affordable, up-todate and readily available guideline for the interpretation of antibiotic susceptibility. In our comparison of CLSI and EUCAST guidelines for interpretation of antibiotic susceptibility, for E. coli, most of the antibiotics had moderate to perfect agreement between the two guidelines with kappa values ranging from 0.581 to 1 and two-thirds of them having almost perfect or perfect agreement with kappa values ranging from 0.859 to 1. Poor agreement was noted with amikacin with EUCAST having a more stringent breakpoint for susceptibility of ≤8 mg/L compared to the CLSI breakpoint of ≤16 mg/L. The major discrepancy was in the intermediate and susceptible categories as 8.8 % of isolates labeled as intermediate by EUCAST were all categorized as susceptible by CLSI. EUCAST guidelines eliminated the intermediate category for some antibiotics and this explains the reduced level of agreement for some of the antibiotics. For example, CLSI categorized 23.8 % of E. coli as having intermediate susceptibility to amoxicillin-clavulanate but were all categorized as resistant by EUCAST while 11.9 % categorized as intermediate susceptibility to nitrofurantoin by CLSI were categorized as susceptible by EUCAST. From a clinical stand point, reclassifying amoxicillin-clavulanate from intermediate to resistant is unlikely to adversely affect the patient as it simply removes it from being a therapeutic consideration. As for nitrofurantoin, its ability to concentrate in urine enables it to achieve significant concentrations and eliminate isolates that may have intermediate susceptibility. Therefore, the reclassification of isolates that are intermediate by CLSI to susceptible by EUCAST is unlikely to contribute to adverse outcomes for patients with urinary tract infections. The EUCAST guidelines have slightly more stringent breakpoints for some antibiotics in an effort to curb the inappropriate use of antibiotics and control the rising rates of antibiotic resistance.

For S. aureus, all except one of the antibiotics had moderate to perfect agreement and three quarters of them had almost perfect or perfect agreement with kappa values ranging from 0.862 to 1. The two guidelines performed equally in the detection of the rate of methicillin resistant Staphylococcus aureus (MRSA) of 7.2 %. Vancomycin susceptible Staphylococcus aureus (VSSA) were also detected equally by the two guidelines. EUCAST eliminated the intermediate category for vancomycin in a bid to discourage the reporting of Glycopeptideintermediate Staphylococcus aureus (GISA) due to poor response even to increased doses of vancomycin [8,16]. In view of the fact that no vancomycin resistant Staphylococcus aureus (VRSA) has been identified in our set up as yet, these changes are unlikely to influence interpretation of breakpoints. The moderate level of agreement for gentamicin is due to the more stringent breakpoints by EUCAST leading to a much higher resistance rate of 8.6 % compared to 3.2 % by CLSI. The difference between the two susceptibility cut-offs is two dilutions and this may require further harmonization. Gentamicin is rarely used as monotherapy in treating gram positive bacteria and as such this difference in MIC cut off is unlikely to be clinically significant.

For P. aeruginosa, five out of seven of the antibiotics had almost perfect agreement with kappa values ranging from 0.830 to 0.912 with the remaining two having moderate agreement with kappa values of 0.762 and 0.734. EUCAST abolished the intermediate category for ceftazidime, cefepime, gentamicin and piperacillin-tazobactum reclassifying the MICs as resistant. This accounts for the slightly reduced level of agreement. For amikacin and ciprofloxacin, with moderate agreement, EUCAST has more stringent breakpoints for susceptibility of ≤8 and ≤0.5 ug/mL compared to CLSI breakpoints of ≤16 and ≤1 ug/mL respectively. These accounted for the reduced level of agreement and may require further harmonization between the two breakpoints. For meropenem, EUCAST uses a resistant breakpoint of >8 ug/ mL while CLSI uses ≥8 ug/mL. In effect this has led to a slightly higher meropenem resistance of 27.6 % compared to 23.1 % for CLSI and EUCAST respectively. This is another aspect that will require harmonization.

Our study is limited by the fact that we only compared the susceptibility for three bacteria whose results may not necessarily be generalizable to all clinically relevant gram positive and negative bacteria. However, these three bacteria represent a significant proportion of common bacterial pathogens both in the community and healthcare settings namely Enterobacteriaceae, non-fermenting gram negative bacteria and Staphylococci. The results obtained are also limited to MICs generated by an automated bacterial identification system which is not widely used in developing countries. For P. aeruginosa, Colistin had not yet been included in the gram negative AST cards being used at the time the study was being conducted. Colistin Etest ® (bioMerieux, Durham, NC, USA) was only run for multi-drug resistant isolates of clinical significance but this data was not available for analysis. In most laboratories in sub-Saharan Africa, disk diffusion is the preferred mode of antibiotic susceptibility testing. However, disk diffusion cut offs generally approximate MIC cut offs fairly well and we think a similar comparison based on disc diffusion cut offs would yield similar results.

Conclusion

Our study shows acceptable level of agreement between EUCAST and CLSI 2015 AST guidelines for E. coli, S. aureus and P. aeruginosa and laboratories with similar antibiotic susceptibility patterns may choose to adopt either guideline without fear of significantly altering reported antibiotic susceptibility. With EUCAST guidelines being freely available it should be considered as an alternative especially in resource poor settings in order to maintain up-to-date antibiotic susceptibility interpretation.

Table 1
1Susceptibilities of E. coli to various antibiotics, concordance and kappa statistics between CLSI 2015 and EUCAST 2015 guidelinesAmox-Clav amoxicillin-clavulanate, TMP/SMX trimethoprim-sulfamethoxazole, S susceptible, I intermediate, R resistantAntibiotic
CLSI (%); n = 5165
EUCAST (%); n = 5165
Concordance (%)
Kappa, κ (95 % CI)

S
I
R
S
I
R

Ampicillin
13.8
0.5
85.7
13.8
0
86.2
99.5
0.985 (0.979, 0.991)

Amox-Clav
55.8
23.8
20.4
55.8
0
44.2
78.2
0.581 (0.567, 0.595)

TMP/SMX
0
0
100
0
0
100
100
1

Nitrofurantoin
84.6
11.9
3.5
96.5
0
3.5
87.6
0.351 (0.314, 0.388)

Ciprofloxacin
57.3
0.2
42.5
55.9
1.4
42.7
98.4
0.969 (0.963, 0.975)

Cefuroxime
63.7
3.5
32.8
63.7
0
36.3
96.5
0.924 (0.914, 0.934)

Gentamicin
78.5
0.2
21.3
78.1
0.4
21.5
99.5
0.979 (0.973, 0.985)

Amikacin
99.3
0.3
0.4
90.5
8.8
0.7
91.1
0.112 (0.079, 0.145)

Cefotaxime
69.7
0.4
29.9
69.7
0.4
29.9
100
1

Ceftazidime
76.3
0.9
22.8
71.5
4.8
23.7
93.7
0.859 (0.843, 0.85)

Ceftriaxone
67.7
0.1
32.2
67.7
0.1
32.2
100
1

Cefepime
80.5
10.0
9.5
72.9
9.8
17.3
84.9
0.600 (0.578, 0.622)

Meropenem
99.7
0.1
0.2
99.8
0.1
0.1
99.7
0.724 (0.573, 0.875)

Table 2
2Susceptibilities of S.aureus to various antibiotics and the concordance and kappa statistics between CLSI 2015 and EUCAST 2015 guidelinesTMP/SMX trimethoprim-sulfamethoxazole, S susceptible, I intermediate, R resistantAntibiotic
CLSI (%); n = 1103
EUCAST (%); n = 1103
Concordance (%)
Kappa, κ (95 % CI)

S
I
R
S
I
R

Penicillin
10.6
0
89.4
10.6
0
89.4
100
1

TMP/SMX
0
0
100
0
0
100
100
1

Clindamycin
98.0
0.3
1.7
98.0
0.1
1.9
99.5
0.904 (0.826, 0.982)

Erythromycin
85.6
0.5
13.9
86.0
0
14.0
99.4
0.978 (0.960, 0.996)

Gentamicin
96.3
0.5
3.2
91.5
0
8.5
94.6
0.537 (0.441, 0.633)

Levofloxacin
90.8
0.3
8.9
90.8
0.3
8.9
100
1

Linezolid
100
0
0
100
0
0
100
1

Oxacillin
93.3
0
6.7
93.3
0
6.7
100
1

Tetracycline
83.2
0.1
16.7
82.4
0.8
16.8
98.9
0.962 (0.940, 0.984)

Vancomycin
100
0
0
100
0
0
100
1

Table 3
3Susceptibilities of P.aeruginosa to various antibiotics and the concordance and kappa statistics between CLSI 2015 and EUCAST 2015 guidelinesPip-Taz piperacillin-tazobactum, CI confidence interval, S susceptible, I intermediate, R resistantCLSI (%); n = 532
EUCAST (%); n = 532
Concordance (%)
Kappa, κ (95 % CI)

S
I
R
S
I
R

Amikacin
79.5
3.0
17.5
72.2
7.3
20.5
89.7
0.734 (0.703; 0.765)

Ceftazidime
70.9
4.7
24.4
70.9
0
29.1
95.3
0.890 (0.870; 0.910)

Cefepime
72.6
4.7
22.7
72.6
0
27.4
95.3
0.886 (0.866; 0.906)

Gentamicin
72.6
6.2
21.2
72.6
0
27.4
93.8
0.851 (0.829; 0.873)

Meropenem
64.1
8.3
27.6
64.1
12.8
23.1
95.5
0.912 (0.895; 0.929)

Pip-taz
64.5
8.3
27.3
64.5
0
35.5
91.7
0.830 (0.808; 0.852)

Ciprofloxacin
71.8
5.3
22.9
66.2
5.6
28.2
89.1
0.762 (0.736; 0.788)

AcknowledgementsNone.Competing interestsThe authors declare that they have no competing interests.Authors' contributions AK and GO contributed equally in conceiving and designing the study, data collection, data analysis and drafting the manuscript. ZP and GR participated in study design, coordination and critical revision of the manuscript. All authors read and approved the final manuscript.
Amox-Clav: amoxicillin-clavulanate; AST: antimicrobial susceptibility testing; CLSI: Clinical Laboratory Standards Institute; ECOFFS: epidemiological cut-offs; ESBL: extended spectrum beta lactamase; Etest: epsilometer test; EUCAST: European Committee on Antimicrobial Susceptibility Testing; E. coli: Escherichia coli; FDA: Food and Drug Administration; GC: General Committee; GISA: Glycopeptideintermediate Staphylococcus aureus. IBM: International Business Machines Corporation. Nairobi; Armonk, New YorkAKUHN: Aga Khan University HospitalMIC: minimum inhibitory concentration; MRSA: methicillin resistant Staphylococcus aureusAKUHN: Aga Khan University Hospital, Nairobi; Amox-Clav: amoxicillin-cla- vulanate; AST: antimicrobial susceptibility testing; CLSI: Clinical Laboratory Standards Institute; ECOFFS: epidemiological cut-offs; ESBL: extended spectrum beta lactamase; Etest: epsilometer test; EUCAST: European Com- mittee on Antimicrobial Susceptibility Testing; E. coli: Escherichia coli; FDA: Food and Drug Administration; GC: General Committee; GISA: Glycopeptide- intermediate Staphylococcus aureus; IBM: International Business Machines Corporation (Armonk, New York, United States of America); MIC: minimum inhibitory concentration; MRSA: methicillin resistant Staphylococcus aureus;

NAC: National Antimicrobial Susceptibility Testing Committees; NTS: nontyphoidal salmonella; Pip-Taz: piperacillin-tazobactum; PK-PD: pharmacokinetic-pharmacodynamics; SPSS: statistical package for the social sciences. S. aureus: Staphylococcus aureusNAC: National Antimicrobial Susceptibility Testing Committees; NTS: non- typhoidal salmonella; Pip-Taz: piperacillin-tazobactum; PK-PD: pharmacoki- netic-pharmacodynamics; SPSS: statistical package for the social sciences; S. aureus: Staphylococcus aureus;

United States of America dollar; VRSA: vancomycin resistant Staphylococcus aureus. ; Tmp/Smx: Trimethoprim-Sulfamethoxazole, Us $, VSSA: vancomycin susceptible Staphylococcus aureusTMP/SMX: trimethoprim-sulfamethoxazole; US $: United States of America dollar; VRSA: vancomycin resistant Staphylococcus aureus; VSSA: vancomycin susceptible Staphylococcus aureus.

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A Pilot Study of Nuclear Instability in Archived Renal and Upper Urinary Tract Tumours with Putative Ochratoxin Aetiology
2010

Peter G Mantle
Centre for Environmental Policy
Imperial College London
SW7 2AZLondonUK

Cyrille Amerasinghe
Department of Histopathology
Ealing Hospital
UB1 3HWSouthallMiddlesexUK

Amy L Brown
Department of Oral Pathology
Kings College London
LondonUK

Diana Herman
Pathology Department
County Hospital Timisoara
Romania

Thomas Horn
Pathology Department
Herlev Hospital
University of Copenhagen
DK-2730HerlevDenmark

Thoger Krogh
Department of Rheumatology
Aarhus University Hospital
8000AarhusDenmark

Edward W Odell
Department of Oral Pathology
Kings College London
LondonUK

Tomas Rosenbaum
Department of Urology
Ealing Hospital
UB1 3HWSouthallMiddlesexUK

Calin A Tatu
Department of Biology
University of Medicine and Pharmacy
TimisoaraRomania

A Pilot Study of Nuclear Instability in Archived Renal and Upper Urinary Tract Tumours with Putative Ochratoxin Aetiology

Toxins
2201010.3390/toxins2030326Received: 21 January 2010; in revised form: 22 February 2010 / Accepted: 24 February 2010 /* Author to whom correspondence should be addressed; E-Mail: email@example.com;DNA ploidyBalkan endemic nephropathyrenal cell carcinomaochratoxin Aporcine nephropathymetastasisnuclear instabilitytransitional cell carcinomaaneuploidisation OPEN ACCESS Toxins 2010, 2 327
DNA ploidy measurement has been applied uniquely to wax-embedded tissue of primary renal cell and metastatic tumours of a key experimental researcher on porcine ochratoxicosis, a control, and four transitional cell carcinomas from cases of Balkan endemic nephropathy. Primary renal tumour was diploid, and hyperdiploid metastasis was within the lower ploidy range for typical renal cell carcinoma. Three Balkan primary tumours showed extensive aneuploidy indicating marked nuclear instability, similar to model rat renal carcinoma caused by ochratoxin A. In contrast, much less nuclear instability in the putative occupational ochratoxicosis case fitted poorly with the ochratoxin A model.

Introduction

The study arises from the potent nephrocarcinogenicity of the mycotoxin ochratoxin A (OTA), the fact that most human renal tumours are idiopathic, and the uncertain designation of dietary OTA as a human health risk.

Ochratoxin A is one of the first-discovered mycotoxins. Recognised in South Africa as a fungal metabolite that was toxic to experimental rats [1], the structure was also determined there [2]. OTA became more widely important in the 1970s when implicated in the aetiology of the chronic porcine nephropathy that was occurring sporadically in Danish pigs [3,4]. OTA occurrence in Denmark was associated particularly with mould spoilage of home-grown cereals destined for pig feed that had not been dried adequately before storage.

A curious human nephropathy was recognised as a discrete entity in the Vratza district of Bulgaria in the mid-1950s [5,6] and gradually became recognised more widely within the Balkans as an idiopathic nosologic entity of mosaic occurrence in certain households in certain agricultural villages. It manifests as a silent progressive bilateral renal atrophy that generally becomes apparent in late middle age. The pathology was addressed formally by the World Health Organisation [7], was a topic for a CIBA Foundation Study [8], and articles on it gradually appeared in Western European literature, summarised in [9]. Concurrently, there was a climate of expanding discovery of fungal metabolites (e.g., aflatoxin B1, zearalenone and several trichothecenes) as potent agents of natural mammalian disease. Since Danish veterinarian Professor Palle Krogh had been much involved in large-scale pig experiments on OTA and renal disease, it was just imaginative lateral-thinking for him to propose that the mycotoxin might also be an agent of human disease, particularly the Balkan endemic nephropathy [10]. Balkan endemic nephropathy (BEN) occurred mainly in flood-plain regions associated with the Danube and its tributaries, but was focused on particular households, irrespective of ethnic origin, in rural communities in which storage conditions for agricultural commodities, predominantly home-grown for humans and for pigs, were susceptible to mould spoilage. Thus causal association with Danish porcine nephropathy was readily made [3] following detection of OTA in cereals [11] and experimental studies based on the natural occurrence of OTA in Danish cereals [12]. Consequently, research was stimulated within and outside Balkan countries into the human disease, the extent and identity of endemic mould sources of OTA, the incidence of the toxin in human diet, and on assessing the potential human risk of traces of OTA in human food.

Whereas, in its original South African discovery, OTA was a metabolite of an isolate of Aspergillus ochraceus, which is not a part of the natural mycobiota in Northern European latitudes (where Penicillium verrucosum was the producing fungus), it was not easy to predict the source in the Balkans, where climate is more temperate although winters can be cold. Studies on moulds contaminating foodstuffs from nephropathy households in hyperendemic villages in Croatia and Bulgaria revealed no evidence of ochratoxinogenic P. verrucosum; A. ochraceus was very rare and usually was not ochratoxinogenic in laboratory culture on cereal substrate [13,14]. Concurrently, A. ochraceus was isolated from 10% of 855 samples of stored cereals and dried meats collected (1980)(1981)(1982)(1983)(1984)(1985)(1986)(1987) from nephropathy and non-nephropathy households in Croatia [15]. One third of isolates were slightly ochratoxinogenic in laboratory culture but only one, from ham, was a prominent producer with yield similar to a reference culture. It was concluded that there was no significant difference in ochratoxinogenic mycobiota between the two regions. Otherwise, the only other systematic mycological study was on 14 samples of poor quality wheat tailings used in feed of pigs expressing typical clinical symptoms of mycotoxic porcine nephropathy in Bulgaria [16];

A. ochraceus was a rare component in only four of these but was not ochratoxinogenic in laboratory culture.

The problem of mycotoxic porcine nephropathy in Denmark became less prevalent as moisture in grain for storage became controlled more rigorously. Consequently, concern over OTA waned somewhat until results of a US National Toxicology Programme (NTP) study on the toxicology of OTA appeared [17], showing it to be the most potent rat renal carcinogen found to date. This distinction still prevails [18]. Additionally, the incidence of upper urinary tract tumours amongst subjects diagnosed with the Balkan nephropathy was found to be up to 100-fold higher than amongst Balkan populations not affected by the bilateral renal atrophy [19]. Naturally, concern then became heightened regarding a putative added aetiological connection between OTA and human urological tumours, most of which were, and still are, of uncertain/unknown cause.

Shortly before the NTP study was published, Palle Krogh was diagnosed with a renal cell carcinoma (RCC) requiring radical nephrectomy in 1988, but tragically he died 2 years later. This might ironically have been a unique case of potential occupational accidental exposure to the potent OTA during his practical involvement in large-scale pig experiments before its carcinogenic potential had been fully realised. To our knowledge, no person who has died from renal cell carcinoma has also been shown specifically to have been exposed to abnormal amounts of OTA. Trace amounts (~1 ppb) of OTA are found widely in human blood but no human morbidity has yet been attributed conclusively to OTA.

Recently, the opportunity arose to measure DNA ploidy distribution in experimental rat renal tumours that had occurred in response to dietary OTA during lifetime studies [20 and associated experiments]. Differentiation of tumours into adenomas and carcinomas by histopathological criteria was consistently correlated with diploid and markedly aneuploid status, respectively [21]. That study has now been extended to include some archived human pathological material, particularly including primary renal tumour and a distant metastatic tumour of Palle Krogh, and a control renal tumour for which abnormal OTA exposure was not suspected. The technique has been extended into a pilot study of four archived upper urinary tract transitional cell carcinomas (TCC) from cases of Balkan endemic nephropathy to test its efficacy and contribute to current debate on the validity of extrapolation from animal experiments with OTA to human risk. The pilot study also assessed potential for meaningful application of DNA ploidy measurement to the extensive histological archives of TCC from Balkan nephropathy cases, as well as to currently-emerging cases.

Results

DNA ploidy distribution in Palle Krogh's RCC, and in the control RCC, was diploid in both regions analysed (Figure 1 A, B). DNA in nuclei of a distant metastasis had an aneuploid distribution, with one main hyperdiploid peak and only occasional nuclei of higher ploidy ( Figure 1D). The primary clear cell tumour ( Figure 3B) had extended into the renal vein; this tissue also had a diploid distribution pattern ( Figure 1C) matching the mass of tumour at the renal pole. However, a histological preparation of a serial section adjacent to those taken for ploidy analysis revealed occasional pleomorphic nuclei ( Figure 2) near to the venous lumen. Whereas their ploidy status is unknown, it is possible that aneuploidisation had already commenced in this part of the primary tumour, from which metastatic dissemination might readily have occurred.   DNA ploidy in one of the Balkan TCC was diploid ( Figure 4C; case 3), as was the skin tumour control ( Figure 4C). However, the other three TCC were markedly aneuploid ( Figure 4A, B, D; cases 1, 2, 4) although none of these distributions contained a significant hyperdiploid component. Case 1 aneuploidy was mainly hypertriploid, but in cases 2 and 4 aneuploidy extended considerably beyond tetraploid. The factors of tumour size and grading and association with pyelonephritis in the 4 tumours did not correlate with the degree of aneuploidisation. There was also considerable variation in the histopathology among the four Balkan TCC ( Figure 4A-D).

Discussion

The renal cancer cases

The Krogh case study focuses on RCC developing silently in a Caucasian male in a developed Western European environment during middle age. The morbidity provoking surgical intervention occurred only when the primary tumour had already become rather large. However, although archived renal tumour had a diploid DNA distribution in the analysed tissue, subsequent survival for only two years after radical nephrectomy pointed to some further clonal selection for metastasis, prior to nephrectomy, as evidenced by the aneuploidy of a metastatic carcinoma discovered subsequently. Unfortunately, metastatic tissue from lung at the terminal stage had not been archived. However, it is tantalising to contemplate whether occasional nuclear instability in tumour invading the renal vein had already been a crucial step spawning aggressive metastasis to distant organs.  The picture fits closely into the current pattern exhibited by West European cases of RCC as shown in a recent publication on clinical and molecular aspects of the disease [25]. The case also conforms to recent surveys of DNA ploidy distribution in RCC cases coming to radical nephrectomy, with aneuploidisation discovered in some part of the primary tumour correlating with tumour classification, so that a switch from diploidy to aneuploidy is a feature developing late within a RCC [26,27]. Specifically, the significant hyperdiploid component in the Krogh metastatic tumour corresponds to the ploidy distribution illustrated as within the typical range for clear cell RCC [27]. The present ploidy analysis of both primary tumour and distant metastasis is possibly the first individual sequence reported for human RCC. For human renal tumours, ploidy analysis of up to five regions of the tissue is desirable for a reliable measure, but in many of the rat renal tumours attributed to OTA [21] analysis was made on all, or a large proportion of, the tumour. Pinto et al. [25] stated 'patients presenting aneuploid tumours are estimated to be 15.6 times more likely to die of the disease than are those with diploid tumours'. The clear sequence from diploid primary tumour to aneuploid secondary tumour in the Krogh case is illustrative of the aneuploidisation principle, and it is tantalising to consider whether renal vein invasion had been involved in this process. In retrospect, a stage of T4N0M1 could be ascribed to the primary renal tumour, consistent with a poor actual outcome and particularly on account also of the venous invasion [28].

The mechanisms of initiation and proliferation of human RCC remain generally obscure. Some predisposing factors such as analgesic abuse (notably phenacetin) are recognised, but otherwise the disease seems just to be linked non-specifically to societies with an affluent lifestyle [29]. That specific DNA damage is necessary for the initial adenoma lesion, and again for differentiation into metastasising carcinoma, has justified study of the rare familial form of RCC, involving homozygous occurrence of the von Hippel Lindau recessive gene mutation, as an interesting putative model. However, study of early stages of human RCC is extremely difficult. Hence in the Krogh case, the close professional involvement in experimental ochratoxicosis A in farm and laboratory animals at a time when the possible human risk, as then perceived, could not have been fully appreciated, might have allowed unwise and un-natural exposure to the toxin. The pathological outcome might therefore be a pointer to OTA as a human carcinogen. For this reason, in the absence of any human cases in which OTA has been shown to cause any morbidity, several putative mechanisms have been proposed to account for carcinogenesis in the specific renal tumours that have been produced experimentally in male rats by OTA [30][31][32][33]. The relative contribution of these remains unclear while official uncertainty about the genotoxicity of OTA as a chemical carcinogen persists [34].

However, the present finding of rather limited aneuploidisation (hyperdiploid) only in the Krogh metastatic tumour does not fit comfortably with the marked nuclear instability implied from the extreme aneuploidy (up to ~32-ploid) in primary rat renal carcinomas caused by OTA [21] and the case can not provide evidence supporting the IARC classification of OTA as a potential human carcinogen [35]. In contrast, the extreme aneuploidy found in the TCC of most of the Romanian BEN patients, matches that found in rat OTA tumours [21] quite closely. Human tumours initially develop with minimal genetic damage, as revealed by the DNA ploidy measurement, but only develop nuclear instability at the stage at which they metastasise. OTA tumours in rats develop marked nuclear instability quite early, often in situ and certainly before metastasis, and develop multiple aneuploid stem lines and aneuploid cells early during proliferation of the mass. The Krogh RCC showed no evidence of this until metastasis and thus this human type is unlikely to be due to OTA. Wider study of nuclear instability in TCC of BEN patients from across the Balkans would assist in consolidating the apparent contrasting situation in RCC and TCC. The technique used here is particularly designed to enable measurements on wax-embedded archival tissue.

Balkan endemic nephropathy: The ochratoxin A hypothesis

The aetiology of the insidious bilateral renal atrophy of BEN, and the TCC with which it is frequently associated, remains at most uncertain, as is also the extent to which the two disparate pathologies of atrophy and proliferation have a common cause. Krogh's perception of OTA's involvement concerned just the very slow human nephropathy, but was based on general extrapolation from the relatively more rapid Danish mycotoxic nephropathy in bacon pig production. However, there has always been the uncomfortable juxtaposition of the marked renal hypertrophy in pigs (also associated with impaired live-weight gain) with the progressive silent renal atrophy in BEN, which are not easy to reconcile through a common pathological mechanism [36]. A more plausible role for OTA might concern the TCC in BEN patients, because of a presumed greater availability of unmetabolised OTA passing through partially dysfunctional fibrotic kidney and thus having access to the transitional cell epithelia of the renal pelvis and ureter. Increased OTA concentration has also been detected in sera of BEN patients from Croatia, compared to controls [37], supporting the idea of a dysfunctional kidney unable to clear the mycotoxin efficiently from the body. Moreover, the principal DNA adduct attributed to covalent binding with OTA was detected in three Balkan renal pelvis tumours, but also in bladder tumours [38,39]. However, it is important to know whether evidence of genuine OTA/DNA adducts in tumour tissue reflects their tumour-causing role for the mycotoxin, or shows an attractive proliferating depot in a morbid ageing subject where traces of circulating OTA can accumulate in malignant tumour. The evidence could just represent genetic damage that was unrepairable by malignant tumour. Study of tumours in non-urological tissues of BEN subjects could reveal data to clarify any specificity to urological tissue. In any case, in establishing an aetiological connection of OTA with all or part of the pathologies of BEN it would be vital to demonstrate consistent correlation of evidence of toxin exposure with disease development; correlation only in a proportion of cases would not suffice for satisfactory proof.

Upper tract urothelial tumours [40] occurring in some BEN patients are all transitional cell neoplasms irrespective of their close proximity to the kidney in the renal pelvis. In contrast, the most potent expression of experimental renal cancer specifically by OTA is in the male rat, but tumours are all closely associated with the renal parenchyma and are found in ageing animals, even if OTA exposure occurs only during the first third of a normal lifespan.

Extensive literature on analysis for OTA in food commodities and human diets, and also human blood, records the widespread occurrence of traces of the toxin, but no notable pattern of exceptional geographic foci [41,42]. However, a dietary study in 16 young healthy adults (gender not specified) in two Bulgarian hyperendemic villages found consistent exposure to OTA [43], but in two subjects the amounts were close to or slightly exceeded the current provisional tolerable weekly intake (100 ng/ kg body weight) recommended at that time by JECFA. By definition, this may not impose a health risk, but these individuals (if identified) may be particularly valuable for long-term assessment of OTA toxicology. However, another survey in the same region had found hardly different mean daily intake of OTA (1.21 µg and 1.03 µg, respectively) in BEN households versus households in non-BEN villages [44].

Even the sources of the OTA detected in some BEN areas are still unclear. The dominant OTA-producing fungus in Northern Europe is P. verrucosum, and of more tropical areas is A.

ochraceus and the black-spored species A. niger and A. carbonarius. Studies on food-borne moulds in the specific houses in the Croatian village of Kaniza [12] revealed none of the above. One isolate of P.

variabile showed blue fluorescence in agar culture, but this was not due to OTA. A more detailed study in 20 BEN households across five Bulgarian hyperendemic villages [13] found one example of A. ochraceus, with a weak expression of OTA biosynthesis, and two atypical forms of P. verrucosum which did not produce OTA. However, two Penicillia ( Figure 5) from maize, P. griseofulvum (IMI 351303) and an atypical P. viridicatum, produced OTA in laboratory culture on shredded wheat (1-2 ppb). Also, an isolate ( Figure 5) assigned as another atypically-ochratoxinogenic species (P. solitum (IMI 351303), supported significant OTA productivity (1.3 ppm) in shredded wheat. These fungi had been isolated from the hyperendemic village of Gorno Peshtene, also included in the study by Vrabcheva et al. [43]. Subsequent study of low-grade local cereals, distant from the Bulgarian BEN endemic area and used for feed in pig production units where mycotoxic porcine nephropathy occurred, revealed no obvious ochratoxinogenic fungi [15]. Clearly there has been considerable underinvestment in understanding the mycological epidemiological aspect of putative mycotoxin involvement in BEN. P. viridicatum (right). The latter three isolates were ochratoxinogenic [14].

Conclusions

Potential for application of the present technique for DNA ploidy measurement in Balkan TCC, archived in wax blocks or preserved in formalin, has been demonstrated in this pilot study and could add valuable information on the contrasting extent of nuclear instability in primary TCC and RCC. However, further analysis would require a well-funded and dedicated programme. It should be remembered that the precise aetiological relationship between the silent bilateral renal atrophy of BEN and the upper urinary tract TCC that arise in some cases remains unclear. To define the extent of nuclear instability across the resource of tumours archived within the Balkan countries could add fundamental information to which mode(s) of mutagenic mechanism of putative toxicants (including OTA) in animal models might be compared for a possible fit.

Experimental Section

Ploidy analysis

DNA ploidy distribution analysis was performed as previously described [22] and applied recently to rat renal tumours [21]. Briefly, several 50 µm sections were cut from selected areas of blocks, dewaxed in xylene, rehydrated and nuclei extracted using protease type XXIV (Sigma Chemical Co., Poole, UK) [23]. Nuclear monolayers were prepared in a cytospin 4 (Thermo Shandon, UK) and stained by the Feulgen-Schiff method and analysed on a Fairfield image-based ploidy analyzer (Medical Solutions, UK). Monolayers were analysed using the automated scanning option. Diagnostic criteria for ploidy status were as previously published for this system. A sample was classified as diploid if only one G0/G1 (2c) peak was present, or if the number of nuclei in the G2 (4c) peak did not exceed 1% of the total number of epithelial cells. A lesion was defined as aneuploid if non-euploid peaks were present, or if the number of nuclei with DNA content greater than 5c exceeded 1%.

Tissues relating to renal cell carcinoma

Tumour details for Palle Krogh ( Figure 6): In June 1988 (age 53) CT-scanning revealed an 8 cm tumour in the lower part of the right kidney. Needle aspiration showed cells suspicious of renal cell carcinoma (RCC). One month later, radical nephrectomy removed a carcinoma (9 × 8 × 8 cm) located at the lower pole. The cut surface had areas of necrosis and haemorrhage. The tumour did not obviously extend beyond the capsule, but did extend into the renal vein. A tumour thrombus (10 × 5 × 5 mm) was removed from the inferior vena cava. Microscopically, it was a typical RCC of clear cell type, moderately differentiated. Tumour tissue with similar morphology was found in the renal vein and in the inferior vena cava. The primary renal cell tumour at operation was staged as T3bN0M0. In September 1989 a solitary metastasis of a clear cell carcinoma was removed from soft tissue in the right femoral region. Notably, recent MRI illustration of a femoral metastatic deposit is shown in [24]. In February 1990 biopsies from the right main bronchus, lower right bronchus and at the bifurcation showed metastasis from RCC; histopathology was of the clear cell type. Control male: Currently resident in England, age 56 at radical left nephrectomy. The tumourous kidney (300 g) had a partially necrotic and haemorrhagic tumour at the upper pole of the kidney (11 × 9 × 6 cm). There were multiple external nodules (largest 1.5 cm in diameter) in perirenal fat.

Microscopically, the tumour was a moderately differentiated RCC with areas of haemorrhage and necrosis; the tumour infiltrated through the capsule into perirenal fat. Areas showed lymphovascular invasion, but renal vein and large arteries were free of tumour. Pathological grade was Fuhrman's grade 2 and Robens stage at least 2. Additionally, bladder mucosa had a well-differentiated papilliary transitional cell carcinoma, pathological grade G1pTa.

Romanian transitional cell tumours (TCC) from female BEN patients (2002-2004):

Case 1; 75 years. Gross: Left kidney, tumour located in the renal hilum, 3 × 1.5 × 1.5 cm, exophytic aspect, friable. Microscopy: TCC, papillary, moderately differentiated G2. Chronic pyelonephritis in the adjacent parenchyma. Proximal ureter and bladder wall not affected. Case 2; 70 years. Gross: Dorso-lumbar wall tumour, infiltrating the fat, muscle and upper dermis, 3 × 3.8 cm, solid, high consistency. Microscopy: TCC, moderately differentiated G2, associated to poorly differentiated adenocarcinoma. Case 3; 65 years. Gross: Left kidney, tumour of pelvic origin, 7 × 6.5 cm, papillary aspect, friable, necrotic-haemorrhagic. Microscopy: TCC, moderately/poorly differentiated G2/3, with foci of squamous differentiation, tumor necrosis, hyalinization, calcifications and vascular invasion. Chronic pyelonephritis in the adjacent parenchyma. No tumour invasion into proximal or distal ureter. Case 4; 74 years. Gross: Left kidney, tumour of pelvic origin, One dilated calyx containing a slightly raised tumour, 0.2 × 1 × 1.5 cm. Microscopy: TCC, poorly differentiated G3, infiltrating the submucosa. Chronic pyelonephritis in the adjacent parenchyma. Tumour invasion of ureter as small, multiple islands of the same cancer type, infiltrating the whole ureter wall.

Control female: 69 years in 2001. Romanian benign papillomatous skin tumour, not associated with BEN

Figure 1 .
1DNA ploidy distribution graphs for nuclei of tumour tissues. A; typical diploid display for the UK renal cell carcinoma (RCC) providing 'control' data for that of three Krogh tumour tissues. B; main mass of Krogh RCC (diploid), C; Krogh tumour extending into renal vein (diploid), D; Krogh distant aneuploid metastasis (hyperdiploid). Ploidy chart nuclear codes: black = renal epithelial cells, white = internal control lymphocytes, shaded = internal control fibroblasts.

Figure 2 .
2H & E stained histopathology of RCC extending into the renal vein, showing occasional pleomorphic nuclei.

Figure 3 .
3H & E stained histopathology of RCC. Left; UK 'control' tissue. Right above; Krogh's typical clear cell renal tumour. Right below; disorganised aneuploid tissue of the distant metastasis. Synchronised magnification from a ×20 objective.

Figure 4 .
4Matched DNA ploidy distribution and H & E stained histopathology of four Romanian transitional cell carcinomas (TCC) from upper urinary tract of subjects diagnosed with BEN. A, B and D: aneuploid tumours of cases 1, 2 and 4, respectively. C: Typical diploid data of a 'control' tumour from skin, adjacent to a similar pattern in a diploid TCC with matching histopathology (case 3). Notably, a hyperdiploid population of nuclei, as in the Krogh distant metastasis, is absent in all cases. Magnifications: A,D x 400 ; B,C x 200. Ploidy chart nuclear codes: green = renal epithelial cells, red = internal control lymphocytes, blue= internal control fibroblasts.

Figure 4 .
4Cont.

Figure 5 .
5Fungi isolated from maize (containing ochratoxin A) in a BEN household in Gorno Peshtene, Bulgaria cultured for taxonomic identification according to Pitt[45] on Czapek-yeast agar (left), G25N agar (centre) and malt extract agar (right) for 7 days at 25 ºC. Within each plate: P. verucosum (left), P. griseofulvum (above), P. solitum (below),

Figure 6 .
6Prof. PalleKrogh (1935Krogh ( -1990.
© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
AcknowledgementsWe are grateful to Guy's and St Thomas' Charity for research funding to E. W. Odell.
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