MALDI-TOF MS identification of Malassezia species isolated from patients with pityriasis versicolor at the seafarers’ medical service in Dakar,Senegal

Pityriasis versicolor (PV) is a superficial mycosis caused by yeast of the genus Malassezia. The most common isolated Malassezia species in PV lesions differ among M. furfur, M. globosa and M. sympodialis. We purpose to determine the distribution of Malassezia species in PV patients at the seafarers’ medical service in Dakar, Senegal and to examine whether any association between identified Malassezia species and patients’ profile. From May 2017 to August 2017, first a questionnaire was filled to get informative data before collection of skin scrapings taken from most scaly site using sterile scalpel blade and application of scotch^® for direct examination (DE). At the laboratory, DE, culture and identification by MALDI-TOF MS were done. One hundred patients with PV – all men – were included with a mean age of 34 years. Among seafarers, 81% were sailors. Clinical prevalence of PV was highest in aged adults patients with ages of 31 to 60 years (56%). Seafarers with high level of education were less representative with only 2%. The mean duration of the PV was 26.83 months. 20% of subjects suffered lesions in more than one location. The chest was the most affected anatomical site. Furthermore, possible predisposing factors associated with PV were also detected. DE was positive in 95% but culture growth only in 46%. MALDI-TOF MS analysis of the positive cultures could be performed in 84.8% (39/46). Only M. furfur was identified in 100% (39/39). In definitive, M. furfur is the only causative agent of PV in Dakar.     

Physiological characterization and molecular identification of some rare yeast species causing onychomycosis

IntroductionOnychomycosis are infections with a variety of etiological agents. Although dermatophytes are responsible for most infections, yeasts are gaining importance as agents of these pathologies. The use of antifungals has increased the incidence of what had been considered rare or novel pathogens. We reidentify three rare yeasts from a culture collection of onychomycosis agents by matrix-assisted laser desorption/ionization time of flight/mass spectrometry (MALDI-TOF/MS) and sequencing the internal transcribed spacer (ITS) regions or the intergenic spacer (IGS) 1 region of ribosomal DNA (rDNA), and present their enzymatic and antifungal susceptibility profiles.Material and methodsWe performed a phenotypical characterization and molecular identification of five yeast isolates. We tested the urease, gelatinase, DNase, phospholipase, protease, and esterase activities, as well as the hemolytic activity. We evaluated the antifungal susceptibility to amphotericin B, fluconazole, anidulafungin and caspofungin.ResultsPhenotypic methods could not identify the isolates. MALDI-TOF/MS was able to properly identify Candida duobushameulonii. The five isolates were successfully identified by sequence analysis as Candida duobushaemulonii, Meyerozyma caribbica and Cutaneotrichosporon dermatis. Candida duobushameulonii showed hemolytic, phospholipase, and protease activities. Meyerozyma caribbica was positive for gelatinase and protease activities. All antifungals exhibited minimum inhibitory concentrations (MICs) ≤ 2 μg/mL against both species. The three isolates of Cutaneotrichosporon dermatis showed urease, DNase, and esterase activities, and resistance to echinocandins (MICs ≥ 8 μg/mL), while amphotericin B and fluconazole exhibited low MICs against these isolates (0.50–2 μg/mL).DiscussionSequencing of the ITS or IGS1 regions of rDNA remains the best method for identifying cryptic species over other commercially available systems. More reports are needed to define the enzymatic and antifungal profiles for these species. This is the first report of Meyerozyma caribbica and Cutaneotrichosporon dermatis as etiological agents of onychomycosis.     

Toe web intertrigo in Kaposi’s sarcoma patients: a microbiological study in a large cohort of patients

Kaposi ‘s sarcoma (KS) is a rare multifocal angioproliferative disease associated with human herpes virus 8 (HHV-8) infection, characterized by cutaneous nodules or plaques especially on the lower limbs. Some skin modifications, such as chronic lymphedema, plantar hyperkeratosis and interdigital desquamation, may be associated with consequent impairment of the local immunosurveillance and increased risk of some bacterial or mycotic infections. With the objective of evaluating if bacterial or mycotic infections in KS patients are supported by different microorganisms compared to control patients, we performed an observational retrospective study, comparing positive cultural swabs of interdigital intertrigo of KS patients with positive cultural swabs of interdigital intertrigo of patients admitted to our dermatologic unit during the last 10 years. One hundred KS patients and 84 control patients were admitted to this study. Some of the skin swabs from interdigital spaces were positive for more than one microorganism, and therefore we found 187 microorganisms among the KS group and 182 microorganisms in the control group. The most common microrganisms among KS patients were T. mentagrophytes (16%), S. aureus (14.9%), P. aeruginosa (13.9%), S. marcescens (5,9%), while among non-KS patients were S. aureus (26,9%), C. albicans (22%), S. agalactiae (7.7%) and E. coli (9.9%). These differences are statistically significant (p < 0.01). KS patients may be more affected by toe web intertrigo due to other bacteria and dermatophytes than the general population. During clinical examination, a careful inspection is necessary for an early diagnosis of toe web intertrigo, in order to prevent serious complications, such as cellulitis and sepsis. Consequently, a cultural examination with antibiogram is required to identify the causative agent of intertrigo and guide antimicrobial therapy.     

Quality of life in Polish patients with multiple sclerosis

PurposeThis study is a pilot evaluation of the quality of life (QoL) in Polish patients with multiple sclerosis (MS).Material/methodsData from 21 centers in Poland were collected from May 2008 to January 2009. QoL was assessed using the questionnaire Euro Quality of Life (EQ-5D), with Polish population norms. Demographic profile of patients, duration/form/relapsing activity of the disease, disability and comorbidity were also analyzed.ResultsData from 3521 patients (F/M ratio 2.4:1) were collected. The average EQ-5D index was 0.8 ± 0.27 and the mean score in a visual analog scale (EQ-VAS) was 65.6 ± 21.5. There was a highly significant positive correlation between both indices (r = 0.7334, p < 0.0001). The mean patient age was 40.7 years (11.2–92.3 years) and disease duration was 10.3 ± 8.8 years (0.04–53 years). 74.2% of subjects had relapsing-remitting form of MS, while 17.2% were classified as secondary progressive and 8.6% as primary progressive. In the group of relapsing-remitting MS subjects there were 2.5% patients with “benign MS”. The average degree of disability on EDSS scale was 3.6 ± 2.2, while disability ≥6 was observed in 20.3% of patients. Most patients did not have other diseases besides MS.ConclusionsThis is the first large study of QoL in patients with MS in Poland (approximately 18% of all patients). Our results confirm a reduction in QoL compared with the general population. Further studies are indicated to identify the modifiable risk factors (e.g. type of treatment) that may affect QoL.     

Influence of metabolic syndrome and its components on subclinical organ damage in hypertensive perimenopausal women

PurposeWe investigated the association between metabolic syndrome (MS), its components and the presence of subclinical organ damage in hypertensive perimenopausal women.Patients/methods152 women with newly diagnosed, untreated arterial hypertension (mean age 51.0 ± 3.5 years) were included in the study. In all subjects anthropometrical measurements, 24-hr blood pressure monitoring, echocardiographic examination, and carotid ultrasound were performed. Carotid-femoral pulse wave velocity (PWV) was measured to obtain data on vascular compliance. As the index of early kidney damage both glomerular filtration rate was calculated and albumin/creatinine ratio in the urine sample was measured. A fasting blood sample was taken to measure glucose and lipid concentration.ResultsMS was found in 41% of patients. Patients with MS exhibited elevated left ventricular mass index (LVMI 84.7 vs. 78.8 g/m^2.7, p = 0.03), higher intima-media thickness (IMT 0.67 vs. 0.62 mm, p = 0.003), greater prevalence of LV hypertrophy (30% vs. 13%, p = 0.01), and carotid plaques (24% vs. 15%, p = 0.01). The multivariate regression analysis revealed that components of MS (systolic blood pressure and waist circumference) are stronger predictors of LVM than MS itself. The relationship between MS and LVMI lost its significance when BMI was included in the model, and remained significant for IMT.ConclusionIn hypertensive perimenopausal women components of MS are stronger predictors of subclinical organ damage than MS itself. Left ventricular mass and hypertrophy are more strongly correlated with increasing body weight than with the presence of MS. MS, independently of BMI, influences the level of subclinical atherosclerosis in the study group.     

Dermatomycosis: comparison of the performance of calcofluor and potassium hydroxide 30% for the direct examination of skin scrapings and nails

Dermatomycosis is a common problem in clinical practice. A definitive diagnosis should be made before initiation of antifungal therapy. Mycological diagnosis is usually based on direct microscopy using potassium hydroxide and fungal culture. The aim of our study was to compare the performance of potassium hydroxide 30% (KOH) with slight heating and staining with 0.01% calcofluor (Fluorescent brightener 28^®) for the detection of fungi by direct microscopy. We evaluated prospectively 241 samples from patients suspected to have dermatomycosis (159 nail samples, 82 skin scrapings). Mycological culture was chosen as the reference method to assess the performance of each test. Out of the 241 samples, 152 mycological cultures were positive, of which 98 were classified as pathogenic fungi (86 dermatophytes, 11 non-dermatophyte molds, 1 Candida albicans). The sensitivity of direct microscopy was significantly higher with calcofluor than with KOH (respectively, 88% and 72%, P = 0.0116). Specificity was 93% with calcofluor and 88% with KOH (P = NS). Calcofluor showed higher specificity on skin scraping than on nails samples (respectively, 100% and 89%, P = 0.0138) while KOH specificity did not differ as a function of the type of specimen (respectively, 92% and 86%). In conclusion, calcofluor is a more sensitive and specific technique than KOH for the microscopic detection of dermatophytes and non-dermatophyte pathogenic fungi in nails and skin scrapings. The results are easy to read by inexperienced staff.     

Modulation of alveolar macrophage innate response in proinflammatory-, pro-oxidant-, and infection- models by mint extract and chemical constituents: Role of MAPKs

There is a continuing need for discovering novel primary or adjunct therapeutic agents to treat inflammatory conditions and infections. Natural products have inspired the discovery of several modern therapeutics; however, there is a paucity of mechanistic information on their mode of action. This study investigated the therapeutic potential and mode of action of corn mint’s (Mentha arvensis) leaf extract (ME) in alveolar macrophages (AMs) challenged with model pro-inflammatory (LPS), pro- oxidant (LPS or H₂O₂), and infection (Mycobacterium) agents and contribution of its dominant constituents rosmarinic acid, l-menthol, and l-menthone. LPS-induced inflammatory response in the murine AM cell line MH-S was significantly reduced in terms of pro-inflammatory cytokines (TNF-α, IL-1α) and nitric oxide (NO) when pre- or post-treated with ME. The ME pretreatment of macrophages led to a significant increase ( P≤ 0.05) in phagocytic activity toward Mycobacterium smegmatis and a greater pathogen clearance in 24 h in both ME pre-treated (P ≤ 0.05) and post-treated cells. Significant attenuation (P ≤ 0.01) of reactive oxygen species (ROS) production in LPS- or H₂O_2-treated macrophages by pretreatment with whole mint extract (ME) was accounted for in part by the mint constituents rosmarinic acid and l-menthone. Attenuation of pro-inflammatory response by ME pretreatment coincided with the significant reduction in total and phosphorylated JNK1/2, decrease in total p38, and increase in phospho-ERK1/2 thereby implying a role of differential modulation of MAPKs. Taken together, the results demonstrate that corn mint leaf components cause potent anti-inflammatory, anti-oxidant, and anti–infection effects in AMs via suppression of the production of cytokines/soluble mediators and ROS and increased pathogen clearance, respectively. To our knowledge, this is the first report on the mode of action of corn mint targeting the alveolar macrophages and on the potential role of MAPKs in immunomodulation by this product.     

Side effect of metenolone enanthate on rats heart in puberty: Morphometrical study

The aim of this study was the investigation of effects of the metenolone enanthate (ME) that is used among athletes as doping and muscle amplifier, on hearts of male and female rats that are in puberty using morphometrical methods.A total of 36 rats which were divided into three separate groups (Experiment, ME; vehicle, PO; control, C) each consisting of 6 male and 6 female rats were used. 0.5 mg/kg metenolone enanthate was applied intraperitoneally into experiment subjects 5 times a week over a period of 4 weeks. At the end of experiment, rats were euthanized and their hearts were cut at the level of musculus papillaris after the fixation in formalin. Hearts were taken out and embedded in paraffin wax. Photos were taken at cut surfaces, and thickness, diameters and surface area levels were measured. Left ventriculus mass (LVM) and left ventriculus mass index (LVMI) were calculated.In the study LVM (p < 0.005) and LVMI (p < 0.05) were found to be significantly higher in the ME group in females whereas left ventricular lumen diameter (LVLD) were found to be significantly lower (p < 0.05). Thus left ventricular hypertrophy development was observed. LVM and LVMI were found to be similar in ME and C groups among male rats and the highest level of these data were found in the group. LVM and LVMI were higher among females (p < 0.006).In conclusion, it has been shown that the adverse effects of ME on heart were developing starting from puberty and resulting with the enlargement of the heart and left ventricular hypertrophy and especially among females this condition was more evident. It has also been discussed that the continuous use of drugs may further enhance this condition.     

SIRT1 as a potential biomarker of response to treatment with glatiramer acetate in multiple sclerosis

SIRT1, a NAD dependent histone and protein deacetylase, is a member of the histone deacetylase class III family. We previously showed that SIRT1 mRNA expression is significantly lower in peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients during relapses than in stable patients. We have now investigated SIRT1 as a possible biomarker to predict relapse as well as responsiveness to glatiramer acetate (GA) treatment in relapsing-remitting MS (RRMS) patients. Over the course of 2 years, a cohort of 15 GA-treated RRMS patients were clinically monitored using the Expanded Disability Status Scale and assessed for MS relapses. Blood samples collected from MS patients were analyzed for levels of SIRT1 and histone H3 lysine 9 (H3K9) acetylation and dimethylation. During relapses, MS patients had a lower expression of SIRT1 mRNA than did stable MS patients. In addition, there was a significant decrease in H3K9 dimethylation (H3K9me2) during relapses in MS patients when compared to stable patients (p = 0.01). Responders to GA treatment had significantly higher SIRT1 mRNA (p = 0.01) and H3K9me2 levels than did non-responders (p = 0.018). Receiver operating characteristic analysis was used to assess the predictive power of SIRT1 and H3K9me2 as putative biomarkers: for SIRT1 mRNA, the predictive value for responsiveness to GA treatment was 70% (p = 0.04) and for H3K9me2 was 71% (p = 0.03). Our data suggest that SIRT1 and H3K9me2 could serve as potential biomarkers for evaluating patients' responsiveness to GA therapy in order to help guide treatment decisions in MS.     

High expression of OX40 (CD134) and 4-1BB (CD137) molecules on CD4+CD25high cells in children with type 1 diabetes

PurposeDespite the rapidly rising incidence of diabetes in children, with the highest rise in children < 5 years of age, data on mechanisms that trigger severe beta-cells damage are limited. The aim of the study was to assess the frequency of OX40 (CD134) or 4-1BB (CD137) positive cells in the peripheral blood of children with newly diagnosed type 1 diabetes (T1D) in comparison to healthy controls.Material/methodsThe study included 33 children (mean age 7.3 ± 5.4 years) with newly diagnosed T1D and 39 age-matched healthy controls. Separate analysis was performed in children < 5 years. Flow cytometric analysis was performed using the following markers: CD4, CD25, CD137, and CD134. Fasting C-peptide level was assessed as well.ResultsThe frequency of CD4⁺CD25^highOX40⁺ was higher in T1D children than in controls (median value 3.58% vs. 1.1%, respectively; p = 0.003). Moreover, T1D children had higher frequency of CD4⁺CD25^high4-1BB⁺ cells than healthy subjects (median value 5.76% vs. 3.74%, respectively; p = 0.037). A significant correlation was noted between the age of diabetic children and the C-peptide level (r = 0.54, 95% CI [0.19–0.77], p = 0.004). In comparison with age-matched controls, children < 5 years had higher frequency of CD4⁺CD25^highOX40⁺ (p = 0.004) and CD4⁺CD25^high4-1BB⁺ cells (p = 0.079).ConclusionsOur study showed higher frequency of both OX40 and 4-1BB positive cells in T1D children in comparison to controls. It seems that observed differences might be more pronounced in children < 5 years of age than in older subjects. Further clinical studies are needed to determine the age-related differences in the immune system, in the pathogenesis of T1D.     

Numeracy of multiple sclerosis patients: A comparison of patients from the PERCEPT study to a German probabilistic sample

Objective

A shared decision-making approach is suggested for multiple sclerosis (MS) patients. To properly evaluate benefits and risks of different treatment options accordingly, MS patients require sufficient numeracy – the ability to understand quantitative information. It is unknown whether MS affects numeracy. Therefore, we investigated whether patients’ numeracy was impaired compared to a probabilistic national sample.

Health-related and socio-demographic correlates of physical activity level amongst urban menopausal women in Nigeria

ObjectivesTo investigate the physical activity (PA) level of Nigerian women aged 40–60 years and examine possible association between the PA level and some health-related and socio-demographic variables.MethodsThis is a cross-sectional study of 547 women in which a purposive sampling method was used to recruit participants in urban centers of three states from three geopolitical zones in Nigeria. The International Physical Activity Questionnaire (IPAQ)-short form, was used to assess PA level. A self-administered 13-item semi-structured questionnaire was used to obtain health-related (menopausal status, perceived health status, health problems, menopausal symptoms) and socio-demographic (age, marital status, educational level, occupation, personal income) information from participants. Chi-square and logistic-regression analysis were used to assess association between PA level and these variables.ResultsThe mean age of participants was 49.21 ± 5.2 years, comprising 184 (33.6%) premenopausal, 129 (23.6%) perimenopausal and 234 (42.8%) postmenopausal women. Most of the women in the three menopausal groups reported moderate PA level. No significant association was observed between PA level and menopausal status (P = 0.348), health problems (P = 0.079) or any of the menopausal symptoms and age-group (P = 0.381) of the women. PA level had a direct significant association with perceived health status (P = 0.001) and educational level (P = 0.000).ConclusionMenopausal women in Nigeria reported a moderate PA level. Self-perception of good health, having secondary/post-secondary education, were directly associated with not being of a low PA level.     

Syndrome de basse T3 chez des patients âgés présentant une dénutrition modérée ou sévère

Under nutrition causes alterations in serum thyroid function tests referred as “non-thyroidal illness syndrome” (NTIS). We determined serum thyroid hormones in elderly patients (≥ 70 years) with mild (25 ≤ albuminemia < 35 g/l, N = 64) or severe (albuminemia < 25 g/l, N = 67) under nutrition, compared to well-nourished patients (albuminemia ≥ 35 g/l, N = 78). Patients with under nutrition had lower (32%) free T3 (FT3) and higher (21%) free T4 (FT4) concentrations than in controls. TSH level was similar in the three groups of patients. Therefore, malnourished elderly patients showed euthyroidism profile, whatever the severity of under nutrition. The decrease in FT3 level was more marked in patients with severe under nutrition. In addition, there was a positive correlation between FT3 and albumin, transthyretin and transferrin and a negative correlation with PINI (“prognostic inflammatory and nutritional index”). These data showed that FT3 is a good marker of severity of under nutrition in elderly patients.     

Tinea capitis at the University Hospital of Tizi-Ouzou,Algeria, and first isolation of Trichophyton tonsurans

Tinea capitis, which are fungal infections caused by some dermatophyte species, are common in developing countries, such as Algeria, where they represent a public health concern. In order to define the epidemiological, clinical and diagnostic features of these infections, a prospective study was conducting from September 2018 to May 2019, at the University Hospital of Tizi-Ouzou (Algeria).PatientsAll patients addressed to the Laboratory of Parasitology–Mycology of the University Hospital of Tizi-Ouzou for a suspected Tinea capitis, were included in this study.Materials and methodsBefore sampling, contact with animals or soil, presence of similar lesions in the family circle, and previous antifungal or corticosteroid treatment were searched. Mycological examination included direct microscopic examination of the samples, and culture on Sabouraud agar slants at 27 °C for up to 4 weeks.ResultsOut of the 87 samples examined, 46 allowed us to confirm the diagnosis of Tinea capitis, representing a positivity rate of 52.9%. The sex ratio was 1.09 (52.2% males vs. 47.8% females among the infected patients), and the infection mainly involved children of 4–6 years (43.3%). Thirty-four strains of dermatophytes were isolated. Microsporum canis was the most frequent species identified (44.1%), followed by Trichophyton mentagrophytes (38.2%). During this study, Trichophyton tonsurans, an unusual dermatophyte species in Algeria, was identified for the first time in our hospital.ConclusionTinea capitis are still common in Algeria, mainly affecting school-aged children and preschool children. Microsporum canis and T. mentagrophytes are the major causative agents, in agreement with previous studies showing a decrease in frequency of anthropophilic species, in favour of zoophilic species.     

Chemokine receptor V Δ32 deletion in multiple sclerosis patients in Csongrád County in Hungary and the North-Bácska region in Serbia

The roles of chemokine receptor V (CCR5) and its polymorphism, rs333 in multiple sclerosis (MS) are controversial. We investigated the receptor and its deletion in a large MS (428) and a numerous control (831) population in Csongrád County (Hungary) and North-Bácska (Serbia). Taqman probes firstly were used for the allele discrimination. There was no significant difference in genotype (OR = 1.092, 95% CI = 0.807–1.478, p = 0.568 for wt/wt (wt = wild type allele) vs wt/Δ32, Δ32/Δ32 (Δ32 = Δ32 base pair deletion allele)) or allele frequency (OR = 0.914, 95% CI = 0.692–1.207, p = 0.525). Neither the deletion nor the wt allele affected the Expanded Disability Status Scale score or the age at onset. Our results indicate no association between the CCR5 Δ32 allele and MS.     

Criterion validity of the California Verbal Learning Test-Second Edition (CVLT-II) after traumatic brain injury

The California Verbal Learning Test-Second Edition (CVLT-II) was performed by 200 participants, divided into moderate–severe traumatic brain injury (n = 43), mild traumatic brain injury (n = 57) and demographically matched control (n = 100) groups. Participants with complicating premorbid histories or who scored below 15/16 on the CVLT-II forced choice recognition trial were excluded. There were statistically significant (p < .0001) effects of group status on the CVLT-II total recall discriminability and recognition discriminability indices. Logistic regression revealed that, in the classification of control versus moderate–severe traumatic brain injury, CVLT-II variables were accurate 66–71% overall, but false positive rates ranged from 49 to 54%. In conclusion, average scores on the CVLT-II differ meaningfully between patients with various degrees of severity of traumatic brain injury and controls, but this test should not be used in isolation to determine the presence or absence of acquired memory impairment.     

Antioxidant and antifungal activities of marrubiin,extracts and essential oil from Marrubium vulgare L. against pathogenic dermatophyte strains

ObjectiveMedicinal plants extracts and plant-derived compounds are one of the natural sources for discovering new antifungal agents, the objectives of this work were to investigate for the first time the antidermatophytic, antipathogenic activities of methanol, acetone extracts, and essential oil of Marrubium vulgare L. grown in Tunisia and its active compound marrubiin on pathogenic for animals and humans, such as some dermatophytes and pathogenic for plants, and to evaluate antioxidant activities of different extracts with consideration to their chemical compositions.Material and methodsAcetone and methanol extracts were evaluated by HPLC, the essential oil was also analyzed by GC/MS. PCL assay was used to determine the antioxidant activity.ResultsResults showed that methanol and acetone extracts exhibited a significant antioxidant activity (261.41 and 272.90 μmol TE/g respectively), while the lowest one was observed in the case of marrubiin and essential oil. The antifungal activity of different extracts, marrubiin and essential oil at two concentrations (20 and 100 μg/mL) were screened against the dermatophytes fungi Microsporum gypseum, Microsporum canis, Arthroderma cajetani, Trichophyton mentagrophytes, Trichophyton tonsurans, Epidermophyton floccosum and against two fungi strains (Botrytis cinerea, Pythium ultimum). Among tested extracts, marrubiin at 100 μg/mL showed about 50% inhibition for T. mentagrophytes and E. floccosum. The anti-phytopathogenic activity was also carried out, only marrubiin had in activity against B. cinerea at the highest dose (32.40%), while methanol extract of M.vulgare and marrubiin are able to increase the mycelial growth of P. ultimum at the highest concentration (45.15 and 40.30% respectively).ConclusionIn our study, we conclude that M.vulgare and marrubiin can be used as natural antioxidants and antifungal agent for treatment of skin dermatophyte infections.     

Rapid determination of C4-acylcarnitine and C5-acylcarnitine isomers in plasma and dried blood spots by UPLC–MS/MS as a second tier test following flow-injection MS/MS acylcarnitine profile analysis

BackgroundFlow-injection MS/MS methods for elevated acylcarnitines are routinely performed in most newborn screening and biochemical genetics laboratories; however this technique cannot distinguish between isobaric compounds; therefore, chromatographic separation is required to quantitate isomers for differential diagnosis of some inborn errors of metabolism.MethodsA UPLC–MS/MS method has been developed for the simultaneous quantitation of isobutyrylcarnitine and butyrylcarnitine, and a second UPLC–MS/MS method for the quantitation of isovalerylcarnitine, (S) and (R) 2-methylbutyrylcarnitine, pivaloylcarnitine and valerylcarnitine. Plasma and dried blood spots samples are extracted with methanol and derivatized with butanolic HCl. Deuterium labeled internal standards are used for quantitation. Separation is obtained using a methanol/water gradient with a C18 BEH, 1 × 100 mm, 1.7 μm UPLC column, at 60 °C; run time is less than 10 min. The isomers are detected with a Quattro Premier triple quadrupole, with electrospray ionization in positive ion mode.ResultsIntra-day precision in plasma and dried blood spots ranged from 1.4% to 14% and accuracy from 88% to 114% respectively for butyrylcarnitine and isobutyrylcarnitine. Precision for the isomers of C5-acylcarnitine ranged from 1.3% to 15% and accuracy 87% to 119%, respectively in plasma or dried blood spots. Inter-day precision was within 20% at each concentration of isobutyrylcarnitine and butyrylcarnitine. Precision for 2-methylbutyrylcarnitine and isovalerylcarnitine at concentrations above the normal range was within 24%.ConclusionsTwo diagnostic tests based on the separation of C4-acylcarnitine and C5-acylcarnitine isomers by UPLC–MS/MS provide fast differential diagnosis of SCAD deficiency versus IBCD deficiency and IVA versus 2-MBCD deficiency. The separation of C5-acylcarnitines can reveal false elevation due to pivalic acid-containing antibiotics. Abnormal newborn screen results due to pivalate-generating prodrug antibiotics of maternal origin were confirmed. This separation of isomers can resolve multiple diagnostic challenges in both newborn screening and in cases with ambiguous metabolic test results.     

The prognosis of highly active antiretroviral therapy (HAART) treated HIV infected patients in Serbia,related to the time of treatment initiation

BackgroundWith the introduction of highly active antiretroviral treatment (HAART) an impressive improvement in patient survival and quality of life has bee observed. However, the optimal timing of initial HAART is still under consideration.ObjectiveTo investigate the prognosis of HAART treated patients in Serbia, related to the timing of HAART initiation.Study designA series of 563 patients on HAART was retrospectively analyzed to investigate treatment response and survival.ResultsAfter a mean of 6 years (range 1–14) of treatment with PI-based and/or NNRTI-based regimens, a favorable response was achieved in 72.4%, treatment failure occurred in 7.9%, while 19.7% had a dissociative immunological/virological response. If treatment was initiated during primary HIV infection it took a shorter time to achieve a favorable response than in patients who began HAART in chronic HIV infection (2.7 ± 2.2 years vs. 6.9 ± 2.7 years, P < 0.01). A higher proportion of patients with primary HIV infection then those treated in the chronic phase achieved a favorable response to HAART (88.4% vs. 71.9%, P = 0.045). Patients who initiated HAART when their CD4 cell counts were below 200 cells/μL needed longer treatment for favorable response (8 years vs. 6 years, log rank P < 0.01). Forty-seven (8.3%) patients died. The overall estimated survival was 13 years. Patients older then 40 and IVDU were more likely to die during HAART (OR 2.6, 95% CI 1.1–5.9, P = 0.016, and OR 2.0, 95% CI 1.0–3.7, P = 0.02, respectively). However, reaching and maintaining undetectable viremia was an independent predictor of longer survival (OR 11.3, 95% CI 4.6–27.7, P < 0.01).ConclusionReaching and maintaining undetectable viremia during HAART predicted longer survival, even if sub-clinical immunodeficiency remained.     

Impact of blood donor characteristics on quality of packed red blood cell concentrates

IntroductionImpact of blood donor characteristics on quality of packed red blood cell concentrates.ObjectiveTo determine the impact of donor factors on the quality of packed red blood cell concentrates.Material and methodsThe analytical cross sectional study was conducted on 505 whole blood donors after approval by the Institutional Ethics Committee and written informed consent from blood donors. Two mL EDTA sample was collected for pre-donation haemoglobin estimation and all relevant donor details were recorded. Whole blood was collected in 350 mL double blood bags. PRBCs were prepared as per the departmental SOP. Volume of each PRBC was recorded and sample from each bag was taken for estimation of total haemoglobin content and haematocrit.ResultsOf 505 blood donors, 459 (90.9%) were males and 324 donors (64.2%) were less than 30 years of age. The majority of the donors were repeat donors (61%, n = 308 repeat donors), vegetarians (52.9%, n = 267 vegetarians) and non-smokers (92.7%, n = 468). Mean haemoglobin was found to be significantly higher in males (14.9 vs. 13.3; P ≤ 0.001), donors more than 30 years of age (15 vs. 14.7; P = 0.042), repeat donors (14.9 vs. 14.7), non-vegetarians (15.1 vs. 14.6; P ≤ 0.001) and smokers (15.3 vs. 14.8 g/dL; P = 0.020). PRBC units prepared from male blood donors, repeat donors and non vegetarians had significantly higher mean volume and mean total haemoglobin content. Strong positive correlation was observed between haemoglobin of the blood donor and total haemoglobin content of the PRBC and volume of blood collected.ConclusionsDonor characteristics do have effect on total haemoglobin content of packed red blood cells.