Evaluation of MALDI-TOF MS for identification of nontuberculous mycobacteria isolated from clinical specimens in mycobacteria growth indicator tube medium.

Nowadays, there is a rising worldwide incidence of diseases caused by nontuberculous mycobacteria (NTM) species, especially in immunocompromised patients and those with underlying chronic pulmonary diseases. Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) became a method of choice for the identification of NTM species. The aim of this study was to evaluate MALDI-TOF MS for the identification of NTM isolates compared to the PCR-restriction enzyme analysis (PRA)-hsp65 method. In this study, a total of 152 NTM strains isolated from various clinical specimens were retrospectively analysed. MALDI-TOF MS successfully identified 148 (97.4%) of the 152 NTM isolates but failed to identify four (2.6%) of them. Bruker mycobacteria library gave spectral scores higher than 2.0 for 45 (29.6%) of NTM isolates, between 1.6 and 2.0 for 98 (64.5%) of NTM isolates, and lower than 1.6 for nine (5.9%) NTM isolates. The discordant results between MALDI-TOF MS and PRA-hsp65 analysis were confirmed by sequence analysis. In conclusion, MALDI-TOF MS is a technique capable of performing accurate, rapid, cost-effective, and easy identification of NTM isolates.

[Evaluation of mycobacterial microscopy and culture results of Sureyyapasa Chest Diseases and Chest Surgery Training and Research Hospital: A 3-year analysis]. / Süreyyapasa Gögüs Hastaliklari ve Gögüs Cerrahisi Egitim ve Arastirma Hastanesi mikobakteri mikroskopi ve kültür sonuçlarinin degerlendirilmesi: Üç yillik analiz.

INTRODUCTION: Effective diagnosis of tuberculosis is of great importance for transmission control and treatment success. The purpose of this study is to evaluate microscopic examination results of Ehrlich-Ziehl Neelsen (EZN) and Auramine-Rhodamine staining methods and automated BACTEC MGIT 960™ system and Löwenstein-Jensen (L-J) culture results of various clinical samples in the light of recent data from the world and Turkey. MATERIALS AND METHODS: Specimens that were sent from various clinics to Sureyyapasa Chest Diseases and Chest Surgery Training and Research Hospital Microbiology Laboratory from January 2012 to December 2015 were evaluated retrospectively. RESULT: From a total of 62456 samples; 60923 (97.5%) were pulmonary and 1533 (2.5%) were non-pulmonary samples, especially pleura. 2853 (4.6%) Acid-resistant bacilli (ARB) positivity was detected and mycobacterial culture positivity was in total 12.2%. 7076 (93%) and 535 (7%) mycobacteria other than tuberculosis (MOTT) strains were isolated. In 356 specimens the cultures were negative in despite the positive ARB results. Considering mycobacterial culture as the gold standard; the sensitivity, specificity, positive and negative predictive values of ARB microscopy were 32.8%, 99.4%, 87.5% and 91.4%, respectively. The contamination rates in total were within acceptable limits being 2.7% for L-J and 3.8% for MGIT. CONCLUSIONS: Analysis of our data indicated that the sensitivity of microscopy is low and it should be evaluated together with the mycobacterial culture to rule out tuberculosis infection. With the use of fluorescent staining and also L-J and MGIT broth together for routine culture since 2013; ARB false negativity rate was observed to fall to 51.7% from 74.1% compared to the years. The follow-up of data such as the sensitivity of microscopy, culture positivity, false-positivity and false-negativity rates and contamination values is of great importance in terms of assessing compliance with laboratory quality standards and contributing to the surveillance studies.

[A case of rib tuberculosis and chest wall abscess with multi-drug hypersensitivity reactions]. / Çok ilaca asiri duyarlilik reaksiyonu görülen bir kot tüberkülozu ve gögüs duvari apsesi olgusu.

Extrapulmonary tuberculosis is the reactivation of the remaining latent organism which spreads during primary infection by the lymphohematogenous way. It should be considered in the differential diagnosis especially in endemic countries for tuberculosis. Tuberculosis (TB) treatment is based on the principle of the combined use of several drugs. As a result of the combination therapy there can be life threatening side effects which can lead to improper use of medications and may also cause drug resistance. In this report, we present an 85-year-old male patient desensitized due to the development of allergy against multi-drugs with rib tuberculosis and chest wall abscess to whom, culture, drug susceptibility and genotypical tests were applied. In November 2012, the patient applied to a medical center with complaints of swelling and pain under the right rib, underwent rib resection and eventually diagnosed as rib TB by histopathological examination. However, the anti-TB treatment was discontinued due to the hypersensitivity reactions in the skin and in addition to the hepatic and renal dysfunction side effects. The patient had widespread redness, rash and pruritus on the body and the laboratory findings were as follows; ALT: 114 U/L, AST: 152 U/L, ALP: 93 U/L, GGT: 26U/L, blood urea nitrogen (BUN): 26 mg/dL and creatinine: 1.7 mg/dL. After the disapperance of the complaints within 3 days of drug discontinuation, isoniazid treatment was initiated. However, the new treatment was also discontinued when the reactions reoccurred. Afterwards, the patient developed hypersensitivity reactions against the combination of streptomycin and ethambutol. The patient refused any further treatment and was discharged from the hospital. The patient was untreated for the last 5 months and admitted to our clinic with a fistulized swelling and abscess in the right chest wall. Bacteria was not detected in the acid-fast staining of the abscess material, however Mycobacterium tuberculosis was isolated from culture by MGIT (Mycobacteria Growth Incubator Tube; BBL MGIT, BD, USA) system. The spoligotyping revealed that the genotype was Haarlem 1. Major drug susceptibility testing against rifampin, streptomycin, ethambutol, isoniazid, and pyrazinamide yielded sensitivity to those drugs. Minor drug susceptibility testing against paraaminosalicylic acid, ethionamide, kanamycin, capreomycin and ofloxacin was found to be sensitive. A regimen of isoniazid 300 mg/day, ethambutol 1000 mg/day and moxifloxacin 400 mg/day was initiated. Rapid oral desensitization against isoniazid and ethambutol were repeated on two consecutive days. The patient continued antituberculosis therapy for 12 months without adverse reactions. The chest wall fistula was closed. Abscess was drained surgically. Clinical and radiological improvements were achieved. The patient remains clinically disease free and continues his regular follow ups. This case is presented to emphasize about the importance of culture and susceptibility testing in extrapulmonary tuberculosis cases and desensitization in drug hypersensitivity reactions.

IgG4 production is confined to human IL-10-producing regulatory B cells that suppress antigen-specific immune responses.

BACKGROUND: IL-10-producing regulatory B cells suppress immune responses, and lack of these cells leads to exacerbated symptoms in mouse models of chronic inflammation, transplantation, and chronic infection. IgG4 is a blocking antibody isotype with anti-inflammatory potential that is induced in human high-dose antigen tolerance models. OBJECTIVE: We sought to characterize human inducible IL-10-secreting B regulatory 1 (BR1) cells and to investigate their immunoregulatory capacity through suppression of cellular immune responses and production of anti-inflammatory immunoglobulins. METHODS: Highly purified IL-10-secreting B cells were phenotypically and functionally characterized by means of whole-genome expression analysis, flow cytometry, suppression assay, and antibody production. B cells specific for the major bee venom allergen phospholipase A2 (PLA) were isolated from beekeepers who displayed tolerance to bee venom antigens and allergic patients before and after specific immunotherapy. RESULTS: Human IL-10+ BR1 cells expressed high surface CD25 and CD71 and low CD73 levels. Sorting of CD73-CD25+CD71+ B cells allowed enrichment of human BR1 cells, which produced high levels of IL-10 and potently suppressed antigen-specific CD4+ T-cell proliferation. IgG4 was selectively confined to human BR1 cells. B cells specific for the major bee venom allergen PLA isolated from nonallergic beekeepers show increased expression of IL-10 and IgG4. Furthermore, the frequency of IL-10+ PLA-specific B cells increased in allergic patients receiving allergen-specific immunotherapy. CONCLUSION: Our data show the characterization of IL-10+ BR1 cells and in vivo evidence for 2 essential features of allergen tolerance: the suppressive B cells and IgG4-expressing B cells that are confined to IL-10+ BR1 cells in human subjects.

[Mycobacterium tuberculosis strains isolated from Van region four different sensitivity detection method antimycobacterial agents]. / Van yöresinde izole edilen Mycobacterium tuberculosis suslarinin dört farkli yöntemle antimikobakteriyel ajanlara duyarlilik tespiti.

INTRODUCTION: The purpose of this study is detecting the susceptibility rates of 58 Mycobacterium tuberculosis complex strains which were isolated from patient specimens sent to our mycobacteriology laboratory, for major anti-tuberculosis drugs like streptomycin, isoniazid, rifampicin and ethambutol with three different systems and agar proportion method and compare the accessibility, speed, specificity and sensitivity of these three systems. MATERIALS AND METHODS: With this purpose, 58 (96.6%) strains out of 60 which were isolated from the patients attended to the mycobacteriology laboratory were identified as M. tuberculosis complex with conventional methods. These strains susceptibilities to four major anti-tuberculosis drugs were detected with Manuel MGIT AST SIRE system, BacT/ALERT 3D system MB/BacT SIRE, TK anti-TB system and compared with reference method in Middlebrook 7H10 media. RESULTS: As a result, INH resistance in Van province with agar proportion method was detected as 12%, followed by INH + RIF resistance of 1.7% and INH + SM resistance of 1.7%. These result compared with other studies conducted country wide are in median range. The systems included in our study were determined to have 100% sensitivity for all of the drugs for detecting resistance and sensitivity rates. Specificities for INH for TK anti-TB, MGIT and MB/BacT were detected as 98%, 96% and 95% respectively. Multidrug resistance rates were detected in 100% sensitivity and specificity with all of the three systems. Only MB/BacT system gave a false negative RIF resistance for 1 strain. Fastest system according to resistance determination times is found to be the MGIT system. CONCLUSION: However, presence of INH + RIF resistance pattern, indicates inadequate treatment programs in our region. As a result these three systems are fast and reliable systems for antimicrobial susceptibility testing of Mycobacterium spp. to be used in routine mycobacteriology laboratories.

Role of Treg in immune regulation of allergic diseases.

Allergy is a Th2-mediated disease that involves the formation of specific IgE antibodies against innocuous environmental substances. The prevalence of allergic diseases has dramatically increased over the past decades, affecting up to 30% of the population in industrialized countries. The understanding of mechanisms underlying allergic diseases as well as those operating in non-allergic healthy responses and allergen-specific immunotherapy has experienced exciting advances over the past 15 years. Studies in healthy non-atopic individuals and several clinical trials of allergen-specific immunotherapy have demonstrated that the induction of a tolerant state in peripheral T cells represent a key step in healthy immune responses to allergens. Both naturally occurring thymus-derived CD4+CD25+FOXP3+ Treg and inducible type 1 Treg inhibit the development of allergy via several mechanisms, including suppression of other effector Th1, Th2, Th17 cells; suppression of eosinophils, mast cells and basophils; Ab isotype change from IgE to IgG4; suppression of inflammatory DC; and suppression of inflammatory cell migration to tissues. The identification of the molecules involved in these processes will contribute to the development of more efficient and safer treatment modalities.

Are physicians aware of current HIV / AIDS diagnostic practices? A study from a tertiary centre in Turkey.

INTRODUCTION: Early diagnosis of HIV infection is essential for the reduction of morbidity/mortality rates, health expenditures and the prevention of infection spread. In this study we aimed to test the knowledge of physicians regarding HIV risk groups, AIDS indicator diseases and their current practices about screening. METHODOLOGY: A questionnaire was used to collect data from physicians working in a multidisciplinary 170-bed tertiary university hospital in Istanbul, Turkey. The questionnaire measured physician knowledge of the above-mentioned points. RESULTS: Ninety-six physicians replied to the questionnaire. "Preoperative screening" was found to be the most common (65.6%) indication for HIV testing. A large portion of physicians (72.9%) felt comfortable with an HIV test and 71.9% of the physicians had no impeding condition for HIV testing. Physicians were mostly (67.7%) unaware of the current guidelines for HIV testing. CONCLUSIONS: Teaching programs are essential to increase knowledge of HIV screening for physicians as this is an essential part of early diagnosis and therefore important for decreasing morbidity and mortality.

An analysis on the roles of angiogenesis-related factors including serum vitamin D, soluble endoglin (sEng), soluble fms-like tyrosine kinase 1 (sFlt1), and vascular endothelial growth factor (VEGF) in the diagnosis and severity of late-onset preeclampsia.

AIM: The aim of this study was to evaluate the roles of proangiogenic factors including serum vitamin D and vascular endothelial growth factor (VEGF) and anti-angiogenic factors including soluble endoglin (sEng) and soluble fms-like tyrosine kinase 1 (sFlt1) in the diagnosis and severity of late-onset preeclampsia. MATERIALS AND METHODS: The study was conducted at Yuzuncu Yil University Research and Education Hospital Department of Gynecology and Obstetrics. The study included a patient group of 40 women with late-onset preeclampsia who were pregnant at ≥32 weeks of gestation according to the last menstrual period (LMP) or ultrasonographic fetal biometric measurement and a control group of 40 healthy pregnant women who presented to our clinic for routine pregnancy examination and were at the same age and gestational period with those in the patient group. The two groups were compared in terms of maternal age, gravida, parity, week of gestation, systolic/diastolic blood pressure, total protein in spot urine sample, 24-h urine protein, white blood cell (WBC), hemoglobin (Hgb), platelet count, urea, creatinine, liver function tests (AST, ALT, LDH), vitamin D3, 25(OH) vitamin D3, 1,25(OH) vitamin D3, sEng, sFlt1, and VEGF levels, mode of delivery, the infant APGAR score at 1 and 5 min after delivery, and infant weight at delivery. RESULTS: The groups were similar in terms of age, gravida, parity, week of gestation, serum vitamin D3, 25(OH) vitamin D3, 1,25(OH)2 vitamin D3 and VEGF levels, and infant weight at delivery (p > 0.05). Systolic/diastolic blood pressure, total protein in spot urine sample, 24-h urine protein, WBC, Hgb, serum urea, creatine, AST, ALT, and LDH were significantly higher in the preeclamptic group compared to the healthy group (p < 0.05). However, thrombocyte level and the APGAR score at 1 and 5 min after delivery were significantly lower in the preeclamptic group compared to the healthy group (p < 0.05). No significant correlation was found between serum sEng, sFlt1, VEGF, vitamin D3, 25(OH) vitamin D3, and 1,25(OH)2 vitamin D3 levels. The sEng level was higher in the women with severe preeclampsia compared to the women with mild preeclampsia (p < 0.05) and no significant difference was observed in serum sFlt1, VEGF, vitamin D3, 25(OH) vitamin D3, and 1,25(OH)2 vitamin D3 levels between the subgroups of preeclampsia (p > 0.05). CONCLUSION: Both sEng and sFlt1 levels are remarkably high in patients with late-onset preeclampsia; however, only sEng may be a useful tool in the determination of the severity of preeclampsia.

Evaluation of related with age of IFN-gamma levels of treated leprosy patients and their healthy relatives compared with a control group.

OBJECTIVE: This study was to search how change with age of IFN-gamma (IFN-gamma) levels of leprosy patients who had received or still undergoing treatment, their healthy close relatives and healthy people. METHOD: From a total of 39 patients, 28 had received leprosy treatment and recovered with some defects [17 Lepromatous Leprosy(LL), 7 Borderline Lepromatous (BL), 3 Borderlien Tuberculoid (BT), 1 Tuberculoid form (TT)] and 11 (9 LL, 2 BL) were undergoing therapy in a hospital at that moment. Close relatives of the patients included 39 people living with treated patients in the same house and the control group consisted of 39 healthy people. Human IFN-gamma kits were used as a solid phase sandwich ELISA method. RESULTS: The IFN-gamma values of treated leprosy patients, therapy receiving patients, relatives group's and control group's were detected as 11-20 pg/mL, 11-18 pg/mL, 12-145 pg/mL and 12-65 pg/mL respectively. CONCLUSION: According to the statistical evaluation of these three groups, IFN-gamma levels of patients were detected lower than other two groups. According to the age, if IFN-gamma levels were compared, it was detected that significant relationship between relatives groups and interferon gamma levels.

Spread of a single clone Acinetobacter baumannii strain in an intensive care unit of a teaching hospital in Turkey.

Acinetobacter baumannii is an important nosocomial pathogen, especially in immunocomprimised patients and those hospitalized in intensive care units. After the first isolation of A. baumannii strains from the bronchial aspirates of two patients in the intensive care unit (ICU) of our hospital as a pure culture, screening studies were performed to define possible source(s). A. baumannii strains isolated from bronchial aspirates and blood cultures of the patients in ICU were collected as a possible part of the outbreak. A total of 23 screening samples collected from equipment (7), hands (4) and gloves (2) of the staff, and from ten different body regions of the patients in the ICU were cultured. Antimicrobial susceptibility test of the isolates was performed by the standardized disk-diffusion method. All isolates were subtyped by antibiogram, arbitrarily primed polymerase chain reaction (AP-PCR) and pulsed-field gel electrophoresis (PFGE) typing methods. A total of 26 A. baumannii strains including eight clinical and 18 screening isolates were identified. All isolates were susceptible only to meropenem, tobramycin, and imipenem. There was at least a 96% resistance rate to the other antibiotics tested. Antibiogram typing showed that 24 of the 26 isolates were epidemiologically related, two were unique. AP-PCR yielded two types, one of which had 21 isolates, the other had five. PFGE fingerprinting revealed that all isolates were clonally related, including four closely related and 22 indistinguishable strains. Based on the results of PFGE which has been accepted as a reference method it can be concluded that A. baumannii strains isolated from our intensive care unit originated from a single type of strain.

Performances and Reliability of Bruker Microflex LT and VITEK MS MALDI-TOF Mass Spectrometry Systems for the Identification of Clinical Microorganisms.

In clinical microbiology laboratories, routine microbial identification is mostly performed using culture based methodologies requiring 24 to 72 hours from culturing to identification. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology has been established as a cost effective, reliable, and faster alternative identification platform. In this study, we evaluated the reliability of the two available MALDI-TOF MS systems for their routine clinical level identification accuracy and efficiency in a clinical microbiology laboratory setting. A total of 1,341 routine phenotypically identified clinical bacterial and fungal isolates were selected and simultaneously analyzed using VITEK MS (bioMérieux, France) and Microflex LT (Bruker Diagnostics, Germany) MALDI-TOF MS systems. For any isolate that could not be identified with either of the systems and for any discordant result, 16S rDNA gene or ITS1/ITS2 sequencing was used. VITEK MS and Microflex LT correctly identified 1,303 (97.17%) and 1,298 (96.79%) isolates to the species level, respectively. In 114 (8.50%) isolates initial phenotypic identification was inaccurate. Both systems showed a similar identification efficiency and workflow robustness, and they were twice as more accurate compared to routine phenotypic identification in our sample pool. MALDITOF systems with their accuracy and robustness offer a good identification platform for routine clinical microbiology laboratories.

Evaluation of the MALDI TOF-MS method for identification of Candida strains isolated from blood cultures.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF-MS) allows rapid and accurate identification of microorganisms. It is being used increasingly and becoming an important tool in clinical laboratories. Phenotypic identification of Candida species remains labor- and time consuming, and the results may sometimes be inconclusive. Rapid and reliable species identification of Candida is essential for antifungal treatment due to species-specific susceptibility patterns. In this study, we evaluated the performance of MALDI TOF-MS for identification of Candida strains. A total of 281 clinical Candida strains isolated from blood cultures were included in this study. Candida species were identified with conventional methods and automated VITEK 2 YST panels as well as with MALDI TOF-MS. Isolates with discrepant results were subjected to DNA sequencing of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2). Ninety-four percent of the isolates were identified correctly by VITEK 2 and MALDI TOF-MS. Altogether, MALDI-TOF MS yielded the correct species identification for 281 (100%) clinical Candida isolates. MALDI-TOF proved to be a rapid and reliable method for identification of Candida strains in the clinical laboratory.

In vitro effects of sulbactam combinations with different antibiotic groups against clinical Acinetobacter baumannii isolates.

Treatment of multidrug resistant (MDR) Acinetobacter baumannii infections causes some problems as a result of possessing various antibacterial resistance mechanisms against available antibiotics. Combination of antibiotics, acting by different mechanisms, is used for the treatment of MDR bacterial infections. It is an important factor to determine synergy or antagonism between agents in the combination for the constitution of effective therapy. The study aimed to determine In vitro interactions interpreted according to calculated fractional inhibitory concentration (FIC) index between sulbactam and ceftazidime, ceftriaxone, cefepime, ciprofloxacin, gentamicin, meropenem, tigecycline, and colistin. Ten clinical isolates of A. baumannii were tested for determination of synergistic effects of sulbactam with different antimicrobial combinations. Minimal inhibitory concentration (MIC) values of both sulbactam and combined antibiotics decreased 2- to 128-fold. Synergy and partial synergy were determined in combination of sulbactam with ceftazidime and gentamicin (FIC index: ≤ 0.5 or >0.5 to <1) and MIC values of both ceftazidime and gentamicin for five isolates fell down below the susceptibility break point. Similarly, MIC value of ciprofloxacin for six ciprofloxacin resistant isolates was determined as below the susceptibility break point in combination. However, all isolates were susceptible to colistin and tigecycline, MIC values of both were decreased in combination with sulbactam. Although synergistic and partial synergistic effects were observed in the combination of sulbactam and ceftriaxone, all isolates remained resistant to ceftriaxone. The effect of cefepime-sulbactam combination was synergy in five, partial synergy in one and indifferent in four isolates. Meropenem and sulbactam showed a partial synergistic effect (FIC index: >0.5 to <1) in three, an additive effect (FIC index: 1) in one and an indifferent effect (FIC index: >1-2) in six isolates. Antagonism was not determined in any combination for clinical A. baumannii isolates in the study. In conclusion, sulbactam is a good candidate for combination treatment regimes for MDR A. baumannii infections.

[Investigation of intestinal parasites in students of Mustafa Cengiz primary school in Van]. / Van mustafa cengiz ilkögretim okulu ögrencilerinde bagirsak parazitlerinin arastirilmasi

Intestinal parasites still maintain as a major public health problem in our country. In this study, we aimed to investigate the distribution of intestinal parasites in 1st and 2nd grade students of Mustafa Cengiz Primary School, aged between 7-9 and to contribute to the parasitological data of our province. For this purpose, stool examinations of a total of 195 students, including 82 boys and 113 girls, were performed. The results of the microscopic analysis of stool samples revealed one or more parasites in a total of 117 (60%) samples including 45 male students (54.8%) and 72 female students (63.7%). The diagnosed parasites and their ratios in children were; Giardia intestinalis 36.4%, Entamoeba coli 17.9%, Blastocystis hominis 14.4%, Hymenolepis nana 10.8%, Chilomastix mesnili 3.6%, Ascaris lumbricoides 2.6%, Entamoeba hartmanni 1.5%, Trichuris trichiura 1%, Iodamoeba butschlii 0.5%, Retortamonas intestinalis 0.5% ve Endolimax nana 0.5%, respectively. From 117 positive samples for parasites, only one parasite was found in 71 (60.7%), and more than one parasites were found in 46 (39.3%). As a result, parasitic infectious diseases still maintain its importance in our region. We conclude that incidence of parasitic infectious diseases will be reduced with education about personal hygiene and improvement of physical conditions.

[Case report: two Plasmodium vivax malaria cases in the Van Province]. / Olgu Sunumu: Van Ilindeki Plasmodium vivax Sitmasi.

Malaria is an important parasitic infection which is endemic in the Eastern Mediterranean and Southeastern Anatolia regions and sporadic in other regions of Turkey, while Plasmodium vivax is the most common cause. Two patients who were admitted to our hospital in October with complaints of high fever, chills, nausea-vomiting, generalized body pain and fatigue and diagnosed as P. Vivax malaria are presented, because they were two brothers with no history of travel outside of Van city.