多重PCR结合核酸侵入反应及纳米金显色技术检测呼吸道病原体

目的 建立一种呼吸道病原体多重PCR结合核酸侵入反应及纳米金显色的检测方法.方法 针对几种重要的呼吸道病原体(甲型流感病毒、乙型流感病毒、SARS冠状病毒、嗜肺军团菌、脑膜炎奈瑟菌以及腺病毒)保守区基因设计引物,进行多重PCR反应、核酸侵入反应及纳米金显色反应,对多种呼吸道病原体同时进行检测,以人偏肺病毒、呼吸道合胞病毒、人鼻病毒、肺炎链球菌4种呼吸道病原核酸评价其检测特异性,以体外转录的病毒RNA或扩增的PCR片段评价其检测敏感性.结果 成功建立了一种呼吸道病原体多重PCR结合核酸侵入反应及纳米金显色的检测技术.建立的检测方法可特异的检测目的病原体,且与人偏肺病毒、呼吸道合胞病毒、人鼻病毒、肺炎链球菌无交叉反应.该方法对不同靶标的检测灵敏度介0.5 ～50拷贝/μL.结论 建立的呼吸道病原体多重PCR结合核酸侵入反应及纳米金显色技术的检测方法,具有较高的检测特异性及灵敏度,检测通量高,肉眼即可观察结果,在传染病病原体检测方面具有广阔的应用前景.     

Use of a New Single Multiplex PCR-Based Assay for Direct Simultaneous Characterization of Six Neisseria meningitidis Serogroups

We developed a new Neisseria meningitidis multiplex PCR to determine six serogroups, including X-specific primers, and to allow direct W135/Y discrimination. This assay offers a simple and low-cost method for serogrouping N. meningitidis from cerebrospinal fluid that could be useful in Africa.Meningitis is one of the various forms of meningococcal diseases, which may occur as epidemics and severe sepsis, often with a fatal outcome (4). An etiologic diagnosis is confirmed by the isolation of Neisseria meningitidis from cerebrospinal fluid (CSF), blood, or other body fluids. Characterization of meningococci by serogroup is compulsory to investigate disease outbreaks and before starting vaccination campaigns. Twelve meningococcal serogroups identified by the capsular polysaccharide antigens are distinguished and chemically defined. Patient strains are nearly always encapsulated, but only six of these groups (A, B, C, W135, Y, and more recently X) (3) have significant pathogenic potential, causing more than 90% of the invasive disease worldwide (12).Vaccines are available against strains of N. meningitidis belonging to serogroups A, C, W135, and Y but not against strains of serogroups B or X. Hence, serogrouping is really necessary to better apply preventive measures (14). Recent outbreaks due to serogroup X have been described in Niger and Ghana (3, 5, 8); thus, the detection of serogroup X becomes essential in African countries and a low-cost assay needs to be developed.Standard PCR-based assays are routinely used for the detection and identification of serogroups A, B, C, W135, and Y. Simultaneous identification of serogroups A, B, C, W135, and Y has been described in one previous report (14) but a subsequent PCR was necessary to distinguish between W135 and Y. Recently, a separate standard multiplex PCR has also been described for the detection of serogroups 29E, X, and Z (1). Alternatively, a real-time quantitative PCR analysis has also been developed for simultaneous serogroup determination (10), but such an assay might be too expensive for African laboratories.Here we report for the first time a standard multiplex PCR-based assay for rapid simultaneous identification of the six serogroups A, B, C, W135, Y, and X, with direct discrimination between W135 and Y. We used oligonucleotides in orf-2 of a gene cassette required for the biosynthesis of the capsule of serogroup A (13) in the synD and synE genes encoding the polysialyltransferase responsible for the polymerization of polymers of sialic acid containing polysaccharides in strains belonging to serogroups B and C, respectively, and in the synF and synG genes, encoding proteins that catalyze the linkage of sialic acid with other sugars in the polysaccharide chains of serogroups Y and W135, respectively (6, 10); these primers were those described by Taha (14). The sizes of the expected amplicons are 450 bp (B), 400 bp (A), 250 bp (C), and 120 bp (W135). For direct identification of serogroup Y, the reverse primer in the synF gene was identical to that previously described (14), but a new forward primer in the synF gene was designed to amplify a Y-specific 75-bp product, distinct from the 120-bp W135-specific amplicon. The 3′ end of the crtA gene, which encodes an outer membrane protein involved in capsule transport, is highly conserved among meningococci irrespective of serogroups, but the 5′ end is very variable (7). Both forward and reverse primers specific to serogroup X were then designed within this variable region, giving rise to a serogroup X-specific 190-bp amplification product (Table ​(Table11).

TABLE 1.

Oligonucleotides used in this study

Simultaneous Identification of 14 Genital Microorganisms in Urine by Use of a Multiplex PCR-Based Reverse Line Blot Assay

The aim of this study was to develop and evaluate a sensitive method for the simultaneous identification of 14 urogenital potential pathogens. A multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to detect 14 urogenital pathogens or putative pathogens, namely Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus type 1 (HSV1) and HSV2, N. meningitidis, Mycoplasma hominis, M. genitalium, and adenovirus, using two species-specific primer pairs and probes for each. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism and was found to be both sensitive and specific. The limits of detection for the mPCR/RLB assay varied among the 14 target organisms from 4.2 × 10⁻¹ to 7.0 × 10⁻¹¹ ng/μl of genomic DNA. There were no cross-reactions among any of the probes. This method was used to test 529 first-voided urine specimens from male patients with and without urethritis attending two Sydney sexual health clinics. One or more target species were detected in 193 (36%) subjects. Of 233 positive results, overall 216 (93%) were concordant between mPCR/RLB and a comparator method (culture and/or species-specific PCR), 9 were positive only by mPCR/RLB, and 8 were positive only by the comparator method. The mPCR/RLB method was an accurate, convenient, and inexpensive method for the detection of multiple potential pathogens in first-voided urine specimens from men.Sexually transmitted infections (STIs) are a major global health problem. Worldwide, an estimated 340 million cases of curable STIs, including chlamydial infection, gonorrhea, trichomoniasis, and syphilis, occur annually, and their incidence is increasing in many parts of the world. In developing countries, their complications rank in the top five disease categories for which adults seek health care (www.who.int/mediacentre/factsheets/fs110/en/). Many STIs cause asymptomatic infection; for example, up to 70% of men and women with gonococcal and/or chlamydial infections are symptom free (www.who.int/mediacentre/factsheets/fs110/en/), which creates the potential for unrecognized transmission with significant implications for both individual and population health.Urethritis is characterized by discharge and dysuria (37) and is broadly classified as nongonococcal (NGU) or gonococcal. It occurs in both men and women but often is unrecognized in women. Acute NGU is one of the commonest STIs affecting heterosexual men, yet a specific pathogen, most commonly Chlamydia trachomatis, is identified in only 50 to 70% of cases (7). Pelvic inflammatory disease is an important complication of STI in women; C. trachomatis and N. gonorrhoeae commonly are implicated, but often the cause is unknown. Bacterial vaginosis is the commonest cause of vaginal discharge and is associated both with recognized STIs and other genital syndromes (3, 18). Additional epidemiological studies are needed to determine the significance of organisms other than recognized genital pathogens in urethral and vaginal syndromes (7, 13-15). In particular, the pathogenic roles, if any, of the two recently defined human Ureaplasma species (10), U. urealyticum (previously U. urealyticum biovar 2) and U. parvum (previously U. urealyticum biovar 1), and several other genital (32, 43, 44) and respiratory pathogens (20, 30, 33, 42) in NGU are unclear.The high level of sensitivity of nucleic acid amplification tests, such as PCR, allows the use of less invasive specimen types, including first-voided urine specimens or self-collected vaginal swabs that are unsuitable for less sensitive methods, such as culture and antigen tests (8). This paper describes the development and evaluation of a multiplex PCR-based reverse line blot (mPCR/RLB) assay (19) that can detect any of 14 recognized and potential genital pathogens in urine specimens for use in clinical and epidemiological studies of genital infections.     

Detection of Highly Prevalent Hepatitis B Virus Coinfection among HIV-Seropositive Persons in Ghana

Simple hepatitis B surface antigen (HBsAg) tests may facilitate ascertainment of hepatitis B virus (HBV) infection in settings with high endemicity but limited infrastructure. We evaluated two rapid HBsAg tests and characterized HBV coinfection in a Ghanaian HIV-positive cohort. Samples from 838 patients were tested by the rapid assays Determine and Vikia and the reference assays Architect, Murex version 3, and Liaison Ultra. The assays were also evaluated using the 2nd International Standard, a seroconversion panel, and two mutant panels. HBsAg-positive samples underwent HBV DNA quantification by real-time PCR and surface and polymerase gene population sequencing. Overall, 140/838 patients (16.7%; 95% confidence interval, 14.2 to 19.2%) were HBsAg positive, and of these, 103/140 (73.6%) were e-antigen negative and 118/140 (84.3%) showed an HBV DNA level of >14 IU/ml (median, 8,279 IU/ml). Assay sensitivities and specificities were as follows: Architect, 97.9 and 99.6%; Liaison, 97.1 and 99.4%; Murex, 98.6 and 99.3%; Determine, 69.3 and 100%; and Vikia, 70.7 and 100%. With Determine, the limit of detection was >1.5 to 3.4 HBsAg IU/ml, and the median HBV DNA loads were 598 and 10,905 IU/ml in Determine-negative and -positive samples, respectively (P = 0.0005). Results were similar with the Vikia assay. HBV DNA sequencing indicated infection with genotype E in 82/86 (95.3%) patients. HBsAg mutations affected assay performance, including a T123A mutant that escaped detection by Architect. Major drug resistance mutations were observed in 4/86 patients (4.6%). The prevalence of HBV coinfection was high in this HIV-positive Ghanaian cohort. The two rapid assays identified HBsAg-positive patients at risk for liver disease with high specificity, albeit with only moderate sensitivity.In recent years, coinfection with hepatitis B virus (HBV) has emerged as a significant cause of morbidity and mortality among HIV-positive patients, due to the promoting effect of HIV on HBV replication and progression of hepatic damage (12, 34, 47, 50). Across Europe, approximately 9% of HIV-positive patients are coinfected with HBV (23). Data from sub-Saharan Africa are limited, primarily due to a lack of routine screening. A number of seroprevalence studies conducted in various HIV-positive populations using various screening and confirmatory modalities have reported coinfection rates of 2 to 20% in east and south Africa (Kenya, Uganda, Rwanda, Malawi, and South Africa) (17, 18, 36, 40) and 12 to 17% in west Africa (Senegal, Burkina Faso, Nigeria, and Ivory Coast) (15, 38, 42, 45). No data are available for Ghana, although a previous national multicenter study described a significant association between HIV and HBV infection among prison inmates and officers (2).Treatment of coinfected patients is facilitated by the availability of nucleoside or nucleotide analogues with dual antiviral activity and can achieve HBV suppression and be of clinical benefit (29, 30, 39). Current guidelines recommend against the use of lamivudine as a single anti-HBV agent due to the high risk of drug resistance (6, 46). Tenofovir, typically used in combination with emtricitabine or lamivudine in the context of highly active antiretroviral therapy (HAART), is currently the preferred option for treating HBV coinfection (46). Expanded access to HAART is dramatically reducing HIV-related mortality in many resource-limited countries, including Ghana (13, 16, 24). In these settings, first-line therapy usually uses lamivudine in combination with either zidovudine or stavudine and nevirapine or efavirenz. Tenofovir is often available but reserved for use after failure of the initial regimen. Hepatitis B testing is not part of routine care. As a result, a substantial proportion of HIV- and HBV-coinfected patients are currently receiving lamivudine as a single anti-HBV agent and are at significant risk of HBV drug resistance and progression of liver disease (46). The lack of routine HBV testing, in turn a reflection of limited laboratory infrastructure, is one obstacle to the improved management of HBV coinfection in these settings.Two previous studies have evaluated the rapid Determine hepatitis B surface antigen (HBsAg) test in African cohorts (36, 41). One small study recently raised significant concerns about the specificity of the assay when used in HIV-infected patients in Malawi (36). The aim of the study was to determine HBsAg seroprevalence in a large cohort of HIV-1-infected patients receiving routine HIV care in Kumasi, Ghana; characterize the HBV virological profile of coinfected patients; and, in this population, evaluate the performance of rapid and simple tests for HBsAg detection, which may allow the introduction of reliable screening in the absence of extensive laboratory infrastructure. The Determine test in particular was evaluated for its performance both on site in Kumasi and by repeat testing of the samples in London, United Kingdom. In addition, the Vikia test, not previously evaluated, was studied as a possible alternative to the Determine test.(This work was presented at the 12th European AIDS Conference, Cologne, Germany, 11 to 14 November 2009, and the 17th Conference on Retroviruses and Opportunistic Infections, San Francisco, CA, 16 to 19 February 2010.)     

Recombinant Helicobacter bilis Protein P167 for Mouse Serodiagnosis in a Multiplex Microbead Assay

Infection of mice with Helicobacter bilis is widespread in research and commercial mouse colonies. Therefore, sensitive, specific, and high-throughput assays are needed for rapid and accurate testing of mice in large numbers. This report describes a novel multiplex assay, based on fluorescent microbeads, for serodetection of H. bilis infection. The assay requires only a few microliters of serum to perform and is amenable to a high-throughput format. Individual microbead sets were conjugated to purified, H. bilis-specific, recombinant proteins P167C and P167D and bacterial membrane extracts from H. bilis and Helicobacter hepaticus. For detecting H. bilis infection in the microbead multiplex assay, P167C and P167D provided significantly higher sensitivities (94 and 100%, respectively) and specificities (100 and 95%, respectively) than membrane extract (78% sensitivity and 65% specificity). Microbead multiplex assay results were validated by enzyme-linked immunosorbent assay. Purified recombinant proteins showed low batch-to-batch variation; this feature allows for ease of quality control, assay robustness, and affordability. Thus, recombinant antigens are highly suitable in the multiplex microbead assay format for serodetection of H. bilis infection.     

Preclinical Assessment of a Fully Automated Multiplex PCR Panel for Detection of Central Nervous System Pathogens

We evaluated a multiplexed PCR panel for the detection of 16 bacterial, viral, and fungal pathogens in cerebrospinal fluid. Panel results were compared to routine testing, and discrepancies were resolved by additional nucleic acid amplification tests or sequencing. Overall, the positive and negative agreements across methods were 92.9% and 91.9%, respectively.     

Utility of a Commercially Available Multiplex Real-Time PCR Assay To Detect Bacterial and Fungal Pathogens in Febrile Neutropenia

Infection is the main treatment-related cause of mortality in cancer patients. Rapid and accurate diagnosis to facilitate specific therapy of febrile neutropenia is therefore urgently warranted. Here, we evaluated a commercial PCR-based kit to detect the DNA of 20 different pathogens (SeptiFast) in the setting of febrile neutropenia after chemotherapy. Seven hundred eighty-four serum samples of 119 febrile neutropenic episodes (FNEs) in 70 patients with hematological malignancies were analyzed and compared with clinical, microbiological, and biochemical findings. In the antibiotic-naïve setting, bacteremia was diagnosed in 34 FNEs and 11 of them yielded the same result in the PCR. Seventy-three FNEs were negative in both systems, leading to an overall agreement in 84 of 119 FNEs (71%). During antibiotic therapy, positivity in blood culture occurred only in 3% of cases, but the PCR yielded a positive result in 15% of cases. In six cases the PCR during antibiotic treatment detected a new pathogen repetitively; this was accompanied by a significant rise in procalcitonin levels, suggestive of a true detection of infection. All patients with probable invasive fungal infection (IFI; n = 3) according to the standards of the European Organization for Research and Treatment of Cancer had a positive PCR result for Aspergillus fumigatus; in contrast there was only one positive result for Aspergillus fumigatus in an episode without signs and symptoms of IFI. Our results demonstrate that the SeptiFast kit cannot replace blood cultures in the diagnostic workup of FNEs. However, it might be helpful in situations where blood cultures remain negative (e.g., during antimicrobial therapy or in IFI).While systemic infection is the most common cause of a febrile neutropenia episode (FNE) with significant effects on morbidity and mortality, only 30% of blood cultures taken at the onset of fever are positive (11, 15). Nonetheless, patients with FNEs are treated with broad-spectrum antimicrobial agents regardless of the result of their blood culture (7) because potentially life-threatening infections need early treatment to ensure better clinical outcome. Noninfective causes of a systemic reaction culminating in a rise in temperature such as tumor fever, drug fever, or transfusion reactions complicate the diagnostic challenge in cancer patients. In addition, the etiology of a deterioration of an FNE during antimicrobial therapy is often difficult to elucidate, since blood cultures are infrequently positive once effective antimicrobial therapy has started (4). Pathogens such as molds which are rarely found in blood cultures are not uncommon in patients with FNEs, particularly if they suffer from hematological malignancies. For these reasons, FNE is one of the conditions where new diagnostic tools to distinguish an infection from a nonmicrobial cause for fever or to identify rare pathogens are most urgently needed. In the past, raised levels of indirect markers such as procalcitonin (PCT) and interleukin 6 (3, 16) have been shown to be associated with bacteremia. Ideally, though, the cause should be identified directly and improvements in the detection of pathogens in the bloodstream should be made. In addition to refinements of the classical blood culture systems, attempts have been made to detect pathogen DNA by means of PCR. Initially, this involved conventional PCR techniques (9) detecting the gene for the 16S subunit of bacterial rRNA for the presence of bacterial DNA. Specification was then carried out by sequencing the PCR product. Later, more rapid methods were developed when real-time PCR became available (1, 14, 17). PCR results are more readily available, and the method also detects remnants of bacteria, which might make it more robust to the influence of antibiotic treatment, while potentially detecting pathogens which do not grow in the blood cultures. However, the main disadvantage, apart from higher costs, a potential for false-positive results during transient bacteremia/fungemia (e.g., during brushing of teeth), and laboratory workload, is the restriction of the spectrum of species detected. In addition, because most PCR methods use smaller sample volumes (commonly 1 to 4 ml [9, 17]), this method depends on a higher concentration of bacteria than that for the blood culture, which theoretically can reveal positive results after one living and propagating bacterial cell has been injected into the culture bottle.A new commercially available kit (SeptiFast) to detect DNA from 20 clinically relevant pathogens has recently been evaluated in a small cohort of neutropenic patients (10) with promising results. The aim of our study was to evaluate the usefulness of the SeptiFast kit in a larger cohort of patients with febrile neutropenia after chemotherapy for hematological malignancies. Also, we sought to determine a correlation between SeptiFast results and clinical findings. Altogether 784 samples from 119 FNEs in 70 patients were analyzed and compared with clinical, microbiological, and biochemical findings in the antibiotic-naïve setting and during antimicrobial therapy.     

Rapid and Simultaneous Detection of blaKPC and blaNDM by Use of Multiplex Real-Time PCR

The increasing incidence of carbapenem nonsusceptibility among clinically important species is of global concern. Identification of the molecular mechanisms underlying carbapenem nonsusceptibility is critical for epidemiological investigations. In this report, we describe a real-time PCR-based assay capable of simultaneously detecting bla_KPC and bla_NDM, two of the most important carbapenemases, directly from culture in less than 90 min. The assay was validated with bla_KPC- and bla_NDM-carrying clinical isolates and demonstrated 100% concordance with the Carba NP test.     

Comparative Evaluation of Two Commercial Multiplex Panels for Detection of Gastrointestinal Pathogens by Use of Clinical Stool Specimens

The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 μl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 μl raw stool; however, 100 μl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.     

Analysis of Dystrophin Gene Deletions by Multiplex PCR in Moroccan Patients

Duchenne and Becker muscular dystrophy (DMD and BMD) are X-linked diseases resulting from a defect in the dystrophin gene located on Xp21. DMD is the most frequent neuromuscular disease in humans (1/3500 male newborn). Deletions in the dystrophin gene represent 65% of mutations in DMD/BMD patients. We have analyzed DNA from 72 Moroccan patients with DMD/BMD using the multiplex polymerase chain reaction (PCR) to screen for exon deletions within the dystrophin gene, and to estimate the frequency of these abnormalities. We found dystrophin gene deletions in 37 cases. Therefore the frequency in Moroccan DMD/BMD patients is about 51.3%. All deletions were clustered in the two known hot-spots regions, and in 81% of cases deletions were detected in the region from exon 43 to exon 52. These findings are comparable to those reported in other studies. It is important to note that in our population, we can first search for deletions of DMD gene in the most frequently deleted exons determined by this study. This may facilitate the molecular diagnosis of DMD and BMD in our country.     

A Mouse with a Monoclonal Primary lmmunoglobulin Repertoire not Further Diversified by V-Gene Replacement

We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional RAG-2 alleles and a λ1 light-chain transgene into the quasi-monoclonal (QM) mouse, which contains a “knocked-in” V_HDJ_H rearrangement. Thus, this mouse, which we call MonoB, is devoid of T cells and contains preformed heavy- and light-chain genes encoding immunoglobulin with an anti-NP specificity. The MonoB mouse allows us to examine immunoglobulin diversity in the absence of processes mediated by V(D)J recombination and T cells. Here we report that not only is the MonoB''s primary immunoglobulin repertoire monoclonal, but also that its secondary repertoire is not further diversified by V-gene replacement or gene conversion. Among 99 heavy-chain and 41 λ light-chain genes from peripheral B cells of the MonoB mouse, there were no V-gene replacements. When compared to the QM mouse, which has RAG activity, and for which V-gene replacement is the major diversifying mechanism, these data suggest that V-gene replacement is mediated by V(D)J recombination and not by other recombination systems.     

本院10年内抗菌药物不良反应的回顾性分析

目的 评估广东省中医院10年间由于抗菌药物引起的药物不良反应情况.方法 收集10年间广东省中医院使用抗菌药物引起的不良反应报告,对其进行回顾性分析.结果 对于222例纳入研究的报告分析显示,静脉用药最容易引起不良反应,占98.2％,其中头孢类引起者占31.5％,发生的不良反应涉及各个系统,表现多样,最常出现在用药后24h内.结论 抗菌药物发生的不良反应表现多样,在用药后24h内最常见,临床应针对预防.     

Simultaneous serodetection of 10 highly prevalent mouse infectious pathogens in a single reaction by multiplex analysis

Under current practices of mouse colony maintenance, sera from mice are analyzed for antibodies against several widespread infectious pathogens by conventional immunoassays, generally enzyme-linked immunosorbent assay (ELISA). To test for multiple agents, these methods consume large volumes of mouse serum and are laborious and time-consuming. More efficient immunoassays, using small amounts of sample, are therefore needed. Accordingly, we have developed a novel multiplex diagnostic system that employs fluorescent microbeads, coated with purified antigens, for simultaneous serodetection of 10 mouse infectious agents. Individually identifiable, fluorescent microbeads were coated with antigens from Sendai virus, mouse hepatitis virus, Theiler's mouse encephalomyelitis virus/GDVII strain, mouse minute virus, mouse cytomegalovirus, respiratory enteric orphan virus (Reo-3 virus), mouse parvovirus, calf rotavirus for epizootic diarrhea virus of infant mice, vaccinia virus for ectromelia virus, and Mycoplasma pulmonis. Standard sera, singly positive for antibodies to individual infectious agents, were generated by inoculation of BALB/cj and C57BL/6j mice. Sera from these experimentally infected mice, as well as sera from naturally infected mice, were analyzed using a mixture of microbeads coated with antigens of the 10 infectious agents listed above. Results demonstrated that the multiplex assay was at least as sensitive and specific as ELISA for serodetection. Importantly, the multiplex assay required only 1 microliter of serum for simultaneous serodetection of the 10 mouse infectious agents in one reaction vessel. Thus, this multiplex microbead assay is a reliable, efficient, and cost-effective diagnostic modality that will impact serosurveillance of mice used in research.     

Multiplex PCR-Ligation Detection Reaction Assay for Simultaneous Detection of Drug Resistance and Toxin Genes from Staphylococcus aureus,Enterococcus faecalis,and Enterococcus faecium

A multiplex PCR-ligation detection reaction (PCR-LDR) assay was developed for rapid detection of methicillin, tetracycline, and vancomycin resistance, as well as toxic shock toxin and Panton-Valentine leukocidin. The assay was tested on 470 positive blood culture bottles containing Staphylococcus aureus or enterococci. PCR-LDR exhibited a sensitivity and specificity of ≥98% for all components except tetracycline resistance, which had a sensitivity of 94.7%. Rapid and sensitive detection of antimicrobial resistance and virulence genes could help guide therapy and appropriate infection control measures.Previous studies have demonstrated the ability of multiplex PCR-ligation detection reaction (PCR-LDR) assays to detect and identify a variety of clinically significant bacteria and flaviviruses (4, 12, 14). We have now developed a multiplex PCR-LDR assay to directly determine antibiotic resistance profiles, as well as the toxic shock syndrome (Tsst-1) and Panton-Valentine leukocidin (PVL) toxins, of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium present in positive blood culture bottles. Rapid identification of antimicrobial resistance and virulence determinants could have a significant impact on reducing morbidity, mortality, and health care costs. Methicillin and vancomycin resistance genes were included in the assay because of the clinical importance of vancomycin-resistant enterococci (VRE), methicillin-resistant S. aureus (MRSA), and most recently vancomycin-resistant S. aureus (VRSA) infections (5, 15, 18). Tetracycline resistance genes were included because of renewed interest in this group of antibiotics due to their activity against several biothreat agents and the increase in community-acquired MRSA (7, 8, 21, 22).Positive blood cultures containing S. aureus, E. faecalis, and/or E. faecium were obtained from patients at Weill Cornell Medical Center, New York Presbyterian Hospital (WCMC-NYPH). Blood cultures were collected and incubated in a BacT/Alert system (bioMérieux, Durham, NC) in accordance with the manufacturer''s instructions. When a blood culture was read as positive by the BacT/Alert, 100-μl aliquots of the blood culture were transferred in quadruplicate to a 96-deep-well plate for subsequent extraction of DNA; negative controls containing only Tris-EDTA buffer were incorporated into each 96-well plate, and the plates were stored at −70°C. An additional 400 μl of the positive blood culture was stored for retesting of any discordant results. Bacteria were identified using standard methods. Additional clinical isolates of S. aureus, E. faecalis, and E. faecium were collected from wound, urine, respiratory, or autopsy samples. These isolates were spiked into negative clinical blood culture bottles as described previously (12). The use of human clinical samples in this research has complied with all relevant federal guidelines and WCMC-NYPH institutional policies.Bacterial isolates previously shown by sequencing to contain the mecA, vanA, vanB, tetK, tetL, tetM, or lukS-lukF (PVL) gene(s) were obtained from the WCMC-NYPH clinical microbiology collection. E. faecium N97-0330 (vanD3) and E. faecium N03-0072 (vanD5) (1) were provided by Michael Mulvey. E. faecium NEF1 (vanD1) (9) was provided by Albert Sotto and Jean Phillippe Lavigne.DNA from clinical specimens was extracted using an ABI 6100 nucleic acid prep system (Applied Biosystems, Foster City, CA) as described previously (12). The procedure used fine grade charcoal to remove sodium polyanetholesulfonate (SPS), an essential component of blood culture media and a potent inhibitor of PCR.PCR and LDR primers were designed based on sequences obtained from the GenBank database (http://www.ncbi.nlm.nih.gov) as previously described (12). In instances where sequences did not share 100% identity, multiple PCR/LDR primers were designed over the selected region. To prevent failure of PCR amplification, two PCR primer pairs were designed for each of the gene amplicons. The 44 PCR primers used in the multiplex PCR assay are presented in Table SA1 in the supplemental material. The 71 LDR primers used in the multiplex LDR assay are presented in Table SA2 in the supplemental material. Two to three LDR primer pairs were designed for each of the PCR amplicons. Identification of at least two LDR signals for any gene was required for a positive result. The assay also incorporated two previously defined positive amplification controls specific to the 16S rRNA genes, identifying the bacteria as Staphylococcus species, E. faecium, or E. faecalis (12). PCRs and LDR were performed as described in Table SA3 in the supplemental material. Capillary electrophoresis (CE) was performed and analyzed as previously described (4, 12, 14) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.(A) Capillary electrophoresis (CE) trace for the antibiotic resistance, and toxin PCR-LDR assay of a vancomycin-resistant E. faecium isolate. The x axis shows the LDR product size, and the y axis shows fluorescence intensity of the bands. Red arrows indicate signals that are unique to the 16S rRNA genes of E. faecium and the vanA gene. (B) The CE data are displayed as a reconstructed gel image generated by a software program developed in our laboratory. The left panel represents the size and fluorescence of the 16S rRNA gene LDR products which act as a positive control and differentiate between the bacteria E. faecalis, E. faecium, and Staphylococcus species. The middle panel represents the LDR signals that identify the presence of methicillin resistance (mecA), vancomycin resistance (vanA, vanB, vanD), tetracycline resistance (tetL tetK, tetM), the toxic shock toxin gene (tst), and the PVL toxin genes (lukS-lukF). Data in the right panel represent two blood cultures. In one, the 16S rRNA gene control identified E. faecium, and the vanA gene was detected, indicating a VRE; in the other the 16S rRNA gene control identified S. aureus, and the mecA and lukS-lukF genes were detected, indicating an MRSA gene encoding the PVL toxin.The antibiotic susceptibility of each organism was determined by VITEK2 (bioMerieux, Durham, NC). If an intermediate or discrepant call was made by the VITEK2, secondary/confirmatory testing was performed by Etest (AB Biodisk, Solna, Sweden). MICs were interpreted based on CLSI guidelines (3). Production of Tsst-1 was confirmed by reverse passive latex agglutination, using the TST-RPLA kit (Oxoid, United Kingdom). S. aureus ATCC 51651 and ATCC 13566 were used as positive and negative controls, respectively. The presence of the PVL toxin genes was confirmed by amplification and sequencing by using the primers luk-PV-1 and luk-PV-2 (10).The resistance and toxin genotypes of 470 blood culture samples (193 positive blood cultures and 277 clinical isolates spiked into negative blood cultures) were examined using the multiplex PCR-LDR assay. Twenty of the positive blood cultures were polymicrobial; fourteen of these contained at least one S. aureus, E. faecium, or E. faecalis resistance gene.Correlation of the multiplex PCR-LDR assay with standard susceptibility testing for pure cultures is presented in Table ​Table1.1. The PCR-LDR assay detected methicillin resistance in S. aureus with 99.2% sensitivity and 99.1% specificity. One isolate was mecA positive and oxacillin susceptible, and one was mecA negative and oxacillin resistant. Vancomycin resistance was detected in E. faecium and E. faecalis with 98.6% sensitivity and specificity compared to susceptibility testing. Almost all (98.4%) vancomycin resistance in E. faecium was due to vanA, whereas vanB was responsible for an appreciable portion (44.4%) of resistance in E. faecalis (see Table SA4 in the supplemental material). Two isolates of E. faecalis were vancomycin susceptible but were positive for vanA or vanB. Low-level expression of the van genes may have failed to raise the MIC above the breakpoint for resistance. One isolate of E. faecium was vancomycin resistant but was negative for vanA, vanB, and vanD. Resistance may have been due to gene sequences not incorporated into this assay, such as vanE or vanG (5).

TABLE 1.

Antibiotic resistance phenotypes and PCR-LDR gene identification in blood cultures containing S. aureus or enterococci

PsrP,a Protective Pneumococcal Antigen,Is Highly Prevalent in Children with Pneumonia and Is Strongly Associated with Clonal Type

Invasive pneumococcal disease (IPD) is a major health problem worldwide. Due to ongoing serotype replacement, current efforts are focused in an attempt to identify the pneumococcal antigens that could be used in a next-generation multivalent protein vaccine. The objective of our study was to use real-time PCR to determine the distribution and clonal type variability of PsrP, a protective pneumococcal antigen, among pneumococcal isolates from children with IPD or healthy nasopharyngeal carriers. psrP was detected in 52.4% of the 441 strains tested. While no differences were determined when the prevalence of psrP in colonizing strains (n = 89) versus that in all invasive strains (n = 352) was compared, a strong trend was observed when the prevalence of psrP in all pneumonia isolates (n = 209) and colonizing isolates (P = 0.067) was compared, and a significant difference was observed when the prevalence in all pneumonia isolates and those causing bacteremia (n = 76) was compared (P = 0.001). An age-dependent distribution of psrP was also observed, with the incidence of psrP being the greatest in strains isolated from children >2 years of age (P = 0.02). Strikingly, the presence of psrP within a serotype was highly dependent on the clonotype, with all isolates of invasive clones such as clonal complex 306 carrying psrP (n = 88), whereas for sequence type 304, only 1 of 19 isolates carried psrP; moreover, this was inversely correlated with antibiotic susceptibility. This finding suggests that inclusion of psrP in a vaccine formulation would not target resistant strains. We conclude that psrP is highly prevalent in strains that cause IPD but is most prevalent in strains isolated from older children with pneumonia. These data support the potential use of PsrP as one component in a multivalent protein-based vaccine.Invasive pneumococcal disease (IPD), defined herein as the isolation of Streptococcus pneumoniae from normally sterile sites during a clinical syndrome of infection such as bacteremia/sepsis, pneumonia, or meningitis, is an important health problem worldwide. In the year 2000, it is estimated that there were 11 million to 18 million episodes/cases of IPD and 0.7 million to 1 million deaths in children younger than 5 years of age as a result (17). Streptococcus pneumoniae is a Gram-positive commensal that colonizes the nasopharynx of healthy children and, less frequently, adults. From the upper respiratory tract, the bacteria can be aspirated into the lungs and can translocate through mucosal cell barriers to the bloodstream and lead to development of IPD (18). This primarily occurs in young children, elderly individuals, and those who are immunocompromised.The ability of S. pneumoniae to cause IPD is dependent on the presence of a polysaccharide capsule that prevents phagocytosis (1). At least 92 chemically and immunologically distinct capsular types (i.e., serotypes) can be produced by the pneumococcus, with certain serotypes more frequently being associated with invasive disease (23). Importantly, while the capsule is requisite for IPD, it is insufficient alone to confer virulence; and an assortment of additional determinants such as adhesins, proteases, toxins, transport systems, and enzymes that modify the extracellular milieu are also required (25). This requirement for noncapsular virulence determinants is proven by human epidemiological studies that show that invasive and noninvasive clonotypes exist within the most invasive serotypes, comparative genomic analyses that find an unequal distribution of noncapsular genes between invasive and noninvasive isolates within the same serotype, and scores of studies that show that deletion of noncapsular genes impact pneumococcal virulence in animal models of pneumonia, sepsis, and meningitis (7, 11, 19, 22).One recently identified pneumococcal virulence determinant is the pneumococcal serine-rich repeat protein (PsrP), a lung cell and intraspecies bacterial adhesin that is encoded within the 37-kb pathogenicity island called psrP-secY2A2 (16). PsrP is an extremely large glycosylated cell surface protein that belongs to the serine-rich repeat protein (SRRP) family of Gram-positive bacteria (22). For the pneumococcus, the presence of PsrP has been positively correlated with strains that cause human disease, and PsrP has been shown to mediate adhesion to keratin 10 on lung cells and to mediate the formation of bacterial aggregates in the nasopharynges and lungs of infected mice (21, 22). Antibodies against PsrP neutralize bacterial adhesion to cells in vitro and inhibit biofilm formation (20, 21). Furthermore, passive immunization of mice with PsrP antiserum or active immunization with recombinant protein protected mice against pneumococcal challenge (20). Thus, PsrP is an important virulence factor by which S. pneumoniae is able to cause IPD and is potentially a vaccine candidate.At this time, considerable resources are being spent in an attempt to identify the pneumococcal antigens that would be used in a next-generation multivalent protein vaccine designed against the pneumococcus. The advantage of such a vaccine is that it would have a lower cost and potentially expanded global coverage compared with the cost and coverage of existing conjugate vaccines. It is generally accepted that multiple antigens will be necessary due to the fact that not all protein determinants are conserved or found within all pneumococcal strains and on their own are not able to confer sufficient protection. To this end, knowledge of the real prevalence of a protein in different clones and serotypes of Streptococcus pneumoniae is necessary to consider any protein as a candidate vaccine antigen. Therefore, the objective of our study was to determine the distribution and clonal type variability of PsrP among pneumococcal isolates from children with IPD or healthy nasopharyngeal carriers.