Monoclonal antibody based capture ELISA/ELIFA for detection of Coxiella burnetii in clinical specimens

A CAPTURE ELISA/ELIFA system based on monoclonal capture and biotinylated monoclonal detection antibody is described. The assay is fast, highly specific and detects a minimum dose of 2500 Coxiella (C.) burnetii particles. In contrast to the sophisticated and cumbersome isolation procedures, even non-specialized laboratories could use this assay system for investigating clinical samples of different origin for C. burnetii within a short period of time.Corresponding author.     

Characterization and protective effect of a 29 kDa protein isolated fromCoxiella burnetii by detergent Empigen BB

A 29 kDa protein, isolated from the outer membrane ofCoxiella burnetii strain Nine Mile phase I by detergent Empigen BB, was characterized. The failure in removing lipopolysaccharides (LPS) from preparations of the protein by the purification method used indicates a strong binding between proteins and LPS in the outer membrane ofC. burnetii. The protein was immunogenic in mice and protected them against virulentC. burnetii challenge.     

Seroprevalence of infection by Coxiella burnetii in Canary Islands (Spain)

Objectives: The aims of our study were: (i) to know the seroprevalence of Coxiella burnetii infection in the Canary Islands, (ii) to evaluate its epidemiologic features and (iii) to compare the rates of seroprevalence using two different cut-offs (1:20 and 1:80) for the diagnosis of past infection. Methods: We analysed a representative sample of the canarian population. 662 sera were tested. For the detection of IgG and IgM antibodies against C. burnetii phase II antigens an immuofluorescence assay was used. The serologic screening for IgG detection begun with a 1:20 dilution. A titer of IgG 1:80 along with a negative IgM were used as criteria for previous infection. Results: At an IgG antibody titer against C. burnetii of 1:80 as diagnostic for past infection, the observed global seroprevalence was 21.5%. If the cut-off used was 1:20, the observed prevalence increased up to 35.8% (p = 0.001). Significantly different seroprevalence rates were obtained at these different cut-offs when results were analysed for groups of age and socioeconomic status, but not for either the island of origin or for farmers. Conclusion: Our results strongly suggest that Coxiella burnetii infection is endemic in all the Canary Islands. Although it is more frequent in males above 30 years old, it do affect people of all ages, and thus it should be borne in mind in the face of any acute febrile syndrome.     

The 16S/23S ribosomal spacer region ofCoxiella burnetii

The 16S/23S spacer region ofCoxiella burnetii isolate Nine Nile, phase 1, was sequenced. Sequence analysis revealed two tRNA coding regions for tRNA^Ile and tRNA^Ala. DNA sequence alignment demonstrated significant homology with tRNA species fromPseudomonas aeruginosa andRhodobacter sphaeroides, respectively. The non-coding tRNA spacer region was unique toCoxiella burnetii, based on database alignment.     

Isolation ofCoxiella burnetii and of an unknown rickettsial organism fromIxodes ricinus ticks collected in Austria

Two strains ofCoxiella burnetii and two strains of an unidentified rickettsial organism were isolated for the first time fromIxodes ricinus ticks collected in the Alpine region of Tirol, Austria. TheC. burnetii strains belong to the group of agents causing acute forms of Q fever. The other two strains of isolated rickettsial agent share some antigenic epitopes withC. burnetii andR. prowazekii but they differ from them by their high sensitivity to freezing and refreezing and by poor multiplication in yolk sacs of chick embryos. There is at present no evidence that these organisms cause human illness and no ecological information is available. We suggest they may be some new species of rickettsiae or rickettsia-like organisms.     

The use of PCR ribotyping for typing strains ofListeria spp.

The potential of PCR ribotyping for discriminating between and within various species ofListeria, as well as strains ofListeria monocytogenes was examined. In total, 49 strains ofListeria monocytogenes and 12 isolates ofListeria spp. were analyzed. The genomic DNA isolated from these strains was subjected to PCR amplification in the regions between 16S and 5S rRNA. Amplifications were performed with both low and high concentrations of Taq polymerase. Length polymorphisms in the amplified DNA products enabled distinction between various strains of Listeria spp. and between various serotypes ofL. monocytogenes. Six composite profiles for serotype 4b strains, 8 for l/2a strains and 11 for l/2b strains, were observed. In addition, several different PCR ribotyping strategies were evaluated. Restriction fragment length polymorphisms of the spacer region between the 16S and 23S rRNA genes of 16 strains ofL. monocytogenes were not observed, except for two isolates. PCR ribotyping analysis displayed promise as an alternative to traditionalL. monocytogenes molecular typing methods.     

Specific primers for PCR amplification of the ITS1 (ribosomal DNA) of Trypanosoma lewisi

Trypanosoma lewisi is a mild or non-pathogenic parasite of the sub-genus Herpetosoma transmitted by fleas to rats. In a previous study we described pan-trypanosome specific primers TRYP1 which amplify the ITS1 of ribosomal DNA by hybridizing in highly conserved regions of 18S and 5.8S genes. These primers proved to be useful for detecting T. lewisi DNA in laboratory rats, but a recent large scale survey in wild rodents demonstrated a lack of specificity. In the present study, we designed and evaluated mono-specific primers LEW1S and LEW1R, for the detection and identification of T. lewisi by a single-step PCR. These primers were designed inside the highly variable region of the ITS1 sequence of T. lewisi ribosomal DNA. The product size of 220 bp is specific to T. lewisi. The sensitivity limit was estimated between 0.055 and 0.55 pg of DNA per reaction, equivalent to 1–10 organisms per reaction. All the PCR products obtained from 6 different T. lewisi isolates were more than 98% similar with each other and similar to the sequences of T. lewisi already published in Genbank. All DNA of 7 T. lewisi stocks from China gave the specific 220 bp product. We showed that LEW1S and LEW1R primers enabled sensitive detection and identification of T. lewisi infection in laboratory and wild rats. This assay is recommended for monitoring T. lewisi infections in rat colonies or for studying infections in the wild fauna. An absence of cross reaction with human DNA means that these primers can be used to investigate atypical trypanosome infections in humans. Given the risk of T. lewisi infection in human, we believe that these primers will be beneficial for public health diagnosis and rodents investigation programmes.     

Is plasmid based differentiation ofCoxiella burnetii in ‘acute’ and ‘chronic’ isolates still valid?

Ten humanCoxiella burnetii isolates from french patients with acute hepatitis or chronic endocarditis were characterized according to their polymorphism in DNA restriction patterns and differentiated by plasmid-specific PCR. The aim of this investigation was to clarify if the present classification of so called acute and chronicCoxiella burnetii isolates — based on plasmid profile of a so far limited number of partly ancient isolates — could be confirmed with lately isolated organisms of this agent. The data obtained in this investigation indicate that this classification based onC. burnetii plasmid content is no longer justified.     

宏基因组二代测序技术辅助诊断中枢神经系统贝氏柯克斯体感染性血管炎1例

Q热是由贝氏柯克斯体感染引起的人畜共患病,临床表现多样且无特异性,贝氏柯克斯体颅内感染罕见,经常被误诊和漏诊,导致部分患者预后不佳。此文报告1例宏基因组二代测序(mNGS)技术辅助诊断的中枢神经系统贝氏柯克斯体颅内感染性血管炎病例,提示mNGS技术在Q热快速诊断中起到重要作用,通过早期诊断,精准治疗,明显改善患者预后。在此基础上回顾国内外相关文献,总结贝氏柯克斯体颅内感染的临床表现和诊治经验,供国内外同行参考。     

Serological examination of human and animal sera from six countries of three continents for the presence of rickettsial antibodies

Forty samples each of human sera collected in Guinea Bissau, Cape Verde, El Salvador and Iran, and animal sera (goat and cattle from Sri Lanka and sheep from Tanzania) were examined for the presence of antibodies to typhus group (TG) rickettsiae, spotted fever group (SFG) rickettsiae and Coxiella burnetii by enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) test. Of human sera tested, a higher proportion of positive sera were found with ELISA and IFA test for TG, SFG rickettsiae and C. burnetii in El Salvador (42.5 vs 20.0%, 40.0 vs 32.5%, and 27.5 vs 27.5%, respectively) and in Iran (25.0 vs 15.0%, 45.0 vs 27.5%, and 27.5 vs 25.0%, respectively), than in Guinea Bissau and Cape Verde, where they were less than 20.0% except for antibodies to SFG rickettsiae in Guinea Bissau (25.0% with ELISA and 20.0% with IFA test). While all animal sera were negative for the presence of antibodies to TG rickettsiae, a high proportion of sera from Sri Lanka reacted in ELISA and IFA test with SFG rickettsiae and C. burnetii (37.5 vs 20.0% and 27.5 vs 25.0% for goat sera, and 40.0 vs 30.0%, and 17.5 vs 15.0% for cattle sera, respectively). The results obtained indicate that the studied rickettsial diseases can be spread in given territories and may pose a public health problem requiring greater attention than has been paid so far. The suitability of ELISA and IFA test for serological survey of rickettsial antibodies is discussed.     

Seroepidemiology of rickettsial infections in Morocco

The prevalence of antibodies reactive withRickettsia conorii, Rickettsia typhi, Coxiella burnetii andEhrlichia chaffeensis was investigated using indirect immunofluorescence (IFA) test on human sera obtained from 300 blood donors in Casablanca and 126 sera obtained from clinical laboratories in Fez. In sera from Casablanca, antibodies reactive at titers >=1: 32 were found againstR. conorii (7%), andR. typhi (1.7%), but not againstE. chaffeensis. In the sera from Fez, antibodies were also detected againstR. conorii (5.6%),R. typhi (4%), but not againstE. chaffeensis. By Western immunoblotting, seroprevalence forR. conorii was in Casablanca and 4.8% in Fez. Antibodies reactive at titers >=1:50 againstC. burnetii (phase II) were present in sera from Casablanca (1%) and Fez (18.3%).Abbreviations IFA Immunofluorescence assay - MSF Mediterranean spotted fever - PBS Phosphate-buffered saline     

A seroepidemiological study of the risks of Q fever infection in Japanese veterinarians

The causative agent of Q fever, a widespread zoonotic disease, is the bacteria Coxiella burnetii. Although cases of Q fever have been documented in countries throughout the world, the prevalence of the disease in Japan is not yet known. Q fever is a demonstrated occupational hazard to those employed in zoological professions, but the risk to Japanese veterinarians has not yet been quantified. In order to evaluate the risk to Japanese veterinarians, we performed a serological survey using serum samples from 267 veterinarians. Two control groups consisting of 352 medical workers and 2003 healthy blood donors were also evaluated. The antibody titers of the serum samples were measured by indirect immunofluorescence assay (IFA) using phase II C. burnetii Nine Mile strain as the antigen. The positive rate of IgG antibody was 13.5% in the veterinarians, which was higher than in the blood donors (3.6%, p < 0.001) and medical workers (5.1%, p < 0.001). These findings suggest that Japanese veterinarians have a higher risk of infection by C. burnetii than other members of the Japanese population. An interesting finding of this study was that positive rates of IgG and IgM antibodies in the blood donor group were higher in younger individuals. The IgM antibody positive rate was the highest in females under 30 years old.     

Prevalence of anti-Toxoplasma gondii antibodies in the population of the area of Parma (Italy)

Between 1987 and 1991, the prevalence of IgG antibodies toToxoplasma gondii was determined by ELISA in 28,247 serum samples belonging to 19,432 subjects of the area of Parma (Italy). The overall prevalence was 48.5%. The correlation of infection with age, performed on 420 sera, showed a significant increase of positivity until 30–40 approximately years. In consecutive sera obtained from 172 subjects, the IgG and IgM production was analyzed for about 8 months, and four different patterns were delineated which were comprehensive of the wide range of immunological responses toToxoplasma gondii exposure observed. Among pregnant females the prevalence ofanti-Toxoplasma gondii antibodies was 48.7%, and 5 cases of seroconversion during the pregnancy were observed (0.27%) from which two cases of congenital toxoplasmosis originated.     

Real-time PCR assay with an endogenous internal amplification control for detection and quantification of Anaplasma marginale in bovine blood

Bovine anaplasmosis is a tick-borne rickettsial disease, causing significant economic losses in many countries. The main causative agent of bovine anaplasmosis is Anaplasma marginale (Rickettsiales, Anaplasmataceae). To date, several PCR assays for A. marginale DNA detection were proposed, but most of them do not provide an internal amplification control, which allows to prevent false-negative results and is required for reliability of the results of pathogen DNA detection by PCR assay. In the present study, a real-time PCR assay based on the species-specific and highly conserved fragment of msp1α gene was developed for detection and quantification of A. marginale in bovine blood. The real-time PCR assay is able to detect as few as one copу of msp1α gene per reaction. To prevent false-negative results, simultaneous amplification and detection of the bovine genomic DNA fragment as an endogenous internal amplification control (IAC) was provided. The assay can be used as a highly specific and sensitive method for detection and quantification of A. marginale in infected cattle, and for the evaluation of the efficacy of anti-rickettsial drugs and anaplasmosis vaccines.     

Adaptation of Lactobacillus rhamnosus riboflavin assay to microtiter plates

Riboflavin (vitamin B2) is essential to humans and must be obtained through the diet. It plays a significant role in the metabolism of carbohydrates, fatty acids and amino acids. The test microorganism, most commonly used to quantify riboflavin is Lactobacillus rhamnosus ATCC 7469 since this bacterium requires external B2 for growth. The objective of the current study was to reduce the time of the assay and volumes of assay media by adaptation to microtiter plates while still maintaining the repeatability of the original tube assay. A previously developed riboflavin tube assay was used as a guideline for adapting the method to a microtiter plate assay. The standard growth curve for the riboflavin assay was linear from 0 to 20 ng/mL (R²=0.99) and from 0 to 10 ng/mL (R²=0.97) when conducted in microtiter plates and tubes, respectively. The data showed no significant difference between the tube assay and microtiter plate assay (P>0.05) for the commercial maize sample. Commercial cereal and grain samples were analyzed to confirm repeatability among multiple independent trials performed with the microtiter plates. The microtiter assay reduced the amount of time required for sufficient bacterial growth response to generate linear standard curves from 16.5 to 10 h.     

High incidence of Coxiella burnetii markers in a rural population in France

Since Coxiella burnetii, the causative agent of Q fever, is often transmitted from goats and sheep to humans through aerosols, we examined the sera from 168 persons involved in goat breeding in the Centre region of France and 40 members of veterinary and medical staff from the same region for the presence of antibodies against C. burnetii. An immunofluorescence assay was used to detect the presence of antibodies of the IgG isotope against epitopes from phase II of C. burnetii, which are the first antibodies to appear in infected people, and from phase I, which reflect more chronic stages of the infection. Our serological survey showed that most of the tested sera were positive for C. burnetii markers, indicating at least an encounter with the bacterium. In the overall population of 208 subjects, 71% of the sera had antibodies against phase II epitopes (titres 1:40). Among the goat farmers and their immediate families, 78% had antibodies against phase II and 33% against phase I (titres 1:40). Considering only high titres ( 1:320), though, only 37% of the farmers had antibodies against phase II and 15% against phase 1. Only 3 out of 12 veterinarians working in the field had high titres of antibodies against phase II and phase I, while none of 28 members of veterinary and medical laboratories had significant levels of antibodies. These results emphasize the need for closer surveillance of populations at risk for Q fever, to prevent the infection by C. burnetii from reaching chronic stages of the disease.     

微量液体培养法快速鉴别分枝杆菌

目的 建立微量液体培养法快速鉴别MTB和非结核分枝杆菌(NTM)方法,探讨其临床应用价值.方法 将不同浓度的对硝基苯甲酸(PNB)及噻吩-2-羧酸肼(TCH)和液体培养基加入96孔板中,种入待检菌株,37 ℃培养7～10 d观察结果.根据15种分枝杆菌标准株和30株已知MTB临床分离株不同PNB和TCH浓度时生长试验结果,确定PNB和TCH最佳浓度.用建立的微量液体培养法对424株分枝杆菌临床分离株进行菌种鉴定,并与PCR鉴定及测序鉴定结果进行比较.结果 微量液体培养法中PNB浓度200μg/ml时可有效区分MTB和NTM标准株;TCH浓度0.5 μg/ml时可有效区分结核分枝杆菌与牛结核分枝杆菌标准株.本法鉴定424株分枝杆菌临床分离株,以PCR法鉴定结果为标准,可鉴定出313株结核分枝杆菌复合群菌株中306株和全部NTM菌株,鉴定结核分枝杆菌复合群灵敏度为97.8%(306/313),特异度为100.0%(107/107);鉴定NTM的灵敏度为100.0%(107/107),特异度为96.5%(306/317);可鉴定出全部的MTB菌株,鉴定MTB的灵敏度可以达到100.0%(305/305),特异度尚待研究.结论 微量液体培养法可以在7～10 d内对分枝杆菌菌种进行快速鉴定,结果准确度高,操作简便,成本低廉,符合临床快速诊断的需要,适合在我国各级医疗机构推广使用.

Abstract：

Objective This research was to establish a method for fast identification of mycobacteria in microtiter liquid culture and to evaluate its clinical value. Methods 2-thiophenecarboxylic acid hydrazide (TCH) and paranitrobenzoic acid(PNB) at different concentrations were added into liquid culture in 96-well plate. Different mycobacterium standard strains were incubated in liquid culture with PNB and TCH for 7 to 10 days. According to the growth assay for 15 mycobacterium strains and 30 mycobacterium tuberculosis strains, the best PNB and TCH concentration were determined. A total of 424 clinical mycobacterium isolates were identified by microtiter liquid culture at the best PNB and TCH concentration. The results of microtiter liquid culture were compared with those of PCR and DNA sequencing. Results The best concentration of PNB was 200 μg/ml in microtiter liquid culture. Compared with the results of PCR, the sensitivity and specificity for identification of mycobacterium tuberculosis complex in microtiter liquid culture were 97. 8%(306/313) and 100. 0% (107/107) respectively and those for non-tuberculosis mycobacteria in microtiter liquid culture were 100. 0% (107/107) and 96. 5% (306/317) respectively. The best concentration of TCH was 0. 5 μg/ml. Compared with the results of PCR,the sensitivity of mycobacterium tuberculosis in microtiter liquid culture was 100.0% (305/305). The specificity remained under and more studies were needed. Conclusion In microtiter liquid culture with PNB and TCH, mycobacteria can be identified in 7 to 10 days. The results were accurate and the process was simple without expensive equipments. This method meets clinical needs and can be used in all level hospitals in China.     

Q fever seroprevalence and associated risk factors among students from the Veterinary School of Zaragoza, Spain

Q fever is a zoonosis related to the existence of Coxiella burnetiiinfected animals. The authors studied the seroprevalence and risk factors associated to C. burnetiiinfection in veterinary students in Zaragoza (Spain). Sera were collected at the beginning and the end of the academic year (1994–1995) and were tested by Complement fixation test to detect antibodies against C. burnetii.10.02 and 11.02% seroprevalences were observed at the beginning and the end of the study respectively. The cumulative incidence through the period of study was 0.0157. Risk factors associated to C. burnetiiwere multiple: students coursing the speciality in Food Inspection and Technology or the speciality of Animal Production; to practise with living animals in general and particularly with ruminants and to contact frequently with persons who worked with animals, particularly with veterinarians, farmers and animal traders. In parallel, the students coursing the first course showed a significant lower seroprevalence. Male students from the fifth course were significantly more seroprevalent than females, where sex was a protection factor. Concerning the clinical signs asked in the questionnaire, cardiovascular disturbances, flu and/or pneumonia, sweating, transient hypertermia or spondylitis were associated factors. Conversely, a good response after treatment of symptoms was a protection factor. The only risk factor associated with incidence along the year of study was practising in farms. The authors recommend a revision of hygiene measures to control risk factors and the diagnostic of C. burnetiiinfection when populations at risk show the associated symptoms.     

应用巢式聚合酶链反应在唾液中检出幽门螺杆菌

用互补于幽门螺杆菌(HP)尿素酶A基因的两对引物行巢式聚合酶链反应(N-PCR),检测1株HP标准菌株、20株HP临床分离株均阳性,而12种肠道菌均阴性,特异性100%。该法敏感性好可检测0.1fg细菌DNA.57例因上消化道症状行胃镜检查者,取粘膜分别做细菌培养、尿素酶试验、组织学检查和N-PCR检测,其中27例在行胃镜前收集其唾液标本做N-PCR。19例胃粘膜HP阳性者中有11例唾液中检出HP,而8例胃粘膜HP阴性者中有1例唾液N-PCR阳性。作者认为口腔中确实存在HP,该菌可能通过口—口途径传播。     

Detection of Coxiella burnetii in ticks collected in Slovakia and Hungary

A total of 235 adult ticks collected from vegetation in Slovakia and Hungary in 1998–2000 were tested for Coxiella burnetii by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). C. burnetii was identified in six ticks of Ixodes ricinus, Dermacentor marginatus, and Haemaphysalis concinna species.