Detecting multiple DNA human profile from a mosquito blood meal.

Criminal traces commonly found at crime scenes may present mixtures from two or more individuals. The scene of the crime is important for the collection of various types of traces in order to find the perpetrator of the crime. Thus, we propose that hematophagous mosquitoes found at crime scenes can be used to perform genetic testing of human blood and aid in suspect investigation. The aim of the study was to obtain a single Aedes aegypti mosquito profile from a human DNA mixture containing genetic materials of four individuals. We also determined the effect of blood acquisition time by setting time intervals of 24, 48, and 72 h after the blood meal. STR loci and amelogenin were analyzed, and the results showed that human DNA profiles could be obtained from hematophagous mosquitos at 24 h following the blood meal. It is possible that hematophagous mosquitoes can be used as biological remains at the scene of the crime, and can be used to detect human DNA profiles of up to four individuals.

Trace samples of human blood in mosquitoes as a forensic investigation tool.

Investigations of any type of crime invariably starts at the crime scene by collecting evidence. Thus, the purpose of this research was to collect and analyze an entomological trace from an environment that is similar to those of indoor crime scenes. Hematophagous mosquitoes were collected from two residential units; saliva of volunteers that were residents in the units was also collected for genetic analysis as reference samples. We examined the allele frequencies of 15 short tandem repeat loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) and amelogenin. A total of 26 female hematophagous mosquitoes were identified as Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus; we were able to obtain 11 forensically valid genetic profiles, with a minimum of 0.028203 ng/µL of human DNA. Thus, the results of this study showed that it was possible to correlate human genetic information from mosquitoes with the volunteer reference samples, which validates the use of this information as forensic evidence. Furthermore, we observed mixed genetic profiles from one mosquito. Therefore, it is clearly important to collect these insects indoors where crimes were committed, because it may be possible to find intact genetic profiles of suspects in the blood found in the digestive tract of hematophagous mosquitoes for later comparison to identify an offender and/or exclude suspects.

Hemolytic uremic syndrome as a primary manifestation of acute human immunodeficiency virus infection.

Hemolytic uremic syndrome may be associated with human immunodeficiency virus infection but it occurs in advanced stages of human immunodeficiency virus disease. As in other forms of hemolytic uremic syndrome plasmapheresis seems to be the treatment of choice. The authors present an unusual case of hemolytic uremic syndrome associated with acute human immunodeficiency virus infection in a 38 year-old black male. The patient was admitted with fever, asthenia, nausea, diarrhea, and reduced urinary output. He was found to have anemia, thrombocytopenia and severe renal failure. Hemolytic uremic syndrome was diagnosed and he was started on plasmapheresis and hemodialysis. Serological tests were consistent with acute human immunodeficiency virus infection: the enzyme linked immunosorbent assay for human immunodeficiency virus was weakly positive, Western Blot test was negative and human immunodeficiency virus RNA quantification was positive, with > 1,000,000 copies/microl. After 4 daily treatment sessions, patient's clinical condition improved and hemoglobin, platelets, lactic dehydrogenase and renal function normalized.

Determination of dimenhydrinate in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a relative bioavailability study.

Here we present a sensitive and specific liquid chromatography-tandem mass spectrometric method for the quantification of dimenhydrinate (I) in human plasma. Sample preparation is conducted using citalopram (II) addition as an internal standard (IS), liquid-liquid extraction with basified plasma using a mixture hexane/acetate (1:1, v/v) as the extracting solvent, and the final extract reconstituted in the mobile phase. I and II (IS) were injected in a C8 column with the mobile phase composed of methanol:isopropanol:water:formic acid (78.00:19.92:2.00:0.08, v/v/v/v) and monitored using a positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 256.0>167.0 and m/z 325.0>109.0 for I and II, respectively. The limit of quantification (LOQ) was 0.4 ng/mL, the dynamic range being 0.4-200 ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of plasma samples taken up to 24 h after oral administration of 100 mg of dimenhydrinate in healthy volunteers demonstrated its applicability to bioavailability studies.

Microbial acid production (Clinpro Cario L-Pop) and dental caries in infants and children.

OBJECTIVE: The purpose of this study was to evaluate the diagnostic utility of the Clinpro Cario-L-Pop test as it relates to dental caries rates and severity in infants and children. METHOD AND MATERIALS: The study population was comprised of 771 infants and children who were on average 5.2 years of age (range of 1.5 to 8 years of age). Examiners conducted dental caries clinical examination using established criteria. In addition, lesion severity was determined be measuring its depth. An indicator swab was applied to the tongue dorsum until completely moistened with saliva. The indicator swab was processed according to the manufacturer's instructions, and acid production was assessed with the aid of a color chart. RESULTS: Twenty-three percent of children were caries free, and 7% (n = 50) of participants were categorized as having low production of lactic acid (scores 1 to 3), 17% (n = 135) moderate production of lactic acid (scores 4 to 6), and 76% (n = 586) high production of lactic acid (scores 7 to 9). There was a tendency for moderate and high lactic acid formers to exhibit higher surface-based caries prevalence rates, higher rates for deep dentinal lesions, and increased lesion severity. There was a linear increase of white spot surface-based lesions from low to high lactic acid formers and for initial dentinal lesions. Clinpro Cario-L-Pop test results, when controlling for age and gender, significantly distinguished caries-free participants from those exhibiting any form of decay. CONCLUSION: These results suggest that Clinpro Cario-L-Pop test was useful in explaining elevated frequency and severity of dental caries in spite of the high levels of decay and of microbial acid production observed in this population.

Evolution of nutritional therapy prescription in critically ill patients.

AIM: The aim of this study was to investigate factors that may affect the evolution of the caloric prescription in critically ill patients. Local: Intensive care unit patients. PATIENTS: 60 patients (33 M and 27 F); median age = 49 (1593) y were followed prospectively. They were divided in three groups according to the diagnostic: (a) trauma (n=20); (b) surgical (n=22), and 3) medical treatment (n=18). Forty-and-one (68.3%) patients received enteral nutrition (EN), 17 (28.3%) parenteral nutrition (TPN), and 2 (3.4%) TNP and EN. Nutritional status was graded B or C by global subjective evaluation. METHODS: Endpoints of the study were the time to begin the nutritional support, success or failure of the caloric prescription, and the evolution of the planned caloric prescription. The caloric evolution was considered as success if the prescription for the patient attained: (a) 25% of the caloric requirements on the 1st day; (b) 50% until the 3rd day; (c) 75% until the 6th day; and (e) 100% until the 10th day of the beginning of the support. RESULTS: In 54 (90%) patients, the nutritional support has begun until 48 h after admission and in 73.3% (44 patients), until the first 24 hours. EN was most prescribed for both trauma and medical patients while NPT was most used for surgical patients (p < 0.01). Success in caloric prescription was obtained in 73.3% (44) of the patients. There was no statistical difference for the success on the evolution of the prescription related to sex, age, diagnostic group, albumin level, type of support, mortality, use of fiber or glutamine. Success was attained earlier in patients without (median = 3.8 [95% CI, 5.7-16.7] days) than with (11.2 [95% CI, 5.7-16.7] days; p < 0.01) mechanical ventilation. CONCLUSIONS: Early nutritional support and success on the evolution of the caloric prescription can be accomplished in most critically ill patients. Evolution of the caloric prescription was slower in mechanical ventilated patients.

Mitochondrial encephalomyopathy and hypoparathyroidism associated with a duplication and a deletion of mitochondrial deoxyribonucleic acid.

Diabetes mellitus is the most frequent endocrinopathy associated with mitochondrial disorders, particularly in patients with duplications of mitochondrial DNA (mtDNA). Although hypoparathyroidism has also been described in mitochondrial diseases, there have been few molecular studies in these cases, most of which identified the presence of single mtDNA deletions in the patients' tissues. We studied muscle DNA of a 12-yr-old patient with incomplete Kearns-Sayre syndrome and hypoparathyroidism. Southern analysis showed that muscle DNA contained three populations of mtDNA: wild type (26%), deleted (65%), and duplicated (9%). To determine the sequence of the breakpoint region from deleted and duplicated mtDNA independently, we isolated the deleted and duplicated mtDNA by gel fractionation of a PstI-digested total DNA. The breakpoint was located at mtDNA positions 5788 and 15,448 for both duplicated and deleted molecules. Our study reinforces the concept that endocrinopathies other than diabetes can be associated with a duplication of mtDNA and gives additional support to the hypothesis that the duplication and deletion of mtDNA are generated from the same recombination event.

Fibrodysplasia ossificans progressiva: report of two cases.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare hereditary connective tissue disease, genetically inherited as an autosomal dominant trait with complete penetrance but variable expressivity. Onset is typically in childhood and progressive involvement of the spine and proximal extremities leads to immobility and articular dysfunction. We present two cases with the typical features of FOP and a review of the pathogenesis, clinical manifestations and treatment options of this rare disease.

Guidelines for clinical studies assessing the efficacy of drugs for the management of acute low back pain.

In this paper we propose guidelines for clinical trials aimed at assessing the efficacy of drugs for acute non-specific low back pain (LBP) with or without radicular pain, preliminary to their approval and registration. To this end, consensus statements were obtained from a group of experts in the fields of rheumatology, clinical medicine, public health and epidemiology. EBM resources were systematically used as references. Four diagnostic categories were defined: type 1--LBP with no radiation; type 2--LBP radiating no further than the knee; type 3--LBP radiating beyond the knee, but with no neurologic signs; and type 4--LBP radiating to a specific and entire leg dermatome, with or without neurologic signs. Studies should be designed on the basis of the claimed indications for the drug, but must be double-blinded whatever the indication. The duration of the study may be shorter for LBP type 1 or 2 (one week) than for LBP types 3 and 4 (up to one month), depending on the aim of the study and the indications for the drug. The comparator may be inactive (placebo) or active (for a superiority trial, e.g., versus paracetamol). Specific inclusion and exclusion criteria have been defined here for each category. An appropriate wash-out period for any drugs that could affect the pain status should be planned. Paracetamol may be allowed as rescue medication. The primary endpoint should be based on a validated pain assessment tool that may be either generic (type 1 or 2) or oriented (back and knee for types 3 and 4). Secondary endpoints could include the assessment of functional performance; the duration of any period of bed-rest; work limitation; a global assessment comprising pain at rest, standing and walking; the time elapsed before epidural injection, the prescription of other therapeutic agents, or surgery; and the use of rescue medication. Adverse events (AE) should be monitored systematically using a methodology that reflects the mode of action of the tested drug. With the application of these guidelines, LBP could serve as an appropriate disease for testing analgesic drugs. Rigorous evaluation may also help to improve the management of acute LBP.

[Serological investigation of influenza type A and B in university students, Brazil]. / Investigação sorológica da influenza tipos A e B em estudantes universitários, Brasil.

Wide serological prevalence of influenza A and B was verified by the serological survey covering 200 students of the University of S. Paulo during the 1984-1985 period. The humoral antibodies were detected by the single radial haemolysis technique, whose arithmetic titres averages were greater for both subtypes, (H1N1) and (H3N2) of the influenza A virus strains recently isolated from the population. However, the situation of this type B virus was not the same as that of type A seeing that the B/Engl/847/73, although an older strain, showed better reactogenicity than the other strains evaluated. It is possible that is was responsible for the primo infection of most of the components of the group surveyed, as the phenomenon of the "Original Antigenic Sin" explains. The antibody responses to subtypes of influenza A and B in this survey demonstrated levels with low SRH titres (2.5 to 3.5 mm) and high SRH titres (> or = 4.0 mm) related to the lowest and highest levels of suggest protection against infection. Individual immunity and persistence of antibody are related to frequency and recent occurrence of exposition to influenza. As concerns the formulation of influenza vaccine it was established that this preserves the antigen selection of the new strains isolated from the population, mainly due to the behavior of the influenza A virus.

Dengue in the south-eastern region of Brazil: historical analysis and epidemiology.

The aim of the study is an historical analysis of the work undertaken by the Public Health organizations dedicated to the combat of the Aedes aegypti, as well as an epidemiological study of persons with unexplained fever, with a view to evaluating the occurrence of dengue within the population. The Mac-Elisa, Gac-Elisa, hemagglutination inhibition, isolation and typage tests were used. Organophosphate intoxication in agricultural workers was also assessed by measuring concentrations of seric cholinesterase. A sera samples of 2,094 were collected in 23 towns, and the type 1 dengue virus was detected in 17 towns and autochthony was confirmed in 12 of them. The cholinesterase was measured in 2,391 sera samples of which 53 cases had abnormal levels. Poisoning was confirmed in 3 cases. Results reveal an epidemic the gravity of which was not officially know. The relationship between levels of IgM and IgG antibodies indicates the outbreak tendency. The widespread distribution of the vector is troubling because of the possibility of the urbanization of wild yellow fever, whereas the absence of A. aegypti in 2 towns with autochthony suggests the existence of another vector. Since there is no vaccine against dengue, the combat of the vector is the most efficient measure for preventing outbreaks. The eradication of the vector depends on government decisions which depend, for their execution, on the organization of the Health System and the propagation of information concerning the prevention of the disease using all possible means because short and long term results depend on the education and the active participation of the entire population.

[Viral infection and fibromyalgia]. / Infecção viral e fibromialgia.

Fibromyalgia (FM) is a very frequent syndrome of unknown cause, characterized by generalized pain, fatigue and a number of tender points to palpation. Among the several etiopathogenic hypotheses discussed, the association of FM with some viral infections has been the object of multiple studies due to its relation and similarity with the chronic fatigue syndrome, acknowledges as being related, although not exclusively, with the chronic infection by the Epstein-Barr virus. Many individual descriptions of association between infection with the human parvovirus B19 and FM led us to carry out this study, comparing the serology for that virus in 52 patients with FM and 39 healthy controls. The titers of specific IgG anti-parvovirus B19 antibodies, indicating previous infection with that virus, were determined in all 91 individuals through ELISA method and at the same laboratory. Results revealed, though not significantly, a greater prevalence of positive titers, of which the mean was also higher, in patients than in controls. When comparing the women from both groups, this tendency was even less perceptible. These data imply that there is no etiologic association between infection with the human parvovirus B19 and FM.

Granulomatosis with polyangiitis initially misdiagnosed as lung cancer.

Granulomatosis with Polyangiitis (GPA), which was formerly named Wegener's Granulomatosis (WG) is a systemic disease characterized by necrotizing granulomatous inflammation and vasculitis that primarily involves upper and lower respiratory tract, as well as kidneys. Diagnosing GPA on the basis of transthoracic fine needle aspiration (TFNA) may be problematic, as it can be misdiagnosed as cancer. We describe a patient with a probable GPA which was originally diagnosed as malignancy, but who responded to lung cancer chemotherapy.

[Myalgia of the girdle in the elderly: an uncommon etiology]. / Mialgias das cinturas no idoso: uma etiologia pouco frequente.

We describe the clinical case of 80 years, caucasian woman, with personal history of osteoporosis and chronic normochromic normocytic anemia (NN). She had a three month history of myalgias of the girdle, stiffness in the morning, exceeding 1 hour, associated with inflammatory arthralgia of the small joints of hands and feet. Complementary exams showed normocytic normochromic anemia with Hg 9.8 g/dL; ESR 44 mm/h; CRP 7 mg/dL. Given the profile suggestive of Polymyalgia Rheumatica started prednisolone 10 mg/day with favorable clinical response. Four months after treatment she started paresthesias of right hand and foot, polaquiuria, petequial lesions in lower limbs and inability to walk; there was worsening of anemia and elevation of the biological parameters of inflammation, beginning of renal insufficiency with creatinine clearance 22 ml/min, proteinuria and eritrocituria. Renal biopsy was compatible with Wegener's granulomatosis/microscopic poliangeite. Vasculitis is a rare disease of the elderly and its clinical presentation is varied. The arteritis of giant cells and Polymyalgia Rheumatica are more common in the elderly. It is not often Polymyalgia-like presentation in cases of Wegener's granulomatosis/microscopic polyangitis.

Separação e identificação de ácidos graxos em formulações cosméticas contendo óleo de castanha do Brasil por eletroforese capilar / Separation and identification of fatty acids in cosmetic formulations containing Brazil nut oil by capillary electrophoresis

An analytical method based on capillary electrophoresis (CE) with a partially aqueous electrolyte system was developed to enable the free fatty acids of Brazil nut oil to be identified in cosmetic formulations. In this study, a gel cream formulation was developed and its oil phase was extracted with a mixture of chloroform-methanol-water (1:2:0.8 v/v/v). The chloroform layer was saponified with a methanolic solution of NaOH (0.5 mol L-1) at 75-80 °C for 25 minutes. Experiments were carried out on a Beckman PACE/MDQ CE system (Fullerton, CA, USA) equipped with an on-column, diode-array detection system set at 254 nm and at 25ºC. The electrolyte consisted of 12.5 mmol L-1 sodium tetraborate buffer pH 7.0, 12.5 mmol L-1 polyoxyethylene 23-lauryl ether, 7.5 mmol L-1 sodium dodecylbenzenesulfonate (used as chromophore for indirect UV detection) and acetonitrile (35% v/v). The proposed method allowed the separation and identification of the fatty acids of Brazil nut oil in a cosmetic gel cream, as well as enabling possible interference by the oily phase components in the formulation to be identified.

Um método de análise por eletroforese capilar com sistema de eletrólito parcialmente aquoso foi desenvolvido para identificar os ácidos graxos livres do óleo de Castanha do Brasil em formulações cosméticas. No presente trabalho foi desenvolvida uma formulação cosmética (gel creme) cuja fase oleosa foi extraída com uma mistura de clorofórmio-metanol-água (1:2:0.8 v/v/v). A camada de clorofórmio, foi saponificada com solução de NaOH em metanol (0,5 mol L-1) a 75-80 °C durante 25 minutos. Os experimentos foram realizados em sistema de eletroforese capilar Beckman PACE/MDQ (Fullerton, CA, USA), com detecção de arranjo de diodos a 254 nm e a 25 ºC. O eletrólito utilizado foi 12,5 mmol L-1 de tetraborato de sódio tampão a pH 7,0, 12,5 mmol L-1 de éter de polioxietileno 23-lauril, 7,5 mmol L-1 de dodecilbenzenosulfonato de sódio (utilizado como agente cromóforo para detecção UV indireta) e acetonitrila (35% v/v). O método proposto permitiu a separação e a identificação dos ácidos graxos do óleo de Castanha do Brasil em formulações cosméticas, bem como possibilitou a identificação de interferências presentes na fase oleosa da formulação.

Association between Pro12Ala polymorphism of the PPAR-gamma2 gene and insulin sensitivity in Brazilian patients with type-2 diabetes mellitus.

AIM: Peroxisome-proliferator-activated receptor-gamma2 (PPAR-gamma2) is a nuclear receptor that plays an important role in lipid metabolism and insulin sensitivity. The purpose of this study is to investigate the association of Pro12Ala polymorphism at the PPAR-gamma2 gene in Brazilian patients with type-2 diabetes mellitus (T2Dm) and controls (CG). METHODS: Genomic DNA was obtained from 207 unrelated white people presenting with T2Dm and from 170 controls. Anthropometric data included body mass index and waist to hip ratio. Biochemical parameters included fasting plasma glucose, total cholesterol, high- and low-density lipoprotein cholesterol, triglycerides, glycated haemoglobin and insulin. Systolic and diastolic blood pressures were also measured. Screening for mutations in the entire coding region of the PPAR-gamma gene was performed by means of polymerase chain reaction (PCR), single-strand conformational polymorphism and sequencing. Pro12Ala polymorphism was analysed by using PCR-RFLP (restriction fragment-length polymorphism). RESULTS: One base substitution was identified - a C to G substitution in exon B of the PPAR-gamma2 gene. The frequency of the Ala12 allele in T2Dm (0.09) was similar to that found in CG (0.06, p = 0.185). In the T2Dm group, Ala12 allele was associated with lower fasting plasma insulin levels (p = 0.036) and higher insulin sensitivity (p = 0.049) by means of homeostasis model assessment. Among obese people, there was no association between any of the T2Dm or obesity-related traits and the Pro12Ala polymorphism. CONCLUSIONS: The results of our study suggest that people with the Ala12 allele of the PPAR-gamma2 gene could be more sensitive to insulin than those carriers of the Pro12 allele among Brazilian Caucasians.

Effect of the peroxisome proliferator-activated receptor-gamma C161T polymorphism on lipid profile in Brazilian patients with Type 2 diabetes mellitus.

AIM: The aim of the present study was to examine the effects of the C161T polymorphism of the peroxisome proliferator-activated receptor gamma (PPARgamma) gene in Brazilian subjects with Type 2 diabetes mellitus (T2DM) and controls residing in Sao Paulo City, Brazil. METHODS: Genomic DNA was obtained from 207 patients with T2DM and 170 unrelated normoglycemic individuals (CG). Anthropometric data included: body mass index, waist, hip, waist-to-hip ratio; biochemical parameters: fasting plasma glucose, total cholesterol, HDL- and LDL-cholesterol, triglycerides, glycated hemoglobin and insulin. Systolic and diastolic blood pressure was also measured. Screening for mutations in the entire coding region of the PPARgamma gene was carried out by PCR, single strand conformational polymorphism analysis (SSCP) and sequencing. C161T polymorphism was analyzed by PCR-RFLP. RESULTS: The C161T polymorphism was the only variant found in exon 6 of the PPARgamma gene. The frequency of the 161T allele in T2DM (0.10) was similar to that found in CG (0.07, p=0.210). Serum triglycerides (p=0.040), VLDL-cholesterol (p=0.040) and Atherogenic Index of Plasma (AIP; p=0.003) were significantly lower in 161T allele carriers than non-carriers in women of the T2DM group. CONCLUSIONS: Our results show that the C161T polymorphism in the PPARgamma gene is not associated with variables related to T2DM or insulin resistance in the Brazilian population. However, a reduction of serum triglycerides and AIP was observed in women with 161T allele of the C161T polymorphism of the PPARgamma gene.