A semi-automated microscopic image analysis method for scoring Ki-67 nuclear immunostaining.

Nuclear proliferation marker MIB-1 (Ki-67) immunohistochemistry (IHC) is used to examine tumor cell proliferation. However, the diagnostic or prognostic value of the Ki-67 nuclear staining intensity and location, defined as nuclear gradient (NG), has not been assessed. This study examined the potential association between Ki-67 NG and cell cycle phases and its effect on the prognosis of pulmonary typical carcinoid (PTC) tumors. We propose a method for classifying the NG of Ki-67 during the cell cycle and compare the results between PTC, pulmonary adenocarcinoma (PAD), and breast ductal carcinoma (BDC). A literature review and objective analysis of IHC-stained paraffin sections were used to determine the Ki-67 labeling index and composed a stratification of the NG into NG1, NG2, and NG3/4 categories. A semi-automated image analysis protocol was established to determine the Ki-67 NG in PTC, PAD, and BDC. High intraobserver consistency and moderate interobserver agreement were achieved in the determination of Ki-67 NG in tumor specimens. NG1 and NG2 were lower in PTC than in PAD and BDC. Cox multivariate analysis of PTC after adjusting for age and number of metastatic lymph nodes showed that Ki-67 NG1 and NG2 significantly predicted clinical outcomes. The semi-automated method for quantification of Ki-67 nuclear immunostaining proposed in this study could become a valuable diagnostic and prognostic tool in PTC.

A semi-automated microscopic image analysis method for scoring Ki-67 nuclear immunostaining

Nuclear proliferation marker MIB-1 (Ki-67) immunohistochemistry (IHC) is used to examine tumor cell proliferation. However, the diagnostic or prognostic value of the Ki-67 nuclear staining intensity and location, defined as nuclear gradient (NG), has not been assessed. This study examined the potential association between Ki-67 NG and cell cycle phases and its effect on the prognosis of pulmonary typical carcinoid (PTC) tumors. We propose a method for classifying the NG of Ki-67 during the cell cycle and compare the results between PTC, pulmonary adenocarcinoma (PAD), and breast ductal carcinoma (BDC). A literature review and objective analysis of IHC-stained paraffin sections were used to determine the Ki-67 labeling index and composed a stratification of the NG into NG1, NG2, and NG3/4 categories. A semi-automated image analysis protocol was established to determine the Ki-67 NG in PTC, PAD, and BDC. High intraobserver consistency and moderate interobserver agreement were achieved in the determination of Ki-67 NG in tumor specimens. NG1 and NG2 were lower in PTC than in PAD and BDC. Cox multivariate analysis of PTC after adjusting for age and number of metastatic lymph nodes showed that Ki-67 NG1 and NG2 significantly predicted clinical outcomes. The semi-automated method for quantification of Ki-67 nuclear immunostaining proposed in this study could become a valuable diagnostic and prognostic tool in PTC.

Monitoring non-small cell lung cancer progression and treatment response through hyaluronic acid in sputum.

We evaluated whether hyaluronan (HA) levels in the sputum could be used as a noninvasive tool to predict progressive disease and treatment response, as detected in a computed tomography scan in non-small cell lung cancer (NSCLC) patients. Sputum samples were collected from 84 patients with histological confirmation of NSCLC, 33 of which were in early-stage and 51 in advanced-stage disease. Patients received systemic chemotherapy (CT) after surgery (n=36), combined CT and immunotherapy (IO) (n=15), or targeted therapy for driver mutation and disease relapse (N=4). The primary end-point was to compare sputum HA levels in two different concentrations of hypertonic saline solution with overall survival (OS) and the secondary and exploratory end-points were radiologic responses to treatment and patient outcome. Higher concentrations of HA in the sputum were significantly associated to factors related to tumor stage, phenotype, response to treatment, and outcome. In the early stage, patients with lower sputum HA levels before treatment achieved a complete tumor response after systemic CT with better progression-free survival (PFS) than those with high HA levels. We also examined the importance of the sputum HA concentration and tumor response in the 51 patients who developed metastatic disease and received CT+IO. Patients with low levels of sputum HA showed a complete tumor response in the computed tomography scan and stable disease after CT+IO treatment, as well as a better PFS than those receiving CT alone. HA levels in sputum of NSCLC patients may serve as a candidate biomarker to detect progressive disease and monitor treatment response in computed tomography scans.

Monitoring non-small cell lung cancer progression and treatment response through hyaluronic acid in sputum

We evaluated whether hyaluronan (HA) levels in the sputum could be used as a noninvasive tool to predict progressive disease and treatment response, as detected in a computed tomography scan in non-small cell lung cancer (NSCLC) patients. Sputum samples were collected from 84 patients with histological confirmation of NSCLC, 33 of which were in early-stage and 51 in advanced-stage disease. Patients received systemic chemotherapy (CT) after surgery (n=36), combined CT and immunotherapy (IO) (n=15), or targeted therapy for driver mutation and disease relapse (N=4). The primary end-point was to compare sputum HA levels in two different concentrations of hypertonic saline solution with overall survival (OS) and the secondary and exploratory end-points were radiologic responses to treatment and patient outcome. Higher concentrations of HA in the sputum were significantly associated to factors related to tumor stage, phenotype, response to treatment, and outcome. In the early stage, patients with lower sputum HA levels before treatment achieved a complete tumor response after systemic CT with better progression-free survival (PFS) than those with high HA levels. We also examined the importance of the sputum HA concentration and tumor response in the 51 patients who developed metastatic disease and received CT+IO. Patients with low levels of sputum HA showed a complete tumor response in the computed tomography scan and stable disease after CT+IO treatment, as well as a better PFS than those receiving CT alone. HA levels in sputum of NSCLC patients may serve as a candidate biomarker to detect progressive disease and monitor treatment response in computed tomography scans.

Determinants of treatment of mental disorders in Lebanon: barriers to treatment and changing patterns of service use.

AIMS: To investigate for the first time the determinants and barriers of seeking help for mental disorders in the Arab world based on a national study: Lebanese Evaluation of the Burden of Ailments and Needs Of the Nation (L.E.B.A.N.O.N). METHODS: A nationally representative (n = 2857) and multistage clustered area probability household sample of adults ≥18 years and older was assessed for lifetime and 12 months mental disorders using the Composite International Diagnostic Interview. In addition, detailed information was obtained on help- seeking behaviour and barriers to treatment. RESULTS: In total, 19.7% of the Lebanese with mental disorders sought any type of treatment: 91% of those who sought treatment did so within the health sector. Severity and perceived severity of disorders predicted seeking help, the highest being for panic disorder. The greatest barrier to seek help was low perceived need for treatment (73.9%). Stigma was reported to be a factor only in 5.9% of those who thought about seeking treatment. Eighty per cent of the Lebanese reported they would not be embarrassed if friends knew they were seeking help from a professional. CONCLUSIONS: A small fraction of Lebanese seek help for their mental health problems: female gender, higher education and income are predictors of positive attitudes to help seeking. Severity and recognition of disorders, more than stigma, to get treatment seem to be the most important factors in determining help seeking. The findings underscore the importance of helping the public recognise mental health disorders.

Detection of sputum cofilin-1 as indicator of malignancy.

Cofilin-1 (CFL1), a small protein of 18 kDa, has been studied as a biomarker due to its involvement in tumor cell migration and invasion. Our aim was to evaluate CFL1 as an indicator of malignancy and aggressiveness in sputum samples. CFL1 was analyzed by ELISA immunoassay in the sputum of 73 lung cancer patients, 13 cancer-free patients, and 6 healthy volunteers. Statistical analyses included ANOVA, ROC curves, Spearman correlation, and logistic regression. Sputum CFL1 levels were increased in cancer patients compared to cancer-free patients and volunteers (P<0.05). High expression of sputum CFL1 was correlated to T4 stage (P=0.01) and N stage (P=0.03), tobacco history (P=0.01), and squamous cell carcinoma histologic type (P=0.04). The accuracy of sputum CFL1 in discriminating cancer patients from cancer-free patients and healthy volunteers were 0.78 and 0.69, respectively. CFL1 at a cut-off value of 415.25 pg/mL showed sensitivity/specificity of 0.80/0.70 in differentiating between healthy volunteers and cancer patients. Sputum CFL1 was also able to identify cancer-free patients from patients with lung cancer. The AUC was 0.70 and, at a cut-off point ≥662.63 pg/mL, we obtained 60% sensitivity and 54% specificity. Logistic regression analysis controlled for tobacco history, histologic types, and N stage showed that cancer cell-associated CFL1 was an independent predictor of death. Smoker patients with squamous cell carcinoma, lymph node metastasis and sputum CFL1>1.475 pg/mL showed augmented chance of death, suggesting lung cancer aggressiveness. CFL1 presented diagnostic value in detecting lung cancer and was associated to tumor aggressiveness.

Detection of sputum cofilin-1 as indicator of malignancy

Cofilin-1 (CFL1), a small protein of 18 kDa, has been studied as a biomarker due to its involvement in tumor cell migration and invasion. Our aim was to evaluate CFL1 as an indicator of malignancy and aggressiveness in sputum samples. CFL1 was analyzed by ELISA immunoassay in the sputum of 73 lung cancer patients, 13 cancer-free patients, and 6 healthy volunteers. Statistical analyses included ANOVA, ROC curves, Spearman correlation, and logistic regression. Sputum CFL1 levels were increased in cancer patients compared to cancer-free patients and volunteers (P<0.05). High expression of sputum CFL1 was correlated to T4 stage (P=0.01) and N stage (P=0.03), tobacco history (P=0.01), and squamous cell carcinoma histologic type (P=0.04). The accuracy of sputum CFL1 in discriminating cancer patients from cancer-free patients and healthy volunteers were 0.78 and 0.69, respectively. CFL1 at a cut-off value of 415.25 pg/mL showed sensitivity/specificity of 0.80/0.70 in differentiating between healthy volunteers and cancer patients. Sputum CFL1 was also able to identify cancer-free patients from patients with lung cancer. The AUC was 0.70 and, at a cut-off point ≥662.63 pg/mL, we obtained 60% sensitivity and 54% specificity. Logistic regression analysis controlled for tobacco history, histologic types, and N stage showed that cancer cell-associated CFL1 was an independent predictor of death. Smoker patients with squamous cell carcinoma, lymph node metastasis and sputum CFL1>1.475 pg/mL showed augmented chance of death, suggesting lung cancer aggressiveness. CFL1 presented diagnostic value in detecting lung cancer and was associated to tumor aggressiveness.

Assessing the impact of the presence of pharmacists in a clinical department of a Lebanese hospital.

BACKGROUND: Clinical pharmacy services are still in their very early implementation stages in Lebanon. The objective of this pilot study was to evaluate the impact of clinical pharmacist's presence at the infectious diseases department of Hôtel-Dieu de France University Hospital of Beirut (HDF) and to evaluate the acceptance of pharmacist's interventions by healthcare providers. MATERIAL AND METHODS: A 21-month prospective analysis was conducted, including 240 hospitalized patients in the infectious diseases department of HDF and 475 interventions performed by the pharmacist. A clinical pharmacist and pharmacy residents were present for 1 to 2hours/day in the ward. A pharmaceutical care plan was established and used to document patients' problems and pharmacist's interventions. Main criteria analyzed were: types and frequencies of pharmaceutical problems detected, types of pharmaceutical interventions performed, their acceptance by the prescribers and/or nurses, and factors affecting the interventions and their acceptance. RESULTS: The most frequent pharmaceutical problem detected was incorrect dosage and the three most frequent interventions performed by the pharmacist were stop/start/substitute a drug, change drug dosage/or daily distribution, and change administration time. The acceptance was the highest for I.TIM (change drug administration time) and the lowest for I.FOL (request a lab test/exam/clinical follow-up). DISCUSSION AND CONCLUSION: Even a short daily pharmacist's presence is an added value in inpatient care at the infectious diseases department of Hôtel-Dieu de France University Hospital. Areas of improvement are a better communication between the pharmacist and the prescribers, a direct contact between pharmacist and patient and a longer presence of the clinical pharmacist in the clinical department.

Associated or combined vaccination of Brazilian infants with a conjugate Haemophilus influenzae type b (Hib) vaccine, a diphtheria-tetanus-whole-cell pertussis vaccine and IPV or OPV elicits protective levels of antibodies against Hib.

This study investigated the immunogenicity and safety of including a Haemophilus influenzae type b vaccine (polyribosylribitol phosphate conjugated to tetanus toxoid, PRP-T) in three different vaccination schemes: (1) PRP-T reconstituted with a combined diphtheria-tetanus-pertussis-inactivated poliovirus vaccine (DTP-IPV//PRP-T); (2) PRP-T reconstituted with DTP and administered concomitantly with an oral poliovirus vaccine (DTP//PRP-T+OPV); and (3) PRP-T administered concomitantly with DTP at a different injection site and OPV (DTP+PRP-T+OPV). Vaccines were given at 2, 4, and 6 months of age. A total of 252 infants were enrolled, and randomly assigned to one of the three vaccination groups (84 infants in each group); 241 infants were followed until the end of the study. Antibody production against PRP, diphtheria, tetanus and pertussis antigens was satisfactory for each vaccination scheme used. A good response to Hib vaccine was elicited in each group, and 3 months after the third vaccine dose, at least 97% of children in each group had levels of PRP antibody considered to be seroprotective (>0.15 microg/ml), and over 90% of children in each group had levels over 1. 0 microg/ml. The solicited local and systemic adverse events following vaccination were mild in all groups and resolved within 4 days without medical intervention. With the exception of fever, which was more common after the second dose in children who received DTP-IPV//PRP-T, local and systemic reactions did not differ between the vaccination groups. Due to the practical advantages of combined vaccines, their use in routine immunization programs in developing countries is highly desirable. Our results show that Hib conjugate vaccine can be included in routine immunization programs that include either OPV or IPV with satisfactory immunogenicity and safety profiles. This flexible approach should facilitate the inclusion of the Hib conjugate vaccine in routine immunization programs on a world-wide scale.

Prognostic values of stromal proportion and PCNA, Ki-67, and p53 proteins in patients with resected adenocarcinoma of the lung.

Data from 64 patients who underwent surgical resection of lung adenocarcinomas were studied to identify clinicopathologic markers that might provide prognostic information on the clinical behavior of this neoplasia Patient staging was performed in accordance with the tumor-node-metastasis system as follows: Stage I (n = 29), Stage II (n = 11), Stage IIIA (n = 21), and Stage IIIB (n = 3). Overall follow-up time corresponded to the follow-up time for patients who were alive and to the survival time for patients who had died, all of them expressed in months. Data included age, staging, histologic type, morphometric assessment of histologic features related to tumor (stroma and vascularization), and immunohistochemical detection of proliferation cell markers (Ki-67 protein and proliferating cell nuclear antigen) and p53 protein. The morphometric assessment was made by the point-counting procedure. Data analysis included Life Tables for Survival and Cox Regression models. Overall follow-up analysis showed that significant univariate predictors (P < .05) were T stage; N stage; tumor stromal proportion; and immunohistochemical indexes of proliferating cell nuclear antigen, Ki-67, and p53 proteins. Variables that presented independent predictive value for overall follow-up with the multivariate model (P < .05) were sex, T stage, N stage, tumor stromal proportion, and immunohistochemical detection of p53 protein. We conclude that tumor stromal proportion and immunohistochemical detection of p53 protein, controlled for sex, T stage, and N stage, may be of critical value in the evaluation of recurrence of lung adenocarcinoma, serving as indicators for a more accurate prognosis.

Stereological estimates of nuclear star volume and vessels as predictors of chemotherapy response in small cell carcinoma of the lung: a preliminary report.

AIMS: This study was designed to evaluate the role of morphometric and clinical parameters in predicting chemotherapy responder patients with small cell carcinoma of the lung. METHODS AND RESULTS: Morphometric studies were performed by means of point counting techniques. Forty-six patients were included in this study. Group 1 patients (n = 19) were those without response to chemotherapy; Group 2 (n = 27) was composed by patients with partial or complete response to chemotherapy. Logistic regression analysis was used to attain the best separation of non-responder from responder patients. Star volume of the nuclei and vessel were selected during the backward procedure as relevant variables to characterize the two groups of patients. The overall sensitivity of the model was 80.43%. CONCLUSIONS: Our results indicate that histopathological data may help to predict the chemotherapy response in patients with small cell lung carcinoma, and encourage the use of morphometric procedures in histopathological analysis of this type of lung tumours.

Evaluation of serum levels of IgG subclasses and anti-ribosyl-ribitolphosphate IgG and IgG2 in children with Haemophilus influenzae b meningitis.

In 40 children with Haemophilus influenzae b (Hib) meningitis, we determined serum levels (mg/dl) of IgG subclasses using the radial immunodiffusion method; 67.8 per cent of these children were less than 24 months old. In 14 children of the sample we measured serum IgG and IgG2 anti-ribosyl-ribitolphosphate (anti-PRP) (by enzyme-linked immunosorbent assay, ELISA) in the acute and convalescent phases of the disease. Lower IgG2 levels than those of the control group were obtained in all age ranges: 3-12 months, 1-2 years (p < 0.01), and 2-5 years (p < 0.001). IgG4 was also present in lower levels in patients of all age ranges (p < 0.05, p < 0.001, and p < 0.01 respectively). Serum levels of IgG anti-PRP and IgG2 anti-PRP measured were very low in the acute phase of the disease in all age ranges and there was no notable increase in levels during the convalescent phase of the disease. This result indicates that children less than 24 months old do not produce sufficient levels of IgG and IgG2 anti-PRP even after Hib meningitis.

Human serum beta2-microglobulin levels: correlation with total serum IgG and placental IgG transfer in HIV-infected and non-HIV infected individuals.

The acquisition of maternal immunoglobulin G (IgG) is fundamental to the immune defence of the neonate. The receptor responsible for IgG transfer across the human placenta has also been implicated in the maintenance of IgG levels in the circulation. beta2-microglobulin is part of the Fc receptor (FcR) that has recently been purified from the human placenta. In HIV infection, increasing serum levels of total IgG and beta2-microglobulin are observed as the disease progresses. Herein, we have investigated the correlation between beta2-microglobulin and total serum IgG levels in HIV-seropositive mothers and their term neonates (HIV group, n = 37), as well as in HIV-seronegative mothers and their term neonates (control group, n = 50). Serum maternal beta2-microglobulin was directly correlated with total serum IgG levels in HIV-infected mothers (r = 0.58; P = 0.0002), but not in healthy HIV-seronegative mothers (r = -0.20; P = 0.16). Maternal serum beta2-microglobulin was also inversely correlated with placental antibody transfer of total IgG in mother-newborn pairs from the HIV group (r = 0.38; P = 0.02), but not from the control group (r = 0.15. P = 0.31). These results seem to indicate that, in HIV infection, elevated serum beta2-microglobulin levels could be involved in maintenance of abnormally high total serum IgG concentrations; by interfering with the binding of IgG to Fc receptors at the maternal-fetal interface, they might also reduce IgG transfer. By contrast, in normal non-HIV infected individuals, serum beta2-microglobulin levels do not appear implicated in regulation of these two phenomena.

[Bacterial meningitis - diagnosis and treatment] / Meningites bacterianas - diagnóstico e conduta.

OBJECTIVE: To present new concepts on diagnosis and treatment of bacterial meningitis regarding etiologic agents, pathophysiology and options about antimicrobial, antiinflammatory and supportive therapy. METHODS: Bibliographic review from MEDLINE data including articles published during the last ten years. One classic article published before this period and chapters of textbooks on infectious diseases were also included. RESULTS: Initial empirical antibiotic therapy is chosen according to probable etiologic agents for the age group. In the CSF microbiological analysis, the gram stains can reveal bacteria in 50% to 80% of the cases and the culture in nearly 85%. The tests for detection of bacterial antigens are useful for the diagnosis but they present low sensitivity. The most common agents during the neonatal period continue to be E. coli, Streptococcus B and L. monocytogenes. Beyond this period, the incidence of meningitis by Haemophilus influenzae b had a significant decrease after the introduction of conjugate vaccines. However, S. pneumoniae and N. meningitidis continue to be frequent agents. Currently, the thirdgeneration cephalosporins, ceftriaxone or cefotaxime, are the antibiotic therapy of choice. They are used with ampicillin up to two months of life and alone beyond this age. Dexamethasone has showed to be effective in reducing the inflammatory response and the sequelae, mainly the auditory sequelae. The fluid restriction doesn't offer advantages in the supportive care. CONCLUSIONS: Early diagnosis and prompt treatment are related to good outcome. The new insights on pathophysiology, the new antibiotics and the increasing bacterial resistance have determined changes in treatment.

[Acquired toxoplasmosis] / Toxoplamose adquirida.

OBJECTIVE: The aim of this review is to present some important aspects of acquired toxoplasmosis to the pediatric practitioner. METHOD: All the articles about acquired toxoplasmosis published during the last decade and indexed in the Index Medicus were revised. From each one, interesting aspects were critically selected. RESULTS: We describe aspects of the epidemiology, pathogenesis, clinical manifestations, diagnosis, treatment and prevention related to acquired toxoplasmosis. CONCLUSION: The content of this article may facilitate the management of patients suspected of having acquired toxoplasmosis.

Seroconversion of a trivalent measles, mumps, and rubella vaccine in children aged 9 and 15 months.

The serological response to MMR vaccine was evaluated in 109 9-month-old infants having no history of measles vaccination, and in 98 15-month-old children who had received monocomponent measles immunisation at 9 months. The combined vaccine contained Schwarz, Urabe Am9, and Wistar RA 27/3 live attenuated virus strains. Preimmunisation antibody levels were extremely low for the 9-month-old children, indicating that maternally-transmitted antibodies do not persist at this age. In the case of mumps, preimmunisation antibody levels were significantly higher in the 15-month-old than in the 9-month-old group. A difference between groups in terms of postimmunisation antibody titres was observed only for rubella, with titres being significantly higher in the older group. Seroconversion rates were high in both groups and no serious events attributable to vaccination were observed. The MMR vaccine can thus be administered to children as young as 9 months of age. Evidence for the efficacy of a two-dose schedule, i.e. at 9 and 15 months, is presented.

[Prevalence of Haemophilus influenzae resistant to ampicillin, cefaclor, cefotaxime, chloramphenicol and cotrimoxazol isolated from laboratories in the city of São Paulo, Brazil]. / Prevalência de Haemophilus influenzae resistentes a ampicilina, cefaclor, cefotaxime, cloranfenicol e cotrimoxazol isolados de laboratórios na cidade de São Paulo.

OBJECTIVE: To determine Haemophilus influenzae resistant to ampicillin and other antibiotics isolated from different clinical specimens. METHODS: Isolates of H. influenzae were identified by culture with V and X factors and the aminolevulinic test. Nitrocefin was used to detect beta-lactamase (betaLac) production isolates were tested for antimicrobial susceptibility by disc diffusion and agar dilution methods. Serotype b was assessed by slide co-agglutination. RESULTS: From 245 H. influenzae identified, 155 were tested for serotype b, 28% (43/155) of which were positive. The global rate of beta-lactamase-positive isolates was 9% (22/245). Resistance was similar among serotype b (11.6%) and non-type b H. influenzae (9.8%) (p>0.05). No difference on betaLac production was found according to specimen's origin or the patients' age. Resistances to other antibiotics (by agar dilution and disc diffusion method, respectively) were: chloramphenicol 3.3 to 7.1%; cefaclor: 1.6 to 3.9% and cotrimoxazol: 9.1 to 10.5%. No resistance to cefotaxime has been detected; 63% (5/8) beta-Lac-positive isolates by agar dilution showed also resistance to chloramphenicol, compared to 3% (4/118) in the ss-Lac-negative group (p<0.001). CONCLUSIONS: H. influenzae ampicillin-resistance has shown to be lower than other hospital-based-studies in São Paulo, and comparable to rates found in healthy carriers. The association between ampicillin and chloramphenicol resistance was significant: where this pattern is frequently found, the initial therapy for severe H. influenzae infections - like meningitis - should include a third generation cephalosporin.