Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats  obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species “Leptospira licerasiae” serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010T, which has been deposited into internationally accessible culture collections. By microscopic agglutination test, “Leptospira licerasiae” serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti–L. fainei serovar Hurstbridge at a titer of 1∶100. LipL32, although not detectable by PCR, was detectable in “Leptospira licerasiae” serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against “Leptospira licerasiae” serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon.As part of a prospective study of leptospirosis and biodiversity of  Leptospira in the highly biodiverse region of the Peruvian Amazon, and demonstrate that the animal source of infection for humans is the domestic rat. Detailed biological characterization of this new species shows that it is antigenically unique and represents a new serogroup and serovar, proposed as Leptospira licerasiae serogroup Iquitos serovar Varillal. Incorporation of this new isolate into serological testing of patients presenting with acute febrile illness in Iquitos, Peru, showed a far higher incidence of leptospirosis than previously suspected, showing the important of using region-specific Leptospira in diagnosis. The field-to-laboratory approach presented here has general application to the discovery of other emerging pathogens and their impact on human health.Leptospirosis has emerged as a globally important infectious disease. Its impact on public health is often difficult to determine, sometimes because of low clinical suspicion, or, as is more common, difficulty in laboratory diagnosis. Gold-standard serology-based diagnosis has a number of important limitations, including the need to use live leptospires that have a sufficient diversity of antigens to be able to detect specific anti-leptospiral antibodies; such antigens vary greatly from region to region. In this paper, we report the discovery of a new species of  LeptospiraLeptospirosis is a zoonotic disease of world-wide distribution caused by pathogenic spirochetes of the genus Leptospira licerasiae” serovar Varillal strain VAR 010T, determined its major mammalian reservoir, and shown its importance in regional diagnosis of acute leptospirosis.With its diverse fauna, tropical climate and the lack of proper sanitation, the Peruvian Amazon region of Iquitos and its surrounding areas provide an ideal ecological setting for the maintenance and transmission of leptospirosis Patients presenting at the Belen, Moralillo, Varillal, Padrecocha and Zungarococha Ministry of Health health posts and the Hospital de Apoyo in the Iquitos region of the Peruvian Amazon with complaint of fever were enrolled in a prospective study after oral assent for adults  or written informed consent from parents or legal guardians for children. Included in the informed consent process was a request to administer a questionnaire that asked for personal, medical, demographic and economic information, and requests for serial samples of blood and urine. Specific dates of the study periods are as follows: Belen, January 2003 to September 2005; Hospital Apoyo de Iquitos, May 2003–April 2006; Zungarococha, November 2002 to July 2005; Moralillo, January 2003 to January 2005; Varillal, November 2002 to July 2005; Padre Cocha February 2004 to May 2005.Leptospira were observed by 12 weeks. A high level of care was taken to avoid contamination by water-borne saprophytic Leptospira; no saprophytes were obtained during the course of the study (as determined by 16S rRNA gene sequencing).Inclusion criteria were a self-reported undifferentiated febrile illness of ≤2 weeks duration with a negative malaria smear. Clinical and demographic data were collected from the patients using a questionnaire. Seven milliliters of whole blood were collected by venipuncture at the time of presentation for culture and serological analysis. Follow-up blood samples for serological analysis and mid-stream urine samples for leptospiral culture were collected 10–70 days after enrollment. For urine culture, the pH of samples was adjusted to ∼7.4 with 10 M NaOH at the time of collection. Two tubes of 5 ml semisolid EMJH  containing 0.01% (w/v) 5-fluorouracil (5-FU) and 300 µg/ml neomycin were inoculated on site with 2 and 4 drops of whole blood, respectively, using strict aseptic techniques. Urine samples were centrifuged briefly at ∼800 rpm and 2 tubes of semisolid EMJH medium (BD Biosciences) containing the same antibiotics and concentrations were inoculated with 2 and 4 drops of clarified urine, respectively. Cultures were examined weekly by darkfield microscopy and classified as negative if no organisms typical of This study was approved by the Human Subjects Protection Program, University of California San Diego, and the Ethical Committees of Asociacion Benefica PRISMA, Lima, Peru, and Universidad Peruana Cayetano Heredia, Lima, Peru.“L. licerasiae” serovar Varillal). Microscopic agglutination testing (MAT) was done using the following antigens (serogroup followed by serovar in parentheses): serogroup Andamana (serovar Andamana), Australis , Ballum , Bataviae (Bataviae), Canicola (Canicola), Celledoni (Celledoni), Cynopteri (Cynopteri), Djasiman (Djasiman), Grippotyphosa (Grippotyphosa), Hebdomadis (Borincana), Icterohaemorrhagiae , Javanica (Javanica), Mini (Georgia), Pomona (Pomona), Pyrogenes (Alexi and Pyrogenes), Sejroe (Hardjo and Wolffi), and Tarassovi (Tarassovi). Sera were screened at a dilution of 1∶100 and positive sera were titrated to endpoint using standard methods Serologic testing of patient samples was performed at the Instituto Nacional de Salud in Lima, Peru using an in-house IgM ELISA Clinical criteria for submitting sera on patients (both in Iquitos and nationwide) for serological diagnosis were undifferentiated fever for 2 weeks or less, malaria smear negative, and no alternative explanation for fever.Serological criteria for diagnosing acute leptospirosis in all areas of Peru other than Iquitos included any one of the following: seroconversion in IgM by ELISA from acute to convalescent sera; seroconversion in MAT from negative to 1∶100 or greater; 4-fold rise in titer between acute and convalescent sera; or a single titer of 1∶400 or greater. The single titer of 1∶400 in non-Iquitos regions was chosen as the seropositivity cutoff because of national data indicating that titers at this level or lower were background in the population in asymptomatic individuals .“L. licerasiae” serovar Varillal antibodies in asymptomatic individuals (data not shown). Serological criteria to assign a diagnosis of acute leptospirosis in Iquitos included any one of the following: IgM positive by ELISA in either acute or convalescent sera; seroconversion in MAT from negative to 1∶100 or greater; 4-fold rise in titer between acute and convalescent sera; or a single titer of 1∶800 or greater.Serological criteria for stating that a specific MAT titer was associated with acute leptospirosis were made more stringent in Iquitos than in other parts of Peru because of the high prevalence of low level (1∶400 or less) anti-Rats were caught live in baited wire-mesh traps  left overnight near dwellings in the urban area of San Juan near the Iquitos airport, in the urban slum of Belen in Iquitos, or the rural area of Moralillo 15 km outside Iquitos removed 1 km from the Iquitos-Nauta road. Dates which animals were captured, according to isolate, are as follows: CEH001, 11/22/02; CEH006, 11/26/02; CEH010 11/26/02; CEH011, 12/14/02; CEH033 12/17/02; CEH044 12/19/02; CEH162 01/21/03; MMD735, 01/19/03. Animals were anesthetized with isoflurane and the kidneys were removed aseptically; blood was collected by cardiac puncture. Excised kidney material was minced using a sterile scalpel blade and cultured in semisolid EMJH containing antibiotics. All cultures were incubated at 30°C for up to 12 weeks and checked bi-weekly for growth. Positive cultures were sub-cultured into liquid EMJH for serological and molecular typing. Animal trapping and use was approved by the Instituto Nacional de Recursos Naturales of Peru (INRENA) and the Institutional Animal Care and Use Committee, University of California San Diego.Salmonella serotype Braenderup H9812 was digested with 50 U XbaI (New England Biolabs) for use as a DNA size standard Agarose blocks containing leptospiral DNA were prepared and then digested with 30 units of NotI restriction enzyme  for 2 hours at 37°C. Plug slices containing the digested DNA were placed in the wells of a 1% agarose gel and electrophoresed in a Bio-Rad CHEF-DRIII for 18 hours at 14°C with recirculating TBE buffer. Initial and final switch times of 2.16 and 35.07 s, respectively, were employed, and voltage was 6 V/cm. 8 leptospires/mL) using the QIAamp DNA extraction kit . Initial PCR amplification was performed using the eubacterial rDNA primers fD1/rD1 as described previously for leptospiral 16S rRNA gene sequencing 5′- GGC GGC GCG TCT TAA ACA TGC - 3′); and lepto16S1388r, a 20 bp reverse primer located at position 1388, (5′-TGT GTA CAA GGT CCG GGA AC - 3′). Additional internal sequencing was done using specific forward primers beginning at position 505 (5′- TCA TTG GGC GTA AAG GGT G – 3′) and position 1006 (5′ - TCA GCT CGT GTC GTG AGA TG – 3′). For clarity, the sequencing strategy is available online  Escherichia coli K-12 as a control.The G+C content (mol%) was determined for strain VAR 0108 cells/mL were used in microscopic agglutination reactions with reference rabbit anti-sera raised against the panel of all leptospiral serogroups except for Lyme and Sehgali shown in Leptospiral isolates at a density of 2×10L. interrogans serovar Icterohaemorrhagiae strain HAI188 L. fainei serovar Hurstbridge strain BUT6T, and L. biflexa serovar Patoc strain Patoc IT were included as representative pathogenic, intermediate and saprophytic strains, respectively. Growth in liquid EMJH, without 8-AZA, was used as a positive control.Growth of the unknown leptospiral isolate was determined in the presence of 225 µg/mL 8-azaguanine (8-AZA) at 30°C LipL32 gene in “L. licerasiae”, we used a modified PCR procedure L. interrogans serovars Copenhageni strain L1-130 To assess the presence of a PCR-amplifiable 7 leptospires/well separated by SDS-PAGE and transferred to a nitrocellulose membrane. The strains studied included “L. licerasiae” serovar Varillal strain VAR 010T, L. interrogans serovar Icterohaemorrhagiae strain HAI188 L. fainei serovar Hurstbridge strain BUT6T (ATCC BAA-1109T), as well as L. broomii ((ATCC BAA-1107T and BAA-1108), L. weilii serovar Celledoni (ATCC 43285T), L. wolbachii serovar Codice (ATCC 43284T), L. biflexa serovar Patoc (ATCC 23482T), and Turneriella parva (ATCC BAA-1111T) were obtained from the American Type Culture Collection, Virginia, USA. 100 µl of cultures were taken directly from the glycerol stock and centrifuged at 14,000 rpm for 30 minutes. The pellets were washed three times with PBS, suspended in 100 µl of 1× SDS sample buffer and incubated in a boiling water bath for 10 min. Ten µl of the SDS-solubilized whole bacterial cell lysate were loaded onto 4–12% Bis-Tris SDS polyacrylamide gels  and transferred to nitrocellulose membrane. The blot was blocked in PBS containing 5% BSA, incubated in anti-LipL32 rabbit polyclonal antiserum  followed by 1 hr incubation with 1∶3000 dilution of phosphatase-labeled anti-rabbit IgG , and development with BCIP/NBT (Kierkegaard and Perry Laboratories).Western blot analysis to detect LipL32 in leptospiral strains was performed using 2×10“L. licerasiae” strain VAR 010T and 2 other isolates from Iquitos, Peru  that were isolated from leptospirosis patients were used to infect hamsters (N = 2 in each group). Organisms fixed with 10% formalin were counted using a Petroff-Hauser counting chamber using dark-field microscopy. Groups of hamsters were inoculated intraperitoneally with 108 organisms for each Leptospira strain; one animal was injected with EMJH leptospiral culture medium alone as a negative control. The animals were observed twice daily for clinical signs of disease . On day 3 following infection, one member of each group was sacrificed and the organs  were removed aseptically to determine the bacterial load by real-time quantitative PCR Outbred female Syrian Golden hamsters were obtained from Charles River Laboratories . Animal experiments were approved by the University of California, San Diego Institutional Animal Care and Use Committee and were performed in Biosafety Level 2 animal facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care under approved biological safety procedures. 5′-CCCGCGCCCGCG TCCGATTAG-3′) and Lepto R (5′ TCCATTGTGGCCGRA/GACAC-3′), allow amplification of the region between the positions 171 and 258 of the rrs (16S) gene, with an expected product size of 87 bp. The FAM-TAMRA labeled probe [5′-CTCACCAAGCTCACCAAG GCGACGATCGGTAGC-3′] spans the region from position 205 to 228. Reaction mixes were prepared using the Platinum Quantitative PCR supermix-UDG kit  with final primer and probe concentrations of 600 nM and 100 nM, respectively, and 5 µl DNA extract. Reactions were performed in triplicate. Amplification and fluorescent monitoring were performed using a DNA Engine Opticon® 2 thermal cycler (MJ Research) using the following protocol: Incubate 2 min at 50.0°C; incubate 2 min at 95.0°C; incubate 30 s at 94.0°C; incubate 1 min at 50.0°C; plate read; repeat steps 2–5 for 44 more cycles.Total DNA for qPCR was prepared from three different pieces of weighed tissue samples using the DNeasy tissue kit  according to the manufacturer's directions. Real-time qPCR was performed using a previously described primer pair and probe 8 leptospires, extracted as described above, and used to prepare a 10-fold dilution series for real-time qPCR. The tissue burden of Leptospira for each sample was quantified by interpolating threshold cycle (Ct) values against the standard curve. Samples with a Ct value >40 were considered negative.To generate a standard curve, 13 mg of uninfected hamster kidney was spiked with 10ligA gene was synthesized by PCR, using the following forward primer, 5′ - CAAAGTTGTATGTCTTGGCCA C 3 - ′ and reverse primer, 5′ - GGAAGACCAAACGATCAG TGG - 3′. DNA from L. interrogans serovar Icterohaemorrhagiae strain HAI0188 was used as template. The PCR cycling profile consisted of 40 cycles of 95°C for 30 s; 49°C for 30 s; 72°C for 40 s; and a final extension of 72°C for 7 min. A 16S rRNA gene probe to be used as a control was generated using primers lepto16S1006f, 5′ - TCAGCTCGTCGTGTCGTGAGATG - 3′, designed from aligned leptospiral 16S sequences retrieved from GenBank, and rD1, 5′- AAGGAGGTGATCCAGCC - 3′“L. licerasiae” strain VAR 010T, L. fainei serovar Hurstbridge strain BUT6T, and L. biflexa serovar Patoc IT using the DNeasy Tissue Kit , and was then digested with BamHI  according to manufacturer's directions. Hybridization was carried out at 42°C. The membrane was washed with 2× SSC at room temperature and 0.1× SSC at 42°C. Bands were detected using anti-DIG-alkaline phosphatase Fab fragments  and CDP-Star chemiluminescence substrate .A dioxigenin (DIG)-labeled 508 bp probe for detection of the pathogenic marker Of 881 patients presenting with a history of undifferentiated fever to a study site, 45 patients' blood cultures yielded leptospires with typical morphology and motility as visualized under darkfield microscopy. Two of these leptospiral isolates from humans, obtained from blood cultures and identified as novel based on results presented below, were studied further. None of the 881 patients had urine cultures positive for this novel leptospire.A 31 year old female food vendor presented at the Varillal health post complaining of 2 days of fever, malaise, chills, headache and dizziness. She denied having gastrointestinal or urinary symptoms. The physical exam was unremarkable. The blood smear was negative for malaria and the patient was sent home with antipyretics. Illness resolved after 5 days without any further treatment or complications.A 19 year-old female student/domestic worker presented at the Hospital de Apoyo in Iquitos with a 5-day history of fever, malaise, headache, dizziness, chills, leg pain and weakness, abdominal pain, anorexia, nausea, and vomiting. Her illness spontaneously resolved with no complications.Leptospira at 3 and 2 weeks after inoculation, respectively. The characterization of these leptospiral isolates as a new species and unique antigenic type is described below. The isolate from patient VAR10 (strain VAR 010T) has been deposited at the American Type Culture Collection as ATCC BAA-1110T, at the U.S. National Veterinary Services Laboratory, Ames, Iowa, USA, and at the WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, Australia and Western Pacific Region, Brisbane, Australia.Neither patients VAR10 nor HAI029 received antibiotic treatment. At 5 week follow up, all signs and symptoms of infection had completely resolved in both patients. Blood cultures from both patients were positive for Leptospira panel was negative. When the isolate from patient VAR10 was used as MAT antigen, the acute sample was negative but the convalescent sample (taken 42 days after the acute sample) was positive at a titer of 1∶400.Serological results for patient VAR10 showed negative ELISA IgM serology on both acute and convalescent samples. MAT using the standard live “L. licerasiae” strain VAR 010T but convalescent samples reacted to serogroups Canicola, Icterohaemorrhagiae, Australis, and Sejroe at a titer of 1∶1600, to serogroup Mini at a titer of 1∶3200, and had a titer of 1∶6400 against her own isolate subsequently identified as “L. licerasiae” serovar Varillal, and identical to strain VAR 010T as determined by PFGE and 16S rRNA gene sequencing (see below).The acute serum sample of patient HAI029 was negative for anti-leptospiral antibodies but was IgM positive by ELISA on a convalescent sample taken 31 days after the acute sample. The acute serum sample of patient HAI029 was negative by MAT by both the standard leptospiral panel plus Leptospira reservoir hosts in the Peruvian Amazon. Of 100 Rattus rattus and R. norvegicus trapped, 55 isolates of Leptospira were obtained from culture of kidney. Of these 55 isolates, 47 proved to be L. interrogans serovar Icterohaemorrhagiae by testing with serovar-specific reference polyclonal antisera; 8 were not agglutinated by a panel of antisera against the standard serogroups and thus were studied further. Six of these non-serologically-typeable isolates came from R. norvegicus, two from R. rattus. Of more than 100 isolates obtained from cattle, pigs and water buffalo in the Iquitos region, none had any genetic relatedness to the novel leptospire (data not shown).Peri-domestic rats were trapped in the context of a mammalian ecology study of T and HAI029 human isolates and the 8 non-serologically-typeable isolates from rats. PFGE fingerprints were compared to PFGE fingerprints from 206 other pathogenic, intermediate  and saprophytic serovars . These rat and human isolates shared a previously undescribed fingerprint pattern  was not carried out. A similar approach was used to designate the Hurstbridge serovar of L. fainei serovar “L. licerasiae” serovar Varillal strain VAR 010T  agglutinated the leptospires from patients VAR10 and HAI029, and the eight rat isolates, at a titer of 1∶51,200. These serological results conclusively demonstrate that the two human and eight rat leptospires represent a new serogroup and a new serovar.The panel of reference rabbit anti-serogroup polyclonal antisera used in this study failed to agglutinate the leptospiral strains isolated from patients VAR10 and HAI029 and the 8 rat isolates in the MAT. T, CEH010, CEH011, CEH033, CEH044, and CEH162 grouped with the intermediate leptospires by 16S rRNA phylogenetic analysis, DNA-DNA hybridization was only carried out on the other intermediates L. broomii 5399T, L. fainei BUT6T and L. inadai LymeT as well as L. interrogans RGAT as an outgroup. As shown by DNA-DNA hybridization analysis .The presence of the lipoprotein Leptospira to have sufficiently diverged so that PCR amplification may have failed because of primer mismatch, we determined whether a LipL32 cross-reactive protein might be present in “L. licerasiae” serovar Varillal strain VAR 010T by Western immunoblot using a rabbit anti-L. kirschneri serovar Grippotyphosa LipL32 polyclonal antiserum. As expected, a protein of ∼32 kDa was seen in L. interrogans serovar Icterohaemorrhagiae strain HAI188 . The unique size of the LipL32-cross reactive protein in “L. licerasiae” serovar Varillal strain VAR 010T supports lack of contamination of this culture by another LipL32-containing leptospire. To further rule out the possibility that the cultures may have been contaminated by a known pathogenic Leptospira known to express LipL32, serological typing and 16S rRNA gene sequencing were repeated on all cultures, which confirmed their expected identities (data not shown).Because of the potential for the phylogenetically distant n HAI188 . SurprisL. interrogans serovar Canicola strain HAI156 and L. interrogans serovar Icterohaemorrhagiae strain HAI188 caused severe disease in hamsters infected intraperitoneally with 108 leptospires. HAI156- and HAI188- infected hamsters were sick on day 3 following challenge and moribund by day 5. In contrast, hamsters infected with “L. licerasiae” serovar Varillal strain VAR 010T showed no sign of illness (data not shown). Quantitative real time PCR detected high levels of leptospires in organs of hamsters infected with HAI156- and HAI188, but leptospires were nearly completely eliminated by day 3 after infection in liver, lungs and kidneys of hamsters infected with “L. licerasiae” serovar Varillal strain VAR 010T .Both VAR 010T , showingL. licerasiae” serovar Varillal strain VAR 010T only or had mixed reactions with “L. licerasiae” serovar Varillal strain VAR 010T and other serovars  with diagnostic titers highest against “L. licerasiae” serovar Varillal strain VAR 010T  was found in 40 patients in the acute sample, 57 in the second sample, and 16 patients had a titer of ≥1∶800 in both.During the study period, 1831 consecutive febrile patients were enrolled. Within these 1831 febrile patients on the data , 881 had a second serum sample available between 10 and 70 days after the first sample. Of these, 516 (58.6%) met criteria for acute leptospirosis. Of these, 367 (41%) reacted to “VAR 010T . The medL. licerasiae” serovar Varillal seroreactivity in the acute febrile population in Iquitos, we found serological evidence of seroreactivity in sera from 11 distinct geographic locations in Peru , did not grow significantly in vivo, and did not cause observable disease in experimentally infected animal. These observations suggest important biological and virulence differences between pathogenic and intermediate Leptospira.The genus 1,22 Neither patient had an occupation that would be considered a risk factor for leptospirosis. Patient A in this study was a food vendor and had contact with obvious risk factors having frequented a market area (Belen) with poor sanitation and a high density of rats, and bathed in a natural pool, as there is no running water in her village. Patient B was a female student/domestic worker who lived in the city, did not frequent Belen (the urban slum area of Iquitos) and did not engage in other behavior that would place her at particular risk for contracting leptospirosis. However, both patients recalled seeing rats in and around their homes. Patient B did raise dogs and chickens, and the dogs urinated within the house. There were no established social or professional links between the patients, and their infections occurred in different places and times. Both cases presented with a mild, self-resolving febrile illness without secondary complications and show the ubiquity of exposure as part of the activities of daily living in this region.Leptospirosis is typically thought of as an occupational disease originating from contact with water, soil or vegetation contaminated with the infected urine of carrier animals. The literature in recent years has shown that in under-developed areas of the world it is associated with environmental exposure during activities of daily living.2 underscores the importance of including region-specific leptospiral isolates in the panel of strains used in MAT for diagnosing leptospirosis and determining its true burden.Both patients initially had negative MAT and IgM results in their acute serum sample. While MAT of convalescent serum from Patient B was initially positive to a variety of serogroups, both acute and convalescent sera from Patient A were negative. However, when the test was repeated with the patient's own isolate, Patient A was found to have circulating leptospiral antibodies. This pattern of leptospiral seroreactivity, known to be a common problem in the diagnosis of leptospirosis,L. licerasiae” serovar Varillal from humans was rare, only being obtained from 2 of 881 febrile patients, despite the far higher seroprevalence rate of antibodies to this serovar. Some might raise the concern that this rare isolation rate could reflect a laboratory contamination with this leptospiral species. We believe this possibility is unlikely for two reasons. First, the patients from whom these isolates were obtained seroconverted to this novel serovar: patient 1 seroconverted to L. licerasiae serovar VAR 010T but to no other leptospiral antigen by MAT, while patient 2 seroconverted with the highest titer against her own isolate of L. licerasiae at a titer higher than other leptospires. Second, we never obtained an isolate of L. licerasiae in any other culture of human or animal specimens other than those reported here, making the possibility of contaminated culture medium negligible. While the biological basis for the rare isolation of L. licerasiae remains speculative, we propose two hypotheses. First, it is possible that the two patients from whom this leptospire was isolated had an undefined, undetermined genetic predisposition that led to higher leptospiremia or failure to clear this relatively less virulent leptospire after exposure. Second, it is possible that varying degrees of heterologous, cross-reacting, anti-leptospiral immunity exist in the study population. This latter hypothesis is supported by the very high prevalence of anti-leptospiral antibodies in the Iquitos region, likely due to ubiquity of leptospiral exposure. It may be that these two patients, for some reason, never had been exposed to Leptospira, and thus were immunologically naïve and thus predisposed to a higher level of leptospiral bacteremia. Further prospective, population-based studies are needed to address these important questions.A curious finding in this study is that isolation of ““L. licerasiae” is an important cause of fever in the Iquitos area and its surroundings, as evidenced by the number of patient sera that reacted predominantly or solely with serovar Varillal . Isolation of “L. licerasiae” from rats suggests that this leptospiral species has at least Rattus spp. as a major reservoir host; we have not found this leptospiral species in other rodent, bat and marsupial species in the Peruvian Amazon . Domestic rats are common in Iquitos: R. norvegicus and R. rattus are closely associated with human settlements in the area. R. norvegicus is more often encountered in urbanized areas, while R. rattus is the predominant rural species (data not shown). Six isolates identical to those isolated from both patients were recovered from rats caught in Belen, a city slum where sanitation is poor, rats are common and the risk of transmission to man is high. A further two VAR 010T-related isolates were recovered from rural rats. The isolation of a strain common to rats and found in 2 clinical cases, the ubiquity of the rat in Iquitos and the poor sanitation in most areas make the rat the likely source of leptospires in Iquitos. In other studies, we have succeeded in isolating L. interrogans serovar Icterohaemorrhagiae from 22 to 48% of peri-domestic Rattus species in villages near to and within urban areas of Iquitos (unpublished observations).This prospective study of acute febrile illness in Peru has shown that L. fainei serovar Hurstbridge L. broomiiL. fainei serovar Hurstbridge L. inadai serovar Lyme Leptospira and, as such, share characteristics similar to both pathogenic and saprophytic leptospires. DNA-DNA hybridization further confirmed that L. licerasiae is a new Leptospira species. Our logic was similar to that taken in using DNA-DNA hybridization to further confirm L. broomii as a new Leptospira species L. licerasiae into the intermediate Leptospira group close to L. fainei, L. broomii, etc., the only relevant DNA-DNA hybridization analysis is to differentiate the closest known clade partners identified by 16S rRNA gene sequence, so as to be able to confirm the distinctness of these 16S-rRNA gene-defined intermediate Leptospira species. The DNA-DNA hybridization analysis reported here does indeed confirm the distinctness of L. licerasiae from the other known intermediate Leptospira.Molecular and serological analysis of human- and rat-derived strains revealed that they comprise a single novel leptospiral species and serovar. The fact that the PFGE fingerprint patterns were found to be identical to each other, but did not match any of the patterns in the CDC database  supports our contention that the strains are novel leptospires. The isolates are members of a new serovar and serogroup as none were agglutinated by any of the reference anti-sera in our panel, although they had trace reactions to serogroup Hurstbridge. Given the lack of reactivity to the leptospiral serogroups represented by the rabbit reference sera used in the present study, the reference serological test, the cross-absorption agglutination test, was not necessary to define Varillal as a new serovar or antigenic type, similar to what was found for Leptospira licerasiae” serovar Varillal, which causes acute leptospirosis in the Peruvian Amazon. We have proposed this name to recognize the contribution of Professor Julia Liceras de Hidalgo who obtained the first leptospiral isolates in Peru We report the existence of a new species of leptospire, provisionally named “Leptospira licerasiae” serovar Varillal appears to be an important cause of leptospirosis in the Peruvian Amazon region, but is uncommon elsewhere in Peru. The peridomestic rat is likely the major reservoir of this new species. Elucidation of virulence differences between pathogenic and intermediate leptospires will provide insight into leptospiral evolution and disease mechanisms, and may contribute to the control and amelioration of leptospirosis in the developing world.Based on serological data that take advantage of its antigenic uniqueness, “To fulfill the rules of the International Code of Nomenclature of Bacteria , we provide the following description of the novel species identified in this report.Description of Leptospira licerasiae sp. nov.Leptospira licerasiae . Isolated from the blood of human patients with febrile illness and from kidneys of rats in Peru. Morphology is as described previously for the genus T ( = ATCC BAA-1110T = WPR VAR 010T), and has been deposited at the American Type Culture Collection, Manassas, Virginia, the National Veterinary Services Laboratory, U.S. Department of Agriculture, Ames, Iowa, and the WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, Australia and Western Pacific Region.Leptospira licerasiae isolates reported in this paper have been deposited in GenBank with the following accession numbers : CEH006, EF612278; CEH011, EF612279; CEH033,EF612280; CEH044, EF612281; CEH162, EF612282; MMD735, EF612283; VAR 010T, EF612284; CEH010, EF612285; CEH174, EF612286; MMD835, EF612287; HAI029, EF612288.The 16S rRNA sequences for the Alternative Lanuage Abstract S1Translation of the Abstract into Spanish by Jessica N. Ricaldi(0.03 MB DOC)Click here for additional data file.Figure S1Schematic of Leptospiral 16rDNA Gene Sequencing Strategy(9.45 MB TIF)Click here for additional data file.Figure S2L. licerasiae, Strain CEH033Assembly of One of 10 Identical 16S rDNA Sequences of (0.06 MB PDF)Click here for additional data file.Table S1T as Determined by the WHO/FAO/OIE Collaborating Centre For Reference & Research on Leptospirosis, Brisbane, AustraliaResults of Serogroup Screening Against VAR 010(0.05 MB DOC)Click here for additional data file.


To compare the efficacy and side effects of 3% hypertonic saline and mannitol in the management of raised intracranial pressure in children.Prospective randomized study.Pediatric intensive care unit (PICU) in a tertiary care hospital.200 patients with raised intracranial pressure.Patients were randomized into two statistically comparable groups; Group A (n = 98) was treated with mannitol while Group B (n = 100) was treated with 3% hypertonic saline. Group C (n = 2) included those members of Group A in whom serum osmolality ≥320 mosmol/kg and were then treated with 3% hypertonic saline. Both Drugs were given at a loading dose of 5 ml/kg stat followed by 2 ml/kg in every 6 h(both have same osmolarity) for two days in their respective groups. Besides monitoring, blood pressure (NIBP), mean arterial pressure (pre and post 30 min of drug), serum sodium, chloride and osmolality were measured. Intracranial pressure was assessed indirectly by measuring mean arterial ressure “MAP”. Student paired ‘t’ test was applied.P<0.001) at 0 h in males 0,6 h in females, and moderately significant at 12,36 h in females and significant(P<0.05) at 6,24,42 h in males of Group B. Decrease in coma hours was a highly significant finding (P<0.001) in Group B. In Group B, serum sodium and chloride increased significantly but remained within acceptable limits. There was no difference in osmolality and mortality (fisher Z).Decrease in MAP was highly significant (Mannitol has several side effects, 3% hypertonic saline is a safe and effective alternative in managing cerebral edema. Hyperosmolar treatment is one of the important methods for treating cerebral edema, and has been employed since early 1960. Urea, glycerol and mannitol were used for the treatment of this condition in the early years, but urea and glycerol were soon abandoned because of low efficacy. Mannitol is still used extensively. Side effects such as rebound effect, serum electrolyte imbalance and hypovolemia have led to the continued search for other osmotically active agents. One of them is hypertonic saline.To study the efficacy of hypertonic saline in the treatment of raised intracranial pressure in children in comparison to mannitol.To study the side effects (if any) of hypertonic saline treatment in the management of raised intracranial pressure.Study design: Prospective randomized comparative studySetting: Pediatric intensive care unit (PICU)Data analysis: Continuous data was analyzed by using Student unpaired ‘t’ testInclusion criteria: 200 Children aged 2 to 18 years, admitted to the emergency Department of Pediatrics between Jan 15th, 2007 and May 31st, 2008 with clinical symptoms and signs of raised intracranial pressure.Exclusion criteriaPatients with:compromised renal function (increased Serum creatinine)hepatic encephalopathyserum Na+ (>150 meq/l)diabetic ketoacidosiscerebral malarian = 98): treated with mannitolGroup A : treated with 3% hypertonic saline.Group B  was later made and Included those members of Group A in whom S. osmolality increased to more than 320 mOsm / kg and were then treated with 3% hypertonic saline.Group C (Loading dose(5ml/kg) was followed by maintenance dose (2 ml/kg) in every 6 h in both groups for two days .Blood pressure: By NIBP, at admission and then in every 6 h, both pre and post drug .S. creatinine: Before starting treatment and after 48 h.Blood urea nitrogen: Before starting treatment and after 48 h.Serum electrolytes: Before starting treatment and then in every 12 h.ABG: Before starting treatment, and after 12, 24, 36 and 48 h.Comparison of average reduction of mean arterial pressure (pre and post drug) at definite time (6h) intervals was done to indirectly assess reduction in intracranial pressure.Mean arterial pressure indirectly represents intracranial pressure = mean arterial pressure – intra cranial pressure (ICP) i.e. MAP = CPP + ICP (where CPP remains constants)Formula applied: MAP α ICP3% hypertonic saline was more efficacious than mannitol in the initial 12 h and equally or more efficacious than mannitol therapy later.Decrease in coma hours was a significant additional finding in the group treated with 3% hypertonic saline (Group B).Change in blood biochemistry was within acceptable limits in Group B. Tables –5et al.[For the first time, in 1919, Weed and McKibben reported4et al. demonstret al.9et al.[et al.[et al.[During the period in which mannitol was used intensively, maintenance of serum osmolarity below 320 mOsm/l was recommended because of complications of acute tubular necrosis (ATN) and renal failure. However, it was later understoodthat this complication developed as a result of dehydration and hypovolemia. Children11et al. showed tl.[et al. using 7.Hypertonic saline has been used more frequently in trauma, intracerebral hemorrhage, burn and stroke patients. Our patient group was different in their etiology of brain edema. Rationale of use of mannitol and HS is similar in both traumatic and non traumatic cerebral edema because all cerebral edemas with varied etiologies usually have vasogenic mechanism. In addition, HS is more effective and safer than mannitol. Greater efficacy of HS compared to mannitol can be speculated by different reflection coefficients of these agents. Because et al.,[In terms of the efficacy and side effect profile of the saline treatment in brain edema, the optimum serum-Na concentration and osmolarity are not known. In a retrospective study Peterson et al., though met al.,–11 Thereet al.,–18 So afPotential side effects of hypertonic saline have been reported: MyelinolRenal failure, congestive heart failure, pulmonary edema, hypokalemia and phlebitis were not observed in any of our patients. There was no significant tendency for hemolysis or hemorrhage associated with acute fall in the hematocrit level.In conclusion, in the treatment of cerebral edema of infectious, anoxic, hemorrhagic and traumatic origin, administration of HS is probably more effective and safer than mannitol. However, to determine just when to initiate treatment, how long to continue treatment, and target serum-Na concentration requires monitoring of the intracranial pressure. Further studies are required to resolve these concerns.


Free prostate-specific antigen levels were significantly higher in women with colorectal carcinoma than healthy women (P=0.006). The percentage of free prostate-specific antigen predominant  subjects was 20% in colorectal carcinoma patients and 3.3% in healthy women (P=0.035). Cut-off values were 0.34 ng ml−1 for total prostate-specific antigen and 0.01 ng ml−1 for free prostate-specific antigen. In women with colorectal carcinoma, total prostate-specific antigen positivity was 20% and free prostate-specific antigen positivity was 34.6%. When compared to negatives, total prostate-specific antigen positive patients had a lower percentage of well-differentiated (P=0.056) and early stage (stages I and II) tumours (P=0.070). However, patients with predominant free prostate-specific antigen, had a higher percentage of well-differentiated (P=0.014) and early stage tumours (P=0.090) than patients with predominant bound prostate-specific antigen. In conclusion, although the sensitivity of free prostate-specific antigen predominancy is low (20%), in distinguishing women with colorectal carcinoma than healthy women, its specificity is high (96.7%). Free prostate-specific antigen predominancy tends to be present in less aggressive tumours. These findings may indicate clinical significance of preoperative measurement of serum total and free prostate-specific antigen in women with colorectal carcinoma.We investigated the diagnostic value and the relationship with clinicopathological features of total and free prostate-specific antigen by measuring the concentrations of these markers in the sera of 75 women with colorectal carcinoma and in 30 healthy women. Measurements were performed by immunoradiometric assay which utilizes monoclonal and polyclonal anti-prostate-specific antigen antibodies; the lowest detection level for both markers was 0.01 ng mlBritish Journal of Cancer (2002) 86, 203–206. DOI: 10.1038/sj/bjc/6600049www.bjcancer.comThe Cancer Research Campaign© 2002  RecentlThe production of PSA in the prostate is up-regulated by androgens, through the androgen receptor . The genIt has been reported that PSA expression is not found  or wheneIn this study, to our knowledge for the first time in the literature, preoperative serum total and free PSA concentrations were measured in women with colorectal carcinoma, and the relationship with clinicopathological features and the diagnostic value of these markers were investigated.Between June 1998 and December 2000, serum total and free PSA levels were measured preoperatively in 75 women with colorectal carcinoma and with no other pathology , who were operated at SSK Okmeydanı Training Hospital, İstanbul, and in 30 ‘healthy’ women . All women included in the study had been informed and their approvals were obtained. In 69 patients, the tumour could be resected while six tumours were unresectable due to adjacent organ and structure invasion. The location, size and histologic features of the tumour were reported by the referred pathologist. The size and histologic features of the unresected tumours were not included in the evaluation. Venous invasion was not examined in one of the resected tumours. Histologic type was divided in two subgroups as well-differentiated adenocarcinoma (15 cases) and the others. In the second group, there were 47 moderately and three poorly differentiated adenocarcinomas, three mucinous carcinomas and one signet ring cell carcinoma. The depth of invasion and staging of the disease were assessed according to TNM system  kit and free PSA measurements were performed with Coat-A-Count® Free PSA IRMA  kit, by utilizing solid-phase sequential immunoradiometric assay which is based on monoclonal and polyclonal anti-PSA antibodies. Lower detection limit for each tumour marker was calculated as 0.01 ng mlThe serum total and free PSA levels of 30 healthy women were arranged in sequence from the lowest to highest and the marker concentrations of the 28th woman were accepted as cut-off values (specificity 93.3%). Marker levels exceeding these cut-off values were considered as positive. Cases with a free PSA/total PSA ratio over 50% were titled ‘free PSA predominant subject’.U-Wilcoxon Rank Sum W test was used for comparisons of total PSA or free PSA levels between colorectal carcinoma patients and healthy women. The effect of age on total PSA and free PSA levels was investigated using Spearman correlation analysis. Fisher exact test was used for comparison of free PSA predominancy between carcinoma patients and healthy women. Chi-square test and Fisher exact test were used to assess the association between clinicopathological factors and total PSA positivity, free PSA positivity or free PSA predominancy. P-values of less than 0.05 were considered to be statistically significant.Mann–Whitney P=0.006) whereas no significant association existed in total PSA levels of both groups. The ratio of free PSA predominant subjects were 20% (15 out of 75) in colorectal carcinoma group and 3.3% (one out of 30) in the healthy group (P=0.035). In colorectal carcinoma patients and healthy women, total PSA and free PSA levels decreased as age increased, though the correlations were not significant in Spearman analysis (data not shown).As shown in Table 1−1 and 0.01 ng ml−1 respectively. Total PSA positivity was 20% (15 out of 75) and free PSA positivity was 34.6% (26 out of 75) in women with colorectal carcinoma. As shown in Table 2P=0.090). In total PSA positive patients, no well-differentiated tumour existed, whereas 26.8% of total PSA negatives had well-differentiated tumours (P=0.056). Compared to negatives, in total PSA positive group, the ratio of the patients with lymph node metastasis and advanced stage disease had a tendency to increase (P=0.062 and P=0.070 respectively). There was no significant association between clinicopathological variables and free PSA positivity (data not shown).The cut-off values for total PSA and free PSA were 0.34 ng ml1-antichymotripsin bound PSA as the predominant molecular form, the ratio of well- differentiated tumours were significantly higher (P=0.014); the ratios of T1–T2 tumours (P=0.052) and early stage disease (stages I and II) (P=0.090) were slightly higher.In this initial report assessing serum total and free PSA levels in women with colorectal carcinoma, total PSA levels were found to be higher than healthy women but the difference was not significant. On the other hand free PSA levels were significantly higher in women with colorectal carcinoma.PSA measurements in nonprostatic carcinomas have been conducted mostly in women with breast cancer. It has been shown that serum total PSA levels in women with breast cancer were significantly higher than healthy women, and in women with breast cysts were significantly higher than women with breast cancer and serum free PSA levels in breast cancer patients were significantly higher than healthy women  PSA and PSA complexed to αiagnosis . Cut-offiagnosis .1-antichymotripsin as the major molecular form, and it was suggested that the ratio of free PSA/bound PSA might have value for diagnosis of breast diseases including breast cancer  showed a sensitivity rate of 20% and a specificity rate of 96.7% in distinguishing women with colorectal carcinoma than healthy women. Although total PSA positivity had a tendency to be present in aggressive histologic types and in advanced stage tumours, free PSA predominancy had a tendency to be present in well-differentiated and early stage tumours.In conclusion, this data reveals that preoperative serum total and free PSA measurements may offer clinical significance in women with colorectal carcinoma and there is need for new studies including wider patient and control groups, utilizing ultrasensitive PSA assays whose lowest detection limit is lower.


Although the potential roles of fruits and vegetables in cancer prevention have been demonstrated at various cancer sites , the assHere, we investigated the association between fruit and vegetable consumption and HCC in a large-scale population-based cohort study in Japan, with due consideration for hepatitis C virus (HCV) and hepatitis B virus (HBV) infection status.n=1219). Among them, a total of 20 406 participants (36%) provided a blood sample. These plasma samples were screened for anti-HCV and for HBV antigen (HBsAg).The Japan Public Health Center-based Prospective Study (JPHC study) Cohort II, initiated during 1993–1994, has been described earlier (−1) was calculated from these responses. We documented the validity of the FFQ in the assessment of vegetable and fruit consumption in subsamples using dietary records. Although validities for vegetables and fruits were relatively low (from 0.22 for vegetables to 0.31 for fruit), correlation coefficients for antioxidant nutrients were considered moderate (from 0.31 for vitamin C to 0.41 for β-carotene).The self-administered food-frequency questionnaire (FFQ) consisted of 52 foods, including beverages. It asked about the usual consumption of six vegetable and three fruit items during the previous year. The vegetables included two pickled vegetables (green leafy vegetables and other vegetables), green leafy vegetables , carrot, tomato and 100% vegetable juice, whereas the fruit items included apple, citrus fruits and 100% fruit juice. The questionnaire contained five frequency categories for vegetable and fruit consumption ranging from ‘never’ to ‘almost every day’, except for juices. Standard portion sizes were specified for each food item, which were then used to determine the three choice amounts of small , medium (same as the standard) and large (50% larger). Six frequency choices for juice ranged from ‘almost never’ to ‘5 or more cups per day’. The consumption of total fruit and total vegetables  were excluded, leaving 19 998 participants for analysis, who were followed from the baseline survey until 31 December 2005. Of these, 5% moved out of a study area and 0.2% were lost to follow-up during the study period.We used Cox regression to compute hazard ratios (HRs) and 95% confidence intervals (CIs) of HCC according to tertiles of consumption of the respective food items or nutrients with adjustment for potential confounders, including HCV or HBV infection status.During 235 811 person-years of follow-up (11.8 years), a total of 101 new HCC cases were identified. The prevalence of chronic HCV and HBV infection in HCC cases was 70.3 and 12.9%, respectively.We observed that participants with higher vegetable and fruit consumption tended to be older, smoke less, drink less alcohol, and consume less coffee and more genistein. Body mass index did not substantially differ according to consumption. The proportion of participants positive for anti-HCV, HBsAg or both among tertiles of vegetable and fruit consumption was similar. The prevalence of positive markers for HCV and HBV in this cohort was 5.3 and 2.5%, respectively.vs lowest tertile of 0.61  and 0.65 , respectively. In particular, green leafy vegetable consumption showed an inverse dose-dependent association with HCC . Results for vegetables excluding pickled vegetables were similar to those for when they were included. In contrast, fruit consumption including fruit juice appeared to increase the risk of HCC, albeit without statistical significance .α-carotene and β-carotene) and vitamin C and HCC risk. A slightly negative association was seen between α- and β-carotene and HCC, with respective multivariable HRs for the highest vs lowest tertile of 0.69 (95% CI=0.42–1.15) and 0.64 (95% CI=0.38–1.08). Multivariable HR for vitamin C was somewhat increased in the highest category .α- and β-carotene on HCC strengthened, with respective multivariable HRs for the highest vs lowest tertile of 0.60  and 0.61  (data not shown).When the analysis was restricted to participants who were either or both anti-HCV- or HBsAg-positive, these results were substantially unchanged. It is worth noting that our study showed that the preventive effects of vs lowest tertile among never smokers were 0.42 for vegetables , 0.30 for green–yellow vegetables  and 0.31 for green leafy vegetables . Regarding nutrients, β-carotene showed a significant inverse association with risk among never smokers . In contrast, vitamin C seemed to be positively associated with HCC risk among current smokers, with an increase in multivariable HR for HCC in the second and highest categories  (data not shown).After participants were stratified by smoking status, multivariable HRs for the highest α- and β-carotene and HCC risk was shown. These results are plausible, given the abundance of these nutrients in vegetables, particularly green–yellow vegetables.Our study identified inverse associations between the consumption of vegetables, green–yellow and green leafy vegetables and HCC. Concomitantly, an inverse association between α- and β-carotene on HCC were strengthened when participants were limited to those who were either or both HBV and HCV positive. Given that inflammation is accompanied by the excess production of free radicals and that carotenoids have antioxidant potential in the scavenging of free radicals . Although vitamin C has antioxidant potential, it also acts to stimulate the absorption of iron from food , and iroα- and β-carotene and restraint in those rich in vitamin C may modify the development of HCC in HBV- and/or HCV-infected participants.Given that the prognosis for HCC is extremely poor, our results would, if confirmed, have important implications for public health. Greater consumption of vegetables that contain


P for trend=0.03). Of interest, the observed association was more prominent in women than in men. The observed ceiling effect associated with higher isoflavone intake suggests that a lower intake of dietary isoflavone might be associated with an increased risk of colorectal adenoma in Japanese populations.We conducted a case–control study in a Japanese population to investigate the association between dietary isoflavone intake and the risk of colorectal adenoma. Participants who underwent magnifying colonoscopy with dye spreading as part of a cancer screening programme responded to a self-administered questionnaire, which included lifestyle information and intake of 145 food items, before the colonoscopy. A total of 721 case and 697 control subjects were enrolled. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression models. We found a significant inverse association between dietary isoflavone intake and the risk of colorectal adenoma in men and women combined. However, the inverse association was not linear; rather, all quartiles above the first showed a similar decrease in risk, with multivariable-adjusted ORs and 95% CIs compared with the lowest quartile of 0.77 (0.57–1.04), 0.76 (0.56–1.02) and 0.70 (0.51–0.96) in the second, third and highest quartiles, respectively ( The incidence of colorectal cancer, a common malignancy in developed countries, is higher in men than in women  consisted of examinees who underwent magnifying colonoscopy with dye spreading as a part of a cancer screening programme provided by the Research Center for Cancer Prevention and Screening, National Cancer Center, between February 2004 and February 2005. Details of this programme have been provided elsewhere  and other lifestyle-related factors.miso (fermented soya bean paste) soup, tofu (soya bean curd) in miso soup, tofu (boiled or cold) in other dishes, atsuage (fried tofu), koyadofu (freeze-dried tofu), aburaage (deep-fried tofu), natto (fermented soya bean) and tonyu (soya milk made from soya bean as major ingredient). The frequency of miso soup consumption was divided into six categories of almost never, 1–3 days per month, 1–2 days per week, 3–4 days per week, 5–6 days per week and daily. Portion size was specified with one bowl of miso soup equalling 150 ml into the nine categories of <1 bowl per day, 1 bowl per day, 2 bowls per day, 3 bowls per day, 4 bowls per day, 5 bowls per day, 6 bowls per day, 7–9 bowls per day and ⩾10 bowls per day. Daily intake of genistein and daidzein was calculated using values in a specially developed food composition table for isoflavone in Japanese foods (The self-administered questionnaire included a food frequency questionnaire (FFQ) that asked about 145 food and beverage items in terms of standard portion sizes and 9 frequency categories. The standard portion sizes of respective food items were specified in the FFQ and the amounts were determined into three categories of less than half, the same as, and more than 1.5 times the reference portion size. The frequency response choices for food items were less than once per month, 1–3 times per month, 1–2 times per week, 3–4 times per week, 5–6 times per week, once per day, 2–3 times per day, 4–6 times per day and ⩾7 times per day. Soya food items included r=0.99), the sum of genistein and daidzein is provided here as representative for total isoflavones. Isoflavone intake was then adjusted for total energy intake using the residual method in the statistical model after log transformation (−2), physical activity (quartiles based on controls), supplement use (yes or no), non-steroidal anti-inflammatory drug use (yes or no). Age at menarche , menopausal status (pre or post) and current use of female hormones (user or non-user) were also adjusted in women. Statistical significance of the interaction terms was evaluated by the likelihood ratio test. Tests for linear trend were estimated using the median value of isoflavone intake and treating this variable as continuous. All statistical tests were two tailed, and P-values <0.05 were considered statistically significant. All analyses were performed using Stata Statistics/Data Analysis Special Edition version 9.2 .After the exclusion of 102 subjects who reported extreme total energy intake (upper and lower 2.5%) or submitted incomplete answers regarding dietary isoflavone intake and other confounding variables, 721 cases (492 men and 229 women) and 697 controls (460 men and 237 women) were entered into the analysis. Because dietary genistein and daidzein intakes were highly correlated (Spearman The demographic characteristics and lifestyle factors according to case–control and sex had mentioned previously (P for trend=0.03). Stratified analysis by sex showed a significant inverse association between isoflavone consumption and colorectal adenoma in women, and a non-significant inverse association in men. The multivariable-adjusted ORs (95% CIs) for women in increasing quartile intake categories were 0.53 (0.28–0.98), 0.44 (0.24–0.80) and 0.49 (0.27–0.90), respectively (P for trend=0.03).n=393 subjects), primarily on the basis that the change might have been related to the presence of adenoma. Results were essentially the same as those above (data not shown). We also investigated the association between colorectal adenoma for quartile categories of dietary isoflavone intake after further adjustment of fibre, folate, calcium and vitamin D in the multivariate model and similar decreased risk of association was observed (data not shown). We further categorised the subjects into octiles. As in the quartile analysis, however, the inverse association was not linear in the octile groups; rather, the third to highest octiles showed a decrease in risk, with the pattern more apparent in women, in whom multivariable-adjusted ORs (95% CIs) in increasing octile categories were 0.67 (0.26–1.70), 0.41 (0.16–1.06), 0.44 (0.18–1.05), 0.40 (0.17–0.97), 0.30 (0.13–0.72), 0.40 (0.17–0.93) and 0.42 (0.18–0.97), respectively (P for trend=0.03).We repeated the above analyses after excluding those who had changed their dietary habits in the past 5 years .Stratified analysis according to site, size and number of colorectal adenomas showed no difference in the association with isoflavone intake by adenoma characteristics in overall men and women . We furtIn this colonoscopy-based case–control study, we found a significant inverse association between dietary isoflavone intake and colorectal adenoma risk in a Japanese population. The inverse association was not linear; with a ceiling effect observed with higher intake, which implied conversely that a lower intake of dietary isoflavone was related to an increased risk.vs 17.22 mg per day for the lowest octile in our study population; it therefore investigated the effect of lower intake levels, which we could not observe in detail in our study, and supported our finding that a lower intake of dietary isoflavone was related to an increase in risk.To our knowledge, this is the first study of this subject. Because adenomas are much more common than cancers, we may have greater power to detect the isoflavone–adenoma association. To date, three studies have examined the association between dietary isoflavone intake and the risk of colon (in vitro and in vivo experimental studies have suggested that phyto-oestrogens protect against colorectal cancer (α and ER-β oestrogen receptors, phyto-oestrogens bind with high affinity to ER-β, and consistently mediate signals to inhibit abnormal cellular proliferation in colon tumour cell lines by blocking tyrosine protein kinases, aromatase and DNA topoisomerases. Phyto-oestrogens also possess antioxidant activity, and inhibit cell-cycle progression and angiogenesis in endothelial cells (Isoflavones may help prevent the development of colorectal neoplasm through their oestrogenic effect. In fact, several We found a stronger inverse association in women than in men, although effect modification by sex was not statistically significant. Nonetheless, this suggested difference by sex is of particular interest, because isoflavones may have a more important function in the prevention of colorectal adenoma in women than in men. Of the five of seven epidemiological studies of the association between soya product intake and colorectal neoplasm, which provided results for men and women separately (Our study has several methodological advantages over previous studies. First, we used a validated FFQ and calculated isoflavone intake from a variety of dietary soya sources. Many different foods contribute to isoflavone intake, and no single soya food category predominates. Moreover, our subjects had relatively large isoflavone intake with substantial variation, making them suitable for the investigation of this association. Second, subjects answered the questionnaire before diagnosis, likely minimising the possibility of recall bias.vs data from our institute showing 95% accuracy, our analysis may have included a few false-positive cases (Several limitations also warrant mention. First, although all examinees underwent total colonoscopy, colorectal adenoma cases were diagnosed according to pit-pattern classification and were not confirmed histologically. Given an overall accuracy of pit-pattern diagnosis of approximately 90% In summary, we found an inverse association between dietary isoflavone intake and the risk of colorectal adenoma in a Japanese population, which was not linear, but rather showed a ceiling effect associated with higher isoflavone intake; it was more prominent in women than in men, possibly reflecting an oestrogenic effect.


Compared to those treated with no/low gonadotoxic chemotherapy patients from all other treatment groups had significantly elevated risk for exocrine hypogonadism. Patients from the other treatment groups, except those in the med-NHL group, also had significantly elevated risk for endocrine hypogonadism compared with the group treated with no/low gonadotoxic chemotherapy. Men aged above 50 years at survey were about five times more likely to have endocrine hypogonadism compared with those less than 40 years. Because of the adverse health effects following long-lasting endocrine hypogonadism, gonadal hormones should be assessed regularly in male lymphoma survivors, especially after treatment with alkylating agents and high-dose chemotherapy with autologous stem cell support and in male patients who are 50 years and older.Gonadal function was assessed in male lymphoma survivors based on serum hormone levels , and was related to treatment, age and observation time. Male patients ⩽50 years at diagnosis treated for Hodgkin's (HL) and/or non-Hodgkin's lymphoma (NHL) at the Norwegian Radium Hospital from 1 January 1980 to 31 December 2002 were included. Five treatment groups were defined: 1: radiotherapy only and/or low gonadotoxic chemotherapy (both HL and NHL)(‘No/low’), 2: medium gonadotoxicity chemotherapy for NHL (‘med-NHL’), 3: medium gonadotoxicity chemotherapy for HL (‘med-HL’), 4: highly gonadotoxic chemotherapy for NHL (‘high-NHL’), 5: highly gonadotoxic chemotherapy for HL (‘high-HL’). Gonadal hormone levels were categorised into three groups: 1: All gonadal hormones within normal range , 2: Isolated elevated FSH, with LH, SHBG and testosterone within normal range (exocrine hypogonadism), 3: Testosterone below and/or LH above normal range (endocrine hypogonadism). One hundred and forty-four (49%) of the patients had normal gonadal hormones, 60 (20%) displayed exocrine hypogonadism and almost one-third ( Testicular dysfunction can be a late side effect after cancer treatment. It is known that post-treatment spermatogenesis in male survivors after malignant lymphoma is dependent on the type of chemotherapy, the drugs’ cumulative dose, the radiation doses to the testicles from scattered or direct irradiation and the time since last treatment  is impaired at a greater degree than in survivors after non-Hodgkin's lymphoma (NHLSs)  and exocrine (spermatogenesis) function, controlled by the hypothalamus – pituitary – gonad axis. Gonadotropin-releasing hormone (GnRH) from the hypothalamus promotes the production of the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary. Simplified, LH stimulates the Leydig cells to produce testosterone; FSH and testosterone stimulate the Sertoli cells, which together provide support for spermatogenesis.Gonadal dysfunction may be classified as primary and/or secondary hypogonadism. Primary hypogonadism is due to testicular damage resulting in impaired spermatogenesis and/or dysfunction of the Leydig cells. As a rule of thumb, elevated FSH indicates impaired spermatogenesis, whereas permanently elevated LH indicates Leydig cell dysfunction. Secondary hypogonadism is caused by damage to the pituitary gland or hypothalamus, that is, after cranial radiotherapy, and leads to decreased LH and FSH followed by both exocrine and endocrine gonadal failure. Male gonadal function can thus be screened by assessment of serum levels of LH, FSH, testosterone, SHBG and sperm cell count and by the consideration of fatherhood. In case of Leydig cell damage the serum levels of testosterone are low and those of LH are often elevated, though With this background, the aim of the present cross-sectional study was to assess post-treatment gonadal function in a large series of lymphoma patients in relation to treatment, age and observation time emphasizing the possible differences between HLSs and NHLSs.Eligible patients for the present postal cross-sectional study fulfilled all the following criteria: 1: male patients ⩽50 years at diagnosis, 2: treated for Hodgkin's lymphoma (HL) and/or non-Hodgkin's lymphoma (NHL), 3: registered in the lymphoma database at the Norwegian Radium Hospital (NRH), 4: Period of diagnosis from 1 January 1980 to 31 December 2002, 5: age above 18 years at the time of the survey (January 2007), 6: valid postal address and 7: alive in June 2007.Patients who had received total brain irradiation or scrotal irradiation were excluded. Patients registered with both HL and NHL was grouped according to their first diagnosis. Totally there were 16 patients, of whom seven had their gonadal hormones assessed. Of these seven, four were initially diagnosed with HL and three with NHL, and later on they were diagnosed with a secondary lymphoma.Treatment strategies of HL and NHL at the NRH for the period 1980–2002 have been described earlier  (‘No/low’), 2: medium gonadotoxicity chemotherapy for NHL (‘med-NHL’), 3: medium gonadotoxicity chemotherapy for HL (‘med-HL’), 4: highly gonadotoxic chemotherapy for NHL (‘high-NHL’), 5: highly gonadotoxic chemotherapy for HL (‘high-HL’). The different chemotherapy regimens were allocated to groups 1–5 as described in The cross-sectional study included serum hormone analyses and a questionnaire assessing various aspects of quality of life. The present analyses are restricted to gonadal hormones, whereas quality of life aspects will be discussed in detail in a separate paper.−1, SHBG: 15–85 nmol l−1, FSH<12.0 U l−1, LH<10.0 U l−1. The testosterone/SHBG ratio was calculated for each patient, as a surrogate for free testosterone, with testosterone/SHBG ratio <0.25 indicating hypogonadism.Blood samples for analyses of gonadal hormones  were to be drawn before noon by the patient's GP and mailed to the Hospital's Department of Medical Biochemistry. Patients with gonadal hormones indicating hypogonadism were invited to an outpatient clinical examination and a control morning blood sample. All analyses were performed at the same laboratory at Rikshospitalet with E170 module for Modular Analytics, Roche Professional Diagnostic, Germany. Normal ranges were: Testosterone: 9.0–31.0 nmol lThe patients’ gonadal hormone levels were categorised into three groups: 1: All gonadal hormones within normal range , 2: Isolated elevated FSH, with LH, SHBG and testosterone within normal range (exocrine hypogonadism), 3: Testosterone below and/or LH above normal range, independent of FSH (endocrine hypogonadism), admitting that this group includes some male patients with both endocrine and exocrine hypogonadism.  was categorised as follows: 1: 21–39 years, 2: 40–49 years, 3: 50–56 years, 4: 57–75 years. Observation time was calculated from date of first diagnosis to 1 January 2007, and was further categorised in three groups: 1: 4–10 years, 2: 11–20 years, 3: 21–28 years.Multinomial regression analyses were performed, the dependent variable being gonadal hormones divided into three categories: , 2=exocrine hypogonadism, 3=endocrine hypogonadism). The model was adjusted for the five treatment groups, age groups and observation time. As initial analyses revealed that observation time was not significant, this variable was excluded from the final model. Finally, two multivariable models were fitted; one including treatment groups and age at survey as independent variables, the other included treatment groups and age at diagnosis. The odds ratios for both exocrine and endocrine hypogonadism were similar in both models, possibly due to a strong correlation between age at diagnosis and age at survey. Only results from the model fitted with treatment and age at survey are presented.P<0.05 were considered statistically significant . All analyses were performed using SPSS 13 for Windows.In all 653 patients met the inclusion criteria and were invited to participate by mail . A total of 294 (45%) had their gonadal hormones assessed and were included in this study (compliers).vs 31 years (P=0.006)) and at survey (median 49 vs 45 years (P<0.001)), and had significantly longer observation time (median 15 vs 13 years (P=0.028)).The compliers and non-compliers (survivors without blood samples) were similar as to lymphoma diagnosis and treatment groups. However, the compliers were older than the non-compliers both at diagnosis (median 33 n=187) had received chemotherapy and radiotherapy, 63 (21%) had had chemotherapy only and 44 patients (15%) had undergone radiotherapy only. Median age at diagnosis was 33 years , median age at survey was 49 years , and median observation time was 15 years   had been treated for HL and 129 (44%) for NHL. Sixty-four percent  .As expected, age at survey and age at diagnosis were higher in NHLSs compared with HLSs, without difference in observation time between these groups.−1 . Forty-one percent had FSH values > 12.0 U l−1, and in 26% the FSH value were > 20.0 U l−1. The median LH value was 6.0 U l−1 , and 16% of the patients had elevated LH (>10.0 U l−1)  .−1 , with 20% of the patients having testosterone value <9.0 nmol l−1. The median value of the testosterone/SHBG-ratio was 0.37. Of all patients, 14% had a testosterone/SHBG ratio <0.25.The median testosterone value was 12.9 nmol lP<0.001). For testosterone, no such association with age at survey was observed. However, the testosterone/SHBG-ratio decreased significantly with increasing age (P<0.001).There was a significant association between age at survey and the individual levels of FSH, LH and SHBG (P<0.001) and SHBG (P=0.042) and treatment groups. The median level of FSH and LH increased significantly in all other treatment groups compared with patients with no/low gonadotoxic chemotherapy (P<0.001–P<0.02). For testosterone and testosterone/SHBG-ratio no association with treatment groups was found.There was also a significant association between the individual levels of both FSH, LH (n=144) had all gonadal hormones within normal ranges, 60 (20%) displayed exocrine hypogonadism  and in almost one-third  the hormone levels were compatible with endocrine hypogonadism . There was a statistically significant association between treatment group and groups of gonadal dysfunction (P<0.001). After treatment with no/low gonadotoxic chemotherapy 79% had normal gonadal hormones, whereas the comparable figures were 62% after med-NHL, 26% after med-HL, 16% after high-NHL and 8% after high-HL (Forty-nine percent ( high-HL .Of the 41 patients treated with high dose chemotherapy and autologous stem cell support (HDT), 19 men had exocrine and 20 endocrine hypogonadism. Only two patients had normal gonadal hormones.P<0.001). In patients with stage I/II at diagnosis 62% had normal gonadal hormones, 11% displayed exocrine hypogonadism and endocrine hypogonadism was demonstrated in 27%. The comparable numbers in patients with stage III/IV were: normal gonadal hormones in 30%, exocrine hypogonadism in 34% and endocrine hypogonadism in 36%.Stage at diagnosis was significantly associated with the group of hormonal dysfunction (P=0.004) and at survey (P<0.001) and they had a longer median observation time (P=0.047) than those with normal gonadal hormones. There was no difference in age at diagnosis or survey and in observation times between patients with exocrine hypogonadism and patients with normal gonadal hormones.The patients with endocrine hypogonadism were older both at diagnosis  for exocrine hypogonadism . PatientIn addition, compared to the group treated with no/low gonadotoxic chemotherapy, patients from the other treatment groups, except those in the med-NHL group, had significantly elevated risk also for endocrine hypogonadism. Patients in the med-HL were 8.0 (95% CI: 3.2–20.4) times more likely to have endocrine hypogonadism compared to those treated with no/low gonadotoxic chemotherapy, whereas the comparable ORs for high-NHL were 10.5 (95% CI: 3.6–30.6) and 37.2 (95% CI: 9.4–147.7) for high-HL.Men aged above 50 years at survey were about five times more likely to have endocrine hypogonadism compared with those less than 40 years at survey.The ORs for both isolated elevated FSH and endocrine hypogonadism increased more in HL survivors compared with NHL survivors in comparable treatment groups.Overall, treatment with medium and highly gonadotoxic chemotherapy for both HL and NHL had a stronger effect on exocrine than on endocrine hypogonadism.P=0.001). This group had also significantly lower testosterone/SHBG ratios than the other subgroups (P=0.003/P=0.001). (Patients with low testosterone combined with elevated LH and/or FSH displayed significantly longer observation times than the other two subgroups (=0.001). .In this cross-sectional observational study gonadal hormones were assessed in 294 male survivors after treatment for both HL and NHL. About half had all gonadal hormones within normal range, 20% had isolated elevated FSH, and almost one-third had gonadal hormones compatible with our definition of endocrine hypogonadism. Compared to those treated with no/low gonadotoxic chemotherapy, patients from all other treatment groups had significantly elevated risk for exocrine hypogonadism. In addition, patients from the other treatment groups, except those in the med-NHL group, had a significantly elevated risk also for endocrine hypogonadism compared to the group treated with no/low gonadotoxic chemotherapy.We confirm the results from our previous study in HLSs, where we observed exocrine gonadal damage related to the type, cumulative dose and intensity of chemotherapy, assessed by post-treatment paternity . Intensification of chemotherapy increased the risk of endocrine gonadal failure more in HLSs than in NHLSs. This probably reflects that chemotherapy regimens used for HL generally are more gonadotoxic than those used for NHL. This difference may also be due to differences in the pre-treatment gonadal function between HLSs and NHLSs, pointed out by −1) in 24 (13.6%) of 176 patients, but only eight of these men had increased LH. This may reflect that causes of endocrine hypogonadism in cancer survivors may be more complex than previously described. Chemotherapy and/or radiation-induced damage may develop either in the gonads, the hypothalamus–pituitary axis or as a combination of these two. To verify the influence of secondary hypogonadism, the pituitary function has to be tested by a gonadotropin-releasing hormone test, which was not performed in our patients. Further, except for the pathway through the hypothalamus–pituitary axis and the release of GnRH, the mechanisms related to Leydig cell dysfunction after cancer treatment is not well characterised. The possible negative influence of germinative dysfunction on Leydig cells suggested by In this paper, we followed the categorisation for endocrine hypogonadism based on elevated LH and normal testosterone (compensated hypogonadism) or low testosterone with or without elevated LH proposed by The blood samples were generally taken during the morning. We were aware of the risk of falsely low testosterone in case of afternoon blood sampling and tried to avoid this possibility as much as possible. However, we completely cannot exclude such sampling as explanation in some patients in subgroup 3A. For most of these patients, the results have been confirmed in a subsequent morning sample taken at the hospital's outpatient clinic, as patients with gonadal hormones indicating hypogonadism were invited to clinical examination and a control morning blood sample. Finally, a low testosterone value with normal LH and FSH may also be due to a random low testosterone value caused by, for instance patients’ stress.Testosterone deficiency in adult males has been associated with declining bone mass, muscle strength, reduced energy levels and altered sexual function (Testosterone replacement therapy is a challenging issue in both male cancer survivors and in the general male population. Age has a significant influence on physiological testosterone levels. To evaluate endocrine hypogonadism at least two morning blood samples are needed and clinical symptoms, age and possible contraindications for testosterone substitution have to be considered. Owing to these caveats our study patients’ with endocrine hypogonadism identified in this study were offered repeated hormone analyses and clinical examination at the outpatient clinic before any testosterone substitution was initiated.There are some limitations to be discussed:The compliers were older at survey than the non-compliers. This may have caused a biased patient sample with a slightly higher proportion of hypogonadism in our patient sample than in the total population of male lymphoma survivors.Our patients’ age ranged from 21 to 73 years at survey and we found a fivefold increase in the risk of endocrine hypogonadism in men aged above 50 years at survey compared to those less than 40 years at survey. Similarly, Seven patients registered in the database with both HL and NHL had their gonadal hormones assessed, and were grouped according to their first diagnosis. This may have biased the results because their treatment consisted of combinations of different types of chemotherapy.The strength of this cross-sectional study is that it consists of a large series of male survivors after both HL and NHL with known treatment details and gonadal hormones assessed by a single laboratory. We achieved a compliance of 45%, which we consider satisfactory taken into account that this long-term follow-up survey implied blood sampling at a GP's office.The results of this study implicate that gonadal hormones should be assessed regularly in male lymphoma survivors, especially after treatment with high-dose alkylating agents and HDT and in male patients 50 years and older. If gonadal hormones indicate hypogonadism, further analyses and clinical evaluation should evaluate the need of testosterone substitution to avoid the development of serious health problems.


P<0.001) and had a significantly better disease-free survival than the infiltrative margin tumours (log rank P=0.01). Primary cancers with a high MVD tended to form high MVD liver metastases (P=0.007). Microvessel density was a significant predictor of disease recurrence in primary CRCs (P=0.006), but not liver metastases. These results suggest that primary CRCs and their liver metastases show common histological features. This may reflect common mechanisms underlying the tumour–host interaction.The nature of the invasive growth pattern and microvessel density (MVD) have been suggested to be predictors of prognosis in primary colorectal cancer (CRC) and colorectal liver metastases. The purpose of the present study was to determine whether these two histological features were interrelated and to assess their relative influence on disease recurrence and survival following surgical resection. Archival tissue was retrieved from 55 patients who had undergone surgical resection for primary CRC and matching liver metastases. The nature of the invasive margin was determined by haematoxylin and eosin (H&E) histochemistry. Microvessel density was visualised using immunohistochemical detection of CD31 antigen and quantified using image capture computer software. Clinical details and outcome data were retrieved by case note review and collated with invasive margin and MVD data in a statistical database. Primary CRCs with a pushing margin tended to form capsulated liver metastases ( Approximately 50% of patients with colorectal cancer (CRC) will be cured by surgical resection. The remainder will develop disease recurrence, with the liver being the commonest site for metastatic spread . In seleSeveral histological variables have been identified as predictors of disease recurrence, including the degree of tumour differentiation, the depth of local invasion, the presence of nodal metastases, and evidence of extramural vascular or perineural invasion. In 1987, Angiogenesis is a fundamental requirement for tumour growth, invasion, and metastasis formation . The assThe nature of the invasive growth pattern and the angiogenic response both influence a tumour's metastatic capability. However, it is not known if these processes are interrelated. It is possible that different invasive phenotypes are associated with different angiogenic responses, which in turn influence their metastatic capability. The current study aimed to investigate the relationship between the invasive growth patterns observed in primary CRC and liver metastases, the association with MVD, and the impact on clinical outcome.Patients who had undergone potentially curative resection for CRC and colorectal liver metastases were identified from histopathology databases at St James's University Hospital, Leeds and the Royal Liverpool University Hospital, Liverpool, between June 1994 and June 2000. Patients with conditions known to predispose to CRC , and those who had undergone preoperative chemo-radiotherapy were excluded. Patients who died as a complication of their surgery were excluded as their death was unrelated to tumour biology and introduced a confounding influence on survival analysis. Patients included had primary CRC resected and were either diagnosed with synchronous liver metastases and underwent resection or developed metachronous liver disease on follow-up surveillance and underwent resection. A large proportion of patients in the study had synchronous metastases. Demographics are shown in Local ethics committee approval was obtained for the study . Patient demographics were entered into a statistical database  and collated with clinical and histological data.μm were prepared, dewaxed in xylene, rehydrated in graded ethanol solutions, and stained with haematoxylin and eosin (H&E) using a standard protocol.Archival paraffin-embedded tissue samples of matched primary CRC and liver metastases were retrieved for all patients. Thin sections of 4 as capsulated if >50% of the margin exhibited a fibrous capsule separating tumour from stroma.The nature of the invasive margin was determined by two- independent observers and checked by a consultant histopathologist (NS). The invasive margin of the primary cancers were classified as either pushing or infiltrative based on the predominant morphology, as defined by μm sections were dewaxed in Histoclear , rehydrated through graded ethanol, and washed with tap water for 10 min. Endogenous peroxidase activity was blocked with 0.6% (v v−1) H2O2 in 100% methanol for 10 min at room temperature. Antigen was retrieved by pressure cooking for 2 min in citrate buffer solution (pH 6.0). Sections were blocked with 5% (v v−1) goat serum  in phosphate-buffered saline (PBS) for 30 min at room temperature, and then incubated with anti-CD31 antibody  at 1 : 20 dilution in PBS overnight at 4°C. Sections were washed twice with PBS and then incubated with alkaline phosphatase–conjugated goat anti-mouse IgG  for 60 min at room temperature. Following two further washes in PBS, the substrate was developed using the Vector Red ALP kit . Sections were counterstained with methyl green  for 10 min, washed in acetone, and dehydrated in a series of graded ethanol and Histoclear. Slides were mounted in diphenylxylene  and cover slipped. Negative controls were performed in parallel, with omission of the primary antibody incubation step.Sections of 4 2. Twenty tissue samples were reanalysed 2 months following the initial analysis to assess intra and inter observer variation.Anti-CD31 treated sections were examined with a Nikon Eclipse, , E1000 microscope. Each section was divided at low power  into three areas representing the stromal interface, tumour margin, and the tumour centre. Five random high-power fields  were captured from each of the three tumour areas using a Basler A113™ Matrix-camera  connected to a computer running Lucia G™ software (Version 4.6 Nikon). A template was created by setting a colour threshold, which clearly identified the CD31-positive endothelial cells and a fixed light intensity was used throughout the analysis. MVD counts were recorded from each of the five high-power fields and averaged to give the mean MVD at the stromal interface, the tumour margin, and the tumour centre. The MVD was calculated as the number of vessels per mmχ2 test. The association between MVD and invasive growth pattern was analysed using the Mann–Whitney U-test. Spearman's correlation was performed to investigate the relationship of MVD in the primary cancer with that in the liver metastases and inter/intra observer variation. The difference in mean MVD between cancers that formed liver metastases and those that did not was tested using the Mann–Whitney U-test. The influence of mean MVD on disease-free and overall survival was analysed using Kaplan–Meier survival curves and log rank test. For all analyses a P value of <0.05 was deemed to be significant.Statistical analysis was performed using SPSS for windows . The relationship between the invasive growth patterns of the primary CRCs and their liver metastases was tested using the Routine histological analyses of the resected primary CRCs are shown in The mean length of follow-up for all patients was 47 months (range 6–171 months). A total of 28/55 (51%) patients were alive on completion of the study. The mean overall survival following resection of the primary cancers was 47 months (range: 6–171 months) and 36.3 months (range: 0–118 months) following liver resection. The mean disease-free survival following resection of primary cancers was 30 months (3–88 months) and 28 months (4–118 months) following liver resection. A total of 17/55 (30.9%) patients developed disease recurrence following liver resection: recurrence at the primary site (2), liver recurrence (7), lung recurrence (5), and multisite recurrence (3).r=0.75, P<0.001) and intra-observer  variation when 20 sections were re-scored.The scoring system for MVD was shown to be reliable and reproducible with significant correlations between the inter-observer . Similarly, primary cancers with an infiltrative growth pattern were significantly more likely to form noncapsulated metastases (P<0.001).Twenty-six (47%) of the primary cancers had a pushing and 29 (53%) an infiltrative growth pattern . A totaltastases  (P<0.001P=0.017). Significant differences were not observed in overall survival (log rank P=0.10). Similarly capsulated and noncapsulated liver metastases in terms of disease-free survival (log rank P=0.32) or overall survival (log rank P=0.26).Colorectal cancers with a pushing margin had a significantly better disease-free survival than the infiltrative margin tumours (log rank P=0.001) (P=0.011), but not on comparison of the stromal margins (P=0.149).The mean MVD was determined at the stromal interface, the tumour margin, and the tumour centre for each CRC and liver metastasis . PrimaryP=0.001) . A simil−2 (95% CI: 52.3–79.5), whereas the liver metastases had a mean MVD of 84.9 vessels mm−2 (95% CI: 66.8–103.1) at the tumour margin. The disease-free survival for the high and low MVD groups were not found to be significant following resection of primary CRC (log rank P=0.13) (P=0.036)  , but sigP=0.036) .−2, 95% CI: 58.6–106.2) compared to infiltrative cancers . A similar, but nonsignificant, trend to higher MVD was observed in the centre of pushing compared to infiltrative cancers , which in turn would upregulate a number of downstream mediators including various proteases, such as MMP2 (α in CRC and liver metastases has been described previously (Although the existence of two invasive phenotypes in CRC is now well- recognised and their prognostic implications proven, the biological explanation for their existence remains unexplained. The finding that pushing cancers tend to form capsulated liver metastases and infiltrative cancers tend to form non-capsulated liver metastases would argue for a genetic basis for these differences. However, one cannot ignore the influence of the tumour–stromal interaction on the invasive process, and this is highlighted by our finding of a significant difference in tumour margin MVD between pushing and infiltrative cancers. Such a difference was not apparent in liver metastases that reside in the highly vascularised liver parenchyma. One might hypothesise that the higher margin MVD observed in pushing cancers was indicative of better tumour oxygenation, with the assumption that microvessels are functionally competent . The corThe current study has highlighted the importance of the invasive margin in CRC progression and the potential value of tumour–stromal interactions, as assessed by MVD assessment, in prognostication. Further investigation is required to determine the biological mediators, which underlie CRC invasion, to enhance our understanding of the metastatic process, and to reveal potential new targets for therapeutic intervention.