Search

To present our validation study of the BD BACTEC FX blood culture system for sterility testing of cardiovascular tissues aimed for human application. For operational qualification, we performed temperature mapping of the system, vacuum test using non-inoculated BACTEC vials, and growth promotion tests by injecting contaminant strains into aerobic and anaerobic bottles. For performance qualification, negative control, assessment of method suitability, evaluation of sensitivity limits, control of neutralization of antibiotics in biopsy samples from allografts and tissue toxicity effects, were performed. Tissue samples and transport/cryopreservation solutions were homogenized in GentleMACS Dissociator and injected into BACTEC Plus aerobic and anaerobic vials for incubation at 35 °C for 14 days. Tissues were spiked with aerobic and anaerobic bacteria and fungi. Growth of contaminants appeared in all aerobic and anaerobic vials for Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa; in anaerobic vials for Cutibacterium (Propionibacterium) acnes and Clostridium sporogenes; and only in aerobic vials for Candida albicans and Aspergillus brasiliensis. The majority of bacterial strains were detected within two days (59-100%), exceptionally between 3 and 14 days. In contrast, fungal contaminations were detected within 2, 3-6, 7-10 and after 10 days of incubation in 33.3, 71.6, 96.6 and 99.9% of cases,respectively. Uninhibited growth appeared in the tissue biopsies and homogenized tissues with and without antibiotics and in other solutions. BD BACTEC blood culture system with GentleMACS Dissociator is a rapid and efficient tool for detection of contamination in cardio-vascular tissues aimed for human application.

Theoretically, Aspergillus spp. grow in culture media, but frequently, blood cultures of patients with invasive Aspergillosis are negative, even if until now, the reasons are not clear. This aspect underlines the lack of a good strategy for the cultivation and isolation of Aspergillus spp. In order to develop a complete analytical method to detect Aspergillus in clinical and pharmaceutical samples, we investigated the growth performance of two blood culture systems versus the pharmacopeia standard method. At

В данном клиническом случае демонстрируется лечение и ведение туберкулеза легких у пациента с Ну‑ТБ. Проведен полный курс химиотерапии согласно приказу МЗРК №214 от 30.11.2020г «Об утверждении правил проведения мероприятий по профилактике туберкулеза». В результате проведенного удалось добиться устранения симптомов интоксикации, нормализации анализов, конверсии мазка мокроты, положительной рентгенологической динамики и выписать пациента в удовлетворительном состоянии для продолжения химиотерапии в амбулаторных условиях под наблюдение участкового фтизиатра. Пациент в амбулаторных условиях закончил полный курс лечения. Выставлен исход «Вылечен»., Isoniazid‑resistant tuberculosis (HR‑TB) is characterized by resistance to isoniazid and sensitivity to rifampicin. This form can be viewed as a potential precursor to multidrug‑resistant tuberculosis (MDR‑TB). According to the WHO, the prevalence of HR‑ TB is 5.0–11.0% on average. Therefore, it is of course important to control the spread of primary isoniazid resistance and prevent the amplification of resistance. WHO currently recommends treatment with rifampicin (R), ethambutol (E), pyrazinamide (Z) and levofloxacin (Lfx) for 6 months for patients with confirmed HR‑TB. Since the end of 2020, this regime has begun to be implemented in the Republic of Kazakhstan. In clinical phthisiology in the country, there is not yet sufficient experience in the treatment of HR‑TB. We observed the successful treatment of a patient with isoniazid‑resistant TB using the 6REZLfx regimen, and the authors felt it necessary to share their experience with a wide range of TB ‑ doctors.

BACKGROUND Tuberculous lymphadenitis is the commonest form of extrapulmonary tuberculosis and tissue diagnosis is the mainstay in the diagnosis of extrapulmonary tuberculosis. This study was conducted to compare cytology, ZN staining, fine-needle aspiration technique and culture findings of clinically suspected tuberculous lymphadenitis cases. MATERIALS AND METHODS This is a descriptive study. Total 300 patients of lymphadenopathy referred to the Department of Microbiology, Anugrah Narayan Magadh Medical College, Gaya, Bihar and Associated Hospital of Bihar between May 2014 and May 2017, were included. Using solid culture (BACTEC) as the gold standard, we assessed the sensitivity, specificity, positive predictive value (PPV) and negative predictive value of the FNAC for detecting MTB and ZN staining for acid-fast bacilli (AFB) respectively. RESULTS A total of 300 fine-needle aspirated specimens from lymph nodes were included in the study. Out of 300 cases, 140 aspirates were reported as cytomorphology suggestive of tuberculous lymphadenitis. The age ranged from 1 to 70 years, with the mean age of 35.5 years. Female preponderance was noted accounting for 57.14% (80/140) of cases. Maximum number of patients were from age group of 10–29 years comprising 42.15% of the group (59/140). Out of 300 cases, 68(48.57%) had lymphadenitis other than tuberculosis, and 16 (11.43%) had malignant lymphadenopathy, including 04 (02.84%) cases of primary malignancy (i.e., lymphoma) and 13 (09.28%) of secondary metastasis to lymph nodes. Though cytology suggestive of tuberculous lymphadenitis was found in 140 (46.67%) cases out of total 300 cases,Ziehl-Neelsen stain demonstrated acid-fast bacilli (AFB) in 60 (20.00%) cases and BACTEC isolated mycobacteria in 80 (26.67%) cases. When culture (BACTEC) is taken as the gold standard, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the FNAC in the diagnosis of TB lymphadenitis results was 96.55%, 96.38%, 95.89% and 96.96% as per this study and if ZN stain is taken as the standard for diagnosis, the sensitivity, specificity, PPV and NPV were 95.23%, 99.17%, 96.77% and 98.76% respectively. CONCLUSION FNAC is an assay which has high sensitivities when optimally selected is efficient and one of the most accurate frontline method for the diagnosis of Tuberculous Lymphadenitis. It is effectively supplemented by Ziehl-Neelsen Staining which is the most simple, quicker, reliable and relatively cheap diagnostic tool . Culture methods (BACTEC) can be reserved to obtain more accurate diagnosis.

Background: Early diagnosis of tuberculosis is critical for its effective management and prevention. Several gene amplification methods are used in the detection of tubercle bacilli from clinical specimens. MPB64 gene and IS6110 region have been identified as potential gene targets for the specific detection of Mycobacterium tuberculosis from direct clinical specimens. Objective: The present study was conducted to evaluate the diagnostic utility of simultaneous application of two nested polymerase chain reaction (nPCRs) targeting MPB64 and IS6110 region for the detection of M. tuberculosis genome. Materials and Methods: A total of 100 and 354 clinical specimens from the control group and clinically suspected tuberculosis patients, respectively, were included in the study. nPCRs targeting MPB64 and IS6110 region were performed. Results and Conclusion: All of the 100 clinical specimens from the control group were negative for both nPCRs. Out of the 354 clinical specimens, 339 were positive for both culture and nPCRs, 10 and 5 were positive for culture, and nPCR targeting IS6110 and MPB64 regions, respectively. To conclude, nPCRs targeting MPB64 and IS6110 region are reliable and specific targets when applied simultaneously on clinical specimens to attain 100% sensitivity for the detection of M. tuberculosis genome.

Objective To highlight the spectrum of clinical manifestations, labs, complications, treatment and outcome of brucellosis. Methods Retrospective study was conducted in Kasturba Medical College, Manipal University, Karnataka, India which included 68 confirmed cases of brucellosis from January 2006- April 2010. Diagnosis of brucellosis was made by culturing the sera/body fluids by standard BACTEC method (or) by testing the sera for Brucella agglutinins using the standard agglutination test (SAT). A titer of 1:320 or more was considered as significant. SPSS 16 was used for statistical analysis and Microsoft Excel for graphical representation. Results Of the 68 patients, 46 (68%) were male and 22 (32%) were female patients with age distribution of 9–75 years. Forty four (64.7%) had history of contact with unpasteurized dairy products or infected animals. Symptoms included fever (68, 100%), myalgia (21, 31%), musculoskeletal symptoms (23, 34%), headache (16, 24%), gastrointestinal symptoms (19, 28%) and altered sensorium (3, 4%). Co-morbidities and associations included HIV positivity (2, 3%), type 2 diabetes mellitus (13, 19%), steroid therapy (3, 4%) and HBsAg positivity (8, 12%). Ten (15%) patients had cervical lymphadenopathy, 4(6%) had splenic enlargement, 6 (7%) had hepatomegaly, 19 (28%) had hepatosplenomegaly and 2(3%) got meningeal signs. Anaemia was observed in 39 (57.3%) cases, high erythrocyte sedimentation rate (ESR) was present in 55 (80.8%) cases, leucocytosis in 10(14.7%), leucopenia in 10(14.7%), thrombocytopenia in 23 (33.82%) and thrombocytosis in 2 (2.94%) cases. Conclusions In countries like India, where brucellosis and tuberculosis are endemic; rapid, sensitive and highly specific diagnostic methods are required to make early diagnosis and prevent resistance as there is an overlap in therapy.

Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been presented as a novel method for the direct identification of bacteria from positive blood culture bottles. The rate of the MALDI TOF MS-based identification in the present study from positive BacT/ALERT (bioMérieux, Marcy l'Etoile, France) blood culture bottles was 30%, which is far below the previously reported sensitivities using the BACTEC (Becton Dickinson, Franklin Lakes, NJ, USA) system. We also found evidence that the Biotyper algorithm did not identify a second pathogen in cases of positive BacT/ALERT blood culture bottles containing two different species.

Objectives To perform a multicentre study to evaluate the performance of the colorimetric redox indicator (CRI) assay and to establish the MICs and critical concentrations of rifampicin, isoniazid, ofloxacin, kanamycin and capreomycin. Methods The study was carried out in two phases. Phase I determined the MIC of each drug. Phase II established critical concentrations for the five drugs tested by the CRI assay compared with the conventional proportion method. Results Phase I: a strain was considered resistant by the CRI assay if the MIC was >/=0.5 mg/L for rifampicin, >/=0.25 mg/L for isoniazid, >/=4.0 mg/L for ofloxacin and >/=5.0 mg/L for kanamycin and capreomycin. Sensitivity was 99.1% for isoniazid and 100% for the other drugs and specificity was 97.9% for capreomycin and 100% for the other drugs. Phase II: the critical concentration was 0.5 mg/L for rifampicin, 0.25 mg/L for isoniazid, 2.0 mg/L for ofloxacin and 2.5 mg/L for kanamycin and capreomycin giving an overall accuracy of 98.4%, 96.6%, 96.7%, 98.3% and 90%, respectively. Conclusions Results demonstrate that the CRI assay is an accurate method for the rapid detection of XDR Mycobacterium tuberculosis. The CRI assay is faster than the conventional drug susceptibility testing method using solid medium, has the same turnaround time as the BACTEC MGIT 960 system, but is less expensive, and could be an adequate method for low-income countries.

Background/PurposeIdentifying the pathogens present in blood stream infections is crucial to initiate appropriate antimicrobial therapy and avoid morbidity and mortality. The aim of this study was to evaluate the effect of overnight storage of aerobic and anaerobic BACTEC 9240 blood culture bottles on the detection time for common pathogens.MethodsFrom November 2007 to July 2008, a total of 2,105 isolates were positively detected using the BACTEC 9240 system. The time to positive detection (TTD) was calculated by subtracting the time of receipt in the laboratory from the time required to detect a positive culture. The mean TTD values were calculated using the TTD value of the first positive culture bottle only. Overnight delay at the National Cheng Kung University Hospital, Taiwan was 15 hours (from 5 pm to 8 am).ResultsOf the 2,105 total isolates, 972 (46.1%) were Gram-positive bacteria, 1,024 (48.6%) were Gram-negative bacteria and 109 (5.1%) were fungi. Among the top 10 pathogens, 24.7% grew only in the aerobic bottle and 15.1% in the anaerobic bottle, including Staphylococcus spp., Enterococcus faecium, Enterobacteriaceae, and Gram-positive bacilli. Due to the overnight delay in loading a blood culture bottle into the instrument, for most of the pathogens (including Staphylococcus spp. and Enterobacteriaceae), a decrease in TTD by £ 4.4 hours was observed. An increase in TTD by 20.8 hours was observed for Gram-positive bacilli. We also found that the difference between TTD in aerobic versus anaerobic bottles during the day was higher in coagulase-negative staphylococcus (12 hours) and lower in Escherichia coli and Staphylococcus aureus (< 2 hours). TTD was longer than 72 hours in 20.5% of Gram-positive bacilli and 7.3% of Candida albicans.ConclusionNo difference in the TTD of major pathogens was observed in bottles processed during the day and after overnight delay, suggesting that the delayed entry of the blood culture bottle into the instrument may affect the detection time. Since high numbers of facultative anaerobes were detected in anaerobic bottles only, use of a single aerobic bottle might have a detrimental effect on the clinical therapy outcome.

The culture results of 4,807 blood and 383 sterile body fluid specimens received in our laboratory during a 54-month period, were analyzed to determine the time required for culture to become positive, time at which a culture could safely be considered negative, and the spectrum of isolated organisms. The specimens were processed by automated BACTEC 9120 culture system. A total of 1,677 clinically significant microorganisms were isolated. Gram positive and negative bacterial isolation rates were found to be 62.55% and 32.20%, respectively. Yeasts were recovered in 5.24%. False positivity rate was 1.5%. Clinically significant isolates recovered on day four and five were 97.81% and 99.88%, respectively. At day five, the sensitivity was 99.94% and negative predictive value 99.96%. Our data support a five-day incubation protocol for recovery of all clinically significant organisms with sensitivity reduced by 0.06%, when compared with a six-day protocol.

Summary Background Pyrazinamide (PZA) is the most important drug against the latent stage of tuberculosis (TB) and is used in both first and second line treatment regimens. The continued increase in multi-drug resistant TB and the prevalence of PZA resistance makes the development of alternative assays for prompt identification of PZA resistance all the more important. Methods We standardized and evaluated a quantitative variant of the Wayne assay (QW) for determining PZA resistance in Mycobacterium tuberculosis strains. This assay quantifies M. tuberculosis metabolism of PZA and production of pyrazinoic acid (POA) using visible spectrophotometry. We evaluated this method using PZA concentrations of 400 μg/ml and 800 μg/ml at incubation periods of 3, 5 and 7 days. M. tuberculosis strains from 68 sputum samples were also tested with the standard Wayne assay, Tetrazolium Microplate Assay (TEMA), Bactec 460TB and pncA sequencing. We compared QW and standard Wayne assay against a dichotomous reference classification using concordant Bactec 460TB and pncA sequencing. Secondarily, we determined the quantitative correlation between both QW values and TEMA's minimum inhibitory concentration (MIC) against Bactec 460TB percentage growth. Results The standard Wayne showed sensitivity of 88% and specificity of 97.5%, giving a Youden Index (YI) of 0.855 against reference tests. The QW showed maximum YI of 0.934 on day 7 at 400 μg/ml PZA with 96% sensitivity and 97.4% specificity. Absorbance OD values for 400 μg/ml PZA were more accurate than 800 μg/ml PZA. Although QW showed high accuracy for PZA susceptibility, it did not correlate quantitatively with Bactec percentage growth. TEMA testing was unreliable and did not correlate with Bactec results. Conclusions The proposed QW assay is an inexpensive method capable of providing standardization and automation of colorimetric PZA resistance testing, with better discriminatory than the standard Wayne assay.

We evaluated the performance of MGIT 960 system in terms of recover rate, detection time of mycobacteria and contamination rate from various human clinical specimens and compared it with already in use BACTEC 460 TB system and conventional LJ medium. This is the first reported study on MGIT 960 and its comparison with BACTEC 460 system in Pakistan. A total of 260 different clinical specimens received for the culture of mycobacteria were dealt during the six months study period. All the specimens were digested and decontaminated according to the standard N-acetyl-Lcysteine NaOH method. All the processed specimens were inoculated on both the liquid systems and solid medium and incubated for six weeks and eight weeks consecutively. A total of 44 mycobacterial isolates (Mycobacterium tuberculosis, n=43; Mycobacteria other than tuberculosis, n=1) were recovered from 260 clinical specimens. The recovery rate of M. tuberculosis complex was 97.6% on BACTEC MGIT 960 system and 93.0% on BACTEC 460 system and 83.7% on LJ medium. The mean detection time of mycobacteria on BACTEC MGIT 960 system was 11.2 days in smear positive cases, 14.2 days in smear negative cases and 14.8 days in smear positive cases on BACTEC 460 system. Contamination rates were 9.6% and 5.6% and 3.4% for BACTEC MGIT 960, BACTEC 460 system and LJ medium respectively. The non-radiometric, fully automated BACTEC MGIT 960 system has better diagnostic ability as compared with radiometric, semi-automated BACTEC 460 system and LJ medium, so it can be used as a reliable alternative in over burden laboratories.

MethodsIn this prospective study, all blood cultures submitted for mycobacteria detection, taken mainly from HIV-infected patients, were incubated for 12 weeks. The clinical impact of a late positive blood culture result was assessed retrospectively.ResultsFrom a total of 750 blood cultures, 68 had a growth index (GI) >10 due to the presence of mycobacteria. Of 545 negative blood cultures with a GI 6 weeks (one M. tuberculosis, three M. genavense, two M. avium intracellulare complex). The prolonged incubation and the systematic terminal Ziehl—Neelsen increased the recovery of M. genavense from 5% to 14.5%. However, for only three patients did the late microbiological result lead to the introduction of antimycobacterial therapy.ConclusionsNeither a prolonged incubation longer than 6 weeks nor a terminal Ziehl—Neelsen-stained smear of the negative blood cultures at 12 weeks seem to be clinically justified.

Objective Infections are the major cause of morbidity and mortality in febrile neutropenic patients with malignancy. Rapid diagnostic tests are needed for prompt diagnosis and early treatment which is crucial for optimal management. We assessed the utility of soluble triggering receptor expressed on myeloid cells (sTREM-1) in the diagnosis of bacteremia and fungemia in febrile neutropenic patients. Methods Sixty-five febrile neutropenic children with malignancy hospitalized in Mofid Children's Hospital during a period of one year from January 2007 were recruited for this cross sectional study (mean age 66.2± 37 months; 35 females and 30 males). Thirty patients (46.2%) had acute lymphoblastic leukemia, 2 (3.1%) acute myeloid leukemia, one (1.5%) lymphoma and 32 (49.2%) were under treatment for solid tumors. Simultaneous blood samples were collected for measurement of serum sTREM-1 levels and for blood cultures which were grown in BACTEC media. Gold standard for the presence of infection was a positive BACTEC culture as a more sensitive method compared to current blood culture techniques. Findings Blood cultures with BACTEC system were positive in 13(20%) patients (12 bacterial and one fungal culture). The mean serum sTREM-1 level in BACTEC positive patients was 948.2±592.9 pg/ml but in BACTEC negative cases it was 76.3±118.8 pg/ml (P