Factors influencing the success of cord blood collection: a tertiary perinatal medicine center's experience

Background/aim: This study aimed to evaluate the effects of certain maternal, fetal, and umbilical cord blood unit factors on storage and/or discard incidence of collected cord blood units from perinatal medicine patients. Materials and methods: A total of 273 cord blood units collected between January 2011 and December 2016 in the Division of Perinatology of Hacettepe University Hospital were evaluated retrospectively in this study. Results: Of the collected cord blood units, 53.8% (147/273) were stored. Infant birth weight, cord blood unit volume, total nucleated cell count, and CD34+ cell count were statistically significantly different between the eligible and discarded cord blood unit groups (P < 0.001 for all). No cord blood units were discarded owing to contamination-related issues. The mean gestational age for pregnant women whose umbilical cord blood was stored was 36.6 ± 1.0 weeks. Conclusion: Infant birth weight, cord blood unit volume, total nucleated cell count, and CD34+ cell count were significantly different between the eligible and discarded cord blood unit groups. The low rate of specimen storage was most likely because of the unique characteristics of perinatal medicine patients. Physicians should choose appropriate donors for cord blood collection to increase the rate of cord blood utilization.

[Evaluation of Tetrazolium Violet and Resazurin Assays for Ciprofloxacin Susceptibility Testing of Resistant Mycobacterium tuberculosis Isolates]. / Dirençli Mycobacterium tuberculosis Izolatlarinda Siprofloksasin Direncinin Saptanmasinda Tetrazolyum Moru ve Resazurin Testlerinin Etkinliginin Degerlendirilmesi.

Tuberculosis, caused by Mycobacterium tuberculosis, is still a serious public health concern. Antimycobacterial drug resistance which is in an increasing trend worldwide aids to the importance of tuberculosis problem. Fluoroquinolones which exhibit in vitro and in vivo anti-mycobacterial activity, are being recommended by World Health Organization as alternative drugs particularly for the treatment of multidrug resistant tuberculosis. Rapid detection of antimycobacterial resistance is of great importance for the effective treatment of patients with tuberculosis. In this study, we evaluated the efficiency of tetrazolium violet (TV) and resazurin (RES) assays in terms of rapid detection of bacterial growth and ciprofloxacin resistance in M.tuberculosis clinical isolates. Thirty M.tuberculosis isolates which were resistant to at least one of the first-line anti-tuberculosis drugs were tested using TV and RES assays in addition to gold standard agar proportion test. Standard strain M.tuberculosis H37Ra was also included in each run. The tests were performed in four sets as TV and RES were added on day 5, 7, 10 and 12. For the TV assay, any change in colour from yellow to dark purple was recorded as bacterial growth. For the RES assay, any change in colour from blue to pink was recorded as bacterial growth. The optimal incubation period for detection of growth and resistance was 7 days for 25 of 30 bacteria. However, results for five isolates with low inoculum rates were detected on 10th and 12th days. Any change in colour in drug containing media was recorded as resistance to ciprofloxacin. All the susceptibility results were consistent with those obtained from agar proportion method. As indicated by our results, TV and RES assays are rapid and simple tests which could be used for detection of bacterial growth and ciprofloxacin resistance in M.tuberculosis clinical isolates. Widespread use of such colorimetric tests will help to minimize the need of sophisticated expensive susceptibilty test systems particularly in low income countries.

Compartment-specific remodeling of splenic micro-architecture during experimental visceral leishmaniasis.

Progressive splenomegaly is a hallmark of visceral leishmaniasis in humans, canids, and rodents. In experimental murine visceral leishmaniasis, splenomegaly is accompanied by pronounced changes in microarchitecture, including expansion of the red pulp vascular system, neovascularization of the white pulp, and remodeling of the stromal cell populations that define the B-cell and T-cell compartments. Here, we show that Ly6C/G(+) (Gr-1(+)) cells, including neutrophils and inflammatory monocytes, accumulate in the splenic red pulp during infection. Cell depletion using monoclonal antibody against either Ly6C/G(+) (Gr-1; RB6) or Ly6G(+) (1A8) cells increased parasite burden. In contrast, depletion of Ly6C/G(+) cells, but not Ly6G(+) cells, halted the progressive remodeling of Meca-32(+) and CD31(+) red pulp vasculature. Strikingly, neither treatment affected white pulp neovascularization or the remodeling of the fibroblastic reticular cell and follicular dendritic cell networks. These findings demonstrate a previously unrecognized compartment-dependent selectivity to the process of splenic vascular remodeling during experimental murine visceral leishmaniasis, attributable to Ly6C(+) inflammatory monocytes.

Influence of serum on in vitro susceptibility testing of echinocandins for Candida parapsilosis and Candida guilliermondii.

Echinocandins are antifungal drugs used for the treatment of invasive candidiasis and aspergillosis. They bind to serum proteins within a rate of 96 to >99%. The effect of serum on in vitro echinocandin susceptibility tests of certain Candida and Aspergillus species was reported. This study was performed to determine the effect of human serum on in vitro susceptibility testing of echinocandins for clinical isolates of Candida parapsilosis and Candida guilliermondii, the species which generally have higher minimum inhibitor concentrations compared with other Candida species. One hundred C. parapsilosis and 20 C. guilliermondii isolates were included in the study. The susceptibility tests of caspofungin, micafungin and anidulafungin were performed using microdilution method, either in the presence or absence of 50% human serum, according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines. It was demonstrated that human serum significantly affects the in vitro susceptibility results of echinocandins for C. parapsilosis and C. guilliermondii isolates, mostly yielding an increase in MICs. The most prominent fold changes were for micafungin and anidulafungin in C. parapsilosis, and for anidulafungin in C. guilliermondii isolates. Serum influences the in vitro echinocandin susceptibility in C. parapsilosis and C. guilliermondii. The mechanism and clinical significance of this in vitro change need to be clarified.

Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood Hematopoietic Stem Cell Assessment.

OBJECTIVE: Colony-forming units of granulocytes/macrophages (CFU-GM) analysis is the most widely used method to determine the hematopoietic stem cell (HSC) content of human umbilical cord blood (CB) for prediction of engraftment potential. The measurement of aldehyde dehydrogenase (ALDH) activity is a more recent method for HSC qualification. Our aim was to correlate phenotypic and functional assays to find the most predictive method. MATERIALS AND METHODS: In this study, flow cytometric quantitation of CD34+ cells and ALDH positivity along with CFU-GM capacity were assessed in fresh and post-thaw CB units. RESULTS: Among 30 post-processing samples, for each CB unit the mean total number of nucleated cells (TNCs) was (93.8±30.1)x107, CD34+ cells were (3.85±2.55)x106, ALDH+ cells were (3.14±2.55)x106, and CFU-GM count was (2.64±1.96)x105. Among an additional 19 post-thaw samples the cell counts were as follows: TNCs, (32.79±17.27)x107; CD34+, (2.18±3.17)x106; ALDH+, (2.01±2.81)x106; CFU-GM, (0.74±0.92)x105. Our findings showed that in fresh samples TNCs, CD34+ cells, and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively. In terms of post-thaw samples, the correlations were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34+, r=0.67, r=0.48, r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM, and BFU-E, respectively. All correlations were statistically significant. CONCLUSION: In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, particularly for post-thaw samples. Thus, this fast, inexpensive method has the potential to overcome the weaknesses of other techniques.

[Isolation and identification of Burkholderia cepacia complex from cystic fibrosis patients]. / Kistik fibrozis hastalarinda Burkholderia cepacia complex izolasyonu ve tanimlanmasi.

In this study, sputum samples collected from cystic fibrosis patients with preliminary diagnosis of pulmonary infection were evaluated for the presence of bacteria belonging to Burkholderia cepacia complex by commercial phenotypic systems and recA polymerase chain reaction (PCR). A total of 85 patients ages between 5-30 years (mean age: 12.8 years) were included to the study with female/male ratio of 44/41. The mostly isolated bacteria from 85 sputum samples were found as Staphylococcus aureus (55.3%), Pseudomonas aeruginosa (48.2%) and Haemophilus influenzae (15.3%), whereas B. cepacia complex was phenotypically identified from two (2.3%) out of the samples. However only one was confirmed by PCR and typed as Burkholderia multivorans by restriction fragment lenght polymorphism method. This study emphasizes the importance of molecular methods as confirmatory tests for accurate identification of B. cepacia complex.

How to Improve Cord Blood Engraftment?

Various factors make cord blood (CB) a significant source of hematopoietic stem cells (HSCs), including ease of procurement and lack of donor attrition, with the ability to process and store the donor cells long term. Importantly, high proliferative potential of the immature HSCs allows one log less use of cells compared to bone marrow or peripheral blood stem cells. As total nucleated cell (TNC) and CD34(+) cell content of CB grafts are correlated to engraftment rate and speed, strategies to expand HSC and homing have been developed. This chapter will focus only on modalities such as intrabone administration, fucosylation, CD26 inhibition, prostaglandin E2 derivative or complement 3 exposure, and SDF-1/CXCR4/CXCL-12 pathway interventions that have been experimented successfully. Furthermore, increasing evidence in line with better recognition of CB progenitors that are involved in engraftment and homing will also be addressed.

[Immunopathogenesis of Leishmania infections]. / Leishmania enfeksiyonlarinin immünopatogenezi.

Leishmaniasis represents a complex of diseases with a clinical and epidemiological diversity. Leishmaniasis remains a severe public health problem and its burden is increasing. The disease is caused by a parasite belonging to the genus Leishmania. Approximately 350 billion people in 88 different countries are thought to be infected with Leishmania spp. Clinical forms of leishmaniasis are particularly diverse representing different diseases: visceral (VL), cutaneous (CL), diffuse cutaneous (DCL) and muco-cutaneous (MCL) leishmaniasis. Being the most important determinant not only cellular immunity plays the essential role in the control of leishmaniasis, but the virulence, tropism and pathogenicity that is modulated by environmental and genetic factors of their mammalian hosts and sandfly vectors, are the key interactions. These eukaryotic pathogens have evolved with the vertebrate immune system and typically produce long lasting chronic infections. A critical step in their host interaction is the evasion of innate immune defenses. The ability to avoid attack by humoral effector mechanisms, such as complement lysis, and to resist killing by lysosomal enzymes and toxic metabolytes is of particular importance. They do so by remodelling the phagosomal compartments in which they reside and by interfering with signalling pathways that lead to cellular activation. In addition they modify the antigen presenting and immunoregulatory functions of dendritic cells, a process that fascilitates their evasion of both innate and adaptive immunity. Experimental animal studies revealed that these modifications and interference mechanisms led to two different pathogenesis schemes. For CL, the polarization of Th2/Th1 cells is responsible for the progression of the disease which than leads to the chronic-persistant state. The Th2/Th1 paradigm does not apply for visceral leishmaniasis. Immunosupression rather than polarization is responsible for the systemic and progressive outcome of the disease in VL. Based on experience with animal models and humans, new vaccine and novel immunotherapy strategies especially for the locations where the disease is endemic, hold promise for the near future. In this review article the immunopathogenesis of leishmaniasis has been discussed under the light of recent literature.

[Ecological factors for host tissue colonization]. / Konak dokularin kolonizasyonunda ekolojik faktörler.

The first stage of microbial infection is colonization; the establishment of the pathogen at the appropriate portal of the entry. Organisms that have the ability to colonize host tissues, have usually developed tissue adherence mechanisms and some ability to overcome or withstand the constant pressure of host defense mechanisms. There are namely 3 adaptations that are important for bacteria to colonize human hosts: Coaggregation, osmo-adaptation, acid tolerance and resistance. In this review article, these adaptation mechanisms have been discussed under the light of literature.

Molecular methods for detection of invasive fungal infections and mycobacteria and their clinical significance in hematopoietic stem cell transplantation.

Infection remains an important source of morbidity and mortality in patients who undergo hematopoietic stem cell transplantation (HSCT). In the immune reconstitution period after transplantation, HSCT recipients are most likely to have bacterial or fungal infections. Invasive fungal infections (IFIs) and mycobacterial infections (MBIs) are among the complications of HSCT, with high morbidity and mortality rates. Early diagnosis of both is crucial in order to manipulate the disease and to avoid fulminant outcomes. This chapter reviews the current knowledge on the molecular diagnosis of IFIs and MBIs in HSCT recipients, describing two different polymerase chain reaction (PCR)-based methods, one commercial (qPCR, Roche) and one in-house IS6110-based protocol.

Predominance of hospital-associated MRSA among cystic fibrosis patients in a Turkish reference cystic fibrosis centre.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are the major causative agents of numerous hospital- and community-acquired infections. Increasing prevalence of MRSA in cystic fibrosis (CF) populations is reported all over the world. Although there are papers reporting the prevalence and genetic backgrounds of MRSA isolates from different settings in Turkey, there is no information regarding the situation in the CF community. This study was conducted to characterize the MRSA strains recovered from CF patients followed-up at a Turkish reference CF centre. Microbiological testing of isolates was performed via conventional microbiological techniques. Molecular characterization of MRSA isolates was carried out by SCCmec typing by multiplex PCR and PVL gene determination. Among a total of 604 CF patients included in the study, 325 patients were found to harbour S. aureus (53.8%). Of those 325 patients, 24 were positive for MRSA during their follow-up (7.4%). Thirty-two MRSA isolates from these patients were chosen for further assessment of molecular characteristics. Twenty-six MRSA isolates exhibited a pattern like SCCmec type III (81.2%) and six consecutive MRSA isolates of a single patient revealed SCCmec type IV (18.7%). Our findings definitely support the need for further surveillance studies for CF-MRSA strains and highlight the need for infection control measures in the setting of CF centres.

Comparison of two methods and three end points in determination of in vitro activity of micafungin against Aspergillus spp.

We investigated the in vitro activity of micafungin against clinical Aspergillus isolates (n = 37) (Aspergillusfumigatus [n = 21], Aspergillusflavus [n = 14], and Aspergillus niger [n = 2]) by using NCCLS M38A microdilution and an investigational disk diffusion assay. Microdilution assay results were evaluated by using the end points of a MIC-2 (measured in micrograms per milliliter) and minimum effective concentration (MEC, measured in micrograms per milliliter; the lowest concentration of micafungin that produces short and aberrant hyphal branchings microscopically). Disk diffusion results were interpreted by measuring the zone(s) of inhibition (ZOI, measured in millimeters). Micafungin proved to be similarly active against all Aspergillus species tested. At 24 h, MIC-2s and MECs were identical. At 48 h, however, MIC-2s increased unpredictably, leading to the loss of a consistent correlation between the two end points. MECs and ZOI remained consistent and correlated at both reading times, suggesting their use as relevant end points in susceptibility testing of micafungin against Aspergillus: All Aspergillus isolates yielded intrazonal growth on disk diffusion agar plates. The intrazonal colonies contained short, aberrant hyphal branchings microscopically. The in vivo significance of these findings remains to be further investigated.

Some fused heterocyclic compounds as eukaryotic topoisomerase II inhibitors.

Our previously synthesized 37 compounds, which are 2,5,6-substituted benzoxazole, benzimidazole, benzothiazole, and oxazolo(4,5-b)pyridine derivatives, were tested for their eukaryotic DNA topoisomerase II inhibitory activity in cell free system and 28 were found to inhibit the topoisomerase II at an initial concentration of 100 microg/ml. After further testing at a lower range of concentrations, 12 derivatives, which were considered as positive topoisomerase inhibitors, exhibited IC50 values between 11.4 and 46.8 microM. Etoposide was used as the standard reference drug to compare the inhibitor activity. Among these compounds, 2-phenoxymethylbenzothiazole (3f), 6-nitro-2-(2-methoxyphenyl)benzoxazole (1a), 5-methylcarboxylate-2-phenylthiomethylbenzimidazole (3c), and 6-methyl-2-(2-nitrophenyl)benzoxazole (1c) were found to be more active than the reference drug etoposide. Present results point out that, besides the very well-known bi- and ter-benzimidazoles, compounds with single bicycle fused ring systems in their structure such as benzimidazole, benzoxazole, benzothiazole, and/or oxazolopyridine derivatives also exhibit significant topoisomerase II inhibitory activity.