Inhibition of fibroblast apoptosis by Borrelia afzelii, Coxiella burnetii and Bartonella henselae

Apoptosis is a genetically controlled mechanism of cell death involved in the regulation of tissue homeostasis. The aim of this study was to investigate the influence of Borrelia afzelii, Coxiella burnetii, and Bartonella henselae bacteria on apoptosis measured as the level of caspase 3 activity in human fibroblast cells HEL-299. Our findings show that C. burnetii bacteria may inhibit the process of apoptosis in the host cells for a long time. This can permit intracellular survival in the host and mediatingthe development of chronic disease.     

Antibodies to Rickettsia rickettsii, Rickettsia typhi, Coxiella burnetii, Bartonella henselae, Bartonella quintana, and Ehrlichia chaffeensis among healthy population in Minas Gerais, Brazil

Rickettsial diseases except those belonging to spotted fever group rickettsioses are poorly studied in South America particularly in Brazil where few epidemiological reports have been published. We describe a serosurvey for Rickettsia rickettsii, R. typhi, Coxiella burnetii, Bartonella henselae, B. quintana, and Ehrlichia chaffeensis in 437 healthy people from a Brazilian rural community. The serum samples were tested by indirected micro-immunoflourescence technique and a cutoff titer of 1:64 was used. The seroprevalence rates for R. rickettsii, R. typhi, C. burnetii, B. henselae, B. quintana, and E. chaffeensis were respectively 1.6% (7 samples); 1.1% (5 samples); 3.9% (17 samples); 13.7% (60 samples); 12.8% (56 samples), and 10.5% (46 samples). Frequent multiple/cross-reactivity was observed in this study. Age over 40 years old, urban profession, and rural residence were significantly associated with some but not all infections rate. Low seropositivity rates for R. rickettsii, R. typhi, and C. burnetii contrasted with higher rates of seropositivity for B. quintana, B. henselae, and E. chaffeensis. These results show that all tested rickettsial species or antigenically closely related possible exist in this particular region.     

Use of molecular biology methods for diagnosing fungal infections

A total of 130 various clinical materials taken from 48 children with suspected systemic fungal infection were used for the study. Clinical samples were tested by use of classical mycological procedures well as by use of molecular technique (PCR assay). The fragments of 125-bp (EO3) and 317 bp (HSP) specific for C. albicans were used for amplification. Fifty seven samples (48%) were positive for Candida albicans and eighty four (68%) by use of PCR. It should be stressed that 4 blood samples, 21 urine samples and 5 other samples were positive by use of molecular technique, only. PCR is sensitive and rapid method for detection and identification of Candida albicans from clinical materials of children with fungal infection. This technique can be applied for monitoring presence of fungal DNA in tested samples during antifungal therapy.     

Towards a rapid molecular diagnostic for melioidosis: Comparison of DNA extraction methods from clinical specimens

Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat.     

桔小实蝇分子生物学研究中样品采集及保存处理的改进方法

介绍了一种果实蝇,桔小实蝇Bactroceradorsalis的虫样采集和保存处理的方法。利用一种简易的诱捕装置对于野外短时间、大面积采集实蝇虫样效果显著;同时,用TE溶液浸泡的干燥虫样适用于桔小实蝇的DNA抽提和PCR扩增。这种虫样采集和保存方法也适用于果实蝇属其它几类实蝇的分子生物学研究。     

Evaluation of anammox pathway recovery after high COD loading using water quality,molecular biology and isotope labelling analysis

Bioprocess and Biosystems Engineering - Anaerobic ammonium oxidation (anammox) pathway is sensitive to organic matter, and its recovery requires reliable evidence regarding the dominance of anammox...     

Evaluation of qPCR-based assays for leprosy diagnosis directly in clinical specimens

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5-10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations.     

The occurrence of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis and Anaplasma phagocytophium in dogs in China

A survey of the occurrence of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis and Anaplasma phagocytophium in dogs was undertaken in the People's Republic of China between October 2008 and October 2009. A total of 600 blood samples were taken from dogs in four cities in China: 300 in Beijing, 150 in Shenzhen, 30 in Shanghai and 120 in Zhengzhou. All samples were tested for the heartworm antigen and antibodies of canine B. burgdorferi, E. canis and A. phagocytophium by using the canine SNAP? 4Dx? test kit. The occurrence of D. immitis, B. burgdorferi, E. canis and A. phagocytophium was 1.17% (7/600), 0.17% (1/600), 2.17% (13/600) and 0.5% (3/600), respectively. In Shenzhen city 2% (3/150), 8.67% (13/150) and 2% (3/150) of samples were positive for D. immitis, E. canis and A. phagocytophium, respectively. The occurrence of heartworm antigen was 0.33% (1/300) in Beijing, 2.00% (3/150) in Shenzhen, 3.33% (1/30) in Shanghai and 1.67% (2/120) in Zhengzhou. We found E. canis and A. phagocytophium only at one site, Shenzhen, while the only occurrence of B. burgdorferi was at Beijing. In conclusion, the dog population in China is at potential risk for D. immitis, B. burgdorferi, E. canis and A. phagocytophium infection, the risk being especially high in southern China.     

Serologic survey of Eptesicus fuscus from Georgia, U.S.A. for Rickettsia and Borrelia and laboratory transmission of a Rickettsia by bat ticks.

Bats and their ectoparasites are associated with bacterial agents of unknown pathogenicity. We tested sera from 56 Eptesicus fuscus from Georgia against Borrelia hermsii, Orientia tsutsugamushi, Rickettsia conorii, and Rickettsia rickettsii. We detected antibodies reactive against a relapsing fever Borrelia and spotted fever group Rickettsia in 3/56 and 1/56 bats, respectively. We attempted to culture Bartonella from the blood of these bats but were unsuccessful. In addition, we fed bat ticks, Carios kelleyi, infected with Rickettsia on a specific pathogen-free guinea pig. The guinea pig had a weak seroconversion to R. rickettsii with a peak titer of 1:32 starting on day 14. Rickettsia was not detected in any of the tissue samples from the guinea pig by molecular means. Our results indicate that E. fuscus is naturally exposed to both a spotted fever group Rickettsia and a relapsing fever group Borrelia. If these agents are transmitted by bat ticks, then people living in close proximity to bat ticks might be exposed.     

Monitoring Ceratomyxa shasta infection during a hatchery rearing cycle: comparison of molecular, serological and histological methods

The prevalence of Ceratomyxa shasta infection in production stocks of steelhead Oncorhynchus mykiss and cutthroat trout O. clarki was monitored using a parasite-specific polymerase chain reaction (PCR) assay. For all 4 stocks of fish followed through their 1 yr rearing cycle, C. shasta infection was detected despite their genetic resistance to the disease and the treatment of the incoming water with ozone. Infection was confirmed using serological methods and standard histological procedures, except when prevalence was low (<10%). This suggests that at the lowest infection levels PCR is more sensitive than other methodologies, and can be used as an early indicator of infection. Results of the PCR assay continued to correlate with histological and serological detection as the numbers of parasites and the lesion severity increased over the rearing cycle. For both steelhead and cutthroat trout, early infections were characterized by large numbers of parasites on the epithelial surface, but with little associated inflammation. At release as yearlings, the infection prevalence in all stocks was greater than 90 % and the inflammatory response in many fish was extensive, with tissue necrosis and mucosal damage. Although C. shasta infections no longer result in high mortality at this facility, results of this study indicate that the parasite remains a contributor to low condition indices in these fish, despite their genetic resistance and ozone disinfection of the water supply.     

Evaluation of the sensitivity of selected serological tests and activity of Coxiella burnetii antigens in the diagnosis of Q fever]

Sensitivity of three serological tests: indirect immunofluorescence assay (If), complement fixation test (CF), and microagglutination test (MA) was evaluated. Sera (118 samples) of humans suspected of C. burnetii infection were tested. Phase II antibodies were detected in 68.6% of sera and phase I antibodies--in 38.2% of sera. Among seropositive to phase II antigen--93.8% of sera reacted in IF, 62.9% in MA, and 32.1% in CF; among seropositive to phase I antigens--100% of samples reacted in IF, 2.6% in MA and 2.6% in CF. Calculated sensitivity of above tests was as followed: IF-93.8%, MA-67.1%, CF-34.2%. Some human sera (6.1%) reacted with hen egg antigens in CF. Reactivity of diagnostic antigens prepared from reference Henzerling strain and four others isolated in Poland with rabbit immune sera and sera of individuals suspected of C. burnetii infection in IF was compared. Generally, the immune sera reacted in highest titres with homologous antigens derived from homologous strains. Human sera showed differentiated activity to particular antigens. The titres of phase I antibodies fluctuated from 0 to 16 depending on the antigen applied. Because of that fact diagnostic antigens should be prepared from the mixture of reference strains and isolates from a region under study.     

Hepatitis A foci in children's collectives studied by using clinico-biochemical, virological and serological diagnostic methods

In 4 foci of hepatitis A (HA) at children's institutions in Moscow 218 children and 30 staff members were examined. Simultaneously with clinico-biochemical studies, specific IgM and IgG in the blood and hepatitis A virus (HAV) antigen in feces were determined by radioimmunoassay. Different forms of HA were detected in 29.1% of children in kindergartens, 19.4% of school children and 3.3% of adults, which was due to great differences in the size of the immune stratum among them (IgG to HAV was detected in 8-10% of children aged 3-6 years and in 83% of adults). In 2/3 of children with HA in the foci the disease was not accompanied by jaundice, 1/5 of them having the inapparent form of this infection. In 45% of the patients HAV antigen was detected in feces, irrespective of the form of HA, 5-14 days before the appearance of hyperfermentemia, and in 2/3 of them this antigen was also detected during the first 6 days after that. Simultaneously with an increase in aminotransferase activity, all of the HA patients showed the presence of specific IgM in their blood.     

Comparison of NaOH-N-acetyl cysteine and sulfuric acid decontamination methods for recovery of mycobacteria from clinical specimens

We compared the NaOH-N-acetyl cysteine (NaOH-NALC) and the sulfuric acid decontamination procedure in the detection of mycobacteria using the Mycobacteria Growth Indicator Tube (MGIT). In total 219 sputum specimens were collected from 142 Zambian patients and subjected to mycobacterial culture. One half of the specimen was decontaminated with NaOH-NALC and the other half was decontaminated with sulfuric acid. From the 438 samples a total of 261 (60%) cultures yielded growth of mycobacteria, consisting of 22 different species. The sulfuric acid method was more successful than the NaOH-NALC method in recovering mycobacteria in MGITs (146 versus 115 respectively, p = 0.001). Of the 146 positive mycobacterial cultures recovered after sulfuric acid decontamination 28 were Mycobacterium tuberculosis, 84 nontuberculous mycobacteria (NTM) and 34 acid fast bacterial isolates which could not be identified to the species level. The 115 mycobacteria recovered by the NaOH-NALC method consisted of 34 M. tuberculosis strains, 55 NTM and 26 acid fast bacteria that could not be identified. The most frequently isolated NTM were Mycobacterium lentiflavum and Mycobacterium intracellulare. Comparing the two decontamination methods the recovery of NTM in the sulfuric acid group was significant higher than in the NaOH-NALC group (p = 0.001). In contrast, no significant difference was found for the recovery of M. tuberculosis. These results show that the decontamination method used affects the recovery of nontuberculous mycobacteria in particular.     

Evaluation of two fluorescent test kits for detection of selectedBacteroides species in clinical specimens

Two pools of isothiocyanate fluorescein-conjugated rabbit antisera toBacteroides fragilis, B. thetaiotaomicron, B. vulgatus, B. distasonis, B. ovatus, and toB. melaninogenicus, subsp.melaninogenicus andintermedius, andB. asaccharolyticus, respectively, were used in immunofluorescent (IF) tests of smears of 122 clinical specimens from a variety of sources, including abscesses and wounds. Smears of broth medium of 14 blood cultures were also tested. Bacteria of the species and subspecies mentioned were detected by the IF tests in 100 and by culture studies in 106 of the 136 speciments tested. Results of the culture studies indicated that 6 tests were false-negative by the IF test, while no false-positive tests were found. The sensitivities of the two test kits were 97% and 85%, respectively, while their specificities were 100%. The predicted value for a positive test with any of the two pools of antisera was 100%, while the predicted value for a negative test was 93% and 97%, respectively. The use of the two test kits (Fluoretec F and M) was found to be a valuable means for a rapid diagnosis of human infections with theBacteroides species mentioned.