Detection of Chlamydia trachomatis by nucleic acid sandwich hybridization

Abstract Chromosomal DNA fragments from Chlamydia trachomatis serotype L2 were shotgun-cloned into pBR322. A C. trachomatis -specific clone was further subcloned to produce specific DNA reagents for the identification of C. trachomatis by nucleic acid sandwich hybridization. The chosen DNA reagents from serotype L2 also hybridized with all the other chlamydial serotypes tested, but not with the DNA from 41 unrelated organisms. The sensitivity of the sandwich hybridization test was 10⁶ DNA molecules. The applicability of the test for routine diagnostic use was demonstrated by a pilot study in which C. trachomatis was directly detected from genital specimens.     

Identification of an iron-responsive protein that is antigenic in patients with Chlamydia trachomatis genital infections

Chlamydia trachomatis is an important cause of immune-mediated damage to the reproductive tract of infected patients. Certain chlamydial antigens and host genetic factors have been identified as contributing to immunopathological events, but a comprehensive understanding of specific components involved in destructive vs. protective immune responses to chlamydial infections is far from clear. In this study, it is shown that C. trachomatis-infected patients generate antibodies against an iron-responsive chlamydial protein, YtgA. The identity of YtgA was confirmed by mass spectrometry following two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. This finding underscores a necessity to examine patient sera samples to identify chlamydial antigens that are likely encountered and important to the immune response during human infections.     

肺炎支原体和沙眼衣原体致儿童急性下呼吸道感染的研究

本文旨在研究儿童社区获得性急性下呼吸道感染(ALRTI)中肺炎支原体(MP)和沙眼衣原体(CT)的感染特征。采用实时荧光定量聚合酶链反应(PCR)检测2006年10月～2008年2月因ALRTI收入复旦大学附属儿科医院的患儿呼吸道标本中MP和CT的DNA,并分析2种病原体感染患儿的临床特征与实验室检查结果。在1312份深部鼻咽分泌物标本中,MP和CT检出率分别为7.85%(103/1312)和2.97%(39/1312)。MP在5岁以上患儿中的检出率为33.33%(30/90),而CT在3个月以内患儿中的检出率为6.28%(31/494)。MP感染后易出现40℃以上高热,较少发生发绀与重症呼吸道感染;白细胞计数常无明显升高,但C反应蛋白升高较多见。CT感染后40℃以上高热和重症呼吸道感染少见,C反应蛋白升高也较少见。结果提示,在5岁以上儿童的社区获得性ALRTI中,MP是重要的病原体;而在3个月以内儿童中,CT为常见病原体之一。     

实时荧光聚合酶链反应检测性病患者泌尿生殖道沙眼衣原体的临床分析

采用实时荧光聚合酶链反应（PCR ）检测性病患者泌尿生殖道沙眼衣原体,进行临床和实验室分析。采集380例男女性病患者泌尿生殖道分泌物,进行多形核白细胞数检测和荧光PCR。结果显示,216例男性患者中,尿道多形核白细胞数≥5个者占92．59％,荧光PCR检测沙眼衣原体阳性62例,阳性率为28．70％;沙眼衣原体合并淋病奈瑟菌感染30例,合并感染率为13．89％。164例女性患者中,87例宫颈管内多形核白细胞数≥10个（占53．05％）,荧光PCR检测沙眼衣原体阳性33例（占20．12％）。结果提示,性病门诊开展实时荧光PCR检测沙眼衣原体可提高检测阳性率和控制沙眼衣原体传播。     

男性生殖道真菌感染菌种构成及耐药分析

目的探讨男性生殖道真菌感染病原菌的种类构成及耐药性,为临床治疗提供依据。方法采集2367份男性患者生殖道分泌物立即送检采用法国生物梅里埃公司Vitek2-Compact进行菌种鉴定以及ATB FUNGUS3真菌药敏试剂条进行药敏试验。结果 2 367例男性生殖道炎症患者中,共检出真菌410例,检出率为17.32%,其中白色假丝酵母菌303株,占73.90%;近平滑假丝酵母菌75株,占18.29%;热带假丝酵母菌16株,占3.90%;4种假丝酵母菌对两性霉素B的敏感率为100.00%;白色假丝酵母菌和热带假丝酵母菌对伏立康唑、氟康唑、依曲康唑耐药率分别为1.65%、1.32%、7.59%和12.50%、12.50%、18.75%。结论假丝酵母菌在男性生殖道感染中占有一定比例;菌种以白色假丝酵母菌为主,近平滑假丝酵母菌次之。因为它们对唑类抗真菌药物有不同程度的耐药,所以临床应加强假丝酵母菌的检测和药敏分析,合理选用药物。     

The recA gene of Chlamydia trachomatis: Cloning, sequence, and characterization in Escherichia coli

Abstract The recA gene of Chlamydia trachomatis was isolated by complementation of an Escherichia coli recA mutant. The cloned gene restored resistance to methyl methanesulfonate in E. coli recA mutants. The DNA sequence of the chlamydial gene was determined and the deduced protein sequence compared with other RecA proteins. In E. coli recA deletion mutants, the cloned gene conferred moderate recombinational activity as assayed by Hfr matings. The chlamydial recA gene was efficient in repairing alkylated DNA but less so in repairing of UV damage when compared with the E. coli homologue. As detected by an SOS gene fusion, a small but measurable amount of LexA co-cleavage was indicated.     

Association of Chlamydia trachomatis,Neisseria gonorrhoeae,Mycoplasma genitalium and Ureaplasma species infection and organism load with cervicitis in north Indian population

Cervicitis is predominantly caused by Neisseria gonorrhoeae and Chlamydia trachomatis, which accounts for almost half of all the cases of cervicitis. The role of newer organisms like Mycoplasma genitalium and Ureaplasma sp. and association of bacterial load with cervicitis are also not well established. So the study aimed to determine the relative frequency of these organisms and their load in association with cervicitis cases from north India. A case–control study involving 300 women was conducted using quantitative real-time PCR from endocervical swabs for identification of organisms and quantification of bacterial load. Among 150 cervicitis cases, C. trachomatis, N. gonorrhoeae, M. genitalium and Ureaplasma parvum were detected in 5 (3·3%), 10 (6·6%), 37(24·6%) and 47 (31·3%) respectively. Old age (<0·001, chi-squared test) and irregular menstrual cycles (<0·001, chi-squared test) were significantly associated with cervicitis. M genitalium was the only organism to be associated significantly with cervicitis with regard to age (<0·031) and symptoms like discharge (P < 0·033, chi-squared test) and dysuria (P < 0·044, chi-squared test) in multivariate analysis. Our finding suggests that the bacterial load of these organisms is not significantly associated with cervicitis. However, we found significant association of M. genitalium infection with clinical characteristics of cervicitis cases.     

Comparison of multiplex PCR, enzyme immunoassay and cell culture methods for the detection of enterotoxinogenic Bacillus cereus

Fast and reliable methods are needed for the detection of pathogenic Bacillus cereus which should provide consistent results. Therefore, we tested a panel of 176 strains, including B. cereus strains, B. cereus group strains and other Bacillus spp. with polymerase chain reaction, immunoassays and cytotoxicity tests and assessed the consistency of the results. A screening multiplex PCR for the detection of hbl, nhe, ces and cytK1 as well as two multiplex PCRs for the differentiation of Hbl genes (hblC, hblD, hblA) and Nhe genes (nheA, nheB, nheC) was applied. All PCRs included an internal amplification control. Component specific antibody based immunoassays were used for the detection of the three components of Hbl and Nhe and the overall cytotoxicity to Vero cells and HEp-2 cells was checked. An overall excellent correlation was obtained for the results of the three, methodically independent assays and no false-negative PCR results were seen for any of the strains tested positive in immunoassays and cytotoxicity tests. The three multiplex PCRs proved to be a facile method for the identification of enterotoxinogenic B. cereus isolates.     

Chlamydia trachomatis regulates growth and development in response to host cell fatty acid availability in the absence of lipid droplets

Chlamydia trachomatis (Ct) is a Gram‐negative obligate intracellular pathogen of humans that causes significant morbidity from sexually transmitted and ocular diseases globally. Ct acquires host fatty acids (FA) to meet the metabolic and growth requirements of the organism. Lipid droplets (LDs) are storehouses of FAs in host cells and have been proposed to be a source of FAs for the parasitophorous vacuole, termed inclusion, in which Ct replicates. Previously, cells devoid of LDs were shown to produce reduced infectious progeny at 24 hr postinfection (hpi). Here, although we also found reduced progeny at 24 hpi, there were significantly more progeny at 48 hpi in the absence of LDs compared to the control wild‐type (WT) cells. These findings were confirmed using transmission electron microscopy where cells without LDs were shown to have significantly more metabolically active reticulate bodies at 24 hpi and significantly more infectious but metabolically inert elementary bodies at 48 hpi than WT cells. Furthermore, by measuring basal oxygen consumption rates (OCR) using extracellular flux analysis, Ct infected cells without LDs had higher OCRs at 24 hpi than cells with LDs, confirming ongoing metabolic activity in the absence of LDs. Although the FA oleic acid is a major source of phospholipids for Ct and stimulates LD synthesis, treatment with oleic acid, but not other FAs, enhanced growth and led to an increase in basal OCR in both LD depleted and WT cells, indicating that FA transport to the inclusion is not affected by the loss of LDs. Our results show that Ct regulates inclusion metabolic activity and growth in response to host FA availability in the absence of LDs.     

New,sensitive, radioactive-free bioluminescence-enhanced detection system in protein blotting and nucleic acid hybridization

A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blotting and nucleic acid hybridization is described. The method utilizes antibodies conjugated with alkaline phosphatase or nucleotide probes complexed with alkaline phosphatase. Then the alkaline phosphatase takes part in a reaction by releasing D -luciferin (Photinus pyralis) from D -luciferin-O-phosphate. Liberated D -luciferin reacts with luciferase, ATP and oxygen under light emission. Light is measured using the Argus-100 a photon counting camera system or photographic films. Bound alkaline phosphatase conjugated antibodies or hybridized nucleotide probes can be visualized. The limit of detection is at present 5 to 50 fg of protein (IgG), corresponding, for example to 30 to 300 × 10^?21 mol. This means a much higher sensitivity of the detection system in comparison to systems used at present. Experiments concerning nucleic acid hybridization and visualization of the emitted light by a photon counting camera (Argus-100) are under investigation.     

Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay

A specific solid-phase enzyme immunoassay for the detection of as little as 3–4 pg of indole-3-acetic acid (IAA) is described. The assay involves minimal procedural efforts and requires only standard laboratory equipment. Up to 50 samples in triplicate, processed simultaneously, can be assayed and evaluated in 2.5 h. As little as 1 mg oat coleoptile tissue is sufficient for a quantitative IAA analysis and little or no extract purification is necessary. Using this assay, levels of IAA have been determined in coleoptiles of maize and oat. The distribution of IAA within single coleoptiles was quantitated and the production of IAA during the regeneration of the physiological tip in Avena coleoptiles was investigated. The changes in levels of IAA and other major phytohormones were quantitated during the growth of oat coleoptiles.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - BSA bovine serum albumin - IAA indole-3-acetic acid - TBS Trishydroxymethylaminomethane buffered saline Part 21 in the series Use of Immunoassay in Plant Science     

Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time,age and genetic background

The hypocretins (1 and 2) have emerged as key regulators of sleep and wakefulness. We developed a high-throughput enzyme immunoassay (EIA) to measure total brain hypocretin levels from large numbers of mice. Hypocretin levels were not altered by circadian time or age. However, significant differences in one or both hypocretin peptides were observed between different mouse strains. We studied hypocretin levels in knockout and transgenic mouse models with obesity, circadian gene mutations or monoaminergic defects. Compared to controls, only histamine receptor knockouts had lower hypocretin levels. This was most pronounced in H1 receptor knockouts suggesting the existence of a positive feedback loop between hypocretin and histaminergic neurons.     

Comparison between standard culture and peptide nucleic acid 16S rRNA hybridization quantification to study the influence of physico-chemical parameters on Legionella pneumophila survival in drinking water biofilms

Legionella pneumophila is a waterborne pathogen that is mainly transmitted by the inhalation of contaminated aerosols. In this article, the influence of several physico-chemical parameters relating to the supply of potable water was studied using a L. pneumophila peptide nucleic acid (PNA) specific probe to quantify total L. pneumophila in addition to standard culture methods. A two-stage chemostat was used to form the heterotrophic biofilms, with biofilm generating vessels fed with naturally occurring L. pneumophila. The substratum was the commonly used potable water pipe material, uPVC. It proved impossible to recover cultivable L. pneumophila due to overgrowth by other microorganisms and/or the loss of cultivability of this pathogen. Nevertheless, results obtained for total L. pneumophila cells in biofilms using a specific PNA probe showed that for the two temperatures studied (15 and 20°C), there were no significant differences when shear stress was increased. However, when a source of carbon was added there was a significant increase in numbers at 20°C. A comparison of the two temperatures showed that at 15°C, the total cell numbers for L. pneumophila were generally higher compared with the total microbial flora, suggesting that lower temperatures support the inclusion of L. pneumophila in drinking water biofilms. The work reported in this article suggests that standard culture methods are not accurate for the evaluation of water quality in terms of L. pneumophila. This raises public health concerns since culture methods are still considered to be the gold standard for assessing the presence of this opportunistic pathogen in water.     

Clinical characteristics and molecular epidemiology of human metapneumovirus in children with acute lower respiratory tract infections in China, 2017 to 2019: A multicentre prospective observational study

Human metapneumovirus (HMPV) infection is one of the leading causes of hospitalization in young children with acute respiratory illness. In this study, we prospectively collected respiratory tract samples from children who were hospitalized with acute lower respiratory tract infection in six hospitals in China from 2017 to 2019. HMPV was detected in 145 out of 2733 samples (5.3%) from the hospitalized children. The majority of HMPV-positive children were under the age of two (67.6%), with a median age of one year. HMPV can independently cause acute lower respiratory tract infection in young children, while all patients showed mild clinical symptoms. Of all the co-infected patients, HMPV was most commonly detected with enterovirus (EV) or rhinovirus (RhV) (38.0%, followed by respiratory syncytial virus (RSV) (32.0%). The highest detection rate occurred from March to May in both northern and southern China. Out of 145 HMPV positive samples, 48 were successfully typed, of which 36 strains were subgrouped into subtypes A2c (75%), eight strains were included in subtype B1 (16.7%), and four strains were included in subtype B2 (8.3%). Moreover, 16 A2c strains contained 111-nucleotide duplications in the G gene. Twenty-seven complete HMPV genomes were successfully obtained, and 25 (92.6%) strains belonged to subtype A2c, whereas one strain was included in subgroup B1 and another was included in subgroup B2. A total of 277 mutations were observed in the complete genomes of 25 A2c strains. All results presented here improve our understanding of clinical characteristics and molecular epidemiology of HMPV infection in children.     

Adhesion of Lactobacillus plantarum 423 and Lactobacillus salivarius 241 to the intestinal tract of piglets, as recorded with fluorescent in situ hybridization (FISH), and production of plantaricin 423 by cells colonized to the ileum

AIMS: To determine which intestinal section of pre and postweaned piglets are colonized by Lactobacillus plantarum 423 and Lactobacillus salivarius 241, and follow production of plantaricin 423 in a gastro-intestinal model. METHODS AND RESULTS: Lactobacillus plantarum 423 and Lact. salivarius 241, single or in combination, were administered to 1-, 14- and 28-day-old (postweaned) piglets. According to results obtained by fluorescent in situ hybridization (FISH), Lact. plantarum 423 adhered strongly to the ileum and posterior colon and Lact. salivarius 241 to the duodenum in preweaned piglets. High numbers of strain 241 were recorded in the duodenum and posterior colon of postweaned piglets, whereas strain 423 remained localized to the ileum. Lowering in Enterococcus faecalis cell numbers were recorded when preweaned piglets were challenged with strain 241. Plantaricin 423 was produced for 96 h in the ileum section of a gastro-intestinal model. CONCLUSIONS: Lactobacillus plantarum 423 and Lact. salivarius 241 adhere to different sections of the intestinal tract, depending on the piglet's age. Ent. faecalis were inhibited in vivo, probably by plantaricin 423. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescent in situ hybridization proved valuable in the detection of probiotic bacteria adhered to the intestine. This is the first report of bacteriocin production in a model simulating the porcine gastro-intestinal tract.     

cDNA cloning and in situ hybridization of Delta11-desaturase, a key enzyme of pheromone biosynthesis in Ostrinia scapulalis (Lepidoptera: Crambidae)

Female sex pheromones are considered to be produced in a "pheromone gland" located in the terminal abdominal segments (8th-10th, TAS) of a moth; however, in many moth species, the cells that produce pheromones have not actually been specified. We investigated cells in the TAS that synthesize pheromones in the adzuki bean borer Ostrinia scapulalis, by locating pheromones and their precursors, and mRNA for Delta11-desaturase, a key enzyme in pheromone biosynthesis. We demonstrated that the pheromone components, (E)-11- and (Z)-11-tetradecenyl acetates, and their fatty acyl precursors were specifically contained in the dorsal part of the TAS. A cDNA (OscaZ/E11) that encodes a Delta11-desaturase was cloned from the TAS. RT-PCR and in situ hybridization unequivocally showed that OscaZ/E11 is specifically expressed in the modified epidermal cells located at the dorsal end of the 8th-9th intersegmental membrane.