Standardization of in-house polymerase chain reaction for the identification of Mycobacterium tuberculosis at the reference tropical disease hospital in the State of Goiás, Brazil

This study compares smear, growth in Lowenstein-Jensen medium, and in-house polymerase chain reaction (PCR) techniques for the detection of Mycobacterium tuberculosis. A total of 72 specimens from 72 patients with clinical symptoms of tuberculosis, including 70 sputum and two bronchial aspirate samples, were tested in parallel by smear, culture, and in-house PCR techniques. From these, 48 (66.6%) were negative by the 3 methods, 2 (2.8%) were smear positive and negative by culture and in-house PCR, 11 (15.3%) were both smear and culture negative, and in-house PCR positive, 7 (9.7%) were positive by the 3 methods, 2 (2.8%) were positive by smear and culture, and negative by PCR, 2 (2.8%) were positive by culture and PCR, but smear negative. After the resolution of discrepancies in PCR results, the sensitivity and specificity for in-house PCR technique to M. tuberculosis relative to the culture, were 81.8% and 81.9%, respectively. These results confirm that this method, in-house PCR, may be a sensitive and specific technique for M. tuberculosis detection, occurring in both positive and negative smear and negative cultures.

Pyrethroid insecticide evaluation on different house structures in a Chagas disease endemic area of the Paraguayan Chaco

Insecticide effects of deltamethrin 2.5% SC (flowable solution) on different substrates and triatomine infestation rates in two indigenous villages (Estancia Salzar and Nueva Promesa) of the Paraguayan Chaco are reported. This field study was carried out to determine the extent to which variability in spray penetration may affect residual action of the insecticide. A total of 117 houses in the two villages were sprayed. Filter papers discs were placed on aluminium foil pinned to walls and roofs in selected houses and the applied insecticide concentration was determined by high pressure liquid chromatography (HPLC). The target dose rate was 25 mg a.i./m². The mean actual applied dose in Estancia Salazar was 11.2 ± 3.1 mg a.i./m² in walls and 11.9 ± 5.6 mg a.i./m² in roofs while in Nueva Promesa, where duplicates were carried out, the mean values were 19.9 ± 6.9 mg a.i./m² and 34.7 ± 10.4 mg a.i./m² in walls and 28.8 ± 19.2 mg a.i./m² and 24.9 ± 21.8 mg a.i./m² in roofs. This shows the unevenness and variability of applied doses during spraying campaigns, and also the reduced coverage over roof surfaces. However, wall bioassays with Triatoma infestans nymphs in a 72 h exposure test showed that deposits of deltamethrin persisted in quantities sufficient to kill triatomines until three months post spraying. Knockdown by deltamethrin on both types of surfaces resulted in 100% final mortality. A lower insecticidal effect was observed on mud walls. However, three months after treatment, sprayed lime-coated mud surfaces displayed a twofold greater capacity (57.5%) to kill triatomines than mud sprayed surfaces (25%). Re-infestation was detected by manual capture only in one locality, six months after spraying,

The number of families of Triatoma dimidiata in a Guatemalan house

Triatoma dimidiata is an important vector of Chagas disease in Guatemala. To help understand the biology and population dynamics of the insect, we estimated the number of full sibling families living in one house. Forty one families with an average size of 2.17 individuals were detected using random amplification of polymorphic DNA-polymerase chain reaction genetic markers. This result suggests high levels of migration of the vector, polyandry, and a significant capability for spreading the disease.

Triatoma mexicana (Hemiptera: Reduviidae) in Guanajuato, Mexico: house infestation and seasonal variation

Triatoma mexicana was described by Herrich-Schaeffer in 1848. In 1940, a male specimen was found in Hidalgo. In 1970, this species was recorded in the state of Queretaro. Later, it was registered in Guanajuato and San Luis Potosi. In the present paper we performed an investigation in 545 dwellings from three counties in the state of Guanajuato, Mexico, from March 2003 to May 2004. The search and capture of triatomines were seasonally performed indoors and outdoors. Entomological indexes were calculated. The risk and no risk relations between triatomine presence and housing construction materials were analyzed. Fourteen triatomines were collected indoors and 151 outdoors. The vectors were collected in houses built with either risky and non-risky materials. Adults go indoors but do not settle there, hence, no relationship was found between the building materials and infestation of houses. Conventional interventions like house improvement or insecticide spraying are not efficient for the control of T. mexicana, because its developmental cycle is accomplished outdoors in the area surrounding the houses.

Use of in-house PCR for identification of Mycobacterium tuberculosis in BACTEC broth cultures of respiratory specimens

We evaluated the ability of a PCR assay to identify Mycobacterium tuberculosis complex (MTBC) from positive BACTEC® 12B broth cultures. A total of 107 sputum samples were processed and inoculated into Ogawa slants and BACTEC® 12B vials. At a growth index (GI) > 30, 1.0 ml of the 12B broth was removed, stored, and assayed with PCR. Molecular results were compared to those obtained by phenotypic identification methods, including the BACTEC® NAP method. The average times required to perform PCR and NAP were compared. Of the 107 broth cultures evaluated, 90 were NAP positive, while 91 were PCR positive for MTBC. Of particular interest were three contaminated BACTEC® 12B broth cultures yielding microorganisms other than acid-fast bacilli growth with a MTBC that were successfully identified by PCR, resulting in a mean time of 14 days to identify MTBC before NAP identification. These results suggest that PCR could be used as an alternative to the NAP test for the rapid identification of MTBC in BACTEC® 12B cultures, particularly in those that contained both MTBC and nontuberculous mycobacteria.

Pigments and plasters discovered in the House of Diana (Cosa, Grosseto, Italy): An integrated study between art history, archaeology and scientific analyses

The pigments and the plasters of the Roman frescoes discovered at the House of Diana (Cosa, Grosseto, Italy) were analysed using non-destructive and destructive mineralogical and chemical techniques. The characterization of both pigments and plasters was performed through optical microscopy, scanning electron microscopy and electron microprobe analysis. The pigments were identified by Raman spectroscopy and submitted to stable isotope analysis. The results were integrated with the archaeological data in order to determine and reconstruct the provenance, trade patterns and the employment of the raw materials used for the elaboration of the frescoes.

In house colorimetric reverse hybridisation assay for detection of the mutation most frequently associated with resistance to isoniazid in Mycobacterium tuberculosis

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100% of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

The peri-urban interface and house infestation with Triatoma infestans in the Argentine Chaco: an underreported process?

Peri-urban infestations with triatomine bugs, their sources and their dynamics have rarely been investigated. Here, we corroborated the reported occurrence of Triatoma infestans in a peri-urban area and in neighbouring rural houses in Pampa del Indio, in the Argentine Chaco, and identified its putative sources using spatial analysis and demographic questionnaires. Peri-urban householders reported that 10% of their premises had triatomines, whereas T. infestans was collected by timed manual searches or community-based surveillance in only nine (3%) houses. Trypanosoma cruzi-infected T. infestans and Triatoma sordida were collected indoors only in peri-urban houses and were infected with TcV and TcI, respectively. The triatomines fed on chickens, cats and humans. Peri-urban infestations were most frequent in a squatter settlement and particularly within the recently built mud houses of rural immigrants, with large-sized households, more dogs and cats and more crowding. Several of the observed infestations were most likely associated with passive bug transport from other sources and with active bug dispersal from neighbouring foci. Thus, the households in the squatter settlement were at a greater risk of bug invasion and colonisation. In sum, the incipient process of domestic colonisation and transmission, along with persistent rural-to-urban migratory flows and unplanned urbanisation, indicate the need for active vector surveillance and control actions at the peri-urban interface of the Gran Chaco.

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

Interactions between feather-degrading bacteria and an avian host : zebra finches (Taeniopygia guttata)

Abstract: Birds harbor a variety of bacteria on their plumage, some of which can degrade feathers in vitro. Whether these keratinolytic bacteria are active on live birds and can effect feather degradation on birds is debatable. The effect of such bacteria on the body condition and behavior of birds, is unknown. Using a community of feather-degrading bacteria (EB), we investigate the interaction between the activity and load of such bacteria, on the morphology, body condition, and behavior of zebra finches (Taeniopygia guttata). In Chapter 2, we find that the elevated loads of such microbes lead to a reduction in the expression of morphological traits, such as male bill color (a sexually selected trait) and uropygial gland volume, without reducing body mass, or evoking a cellular immune response. We also suggest the presence of a carotenoid based defense response in hosts, to such elevated loads of microbes and document a sex-based difference in the source of carotenoids used for such a response. In Chapter 3, we investigated the effect of EB loads on male mate choice of zebra finches, wherein male choice of females with elevated and un-altered bacterial loads, varied with male size. We found that larger males preferred females with higher bacterial load and smaller males preferred females with lower bacterial load. Chapter 4 demonstrates that the presence of melanin in feathers reduces the growth and activity of the community of feather-degrading bacteria (EB) and that the EB community can effect feather degradation in humid conditions, without broth. Additional results also demonstrate that the EB community consists of bacteria that can attach themselves to feathers on live birds and those that can live freely on avian plumage. Finally, chapter 5 demonstrates that the self-maintenance, social and sexual behaviors of birds are implicated in the infection and horizontal transmission of bacteria. It also suggests a linked oral - faecal - genital mode of transmission of pathogens in birds. These results demonstrate that differential loads of normal flora of vertebrate hosts can effect changes in their morphology and behavior. They also shed light on the role of feather-degrading bacteria in the evolution of melanin polymorphism in birds and suggest that bacteria can be active on live birds. This thesis also highlights the importance of social and, sexual behaviors of birds, in epidemiology. Résumé: Les Oiseaux ont dans leur plumage diverses bactéries dont certaines dégradent les plumes in vitro, néanmoins. Il n'est pas clair, au vu de précédentes études, si ces bactéries kératinolytiques sont actives sur des oiseaux vivants, et si celles-ci dégradent effectivement le plumage de leur hôte, L'effet de ces bactéries sur la condition corporelle ainsi que le comportement des oiseaux n'est pas connu. A l'aide d'une communauté de bactéries dégradant les plumes (EB), non pathogènes, nous examinons les interactions entre l'activité et la charge bactérienne sur la morphologie, la condition corporelle et le comportement du diamant mandarins (Taeniopygia guttata). Dans le chapitre 2, nous montrons qu'une charge élevée de ces microbes mène à une réduction de l'expression de certains traits morphologiques, tels que la couleur du bec chez le mâle (un trait soumis à sélection sexuelle), ainsi que le volume de la glande uropygienne, sans qu'il y ait une réduction de la masse corporelle, ni déclenchement d'une réponse immune cellulaire. Nos données suggèrent la présence d'une défense chez l'hôte à des charges élevées de bactéries basée sur la présence de caroténoïdes. Nous montrons, de plus une différence liée au sexe dans la source des caroténoïdes utilisé pour cette réponse. Dans le chapitre 3 nous examinons l'influence de la charge bactérienne EB sur le choix des mâles chez le diamant mandarins. Des femelles avec une charge bactérienne normale et augmentée sont choisies par les mâles et ce choix varie avec la taille des mâles. Nous avons mis en évidence que les grands mâles préfèrent les femelles avec une charge bactérienne plus élevée. Les petits mâles préfèrent les femelles avec une charge bactérienne réduite. Le chapitre 4 démontre que la présence de mélanine dans les plumes réduit la croissance et l'activité de la communauté de bactéries dégradant le plumage (EB), et que cette communauté EB peut dégrader les plumes dans des conditions humides, sans milieu de culture liquide. De plus nous montrons que cette communauté consiste en des bactéries qui peuvent s'attacher sur les plumes d'oiseaux vivants ainsi que des bactéries libres. Pour finir nous montrons dans le chapitre 5 que la maintenance corporelle, l'interaction sociale et le comportement sexuel de ces oiseaux sont impliqués dans l'infection et la transmission horizontale de ces bactéries. Nos données suggèrent une transmission orale-fécale-génitale des pathogènes chez les oiseaux. Ces résultats montrent que des charges différentes de la flore bactérienne habituelle et non pathogène de vertébrés peuvent affecter leur morphologie et leur comportement. Ils éclaircissent également le rôle des bactéries dégradant les plumes dans l'évolution du polymorphisme mélanique chez les oiseaux et suggèrent que ces bactéries peuvent être actives sur des oiseaux vivants. Cette thèse souligne également l'importance du comportement social et sexuel des oiseaux dans l'épidémiologie.

Buscador de vehicles in House

Aplicació creada per ser utilitzada en una empresa d'automoció per a la localització de vehicles dins de la factoria.

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA from Bordetella pertussis.

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed.

Leading Double Lives: The History of the Double House in Des Moines, 2005

How people choose to live depends on a variety of social and economic circumstances. Single family dwellings, extended family compounds, and communal apartment blocks are all forms of residential architecture that have ancient roots and occur in every culture. Each form both reflects and affects the living styles of the people who reside there. The double house, which shelters two families in units separated by a wall or floor, balances the convenience of an apartment with the psychological comforts of a home. During the mid to late nineteenth and early twentieth centuries in the United States, the double house was hugely popular in some cities, such as Minneapolis and Milwaukee, but only a minimal presence in Des Moines