An evaluation of false-positive rifampicin resistance on the Xpert MTB/RIF

BACKGROUND Mycobacterium tuberculosis (MTB) is one of the most significant causes of mortality and morbidity. Early diagnose is important especially in multiple drug resistant tuberculosis to avoid transmission. Traditional techniques requires at least one to three weeks for diagnosis of tuberculosis. Diagnostic delays with multiple drug resistant tuberculosis are associated with worse clinical outcomes and increased transmission The Xpert MTB/RIF assay is one of the new diagnostic device for the diagnosis of tuberculosis and rapid detection of rifampicin resistance. OBJECTIVE We assessed the performance of Xpert MTB/RIF assay for detecting rifampicin resistance using phenotypic drug susceptibility tests as automated BD MGIT 960. METHODS Total of 2136 specimens were included in the study. Xpert MTB/RIF testing was performed on samples, using version 4 cartridges, according to the manufacturer's recommendations. The MTBC culture and first-line phenotypic DST were performed in automated BD MGIT 960 (Becton & Dickinson, USA) according to the recommendations of the manufacturer. Agar proportion was used in the case of inconsistency for rifampicin resistance. FINDINGS Thirty-four samples (19 respiratory and 15 nonrespiratory samples) were determined as positive for M. tuberculosis complex by Xpert MTB/RIF (Cepheid GeneXpert® System, USA). Xpert MTB/RIF assay detected 4/34 (11.7%) specimens as rifampicin resistant. One of the rifampicin resistant isolates was determined susceptible in MGIT 960 automated system. This isolate was also tested with agar proportion method and found susceptible to rifampicin. MAIN CONCLUSION The Xpert MTB/RIF assay can be used as first-line assay for the detection of M. tuberculosis. However, microbiologists must be aware of the limitations of the assay.

Associated posterior pelvic injury patterns in transverse-oriented acetabular fracture / Padrões de lesão pélvica posterior associada em fratura acetabular com orientação transversal

ABSTRACT Objective: Our study analyzed the incidence of posterior pelvic injury patterns and their influence on the surgical treatment of transverse-oriented acetabular fractures . Methods: Fifty-one transverse-oriented acetabular fracture cases admitted between 1999 and 2013 were evaluated retrospectively. Comparative studies were performed for groups organized by acetabular fracture type, degree of sacroiliac separation, and postoperative reduction quality . Results: Associated posterior pelvic injuries were found in 34 (66.7%) of the 51 patients. There were 32 sacroiliac separations in the 34 patients with associated posterior pelvic injury, and ipsilateral sacroiliac separations were more frequent in this subgroup. Measurements guided by computerized tomography showed that 16 sacroiliac separations were ≤0.5 cm (mean=0.43±0.14 cm), 10 were 0.5-1 cm (mean=0.73±0.17 cm), and the remaining 6 were >1 cm (mean=1.55±0.15 cm). In the group of 34 patients with associated posterior pelvic injury, acetabular reduction was anatomic in 19 (55.9%) patients, imperfect in 10 (29.4%) patients, and poor in 5 (14.7%) patients. For isolated acetabular fractures, reduction rates were as follows: 12 (70.6%) anatomic, 3 (17.6%) imperfect, and 2 (11.8%) poor. The rate of anatomic reduction was significantly higher when sacroiliac separation was ≤0.5 cm (p=0.027) . Conclusion: Associated posterior pelvic injuries, especially ipsilateral sacroiliac joint separation, accompany most transverse-oriented acetabular fractures and may influence the quality of acetabular reduction. Level of Evidence III, Therapeutic Studies Investigating the Results of Treatment.

RESUMO Objetivo: Nosso estudo analisou a incidência de padrões de lesão pélvica posterior e sua influência no tratamento cirúrgico das fraturas do acetábulo com orientação transversal. Métodos: Cinquenta e um casos de fratura acetabular com orientação transversal foram avaliados retrospectivamente entre 1999 e 2013. Foram realizados estudos comparativos para grupos formados de acordo com o tipo de fratura acetabular, grau de separação sacroilíaca e qualidade da redução no pós-operatório. Resultados: Constataram-se lesões pélvicas posteriores associadas em 34 (66,7%) dos 51 pacientes. Havia 32 separações sacroilíacas nos 34 pacientes com lesão pélvica posterior associada, e as separações sacroilíacas ipsilaterais foram mais frequentes nesse subgrupo. De acordo com medições guiadas por tomografia computadorizada, 16 separações sacroilíacas foram ≤ 0,5 cm (média = 0,43 ± 0,14 cm), 10 estavam entre 0,5 e 1 cm (média = 0,73 ± 0,17 cm) e os 6 restantes foram >1 cm (média = 1,55 ± 0,15 cm). No grupo de 34 pacientes com lesão pélvica posterior, a redução acetabular foi anatômica em 19 (55,9%) pacientes, imperfeita em 10 (29,4%) pacientes e deficiente em5 (14,7%) pacientes. Nas fraturas acetabulares, as taxas de redução foram as seguintes: 12 (70,6%) anatômicas, 3 (17,6%) imperfeitas e 2 (11,8%) deficientes. A taxa de redução anatômica foi significativamente maior quando o grau de separação sacroilíaca foi ≤ 0,5 cm (p = 0,027). Conclusão: As lesões pélvicas posteriores associadas, especialmente a separação da articulação sacroilíaca ipsilateral, acompanham a maioria das fraturas do acetábulo com orientação transversal e podem influenciar a qualidade da redução acetabular. Nivel de Evidência III, Estudos Terapêuticos - Investigação dos Resultados do Tratamento.

Evaluation of crystal violet decolorization assay for minimal inhibitory concentration detection of primary antituberculosis drugs against Mycobacterium tuberculosis isolates

In this study we evaluated the crystal violet decolorization assay (CVDA) for detection of minimum inhibitory concentration (MIC) of antituberculosis drugs. 53 isolates were tested in this study and 13 of them were multidrug resistant (MDR) isolates. The antibiotics concentrations were 2-0.06 mg/L for isoniazid (INH) and rifampicin (RIF) and were 16-0.25 mg/L for streptomycin (STM) and ethambutol (EMB). Crystal violet (CV-25 mg/L) was added into the microwells on the seventh day of incubation and incubation was continued until decolorization. Decolorization of CV was the predictor of bacterial growth. Overall agreements for four drugs were detected as 98.1%, and the average time was detected as 9.5 ± 0.89 day after inoculation. One isolate for INH and two isolates for STM were determined resistant in the reference method, but susceptible by the CVDA. One isolate was susceptible to EMB by the reference method, but resistant by the CVDA. All results were concordant for RIF. This study shows that CVDA is a rapid, reliable and suitable for determination of MIC values of Mycobacterium tuberculosis. And it can be used easily especially in countries with limited-sources.

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosisisolates

The purpose of this study is to evaluate four rapid colourimetric methods, including the resazurin microtitre assay (REMA), malachite green decolourisation assay (MGDA), microplate nitrate reductase assay (MNRA) and crystal violet decolourisation assay (CVDA), for the rapid detection of multidrug-resistant (MDR) tuberculosis. Fifty Mycobacterium tuberculosisisolates were used in this study. Eighteen isolates were MDR, two isolates were only resistant to isoniazid (INH) and the remaining isolates were susceptible to both INH and rifampicin (RIF). INH and RIF were tested in 0.25 µg/mL and 0.5 µg/mL, respectively. The agar proportion method was used as a reference method. MNRA and REMA were performed with some modifications. MGDA and CVDA were performed as defined in the literature. The agreements of the MNRA for INH and RIF were 96% and 94%, respectively, while the agreement of the other assays for INH and RIF were 98%. In this study, while the specificities of the REMA, MGDA and CVDA were 100%, the specificity of the MNRA was lower than the others (93.3% for INH and 90.9% for RIF). In addition, while the sensitivity of the MNRA was 100%, the sensitivities of the others were lower than that of the MNRA (from 94.1-95%). The results were reported on the seventh-10th day of the incubation. All methods are reliable, easy to perform, inexpensive and easy to evaluate and do not require special equipment.

A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay

The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.

Resazurin microtiter assay for isoniazid, rifampicin, ethambutol and streptomycin resistance detection in Mycobacterium tuberculosis: Updated meta-analysis

The present meta-analysis aims to assess the evidence regarding the diagnostic accuracy and performance characteristics of the colorimetric redox indicator [CRI] assay with a special emphasis on the use of the resazurin microtiter assay [REMA] for determination of primary anti-tuberculosis drug resistance. By updating previous literature searches in Medline PubMed, ISI Web, Web of Science and Google academic databases of the REMA test for determination of primary anti-tuberculosis drug resistance, this meta-analysis includes 14 studies for isoniazid [INH]; 15 studies for rifampicin [RIF]; 6 studies for streptomycin [STR]; and 5 studies for ethambutol [EMB]. SROC curve analysis was performed for meta-analysis and diagnostic accuracy was summarized.: Pooled sensitivity was 96% [94-98%] for INH, 97% [95-98%] for RIF, 92% [87-96%] for EMB and 92% [88-95%] for STR. Pooled specificity for INH, RIF, EMB and STR was 96% [95-98%], 99% [98-99%], 86% [81-89%] and 90% [87-93%], respectively. Susceptibility testing results had been obtained in 8-9 days. In conclusion, REMA seems to be a reliable test for the determination of multidrug resistant [MDR] isolates in laboratories with limited resources. However, few studies for STR and EMB have been found, and costeffectiveness studies need to be determined to recommend its widespread use

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.

Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 µg/mL and 2.0-0.03 µg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 µg/mL and the RIF concentration was between 2.0-0.06 µg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.

A rapid detection of multidrug-resistant Mycobacterium tuberculosis by a nitrate reductase assay on blood agar

The susceptibility of 49 Mycobacterium tuberculosis clinical isolates to isoniazid (INH) and rifampisin (RIF) (28 multi-drug resistant-tuberculosis samples) was determined by a nitrate reductase assay (NRA) on blood agar. Agreement between the NRA and other testing methods was found to be 93.8 percent for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8 percent, 94.2 percent, 86.6 percent and 97 percent, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4 percent, 96.4 percent, 95 percent and 93.1 percent. In conclusion, we show here that blood agar can be used effectively for the NRA test.

Spontaneous rupture of a giant diaphragmatic hydatid cyst into the intrapleural space

We report a case of giant diaphragmatic hydatid cyst which ruptured spontaneously into the intrapleural space in a patient with coexistent giant hepatic hydatid cyst. A 62-year-old female was admitted for dyspnea, nausea, vomiting, and right thoracic pain. Clinical findings, laboratory and radiological examinations including multislice computed tomography scan were consistent with the diagnosis of a giant diaphragmatic hydatid cyst which ruptured into the intrapleural space. Surgical intervention was performed through thoracotomy and phrenotomy in a one-stage operation for both cysts. This case shows that hydatid cysts of the diaphragm can rupture into the intrapleural space spontaneously. One-stage operation through thoracotomy may be successful for the surgical intervention for diaphragmatic hydatid cysts with coexistent hepatic cyst

Drug susceptibility testing of Mycobacterium tuberculosis by the broth microdilution method with 7H9 broth

In this study, we have evaluated the broth microdilution method (BMM) for susceptibility testing of Mycobacterium tuberculosis. A total of 43 clinical isolates of M. tuberculosis and H37Rv as a control strain were studied. All isolates were tested by the proportion method and the BMM for isoniazid (INH), rifampicin (RIF), streptomycin (STR), and ethambutol (ETM). The proportion method was carried out according to the National Committee for Clinical Laboratory Standards (NCCLS) on Lõwenstein-Jensen (LJ) medium. The BMM was carried out using 7H9 broth with 96 well-plates. All strains were tested at 3.2-0.05 µg/ml, 16-0.25 µg/ml, 32-0.5 µg/ml, and 32-0.5 µg/ml concentrations for INH, RIF, STR, and ETM, respectively. When the BMM was compared with the proportion method, sensitivity was 100, 100, 96.9, and 90.2 percent, while specificity was 100, 85.7, 90.9, and 100 percent for INH, RIF, STR, and ETM, respectively. The plates were examined 7, 10, 14, and 21 days after incubation. The majority of the result were obtained at 14th days after incubation, while the proportion method result were ended in 21-28 days. According to our results, it may be suggested that the BMM is suitable for early determining of multidrug-resistance-M. tuberculosis strains in developed or developing countries.

HLA alleles and lung cancer in a Turkish population

Background: The concept of genetic factors playing a role in the pathogenesis of lung cancer has gained increased attention. The present study was undertaken to examine the question of HLA association with lung cancer and to investigate the effects of HLA on survival time

Methods: The distribution of HLA class I [A, B, C] antigens and class II [DR, DQ] alleles were studied in 81 unrelated Turkish patients with lung cancer. The HLA status of patients was compared with that of a control group consisting of 117 ethnically matched healthy donors. HLA class I antigens were studied by Terasaki?s microlymphocytotoxicity test and HLA class II alleles were studied by polymerase chain reaction with the sequence specific primer [PCR-SSP] low resolution method

Results: Only the frequencies of HLA-B51 and -DRB1x15 were lower in the lung cancer group compared with the healthy control patients. In a univariate analysis, age [P=0.03], Karnofsky Performance Status [P=0.0001], stage [P=0.01], HLAA24[9] [P=0.008], HLA B53 [P=0.0006], HLA B63[15] [P=0.01], HLA B64[14] [P=0.01], HLA B65[14] [P=0.01] and HLA CW5 [P=0.01] were significant prognostic factors. In a multivariate analysis, Karnofsky Performance Status [P=0.001], stage [P=0.02], HLA B53 [P=0.03] and HLA B64[14] [P=0.03] were independent prognostic variables

Conclusion: This study demonstrates different HLA types among patients with lung cancer and healthy control subjects. Our results suggest that HLA antigens might affect the prognosis in lung cancer. Further investigations are warranted to delineate any possible role of the HLA system in the pathogenesis and prognosis of lung cancer

Comparison of the Proportion Method With Mycobacteria Growth Indicator Tube and E-test for Susceptibility Testing of Mycobacterium tuberculosis

The aim of this study was to investigate the correlation between proportion method with mycobacteria growth indicator tube (MGIT) and E-test for Mycobacterium tuberculosis. Forty clinical isolates were tested. MGIT and E-test with the first line antituberculous drugs correlated with the proportion method. Our results suggested that MGIT and E-test methods can be routinely used instead of the proportion method