Our aim was to describe our experience with infrapopliteal endovascular procedures performed in diabetic patients with ischemic ulcers and critical ischemia (CLI). A retrospective study of 101 procedures was performed. Our cohort was divided into groups according to the number of tibial vessels attempted and the number of patent tibial vessels achieved to the foot. An angiosome anatomical classification of ulcers were used to describe the local perfusion obtained after revascularization. Ischemic ulcer healing and limb salvage rates were measured. Ischemic ulcer healing at 12 months and limb salvage at 24 months was similar between a single revascularization and multiple revascularization attempts. The group in whom none patent tibial vessel to the foot was obtained presented lower healing and limb salvage rates. No differences were observed between obtaining a single patent tibial vessel versus more than one tibial vessel. Indirect revascularization of the ulcer through arterial-arterial connections provided similar results than those obtained after direct revascularization via its specific angiosome tibial artery. Our results suggest that, in CLI diabetic patients with ischemic ulcers that undergo infrapopliteal endovascular procedures, better results are expected if at least one patent vessel is obtained and flow is restored to the local ischemic area of the foot. Critical limb ischemia (CLI) mainly affects elderly patients with important comorbidities and significant diffuse multilevel vascular lesions , 2. ThesIn tibial vein bypass planning, it has been generally accepted that the decision of which tibial outflow vessel should be treated must be taken according to angiographic considerations of the target distal vessel quality, since better patency rates are expected if less diseased runoff artery is used for distal anastomosis. However, the endovascular approach offers the possibility of treating more than one tibial vessel. In addition, data shown in several recent reports suggest that improved clinical outcomes could be achieved when blood flow is directed to the local ischemic area of the foot through its specific source tibial artery using and angiosome anatomical model –9, 13. TOur aim is to analyze the clinical and hemodynamic results of infrapopliteal endovascular procedures applied to CLI diabetic patients with ischemic ulcers according to the number of tibial vessels attempted for revascularization, the number of tibial arteries finally achieved to the foot and the local perfusion of the ischemic ulcer obtained after revascularization in order to describe the usefulness of these recent techniques in new reperfusion strategies.We carried out a retrospective study of consecutive primary infrapopliteal endoluminal techniques performed at our Vascular Surgery Department for CLI and ischemic ulcers in diabetic patients over a ten-year period (January 1999–December 2009). All patients with end-stage renal disease were excluded from the analysis since this specific condition is associated with worst outcomes due to the extensive arterial calcification found in these subjects. During this period, endovascular procedures were progressively implemented as the first treatment option for CLI whenever technically feasible. The clinical diagnosis of CLI was confirmed by objective documentation of severe hemodynamic compromise according to TASC (TransAtlantic InterSociety Concensus) criteria [Demographic details, atherosclerosis risk factors, basal and postoperative hemodynamic data, and TASC classification for the worse lesion treated were entered in our database. Femoropopliteal lesions were classified using the TASC-II criteria  and infrOur cohort was divided in groups according to the number of tibial vessels attempted for treatment, the number of patent tibial vessels finally achieved to the foot and the local perfusion of the ischemic ulcer obtained after revascularization. Thus, the infrapopliteal interventions were classified as “single revascularization” if only one tibial vessel was treated and “multiple revascularization” if more than one infrapopliteal vessel was attempted for revascularization.We considered that a patent tibial vessel was finally achieved if lesion recanalization was successful and morphological result of the procedure on target lesion was optimal . Endovascular treatment of the peroneal artery also required at least one patent distal peroneal branch to the foot to consider the artery successfully revascularizated. Those procedures in which none patent tibial arteries were obtained were classified as “runoff 0 group.” Interventions in which a single outflow vessel to the foot was obtained with an optimal morphological result were classified as “runoff 1 group.” Procedures in which more than one outflow tibial artery was achieved were classified as “runoff >1 group.”n = 3), the anterior tibial artery (n = 1), and the peroneal artery (n = 2) [Local perfusion of the ischemic ulcer obtained after revascularization was analyzed according to an angiosome model. Taylor and Palmer introduced this anatomical concept in 1987, widely used in modern plastic surgery, which is defined as 3-dimensional block of tissue supplied by a specific source artery and drained by a specific vein . The foo (n = 2) , 16  , 16. ToeThe analysis according to the postoperative ischemic ulcer local perfusion was only performed in those procedures in which at least one patent tibial vessel was obtained. We excluded the “runoff 0” interventions in order to evaluate the influence of foot vessels patency on our cohort results. Preoperative and intraoperative angiographies were reviewed and procedures were classified using an angiosome anatomical model as ‘‘direct revascularization” (DR) if at least one feeding vessel supplied the injured angiosome and as ‘‘indirect revascularization” (IR) if it fed an unrelated angiosome. Indirect revascularization was subdivided into IR ‘‘through collaterals” (IRc) and ‘‘without collaterals” depending on the presence of collateral vessels to the affected angiosome . The ulcer-angiosome and angiogram-angiosome assignment were done in a blinded fashion. Revascularization of digit ulcers by the ‘‘dorsalis pedis” or the plantar artery was considered as DR. The same exception was applied in heel ulcers perfused by the plantar artery or the calcaneal branch of the peroneal artery.All patients were called for follow-up at 1 month, 3 months, and then every 6 months after the procedure was performed. Patency was assessed by ABI in all cases. Color duplex scanning was performed in those patients with noncompressible ABI, with stent implants or those who required complex endovascular revascularization procedures. Patients who showed clinical worsening or those with a drop in ABI >0.15 were initially assessed by color duplex scanning. Echographic restenosis was defined as the finding of a peak systolic velocity ratio >2.5 in the target lesion and only symptomatic restenosis were treated. Lower limbs angiography was performed prior to reintervention. Reinterventions were classified as major  and minor (new endovascular procedure without the need for thrombectomy or thrombolysis).The main objectives of the study were ischemic ulcer healing at 12 months and limb salvage at 24 months. Foot tissue lesions were considered as infected according to the CDC/NHSN surveillance definition of health care-associated infection criteria . Local tThe results of our cohort were also analyzed according to the definition of the different Objective Performance Goals proposed by the Society for Vascular Surgery for evaluating catheter-based treatment of CLI . The incFreedom from any MALE or perioperative death (freedom from MALE+POD), amputation-free survival, and overall survival at 24 months were calculated to discriminate between limb-specific results and survival-related results as CLI patients use to develop multiple adverse events that can greatly influence the revascularization outcomes and patients survival. Freedom from any reintervention or amputation (freedom from RAO) and freedom from restenosis, any reintervention or amputation (freedom from RAS), were measured as estimators of hemodynamic failure . In thisAll the previously mentioned endpoints were compared according to the number of tibial vessels attempted for revascularization, the number of tibial vessels finally achieved to the foot, and the local perfusion of the ischemic ulcer obtained after revascularization.Statistical Analysis. Data was processed using the software packages SPSS 15.0 (Microsoft). Differences between groups were considered statistically significant for a P < 0.05 in 2-tailed test. The analysis of normality was carried out using the Kolmogorov-Smirnov and Shapiro-Wilk tests and continuous variables were compared using the Mann-Whitney U-test. Wilcoxon signed rank test was performed to measure differences in repeated measurements on a single continuous variable. The Chi-square and Fisher exact test were used to explore the differences between categorical variables. Ischemic ulcer healing at 12 months, limb salvage, and the different Objective Performance Goals-based endpoints at 24 months were measured by the Kaplan Meier method. Survival curves were compared using the Log-Rank test. Continuous variables are expressed as the median (interquartile range (p25–p75)) and the categoricals as percentages.One hundred and one primary infrapopliteal endovascular procedures performed in 92 diabetic patients with ischemic ulcers between January 1999 and December 2009 were retrospectively followed. All the revascularizated ischemic ulcers were limited to the foot. Most of the procedures were performed between 2005 and 2009 (74 (73%) endovascular techniques) and only 8 (8%) between 1999 and 2000. Median follow-up was 19 (9–38) months and 11 (10.8%) patients were lost. Demographic characteristics, comorbidities, ischemic ulcer description, and basal hemodynamic data are described in P < 0.001). Ankle-brachial index was noncompressible in 54 (53.5%) procedures.The TASC-II classification of the worst lesion treated was TASC-D in 79.2% surgeries. In 56 (55.4%) endovascular procedures the tibial intervention was combined with a femoro-popliteal angioplasty. Stents were used selectively in 9 (8.9%) procedures. Seven (6.8%) stents were implanted in the femoro-popliteal sector and 2 (1.9%) in the tibial sector. Multiple revascularization of the tibial sector was attempted in 52 (51.5%) interventions and single revascularization in 49 (48.5%). In 16 (15.8%) procedures a direct line of blood flow to the foot was not achieved (runoff 0 group). A single patent tibial vessel was obtained in 64 (63.4%) interventions (runoff 1 group) and more than one in 21 (20.8%) procedures (runoff >1 group). After excluding the runoff 0 group, DR of the ischemic ulcer was achieved in 46 (54.1%) procedures, IR “through collaterals” in 22 (25.9%) and IR “without collaterals” in 17 (20%) interventions . PostopeThe incidence of MACE, MALE, and major amputation at 30 days was 3%, 3%, and 2%, respectively. Ischemic ulcer healing at 12 months was 55.0% and limb salvage at 24 months was 74.9%. Freedom from MALE+POD at 24 months was 64.8%. We recorded an amputation-free survival rate of 63.3% after 2 years of follow-up. Overall survival at 24 months was 76.9%. Freedom from RAS and freedom from RAO at 24 months were 37.6% and 58.9%, respectively.P = 0.22). More than one patent tibial vessel to foot was finally obtained in 18 (34.6%) procedures of the multiple revascularization group and in 3 (6.1%) interventions of the single revascularization group (P < 0.01)  .P = 0.61), MALE (2.0% versus 3.8% P = 0.59), and major amputation at 30 days (0% versus 3.8% P = 0.49) was similar between single revascularization and multiple revascularization groups (The incidence of MACE (4.1% versus 1.9% n groups .P = 0.18). Limb salvage at 24 months was similar between single revascularization and multiple revascularization groups (72.2% versus 77.6% P = 0.62)  .P = 0.33). There were no statistical differences in amputation-free survival at 24 months between single and multiple revascularization attempt strategy (62.4% versus 64.3% P = 0.96). Overall survival at 24 months was 78.7% in the single revascularization group and 75.2% in the multiple revascularization group (P = 0.99).Freedom from MALE-POD at 24 months was 62.1% in the single revascularization group and 71.3% in the multiple revascularization group (P = 0.35). There were no statistical differences between single and multiple revascularization attempt strategy in terms of freedom from RAO (50.5% versus 67.6% P = 0.25).Freedom from RAS at 24 months was 24% in the single revascularization group and 43% in the multiple revascularization group (P = 0.01) or the runoff >1 group (0.52 (0.57–0.66) versus 0.90 (0.77–0.97)). ). .The incidence of MACE, MALE, and major amputation at 30 days was similar between runoff 0 group, runoff 1 group, and runoff >1 group .P = 0.01) or the runoff >1 group (14.3% versus 64.6% P = 0.003). There were no statistical differences in ulcer healing between runoff 1 group and runoff >1 group (60.3% versus 64.6% P = 0.43). Limb salvage at 24 months was lower in the runoff 0 group than in the runoff 1 group (43.8% versus 80.5% P = 0.001) or the runoff >1 group (43.8% versus 85.5% P = 0.006). There were no differences in terms of limb salvage at 24 months between runoff 1 group and runoff >1 group (80.5% versus 85.5% P = 0.50)  .P > 0.001) or the runoff >1 group (25.0% versus 70.7% P = 0.006) and no differences were observed between runoff 1 group and runoff >1 group (74.9% versus 70.7% P = 0.62).Freedom from MALE+POD at 24 months was lower in the runoff 0 group when it was compared to the runoff 1 group (25.0% versus 74.9% P = 0.02) or runoff >1 group (43.8% versus 76.6% P = 0.04). There were no differences in terms of amputation-free survival at 24 months between runoff 1 group and runoff >1 group (64.3% versus 76.6% P = 0.50).Amputation-free survival at 24 months was lower in runoff 0 group than in runoff 1 group .Overall survival at 24 months was 84.4% in runoff 0 group, 72.9% in runoff 1 group and 83.6% in runoff >1 group and no statistical differences were observed (runoff >1 versus runoff 1 P = 0.01) and runoff >1 group (18.8% versus 42.9% P = 0.08). There were no differences between runoff 1 group and runoff >1 group (42.8% versus 42.9% P = 0.70).Freedom from RAS at 24 months was lower in the runoff 0 group when it was compared with runoff 1 group (18.8% versus 42.8% P > 0.001) and the runoff >1 group (25.0% versus 65.8% P = 0.02). There were no differences between runoff 1 group and runoff >1 group (66.5% versus 65.8% P = 0.50).Freedom from RAO at 24 months in the runoff 0 group was lower than the rates obtained in the runoff 1 group  or IR “without collaterals” (43.5% versus 17.6% P = 0.009) was obtained. A multiple tibial revascularization attempt was more frequently performed in those procedures in which a DR was obtained than in those in which an IR “through collaterals,” (67.4% versus 36.4% P = 0.01) or an IR “without collaterals” (67.4% versus 41.2% P = 0.06) was achieved. DR of the ischemic ulcer was more frequent in procedures that obtained more than one patent tibial vessel and IR “through collaterals” was more frequent in those that achieved only a single patent tibial artery to the foot. Postoperative ankle-brachial index was similar between DR, IR “through collaterals” and IR “without collaterals” groups as it is expected because at least one patent tibial artery to the foot was obtained in all the analyzed groups  or IR “through collaterals” groups (7.1% versus 68.0% P < 0.001). Ischemic ulcer healing rate at 12 months was similar between DR and IR “through collaterals” groups (66.0% versus 68.0% P = 0.38). Limb salvage at 24 months was lower in the procedures in which an IR “without collaterals” of the ischemic ulcer was achieved when it was compared with DR (59.0% versus 88.9% P = 0.04) or IR “through collaterals” groups (59.0% versus 84.8% P = 0.06). Limb salvage at 24 months was similar between DR and IR “through collaterals” groups (88.9% versus 84.8% P = 0.45)  .P = 0.005) or IR “through collaterals” groups (45.9% versus 80.6% P = 0.06). There were no differences between DR and IR “through collaterals” groups (80.9% versus 80.6% P = 0.38).Freedom from MALE+POD at 24 months was lower in IR “without collaterals” group when it was compared with DR . Overall survival at 24 months was 73.6% in the DR group, 76.1% in the IR “through collaterals” group and 84.8% in the IR “without collaterals” group .Amputation-free survival at 24 months was 67.5% in the DR group, 73.3% in the IR “through collaterals” group, and 61.9% in IR “without collaterals” group, and no statistical differences were observed . No differences in terms of freedom from RAS were observed between DR and IR “through collaterals”(53.0% versus 43.9% P = 0.19) and between IR “through collaterals” and IR “without collaterals” (43.9% versus 22.5% P = 0.66).There was a tendency towards a lower freedom from RAS rate at 24 months in the IR “without collaterals” group when it was compared with DR group (22.5% versus 53.0% P = 0.02). There was a tendency towards a lower freedom from RAS rate in the IR “without collaterals” group when it was compared with IR “through collaterals” group (38.5% versus 77% P = 0.08). No differences were observed between DR and IR “through collaterals” groups (74.0% versus 77.0% P = 0.78).Freedom from RAO at 24 months was lower in the IR “without collaterals” group than in DR group (38.5% versus 74.0% The care of reperfused ischemic ulcers is a major postoperative problem due to the slow tissue healing time and the frequent need for associated techniques on the ischemic wound. Delayed healing is especially important in patients with diabetes mellitus. This disease produces a number of complex biomechanical, neuropathogenic, and immunogenic foot disorders, which are able to reduce the capacity of tissue regeneration . In our One of the main advantages of the endovascular treatment is the possibility to “navigate” through the patient's infragenicular vascular anatomy. Endoluminal techniques offer the chance of treating more than one tibial vessel and this strategy could be associated with better clinical outcomes due to a better hypothetical hemodynamic improvement. According to Faglia et al., the risk of major amputation could increase up to eight times for each obstructed crural artery with alarming proportions in those procedures in which all three crural arteries were occluded . HoweverOn the other hand, the success of revascularization, although essential to ensure limb salvage, does not completely reduce the risk of delayed ischemic ulcer healing and major amputation. A healing failure rate and limb loss of around 15% have been reported in patients with pedal tissue loss and successful tibial vein bypasses . SystemiTherefore, according to these results, the depleted choke collateral network seems not to be able to successfully reperfuse foot ischemic wounds in CLI diabetic patients. However, blood flow restoration to the ulcer through patent arterial-arterial connections like pedal arch or distal peroneal branches provided similar results to those obtained after reperfusion of the ulcer through its specific tibial artery in our cohort. The influence of these collateral vessels on distal revascularization results has been widely discussed with contradictory conclusions. Berceli et al. in a retrospective cohort of pedal distal bypasses performed for heel ulcerations described a tendency towards a higher ulcer healing rate in those with a patent pedal arch though no statistical differences were recorded . In the This study has some limitations. It is a retrospective analysis; therefore the groups compared are not homologous, as they were not randomly assigned, situation that prevents from firm conclusions. We could not assess the size of the wounds and the different local treatment applied to the ischemic ulcer. Toe pressure was not routinely measured in those patients with noncompressible ABI. Therefore, hemodynamic data should be interpreted with caution. Most of the procedures were performed during the last 5 years of follow-up. This imbalance could be explained by our learning curve and the progressive technical improvement that experience the endovascular techniques in last years making this type of treatment more accessible for patients with CLI. As a result, the procedures indication may be varied over the years as we covered a wide enrollment period. The number of cases of some of the analyzed groups was small due to the limited sample size obtained following our inclusion criteria. This limitation explains why standard errors of some of the Kaplan-Meier curves exceeded the 10% standard error.With these limitations in mind, our results suggest that obtaining a direct line of blood flow to the foot during a infrapopliteal endovascular procedure performed in diabetics for CLI and tissue loss is mandatory to achieve ischemic ulcer healing and limb salvage. A multiple endovascular revascularization attempt seems secure, although this strategy does not improve the revascularization results. Clinical outcomes are subject to the restoration of blood flow to the local ischemic and are independent of the number of patent tibial vessels obtained after revascularization. In this context, the restoration of blood flow to the ischemic ulcer through a medium or large size collateral vessel could provide similar results in terms of ulcer healing and limb salvage rates to those obtained via its specific source tibial artery. Therefore, a careful study of the foot collateral vessels anatomy should be done before an endovascular infrapopliteal procedure is performed.According to the data draw in this study we suggest a distal endovascular planning in three steps. First, it is mandatory to obtain a direct straight line to the foot through the easier-to-treat tibial artery, even if the injured angiosome is not anatomically fed because this circumstance improves the results of the revascularization significantly. Second, if in the first step we did not achieve blood flow to the ulcer it is advisable to attempt the revascularization of another tibial vessel specifically related to the ulcer or indirectly-related through collateral vessels. Third, if it is not possible to obtain a direct line to the foot, a distal vein bypass has to be considered if feasible.


B. lusitaniae.TOC summary line: The green lizard is implicated in the transmission cycle of  In Europe, spirochetes within the Borrelia burgdorferi sensu lato complex are transmitted by Ixodes ricinus ticks. Specific associations are described between reservoir hosts and individual genospecies. We focused on green lizard (Lacerta viridis) as a host for ticks and potential host for borreliae. In 2004 and 2005, a total of 146 green lizards infested by ticks were captured, and 469 I. ricinus ticks were removed. Borrelial infection was detected in 16.6% of ticks from lizards. Of 102 skin biopsy specimens collected from lizards, 18.6% tested positive. The most frequently detected genospecies was B. lusitaniae (77.9%–94.7%). More than 19% of questing I. ricinus collected in areas where lizards were sampled tested positive for borreliae. B. garinii was the dominant species, and B. lusitaniae represented 11.1%. The presence of B. lusitaniae in skin biopsy specimens and in ticks that had fed on green lizards implicates this species in the transmission cycle of B. lusitaniae. The B. burgdorferi s.l. complex encompasses 12 species , the green lizard is the major host for immature stages of I. ricinus ticks . The climate is warm with low humidity and average temperatures of -4°C in January and 18°C in July. The average rainfall is 700 mm/year.This survey was conducted in 2004–2005, from May to September, when lizards and ticks are active. Questing I. ricinus nymphs and adults were collected by flagging the vegetation in areas where lizards were sampled. Ticks were immediately stored in 70% ethanol.Green lizards were captured along hiking paths by hand or by noosing, in which a loop made from fishing nylon was attached to the end of a wooden stick and dangled in front of a lizard, which would be captured as it walked through the loop. Animals were characterized by sex and age  and examined for ticks.Ticks were removed with forceps immediately after capture and stored in 70% ethanol. Biopsy specimens  were taken from each lizard with sterile scissors and put in separate vials with 70% ethanol. Ticks were identified to the species and sex. Only I. ricinus ticks were further examined for B. burgdorferi sensu lato.Immediately before extraction, ticks and tissues were dried for 30 min to evaporate the ethanol. Each sample was cut with a disposable sterile scalpel. Tissue DNA from lizards' tails and scales was extracted by using DNeasy tissue kit . Extraction steps were conducted according to the manufacturer's protocol. Genomic DNA from ticks was isolated by alkaline hydrolysis (2+), 1.5 μL 25-mmol/L solution of Mg (OAc)2, 0.1 μL Taq DNA polymerase (5 U/μL), 0.5 μL deoxynucleoside triphosphate (dNTP) mix (10 mmol/L) , 1.25 μL of each primer (10 pmol/μL) , and 2.5 μL DNA template.PCR amplification was performed in a 25-μL reaction mixture from the MasterTaq DNA polymerase kit  containing 10.4 μL deionized water, 5 μL 5× TaqMaster PCR Enhancer, 2.5 μL 10× Taq buffer  were used . Previously extracted DNA of B. afzelii, B. garinii, B. valaisiana, and B. burgdorferi s.s. were used as positive controls. For each positive sample, 13 μL amplified DNA was digested at 65°C overnight in a solution containing 5 U of Tru1I (300 U/mL) and 1× Buffer R (Fermentas). Electrophoresis was conducted in 16% polyacrylamide gel at 150 V for 3 h. The gels were stained with SYBR gold nucleic acid gel stain  for 20 min, and bands were visualized with a UV transilluminator. RFLP profiles that differed from the known profiles of positive controls were further analyzed by sequence analysis.http://www.ebi.ac.uk/emboss/align).Sequencing was performed at the Department of Molecular Biology . PCR of the 5S-23S rDNA intergenic spacer was conducted according to the protocol described above. For the fla gene amplicons, DNA strands were sequenced as described previously . The specific infectivity (Is) of a reservoir host species is defined as the sum of individual infectivities and number of individual lizards sampled . The host-to-tick transmission coefficient (βH–T) is defined as the portion of the sum of individual infectivities and the number of lizards that infected >1 larva . Differences in the prevalence of B. burgdorferi s.l. in I. ricinus were evaluated statistically with the 2-tailed χ2 test (degrees of freedom [df] = 1). A value of p<0.05 was considered statistically significant.To estimate the probability of a tick's becoming infected after engorging on a green lizard and to measure the degree of infectiousness of infected animals, specific infectivity IOne hundred forty-six  of 165  captured green lizards were infested by ticks during the study period. In total, 469 (199 larvae and 270 nymphs) ticks were removed and further identified as I. ricinus. Male lizards were infested with 363 ticks (131 larvae and 232 nymphs), which represented 77.4% of all collected ticks. Moreover, 53 tails and 102 skin biopsy specimens were taken from the captured lizards.>1 infected tick. Infected lizards yielded ≈2 infected larvae per host.DNA isolation was successful in 464 ticks (197 larvae and 267 nymphs), from which the fragment of cytochrome b gene was amplified. These ticks were further analyzed for the presence of B. burgdorferi s.l. In total, 77 (16.6%) ticks carried borreliae. The infection prevalence between nymphs (15.2%) and larvae (17.6%) did not differ significantly  . Twenty-<0.001). B. lusitaniae was detected in 26 (86.7%) larvae. B. afzelii, B. garinii, and B. burgdorferi s. s. each were found in 1 larva. Of the 47 B. burgdorferi–infected nymphs, 34 (72.3%) were infected with B. lusitaniae, 5 (10.6%) with B. afzelii, 2 (4.3%) with B. burgdorferi s. s., and 1 (2.1%) with B. garinii. A mixed infection of B. lusitaniae and B. burgdorferi sensu stricto was found in 5 (10.6%) nymphs  were infected with B. lusitaniae. The presence of this species was significantly higher than that of other species (p) nymphs . Nymphs Male lizards were parasitized by 61 (79.2%) of 77 infected ticks. Variability of detected genospecies was higher in ticks collected from male than from female lizards. Larvae that fed on female lizards were only infected with B. lusitaniae. Out of 7 infected nymphs collected from females, B. lusitaniae was present in 5 and B. afzelii in 1 tick; B. lusitaniae and B. burgdorferi s.s. were detected as mixed infection in 1 nymph. The specific infectivity from lizards to larval ticks was highest for B. lusitaniae. The specific infectivity of female lizards was slightly higher than that of males .Isolated genomic DNA from tails and skin biopsy specimens from collar scales was tested for the presence of B. burgdorferi sensu lato. None of 53 tested tail samples was positive. Of 102 skin biopsy specimens collected from green lizards, 19 (18.6%) tested positive. Differences in infection prevalence between sexes  were not significant. Of 9 skin biopsy specimens from subadult individual lizards, 2 (22.2%) were borrelia positive. The most frequently detected genospecies was B. lusitaniae (94.7%), which was present in 18 samples. One lizard was infected with B. afzelii.Cytochrome b was amplified in 325 of 331  questing ticks. Therefore, only these 325 ticks  were analyzed further for the presence of B. burgdorferi s.l. Sixty-three (19.3%) ticks tested positive. B. burgdorferi prevalence in female ticks was the same as in male ticks (22.5%), and it was lower in nymphs (16.9%).RFLP analysis of the amplified products resulted in 5 distinct profiles. Of the 63 positive ticks, 21 (33.3%) were infected with B. garinii, 19 (30.2%) were infected with B. afzelii, 8 (12.7%) were infected with B. burgdorferi s. s., 7 (11.1%) were infected with B. lusitaniae, and 7 (11.1%) were infected with B. valaisiana. One nymph was infected simultaneously with B. garinii and B. valaisiana .Representative samples of RFLP profiles that were different from the known profiles of positive controls were sequenced. The fragment of the 5S-23S rDNA intergenic spacer obtained from the B. burgdorferi s.l.–positive nymph (538N) from lizard belonged to B. lusitaniae. It was 100% identical to a Borrelia-positive skin biopsy specimen (277S) sampled from a lizard. Both obtained sequences were 100% identical with the Turkish B. lusitaniae strain Tr213 (AB 091802) and 98.9%, 98.4%, and 94.5% similar to PotiBL37 (AY 463167), PotiB2 (L30131), and PotiB3 (L30132) strains from Portugal, respectively. To better characterize B. lusitaniae circulating in ticks and lizards from Slovak Karst, the fla gene from a B. burgdorferi s. l.–positive questing adult tick and skin biopsy specimen from collar scale was amplified and sequenced. The flagellin sequence of B. lusitaniae detected in a skin biopsy specimen (277 S) was 100% identical and 99.6% similar to B. lusitaniae detected in questing adult ticks , respectively. Genotypes 277S and 43ZLIF were 100% identical with the Turkish B. lusitaniae strain Tr213 (AB091812) as well as with the Polish strain D23–04 (DQ 016623). Genotype 47ZLIM was 99.6%, 99.6%, and 99.4% similar to Tr213, D23–04, and PotiB2 (DQ111036), respectively.,,The role of lizard species in maintaining B. burgdorferi s.l. has not been clearly elucidated yet. In United States, some lizard species have sustained borrelial infection  were infected with B. lusitaniae. Similarly, B. lusitaniae have been detected in blood and tissue samples of Podarcis muralis in Tuscany in Italy, where borreliae were detected in 2 of 14 tested whole tails from lizards . Larger numbers of ticks feeding on male lizards are associated with higher male activity in spring months, when tick activity peaks as well. Male lizards end hibernation first and are active when the air temperature reaches 10°C–12°C. They need to restock the energy that was depleted during winter and to gain energy for fighting other male lizards to compete for territory and females, for seeking female lizards, and for mating. Female activity increases in summer months when they are incubating eggs.As determined by PCR, the overall prevalence of infection in our sample of questing ticks (19.3%) is consistent with 20.5% found in southern Czech Republic  on the transmission of spirochetes was observed ; none of 183 examined nymphs was infected by this genospecies. This finding raises the questions of whether borreliae are eliminated during molting and thus do not contribute to the transmission cycle or whether we were just unable to detect it. Significant differences were found in B. lusitaniae prevalence also in fed nymphs compared with questing adults . The infection prevalence decreased from 74.5% in fed nymphs to 5% in questing adults. Reduction of infection prevalence has been observed in B. afzelii from 47% in nymphs engorged on the rodents to 7% in questing nymphs (Significant differences were found in B. lusitaniae prevalence in fed larvae compared with questing nymphs (p,,,The occurrence of B. lusitaniae in ticks is frequent in some areas of the Iberian Peninsula and North Africa, where the organism often represents the only species of B. burgdorferi s.l. complex (


The exploitation and utilization of vast varieties of herbal extracts may serve as alternative measures to deter aggregation of deoxygenated sickle cell hemoglobin (deoxyHbS) molecules.in vitro study ascertained the capacity of three medicinal plants, namely, Anacardium occidentale, Psidium guajava, and Terminalia catappa, to alter polymerization of HbS.The present 2 S2 O5) at a regular interval of 30 s for a period of 180 s in the presence of separate aqueous extracts of A. occidentale, P. guajava, and T. catappa. At time intervals of 30 s, the level of polymerization was expressed as percentage of absorbance relative to the control sample at the 180th s.Spectrophotometric method was used to monitor the level of polymerization of hemolysate HbS molecules treated with sodium metabisulfite (NaP < 0.05) reduction in polymerization of deoxyHbS molecules, the corresponding capacity in this regard diminished with increase in incubation time. Aqueous extract of P. guajava exhibited the highest capacity to reduced polymerization of deoxyHbS molecules. Whereas at t > 60 s, extract concentration of 400 mg% of A. occidentale activated polymerization of deoxyHbS molecules by 6.23±1.34, 14.53±1.67, 21.15±1.89, and 24.42±1.09%, 800 mg% of T. catappa at t > 30 s gave values of 2.50±1.93, 5.09±1.96, 10.00±0.99, 15.38±1.33, and 17.31±0.97%.Although extracts of the three medicinal plants caused significant (The capacity of the three medicinal plants to interfere with polymerization of deoxyHbS molecules depended on the duration of incubation and concentration of the extracts. Anacardium occidentale) is a multipurpose tree of the Amazon and African rainforest that grows up to 15 m high. It has a thick and tortuous trunk with branches so winding that they frequently reach the ground. The cashew fruit is a rich source of vitamins, minerals, and other essential nutrients.[Leishmania (Viannia)[A. occidentale against E. coli,[Pseudomonas,[Helicobacter pylori considered to cause acute gastritis and stomach ulcers have been reported.[Cashew  and are  E. coli,9 Pseudomudomonas, and Helireported.Psidium guajava) is a tropical and semi-tropical plant. The branches are crooked, causing opposite leaves to overlap. The flowers are white, incurved petals, 2 or 3 in the leaf axils. The fruit is small, 3–6 cm long, pear-shaped, reddish-yellow when ripe.[P. guajava have been shown to bind to E. coli,[P. guajava fruit intake decreases blood pressure and serum high-density lipoprotein/cholesterol levels.[Guava  tree can reach a height of 35 m (110 ft). It grows upright and forms a symmetrical crown with horizontal branches distinctively arranged in tiers. As the tree ages, the crown will become increasingly flattened and eventually take on a vase shape.[Indian almond  is a product of a defective genetic code of hemoglobin molecule. The HbS 26molecule. The normmolecule.30 CharacIn vitro studies by Abdulmalik et al[The development of chemical modification agents that reduce the tendency of deoxyHbS to aggregate representsan important chemotherapeutic goal. Whereas hemoglobin carbamylation by cyanate32 is a plik et al reportedlik et al and 2-imlik et al are amonA. occidentale, P. guajava, and T. catappa) to interfere with polymerization of HbS molecules.However, limitations of these antisickling agents are undesirable structural andfunctional changes of hemoglobin as well as toxicity resulting from modifications of other protein molecules.31–33 TheA. occidentale, P. guajava, and T. catappa leaves were harvested between July and August 2010, from trees within the environment of Imo State University, Owerri, Nigeria. The plant specimens were identified and authenticated by Dr. F. N. Mbagwu at the Herbarium in the Department of Plant Science and Biotechnology. A voucher specimen was deposited at the Herbarium for reference purposes.Fresh samples of A. occidentale, P. guajava, and T. catappa leaves were obtained by filteration with Whatman No. 2 filter paper. The prepared extracts were kept at 4°C in a refrigerator for at least 24 h before subsequent tests. Serial dilutions of the aqueous extracts in the order of 200, 400, 600, and 800 mg% were used for polymerization analyses.The samples were washed under continuous current of distilled water for 15 min and air dried at room temperature for 60 min. The separate leaves were dried for 5 h in an oven at 60°C to become crispy, and ground with ceramic mortar and pestle. Two grams (2 g) each, of the pulverized specimen was suspended in 100 mL of distilled water and allowed to stand for 6 h at 37°C. The aqueous extracts (2 g%) of Five milliliters (5.0 mL) of human venous blood samples of HbSS genotype were collected by venipuncture and stored in EDTA anticoagulant tubes. The blood samples were obtained between July and August 2010, from 9 male volunteers (59-79 kg) in the age group of 21–34 years attending clinics at the Federal Medical Center (FMC), Imo State University Teaching Hospital (IMSUTH), Orlu, St. John Clinic/Medical Diagnostic Laboratories, Avigram Medical Diagnostic Laboratories, and Qualitech Medical Diagnostic Laboratories. These centers are located in Owerri, Imo State, Nigeria. The Institutional Review Board of the Department of Biochemistry, Imo State University, Owerri, Nigeria, granted approval for this study and all volunteers involved signed an informed consent form. This study was in accordance with the ethical principles that have their origins in the Declaration of Helsinki.et al.[2/10 mM glucose). The erythrocytes were separated from plasma by centrifugation at 1200g for 10 min, washed 3 times by the same centrifugation method with the buffer solution. The erythrocytes were finally resuspended in 1.0 mL of this buffer and stored at 4°C. The washed erythrocytes were lysed by freezing/thawing as described by Galbraith and Watts,[et al.[The erythrocytes were washed by centrifugation methods as described by Tsakiris et al. Within 2nd Watts, and Kambs,[et al. The eryt2 S2 O5; }-induced polymerization of HbS molecules was ascertained as described previously by Iwu et al[et al.[2S2O3 was used as a reductant. The level of polymerization was monitored by recording increasing absorbance of the assay mixture with time. A 0.1 mL of HbS hemolysate was introduced into a test tube, followed by 0.5 mL of phosphate-buffered saline solution  and 1.0 mL of distilled water. The mixture was transferred into a cuvette and 3.4 mL of 2 g% aqueous solution of Na2S2O5 was added. The absorbance of the assay mixture was recorded with a spectrophotometer  at every 30 s for 180 s at λmax = 700 nm (control sample). This procedure was repeated by substituting the distilled water with 1.0 mL of corresponding four increasing concentrations of the separate extracts (test sample).Sodium metabisulfite {NaIwu et al with minal[et al. The undeWheret s.At/c =Absorbance of test/control sample at time = 180th s = Absorbance of control sample at the 180th s.Act test.[The results were expressed in terms of arithmetic means (X) ± standard deviation (SD). The statistical significance of the difference between the means was evaluated by Student’s t test.The pattern of increase in absorbance of the assay mixture with experimental time is illustrated in Figures t < 30 s than subsequent time intervals. The control sample gave absorbance of 0.052 ± 0.05 units at t = 180 s representing 100% polymerization of deoxyHbS molecules. However, the test sample containing 400 mg% of A. occidentale gave a maximum absorbance of 0.0647 ± 0.004 units at the 180th s (polymerization = 126.86%). Furthermore, P. guajava exhibited the lowest absorbance value of 0/0073 ± 0.01 units at t = 30 s corresponding to 14.31 ± 2.11% polymerization [A cursory look at Figures rization .t = 90, 120, and 150 s, they were not significantly different when compared with the value at t = 180 s (P < 0.05). Addition of 200, 600, and 800 mg% aqueous extracts of A. occidentale to the assay mixture caused reduction of deoxyHbS polymerization within the experimental period (t = 0–180 s). However, the capacity of the three mentioned concentrations of A. occidentale to inhibit polymerization of deoxyHbS molecules diminished as the experimental time progressed. Specifically, A. occidentale to inhibit deoxyHbS polymerization fell within the ranges of 33.96%-1.92%, 45.83%-14.81%, and 34.79%-17.88%, respectively. Extract concentration of 400 mg% of A. occidentale caused reduction in polymerization of deoxyHbS molecules at t = 30 s; representing 11.04% ± 1.43% inhibition [t = 180 s; 100% polymerization; t > 60 s, the levels of activation gave 6.23% ± 1.34%, 14.53% ± 1.67%, 21.15% ± 1.89%, and 24.42% ± 1.09%.The levels of polymerization of deoxyHbS at specific time intervals compared with the control sample at the 180th s are presented in hibition . ParadoxP. guajava reduced polymerization of deoxyHbS molecules throughout the experimental time [t = 180 s [t = 30-180 s, whereas the diminishing capacity of P. guajava extract to inhibit polymerization of deoxyHbS molecules were as follows: 600 mg% (4.44%) and 800 mg% (7.87%), inhibition was sustained and increased by 200 mg% (2.48%) and 400 mg% (1.84%).The four concentrations of tal time . Specifital time , which c = 180 s . GeneralT. catappa hindered polymerization of deoxyHbS by 1.46% at t = 30 s, further increases in experimental time (t > 60 s), engendered activation of the polymerization process. The levels of polymerization were 2.50%, 5.09%, 10.00%, 15.38%, and 17.31% greater than the control sample at corresponding time intervals of 30 s [T. catappa to inhibit polymerization of deoxyHbS molecules occurred at t = 30 s, in the presence of extract concentration of 600 mg%. However, the level of inhibition attenuated as experimental time progressed.Although 800 mg% extract concentration of  of 30 s . Also, b of 30 s . The maxet al,[Noteworthy, polymerization of deoxyHbS is dependent on physiologic concentration of 2,3-DPG. However,et al, 2,3-DPG In vitro deoxygenation of hemolysate HbS molecules by sodium metabisulfite caused progressive aggregation and polymerization of the individual hemoglobin molecules. The process of gelation (polymerization) of hemoglobin molecules resulted in increasing absorbance of the assay solution with time.An overview of the present study showed increasing level of polymerization of deoxyHbS molecules with time that is consistent with the pattern described elsewhere.4546 In v45A. occidentale and 800 mg% of T. catappa activated polymerization of deoxyHbS molecules, the present study showed that aqueous extracts of the three medicinal plants  exhibited variable capacity to hinder polymerization of deoxyHbS molecules. In conformity with earlier reports of Oyewole et al,[in vitro studies, exhibited antisickling/polymerization property. The findings of this research are comparable to those previous reports. Research findings have established that the capability of a biomolecule to impede in vitro polymerization depends on one or combinations of the following options.Although after 60 s, extract concentrations of 400 mg% of le et al, a measurle et al,–52 TheirThe tendency and efficiency to bind to the complimentary contact region/site of deoxyHbS monomers.–36Modification of amino acid residues that contribute to the three-dimensional structures of HbS contact region and other critical sites.334733Stabilization of the R (relaxed) state of HbS molecule.4753544753The diminishing capacity of the three plant extracts to inhibit polymerization of deoxyHbS molecule with progression of experimental time suggest that the constituents of the extracts did not covalently modify the amino acid residues unlike other reported compounds.31–36 Rat


Toxoplasma gondii. Toxoplasma encephalitis (TE), which occurs as a reactivation of quiescent chronic infection, is one of the leading causes of central nervous system (CNS) infection in AIDS. Its diagnosis in most centres still remains difficult. We opted to describe the clinical and radiological features of TE as well as in-hospital outcome and its associated factors.It is estimated that about a third of the world’s population is chronically infected with st 2004 to December 31st 2009.We carried out a cross sectional study on the clinical case notes of adult patients admitted and treated for TE at the Douala General Hospital, Cameroon between January 13 (IQR): 43 – 103). Headache and fever were the most common presenting symptoms in 92.8% (90/97) and 87.6% (85/97) of patients. Annular contrast enhanced lesions were the most common brain scan finding in 81.4% (79/97) of patients. In-hospital mortality was 29.9% (29/97). Altered sensorium, presence of focal signs, neck stiffness and low CD4 cell count were factors associated with mortality. Adjusting for low CD4 count, altered sensorium remained strongly associated with fatality, adjusted odd ratio (AOR) 3.5 (95% CI 1.2 – 10.5).Of 672 patients admitted during the study period, 14.4% (97/672) had TE. The mean age was 36.9 ± 14.1 years and the median CD4 cell count was 68/mmToxoplasma encephalitis is common among AIDS patients in Douala. Its high case fatality warrants adequate and compliant prophylactic therapy in severely immune depressed patients as well as early initiation of antiretroviral therapy in HIV-infected patients. Toxoplasma gondii, with 73.2% (71/97) having less than 100 cells/mm3. Men had lower mean CD4 cell counts than women (p < 0.001).During the study period, 672 HIV infected patients were admitted to the Internal Medicine unit of who 97 fulfilled eligibility for the study, giving a prevalence of toxoplasma encephalitis of 14.4% (97/672). Of the 97, 52.6% (51/97) were females (Table Fever and headache were the most common symptoms Table . Neck st3 (IQR: 24 – 75). Men were more likely to die than women though this was not statistically significant (Table A total of 29 patients died giving a case fatality rate of 29.9% Table . Their mOur study showed that the prevalence of TE among HIV infected patients was 14.4%, a finding similar to that of another study . SeropreClinically, patients with TE usually have a sub-acute presentation over several weeks with symptoms and signs usually limited to the CNS including headaches, fever, psychomotor or behavioural abnormalities, confusion, focal signs and seizures . In our Brain CT scan examination has been shown to be the most useful means by which the diagnosis of TE could be presumed . All our3[3 and the development of TE [3.TE is more common in the advanced stage of HIV disease when CD4 count is low ,7. The r3. Many stnt of TE ,7: 73.2%The case fatality was 29.9% in our study. Though similar to that in other studies ,22 such There were some limitations in our study. The diagnosis of TE which remains presumptive may have led to under estimation or over estimation of the burden of TE. Response to treatment which was used as an index to confirm diagnosis of TE was useful only for patients with good response suggesting that some deaths might have been due to a cause other than TE. Moreover, information on anti-toxoplasma prophylaxis and HAART were not included in our analysis because they were consistently not available in patient records.TE is a common cause of morbidity and mortality among severely immune depressed HIV-infected patients in Cameroon. Though relatively easy to treat, a definitive diagnosis of TE is still difficult in most centres in Cameroon. The case fatality rate of TE is high; hence primary prophylaxis with adequate compliance should be instituted among patients with severe immune depression as well as early initiation of antiretroviral therapy in HIV-infected patients to avoid severe immune depression.The authors declare no conflict of interest.HNL, BCNT, YNM, ET and SKS designed the study. BCNT, YNM and HAJ collected the data. HNL and ET analysed the data and did manuscript write up, assisted by MPH and MSD. SKS, MSD and HNL proofread the manuscript. All authors agreed with the final manuscript to be submitted.


Chlorociboria aeruginascens were tested for possible stimulatory effects when grown on malt agar media containing wood additives. The addition of any of the four types of test wood , stimulated colony growth and xylindein production in C. aeruginascens. Addition of any amount of wood produced more growth than no wood additions, while ground wood produced more growth than chopped wood. Of the wood types tested, A. saccharum wood stimulated all four isolates, while spalted Populus tremuloides stimulated three of the four isolates. High glucose and sucrose amounts may be partially responsible for the greater stimulatory affect of some woods over others. The development of this simple and reliable method for growth and pigment stimulation of C. aeruginascens in laboratory conditions will allow for further development of this fungus for decorative and commercial use.Four isolates of  Chlorociboria genus is widely distributed throughout the world; Chlorociboria aeruginascens (Nyl.) Kanouse and Chlorociboria aeruginosa (Oeder) Seaver are native to North America , development of yed wood , and groyed wood . Chlorocicana L. .Chlorociboria sp. was shown to have a maximum growth diameter of 18 mm in four weeks, with neither pigment nor mycelium completely covering the plate during the entire six-month trial period , sugar maple (Acer saccharum Marsh.), tree of heaven  Swingle), and spalted aspen ) were chopped into roughly 5 mm × 5 mm × 2 mm thick squares. These squares were suspended in 2% malt extract agar media (100 mm × 15 mm Fisher brand polystyrene petri dishes) in either a high concentration (15-20 squares) or a low concentration (five to seven squares). Suspension was achieved by first pouring the hot media into the Petri plates, allowing the media to cool for approximately ten minutes in a laminar flow hood, then using tweezers to drop the sterilized wood squares into the media plates.Several sizes and dispersion methods were utilized when making the wood-media plates. Aspen . The wood was stored in glass jars with screw caps and was autoclaved just prior to being mixed with 2% malt agar media. Two different types of wood dust plates were made: plates with a high concentration of wood and plates with a low concentration of wood. Plates containing a high amount of wood were made by adding 7.5 ml of ground wood into each Petri plate, after which the hot malt agar was poured on top and then agitated by hand using a sterilized spatula for 10 seconds. Plates containing a low amount of wood were made by mixing 2.5 ml of ground wood directly into the pre-autoclaved media , autoclaving for 15 minutes, then pouring the sterilized media into the Petri plates. Plates containing only the agar media with no wood added acted as controls. Plates were left to cool in a laminar flow hood for 24 hours prior to inoculation.The same wood species were also ground using a Wiley mill, using both the 20 and 30 mesh screens . After five days of initial growth, the widest diameter of each colony was measured using a caliper. This size was measured as the base size of the colony. Each colony was then re-measured every three to four days, and the new width of the colony was recorded. Recording was completed when inoculation points on the same media plate touched (22 days). Six replicate plates were made per wood species, per mesh size, per wood amount for all preliminary tests.In the second round of testing, the square piece suspension was dropped from testing, and only ground wood plates were tested. In this second round of testing, two points on each plate were inoculated from the The size of white colonies was also measured, as was any green pigment produced by the white colonies during the incubation cycle. Final colony color was recorded after three weeks. Colonies were counted as green if the green pigment (assumed to be xylindein) appeared anywhere within the colony.Based upon the results from the primary testing, aspen, spalted aspen, sugar maple, and tree of heaven were ground on a Wiley Mill using both a 20 and 30 mesh screen. For the secondary testing, the sterilized wood was poured directly into an empty Petri plate until the plate was half-filled with wood. Autoclaved 2% malt agar was then poured onto the wood so that the agar just covered the bottom of the Petri plate.C. aeruginascens  were tested, including two that had been kept previously in refrigerated storage and were not producing xylindein on 2% malt agar plates . A two-point inoculation was used, and the same recording system as mentioned above was utilized, and data collection continued until colonies began to touch at 26 days. Three replicate plates were used per wood species per mesh size per isolate. A simple sugar analysis of test wood was conducted following the methods presented in Robinson et al.  as the independent variables. Follow-up testing on mesh-only plates was analyzed using a 4-way ANOVA, with wood species, wood amount (high or low), mesh size (20 or 30), and time as independent variables, and the diameter of the colony as the dependent variable. Tukey's HSD was then used to discern the location of the differences.Data analysis on the white colonies was done using a 3-way ANOVA, with wood species, wood amount (high or low), and mesh (20 or 30) as the independent variables. Time was not used as a variable, instead readings were based upon final colony color at 45 days incubation.For secondary testing, strains were compared using a 1-way ANOVA, followed by Tukey's HSD. Both UAMH strains were then combined into a single strain and run in a 4-way ANOVA, with strain, wood species, mesh size, and time as independent variables and colony diameter as the dependent variable. This test was followed by Tukey's HSD.Square section suspension plates did not differ significantly from the controls in terms of colony diameter at the two week evaluation point, while both the 20 mesh and 30 mesh were significantly different from the control (P < 0.0001), but not from each other.Within the follow-up 4-way ANOVA, mesh size was insignificant (P = 0.08). For the green colonies, wood species, time, and wood amount were highly significant P < 0.0001), as were their interactions. Within separate 1-way ANOVAs, spalted aspen (11.2 ± 4.9 mm) and sugar maple (10.7 ± 5.9 mm) plates had significantly more xylindein than the controls (7.5 ± 1.5 mm), while aspen (9.2 ± 3.3 mm) and tree of heaven (8.6 ± 3.4 mm) did not differ significantly from the controls (P = 0.0003). Plates with the high amount of wood did not have significantly larger green colonies diameters (10.8 ± 4.7 mm) than those with the low amount (9.1 ± 4.6 mm) (P = 0.0005), although both had significantly larger diameters than the controls (7.5 ± 1.5 mm). The interaction of wood species and time shows that compared to the control plates at the final measurement period (22 days), plates containing sugar maple (at 22 days and 19 days), spalted aspen (at 22 days and 19 days), aspen at 22 days, or tree of heaven at 22 days all contained significantly larger colonies (P < 0.0001) than any other time and wood species, including the controls  was insignificant, as was mesh size or wood species. All wood species significantly increased the number of green colonies as compared to the control (P = 0.0004) Figure .Secondary testing found a difference in colony diameter by strain, with the wild strain DT8315 having larger colonies (14.4 ± 6.8 mm) than either UAMH strain (7.1 ± 4.4 mm) (P < 0.0001), which were not significantly different from each other. Within the 4-way ANOVA, mesh size was not statistically significant. Fungus strain, wood species, and time were all highly significant (P < 0.0001), as were their interactions. Sugar maple (11.8 ± 7.1 mm), spalted aspen (10.7 ± 8.0 mm), and unspalted aspen (9.5 ± 5.5 mm) plates had significantly larger colonies compared to the control (7.6 ± 2.9 mm) regardless of time or strain. Colony size increased significantly with time, although there was no significant increase in colony size between days 19 and 22, days 12 and 15, and days 8 and 12.Chlorociboria DT8315 consistently outperformed the two UAMH strains. For the wild strain, all wood treatments resulted in significantly larger green colonies than the control (9.6 ± 2.6 mm), with sugar maple (17.6 ± 7.7 mm) and spalted aspen (17.0 ± 7.0 mm) producing the largest (P < 0.0001). With the UAMH strains, the controls (6.6 ± 2.6 mm) did not differ from tree of heaven, spalted aspen, or aspen; only sugar maple (8.9 ± 4.6 mm) caused a significant increase in colony diameter over the control. The interaction of strain and time also showed the wild strain generally producing larger colonies than the UAMH strains, although this interaction showed areas of overlap of similar size colony production based on time    and incrra (Fr.) . Howeverra (Fr.) . It is aC. aeruginascens requires a nutrient not found in malt agar media; however continued stimulation with increasing wood amounts suggests an additional unknown element at play. A similar phenomenon was found by French and Manion  Lloyd, Xylaria polymorpha (Pers.) Grev.), the ability to achieve full plate colonization and pigmentation of albino colonies will allow log and lumber scale use of C. aeruginascens as a spalting fungus to develop.The addition of sugar maple as an additive to agar-based growth media for The authors declare that they have no competing interests.


Increased level of serum S100B can serve as a marker of metastatic spread in patients with cutaneous melanoma (CM). In patients with elevated S100 B and/or clinical signs of disease progression PET-CT scan is a valuable tool for discovering metastases and planning treatment.The aims of this study were to determine whether regular measurements of serum S100B are a useful tool for discovering patients with CM metastases and to evaluate the diagnostic value of PET-CT during the follow-up.From September 2007 to February 2010, 115 CM patients included in regular follow up at the Institute of Oncology Ljubljana were appointed to PET-CT. There were 82 (71.3%) patients with clinical signs of disease progression and 33 (28.7%) asymptomatic patients with two subsequent elevated values of S100B. Sensitivity, specificity, positive and negative predictive value  of S100B and PET-CT were calculated using standard procedures.Disease progression was confirmed in 81.7% of patients . Sensitivity, specificity, PPV and NPV of S100B was 33.8%, 90.9%, 96.0% and 17.5% in patients with clinical signs of disease progression. In 20.0% of patients increased serum S100B was the only sign of disease progression. Sensitivity and PPV of S100 in this group of patients were 100.0% and 69.7%.With PET-CT disease progression was diagnosed in 84.2% of symptomatic patients and in 72.7% of asymptomatic patients with elevated S100B. The sensitivity, specificity, PPV and NPV of PET-CT for symptomatic patients was 98.5%, 90.9%, 98.5% and 90.9% and 100%, 90.0%, 95.8% and 100% for asymptomatic patients with elevated S100.Measurements of serum S100B during regular follow-up of patients with CM are a useful tool for discovering disease progression in asymptomatic patients. The value of its use increases if measurements are followed by extended whole body PET-CT. The prognosis of patients with cutaneous melanoma (CM) is determined by histology of the primary tumour and by the presence and extent of metastatic disease. Available laboratory, radiographic and nuclear medicine techniques are important components of patient evaluation but their usefulness is being constantly questioned by clinicians.During the last decade many CM associated blood compounds have been examined including melanoma associated antigens, cytokines, metalloproteinase, angiogenesis factor and others. Of all these compounds, S100 is at the moment the only widely applied CM biomarker . The terS100B, formed by two B or A and B subunits, is currently best-studied CM biomarker. Increased serum levels of S100B are associated with presence of metastases and are described to correlate with reduced survival. They reflect tumour load, stage and prognosis and are believed to be an independent poor prognostic factor in CM . FindingPET-CT is increasingly used for staging of melanoma patients. With its combination of metabolic and morphologic information, PET-CT has high accuracy for metastases and is superior to CT or MRI alone . SensitiIn Slovenia, a country with a population of two million, the prevalence of CM was 1302 male and 1920 female patients in December 2009. All patients with stage III and IV CM and a proportion of patients with stage I and II melanoma are engaged in regular follow-up at the Institute of Oncology Ljubljana, the only comprehensive cancer centre in the country.The aims of this study were to determine whether regular measurements of serum S100B are a useful tool for discovering patients with CM metastases and to evaluate the diagnostic value of subsequent PET-CT during the follow-up.From September 2007 to February 2010, 115 patients  with CM were directed to extended whole body PET-CT, based on clinical signs of recurrent disease, increased serum S100B or both. All patients were engaged in a regular follow-up at the Institute of Oncology Ljubljana after being diagnosed with CM. Clinical stage of the patients at the time of diagnosis of CM was classified according to American Joint Committee on Cancer (AJCC) criteria. Most patients were diagnosed with localized stage I and II of CM (21 or 18.3% and 47 or 40.9% respectively), but 40 (34.8%) of patients were stage III and 5 (4.3%) were stage IV at the time of diagnosis. For two patients (1.7%) stage of the disease at the time of diagnosis could not be assessed reliably.Initially 33 (28.7%) patients were treated only by wide excision, 39 (33.9%) patients with wide excision and sentinel node biopsy, while in 40 cases (34.8%) limphadenectomy was performed. There was a case of locally advanced anal melanoma treated with abdominoperineal resection, a case of isolated limb perfusion and a patient refusing all treatment after initial excision. Three patients diagnosed with stage IV disease received dacarbazine and two patients received only symptomatic treatment. All patients were followed at regular intervals, i.e. every 3 months in the first and second year after the diagnosis, every 6 months in the third to fifth year, and once a year after first 5 years. Patients were monitored at each visit for relapse by history, physical examination and laboratory tests including S100B. Disease progression was suspected based on clinical signs in 82 (71.3%) patients and based on increased S100B in 33 (28.7%) asymptomatic patients. There were 25 (21.7%) patients with both, increased S100B and clinical signs of advancing disease. In majority of patients  skin metastases were the first sign of progression. Twenty-four patients (20.9%) had palpable lymph nodes, 2.6% complained of persistent cough (3/115), 9.6% (11/115) of pain and in two cases (1.7%) mild gastro intestinal bleeding was present. In two patients, accidental radiological findings could not be ruled out as CM metastases by treating physicians. Mean time at which PET-CT followed measurement of serum S100B was 21.19 days.All patients were informed about the research protocol and signed a consent form. The study was reviewed and approved by institutional Ethics Committee.Serum concentrations of S100B (heterodimers S100 A1B and S100 BB) were determined by the use of the electrochemiluminescence immunoassay (ECLIA). The immunoassays were analyzed on the Modular Analytics E170 immunoassay analyzer (Roche Diagnostics). Reference values were obtained from apparently healthy volunteers: 0.105 μg/L was determined as the upper normal value (Roche Diagnostics). In cases of increased tumour marker values, sampling and determination of the serum S100B were repeated for confirmation after two weeks. Only patients with elevated repeated values were appointed to subsequent PET-CT scan.Extended whole body PET-CT scan was performed in cases where distant metastases were suspected due to elevated serum S100B or due to clinical signs of the disease. The scans were performed after a six-hour fast and when the patients were prepared according to recommendations (EANM guidelines) . A hybriThe McNemar symmetry chi square test was used to compare performance of the S100B with that of PET-CT. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of S100B and PET-CT were calculated using standard procedures. Separate analyses were made for patients with no clinical signs of the disease and for patients with CM stage I and II at the time of diagnosis. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) for Windows version 15.0.115 patients suspected of disease progression based on clinical signs  and/or increased levels of serum S100B  were appointed to extended whole body PET-CT at the Institute of Oncology Ljubljana from 2007 until the beginning of 2010. The mean age of the patients at the time PET-CT was performed was 60.8 (ranged from 16.3 to 86.8) years. The group comprised of 39.1% female and of 60.9% male patients.Out of 115 patients, disease progression was diagnosed in 94 (81.7%) patients. Metastases were confirmed by fine needle aspiration cytology in 34 (36.2%), with CT or MRI in 11 cases (11.7%), with ultrasound or radiological finding in 11 cases (11.7%) and based on the histology report in 38 cases (40.4%) prior to implementation of appropriate treatment.As far as the rest of the patients are concerned, there were 3 patients with palpable lymph nodes, 6 patients with pain and 2 patients with suspicious radiological findings, however the metastases could not be confirmed. Altogether 21 patients proved to be disease free at that time (18.3%).The majority of patients with skin metastases (distant and in transit included) had normal level of S100B. Patients with lymph node metastases had increased S100B in 50.0% of cases compared to 57.1% of patients with distant visceral metastases . The majority of these patients did not have increased values of serum S100B . In eleven patients presented with clinical signs, metastases were not discovered.Symptomatic patients had elevated S100B levels in 30.5% of cases (25/82). The mean value of S100B marker in that group of patients was 0.603 μg/L . For symptomatic patients without increased S100B, the mean value of serum marker was 0.051 μg/L . In 33 patients without clinical signs of disease progression, the mean value of S100B was 0.652 μg/L .In 33 patients with no symptoms but with an increased S100B, metastatic disease was confirmed in twenty three patients (69.7%) and 10 patients were healthy. Based solely on increased S100B, metastases were discovered in 20.0% (23/115) of all cases.2 value for McNemar test comparing S100B and PET-CT was 21.9 with p < 0.0001 respectively.The PET-CT pointed out suspicious lesions in 95/115 (82.6%) patients. In 20 (17.4%) patients PET-CT was negative. False positive results with SUV 15.2 and 8.0 were obtained in two patients. Metastases were actually confirmed in 94 (81.7%) out of 115 patients. The χPatients with normal S100B level but suspicious clinical signs, had metastases diagnosed with PET-CT in 84.2% of cases (48/57). Disease progression was later actually confirmed in 47 of those patients. In 9 cases PET-CT was negative and patients were proved to be disease free.If patients had increased S100B level , PET-CT diagnosed suspicious lesions in 47 (81.0%) cases. In 11 cases with increased S100B, PET-CT was negative. In only one of those cases metastases were present later on follow-up. Out of 33 asymptomatic patients with increased S100B suspicious lesions were diagnosed in 72.7% of cases.Based on those results 98.9% sensitivity, 90.4% specificity, 97.8% PPV and 95.0% NPV of PET-CT was calculated for the whole group of patients Table .Analysis was repeated for 68 patients with localized disease (stage I or II) at the time of the first visit. In 54/68 patients disease progression was confirmed during the follow-up. Among 48 patients with clinical signs of disease progression, there were 15 (31.2%) individuals where S100B was also increased. In majority of patients with clinical signs, metastases were confirmed .Table Since the prognosis of patients with CM is determined by the extent of metastatic disease, serum biomarker and imaging studies using radiographic and nuclear medicine techniques are an important component of patient evaluation .Almost 10 years ago researchers suggested that measuring S100B after primary local surgical therapy may allow early recognition of disease progression. It was also suggested that an increasing S100B, followed by a positive emission tomography (PET) scan and appropriate treatment, could lead to an increased life expectancy of patients with CM . DespiteWe analyzed a group of 115 patients appointed to PET-CT due to increased serum S100B value (33 pts) or clinical signs of disease progression encountered during regular follow-up (82 pts). In our study patients with stage I or II of CM had 51.8% sensitivity and 50.0% specificity of S100B. PPV of S100B for this group of patients was 80.0% and NPV was only 21.2%. A similar study conducted in 2002 showed sensitivity between 3% and 9% for stage I and II. The study described a PPV of 17% for stage I and 37% for stage II and specificity was 92%. Based on that, the authors concluded that S100B could not be a reliable marker of disease dissemination at early stages [The sensitivity of increased serum S100B for the whole group of patients 115 pts) regardless of the stage of the disease in our study was 50.0%, specificity 47.6%, PPV 81.0% and NPV 17.5%. In studies which included patients with clinical symptoms being either present or absent the sensitivity was 38% with PPV 93% and 37% respectively 5 pts reg.It has been observed by other authors that location of the metastases influences the level of S100B. Authors report that between 41 to 68% of patients with stage III disease do not have an increased serum marker ,13-15. IComparing increased levels of serum S100B from different studies has one major obstacle, namely researchers are using different immunoassays with slightly different cut off values of serum S100B. Different assays used in clinical practice were compared and it was determined that the correlation among assays is satisfactory with correlation index of 0.9 or higher for most assays. Nevertheless, data from the assays using bovine brain solution and those standardized against human recombinant S100B can not be superimposed. Difficulties are also encountered comparing upper cut off values ranging from 0.09 to 0.13 μg/l, which indicates the need for international agreement regarding the use of uniform reference standards for successful comparison of different studies .All patients in our study were appointed to extended whole body PET-CT. Eleven patients 11/115, 9.5%) had increased level of S100B, but no metastases were proven on PET-CT or by clinical follow up. The number of reported false positive results of S100B in other studies ranges from 17 to 41% of cases [, 9.5% haThe main limitation of our study is that patients were appointed to PET-CT based on the increased S100B or clinical suspicion of disease progression which represents a selection bias influencing NPV and specificity of S100B. Published retrospective studies also describe groups of patients inspected with PET-CT due to increased S100B and report similar bias . Our groThe sensitivity of PET-CT in our study was 98.9% with 90.4% specificity for the whole group of patients and 100% with 90.0% specificity if clinical signs were absent. The accompanying PPV was 97.8% in the first group and 95.8% in the subgroup of patients. Aukema reported a sensitivity of 100%, specificity of 83%, PPV of 85% and NPV of 100% . The rolDue to short follow-up, we did not investigate how the outcome of regular measurements of serum S100B and subsequent PET-CT influences overall survival of our patients. It is known that increased S100B levels are related to prognosis and survival of patients with CM. A meta-analysis of twenty-two articles published until 2008 proved S100B as an independent prognostic factor of survival with special regard to stage I-III patients . In advaWe confirmed metastases in 20.0% of patients based on the increased serum S100B as the only preceding sign of disease progression (23/115). Since the analysis of serum S100B showed the highest sensitivity and specificity of all tumour markers examined in CM and it is fairly inexpensive, numerous clinicians include its measurement in routine follow up of CM patients ,19. In tIf there is an actual benefit of carefully planned follow-up schedules for CM patients remains to be elucidated. A study conducted by German researchers proved a gain in survival time for the detection of metastasis at an early phase of development beyond the effect of lead time bias. Namely the 10-year overall survival probability was increased significantly for patients with early phase metastases .Measurements of serum S100B during regular follow-up of patients with CM are a useful tool for discovering disease progression in asymptomatic patients. This applies equally to patients with localized disease (stage I and II) as to patients with stage III or IV. The value of its use increases if measurements are followed by extended whole body PET-CT. If the approach described in our study has any influence on patients' survival will be validated in the future.The authors declare that they have no competing interests.BP participated in patient recruitment and data set up, performed the statistical analysis and drafted the manuscript. IZ interpreted the PET-CT scans and helped to draft the manuscript. SN participated in the design of the study and helped to draft the manuscript. JZ participated in patient recruitment, study design and helped to draft the manuscript. MH conceived of the study, participated in its design, coordination and patient recruitment and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/328/prepub


Dear Editor,Proximal femoral fracture (PFF) is one of the most important etiologies of mortality and morbidity in elderly. Reported mortality rate is in a range of 1.3 - 16% pre-operatively up to 22 - 42% postoperatively . One of Primary surgical goal is retaining functional position after reduction of fracture and preventing avascular necrosis (AVN) of femoral neck, but it should be considered that reduction of mortality rate and movement capability is more important .We studied records of patients who were hospitalized for PFF between March 2007 and March 2009 in Rasoul-Akram hospital . According to BMI, patients were classified in four groups; including group 1 (BMI<20), group 2 (20≤ BMI < 25), group 3 (26≤BMI<29) and group 4 (BMI ≥ 30). Mobility of patients before and after fracture was compared. Three levels of movement were considered for these patients: complete movement, movement with crutch and immobility. Data were entered in a check list and analyzed using SPSS 16. Relationship between mobility (ability to move by feet) and BMI in these groups were calculated by Chi-square analysis. Among 94 patients, 51 (54.7%) were male and 43 (46%) were female. Average age of the patients was 48.4 ± 7.02 . 16 patients had BMI less than 20, 35 patients had BMI between 20 - 25  and 16 patients had BMI > 29.Three different kinds of fractures were evaluated: femoral neck, intertrochanteric and sub trochanteric.Hospitalization period was recorded for each patient. Initiation of therapy four days after fracture is considered as a therapeutic delay which was observed in 68 patients (30%). Average therapeutic delay was 5.5 ± 4 days (at least 1 day and at most 17 days). Mortality rate among these groups were compared to each other, and qualitative analysis revealed no significant correlation .In previous studies, there was less emphasis on BMI as a probable risk factor, and its effect on mobility after treatment was not well punctuated.It has been shown that mortality rate of patients with very low BMI in PFF is increased and this may be due to cardiovascular complications . In our


Constipation is one of the most common gastrointestinal disorders particularly in industrialized countries. Incidence of constipation varies from 3.4 % to 27.2% in different societies. Increase in urban population, industrialization of communities, changes in behavioral and nutritional habits and inactivity have increased the number of patients suffering from constipation.The aim of the study was to measure colonic transit time in patients with chronic idiopathic constipation.Fifty-two patients with chronic idiopathic constipation (according to ROME III criteria) were selected. Patients with diabetes mellitus, hypothyroidism, and hypoparathyroidism were excluded. Each patient took a capsule containing ten 1-3 mm long angiographic guide daily for 6 days. Abdominal x-ray was performed on the seventh day. Remaining markers in each segment were counted and segmental and total colonic transit time was calculated. The analysis was performed by SPSS version 18. In all tests, a P-value less than 0.05 was considered statistically significant.The mean age of patients was 36.9 ± 10.1 years. 80.8 % of patients were female. The mean total colonic transit time was 40.8 ±35.1 hours. 34.6 % of patients and 36.5 % of them had prolonged total and segmental colonic transit time.We found that prolonged total and segmental colonic transit time is common in patients with chronic idiopathic constipation. Constipation is one of the most common gastrointestinal disorders particularly in industrial countries , 2. InciIdiopathic constipation is divided into three subtypes: 1) Constipation with normal colonic transit, which is often associates with psychosocial abnormalities , 18; 2) Other diagnostic evaluations include colonoscopy, barium enema, and evaluation of colonic transit time (CTT) by marker and anorectal manometer -23. EvalWe aimed to distinguish subtypes of chronic idiopathic constipation by evaluating the colonic transit time, so that the appropriate treatment would be assigned to each patient.In this cross-sectional study, we evaluated patients with constipation that were referred to the gastrointestinal and surgical clinics of Imam Khomeini hospital complex, Tehran, Iran. Patients with the history of known diabetes mellitus, hyperparathyroidism, hypoparathyroidism, and hypothyroidism were excluded. Patients were included according to ROME III criteria Although CTT could be performed in different ways, we chose the Arhan method because of single X-ray exposure and no need for repeated referal to the radiology department. In this method, the patients were given capsules containing ten 1-3 millimeter-long marker made of angiographic catheter. Patients should not have used laxatives during the week before CTT study. They took a capsule at 9 am for six consecutive days and came for taking abdominal X-ray in the 7th day morning.All the plain abdominal X-rays were evaluated by a single radiologistand the number of markers in each segment  was counted as shown in Total, right, left and rectosigmoid colonic transit time was computed using this formula:N= number of available markers in each segment or totallyThe data was then analyzed using the statistical software SPSS 18.0.0. . The t test was used to compare two groups and P-value lower than 0.05 was considered significant.Fifty-two patients fulfilled the inclusion criteria. The mean age of patients was 36.9 ± 10.1 years. (Range: 18-66 years). Forty-two patients (80.8 %) were female. We also found that 28 patients (53.8%) had history of chronic constipation for more than 3 years. In this study, the mean total colonic transit time was 40.8±35.1 hours while in rectosigmoid, ascending colon and descending colon, it was 15.5 ± 12.4 , 14.7 ± 10.8 and 12.9 ± 9.5 hours, respectively . In 18 pFrom 34 patients with normal total colonic transit time (13), 19 patients (36.5 %) had abnormal segmental transit time (≥ 15 hours). among these patients, 10 had prolonged right colonic transit time, while 6 and 3 patients had prolonged rectosigmoid and left colonic transit time, respectively.The mean total colonic transit time was 42 ± 35.4 and 35.2 ± 32.1 hours in women and men, respectively, with no statistically significant difference between them (P = 0.57). The mean total colonic transit time was highest in patients older than 50 years and was lowest in patients less than 30 years old . Regarding the mean total colonic transit time, no significant difference was found among different age groups and pair comparing (P > 0.05). there was no statistically significant difference with regard to the mean total colonic transit time between patients with less or equal to three years history of constipation (38.6 ± 17.08 hours) and those with more than three years history of constipation (P = 0.67).To put in a nutshell, the aim of this study was to evaluate colonic transit time in patients with chronic idiopathic constipation.In our study, among 52 patients who entered the study, 18 (34.6%) had prolonged total colonic transit time. In similar studies, the frequency was different in patients with idiopathic constipation . In a stLopes et al.  found thWith regard to longer mean transit time in right colon, subtotal surgery could be more helpful in the case of resistance to medical treatment. The mean total colonic transit time was higher in women in our study but this was not statistically significant (P ≥ 0.05). Danquechin et al. , measureIn conclusion, abnormal total and segmental colonic transit time is common in patients with chronic idiopathic constipation; hence, in the case of inappropriate response to medical treatment, subtotal colectomy would be helpful.


The mean ± Standard Deviation (SD) serum ferritin level was 452.4 ± 312.60 μg/L. Mean ± SD hemoglobin concentration was 9 ± 1 g/dl. Short stature was present in 46% of patients. Growth hormone deficiency was one of the most frequent (31%) endocrine abnormalities in these patients. Primary hypothyroidism was observed in 21.5% of patients. Hypoparathyroidism was found in one patient (1%). Failure of puberty was present in 2% of patients, secondary ammenorrhea was observed in 6.4% of patients and diabetes mellitus (DM) in 2% of patients. Conclusion: Growth retardation and GH deficiency should be considered as common finding in TI. Therefore endocrine evaluation of these patients is suggested to prevent complications and to improve the overall quality of life.Thalassemias are the most common genetic disorder on a wordwide basis. β-thalassemia is a severe hemolytic anemia which results from genetic defects in the synthesis of the hemoglobin β-chain. Various endocrine abnormalities have been described in patients with thalassemia major. Endocrine disturbances have also been observed in patients with thalassemia intermedia (TI). In this study endocrine functions were investigated in TI and here the frequency of different abnormalities is reported. Ninety-three patients  with thalassemia intermedia, 11-40 years old (mean 19.4 yr) were studied. Medical history was obtained and a complete physical examination was done for each patient. The age, sex, weight, height and serum ferritin levels were recorded using a questionnaire. Growth Hormone (GH) secretion, thyrotropin (TSH), T Thalassemias are the most common genetic disorder on a worldwide basis , β-thalaVarious endocrine abnormalities have been described in patients with thalassemia major and β-thalassemia intermedia , 5 and m++), phosphorus and blood sugar were measured. Blood samples were taken at least 2 weeks after the last blood transfusion. Serum ferritin level was determined by Elisa method. Serum cortisol and T4 were measured by Radioimmune Assay (RIA) standard commercial kit  and TSH by Immunoradiometric Assay (IRMA) with standard commercial kit . The GH secretion was determined after provocative L-Dopa tests with levodopa (500 mg/1.73m2) primed by propranolol 0.75 mg/kg, maximum 40 mg orally. Blood samples were taken at 0 (baseline), 30, 60, 90 and 120 minutes after an overnight fasting and GH was measured by IRMA with standard commercial kit . Girls and boys over 11 and 13 years of age received ethinyl estradiol (0.02 mg) and methyl testosterone 10 mg respectively three times a day for 2 days before L-Dopa test. Growth hormone deficiency was diagnosed if GH level in all sample was below 10 ng/ml.Ninety-three patients  with thalassernia intermedia, aged 11-40 years old (mean age 19.45 ± 5.40 yr) were selected out of 220 TI patients randomly in this study. Medical history was obtained and a complete physical examination was done for each patient. Age, sex, weight and height, age of initiation of blood transfusion and age of initiation of desferrioxamine, its regular or irregular administration were recorded using a questionnaire. Height was measured by a single observer using a stadiometer. Pubertal staging was performed by the same observer and was assessed according to Tanner staging .Serum PTH was determined in those patients whose serum Ca++ below 8 mg/dl and phosphorus over 6.5 mg/d1.Hypoparathyroidism was diagnosed if PTH level was below 13 Pg/ml  in patients with a serum Ca4 was < 4.5 μg/dl  and TSII >4 mlU/1 (0.3-3.8 mIU/1). Compensated hypothyroidism  defined as an elevated serum TSH level but normal concentration of T4 .A total of 93 β-thalassemia intermedia patients were studied. The age of the patients ranged from 11-40 years (mean 19.4 ± 5.40). Characteristics of patients data are summerized in Table P=0.9). There was no significant difference between height of patients with GH deficient and GH sufficient (P=0.3).Short stature was found in 46% of patients defined by height below the 3rd percentile for age. Short stature was found in 43% of patients with multiple transfusions and 51% of patients without transfusions. This difference was not statistically significant (P=0.6).Table P=0.03).The mean age of patients with GH sufficiency was 18.7 ± 5.5 years and in patients with GH deficiency was 21 ± 4.9 years, the difference was statistically significant (P=1).Compensated hypothyroidism was present in 19% of patients and primary hypothyroidism in 2% of patients. Hypothyroidism in patients with blood transfusion was 22% and in patients without transfusion was 21% and the difference was not significant statistically (Hypoparathyroidism was present in only one patient (1%) , who had history of blood transfusion. No adrenal insufficiency was found in these patients. The mean serum cortisol was 15.8 ± 4.8μg/dl.P=0.5).Diabetes mellitus was found in 2% of patients with the mean age of 21 years. Diabetes mellitus in patients with blood transfusion was 3.6% and there was no DM in patients without blood transfusion (P=0.3).Failure of puberty was present in 2% of the patients. Secondary amenorrhea was present in 6.4% of the patients. The mean serum ferritin level in patients with secondary amenorrhea was 503.6 ± 284 μg/l and in patients without amenorrhea was 393.5 ± 287.2 μg/l (P<0.001).Table P=0.6).Table This study was designed to evaluate the endocrine complications in patients with TI from Iran. Iron overload and secondary hemochromatosis have been described in several types of hemolytic anemias including TI , 6. Thalet al. . De Sanctis et al.  reportedGrowth Hormone deficiency was common in the present report. There were no relationship between GH deficiency and serum ferritin level. To the best of our knowledge there is no study about GH deficiency in TI patients. Growth hormone deficiency seems to be a frequent complication of TI in patients treated according to current protocols. Forty-one percent of the patients had at least one endocrine dysfunction and there were no relationship between endocrine dysfunction and serum ferritin level, age of start of desferrioxamine and hemoglubin level. De Sanctis  reportedIn the study of De-Sanctis, hypothyroidism detected in 5.7% of patients. The difference in incidence of hypothyroidism noted is probably related to differences in transfusion.None of the De-Sanctis patients had hypoparathyroidism. The prevalence of delayed puberty in the present series was very lower than those reported .Lack of concordance of ferritin concentrations and endocrine dysfunction is in part due to the fact that serum ferritin levels increase linearly with the transfusion load up to 100 units of transfused blood, but thereafter there is no simple relationship. Misleading ferritin levels also occur with chronic inflammatory liver diseases . There iAbnormal glucose tolerance test (OGTT) was found in 24% of patients in De Sanctis study . The thaWe concluded that growth retardation and GH deficiency should be considered as common finding in TI. Therefore endocrine evaluation of these patients is suggested to prevent complications and to improve the overall quality of life.