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.

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.

Biological activity of the mite Sancassania sp. (Acari: Acaridae) from bat guano associated with the pathogenic fungus Histoplasma capsulatum

Mites and the mammal pathogenic fungus Histoplasma capsulatum are the major components of bat guano microbiota. Interactions between mites and H. capsulatum were evaluated under laboratory conditions. Acarid mites, mainly Sancassania sp., were the most abundant microarthropod in the sampled guano of the Mexican bat Tadarida brasiliensis mexicana and, based on its morphology, Sancassania sp. was similar to the cosmopolitan species Sancassania sphaerogaster. The mycophagous and vectoring activities of this mite were tested for H. capsulatum and two other fungal species, Sporothrix schenckii (pathogenic) and Aspergillus sclerotiorum (non-pathogenic). S. ca. sphaerogaster was able to reproduce in H. capsulatum and S. schenckii colonies, multiplying in great numbers under controlled fungal mycelial-phase culture conditions. H. capsulatum colonies were completely destroyed after 14 days of in vitro interaction with mites. In contrast, S. ca. sphaerogaster did not reproduce in A. sclerotiorum cultures. S. ca. sphaerogaster was found vectoring H. capsulatum, but not the two other fungal species studied.

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.

Parasitism of the mite Acarophenax lacunatus on Tribolium castaneum

The objective of this work was to assess the parasitism potential of Acarophenax lacunatus (Cross & Krantz) on Tribolium castaneum (Herbst). The experimental units encompassed Petri dishes containing 25 g of wheat infested with 20 adults of this beetle species. After seven days of infestation, different mite densities (0, 2, 4, 6, 8 and 10 females) were inoculated into the Petri dishes. The increase in mite density led to a linear increase in parasitism, and a consequent reduction in populations of this insect. It is possible to use this mite species in the biological control of T. castaneum.

Soil oribatid mite in four typical vegetation communities of Misiones forest in the Iguazú area, Argentina

The objective of this work was to assess the soil oribatid mite communities in four sites of the Upper Paraná Bosque Atlántico, in the Iguazú National Park, Argentina and in surrounding areas: bamboo forest, palm forest and two mixed forests. A comparison between each pair of sites, based on the presence-absence of oribatid species, was performed using Jaccard's index. This is the first systematic sampling of oribatid mites in this area. A total of 56 genera and 96 oribatid species were found, 25 and 49 of them, respectively, are new citation for Argentina. The highest similarity was found between mixed forests. Almost 68% and 34% of the genera were cited for similar biotopes in Brazil and Paraguay, respectively.

Ion pair-dispersive liquid-liquid microextraction of trace amount of rhodium ion in water and road dust samples prior to flame atomic absorption spectrometry determination

A simple ion pair-dispersive liquid-liquid microextraction method was proposed for preconcentration trace amounts of rhodium. An ion association complex of RhCl4- and tetradecyldimetylbenzylamonium was extracted into cholorobenzene. The volume and the type of extractive and dispersive solvents, the extraction time and the pH of the aqueous solutions were optimized. The calibration curve was linear in the range of 0.6-500 ng mL-1 of rhodium. The limit of detection was 0.10 ng mL-1 in initial solution and preconcentration factor was 40. The proposed method was successfully applied to the extraction and determination of rhodium in road dust and water samples.

Ligandless cloud point extraction of trace amounts of palladium and rhodium in road dust samples using Span 80 prior to their determination by flame atomic absorption spectrometry

In this study, a procedure is developed for cloud point extraction of Pd(II) and Rh(III) ions in aqueous solution using Span 80 (non-ionic surfactant) prior to their determination by flame atomic absorption spectroscopy. This method is based on the extraction of Pd(II) and Rh(III) ions at a pH of 10 using Span 80 with no chelating agent. We investigated the effect of various parameters on the recovery of the analyte ions, including pH, equilibration temperature and time, concentration of Span 80, and ionic strength. Under the best experimental conditions, the limits of detection based on 3Sb for Pd(II) and Rh(III) ions were 1.3 and 1.2 ng mL-1, respectively. Seven replicate determinations of a mixture of 0.5 µg mL-1 palladium and rhodium ions gave a mean absorbance of 0.058 and 0.053 with relative standard deviations of 1.8 and 1.6%, respectively. The developed method was successfully applied to the extraction and determination of the palladium and rhodium ions in road dust and standard samples and satisfactory results were obtained.

Spatial variability of air dry bulb temperature and black globe humidity index in a broiler house during the heating phase

The air dry-bulb temperature (t db),as well as the black globe humidity index (BGHI), exert great influence on the development of broiler chickens during their heating phase. Therefore, the aim of this study was to analyze the structure and the magnitude of the t db and BGHI spatial variability, using geostatistics tools such as semivariogram analysis and also producing kriging maps. The experiment was conducted in the west mesoregion of the states of Minas Gerais in 2010, in a commercial broiler house with heating system consisting of two furnaces that heat the air indirectly, in the firsts 14 days of the birds' life. The data were registered at intervals of five minutes in the period from 8 a.m. to 10 a.m. The variables were evaluated by variograms fitted by residual maximum likelihood (REML) testing the Spherical and Exponential models. Kriging maps were generated based on the best model used to fit the variogram. It was possible to characterize the variability of the t db and BGHI, which allowed observing the spatial dependence by using geostatistics techniques. In addition, the use of geostatistics and distribution maps made possible to identify problems in the heating system in regions inside the broiler house that may harm the development of chicks.