Rapid latex agglutination test for the serodiagnosis of human brucellosis

We developed and evaluated a user-friendly latex agglutination assay for the serodiagnosis of human brucellosis. The assay was obtained by coating colored latex beads with Brucella lipopolysaccharides and drying of the activated beads onto white agglutination cards. Individual cards were sealed in a protective foil to secure stability of the dried reagent and to obtain a test in a single assay format. The latex agglutination assay is simply performed by suspending the dried latex reagent in a drop of serum and looking for macroscopic agglutination of the latex beads by visual inspection. Results are obtained within 30 s after mixing the sample with the test reagent. The sensitivity of the assay was determined to be 89.1% (95% confidence interval [CI], 76-96) for the initial serum samples collected from patients with culture-confirmed brucellosis and the specificity is 98.2% (95% CI, 96-99). The assay is ideal for use as a field test in remote areas and as point-of-care test in hospitals and health care centers that lack the expertise and facilities to perform the more demanding classic serologic tests.     

Simple, rapid, and affordable point-of-care test for the serodiagnosis of typhoid fever

We developed a point-of-care test for the serodiagnosis of typhoid fever in the format of an immunochromatographic lateral flow assay. The flow assay for typhoid fever is based on the detection of Salmonella enterica serotype Typhi lipopolysaccharide-specific immunoglobulin M (IgM) antibodies. The assay was evaluated on serum samples collected in a hospital in South Sulawesi, Indonesia, where typhoid fever is endemic, and the results were compared with culture and Widal test. The sensitivity of this typhoid fever IgM flow assay for samples collected at 1st diagnosis from patients with culture-confirmed typhoid fever was determined to be 59.3%. The sensitivity ranged from 41.2% to 89.5%, depending on the duration of illness. A specificity of 97.8% was calculated based on results obtained for patients with clinical suspicion of typhoid fever that was later excluded. The assay is ideal for use as a point-of-care test in health care centers that lack the expertise and facilities to perform culture or the less specific Widal test. Because of its simplicity, the assay may also be used as a field test in remote areas.     

Evaluation of Brucella dipstick assay for the diagnosis of acute brucellosis

The diagnostic value of the dipstick assay was evaluated by comparison with Rose Bengal (RB), serum aglutination tests (SAT) and 2 mercaptoethanol test (2-ME) on consecutive serum samples submitted because of suspicion of brucellosis. Serum samples of 232 patients with suspected brucellosis that were submitted for laboratory confirmation were included in the study. Twelve out of 232 serum samples were detected as positive with the dipstick assay. All of these 12 patients had positive RB and SAT results. In 16 RB positive samples dipstick test was negative. Fifteen of these samples had insignificant (titer<1/160) or borderline (titer 1/160) SAT results and the clinical symptoms of these patients were consistent with chronic brucellosis rather than acute or recent-onset brucellosis. Dipstick assay is an easy-to-perform assay that can be used for the diagnosis of acute brucellosis especially in rural areas where brucellosis is widespread and in settings where well-equipped laboratories are not available.     

Development and evaluation of real-time polymerase chain reaction assays on whole blood and paraffin-embedded tissues for rapid diagnosis of human brucellosis

This study aimed at developing a real-time polymerase chain reaction (PCR) assay for the rapid diagnosis of human brucellosis on clinical specimens. Three assays with hybridization probe detection on the LightCycler instrument were developed and compared targeting the 16S-23S internal transcribed spacer region (ITS) and the genes encoding for omp25 and omp31. All assays showed 100% analytical sensitivity and 100% specificity when tested on 28 consecutive clinical isolates of Brucella sp. and 19 clinical isolates of common Gram-negative and Gram-positive bacterial pathogens, respectively. The ITS assay was the most sensitive with a limit of detection of 2 genome equivalents per PCR reaction. This assay was then clinically validated prospectively with 354 samples (351 whole blood samples and 3 paraffin-embedded tissues) collected from 340 patients, 24 samples from patients with active brucellosis including 2 relapsing cases, 31 with treated brucellosis, and 299 seronegative patients where brucellosis was initially suspected. The clinical sensitivity, specificity, and positive and negative predictive values of the ITS assay were 66.7%, 99.7%, 94.1%, and 97.6%, compared with culture at 77%, 100%, 100%, and 97.3%, respectively. The difference in sensitivity between culture and ITS-PCR was not statistically significant (P = 0.71). Both relapsing cases were PCR positive. Treated patients were PCR negative. All 3 assays were positive on tissue samples, but the omp25 and omp31 assays did not detect Brucella sp. DNA in blood samples. Because omp31 is not present in Brucella abortus, these data indicate that the 28 tested isolates are most likely Brucella melitensis. ITS-PCR is rapid and could augment the clinical laboratory diagnosis of human brucellosis.     

Diagnostic performance of whole-blood interferon-γ assay and enzyme-linked immunospot assay for active tuberculosis'

The aim of this study was to compare the diagnostic performance of 2 interferon-γ release assays, an enzyme-linked immunospot assay (T-SPOT.TB; Oxford Immunotec Ltd., Oxford, UK) and the QuantiFERON-TB Gold in-Tube assay (QFT-GIT; Cellestis Ltd., Carnegie, Australia), in patients with suspected active tuberculosis (TB). From October 2009 to October 2011, a total of 200 patients with suspected TB were enrolled. Clinical and microbiological characteristics of the patients were collected and blood samples were obtained for T-SPOT.TB and QFT-GIT assays. Among the 200 subjects, 98 (49%) had culture-confirmed TB, 18 (9%) had probable TB, and the remaining 84 (42%) subjects did not have TB. The sensitivity, specificity, positive predictive value, and negative predictive value for active TB diagnosis by the T SPOT. TB were 83%, 71%, 81%, and 75%, respectively. For QFT-GIT, the sensitivity, specificity, positive predictive value, and negative predictive value for active TB diagnosis were 66%, 76%, 80%, and 62%, respectively. The QFT-GIT assay resulted in more indeterminate and false-negative results than the T-SPOT.TB assay, especially in immunocompromised patients. In conclusion, T-SPOT.TB had a higher sensitivity and resulted in fewer indeterminate results than the QFT-GIT assay for diagnosing active TB.     

A modified indirect fluorescent antibody test for the diagnosis of brucellosis

We adapted the conventional indirect fluorescent antibody (IFA) test to assay IgM and IgG Brucella-specific antibodies to differentiate acute from chronic infections rather than measure total antihuman globulin specific antibodies. The results were compared with the slide agglutination test (SAT) used for screening and the quantitative microagglutination test (MAT). Of a total of 118 randomly selected samples sent for anti-Brucella antibodies received at a general hospital laboratory, 58 (47.9%) were found to be positive for IFA-IgG test but not necessarily by other tests. Eleven of these cases were positive for Brucella melitensis by culture. Sixty serum samples found negative for Brucella antibodies by IFA and other tests were of patients with medical conditions other than brucellosis. Fifty serum samples from healthy blood donors were negative for Brucella spp. antibodies by all the three tests. The IFA test was found to be a more sensitive test than MAT and distinguished an acute infection from chronic disease.     

Comparison of Brucella immunoglobulin M and G flow assays with serum agglutination and 2-mercaptoethanol tests in the diagnosis of brucellosis.

The diagnostic value of Brucella IgM/IgG flow assays was evaluated in comparison with serum agglutination and 2-mercaptoethanol tests by testing a selection of serum samples submitted to the laboratory because of clinical suspicion of brucellosis. All 39 admission and 11 follow-up samples that agglutinated in the serum agglutination test tested positive in the flow assay, whereas all 20 serum agglutination negative samples with clinical suspicion of brucellosis, 23 control samples from healthy individuals and 20 control samples from cases with chronic hepatitis tested negative in the flow assay. The Brucella IgM and IgG flow assays were slightly more sensitive than the agglutination tests in discriminating between specific IgM and IgG antibodies. The Brucella IgM and IgG flow assays are easy-to-perform and quick assays that can be used for the diagnosis of brucellosis. The flow assays are very useful, especially in rural settings where brucellosis is widespread and where well-equipped laboratories to perform the laboratory tests are not readily available.     

Diagnosis of typhoid fever by two serologic methods. Enzyme-linked immunosorbent assay of antilipopolysaccharide of Salmonella typhi antibodies and Widal test.

Serum samples from 85 patients with proven typhoid fever, 11 patients with p-typhoidal fever, 101 patients with febrile non-typhoidal, and 130 healthy subjects were tested for immunoglobulin G (IgG), IgA, and IgM antilipopolysaccharide (LPS) of Salmonella typhi antibodies by enzyme-linked immunosorbent assay (ELISA) and Widal test. The levels of all three classes of immunoglobulin anti-LPS of S. typhi were higher in typhoid patients than in healthy or febrile nontyphoidal groups; we selected various combinations between the three classes of immunoglobulin to obtain the best combination of sensitivity and specificity. The sum of the absorbance values obtained from the ELISA assay for IgG+IgA+IgM (sigma lgs) was the best choice for diagnostic utility for typhoid fever. We selected a positive test at a decision level of sigma lgs > or = 1.2 with a sensitivity of 94% and a specificity of 92% with a frequency of false negative of 5.9%. The frequency of false positives for healthy controls was 7.7% and, for the febrile nontyphoidal group, it was 7.9%. We also compared receiver (or relative) operating characteristic (ROC) curves for the diagnostic usefulness of the ELISA with that of the Widal test, whose merits and limitations, especially in endemic regions, are discussed. The ELISA assay was much more sensitive and specific than any combination of the Widal test, and hence it could be a useful tool for the serologic diagnosis of typhoidal fever with a single blood sample.     

The simultaneous detection of the squamous cell carcinoma antigen and cancer antigen 125 in the cervical cancer serum using nano-Ag polydopamine nanospheres in an SERS-based lateral flow immunoassay

The accurate analysis of tumor related biomarkers is extremely critical in the diagnosis of the early stage cervical cancer. Herein, we designed a novel and inexpensive surface-enhanced Raman scattering-based lateral flow assay (SERS-based LFA) strip with a single test line, which was applied for the rapid and sensitive quantitative simultaneous analysis of SCCA and CA125 in serum samples from patients with cervical cancer. In the presence of target antigens, the monoclonal antibody-coupled and Raman reporter-labeled nano-Ag polydopamine nanospheres (PDA@Ag-NPs) aggregated on the test line modified by the polyclonal antibody to form a double-antibody sandwich structure. The finite difference time domain simulation demonstrated that large number of “hot spots” was generated among the nanogaps of aggregated PDA@AgNPs, which resulted in a huge enhancement of the signal of the Raman reporters. Accordingly, the limit of detection was determined to be 7.156 pg mL⁻¹ for SCCA and 7.182 pg mL⁻¹ for CA125 in phosphate buffer and 8.093 pg mL⁻¹ for SCCA and 7.370 pg mL⁻¹ for CA125 in human serum, revealing high sensitivity of this SERS-based LFA strip. Significantly, the detection of SCCA and CA125 using the SERS-based LFA was observed to have high specificity and reproducibility, and the whole detection was completed within 20 min. Furthermore, the SERS-based LFA and enzyme-linked immunosorbent assay were also employed in serum samples obtained from patients with cervical cancer, cervical intraepithelial neoplasia and healthy subjects, and perfect agreement existed between both the methods. Thus, clinically, the developed SERS-based LFA strip has strong potential for the simultaneous detection of multiple cancer biomarkers in serum.

Based on SERS-based lateral flow immunoassay, nano-Ag polydopamine nanospheres was used for detecting squamous cell carcinoma antigen and cancer antigen 125 simultaneously in cervical cancer serum.     

Evaluation of a SARS-CoV-2 lateral flow assay using the plaque reduction neutralization test

As new tests and technologies advance our understanding and diagnostic capabilities of the severe acute respiratory syndrome coronavirus 2 and the coronavirus disease 2019, they must be appropriately validated to make sure test performance is following manufacturer claims. In this study, we evaluated the Vazyme 2019-nCoV IgG/IgM Detection Kit, which is a lateral flow assay (LFA), by the plaque reduction neutralization test (PRNT) using 100 patient plasma/serum samples. As compared to the PRNT results, the Vazyme LFA had 95.9% sensitivity and 96.1% specificity. Along with the increased need for rapid, effective, and affordable point of care tests to help provide meaningful epidemiological data, we demonstrated that the Vazyme LFA performed well on IgG detection but cannot be judged on the performance of IgM detection using PRNT alone. However, our observation of the low IgM-positive rate supported the poor performance of IgM detection of this LFA which led to the disapproval of its Emergency Use Authorization recently.     

Recombinant VirB5 protein as a potential serological marker for the diagnosis of bovine brucellosis

The molecular tag vaccine against Brucella abortus and serological testing are the main methods of prevention of brucellosis used currently. They can discriminate vaccinated animals and humans from those naturally infected. In this study, we constructed a gene deletion mutant strain, B. abortus S19 virB5 with a molecular tag. Recombinant VirB5 was expressed and purified for evaluation as a diagnostic reagent for bovine brucellosis. In total, 400 sera samples were tested using a VirB5 antigen-based enzyme-linked immunosorbent assay (ELISA) and the results were compared with those of the standard tube agglutination test (SAT). This showed that the sensitivity was 88.2%, specificity was 97.8% and accuracy was 94.8%. Recombinant VirB5 could also be used to discriminate B. abortus-infected mice from mice infected with the B. abortus S19 virB5 mutant strain. It was concluded that recombinant VirB5 could be used as a potential antigen and serological marker for the diagnosis of bovine brucellosis.     

试管凝集试验与虎红平板凝集试验对布鲁氏杆菌病的诊断价值

目的研究试管凝集试验与虎红平板凝集试验对布鲁氏杆菌病的诊断价值。方法于2017年1月至2019年12月将清丰县疾病预防控制中心接受布鲁氏杆菌病筛查的120例疑似布鲁氏杆菌病患者纳入研究,对血液标本进行试管凝集试验、虎红平板凝集试验,比较试管凝集试验与虎红平板凝集试验的诊断结果。结果在布鲁氏杆菌病诊断中,参照布鲁氏杆菌病综合检测结果,虎红平板凝集试验的灵敏度、阴性预测值均高于试管凝集试验,而试管凝集试验的特异度、阳性预测值均高于虎红平板凝集试验(P<0.05),试管凝集试验与虎红平板凝集试验的准确度无显著差异(P>0.05)。试管凝集试验、虎红平板凝集试验诊断结果与布鲁氏杆菌病综合检测结果之间均呈高度一致(Kappa=0.807、0.823)。结论试管凝集试验、虎红平板凝集试验在布鲁氏杆菌病诊断中具有各自优势,临床诊断时可结合两种凝集试验方法进行综合诊断,以提高诊断的准确性。     

Evaluation of a commercial immunochromatographic assay for the serologic diagnosis of tularemia

Tularemia is an infection caused by Francisella tularensis with a worldwide distribution in the northern hemisphere and diverse clinical manifestations. Serology plays an important role in the diagnosis of tularemia. A commercially available immunochromatographic assay (ICA) for the serologic diagnosis of tularemia (VIRapid Tularemia, Vircell, Granada, Spain) was evaluated, and the performance was compared with that of the current standard, the microagglutination test (MA). A panel of 221 sera from 109 cases of tularemia was tested as well as 236 sera from normal individuals or individuals with other infectious or autoimmune diseases. The ICA demonstrated 91.5% (κ = 0.91) agreement with the reference method (MA) and gave an overall sensitivity of 99.3% and a specificity of 94.6%. No cross-reactivity was observed in the ICA with serum samples from normal individuals and patients with autoimmune diseases and bacterial, viral, and parasitic infections, although 4 of 50 patients with brucellosis demonstrated positive results in the ICA. The performance of ICA was simple, and it requires no specialized equipment. The ease of use and significantly high sensitivity and specificity of ICA make it a good choice for diagnostic testing and a valuable field test to support a presumptive diagnosis of tularemia in remote areas.     

Diagnostic value of an enzyme-linked immunospot assay for interferon-γ in cutaneous tuberculosis

The aim of this study was to evaluate the diagnostic performance of an enzyme-linked immunospot (ELISPOT) assay (T-SPOT.TB; Oxford Immunotec, Oxford, UK) for interferon-γ in patients with suspected cutaneous tuberculosis (TB). From March 2007 to June 2010, a total of 45 patients with suspected cutaneous TB were enrolled. Data on clinical characteristics of the patients and conventional laboratory results were collected, and blood samples were obtained for ELISPOT assay. Ten subjects (22.2%) had culture-confirmed TB, 2 (4.4%) subjects had probable TB, and the remaining 33 (73.3%) subjects did not have TB. Twenty-one patients with mycobacterial infection had available biopsy or surgical specimens for histopathologic examination and 16 (76.2%) specimens had pathologic features (granulomatous inflammation, caseating necrosis, or acid-fast bacilli) consistent with mycobacterial infection. Among these 16 patients, all 6 patients with TB and 3 patients with Mycobacterium marinum infection had positive ELISPOT results, but none of the patients with Mycobacterium abscessus, Mycobacterium chelonae, or Mycobacterium avium infection had positive ELISPOT results. Overall, 9 of 10 patients with culture-confirmed TB and both patients with probable TB had positive ELISPOT assay. For patients with nontuberculous mycobacteria infections, ELISPOT assay was positive only in patients with M. marinum and M. kansasii infection. The sensitivity, specificity, positive predictive value, and negative predictive value for cutaneous TB diagnosis by the ELISPOT assay were 91.6% (95% CI, 64.6-98.5%), 75.8% (95% CI, 59.0-87.2%), 57.9% (95% CI, 34.0-78.9%), and 96.2% (95% CI, 59.0-87.2%), respectively. In conclusion, ELISPOT assay can provide useful support in the diagnosis of cutaneous TB.     

布鲁氏菌病患者血液中布鲁氏菌与临床症状和抗体水平关联性研究

目的 应用巢式聚合酶链式反应（PCR）方法检测布鲁氏菌病（布病）患者血液中布鲁氏菌DNA,分析其与临床症状和抗体水平之间的关系。方法 对门诊就诊患者进行个案调查,包括临床症状、既往史、接触史、用药史等。布病可疑患者,采集血液,分离血清,进行血清试管凝集试验（SAT）,检测布鲁氏菌抗体,按照《布鲁氏菌病诊断》（WS 269—2019）进行布病临床诊断。同时对患者抗凝全血进行细菌DNA提取,应用巢式PCR方法检测血液中布鲁氏菌DNA。对不同症状、抗体水平血液中布鲁氏菌细菌数之间的关系进行统计学分析。结果 118例布病患者中,男性78例,女性40例。患者的临床症状主要有发热、乏力、头痛、胸背酸痛、腰痛、四肢酸痛、关节肌肉疼痛、出汗、失眠和睾丸痛等。118例布病患者血液标本中,布鲁氏菌DNA阳性率为63.56%(75/118),布鲁氏菌细菌数中位数(MED)为7拷贝/mL血液,四分位数(IQR)为2～17拷贝/mL血液。118例布病患者中,SAT检测阳性率为38.14%(45/118)。118例布病患者中,新发病例和复诊病例分别占55.08%(65/118)和44.92%(53/118),新发...     

Development of BruAb2_0168 based isothermal polymerase spiral reaction assay for specific detection of Brucella abortus in clinical samples

Bovine brucellosis, predominantly caused by Brucella abortus is one of the most neglected zoonotic diseases causing severe economic losses in the dairy industry. The early and precise diagnosis of the disease is required to reduce the transmission of infection in humans as well as animals. In the current study, a rapid and novel isothermal amplification-based polymerase spiral reaction (PSR) was developed for the specific detection of Brucella abortus by targeting the BruAb2_0168 gene. The assay could be conducted at 65 °C in a water bath and results can be obtained after 60 min. The detection limit of the PSR assay was found to be 1.33 fg. The sensitivity of the assay was found to be 10⁴ fold higher than conventional PCR and equivalent to real-time PCR (RT-PCR). The assay didn't exhibit cross-reaction with selected pathogenic non-Brucella bacteria and Brucella spp. other than B. abortus. Forty clinical samples were also tested using this novel assay and it was able to detect 25 samples as positive, however, conventional PCR could detect the targeted organism in 22 samples only.To the extent of our knowledge, this is the first report towards the development of a PSR assay for specific detection of B. abortus. The assay can be used as a quick, sensitive and accurate test for the diagnosis of bovine brucellosis in the field setting. Relatively one of the paradigm-shifting aspects of this assay would be it does not require any expensive equipment and the results can be easily visualized by the unaided eye, therefore making PSR a valuable diagnostic tool in field conditions.     

A urine-based polymerase chain reaction method for the diagnosis of visceral leishmaniasis in immunocompetent patients

In the Mediterranean basin and Middle East, including Iran, visceral leishmaniasis (VL), also known as kala-azar, is caused by Leishmania donovani infantum. For the first time, the use of urine samples for the diagnosis of VL in immunocompetent patients has been used in this study. Based on its high sensitivity and specificity, as well as simplicity, this approach can serve as a valuable tool in the diagnosis of VL. We studied 60 urine samples from 60 individuals, 30 of which were patients with VL confirmed by parasitology, serology, or molecular methods, 5 were from healthy individuals, and 25 were from patients with cutaneous leishmaniasis, malaria, brucellosis, and hydatid cyst. Out of 30 samples from confirmed VL immunocompetent patients, 29 were positive (sensitivity, 96.8%) by polymerase chain reaction (RV1 and RV2 primers), and all the remaining 30 samples either from healthy individuals or patients with other diseases were negative (specificity, 100%). High sensitivity, specificity, and simplicity of the test can serve as a valuable tool in the diagnosis of VL.     

Implications of laboratory diagnosis on brucellosis therapy

Brucellosis is a worldwide zoonosis with a huge economic impact on animal husbandry and public health. The diagnosis of human brucellosis can be protracted because the disease primarily presents as fever of unknown origin with unspecific clinical signs and symptoms. The isolation rate of the fastidious etiologic agent from blood cultures is low, and therefore laboratory diagnosis is mainly based on serologic and molecular testing. However, seronegative brucellosis patients have been described, and antibody titers of diagnostic significance are difficult to define. Whether the molecular detection of Brucella DNA in clinical samples should be followed by long-term antibiotic treatment or not is also a matter of debate. The aim of this article is to review and discuss the implications of laboratory test results in the diagnosis of human brucellosis on disease therapy.     

Monovalent latex agglutination reagents for the diagnosis of nonmeningitic pneumococcal infection

The pneumococcus is a leading cause of serious bacterial infection worldwide. Given the difficulties with available assays for the diagnosis of invasive nonmeningitic pneumococcal infection, we evaluated monovalent slide latex agglutination reagents among patients with blood culture-confirmed pneumococcal infection and control patients in Baltimore, Maryland, USA; São Paulo, Brazil; and Cairo, Egypt. Among 50 patients with invasive nonmeningitic pneumococcal infection, 23 had a positive urine test for a sensitivity of 46% (95% confidence intervals of 32% and 61%). Among 39 healthy children, 36 had a negative assay, for a specificity of 92% (95% confidence intervals of 78% and 98%). Among 80 children with pneumonia without a positive blood culture for Streptococcus pneumoniae, the specificity was 88% (95% confidence intervals of 78% and 94%). Although the assay was fairly specific, the positive predictive value using optimistic assumptions was only 73%–83%. This study suggests that this assay has a sensitivity and positive predictive value that may limit its value in some settings.