Diagnosis of acute Q fever with emphasis on enzyme-linked immunosorbent assay and nested polymerase chain reaction regarding the time of serum collection

A commercially available enzyme-linked immunosorbent assay (ELISA) (Virion/Serion [Wuerzburg, Germany]), an indirect fluorescent antibody test (IFAT) (BIOS/Focus [Cypress, CA]), and a nested polymerase chain reaction (PCR) were explored for diagnosis of acute Q fever in reference to time of serum collection. Serum samples of 22 patients with acute Q fever collected around the fifth day of illness were included. A sensitivity of 30% by ELISA and 80% by IFAT (P = 0.1) was found for the first 5 days of illness and 92% by ELISA and 83% by IFAT during the sixth and eleventh day. PCR revealed a positive result in 8 cases (36%) with 6 cases deriving from the first 5 days of illness. We conclude that ELISA aids especially in the diagnosis of Q fever after 5 days of illness. The benefit of PCR as an additional tool to ELISA was especially evident in the early days of serum sampling.     

Evaluation of a modified Taqman assay detecting pathogenic Leptospira spp. against culture and Leptospira-specific IgM enzyme-linked immunosorbent assay in a clinical environment

A Taqman assay for the detection of pathogenic Leptospira was modified to suit the LightCycler instrument. The modified assay was found to have an analytical sensitivity of 10 copies/reaction. The assay was then compared to the current gold standard for acute phase detection, culture, and to a commercially available Leptospira-specific IgM enzyme-linked immunosorbent assay (ELISA). Of the 236 samples including serum and ethylenediaminetetraacetic acid anticoagulated blood sample submitted for testing, polymerase chain reaction (PCR) was able to detect Leptospira DNA in 27 of the 28 culture positives and in 1 negative culture. Discrepant results were resolved by using the microscopic agglutination test on convalescent sera. The PCR was found to have a clinical specificity and sensitivity of 99.5% and 96.4%, respectively, when compared to the culture. Comparisons between culture and ELISA showed that the ELISA lack sensitivity (4.2%) and positive predictability (3.6%) for the detection of acute phase Leptospira infections. These results show that the modified LightCycler Taqman assay could be used as a replacement of culture for the detection of pathogenic Leptospira in a clinical setting.     

Acute Q fever in southern Taiwan: atypical manifestations of hyperbilirubinemia and prolonged fever

The clinical information of acute Q fever in Taiwan was limited. A clinical study of 109 adults with serologically documented acute Q fever in the past decade (1994-2005) at 3 referral hospitals in southern Taiwan was reported. Their clinical manifestations, laboratory findings, and clinical outcomes were analyzed. Males predominated (98, 90%). There is a significant correlation between monthly average temperature and case numbers of acute Q fever (r = 0.74, P = 0.006). Fever (99%), chills (69%), and headache (45%) were the common symptoms, and relative bradycardia (44/60, 73 %) was often noted. Acute hepatitis, defined as either serum aspartate aminotransferase >or=60 IU/L or alanine aminotransferase >or=78 IU/L, was found in 88 (85%) cases, and more than one-third (31/87, 36%) had hyperbilirubinemia (serum total bilirubin >or=1.4 mg/dL) at initial presentation. The intervals between initiation of appropriate therapy to defervescence were longer in patients with hyperbilirubinemia than those without hyperbilirubinemia, irrespective of tetracycline or fluoroquinolone therapy. Of note, 8 (7.3%) cases experienced a prolonged period of fever (>28 days). In southern Taiwan, the predominant presentation of acute Q fever is acute febrile illness with hepatitis with or without jaundice. Acute Q fever should be added to the list of differential diagnoses of patients with fever, headache, relative bradycardia, elevated serum aminotransferase levels, or prolongation of activated partial thromboplastin time, irrespective of jaundice.     

Evaluation of a real-time polymerase chain reaction assay for the laboratory diagnosis of giardiasis

A real-time polymerase chain reaction (PCR) assay was evaluated in comparison with the combination of conventional methods (microscopic examination and antigen detection assay) during the period 2006 to 2008 on 771 fecal samples belonging to 386 patients to assess its usefulness for an accurate laboratory diagnosis of giardiasis. The real-time PCR assay detected Giardia intestinalis DNA in 195 samples (106 patients), including 26 samples (21 patients) negative by the conventional assays. Among the 21 patients, in 8 cases, giardiasis was previously diagnosed also by conventional methods in additional samples of the same patients, whereas in 13, it would have been undiagnosed if real-time PCR assay was not used. The real-time PCR assay demonstrated a detection limit of 2 cysts per reaction and 100% specificity and sensitivity compared to conventional methods. A genotype analysis targeting the β-giardin gene allowed to identify 53 samples (23 patients) containing genotype A and 59 samples (45 patients) containing genotype B.     

Epidemiological, clinical and laboratory characteristics of patients with human granulocytic anaplasmosis in Slovenia

Human granulocytic anaplasmosis (HGA) has been recently recognized as an emerging tick-borne disease. Several reports indicate the presence of infection with Anaplasma phagocytophilum in Europe. Between January 1996 and December 2004, 24 adult patients with proven HGA were identified in a prospective study conducted at the Department of Infectious Diseases, University Medical Center Ljubljana, Slovenia, on the etiology of febrile illnesses occurring within 30 days after a tick bite. The diagnosis of acute HGA was established from seroconversion in 18 (75%) patients or at least a four-fold increase in antibody titers to A. phagocytophilum antigens in six (25%) patients and molecular identification of ehrlichial organisms in 15 (62.5%) patients. Clinical characteristics and laboratory findings were similar to those reported from the other European countries. All the patients had an acute febrile illness with headache, malaise, myalgia and/or arthralgia. Leukopenia was found in 16 (66.7%) patients, thrombocytopenia in 20 (83.3%), abnormal liver function test results in 23 (95.8%), elevated erythrocyte sedimentation rates in 18 (75%), and elevated concentration of C-reactive protein in 23 (95.8%). The disease course was relatively mild; none of the patients died and no long-term sequelae were found during a follow-up of one year even though only 15 (62.5%) were treated with doxycycline. At the examination one year after the first visit, 16/24 (66.7%) patients tested seropositive (> or =1 : 256) for A. phagocytophilum antibody, and two years after the first visit positive titers were still present in 10/18 (55.6%) patients.     

Rapid and sensitive detection of methicillin-resistant Staphylococcus periprosthetic infections using real-time polymerase chain reaction

The aim of this study was to validate the accuracy, sensitivity, and specificity of a methicillin-resistant Staphylococcus (MRS) real-time polymerase chain reaction (PCR) assay in clinical periprosthetic infection cases.     

疑似人粒细胞无形体病12例分析

目的研究人粒细胞无形体病疑似病例的流行病学和临床特点。方法回顾性分析12例人粒细胞无形体病疑似病例的临床资料。结果本组人粒细胞无形体病疑似病例流行地区位于胶东半岛地区,病人均为农民,年龄中位数为57岁,其中2例在起病前1周有明确蜱咬史。常见症状包括发热、腹泻、恶心等,并发症有出血、中毒性脑病、急性肾衰竭等,常见实验室检查指标异常有白细胞、血小板减少,转氨酶升高,尿蛋白、尿潜血,9例行人粒细胞无形体套式PCR检测均为阴性。经过抗生素和对症支持治疗痊愈11例,死亡1例。结论人粒细胞无形体病是一种蜱传急性自然疫源性疾病,可累及多脏器,高龄及有并发症病人易发生严重并发症,预后较差。     

Detection of Yersinia pestis using real-time PCR in patients with suspected bubonic plague

Yersinia (Y.) pestis, the causative agent of plague, is endemic in natural foci of Asia, Africa, and America. Real-time PCR assays have been described as rapid diagnostic tools, but so far none has been validated for its clinical use. In a retrospective clinical study we evaluated three real-time PCR assays in two different assay formats, 5′-nuclease and hybridization probes assays. Lymph node aspirates from 149 patients from Madagascar with the clinical diagnosis of bubonic plague were investigated for the detection of Y. pestis DNA. Results of real-time PCR assays targeting the virulence plasmids pPCP1 (pla gene), and pMT1 (caf1, Ymt genes) were compared with an F1-antigen immunochromatographic test (ICT) and cultivation of the organism. Out of the 149 samples an infection with Y. pestis was confirmed by culture in 47 patients while ICT was positive in 88 including all culture proven cases. The best real-time PCR assay was the 5′-nuclease assay targeting pla which was positive in 120 cases. In conclusion, the 5′-nuclease assay targeting pla can be recommended as diagnostic tool for establishing a presumptive diagnosis when bubonic plague is clinically suspected.     

Development of a multiplex polymerase chain reaction assay for diarrheagenic Escherichia coli and Shigella spp. and its evaluation on colonies, culture broths, and stool

Detection of diarrheagenic Escherichia coli (DEC) typically depends on identification of virulence genes from stool cultures, not on stool itself. We developed a multiplex polymerase chain reaction (PCR) assay that detects key DEC virulence genes (stx1, stx2, eae, bfpA, ipaH, LT, STh, aaiC, aatA). The assay involved a multiplex PCR reaction followed by detection of amplicon(s) using Luminex beads. The assay was evaluated on over 100 colony and broth specimens. We then evaluated the assay using DNA extracted from stool, colony pools, and Gram-negative broths, using stool spiked with known quantities of DEC. Performance of the assay on stool DNA was most quantitative, while stool broth DNA offered the lowest limit of detection. The assay was prospectively evaluated on clinical specimens in Tanzania. Stool DNA yielded higher sensitivity than colony pools compared with broth DNA as the standard. We propose using this assay to screen for DEC directly in stool or stool broths.     

Direct detection and analysis of vacA genotypes and cagA gene of Helicobacter pylori from gastric biopsies by a novel multiplex polymerase chain reaction assay

Several tests/methods for the infection, detection, and genotyping of Helicobacter pylori have been developed in the past; all these differ in specificity and sensitivity and thereby could not be routinely used in clinical study especially in resource-poor developing countries. In the present study, a novel method based on multiplex polymerase chain reaction (PCR) assay was developed to detect H. pylori in patients suffering from gastrointestinal diseases. This method does not require steps of sonication, boiling, or centrifugation for obtaining DNA from biopsy samples, which are otherwise prerequisite in detecting H. pylori by PCR assay. Two hundred seventy-six patients were examined, of which 182 cases (excluding 36 patients having multiple H. pylori strain infection and 8 showing amplification of 16S rDNA only) were H. pylori positive. The dominant vacA genotype was s1 and m1 being present in 168 (92.3%) and 106 (58.2%) patients, respectively, followed by m2 (41.7%), and the lowest being s2 (7.7%). Detection of H. pylori by this method seems rapid, simpler, and suitable for both types 1 and 2 bacteria. Furthermore, genotyping of vacA and cagA genes could also be routinely performed in a large number of patients for diagnostic purposes.     

Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples

Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF.     

Diagnostic significance of nested polymerase chain reaction for sensitive detection of Pneumocystis jirovecii in respiratory clinical specimens

A total of 327 clinical specimens, including both invasive and noninvasive samples, obtained from 275 patients with various types of underlying immunocompromised conditions and a clinical suspicion of Pneumocystis pneumonia (PCP) were subjected to 2 different nested polymerase chain reaction (PCR) assays. The target genes used for nested PCR were mitochondrial large subunit ribosomal RNA (mtLSU rRNA) and internal transcribed spacer (ITS) region. The results were compared with a single-round PCR targeting major surface glycoprotein (MSG) gene. Amplification was successful in 16% of cases by mtLSU rRNA nested PCR, in 14.5% by ITS nested PCR, and in 10.9% by MSG PCR. The nested mtLSU rRNA PCR was found to be more sensitive (100% sensitive and 98.7% specific) and useful in detecting PCP for its use in routine diagnosis in our settings. Thus, this assay may be quite useful in the identification of patients who are in the early stage of Pneumocystis jirovecii infection with an organism load that could not be easily detected by the single-step PCR.     

引起一起发热疫情的人腺病毒4a型基因特征分析

目的了解引起一起发热疫情的人腺病毒4a型基因特征。方法2019年10月,北京市通州区某小学出现发热疫情,采集病例的咽拭子标本,提取标本核酸作为模板,利用实时荧光PCR方法检测32种呼吸道病毒和细菌。 对于腺病毒检测阳性的标本核酸,利用腺病毒特异性引物扩增3个基因（Fiber、Hexon和Penton）的片段并测序,采用局部序列比对基本检索工具（BLAST）比对序列,进行进化关系分析。结果采集的6份病例标本中,腺病毒全部阳性,其他检测的呼吸道病毒和细菌均为阴性。 Fiber、Hexon和Penton基因序列比对结果显示,6份标本中的腺病毒均为4a基因型。进化分析显示,该病毒与近年来美国流行的4a型腺病毒高度同源。结论人腺病毒4a型在北京市可以引起呼吸道发热疫情。     

Development of real-time PCR assay for differential detection of Bordetella bronchiseptica and Bordetella parapertussis

Bordetella parapertussis is a causative agent of whooping cough in humans, and B. bronchiseptica is causing wide variety of respiratory infections in mammals, including humans. Specific diagnostic tests are not currently available. Our first objective was to develop a real-time PCR test for the specific detection of B. bronchiseptica based on the previously described end-point PCR, targeting an intergenomic sequence of the fla gene locus, but it has not been reached. However, there is cross-reactivity between B. parapertussis and B. bronchiseptica. Therefore, the targeted region of several clinical isolates of both species was sequenced, and alignment of the sequences allowed the development of a 2-step real-time PCR assay. The first PCR assay detected the DNA of all clinical isolates of both B. bronchiseptica and B. parapertussis tested. The second PCR assay detected only the DNA of B. parapertussis clinical isolates, thereby allowing discrimination between B. parapertussis and B. bronchiseptica.     

Detection of Mycobacterium avium complex DNA directly in clinical respiratory specimens: opportunities for improved turn-around time and cost savings

We developed, evaluated, and implemented a Taqman multiplex real-time polymerase chain reaction (PCR) assay for the detection of Mycobacterium avium complex (MAC), targeting the 16S-23S rRNA internal transcribed spacer, which we have combined with an existing Mycobacterium tuberculosis complex assay for use directly in clinical respiratory specimens. Evaluation of the performance of this assay for MAC detection included 464 clinical respiratory specimens tested prospectively. This real-time PCR assay was found overall to have a sensitivity of 71.1%, a specificity of 99.5%, a positive predictive value of 98.0%, and a negative predictive value of 90.2% for MAC. The assay provides results prior to the availability of cultured material and identification, most within 24 h of specimen receipt, and may reduce the need to culture MAC-PCR–positive specimens when susceptibility testing is not requested. Additionally, we have found significant cost savings of approximately $21.00 per specimen and staff time reductions of 3.75 h per specimen with implementation of this assay.     

Real-time PCR detection of Mycoplasma pneumoniae in respiratory specimens

A real-time PCR assay targeting the phosphotransferase system I gene (ptsI) of Mycoplasma pneumoniae was compared to 2 commercially available PCR assays targeting the P1 cytadhesion gene (the LightMix®Kit Mycoplasma pneumoniae [TIB MOLBIOL, Adelphia, NJ, USA] and M. pneumoniae Analyte Specific Reagent [Focus Diagnostics, Cypress, CA, USA] assays) and to a PCR assay targeting the M. pneumoniae repetitive element, RepMP1. Thirty previously positive specimens including 15 throat swab, 10 bronchoalveolar lavage, and 5 sputum specimens, all tested positive with the ptsI and M. pneumoniae ASR assays. Among the previously positive specimens, 14/15 throat swab, 9/10 bronchoalveolar lavage, and 4/5 sputum specimens tested positive with the LightMix®Kit Mycoplasma pneumoniae assay and 13/15 throat swab, 10/10 bronchoalveolar lavage, and 4/5 sputum specimens tested positive with the RepMP1 assay. Forty previously negative clinical specimens tested negative using the ptsI assay. A PCR assay targeting M. pneumoniae ptsI performs equivalently to assays targeting the P1 cytadhesion gene or RepMP1.     

Evaluation of a new multiplex polymerase chain reaction assay STDFinder for the simultaneous detection of 7 sexually transmitted disease pathogens

We evaluated a new multiplex polymerase chain reaction (mPCR), “STDFinder assay”, a novel multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 7 clinically relevant pathogens of STDs, i.e., Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum, and herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). An internal amplification control was included in the mPCR reaction. The limits of detection for the STDFinder assay varied among the 7 target organisms from 1 to 20 copies per MLPA assay. There were no cross-reactions among any of the probes. Two hundred and forty-two vaginal swabs and an additional 80 specimens with known results for N. gonorrhoeae and C. trachomatis, obtained from infertile women seen at an infertility research clinic at the Kigali Teaching Hospital in Rwanda, were tested by STDFinder assay and the results were confirmed by single real-time PCR using different species-specific targets. Compared to the reference standard, the STDFinder assay showed specificities and sensitivities of 100% and 100%, respectively, for N. gonorrhoeae, C. trachomatis, and M. genitalium; 90.2% and 100%, respectively, for Trichomonas vaginalis; and 96.1% and 100%, respectively, for HSV-2. No specimen was found to be positive for HSV-1 by either the STDFinder assay or the comparator method. Similarly, the sensitivity for Treponema pallidum could not be calculated due to the absence of any Treponema pallidum-positive samples. In conclusion, the STDFinder assays have comparable clinical sensitivity to the conventional mono and duplex real-time PCR assay and are suitable for the routine detection of a broad spectrum of these STDs at relatively low cost due to multiplexing.     

Loop-mediated isothermal amplification assay targeting the ompA gene for rapid detection of Riemerella anatipestifer

A novel loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of Riemerella anatipestifer (RA) infection. The LAMP assay exhibited a higher sensitivity than conventional polymerase chain reaction (PCR) and microbial isolation. The specificity of the assay was determined by restriction enzyme digestion of the LAMP products and detection of Escherichia coli, Salmonella enterica and Pasteurella multocida. The LAMP assay was able to detect RA effectively in samples of the reference strains, isolated strains and infected duck brains. This assay is a useful tool for the diagnosis of RA infection in the clinical setting.     

Identification and molecular discrimination of toxigenic and nontoxigenic diphtheria Corynebacterium strains by combined real-time polymerase chain reaction assays

With the recognition of several diphtheria outbreaks and the emergence of nontoxigenic corynebacteria strains, there has been renewed interest in the development of laboratory diagnostic methods. Previously reported polymerase chain reaction (PCR) assays can have low diagnostic sensitivity or give species misidentifications among clinical isolates. The aim of the present study was the development of combined real-time PCR assays, based on the tox and rpoB genes, for the detection and differentiation of toxigenic and nontoxigenic corynebacteria. By the PCR tox assay, it was possible to perform the direct identification of DT tox gene of Corynebacterium diphtheriae and Corynebacterium ulcerans, while the PCR rpoB assay differentiated C. diphtheriae from C. ulcerans, irrespective of their toxigenic status. In addition, we detected the DT toxin of Corynebacterium pseudotuberculosis for the first time. These assays revealed high sensitivity, specificity, and reproducibility, and the availability of plasmid controls will facilitate further research into the diagnostics of diphtheria corynebacteria.     

Rapid detection of early typhoid fever in endemic community children by the TUBEX O9-antibody test

Typhoid remains a global public health problem, and quick accurate immunodiagnosis is needed. Here, we examined the performance of the 5-min TUBEX O9-antibody detection kit in 243 outpatients (mostly children and infants) in their first week of fever and 57 healthy subjects in the Bangladesh community. Based on culture results, TUBEX was 91.2% (31/34) sensitive and 82.3% (172/209) specific in febrile subjects. However, specificity was better in nonfebrile healthy subjects (89.5%, 51/57) or in febrile individuals who serologically had dengue fever (90.5%, 57/63), suggesting that some culture-negative febrile individuals could be truly typhoidal. These individuals were also positive in an anti-crude O9 enzyme-linked immunosorbent assay (ELISA) and the Widal test. Regression analysis of the TUBEX and ELISA results showed good concordance between them, better with the combined IgM-IgG ELISA than with IgM alone, suggesting that TUBEX detects IgM antibodies not necessarily by themselves, as previously reported, but with the help of IgG antibodies.