Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

The efficiency of different estimation methods of hydro-physical limits

The soil water available to crops is defined by specific values of water potential limits. Underlying the estimation of hydro-physical limits, identified as permanent wilting point (PWP) and field capacity (FC), is the selection of a suitable method based on a multi-criteria analysis that is not always clear and defined. In this kind of analysis, the time required for measurements must be taken into consideration as well as other external measurement factors, e.g., the reliability and suitability of the study area, measurement uncertainty, cost, effort and labour invested. In this paper, the efficiency of different methods for determining hydro-physical limits is evaluated by using indices that allow for the calculation of efficiency in terms of effort and cost. The analysis evaluates both direct determination methods (pressure plate - PP and water activity meter - WAM) and indirect estimation methods (pedotransfer functions - PTFs). The PTFs must be validated for the area of interest before use, but the time and cost associated with this validation are not included in the cost of analysis. Compared to the other methods, the combined use of PP and WAM to determine hydro-physical limits differs significantly in time and cost required and quality of information. For direct methods, increasing sample size significantly reduces cost and time. This paper assesses the effectiveness of combining a general analysis based on efficiency indices and more specific analyses based on the different influencing factors, which were considered separately so as not to mask potential benefits or drawbacks that are not evidenced in efficiency estimation.

Quantification of Trypanosoma cruzi in the heart, lymph nodes and liver of experimentally inffected mice, using limiting dilution analysis

Limiting dilution analysis was used to quantify Trypanosoma cruzi in the lymph nodes, liver and heart of Swiss and C57 B1/10 mice. The results showed that, in Swiss and B1/10 mice infected with T. cruzi Y strain, the number of parasites/mg of tissue increased during the course of the infection in both types of mice, although a grater number of parasites were observed in heart tissue from Swiss mice than from B1/10. With regard to liver tissue, it was observed that the parasite load in the initial phase of infection was higher than in heart. In experiments using T. cruzi Colombian strain, the parasite load in the heart of Swiss and B1/10 mice increased relatively slowly, although high levels of parasitization were nonetheless observable by the end of the infection. As for the liver and lymph nodes, the concentration of parasites was lower over the entire course of infection than in heart. Both strains thus maintained their characteristic tissue tropisms. The limiting dilution assay (LDA) proved to be an appropriate method for more precise quantification of T. cruzi, comparing favorably with other direct microscopic methods that only give approximate scores.

Direct methods for detecting picorna-like virus from dead and alive Triatomine insects

In this work we report four different destructive and non-destructive methods for detecting picorna-like virus particles in triatomines. The methods are based on direct observation under transmission electron microscope and they consist of four ways to prepare samples of presumable infected material. The samples are prepared processing dead or alive insect parts, or even dry or fresh insect feces. The methods can be used as analytical or preparative techniques, for quantifying virus infection and checking virus integrity as well. In this work the four methods are applied in order to detect Triatoma virus (TrV) particles in T. infestans colonies.

Cost of schizophrenia: direct costs and use of resources in the State of São Paulo

OBJECTIVE: To estimate the direct costs of schizophrenia for the public sector. METHODS: A study was carried out in the state of São Paulo, Brazil, during 1998. Data from the medical literature and governmental research bodies were gathered for estimating the total number of schizophrenia patients covered by the Brazilian Unified Health System. A decision tree was built based on an estimated distribution of patients under different types of psychiatric care. Medical charts from public hospitals and outpatient services were used to estimate the resources used over a one-year period. Direct costs were calculated by attributing monetary values for each resource used. RESULTS: Of all patients, 81.5% were covered by the public sector and distributed as follows: 6.0% in psychiatric hospital admissions, 23.0% in outpatient care, and 71.0% without regular treatment. The total direct cost of schizophrenia was US$191,781,327 (2.2% of the total health care expenditure in the state). Of this total, 11.0% was spent on outpatient care and 79.2% went for inpatient care. CONCLUSIONS: Most schizophrenia patients in the state of São Paulo receive no regular treatment. The study findings point out to the importance of investing in research aimed at improving the resource allocation for the treatment of mental disorders in Brazil.

Direct medical costs associated with schizophrenia relapses in health care services in the city of São Paulo

OBJECTIVE: To assess direct medical costs associated with schizophrenia relapses in mental health services. METHODS: The study was conducted in three health facilities in the city of São Paulo: a public state hospital; a Brazilian National Health System (SUS)-contracted hospital; and a community mental health center. Medical records of 90 patients with schizophrenia who received care in 2006 were reviewed. Information on inpatient expenditures was collected and used for cost estimates. RESULTS: Mean direct medical cost of schizophrenia relapses per patient was US$ 4,083.50 (R$ 8,167.58) in the public state hospital; US$ 2,302.76 (R$ 4,605.46) in the community mental health center; and US$ 1,198.50 (R$ 2,397.74) in the SUS-affiliated hospital. The main component was daily inpatient room rates (87% - 98%). Medication costs varied depending on the use of typical or atypical antipsychotic drugs. Atypical antipsychotic drugs were more often used in the community mental health center. CONCLUSIONS: Costs associated with schizophrenia relapses support investments in antipsychotic drugs and strategies to reduce disease relapse and the need for mental health inpatient services. Treating patients in a community mental health center was associated with medium costs and added the benefit of not depriving these patients from family life.

Direct treatment costs of HIV/AIDS in Portugal

OBJECTIVE To analyze the direct medical costs of HIV/AIDS in Portugal from the perspective of the National Health Service. METHODS A retrospective analysis of medical records was conducted for 150 patients from five specialized centers in Portugal in 2008. Data on utilization of medical resources during 12 months and patients’ characteristics were collected. A unit cost was applied to each care component using official sources and accounting data from National Health Service hospitals. RESULTS The average cost of treatment was 14,277 €/patient/year. The main cost-driver was antiretroviral treatment (€ 9,598), followed by hospitalization costs (€ 1,323). Treatment costs increased with the severity of disease from € 11,901 (> 500 CD4 cells/µl) to € 23,351 (CD4 count ≤ 50 cells/ µl). Cost progression was mainly due to the increase in hospitalization costs, while antiretroviral treatment costs remained stable over disease stages. CONCLUSIONS The high burden related to antiretroviral treatment is counterbalanced by relatively low hospitalization costs, which, however, increase with severity of disease. The relatively modest progression of total costs highlights that alternative public health strategies that do not affect transmission of disease may only have a limited impact on expenditure, since treatment costs are largely dominated by constant antiretroviral treatment costs.

Lifestyle factors, direct and indirect costs for a Brazilian airline company

OBJECTIVE To analyze lifestyle risk factors related to direct healthcare costs and the indirect costs due to sick leave among workers of an airline company in Brazil. METHODS In this longitudinal 12-month study of 2,201 employees of a Brazilian airline company, the costs of sick leave and healthcare were the primary outcomes of interest. Information on the independent variables, such as gender, age, educational level, type of work, stress, and lifestyle-related factors (body mass index, physical activity, and smoking), was collected using a questionnaire on enrolment in the study. Data on sick leave days were available from the company register, and data on healthcare costs were obtained from insurance records. Multivariate linear regression analysis was used to investigate the association between direct and indirect healthcare costs with sociodemographic, work, and lifestyle-related factors. RESULTS Over the 12-month study period, the average direct healthcare expenditure per worker was US$505.00 and the average indirect cost because of sick leave was US$249.00 per worker. Direct costs were more than twice the indirect costs and both were higher in women. Body mass index was a determinant of direct costs and smoking was a determinant of indirect costs. CONCLUSIONS Obesity and smoking among workers in a Brazilian airline company were associated with increased health costs. Therefore, promoting a healthy diet, physical activity, and anti-tobacco campaigns are important targets for health promotion in this study population.

Identification of respiratory virus in infants with congenital heart disease by comparison of different methods

Respiratory virus infections are the main cause of infant hospitalization and are potentially severe in children with congenital heart disease (CHD). Rapid and sensitive diagnosis is very important to early introduction of antiviral treatment and implementation of precautions to control transmission, reducing the risk of nosocomial infections. In the present study we compare different techniques in the diagnosis of respiratory viruses in CHD infants. Thirty-nine samples of nasopharyngeal aspirate were obtained from CHD infants with symptoms of respiratory infection. The Multiplex PCR (Seeplex® RV 12 ACE Detection) driven to the detection of 12 respiratory viruses was compared with the direct immunofluorescence assay (DFA) and PCR, both targeting seven respiratory viruses. The positivity found by DFA, Multiplex and PCR was 33.3%, 51.3% and 48.7%, respectively. Kappa index comparing DFA and Multiplex, DFA and PCR and PCR and Multiplex PCR was 0.542, 0.483 and 0.539, respectively. The concordance between techniques was considered moderate. Both Multiplex PCR (p = 0.001) and PCR (p = 0.002) detected significantly more respiratory virus than DFA. As the performance of the tests may vary, the combination of two or more techniques may increase diagnostic sensitivity favoring the diagnosis of co-infections, early introduction of antiviral therapy and implementation of appropriate measures.

COMPARISON OF PERMANENT STAINING METHODS FOR THE LABORATORY DIAGNOSIS OF TRICHOMONIASIS

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in the world. The diagnosis is based on wet mount preparation and direct microscopy on fixed and stained clinical specimens. The aim of this study was to compare the performance of different fixing and staining techniques used in the detection of T. vaginalis in urine. The smears were fixed and submitted to different methods of permanent staining and then, the morphological aspects of the parasites were analyzed and compared. The Papanicolaou staining with ethanol as the fixative solution showed to be the best method of permanent staining. Our data suggest that staining techniques in association with wet mount examination of fresh specimens contribute to increase the sensitivity in the diagnosis of trichomoniasis.

Evaluation of a direct immunofluorescent antibody (difma) test using Leishmania genus - specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis

A direct immunofluorescent antibody (DIFMA) test using a Leishmania genus- specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL) in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.

Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran

INTRODUCTION: Various methods are used for the diagnosis of visceral leishmaniasis (VL), such as microscopic examination, culture and inoculation of laboratory animals; however, serological assays are commonly used for the detection of antibodies in serum samples with a wide range of specificity and sensitivity. METHODS: The purpose of this study was to compare three serological methods, including rA2-ELISA, the recombinant KE16 (rKE16) dipstick test and the direct agglutination test (DAT), for the detection of antibodies against VL antigens. The assays utilized 350 statistically based random serum samples from domestic dogs with clinical symptoms as well as samples from asymptomatic and healthy dogs from rural and urban areas of the Meshkinshahr district, northwestern Iran. RESULTS: Samples were assessed, and the following positive rates were obtained: 11.5% by rKE16, 26.9% by DAT and 49.8% by ELISA. The sensitivity among symptomatic dogs was 32.4% with rKE16, 100% with DAT and 52.9% with ELISA. Conversely, rA2-ELISA was less specific for asymptomatic dogs, at 46.5%, compared with DAT, at 88.9%. CONCLUSIONS : This study recommends rA2-ELISA as a parallel assay combined with DAT to detect VL infection among dogs. Further evaluations should be performed to develop an inexpensive and reliable serologic test for the detection of Leishmania infantum among infected dogs.

Performance of direct immunofluorescence assay for the detection of human metapneumovirus under clinical laboratory settings

ABSTRACT INTRODUCTION: Human metapneumovirus (hMPV) is an emergent human respiratory pathogen. This study aimed to evaluate the performance of direct immunofluorescence (DIF) to detect hMPV in a clinical laboratory setting. METHODS: Nasopharyngeal aspirate samples (448) of children and adults with respiratory illness were used to detect hMPV by using DIF and real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. RESULTS: In all, 36 (8%) samples were positive by DIF and 94 (21%) were positive by qRT-PCR. Direct immunofluorescence specificity was 99% and sensitivity was 38%. CONCLUSIONS: DIF is not very sensitive under clinical laboratory settings.

Quantitative analysis of collagen and elastic fibers in the transversalis fascia in direct and indirect inguinal hernia

PURPOSE: Our previous studies demonstrated structural and quantitative age-related changes of the elastic fibers in transversalis fascia, which may play a role in inguinal hernia formation. To verify whether there were differences in the extracellular matrix between direct and indirect inguinal hernia, we studied the amount of collagen and elastic fibers in the transversalis fascia of 36 male patients with indirect inguinal hernia and 21 with direct inguinal hernia. MATERIAL AND METHODS: Transversalis fascia fragments were obtained during surgical intervention and underwent histological quantitative analysis of collagen by colorimetry and analysis of elastic fibers by histomorphometry. RESULTS: We demonstrated significantly lower amounts of collagen and higher amounts of elastic fibers in transversalis fascia from patients with direct inguinal hernia compared to indirect inguinal hernia patients. The transversalis fascia from direct inguinal hernia patients showed structural changes of the mature and elaunin elastic fibers, which are responsible for elasticity, and lower density of oxytalan elastic fibers, which are responsible for resistance. These changes promoted loss of resiliency of the transversalis fascia. CONCLUSION: These results improve our understanding of the participation of the extracellular matrix in the genesis of direct inguinal hernia, suggesting a relationship with genetic defects of the elastic fiber and collagen synthesis.