Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results.     

Rapid detection and monitoring therapeutic efficacy of Mycobacterium tuberculosis complex using a novel real-time assay

We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 nonrespiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was < or =35 and the ratio of real-time RT-PCR and real-time PCR load was > or =1.51. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including nonrespiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.     

Diagnostic Value of T-Cell Interferon-γ Release Assays on Cerebrospinal Fluid for Tuberculous Meningitis

Diagnosis of tuberculous meningitis (TBM) remains a challenge. This study aimed to evaluate the performance of T-SPOT.TB test on cerebrospinal fluid mononuclear cells (CSFMCs) for suspected TBM patients. 43 consecutive patients with suspected TBM were enrolled in the study from June 2011 to September 2014. T-SPOT.TB was performed on both CSFMCs and peripheral blood mononuclear cells (PBMCs). The final diagnosis of TBM was independent of the T-SPOT.TB result. The diagnostic sensitivity, specificity, predictive value, and likelihood ratio of T-SPOT.TB on CSFMCs and PBMCs were analyzed. Of the 43 patients, 12 (27.9%) were finally diagnosed with TBM, 28 (65.1%) with non-TBM, and 3 (7.0%) with indeterminate diagnoses. Of 40 cases with definite diagnoses, the sensitivity and specificity were 92.0% and 96.0% for T-SPOT.TB on CSFMCs, and 83.0% and 82.0% for T-SPOT.TB on PBMCs, respectively. The positive predictive value (PPV) and negative predictive value (NPV) of T-SPOT.TB on CSFMCs were 85.0% and 96.0%, respectively. The PPV and NPV were 67.0% and 92.0% for T-SPOT.TB on PBMCs. The difference of T-SPOT.TB between CSFMCs and PBMCs was not significant so far as sensitivity, specificity, PPV, and NPV were concerned (P>0.05 for each). However, T-SPOT.TB on CSFMC and CSFMC: PBMC in TBM cases seemed higher than that in non-TBM cases. Our study further showed that T-SPOT.TB on CSFMCs might be a rapid and accurate diagnostic test for TBM. CSFMC: PBMC T-SPOT.TB ratio might be useful for the early diagnosis of TBM.     

评估国产（1,3）-β-D-葡聚糖检测试剂的诊断价值

目的比较国产与进口（1,3）-β-D-葡聚糖（BG）检测试验并对国产试剂在侵袭性真菌感染中的诊断价值进行评估。方法回顾性调查2008年4月～2009年5月我院203例（342份样本）怀疑侵袭性真菌感染的住院患者,根据诊断标准分为感染组和非感染组,对其中同时进行国产与日本试剂检测的100份样本进行配对t检验比较;在不同的判断标准下计算国产试剂的灵敏度、特异度、阳性预测值和阴性预测值等,采用McNemar配对χ2检验对不同方法进行比较。结果国产和日本试剂在感染组和非感染组配对t检验结果分别为P=0.235,P=0.076;国产试剂以单次≥20pg/mL和≥50pg/mL为界值的灵敏度、特异度分别为78.3%,67.2%和56.5%,85.0%,双次≥20pg/mL和≥50pg/mL为界值的灵敏度、特异度分别为25.0%,64.9%和25.0%,87.7%。结论国产与日本试剂检测值无统计学差异;国产试剂单次≥50pg/mL为界值较20pg/mL可以明显提高特异度,降低假阳性率,而不影响灵敏度。连续双份血清阴性（BG〈50pg/mL）具有较高的特异度和阴性预测值。     

Development and application of a simple loop-mediated isothermal amplification method on rapid detection of Listeria monocytogenes strains

A loop-mediated isothermal amplification (LAMP) method for rapid detection of the food-borne L. monocytogenes strains had been developed and evaluated in this study. The optimal reaction condition was 65°C for 45 min, with the detection limit as 1 pg DNA/tube and 100 CFU/reaction. Application of the established LAMP assay was performed on 182 food-borne L. monocytogenes strains using a rapid procedure and easy result confirmation, with the sensitivity of LAMP versus PCR assays as 96.7% (176/182) and 91.2% (166/182), respectively; with 100% specificity, positive predictive value (PPV) and negative predictive value (NPV) for both assays.     

Rapid dot sputum and serum assay in pulmonary tuberculosis

A rapid direct sputum (Sp.) and/or antibody assay, based on immunoblotting and enzyme immunoassay is described. The test can detect mycobacterial antigens or antibodies in clinical specimens from pulmonary tuberculosis (TB) patients. In this study, 87 sputa, 87 sera and 40 paired sputa and sera were utilized from smear-positive and smear-negative, culture-positive patients; 59 sputa, 37 sera and 22 paired sputa and sera from nontuberculosis respiratory disease patients and 68 sera from healthy controls. The antigen detection in sputum by dot assay has 86.1% sensitivity on active tuberculosis patients, 92.9% specificity, 91.6% positive predictive value (PPV), 88.2% negative predictive value (NPV) and 10.3% error. The antibody assay has 83.6% sensitivity, 95.4% specificity, 94.4% positive predictive value, 85.6% negative predictive value and 11% error. The test performed on paired sputum and serum (Sr.) samples has a sensitivity of 93.3%, which rose to 96.1% on smear-positive and culture-positive patients, but the specificity decreased to 83% in sputum, whereas in serum it was 92%. The results of the assay, combined with clinical and radiological data, could form the basis for starting an earlier course of treatment for tuberculosis.     

Polymerase chain reaction for detection of endocervical Chlamydia trachomatis infection in women attending a gynecology outpatient department in India

OBJECTIVES: To detect Chlamydia trachomatis infection by polymerase chain reaction (PCR) in symptomatic women attending a gynecology clinic in a city hospital and in randomly selected slum dwellers. STUDY DESIGN: Endocervical specimens were collected from 350 women with genitourinary complaints (group I) and 53 slum dwellers (group II). Samples were analyzed by PCR, direct fluorescence assay (DFA) and Giemsa stain cytology for detection of C trachomatis and compared for their sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). RESULTS: The prevalence of endocervical C trachomatis infection was 43.1% and 24.5% in groups I and II, respectively. The sensitivity, specificity, PPV and NPV of PCR were 80.0%, 75.0%, 66.6% and 85.7%, respectively, when DFA was considered true positive. The percent increment in detection of C trachomatis by PCR was 15.3%. CONCLUSION: Giemsa stain cytology has low sensitivity and specificity; hence, it cannot be recommended for use as a diagnostic technique. It appears that PCR can be used routinely in Chlamydia diagnosis and in screening selected populations. The high positivity of C trachomatis infection in urban slum dwellers is cause for concern.     

Diagnostic Accuracy and Turnaround Time of the Xpert MTB/RIF Assay in Routine Clinical Practice

The Xpert MTB/RIF assay was introduced for timely and accurate detection of tuberculosis (TB). The aim of this study was to determine the diagnostic accuracy and turnaround time (TAT) of Xpert MTB/RIF assay in clinical practice in South Korea. We retrospectively reviewed the medical records of patients in whom Xpert MTB/RIF assay using sputum were requested. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of pulmonary tuberculosis (PTB) and detection of rifampicin resistance were calculated. In addition, TAT of Xpert MTB/RIF assay was compared with those of other tests. Total 681 patients in whom Xpert MTB/RIF assay was requested were included in the analysis. The sensitivity, specificity, PPV and NPV of Xpert MTB/RIF assay for diagnosis of PTB were 79.5% (124/156), 100.0% (505/505), 100.0% (124/124) and 94.0% (505/537), respectively. Those for the detection of rifampicin resistance were 57.1% (8/14), 100.0% (113/113), 100.0% (8/8) and 94.9% (113/119), respectively. The median TAT of Xpert MTB/RIF assay to the report of results and results confirmed by physicians in outpatient settings were 0 (0–1) and 6 (3–7) days, respectively. Median time to treatment after initial evaluation was 7 (4–9) days in patients with Xpert MTB/RIF assay, but was 21 (7–33.5) days in patients without Xpert MTB/RIF assay. Xpert MTB/RIF assay showed acceptable sensitivity and excellent specificity for the diagnosis of PTB and detection of rifampicin resistance in areas with intermediate TB burden. Additionally, the assay decreased time to the initiation of anti-TB drugs through shorter TAT.     

Ultrasound diagnostic schema for the determination of increased risk for chromosomal fetal aneuploidies in the first half of pregnancy

The aim of the study was to develop an early ultrasound diagnostic schema for the determination of increased risk for fetal chromosomal aneuploidies. The study was conducted on a population of 1318 pregnant women divided into 2 groups: 1255 women with the normal course of pregnancy and 63 women with diagnosed fetal abnormalities. There were 34 cases of chromosomal abnormalities (trisomy 21, 18, 13; triploidy; unbalanced inversion 9; deletion 16) and 29 cases of structural malformations. The estimation of the range of normal values was performed for the nuchal translucency (NT) measurement between 11 and 13 weeks and the nasal bone length (NB) measurement between 12 and 20 week. The results obtained in the collective set of normal pregnancies constituted the basis for the calculation of the range of normal values. The measurements of NB and NT showed a linear value increase with the pregnancy course. The following test characteristics (correlation to CRL) were recorded: NB - sensitivity 60%, specificity 98%, positive predictive value (PPV+) 43%, negative predictive value (NPV-) 98.9%. For the assumption that the test outcome means the presence or absence of the nasal bone in the ultrasound scan the sensitivity was 40%, but specificity 100%; NT - sensitivity 63.6%, specificity 98.2%, PPV+ 38.9%, NPV - 98.2%; NT + NB - presents similar characteristic to the NB or NT alone - sensitivity 55.6%, specificity 98.6%, PPV+ 50%, NPV - 98.9%. The following test characteristics for chromosomal aberration markers (correlation to BPD) were observed: NB - sensitivity 68.4%, specificity 97.4%, PPV+ 56.5%, NPV - 98.4%; NT - sensitivity 73.9%, specificity 97.9%, PPV+ 54.8%, NPV- 99.2%; NT + NB - sensitivity 94.7%, specificity 98.9%, PPV+ 90%, NPV - 99.7%, respectively. The "genetic sonogram" protocol for the structural defect detection was analysed: sensitivity was 80%, specificity 100%, PPV+ 100%, NPV - 99.7%. It is concluded that the new biometric parameter--nasal bone length (NB) and the corrected one--nuchal translucency thickness (NT) are useful markers for fetal abnormalities, especially for chromosomal aberrations. High predictive values of the diagnostic schema for the detection of aneuploidies and structural defects indicate that its application in correlation with the biparietal diameter (BPD) is highly recommended. The proposed schema is an effective algorithm for prenatal diagnostics characterised by high prognostic values. The possible introduction of the schema could result in a decrease of the invasive procedure rates, which could minimise the rate of miscarriages as a complication of amniocenteses.     

A machine learning tool for the diagnosis of SARS-CoV-2 infection from hemogram parameters

Monocytes and neutrophils play key roles in the cytokine storm triggered by SARS-CoV-2 infection, which changes their conformation and function. These changes are detectable at the cellular and molecular level and may be different to what is observed in other respiratory infections. Here, we applied machine learning (ML) to develop and validate an algorithm to diagnose COVID-19 using blood parameters. In this retrospective single-center study, 49 hemogram parameters from 12,321 patients with clinical suspicion of COVID-19 and tested by RT-PCR (4239 positive and 8082 negative) were analysed. The dataset was randomly divided into training and validation sets. Blood cell parameters and patient age were used to construct the predictive model with the support vector machine (SVM) tool. The model constructed from the training set (5936 patients) achieved an accuracy for diagnosis of SARS-CoV-2 infection of 0.952 (95% CI: 0.875–0.892). Test sensitivity and specificity was 0.868 and 0.899, respectively, with a positive (PPV) and negative (NPV) predictive value of 0.896 and 0.872, respectively (prevalence 0.50). The validation set model (4964 patients) achieved an accuracy of 0.894 (95% CI: 0.883–0.903). Test sensitivity and specificity was 0.8922 and 0.8951, respectively, with a positive (PPV) and negative (NPV) predictive value of 0.817 and 0.94, respectively (prevalence 0.34). The area under the receiver operating characteristic curve was 0.952 for the algorithm performance. This algorithm may allow to rule out COVID-19 diagnosis with 94% of probability. This represents a great advance for early diagnostic orientation and guiding clinical decisions.     

Heat denaturation increases the sensitivity of the cytomegalovirus loop‐mediated isothermal amplification method

A new protocol for CMV LAMP with an additional heat denaturation step was developed. While the sensitivity of the original CMV LAMP method was 500 copies/tube, sensitivity was increased by up to 100 copies/tube by additional heat denaturation. CMV DNA was detected in 103 of 350 samples (29.4%) by the original CMV LAMP procedure and 148 of 350 samples (42.3%) by the new CMV LAMP protocol. When the pp65 antigenemia assay was used as the standard method, the sensitivity, specificity, PPV, and NPV of the new protocol were 92.9%, 77.7%, 62.2%, and 96.5%, respectively.     

ERCP-directed brush cytology prepared by the Thinprep method: test performance and morphology of 149 cases.

Conventionally prepared endobiliary brushings are moderately (42%) sensitive and highly (98%) specific in detecting malignancy. The performance and morphological features of brushings prepared by Thinprep, a liquid-based method are mostly unknown. All brushings were retrieved from the laboratory files. Disease was classified as benign or malignant by linkage with the provincial cancer registry and sensitivity, specificity, positive (PPV) and negative predictive values (NPV) calculated. True positives and negatives were reviewed and predictive morphological features analysed by regression tree analysis. Out of 149 brushings, 55 (37%) were positive and 94 (63%) negative. Malignancy was identified in 86 (58%) and benign disease in 63 (42%) of the cases. The sensitivity was 51%, specificity 83%, PPV 80% and NPV 55%. Absolute discriminants of positive and negative brushings were not found, but nuclear cytoplasmic ratio was a useful feature. The performance of Thinprep-prepared brushings from this anatomical site was comparable with conventional preparations.     

The inclusion of N-terminal pro-brain natriuretic peptide in a sensitive screening strategy for systemic sclerosis-related pulmonary arterial hypertension: a cohort study

Introduction

Pulmonary arterial hypertension (PAH) is a major cause of mortality in systemic sclerosis (SSc). Screening guidelines for PAH recommend multiple investigations, including annual echocardiography, which together have low specificity and may not be cost-effective. We sought to evaluate the predictive accuracy of serum N-terminal pro-brain natriuretic peptide (NT-proBNP) in combination with pulmonary function tests (PFT) (‘proposed’ algorithm) in a screening algorithm for SSc-PAH.

Methods

We evaluated our proposed algorithm (PFT with NT-proBNP) on 49 consecutive SSc patients with suspected pulmonary hypertension undergoing right heart catherisation (RHC). The predictive accuracy of the proposed algorithm was compared with existing screening recommendations, and is presented as sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).

Results

Overall, 27 patients were found to have pulmonary hypertension (PH) at RHC, while 22 had no PH. The sensitivity, specificity, PPV and NPV of the proposed algorithm for PAH was 94.1%, 54.5%, 61.5% and 92.3%, respectively; current European Society of Cardiology (ESC)/European Respiratory Society (ERS) guidelines achieved a sensitivity, specificity, PPV and NPV of 94.1%, 31.8%, 51.6% and 87.5%, respectively. In an alternate case scenario analysis, estimating a PAH prevalence of 10%, the proposed algorithm achieved a sensitivity, specificity, PPV and NPV for PAH of 94.1%, 54.5%, 18.7% and 98.8%, respectively.

Conclusions

The combination of NT-proBNP with PFT is a sensitive, yet simple and non-invasive, screening strategy for SSc-PAH. Patients with a positive screening result can be referred for echocardiography, and further confirmatory testing for PAH. In this way, it may be possible to shift the burden of routine screening away from echocardiography. The findings of this study should be confirmed in larger studies.     

Role of urinary NMP-22 combined with urine cytology in follow-up surveillance of recurring superficial bladder urothelial carcinoma

OBJECTIVE: To determine efficacy and utility of NMP-22 in follow-up of bladder urothelial carcinoma (UC) and compare NMP-22 as a single evaluating test vs combination with cytology. STUDY DESIGN: Ninety-four consecutive urine cytology samples of bladder UC were identified. Patients received follow-up urine cytology, NMP-22 testing and cystoscopy with surgical biopsy. RESULTS: NMP-22 specificity was 100%, sensitivity 45%, positive predictive value (PPV) 100% and negative predictive value (NPV) 87%. NMP-22 showed lower sensitivity for high-grade lesions and higher for low-grade lesions. Cytologic diagnosis had a high inconclusive rate; when regarded as positive, it resulted in 75% sensitivity, 58% specificity, 33% PPV and 89% NPV. NMP-22 correctly classified 60% of false negative cases diagnosed by cytology with low-grade UC and clarified 27 inconclusive cytologic diagnoses. NMP-22 misclassified 9 cases as false negative, all with high-grade UC; all were correctly identified on cytology as true positive. Combined interpretation showed 90% sensitivity, 92% specificity, 75% PPV and 98% NPV. CONCLUSION: NMP-22 complements cytology by its higher sensitivity for low-grade lesions; its values are not affected by bacillus Calmette Guérin therapy changes, which are limiting in cytology. Combined interpretation of NMP-22 and cytology shows promise as an effective, noninvasive method for surveillance of UC.     

Bruikbaarheid en validiteit van de Nederlandse versie van de Montreal Cognitive Assessment (MoCA-D) bij het diagnosticeren van Mild Cognitive Impairment

Objective: The MoCA is a new screening test to detect Mild Cognitive Impairment (MCI). Purpose of this study is validating the Dutch version (MoCA-D). Method: We administered the MoCA-D to healthy control subjects and to elderly with MCI or dementia from a memory disorder outpatient clinic and a geriatric (outpatient) clinic (n=30, 32, 37 respectively, age≥60). Neuropsychological testing was part of the standard procedure for patients to diagnose MCI. Sensitivity, specificity and predictive values (positive: PPV and negative: NPV) of the MoCA-D were assessed. Results: A significant effect of group was found on MoCA-D total score (F(2,95)=67,9; p<0,01). With a cutoff score of d25, sensitivity and specificity to detect MCI in relation to healthy controls were 72% and 73%, respectively. PPV and NPV were 84% and 56%, respectively. With a cut-off score of d20, sensitivity to detect dementia in relation to MCI was 100% for severe dementia and 75% for mild dementia. Specificity for dementia was 81%, PPV 94% and NPV 55%. Conclusion: The MoCA-D distinguishes between healthy elderly, MCI patients and dementia patients. However, in this study, insufficient sensitivity and poor specificity were found. For the present, applying a broader and flexible screening procedure in order to detect MCI seems a more useful method than the interpretation of one test result in particular.     

Predictive power of cytomorphological features in equivocal (C3, C4) breast FNAC

Definitive immediate diagnosis in breast fine needle aspiration cytology (FNAC) remains the aim for cytopathologists. We reviewed 72 consecutive equivocal (C3 and C4) aspirates with respect to 16 cytomorphological criteria. We assessed the power of each criterion at predicting either a malignant [positive predictive value (PPV)] or a benign [negative predictive value (NPV)] diagnosis by correlation with follow-up histology. Blind review led to 34% of cases being correctly definitively diagnosed. Eccentrically placed epithelial cell nuclei (PPV = 88%, sensitivity = 67%, specificity = 87%) and coarse nuclear chromatin (PPV = 81%, sensitivity = 72%, specificity = 83%) are the features that are most useful in predicting malignancy in this selected series. The presence of myoepithelial cells within epithelial groups is not a good indicator of a benign diagnosis (NPV = 24%, sensitivity = 80%, specificity = 53%).     

国产血清半乳甘露聚糖检测试剂对侵袭性肺曲霉菌病的诊断价值评估

目的：评估国产血清半乳甘露聚糖（Galactomannan,GM）检测试剂对侵袭性肺曲霉菌病的诊断价值。方法根据血液病/恶性肿瘤患者侵袭性真菌病的诊断标准与治疗原则（第四次修订版）[1]收集临床确诊侵袭性肺曲霉菌病（inva-sive pulmonary aspergillosis,IPA）、临床诊断IPA、拟诊IPA、排除IPA四组病例。采用天津贻诺琦公司酶联免疫吸附法（ELISA）试剂检测纳入的86例患者血清标本的GM浓度,分析其敏感性、特异性、阳性预测值（PPV）、阴性预测值（NPV）。结果86例病例中,临床诊断27例、拟诊12例、排除47例。在3种不同的阳性判断标准下,敏感性：9444%、9630%、6296%;特异性：5625%、4576%、6441%;PPV：4474%、4483%、4474%;NPV：9643%、9643%、7917%。统计学分析证实标准1（即血清GM值〉095μg/L为阳性,〈075μg/L为阴性,075~095μg/L为灰区,未将灰区加入计算）在3种判断标准中最优,故选择其为最终判断标准。结论该血清GM检测试剂盒诊断性能较好,可以用于侵袭性肺曲霉菌病的辅助诊断。