Quillaja saponin mitigates methotrexate-provoked renal injury; insight into Nrf-2/Keap-1 pathway modulation with suppression of oxidative stress and inflammation.

BACKGROUND: Methotrexate (MTX) is an antineoplastic/immunosuppressive drug, whose clinical use is impeded owing to its serious adverse effects; one of which is acute kidney injury (AKI). Most of MTX complications emerged from the provoked pro-oxidant-, pro-inflammatory- and pro-apoptotic effects. Quillaja saponaria bark saponin (QBS) is a bioactive triterpene that has been traditionally used as an antitussive, anti-inflammatory supplement, and to boost the immune system due to its potent antioxidant- and anti-inflammatory activities. However, the protective/therapeutic potential of QBS against AKI has not been previously evaluated. This study aimed to assess the modulatory effect of QBS on MTX-induced reno-toxicity. METHODS: Thirty-two male rats were divided into 4-groups. Control rats received oral saline (group-I). In group-II, rats administered QBS orally for 10-days. In group-III, rats were injected with single i.p. MTX (20 mg/kg) on day-5. Rats in group-IV received QBS and MTX. Serum BUN/creatinine levels were measured, as kidney-damage-indicating biomarkers. Renal malondialdehyde (MDA), reduced-glutathione (GSH) and nitric-oxide (NOx) were determined, as oxidative-stress indices. Renal expression of TNF-α protein and Nrf-2/Keap-1 mRNAs were evaluated as regulators of inflammation. Renal Bcl-2/cleaved caspase-3 immunoreactivities were evaluated as apoptosis indicators. RESULTS: Exaggerated kidney injury upon MTX treatment was evidenced histologically and biochemically. QBS attenuated MTX-mediated renal degeneration, oxidant-burden enhancement, excessive inflammation, and proapoptotic induction. Histopathological analysis further confirmed the reno-protective microenvironment rendered by QBS. CONCLUSIONS: In conclusion, our results suggest the prophylactic and/or therapeutic effects of QBS in treating MTX-induced AKI. Such reno-protection is most-likely mediated via Nrf-2 induction that interferes with oxidant load, inflammatory pathways, and proapoptotic signaling.

Effect of intracorneal ring segments on posterior corneal topography in eyes with keratoconus.

PURPOSE: To analyze the changes in the anterior and posterior corneal surfaces measured with a Scheimpflug imaging device in keratoconus patients implanted with intracorneal ring segments (ICRS) and to correlate those changes with the visual outcomes. METHODS: This prospective interventional case series study included 92 eyes of 60 patients with keratoconus who underwent Kerarings (Mediphacos, Belo Horizonte, Brazil) ICRS implantation. Keratometric (K) readings, corneal asphericity (Q value) and elevations of both anterior and posterior corneal surfaces were evaluated using a Scheimpflug imaging device preoperatively and at 1, 3, 6 and 12 months after surgery. RESULTS: The mean best corrected visual acuity (BCVA) improved from 0.61 to 0.19 logMAR at 12 months after surgery. Both anterior and posterior corneal surfaces showed significant flattening with statistically significant reduction of the mean anterior K reading by 3.39 D and the mean posterior K reading by 0.39 D (p<0.001) at 12 months. Statistically significant change of the anterior Q to a less negative value (from -1.05 to -0.36) was observed (p<0.001) with no significant change of the posterior Q value. Improvement of the anterior Q was significantly correlated to better postoperative BCVA (p=0.03). Better postoperative BCVA significantly correlated to better preoperative BCVA, flatter preoperative anterior and posterior K, less prolate anterior Q value and lower anterior elevations. CONCLUSIONS: ICRS implantation has a significant flattening effect on both anterior and posterior corneal surfaces. Improvement of corneal asphericity is correlated to better visual outcome. Certain preoperative parameters were predictive factors of the postoperative visual improvement.

The role of platelet-rich plasma in microlaryngeal surgery: a randomised, controlled trial.

OBJECTIVE: Platelet-rich plasma has gained interest over the two last decades, mainly because of its role in regenerative medicine. This work aimed to assess the role of intra-operative local application of platelet-rich plasma gel in the improvement of quality of voice after microlaryngeal surgery. METHOD: This was a prospective comparative study that included 40 patients undergoing microlaryngeal surgery for benign vocal fold lesions. There were two groups divided equally into study group A and control group B. The assessment of voice was performed by videostroboscopy and acoustic analysis pre-operatively and at two weeks and one and three months post-operatively. RESULTS: The data demonstrated that all the stroboscopic and acoustic parameters showed significant improvement in both groups. Group A showed significant improvement regarding acoustic parameters at the third post-operative follow up when compared with group B. CONCLUSION: Platelet-rich plasma has a beneficial effect on voice quality following microlaryngeal surgery based in particular on acoustic parameters.

High intensity interval training exercise as a novel protocol for cardiac rehabilitation program in ischemic Egyptian patients with mild left ventricular dysfunction.

BACKGROUND: Exercise-based Cardiac rehabilitation (CR) plays a major role in reducing mortality and morbidity in patients with coronary artery disease (CAD). The standard protocol is usually of moderate intensity exercise. High-intensity interval training (HIIT) consists of alternating periods of intensive aerobic exercise with periods of passive or active moderate/mild intensity recovery. AIM: This study aimed to assess HIIT program for ischemic patients attending CR after percutaneous coronary intervention (PCI) who have mild left ventricular dysfunction and to compare its effect on the functional capacity and quality of life with standard exercise CR program. PATIENTS AND METHODS: Our study included 40 patients with documented CAD, who participated in the outpatient CR program in Ain Shams University hospital (Al-Demerdash Hospital) divided into two equal groups, each included 20 patients. Group A included the patients who underwent standard cardiac rehabilitation program, while group B joined the high intensity interval training exercise protocol. RESULTS: Groups A and B showed significant improvement in all items of comparison; especially functional capacity, lipid profile and quality of life. Group B showed better improvements in the emotional well-being items of QOL parameters. CONCLUSION: We emphasize the positive effects of exercise-based CR program on patients with CAD and mild left ventricular dysfunction after PCI. The novel high intensity cardiac training proved to be safe and at least as beneficial as the standard moderate intensity cardiac training protocols, with better quality of life improvement.

Selection of DNA barcoding loci for Nepeta deflersiana Schweinf. ex Hedge from chloroplast and nuclear DNA genomes.

Molecular markers, mainly DNA-based are potential tools for DNA barcoding and phylogenetic study. The plant species belonging to the Nepeta genus have important medicinal value because of the presence of nepetalactones, and they have been used to treat human diseases. We amplified nuclear and chloroplast gene loci to develop a DNA barcode and phylogenetic study of Nepeta deflersiana. Among the studied loci, psbA-trnH and rps16 showed less identity within the genus than the other loci using the Basic Local Alignment Search Tool of the National Center for Biotechnology Information GenBank database. These loci can be used for the development of a DNA barcode to identify and preserve the identity of this species. We also constructed the phylogram of N. deflersiana and other Nepeta species retrieved from the GenBank database (nonredundant DNA-internal transcribed spacer). N. deflersiana was placed in the same clade as N. insaurica with a 99% bootstrap value.

Bronchoscopy versus multi-detector computed tomography in the diagnosis of congenital vascular ring.

BACKGROUND: Vascular rings are congenital vascular anomalies of the aortic arch complex which cause compression of the trachea and/or oesophagus. A variety of investigations may lead to diagnosis of these anomalies, including bronchoscopy and computed tomography. During the latter, image acquisition and processing use the multi-detector row technique and new reconstruction algorithms, producing high-resolution images which can be visualised as complex, three-dimensional renditions. OBJECTIVE: This study aimed to assess and compare the roles of bronchoscopy and multi-detector row computed tomography in the diagnosis of congenital vascular ring. PATIENTS AND METHODS: We included infants and children below the age of 16 years who presented with congenital vascular ring. All patients underwent rigid bronchoscopy under general anaesthesia, with spontaneous respiration. All computed tomography scans were obtained using a 16 multi-detector row computed tomography system, followed by data reconstruction on a three-dimensional workstation. RESULTS: Ten patients with congenital vascular ring were identified (six boys and four girls). Fifty per cent of cases presented within the first year of life. Double aortic arch was the most common anomaly (40 per cent). Bronchoscopy detected external tracheal compression in nine cases (90 per cent). Associated airway lesions were detected endoscopically in three cases. In contrast, multi-detector row computed tomography detected the vascular ring in all cases, with accurate detection of the compressing vessel; however, it did not detect any associated airway lesions. CONCLUSION: Bronchoscopy and radiological evaluation are essential for the diagnosis and pre-operative evaluation of congenital vascular ring. Multi-detector row computed tomography can provide more information about the nature of the disease, and can facilitate better communication between clinicians, compared with conventional computed tomography.

[Plasmacytoid urothelial carcinoma of the urinary bladder. A study of 7 cases]. / Carcinoma urotelial plasmocitoide de vejiga urinaria. Estudio de 7 casos.

OBJECTIVE: Plasmacytoid urothelial carcinoma is a rare and aggressive variant of bladder cancer that mimics plasmacytoma histologically and that can be confused with hemolymphoid neoplasms secondarily affecting the urinary bladder. Only single cases and short series have been described so far. PATIENTS AND METHODS: Seven cases of plasmacytoid urothelial carcinoma have been found among 720 high grade urothelial carcinomas of the urinary bladder. RESULTS: In our series, 0.97% of high grade urothelial carcinomas of the urinary bladder show plasmacytoid phenotype. All the cases were smoking males between 58 and 75 years old. Histologically, two cases showed pure plasmacytoid features, while in the other five cases the plasmacytoid phenotype was mixed with conventional transitional cell or glandular histologies. By immunohistochemistry, all the plasmacytoid areas showed fair epithelial differentiation. The clinical behaviour was aggressive in all the cases, with distant metastases at diagnosis in three cases and early tumor recurrence after chemotherapy in four of them. CONCLUSIONS: In our experience, the plasmacytoid urothelial carcinoma of the urinary bladder is a rare tumor that can also be detected in association with areas of conventional urothelial carcinoma. It is mandatory to recognize this histological subtype due to the clinical and prognostic implications of this diagnosis.

Laryngotracheal augmentation using titanium mesh.

BACKGROUND: The management of laryngotracheal stenosis is still a serious surgical challenge. The fact that there are currently numerous reconstruction procedures indicates that there is at present no standard treatment. STUDY DESIGN: Titanium mesh was used instead of traditional homografts in reconstruction of the anterior laryngotracheal wall in 12 tracheostomised patients with benign chronic laryngotracheal stenosis. The anterior laryngotracheal wall was split, followed by excision of scar tissue and fixation of the titanium plate at the split end. A Silastic stent was inserted above the tracheostomy tube and fixed in place by running sutures fixed to the skin by buttons. The stent was removed endoscopically six weeks later and a trial of decannulation was undertaken. RESULTS: Endoscopically, good epithelisation was seen on the inner surface of the mesh in 10 cases and decannulation was possible. Four of these patients required endoscopic debulking of granulation tissue. Decannulation was impossible in two cases, one due to excessive granulation tissue and the other due to prolapse of the titanium mesh into the tracheal lumen (the mesh was removed endoscopically and a Montgomery T-tube inserted). CONCLUSION: Titanium mesh was found to be a good alternative for augmentation of the anterior laryngotracheal wall. It offered rigid support, with fewer of the complications reported with other grafts.

[Clinical and epidemiological study of disease caused by Mycobacterium kansasii in the metropolitan area of Bilbao, Spain]. / Estudio clinicoepidemiológico de la enfermedad por Mycobacterium kansasii en el área urbana de Bilbao.

OBJECTIVE: Epidemiological description of individuals from whom Mycobacterium kansasii isolates were obtained in respiratory samples, and analysis of the isolates using molecular biological techniques. MATERIAL AND METHODS: A descriptive retrospective/ prospective study was carried out from January 1994 to April 2002 in Basurto Hospital and Santa Marina Hospital and from January 2000 to April 2002 in Cruces Hospital, Galdakao Hospital, and San Eloy Hospital. Diagnosis of the disease was performed according to American Thoracic Society criteria; other definitions were also applied to allow inclusion of all cases. Disease caused by M. kansasii in patients who were not infected with the human immunodeficiency virus (HIV) was compared with disease caused by Mycobacterium tuberculosis in a control group. Polymerase chain reaction was applied with analysis of restriction fragment length polymorphisms to differentiate between species of mycobacteria and classify them into genotypes. Amplified fragment length polymorphisms were used to recognize clones within each genotype. RESULTS: The patient charts of 334 patients in which an isolate of M. kansasii had been recorded were reviewed. We considered 220 patients to be suffering from disease caused by M. kansasii (American Thoracic Society criteria along with probable disease according to established definitions). The disease was more frequent in male patients (n=185; 84.1%) and in individuals who were not HIV positive (n=184; 83.6%). The highest incidence of disease in the Bizkaia region was found in Margen Izquierda-Encartaciones, where the rate was 8.05 per 100 000 inhabitants. In the Bilbao area, the highest rate was found in the districts lying on the outskirts. The underlying diseases were tuberculosis (20.5%), chronic obstructive pulmonary disease (25.9%), pulmonary neoplasia (7.7%), silicosis (0.9%), chronic liver disease (11.4%), and duodenal ulcer (8.6%). The most frequent constitutional symptoms were fever (39.1%), loss of appetite (23.2%), and weight loss (33.3%). Among the respiratory symptoms, the most outstanding were cough (70.9%) and expectoration (62.3%). The most frequent radiographic patterns were cavitation and pulmonary infiltration. The most common treatment regimen was rifampicin, isoniazid, and ethambutol (43.4%), and the average duration was 12 months in patients who were HIV negative. Analysis of antibiotic sensitivity, performed on 56 strains, revealed that 100% were resistant to isoniazid, while none displayed rifampicin resistance. Thirty-four cases of disease caused by M. kansasii were compared with 68 cases of tuberculosis, all of them without HIV infection. The comparison revealed a predominance of smokers, respiratory symptoms, and cavitation in patients with disease caused by M. kansasii. The majority of the isolates (98.5%) corresponded to genotype I. A total of 8 clones were obtained; the clones designated 1 and 3 were more common in HIV-positive and HIV-negative individuals respectively. CONCLUSIONS: In recent years, there has been an increase in the number of patients with disease caused by M. kansasii in the province of Bizkaia. The disease is more frequent in male patients, individuals who are HIV negative, and in urban areas. In addition, more respiratory symptoms and a higher incidence of cavitation were found in patients with disease caused by M. kansasii than in those with tuberculosis. Genotype I is the most common isolate, and clones 1 and 3 affect 80% of patients suffering from the disease.

Role of some vasoactive mediators in patients with erectile dysfunction: their relationship with angiotensin-converting enzyme and growth hormone.

The imbalance between vasoconstrictors and vasodilators may play an important role in the pathogenesis of erectile dysfunction (ED). A total of 36 patients with ED, organogenic [diabetic (n=12) and nondiabetic (n=12)] and psychogenic (n=12) etiology, and 12 healthy adult men as controls were included. The levels of endothelin-1 (ET-1), growth hormone (GH), angiotensin-converting enzyme activity (ACE), nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) were determined in the flaccid penis cavernosal blood of patients and in cubital blood of patients and controls. In psychogenic ED, systemic ACE activity was elevated compared to controls (P<0.05). In diabetic and nondiabetic ED patients, systemic levels of ET-1 (P<0.0001 for both) and ACE activity (P<0.01 and <0.05) were higher while GH (P<0.0001 and <0.001), NO (P<0.0001 for both) and cGMP (P<0.01 for both) levels were lower compared to controls. In diabetic patients, systemic and cavernosal ET-1 levels (P<0.0001 for both) and cavernosal ACE activity levels (P<0.05) were significantly elevated while systemic and cavernosal NO (P<0.0001 for both) and GH (<0.001 and <0.05) levels were declined compared to psychogenic. In nondiabetic patients, systemic and cavernosal ET-1 levels (P<0.0001 for both) were significantly elevated while systemic and cavernosal NO (P<0.0001 for both) and systemic GH levels (P<0.05) were declined compared to psychogenic. Systemic NO was positively correlated with GH in psychogenic (r=0.616, P<0.05), diabetic (r=0.583, P<0.05) and nondiabetic (r=0.615, P<0.05) patients and correlated positively with cGMP (r=0.605, P<0.05) but negatively with ACE activities (r=-0.585, P<0.05) in diabetic patients. In conclusion, plasma levels of ET-1, ACE activities are elevated and associated with reduction of GH, NO and cGMP levels in the systemic and cavernous blood of ED patients. This disturbance may indicate endothelial dysfunction that may hind at their significance in the pathophysiology of ED.

Segmental tracheal replacement in mongrel dogs.

OBJECTIVE: To evaluate the use of alcohol- and detergent-preserved tracheal allografts in dogs. MATERIAL AND METHODS: Experimental segmental tracheal replacement was performed in 18 adult mongrel dogs. Three different techniques were tried in three groups of dogs. In the first group, a four-ring cervical tracheal segment was dissected out and implanted in the trachea of another dog. In the second group, the procedure was performed using a tracheal allograft that had been preserved in 70% ethyl alcohol for 20 days. In the third group, tracheal grafts were previously impregnated in 10% povidone iodine for 72 h before being implanted. Allografts were harvested 60 days after transplantation and assessed both histologically and in terms of the percentage patency. Dogs that died within 60 days were also included in the analysis. RESULTS: The best results were found in the group in which the tracheal allograft had been preserved in ethyl alcohol and this was explained by the reduced antigenicity of the graft in this group. CONCLUSIONS: The use of alcohol-preserved allografts is a practical method of tracheal transplantation, and the alcohol-preservation technique markedly reduces the immunogenicity of the grafts.

Palliative treatment for tracheal stenoses using carbon dioxide laser and the Gianturco stent. Long-term results.

Between September 1992 and March 1998, the self-expandable Gianturco prosthesis was inserted in 23 patients suffering from tracheal stenosis. After radial incision and dilation of the stenosis as described by Shapshay, the positioning of the stent was performed during an endoscopic procedure under optical control. The prosthesis used was a double-ring stent 50 mm long and 20 mm in diameter. The follow-up period ranged between 0.5 and 67 months with an average of 31 +/- 18 months. Pulmonary function tests showed an average improvement of the peak expiratory flow (50%) from preoperative results of 1.06 +/- 0.60 L/s to short-term postoperative results of 2.08 +/- 0.78 L/s and long-term postoperative results of 2.11 +/- 0.78 L/s. The mean peak inspiratory flow (50%) improved from 1.43 +/- 0.85 L/s to 2.40 +/- 1.29 L/s at short term and to 2.56 +/- 1.20 L/s at long term. Eight patients out of the 23 had to undergo a second endoscopic procedure: 3 patients for granuloma vaporization; 1 patient to change a malpositioned stent; 2 patients to add a second stent because of insufficient tracheal enlargement; and 2 patients to resect mucosal membranes between the 2 stent rings and to place a second stent. Optical control of the accurate positioning and use of this model of Gianturco prosthesis helped to avoid the severe complications described in the literature (migration, extrusion, fracture, wall erosion. and hemorrhage). The follow-up must particularly target the prevention of granulomas. The self-expandable Gianturco prosthesis can be advocated for long-term palliative treatment of tracheal stenoses that are inoperable by an external surgical approach.

Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP.

The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L. donovani GBP (GBPP) in the diagnosis of L. donovani infections in Sudan. The sensitivity of the rGBP ELISA in diagnosing visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) was 92% and 93%, respectively. In contrast, the sensitivity of the GBPP ELISA was 55% for VL and 63% for PKDL. Plasma antibody reactivity of donors with VL and PKDL remained high for an extended period after the end of treatment. Antibody-reactivity to rGBP and GBPP was detected in 71% and 14% of plasma samples from CL patients, respectively. Plasma from healthy Sudanese donors living in an area endemic for malaria but free of leishmaniasis was negative in both assays.

Characterization of the local and systemic immune responses in patients with cutaneous leishmaniasis due to Leishmania major.

In this study skin biopsies and peripheral blood samples were obtained from patients with cutaneous leishmaniasis caused by Leishmania major. Samples were obtained at diagnosis and during healing when the lesions had regressed to half the original size. At diagnosis most of the cells expressed HLA-DR. The numbers of CD8+ cells in the lesions were higher at diagnosis than during healing. By contrast, a lower percentage of PBMC expressed CD8+ cells at diagnosis probably due to sequestration in the lesion. In the lesion, in situ staining for IFN-gamma, IL-10, and IL-4 showed marked variation between all patients in the number of positive cells for a particular cytokine. The proliferative response of PBMC to leishmanial antigens and IFN-gamma production tended to increase during healing. Cytokine patterns in the PBMC in response to Leishmania antigen was more specific than in the lesion and correlated better with the clinical manifestations. Possible reasons for this are discussed.

T-cell response in human leishmaniasis.

In the present communication we provide evidence for the existence of a Th1/Th2 dichotomy in the T-cell response to Leishmania antigens in human leishmaniasis. Our data suggest that the pattern of IL-4 and IFN-gamma response is polarised in these patients. Lymphocytes from individuals recovered from cutaneous leishmaniasis (CL) responded by IFN-gamma production following stimulation with Leishmania antigens whereas cells from patients recovered from visceral leishmaniasis (VL) showed a mixed pattern of IFN-gamma and IL-4 responses. The cells producing these cytokines were predominantly CD4+. Furthermore, IL-10 plays an important role in the development of post kala azar dermal leishmaniasis (PKDL) from VL. The balance between the parasitic-specific T-cell response plays an important regulatory role in determining the outcome of Leishmania infections in humans.

Humoral and cellular immune responses to synthetic peptides of the Leishmania donovani kinetoplastid membrane protein-11.

Native kinetoplastid membrane protein-11 (KMP-11), purified from crude extracts of Leishmania donovani parasites, activates T cells from individuals who have recovered from visceral leishmaniasis. In this work we used three 38-mer peptides spanning the amino acid sequence of the L. donovani KMP-11 as solid-phase ligands in enzyme-linked immunosorbent assays (ELISAs) and as stimulating antigens in lymphoproliferative assays in order to evaluate humoral and cellular immune responses to well-defined sequences of the protein. Antibody reactivity against the three peptides was measured in plasma from 63 Sudanese visceral leishmaniasis patients (VL) and the percentage of patients with anti-KMP-11 antibodies in ELISA were 37% (KMP-11-1), 30% (KMP-11-2) and 58% (KMP-11-3). The fraction of VL patients with measurable antibody reactivity in one or more of the three ELISAs was 79%. Cross-reactivity to the KMP-11 peptides was detected in plasma from Sudanese patients suffering from Leishmania major infections and in plasma from Sudanese and Danish patients infected with Plasmodium falciparum. In lymphoproliferative assays, 10 of 17 PBMC isolates from donors previously infected with L. donovani showed a response to one or more of the three KMP-11 peptides.

Serodiagnosis of cutaneous leishmaniasis: assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein.

An enzyme-linked immunosorbent assay (ELISA) using a 28 amino acid sequence of the repetitive element of gene B protein (GBP) from Leishmania major was developed for serodiagnosis of cutaneous leishmaniasis (CL). The assay was compared to ELISAs using crude amastigote and promastigote antigens from L. donovani and the major surface glycoprotein (Gp63) from either L. donovani or L. major as a solid-phase ligand. The sensitivity of the assays was tested in 33 patients suffering from CL caused by L. major. The sensitivity of the GBP peptide (GBPP) ELISA was 82%. This was higher than in the assays using crude amastigote (67%) or promastigote (67%) antigens, but the difference was not statistically significant. The sensitivity in the assays using Gp63 from L. donovani (52%) or L. major (39%) was significantly lower than in the assay using GBPP (P = 0.019 and P < 0.001, respectively). Plasma samples from healthy Sudanese individuals living in an area endemic for malaria but free of leish-maniasis were negative in all the assays. Significantly higher levels of antibodies were found in the patients who had suffered from the disease for more than eight weeks than in patients with a shorter clinical history (GBPP ELISA; P = 0.038; amastigote ELISA; P = 0.004; and promastigote ELISA; P = 0.017). In the former group, the sensitivities of the five ELISAs were 100% (GBPP), 87% (amastigote), 93% (promastigote), 67% (L. donovani), and 53% (L. major), respectively.