Is European Medicines Agency (EMA) sepsis criteria accurate for neonatal sepsis diagnosis or do we need new criteria?

BACKGROUND: Currently, there is a lack of clear definition for neonatal sepsis. The Pediatric Committee of the European Medicines Agency (EMA) developed consensus criteria to ensure a standardization for neonatal sepsis definition. However, there is no evidence supporting the accuracy of the EMA sepsis criteria in neonatal sepsis diagnosis. The main objective of this study was to evaluate the diagnostic accuracy of EMA sepsis criteria for proven neonatal sepsis. METHODS: A multicenter prospective cohort study was conducted from October 2015 to November 2018. Infants with a gestational age over 34th weeks, diagnosed with clinical sepsis and received antibiotics according to the EMA criteria or experienced neonatologists' opinion were included. Blood culture or multiplex real time-PCR or 16S-rRNA positive infants were accepted as "proven sepsis". The predictive performance of EMA criteria for proven sepsis was evaluated by sensitivity, specificity, accuracy, and area under the curve measures of receiver operator characteristic curves. Data-mining methods were used for further analysis. RESULTS: Among the 245 included infants, the EMA criteria were positive in 97 infants (39.6%), while proven sepsis was diagnosed in 113 infants (46.1%). The sensitivity, specificity, and accuracy of the EMA criteria for proven sepsis were 44.2% (95%CI: 34.9-53.9), 64.4% (95%CI: 55.6-72.5), 55.1% (95%CI: 46.6-59.4) respectively. None of the clinical and laboratory parameters had sufficient performance individually in terms of sensitivity, specificity and accuracy measures. The diagnostic performance was similar when different clinical findings were added to the EMA sepsis criteria or assessment of the score was interpreted in different ways. CONCLUSIONS: Results highlighted that clinician opinion and standard laboratory tests are limited in the neonatal sepsis diagnosis. The EMA criteria also did not efficiently meet the diagnostic accuracy measures for neonatal sepsis. A predictive sepsis definition and rapid bedside point-of care tests are urgently needed.

Multicenter evaluation of crystal violet decolorization assay (CVDA) for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis.

The aim of this multicenter study was to evaluate the performance of the crystal violet decolorization assay (CVDA) for detection of multidrug resistant tuberculosis (MDR-TB). This study was performed in 11 centers in two phases. A total of 156 isolates were tested for INH and RIF resistance. In the phase I, 106 clinical isolates were tested in the Center 1-7. In the phase 2, 156 clinical isolates were tested in the center 1-6, center 8-11. Eighty six of 156 tested isolates were the same in phase I. Agreements were 96.2-96.8% for INH and 98.1-98.7% for RIF in the phase I-II, respectively. Mean time to obtain the results in the phase I was 14.3 ± 5.4 days. In the phase II, mean time to obtain the results was 11.6 ± 3.5 days. Test results were obtained within 14days for 62.3% (66/106) of isolates in the phase I and 81.4% (127/156) of isolates in the phase II. In conclusion, CVDA is rapid, reliable, inexpensive, and easy to perform for rapid detection of MDR-TB isolates. In addition, it could be adapted for drug susceptibility testing with all drugs both in developed and developing countries.

[Multicenter evaluation of the indirect nitrate reductase assay for the rapid detection of multidrug-resistant tuberculosis]. / Çok ilaca dirençli tüberkülozun hizli tespiti için dolayli nitrat redüktaz testinin çok merkezli degerlendirilmesi.

Multidrug-resistant tuberculosis (MDR-TB) is defined as resistance to at least isoniazid (INH) and rifampicin (RIF), and it complicates the implementation of tuberculosis control programmes. The rapid detection of MDR-TB is crucial to reduce the transmission of disease. The nitrate reductase assay (NRA) is one of the colorimetric susceptibility test methods for rapid detection of MDR-TB and based on the ability of reduction of nitrate to nitrite by Mycobacterium tuberculosis. The aim of this study was to evaluate the performance of the NRA for the rapid detection of MDR-TB. A total of 237 M.tuberculosis complex (MTC) isolates that were identified by the same method (BD MGIT(TM) TBc Identification Test, USA) from nine different medical centers in Turkey were included in the study. The susceptibility results of the isolates against INH and RIF obtained by reference test (Bactec MGIT(TM) 960, BD, USA) were then compared with NRA. In order to ensure consistency between centers, Löwenstein-Jensen (LJ) medium with antibiotics and without antibiotics (growth control) and Griess reagent solution were prepared in a single center (Ondokuz Mayis University School of Medicine, Medical Microbiology Department) and sent to all participant centers with the standardized test procedure. After the inoculation of bacteria into the test tubes, the tubes were incubated at 37°C, and after seven days of incubation, 500 µl Griess reagent was added to the LJ medium without antibiotics. If a color change was observed, an equal volume of Griess reagent was added to test LJ media with antibiotics. When a color change was observed in LJ media with antibiotics, it was considered that the isolate was resistant to tested antibiotics. Among 237 MTC isolates, 16 were resistant only to INH and nine were resistant only to RIF; 93 isolates (39.2%) were resistant (MDR) and 119 isolates (50.2%) were susceptible to both of the drugs determined with the reference susceptibility test. In the study, five INH-resistant isolates determined with reference method were found susceptible with NRT and eight INH-susceptible isolates determined with reference method were found resistant with NRT. In contrast, one RIF-resistant isolate determined with reference method was found susceptible with NRT and three RIF-susceptible determined isolates were found resistant with NRT. Accordingly, the concordance rate between the reference method and NRA were estimated as 94.5% for INH and 98.3% for RIF. The sensitivity, specificity, positive and negative predictive values of NRA were detected as 95.4%, 93.7%, 92.8% and 96% for INH, and 99%, 97.8%, 97.1% and 99.2% for RIF, respectively. The results of the 111 isolates were obtained on the seventh day, while the rest of the results were obtained between 10-14 days. In conclusion, the data of this multicenter study showed that NRA is a reliable, relatively inexpensive and practical method to perform for the rapid detection of MDR-TB.

The effects of oral antibiotics on infection prophylaxis in traumatic wounds.

BACKGROUND: The objective of this study is to examine the effectiveness of oral antibiotics in the prevention of infection development in traumatic wounds. METHODS: Forty Wistar albino rats were divided into five groups of eight animals. After the crushed wound model was made on the back of the rats, wounds were closed with a simple suture and Staphylococcus aureus ATCC 29213 strain was used to create infection. All rats apart from the controls were given oral gavage with antibiotics, including cephalexin, amoxicillin-clavulanate, clarithromycin (CAM), or levofloxacin for 5 days. Wounds were evaluated qualitatively and quantitatively on 5th day approximately 18 h after the last treatment. RESULTS: In the quantitative evaluation, no infection was observed in the treatment groups with amoxicillin-clavulanate, CAM, cephalexin, or levofloxacin. There was no significant difference on the numbers of bacteria found in the wounds among the groups. In terms of quantitative inflammation findings, no hyperemia or pus was detected in the groups that were given medication. Furthermore, no statistically significant difference was found among the groups in terms of induration. CONCLUSION: Oral prophylactic antibiotics have been found to be effective in the prevention of wound infection in the traumatic crushed wound model infected with S. aureus in rats.

[Implementation of "5S" methodology in laboratory safety and its effect on employee satisfaction]. / Laboratuvar güvenliginde "5S" yönteminin uygulamasi ve çalisan memnuniyeti üzerine etkisi.

Health institutions use the accreditation process to achieve improvement across the organization and management of the health care system. An ISO 15189 quality and efficiency standard is the recommended standard for medical laboratories qualification. The "safety and accommodation conditions" of this standard covers the requirement to improve working conditions and maintain the necessary safety precautions. The most inevitable precaution for ensuring a safe environment is the creation of a clean and orderly environment to maintain a potentially safe surroundings. In this context, the 5S application which is a superior improvement tool that has been used by the industry, includes some advantages such as encouraging employees to participate in and to help increase the productivity. The main target of this study was to implement 5S methods in a clinical laboratory of a university hospital for evaluating its effect on employees' satisfaction, and correction of non-compliance in terms of the working environment. To start with, first, 5S education was given to management and employees. Secondly, a 5S team was formed and then the main steps of 5S (Seiri: Sort, Seiton: Set in order, Seiso: Shine, Seiketsu: Standardize, and Shitsuke: Systematize) were implemented for a duration of 3 months. A five-point likert scale questionnaire was used in order to determine and assess the impact of 5S on employees' satisfaction considering the areas such as facilitating the job, the job satisfaction, setting up a safe environment, and the effect of participation in management. Questionnaire form was given to 114 employees who actively worked during the 5S implementation period, and the data obtained from 63 (52.3%) participants (16 male, 47 female) were evaluated. The reliability of the questionnaire's Cronbach's alpha value was determined as 0.858 (p< 0.001). After the implementation of 5S it was observed and determined that facilitating the job and setting up a safe environment created a statistically significant effect on employees, and some sufficient satisfaction was observed. In addition, the non-conformity score, which was identified in the laboratory during the previous years, was significantly reduced at a rate of 69.7% after the implementation of 5S. 5S practices have successfully contributed to the establishment and to the sustainability of laboratory safety systems in the first public ISO 15189 accredited public clinical laboratory in Turkey. It is concluded that 5S methods can be used as an effective improvement tool in order to maintain a safe environment, to facilitate the job, and to encourage employees to participate in the management process.

[Three step MIRU-VNTR for routine mycobacteriology laboratory practice]. / Rutin mikobakteriyoloji laboratuvarlari için üç adimda MIRU-VNTR.

Several methods are available for the molecular typing of Mycobacterium tuberculosis complex isolates. The results of the recent research demonstrated that Mycobacterial Interspersed Repetitive Unit (MIRU)-Variable Number Tandem Repeats (VNTR) method has high discriminatory power and reproducibility, is easy to perform, and available for multi-center studies and automation. However, there is insufficient data about the MIRU-VNTR profiles in Turkey. The aim of this study was to determine the most appropriate MIRU-VNTR combinations to distinguish cross contaminations and nosocomial infections in routine mycobacteriology laboratory practice. Following molecular typing of 152 clinical isolates which were consecutively isolated from different patients in two years period (August 2004-July 2006) in our laboratory, a retrospective analysis of MIRU-VNTR data of 12 loci primers was performed by an "in-house" computer based programme. The programme was prepared by using Microsoft QuickBASIC programming language and all of the data were calculated by the help of this programme. The best combinations to differentiate the clusters and to identify the unique isolates were determined out of 4095 possible results of 12 different primer pairs. According to our 152 MIRU-VNTR results, to determine cross contaminations and nosocomial infections in routine mycobacteriology laboratory practice, we recommend to use primers 26, 40, 16, 10 and 23 in the first step; primers 31, 27, 20 and 2 in the second step, and primers 4, 24 and 39 in the third step. The created software is user friendly, fast and meets the requirements of routine clinical mycobacteriology laboratories. Besides its discriminatory power, the speed and cost-effectiveness of a typing method is also considerable. According to the results of this study it was suggested that for more rapid and economic molecular typing of M.tuberculosis and related epidemiological investigations, MIRU-VNTR should be performed in a stepwise manner.

[Evaluation of a rapid trehalase test for the identification of Candida glabrata]. / Candida glabrata'nin tanisinda hizli trehalaz testinin degerlendirilmesi.

Candida species which cause local infections, may also lead to fatal systemic infections. The increasing incidence of non-albicans Candida, especially fluconazole susceptible or resistant dose-dependent C. glabrata, increased the importance of rapid and accurate species level identification for Candida. Rapid and correct identification of C. glabrata is essential for the initiation of the appropriate antifungal therapy. This study was conducted to evaluate the performance of the rapid trehalase test in the diagnosis of C. glabrata isolates. A total of 173 Candida strains isolated from various clinical specimens and identified according to germ tube test, growth on cornmeal Tween 80 agar and the colony morphologies on Mast-CHROMagar Candida medium (Mast Diagnostics, UK), were included to the study. The identification of non-albicans Candida species were also confirmed by API 20CAUX (BioMerieux, France) system. Accordingly 86 (50%) of the isolates were identified as C. glabrata, 48 (28%) C. albicans, 17 (10%) C. krusei, 13 (8%) C. tropicalis, 5 (3%) C. parapsilosis, 3 (2%) C. kefyr and 1 (1%) Cutilis. In order to detect the presence of trehalase enzyme in Condida strains, all isolates were grown on Sabouraud dextrose agar containing 4% glucose and then one yeast colony was emulsified in 50 microl of citrate buffer containing 4% (wt/vol) trehalose for 3 h at 37 degrees C. Presence of glucose which emerged after the action of trehalase on trehalose, was detected by a commercial "urinary glucose detection dipstick" (Spinreacta, Spain). All C. glabrata strains yielded positive result by trehalase test. None C. glabrata isolates were found negative by trehalase test except for one strain of C. tropicalis. In this study, the trehalase test allowed identification of C. globrata with 100% sensitivity and 98.9% specificity. It was concluded that trehalase test is a rapid, cost-effective and simple test that can be used for the accurate identification of C. glabrata.

[Quality assurance in tuberculosis laboratories]. / Tüberküloz laboratuvarlarinda kalite güvencesi.

The laboratory has always played a critical role in diagnosis of tuberculosis and monitoring treatment. The basic aim in tuberculosis laboratory is to generate accurate and reliable test results for clinicians. This aim can be achieved by using quality assurance programs. Three main components of quality assurance are, internal quality control, a process by which each laboratory continuously monitors its own performance (guidelines, education and check of staff); external quality control, a process whereby laboratory performance between the other laboratories is assessed by external quality control organization (accuracy and efficiency) and quality improvement (continuous development of quality which involves identification of errors and remedial action). In this article, quality control procedures which are followed in tuberculosis laboratory to ensure the quality assurance, have been reviewed.

A comparison of the prophylactic uses of topical mupirocin and nitrofurazone in murine crush contaminated wounds.

OBJECTIVES: This work was conducted to study the prophylactic efficacy of 2 topical antibiotic ointments (mupirocin and nitrofurazone) against wound infection in experimental contaminated crush wounds. METHODS: Male Wistar rats underwent two 2-cm incisions at the back side and randomized into 3 groups--placebo (n = 14), mupirocin (n = 14), and nitrofurazone (n = 14)--and infected with either Staphylococcus aureus or S. pyogenes. All wound edges were crushed for 5 seconds with hemostats to simulate crush injury before inoculation of the microorganisms. Half of the wounds were sutured and the other half left open. These wounds were treated 3 times daily for 6 days with topical mupirocin, nitrofurazone, or petrolatum (as placebo). At the end of 6 days, excisional biopsies were taken from wound edges and histopathologic assessments were made based on neutrophilic infiltration, edema formation, myofibroblastic proliferation, and granulation tissue formation. For the microbiologic assessments, quantitative tissue cultures were made. RESULTS: In S. aureus-inoculated wounds, mupirocin showed higher antibacterial activity against bacterial colonization and reduced infection rates compared to placebo groups. The same effect was observed for the infection rates in S. pyogenes-inoculated wounds. In S. pyogenes-inoculated open wounds, nitrofurazone showed higher antibacterial activity against infection, but this effect was not observed in closed wounds. In S. pyogenes- and S. aureus-infected wounds, mupirocin treatment significantly lowered infection rates compared to nitrofurazone treatment. Histopathologic examination showed higher myofibroblastic proliferation and higher volume of granulation tissue in the nitrofurazone groups compared to the mupirocin groups. CONCLUSION: Topical mupirocin application was effective against crush wound infections inoculated with S. pyogenes and S. aureus. Nitrofurazone provides better granulation tissue formation, but did not effectively prevent bacterial colonization and infection in crush contaminated wounds.

[Comparison of minimum inhibitory and minimum effective concentration values for the detection of in vitro susceptibilities of Aspergillus species against caspofungin]. / Aspergillus türlerinin kaspofungine karsi in vitro duyarliliginin saptanmasinda minimum inhibitör ve minimum etkili konsantrasyon degerlerinin karsilastirilmasi.

Caspofungin is a promising echinocandin-group antifungal agent used especially in the treatment of resistant invasive aspergillosis. The guidelines for in vitro susceptibility testing of Aspergillus species against caspofungin are not described by the Clinical and Laboratory Standards Institute (CLSI). The minimum inhibitory concentration that showed a prominent reduction of growth (MIC-2) and minimum effective concentration (MEC) endpoints are frequently used for the susceptibility testing of caspofungin as MIC determination criteria. The aim of this study was to evaluate the in vitro activity of caspofungin against Aspergillus species and to compare MIC-2 and MEC endpoints in the determination of MICs. A total of 32 Aspergillus species (18 A. fumigatus, seven A. flavus, five A. niger, and two A. versicolor) isolated from different clinical samples were included to the study. In vitro susceptibilities of the strains against 0.03-16 microg/ml caspofungin concentrations were searched by broth microdilution method as recommended by CLSI M-38A document, with the use of glucose supplemented 2% RPMI 1640 media. The MIC-2 and MEC endpoints were determined both at 24 and 48 hours. The concordance between MIC-2 and MEC endpoints of the strains at 24 and 48 hours incubations was found as 53% and 100%, respectively, with the difference of +/- 1 dilution. MIC-2 and MEC measurements showed the same values at the end of 48 hours, whereas 7% showed differences in +/- 1 dilution. MEC endpoints were also found to be more stable than MIC-2 in both of the incubation periods. In conclusion, MEC value is a more objective and stable endpoint and easier to use than MIC-2 for testing in vitro caspofungin activity against Aspergillus species.

Is it valuable to examine more than one sputum smear per patient for the diagnosis of pulmonary tuberculosis?

The simplest, cheapest, and fastest diagnostic method for tuberculosis (TB) is the detection of acid-fast bacilli (AFB) by microscopy. The algorithm advised for the diagnosis of TB recommends examination of three consecutive sputum specimens from TB suspects for the presence of AFB. In the present study, we evaluated the contribution of each specimen to the final detection of TB suspect patients with culture-proven disease. The collection and analysis of retrospective data on patients with culture-proven pulmonary TB, from June 2002 to August 2006 at Dokuz Eylul University Hospital, Turkey, have enabled us to assess the value of examining two sputum specimens in diagnosing this disease. AFB were detected from one or more sputum specimens with direct microscopy in 42% of the cases. An analysis of results of smear examination showed that 97% of AFB were detected from the first specimen and only 3% were obtained from the second smear. The third specimen did not have any additional diagnostic value for the detection of AFB by microscopy. As a conclusion the present study shows that examining two sputum smears is sufficient for the early detection of AFB in our laboratory.

Species distribution and antifungal susceptibilities of dermatophytes during a one year period at a university hospital in Turkey.

Dermatophyte infections have been considered to be a major public health problem in many parts of the world. The aim of this study was to determine the causative agents of dermatophytoses and their antifungal susceptibilities in a Turkish University Hospital, west of Turkey. A total of 926 patients suspected to have dermatophytic lesions were examined over a period of 1 year (2001-2002). Samples collected from skin, hair and nails were submitted to direct microscopical examination using KOH and Calcofluor white stain, cultured on Sabouraud dextrose agar and Mycosel agar. The prevalence of dermatophytoses was 7.34% (68/926). Trichophyton rubrum was the most frequent dermatophyte isolated (56%) followed by T. mentagrophytes (38%), T. violaceum (1.5%), T. verrucosum (1.5%), Microsporum canis (1.5%) and Epidermophyton floccosum (1.5%). Tinea pedis (47%) was the most common type of infection, followed by tinea unguium (29%), tinea inguinalis (15%), tinea corporis (7.4%) and tinea capitis (1.6%). Secondary, we have tested 68 strains of dermatophytes against four antifungal agents following mainly the National Committee for Clinical Laboratory Standards M38-P standard for filamentous fungi. In general, all antifungals were shown to be highly effective and itraconazole and naftifine appeared more active than ketoconazole and oxiconazole.

Fungi and indoor conditions in asthma patients.

This study was carried out with 127 asthmatic patients and 127 controls, which aimed to compare and evaluate the environmental conditions in the homes of asthmatic patients and the control group. Air samples were obtained by using an air sampler and the mean mould colony counts were established. Aspergillus and Penicillium were the most common isolated species. No significant difference was observed with regard to various house conditions and the mean mould colony counts between the houses of patients and controls. The mould colony counts were found to be lower in houses with wooden parquet flooring. The odds ratio for stone floors vs. wood floors was 2.3 (95% CI 1.08-4.98) for mould growth.

Evaluation of Cobas Amplicor MTB test to detect Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens.

Tuberculosis (TB) is one of the major public health problems in the world. Effective control of TB depends on rapid and correct diagnosis and appropriate treatment. The aim of this study was to evaluate the performance of Cobas Amplicor MTB (CA-MTB) test for pulmonary and extrapulmonary specimens isolated in our laboratory. A total of 424 specimens obtained from the suspected TB patients from January 2003 to August 2004 were included in this study. All specimens (173 pulmonary and 251 extrapulmonary specimens) were processed, stained, cultured and assayed using the CA-MTB test for identification of Mycobacterium tuberculosis. The CA-MTB test results were compared to culture and acid-fast staining as gold standard. The sensitivity, specificity, positive and negative predictive values of CA-MTB were determined as 73%, 100%, 100%, and 97% for pulmonary specimens, and 45%, 100%, 100% and 96% for extrapulmonary specimens respectively. The sensitivity of the test for acid-fast bacilli (AFB) smear positive pulmonary and extrapulmonary specimens was 92% and 75%. These results indicate that the CA-MTB is a rapid test for detection of tuberculosis in pulmonary specimens, but does not perform well enough in extrapulmonary specimens.

[Molecular epidemiologic surveillance and antifungal agent sensitivity of Candida albicans isolated from anesthesia intensive care units]. / Anestezi yogun bakim ünitesi hastalarindan izole edilen Candida albicans suslarinin antifungal ajanlara duyarliligi ve moleküler epidemiyolojik izlemi.

Patients in intensive care units (ICU) are at risk of nosocomial infections. The incidence of nosocomial fungal infections has increased in parallel with the increase of nosocomial infections. Candida albicans is the most frequent pathogenic species among the fungi. The aim of this study was to make an epidemiological surveillance of C. albicans urine isolates which were isolated from patients who were hospitalized in ICU between June 2000 and October 2001 by antifungal susceptibility testing and Randomly Amplified Polymorphic DNA (RAPD) analysis. For this purpose, 38 C. albicans which were isolated from 29 patients were investigated for amphotericin B and fluconazole susceptibility with the microdilution method. The range of minimal inhibitory concentration (MIC) of amphotericin B was between 0.25-1 microgram/ml and MIC50 value was 0.5 microgram/ml and none of the isolates had high (MIC > 1 microgram/ml) MIC values. The MIC values for fluconazole varied between 0.25-16 micrograms/ml and MIC50 value was 1 microgram/ml. While none of the isolates was resistant to fluconazole, two isolates were detected as dose dependent susceptible. RAPD analysis was performed with two different primers in order to investigate clonal relationship, and 22 patterns were detected with one of the primers and 24 patterns were detected with the other. In conclusion, it is thought that the origin of the C. albicans urine isolates were mostly endogenous but exogenous spread might also be considered as isolates that were clonally related were isolated from different patients at the same time interval.