Dental status, dental treatment procedures and radiotherapy dosage as potential risk factors for an infected osteoradionecrosis (IORN) in patients with oral cancers: Retrospective evaluation of 204 patients treated in two observation periods of approximately ten years each.In group A, 90 patients were treated in the years 1993-2003, in group B 114 patients in the years 1983-1992 (data in brackets). All patients had histopathologically proven squamous cell cancers, mainly UICC stages III and IV. 70%  had undergone surgery before radiotherapy. All patients were referred to the oral and maxillofacial surgeon for dental rehabilitation before further treatment.Radiotherapy was performed using a 3D-conformal technique with 4-6MV photons of a linear accelerator (Co-60 device up to 1987). The majority of patients were treated using conventional fractionation with total doses of 60-70 Gy in daily fractions of 2 Gy. Additionally, in group A, hyperfractionation was used applying a total dose of 72 Gy in fractions of 1.2 Gy twice daily . In group B, a similar schedule was used up to a total dose of 82.8 Gy . 14 (0) patients had radiochemotherapy simultaneously. After therapy, the patients were seen regularly by the radiooncologist and – if necessary – by the oral and maxillofacial surgeon. The duration of follow-up was 3.64 years .Before radiotherapy, the dental health status was very poor. On average, 21.5  teeth were missing. Further 2.04 teeth  were carious, 1.4  destroyed.Extractions were necessary in 3.6 teeth , conserving treatment in 0.4  teeth. After dental treatment, 6.30  teeth remained.IORN was diagnosed after conventionally fractionated radiotherapy in 15% , after hyperfractionation in 0% .Within more than 20 years there was no improvement in dental status of oral cancer patients. Extensive dental treatment procedures remained necessary. There was an impressive reduction of the IORN frequency in patients treated in a hyperfractionated manner probably resulting from a dose reduction and an extension of the interfraction time. Infected osteoradionecrosis (IORN) is still one of the major problems after radiotherapy for neoplasms in the oral cavity. The chewing and swallowing functions of the patients are impaired, long-lasting conservative and surgical interventions may become necessary. In the last decades, there was an ample discussion about potential risk factors for the development of IORN. Besides the kind of surgical procedures, dosage and fractionation of radiotherapy and the simultaneous application of chemotherapy, the patients’ dental status before treatment and the extent of dental treatment procedures were regarded significant.Thus, more than 20 years ago, the departments of Radiotherapy and of Oral and Maxillofacial Surgery of the Saarland University Medical School together started a dental examination and rehabilitation program with the aim to prevent IORN as far as possible. All patients referred for radiotherapy for cancer in the oral cavity were examined by the oral and maxillofacial surgeon, the exact dental findings were recorded and – if necessary, specific rehabilitation procedures were performed. Radiotherapy was started only after approval by the Oral and maxillofacial surgeon (OMF surgeon). After therapy, the patients were seen regularly by the radiooncologist, diagnosis and treatment of IORN were performed by the oral and maxillofacial surgeon.After completion of two theses Barbie , publishTwo groups of patients who had undergone radiotherapy for neoplasms of the oral cavity have been reanalyzed retrospectively:Group A consists of 90 consecutive patients having been treated in the years 1993-2003.Group B consists of 114 consecutive patients having been treated in the years 1983-1992 (data in brackets).All patients suffered from squamous cell carcinoma of the oral cavity mainly in stages III and IV according to the Union Internationale contre le Cancer-(UICC)-definition, one patient had a local recurrence but had not been irradiated before. Patients with treated local or regional recurrences, distant metastases or with insufficient data were excluded. In group B, it seemed impossible to actualize the follow-up data, so that the data from the former analysis were taken. For this reason, no comparison of oncological results was attempted. In order to improve comparability, the inclusion criteria mentioned above were applied to both groups retrospectively which lead to the exclusion of several patients and a complete re-analysis of the data.70% of the patients in group A  had undergone surgery for the primary tumor and the regional lymph node regions. After surgery or after biopsy all patients were referred to the oral and maxillofacial surgeon for assessment of the dental status including an meticulous clinical and x-ray examination. The dental treatment procedures were performed as early as possible with a minimal time interval of 7-10 days from the last procedure to the beginning of radiotherapy. All dental extractions were performed according to a written protocol under “special care” . In the nineties all patients were advised not to wear their dental prostheses up to 6-12 months after radiotherapy   or three-dimensional dose distribution was computed after target volume delineation. Radiotherapy was applied using 4-6MV photons (electrons for level V lymph node region) of a linear accelerator; a 60-Cobalt machine was in use additionally until 1987. In the majority of patients , conventionally fractionated radiotherapy was applied with total doses of 60-70 Gy (details see Table 14(0) patients received chemotherapy consisting of cis-platinum and 5-FU simultaneously due to their unfavourable tumour and nodal stage. No patients with chemotherapy were excluded from the evalution. During therapy, the patients received dental care by the local dental colleagues. Fluoridation was used according to dental advice. Splints were not normally used because of unfavourable experience of patients with aggravating radiation mucositis by applying fluoride jelly to the gingiva using these splints.After radiotherapy, the patients were examined for locoregional result and possible side effects in the Department of Radiotherapy and Radiooncology. Dental follow-up was performed by their local dentists. Consequently, detailed data about this phase are not available. Patients with a suspicion of IORN were referred to the Dept. of Oral and Maxillofacial Surgery for further diagnosis and treatment.The mean duration of follow-up was 3.64 years .Infected osteoradionecrosis was minimally diagnosed when necrosis of the gingiva on top of the eroded bone became visible as infected mucosal ulcers with eroded mandibular bone underneath according to grade 2 or higher of the classification published by Schwartz et al.  examinations available have been reviewed, thus we are quite sure that a potential local recurrence has not been misdiagnosed as an IORN. Furthermore, standardized questionnaires were mailed to the patients’ general medical practitioners and general dentists as well as the local authorities five times within the observation period in order to get additional data about freedom of local or regional recurrence, survival or the onset of IORN.All data were entered into a medical databank . Frequency distributions, means and standard deviations were computed. The groups were compared using the t-test (means) and the Kruskal-Wallace test (distributions). Overall survival and occurrence of IORN over time were computed using the Kaplan-Meier estimate, the comparison of the groups was performed using the Mantel-Haenszel test. Prognostic parameters for IORN were analyzed univariately by comparison of means and distributions in a group containing the patients with IORN compared to another group with the patients who never experienced IORN using the t-test, u-test and chi-square test in the appropriate variables. Multivariate search for independent prognostic factors was performed by logistic regression.Detailed biographical and oncological data have been summarized in Table All patients had given their written informed consent before dental examination and treatment as well as radiotherapy. The approval by the local ethics committee was dispensable due to the retrospective nature of this evaluation. This research is in compliance with the Declaration of Helsinki in its actual version.In group A, up to July 2013, 58 patients were dead with a mean follow-up of 2.4 [0-8.8] years. The patients known to be alive were seen irregularly, the most recent information resulted from questionnaires, nearly all patients were lost to follow-up after on average 7.4 [0-15] years.In group B, 77/114 patients were dead with a mean follow-up of 3.4 [0-11.7] years. The patients known alive were lost to follow-up after on average 8.5 [4.3-13.3] years.The patients’ dental status was generally poor. On average 10.1 (10.8) teeth were found present at the time of initial dental examination. For most of the criteria there was no statistically significant difference between the groups. However, we found significantly more destroyed teeth  in the more recent patient collective, Furthermore there were more roots filled incompletely . However, avital teeth were found less frequently in group A . Chronic periodontitis with less to moderate attachment loss was found less frequently (p = 0.01) whereas chronic periodontitis with severe attachment loss was seen more frequently (p < 0.001) in group A compared to group B. Detailed data have been depicted in Table We can summarize that dental status in these special patients has hardly changed over the decades. Data concerning dental biofilm or the use of dental prostheses had not been collected in group B, thus a comparison could not be performed.In the majority of criteria, the extent of dental rehabilitation procedures was identical in both groups. Tooth extractions were found more frequently in group B  whereas conserving treatment was performed more frequently in Group A . Detailed data are summarized in Table IORN was found in the corpus region of the mandible in 11/90 patients (12%) of group A and 22/114 patients (19%) of group B (n.s.). The one-year prevalence was 5%, the two- and three-year prevalence 15%. A subgroup analysis dividing the collectives into two groups each with the patients having been treated with conventional fractionation or with hyperfractionation yielded the following results:After conventional fractionation IORN was found in group A in 11/74 patients (15%), in group B in 8/73 patients . After hyperfractionation, IORN was not diagnosed in group A wheras it was observed in 14/41 patients (34%) in group B (p = 0.01).The Kaplan-Meier estimate showed that IORN normally occurred in the first two years in group A (first five years in group B) after radiotherapy (differences n.s.), after that time the risk remained stable Figure . The subThe search for prognostically significant factors for the occurrence of IORN was performed using the whole patient collective consisting of 204 patients. The number of carious teeth, the N-stage, the total dose, the size of the daily fractions and the BED2  were found prognostically significant in univariate analysis. These factors were entered into the multivariate analysis where solely the number of carious teeth was found significant .In the literature, the incidence of IORN varied widely (0-74%) as depicted in Table Numerous prognostic factors for the development of IORN have been tested and published. A selection of these is summarized in Table Radiation dose should not exceed 60 – 66 Gy to the mandibular bone whenever possible, the target volume extending to the bone should be limited. Some authors regard hyperfractionation as a risk factor for IORN. In our ancient publication on this topic  has been found advantageous compared to conventional 3D-planned radiotherapy. Additional factors may be chemotherapy, higher body mass index and the use of steroids.An important paper has been published by Tsai et al. in 2013 (Tsai et al. Unfortunately, we did not succeed in identifying clearly significant independent prognostic factors for the development of IORN. In our patient collective, hyperfractionation seemed to have a protective effect whereas this could not be examined further due to the small number of events. In our dataset the number of carious teeth was found to be the only independent prognostic factor after multivariate analysis. Univariately, total dose and BED2 were significant which could be expected. In majority of patients, the total doses lie in a narrow range of 60-82 Gy which may have been a reason for the result, additionally the fact that few IORN cases have been observed.The authors are well aware of the limitations of this retrospective evaluation. In this nearly homogenous collective of patients with oral cavity cancer having undergone radiotherapy +/- surgery, we have found complete data sets with respect to the dental status and restoration procedures of nearly all patients. The IORN data have been investigated meticulously, but due to the known incompliance of head and neck patients we could not exclude that single events did not become known to the authors.The patients’ dental status before radiotherapy was very poor compared to an otherwise healthy population. Apparently we did not succeed in improving these findings over the decades despite all effort in terms of dental prophylaxis. Consequently, extensive dental rehabilitation procedures had to be performed which did not change over time as well.Examining patients irradiated with conventional fractionation, the incidence of IORN was found constant in a range of 10-15% over time. As stated earlier, the influence of the interfraction interval and of very high doses became known after the patients in group B had been irradiated, we thus diagnosed an unacceptably high frequency of IORN which became virtually zero after reduction of the total dose and extension of the interfraction interval. The multivariate search for prognostic factors only resulted in the assumption that dental status and fractionation could influence the occurrence of IORN.


Our purpose was to reveal the efficiency of diffusion weighted imaging (DWI) in the diagnosis of encephalitis, and to determine the relation between the apparent diffusion coefficient (ADC) values, the onset of the clinical symptoms, and the lesion extent.Conventional magnetic resonance imaging (MRI) was performed in 17 patients with encephalitis diagnosed on the basis of laboratory, clinical and radiologic findings during 2009 and 2015. Based on the duration between the onset of the symptoms and the brain MRI findings, the patients were divided into three groups. ADC values of the encephalitis lesion, the lesions’ topographic analysis score, deep gray matter involvement, patients’ clinical situation and the duration of the arrival to the clinic was examined.-3 mm2/s in group I (0-2 days), 1,045±0,347 x10-3 mm2/s in Group-II (3-7 days), 1,451±0,225 x10-3 mm2/s in Group-III (8 days and over). The relation between the ADC values and the duration of the arrival, topographic analysis score, the relation between the patients’ clinical situation and the deep gray matter involvement were found to be statistically significant. The deep gray matter involvement was demonstrated more clearly by FLAIR images when compared with DWI.Mean ADC values were 0,988±0,335 x10Conventional MRI sequences may be insufficient in showing the encephalitis lesion. DWI must be added to the imaging modalities immediately in the cases suspected of having encephalitis. Encephalitis is a life-threatening inflammatory disease of the brain which may be seen at any age. Various agents, as viral agents being in the first line, cause this disease.6Magnetic resonance imaging (MRI) is widely accepted as a sensitive imaging method used in detection of early changes in encephalitis. Routine MRI sequences include T1, T2 and Fluid Attenuated Inversion Recovery (FLAIR) images which are applied in order to define abnormal areas related with viral encephalitis. These sequences, however, may remain insufficient in demonstrating encephalitic lesions at very early stages.8Diffusion-weighted imaging (DWI) is ever-increasingly used in various diseases, mostly in early diagnosis of cerebral ischemia; however its role in other conditions like infections is in phase of discovery.Patients which came to department of radiology in our hospital with the pre-diagnosis of encephalitis were enrolled in the study. MRI work-up with standard sequences were performed to these patients between November 2009 and April 2015. Approval from ethics committee of the university was received for the study (2015/09-234).Diagnosis of encephalitis was reached through evaluation of laboratory analyses of blood and cerebrospinal fluid (CSF), culture results, clinical and radiological findings of the patients by the neurology and infectious diseases departments of our hospital. Cases in which ADC and FLAIR sequence images were missing and images were obtained through inappropriate methods were excluded from the study.2 of ROI corresponding to 1 cm2 of area, regardless of white and gray matters  onto areas with signal alteration as 2-3 mm matters . ApproxiPatients were grouped in the consideration of the time from being symptomatic to cranial MRI work-up. Those patients for which this duration was between 0-2 days were classified as Group- I, ones for which this duration was between 3-7 days were classified as Group-II and patients for which this duration was 8 or more were classified as Group-III.For areas with hemorrhage, ROI was not placed and this area was carefully excluded from the area of interest. Involvement of deep brain structures were recorded separately. Follow-up MRI images of the patients were examined and ADC values were recorded. Follow-up MRI images of four patients could not be found in the archive. One patient in group I did not have FLAIR sequence and he was excluded from evaluation. Evaluation of clinical presentations of patients was based upon status of consciousness. Clinical presentations of patients without deterioration of consciousness were classified as “fine” and clinical presentations of patients with deterioration of consciousness were classified as “moderate-poor”.The relationship among mean ADC values and topographical analysis scores at the admission of the patients with admission time, extensiveness of parenchymal involvement, deep gray matter involvement and clinical findings was examined.Statistical analyses of obtained data were performed using SPSS 18 program. In analysis of obtained data, along with descriptive statistics, relationship between initial ADC values and the involvement of brain parenchyma were analysed using Spearman’s Rho correlation test. Relationship among mean ADC values and the groups was analysed using Kruskal Wallis test. Since the relationship among groups was found to be statistically significant, relationship among groups was examined with post hoc (Conover-Iman test of multiple comparison) test. Relationship between admission time and involvement of brain parenchyma was examined with Man Whitney U test. The relationship between topographical analysis score and clinical presentation was examined with Kruskal Wallis test. P<0.05 was considered as significant.A total of 17 patients whose ages ranged from 0 to 85 years old with a mean age of 37 were evaluated. According to the gender of the patients 15 (88.2%) were male and 2 (11.8%) were female. When patients were classified for admission time; there were 5 patients in Group-I (0-2 days), 6 patients in each of Group-II (3-7 days) and Group-III (8 days and more) .Relationship among mean ADC values and groups was found to be significant (p<0.05). When the relationship among groups was considered; whereas no statistically significant difference was found between group I and II, a statistically significant difference was found between group 2 and 3 (p=0.042). Although there was an obvious difference in ADC values between Group-1 and 3, it was not found to be statistically significant due to low number of patients (p=0.085) . RelatioWhen the relationship between topographical analysis scores and clinical presentation of the patients was examined; topographical analysis score was found to be 9.37±6.69 in 8 patients with fine clinical presentation and to be 9.44±4.97 in 9 patients with moderate-poor clinical presentation. However no statistically significant difference was found (p>0.05). Relationship among clinical presentation of the patients and deep gray matter involvement was found to be statistically significant (p:0.009). All the patients with moderate-poor clinical presentation, but only three out of 11 patients with fine clinical presentation had deep gray matter involvement.Thirteen of 17 patients had follow-up MRI images from different time intervals. It was observed that in ADC measurements performed on follow-up MRI images were eventually increased .When topographical analysis scoring was performed on DWI and FLAIR images, no significant difference was observed in terms of lesional extensiveness, however 1 score difference was found in 4 patients due to the superiority of FLAIR sequences in thalamic involvement. However, lesions seemed to be different in DWI and FLAIR sequences according to the time in terms of resolution. In group I; whereas signal changes seemed more obvious in DWI, in one of the patients FLAIR images were superior to DWI, in 2 patients DWI and FLAIR have similar resolution. In Group-II; in 2 patients DWI and in the remaining 4 patients FLAIR had higher resolution. In Group-III; lesion seemed to be obvious in FLAIR images in all of 6 patients. Statistical analysis could not be performed due to limited number of the patients.Central nervous system infection represents itself with different symptoms which usually present as meningitis or encephalitis. Fate of the disease may range from a benign clinical presentation to encephalitis in which life-threatening neurological symptoms develop due to the factors specific to the host and organism.16Role of imaging methods in the management of encephalitis remains limited to confirmation of diagnosis and follow-up of complications. Imaging patterns demonstrate lesion extension, type of edema and frequently contribute to determine the underlying type of infection; however, clinical and laboratory analyses are required for characterization of infectious agents.6MRI is obviously superior to other radiological modalities in demonstrating brain parenchyma.12In the study conducted by Kiroglu et al. in which they quantitatively evaluated DWI and conventional MRI in determination of the encephalitic lesions in 18 patients, they found that DWI was superior to conventional series in early-stage encephalitic lesions and conventional MRI was superior to DWI in late-stage disease.Prakash et al. reported that T2 imaging was superior to DWI in two patients with encephalitis whose images were taken at 3-7 weeks after neurological symptoms were obvious.Tsuchiya et al. reported that some patients with encephalitis presented with extensive vasogenic edema in acute stage and FLAIR imaging was equal or superior to DWI in these patients.In the study of Renard et al. in which they compared DWI and FLAIR imaging in initial and follow-up MRIs of 11 patients with HSE, topographical analysis scoring was applied, as in our study.We had limited number of patients. Due to the retrospective design of the study, encephalitis diagnosis was based on clinical, laboratory, CSF and radiological findings. Advanced microbiological examinations such as polymerase chain reaction (PCR) analysis may not be available in our country sometimes. Nevertheless, we are in thought of that our results will be a guide in approach to this destructive pathology that may be encountered in daily life at any time.Radiological imaging has to be considered for exclusion of an intracranial pathology in a patient that admits with presentation of acute encephalitis. Early diagnosis and rapid treatment are essential in this disease in order to reduce mortality and morbidity. Therefore, DWI must be added to the imaging modalities in the first touch of the cases suspected of having encephalitis.


Adequate levels of succinyl-coenzyme A (CoA) cannot be synthesized from methylmalonyl-CoA because of the decreased activity of the methylmalonyl-CoA mutase enzyme that uses Cbl as the cofactor. Succinyl-CoA synthesis deficiency leads to decreased heme synthesis and gluconeogenesis. The reason of growth retardation can be gluconeogenesis deficiency together with heme synthesis deficiency whereas the reason of the neurological abnormalities can be glycine increase in the tissue due to decreased heme synthesis. We present 7 infants diagnosed with severe nutritional Cbl deficiency and discuss the role of succinyl-CoA and glycine in the possible pathogenesis in this article. Patients brought to our clinic with a complaint of growth retardation and diagnosed with nutritional Cbl deficiency were included in the study. There were 5 females and 2 males. The mean age was 11 ± 2.30 (range 6–13) months. All patients had general muscular hypotonia and 4 had growth retardation. Neuromotor growth retardation was found in 4 of the children who had previously shown normal neuromotor development for age. The mean Cbl level was 83.8 ± 27.6 (45.6–114) pg/mL. The mean Cbl level of the mothers was 155 ± 56.6 (88–258) pg/mL. Six of the patients had anemia and 1 had thrombocytopenia. Mean corpuscular volume value was 91.5 ± 12.2 fL. Following treatment, the muscle tonus of the patients improved, the anemia and growth retardation decreased, and the lost neuromotor abilities were recovered. Severe nutritional Cbl deficiency is an important nutritional disease where complications can be prevented with early treatment. When evaluating the pathogenesis, it should be noted that nutritional Cbl deficiency is a succinyl-CoA synthesis deficiency.Vitamin B The most common reason of Cbl deficiency in infancy is being fed only with milk of a mother with Cbl deficiency.1 Nutritional Cbl deficiency usually starts in infants aged 6 to 12 months with vomiting, lethargy, tremors, abnormal movements, growth retardation, hypotonia, and loss of developmental skills. There may be increased methylmalonic acid and homocysteine in the blood and sometimes hyperglycinuria.4 Methylmalonic acidemia (MMA) results from deficiency of either methylmalonyl-CoA mutase (MCM) or defects in the production of adenosylcobalamin (AdoCbl). Deficiency of Cbl, a cofactor for MCM, also produces signs and symptoms consistent with MMA. Other biochemically closely related disorders are the Cbl defects, classified as forms A through G. Clinical signs and symptoms include failure to thrive, metabolic acidosis, persistent ketotic episodes, hypoglycemia, hypotonia, hyperammonemia, and neurologic symptoms. Increased methylmalonic acid, homocysteine, and glycine may be present in the blood and urine.5Vitamin B8 Clinical signs and symptoms are similar in severe nutritional Cbl deficiency and MMA. Hyperglycinurina is seen in both cases due to the same enzymes/cofactors whereas the hyperglycinemia seen in MMA is also expected in severe nutritional Cbl deficiency. Glycine and succinyl-CoA are combined with a reaction catalyzed by δ-amino levulinic acid (ALA) synthase and δ-ALA is formed.9 The synthesis of succinyl-CoA from methylmalonyl-CoA decreases because of the level of MCM enzyme activity to (enzyme/AdoCbl) Cbl deficiency.11 The insufficient synthesis of succinyl-CoA cannot fully compensate for the sufficient synthesis of glycine and can lead to insufficient heme synthesis. Therefore, the glycine that cannot combine and react with succinyl-CoA and that possibly increases above the elimination capacity can deposit in the tissues.Although hyperglycinemia and hyperglycinuria are frequently reported in MMA, only hyperglycinuria is reported in severe nutritional Cbl deficiency.12 Cbl deficiency causes a severe growth retardation and various metabolic disorders in animals.13 Growth retardation is commonly reported in nutritional Cbl deficiency.16 However, there is little emphasis on the reason(s). Iron or Cbl stores are consumed approximately in 6 months, with nutritional anemia possibly developing afterward, in infants who have mothers with iron and Cbl deficiency and are only fed breast milk.19 Although rapid growth increases the need for iron and causes iron deficiency anemia in infants, Cbl deficiency in the same period can slow the growth rate and cause growth retardation and megaloblastic anemia.17 With regard to the contrasting effect on growth of these 2 causes of anemia, the insufficient energy (glucose) production as a result of the gluconeogenesis deficiency could be a factor increasing growth retardation in addition to the effect of the Cbl deficiency anemia.12Cbl plays an essential role in mitochondrial energy production (gluconeogenesis) and cellular functions.18 Although there are a large number of publications on abnormal nervous system findings in nutritional Cbl deficiency, only a few publications address the role of glycine in the pathogenesis of these abnormalities.20The increase of glycine in tissues such as the central nervous system (CNS) due to insufficiency of succinyl-CoA may lead to the emergence of abnormal nervous system findings.We present 7 infants diagnosed with severe nutritional Cbl deficiency who suffered from growth retardation and general muscle hypotonia and had lost acquired neuromotor skills, and discuss the role of succinyl-CoA and glycine in the possible pathogenesis in this article.21 Iron and folic acid were not administered. We did not check MMA and homocysteine levels in the patients and therefore confirmed our diagnosis by comparing the laboratory values at the time of diagnosis and 1 year later (after treatment).Kars is a province in the Northeastern Anatolia region of Turkey and is about 1800 to 2000 m above the sea level. It is relatively undeveloped socially and financially. The main source of income is agriculture and livestock. The patients had come from various villages of Kars. This study was designed as a retrospective trial. For that reason, we took a permission in order to scan the patients’ files from our institutional committee. Patients who were brought to our clinic for growth retardation between 2006 and 2013 and were diagnosed with nutritional Cbl deficiency were included in the study. The age ranged from 6 to 13 months. All patients were evaluated with a full history, and physical and neurological examination. The Cbl, folic acid, complete blood count, peripheral blood smear, serum iron (SI), serum iron-binding capacity (SIBC), ferritin, total bilirubin (TB) and direct bilirubin (DB), lactate dehydrogenase (LDH) levels of the patients, and the Cbl and folic acid levels of the mothers were studied. Megaloblastic anemia related to Cbl deficiency was diagnosed with a combination of clinical and laboratory findings, including low Cbl level, neutrophil hypersegmentation, and increased mean corpuscular volume (MCV) (macrocytosis). Growth retardation was described as weight under 3% for the patient's age. Neuromotor growth retardation was described as a general slowing of mental and physical activity and a fundamental failure to reach the developmental stages. The loss of acquired neuromotor skills was described as the inability to hold the head straight, inability to sit without support, inability to crawl, and inability to walk in children who had previously shown normal neuromotor development for their age. Cbl treatment was administered intramuscularly at a dose of 100 μg/d for 1 week, followed by every 2 days for 1 week, 3 days a week for 1 week, once a week for 1 week, and then 1000 μg once a month for 6 months.There were 5 females and 2 males. The mean age was 11 ± 2.30 (range 6–13) months. All patients were fed breast milk. The families of the patients were from a low socioeconomic level. The mothers of all patients had an inadequate intake of animal products. Length, head circumference, and body weight were under 3% in 4 patients and between 3% and 10% in 3 patients according to age. All patients had loss of appetite, pallor, and general muscular hypotonia. Four had loss of deep tendon reflexes. Four children had previously shown normal neuromotor development for age but were now unable to hold the head straight, sit without support, crawl, or walk  and CoA is hydrolyzed. Succinyl-CoA and glycine are also combined by δ-ALA synthase to form δ-ALA for the synthesis of porphyrin (heme), ensuring the homeostasis of succinyl-CoA.d-methylmalonyl-CoA. d-methylmalonyl-CoA is then converted to l-methylmalonyl-CoA by methylmalonyl-CoA racemase. l-methylmalonyl-CoA is converted into succinyl-CoA molecules with the MCM enzyme for which the cofactor is AdoCbl.25Gluconeogenesis is the process of the synthesis of glycogen or glucose from noncarbohydrate precursors. The major substrates are the glycogenic amino acids, lactate, glycerol, and propionate. The liver and kidneys are the major glucogenic tissues, but the small intestines can also be a source of glucose in the fasting state. Glycogenic amino acids are converted to pyruvate or citric acid cycle intermediate metabolites after transamination or deamination. Gluconeogenesis reactions are therefore responsible for the conversion of both lactate and glycogenic amino acids into glucose or glycogen. Propionic acid is the major precursor of glucose in ruminants. Propionic acid is a relatively minor precursor of glucose in humans. Propionic acid is an intermediate metabolite of isoleucine, valine, threonine, methionine, single-chain fatty acids, and cholesterol catabolism. It is also synthesized by intestinal bacteria. Propionic acid enters gluconeogenesis with the citric acid cycle. Propionyl-CoA is formed after the esterification of propionic acid and CoA by acyl-CoA synthase. Propionyl-CoA becomes carboxylated with a reaction catalyzed by biotin-dependent propionyl-CoA to create cblA to cblH; cblA, cblH, and cblB cause MMA. The cblC, cblD, and cblF defects cause homocystinuria in addition to MMA as both AdoCbl and methylcobalamin syntheses are disturbed. The cblE and cblG defects only involve methylcobalamin synthesis and cause homocystinuria without MMA. Megaloblastic anemia is usually present with these defects. The serum Cbl and folic acid levels are normal.26The Cbl in food enters cells as hydroxycobalamin. It is processed in the lysosomes and metabolized into the methionine synthase cofactor methylcobalamin or the methlmalonyl-CoA mutase cofactor AdoCbl. Patients with a Cbl metabolism disorder can develop homocystinuria alone, methylmalonic aciduria alone, or homocystinuria and methylmalonic aciduria in combination instead of a metabolic block. At least 8 different defects in the intracellular metabolism of Cbl have been defined. These are 27A decrease in this enzyme's activity can frequently cause feeding disorders, lethargy, developmental delay, and megaloblastic anemia in the patients. Neurological problems and macrocytic anemia have been reported in adult patients. The serum Cbl and folic acid levels are normal.0” form. There is a structural defect in the enzyme but some residual enzyme activity in the “MUT−” form.28 The deficiency of MCM or its cofactor AdoCbl leads to MMA. Disorder in MCM activity leads to methylmalonyl-CoA accumulation and this in turn causes an increase in propionyl-CoA and its metabolites by suppressing propionyl-CoA carboxylase, whereas both conditions cause methylmalonic and propionic acid accumulation in the blood and excretion through the urine. Laboratory findings are ketosis, acidosis, anemia, neutropenia, thrombocytopenia, hyperglycinemia, hyperammonemia, hypoglycemia, and excess methylmalonic acid in all body fluids. Propionic acid and its metabolites 3-hydroxypropionate and methylcitrate are also present in urine.29 The synthesis of succinyl-CoA from methylmalonyl-CoA decreases because of the level of MCM enzyme activity to (enzyme/AdoCbl) in nutritional Cbl deficiency.11 The inadequate synthesis of succinyl-CoA cannot fully meet the adequate synthesis of glycine and may result in inadequate heme synthesis. We believe that the glycine that cannot combine and react with succinyl-CoA probably increases over the elimination capacity  and deposits in the tissues.There are many clinical pictures from very severely affected newborns to asymptomatic adults, independent of the nature of the enzymatic defect or the biochemical abnormality. Isolated MMA develops as a result of mutations in genes related to MMA, the MUT locus that encodes the MCM apoenzyme, or AdoCbl that is the cofactor of this enzyme. Two types of MCM deficiency are recognized. There is no enzyme activity in the “MUT30 Growth retardation  is therefore observed as the first and striking finding in MMA, an energy production disorder.29Growth is regulated by genetic factors as well as hormones, tissue-specific growth factors, nutrition, and the interaction of many other internal and external environment factors. Normal growth can only be achieved by adequate nutrition. More than 10% of the energy received from food is spent for growth during rapid growth periods. Protein intake is also important for normal cell growth. A child organism where the structural and energy-providing materials are not adequate first tries to survive by stopping growth and development.16 A study has reported plasma methylmalonic acid and homocysteine levels 2 to 8 times of control values that were inversely associated with the significantly low Cbl levels in 41 infants on a macrobiotic diet. Although there was no neurological or hematological deficiency, these children were reported to show psychomotor development disorders and growth retardation when compared with the control group children who received a balanced diet.31 The mean values of all anthropometric parameters were low in infants aged 4 to 18 months of mothers on a macrobiotic diet in another study. Weight and arm circumference growth rate have been found to be independent of the protein content of the macrobiotic diet and the energy intake. Growth has been reported to be positively associated with the protein content of the diet but not with energy intake.32 The presence of severe growth retardation  in almost all the patients in our study, improvement after treatment, and catching up with normal growth and development values within the first year of treatment can be explained with the effects of Cbl at the molecular level.Growth retardation is reported as the most striking clinical finding in infants with nutritional Cbl deficiency.9 Inadequate heme/HGB synthesis (the effect of chronic anemia) leads to inadequate oxygenation (hypoxia), which, in turn, causes growth retardation with inadequate function in many tissues. The severe growth retardation observed in Cbl deficiency cannot only be explained by chronic anemia. Iron-deficiency anemia usually occurs as a result of rapid growth in infants fed only breast milk, and usually does not cause severe growth retardation. However, the cause of severe Cbl deficiency is nutritional deficiency and not rapid growth. Megaloblastic anemia and severe growth retardation are often observed as a result of severe Cbl deficiency.16 We therefore believe that the reason of the growth retardation in Cbl deficiency could be other factors, in addition to the anemia, that cause growth retardation, such as gluconeogenesis deficiency. Cbl deficiency leads to decreased/inadequate succinyl-CoA synthesis. This decrease leads to less energy (glucose) production by the citric acid cycle.12 The reason of the growth retardation could be gluconeogenesis deficiency together with heme synthesis deficiency. The presence of severe growth retardation and hypoglycemia in MMA, and the positive correlation of growth with the protein (and Cbl) content of the diet but not with energy intake may be further proof of a role for gluconeogenesis deficiency.33Insufficient synthesis of heme-containing proteins, primarily HGB, and insufficient gluconeogenesis may cause growth retardation because of insufficient succinyl-CoA production in Cbl deficiency. Heme-containing proteins  are synthesized in all cells except mature erythrocytes. Most of the heme synthesis in the body takes place in the bone marrow erythroid cells and most of the remaining part is in the liver hepatocytes and other heme-synthesizing cells.N5-methyltetrahydrofolate as the methyl (CH3) donor in common bacteria. Another form of the enzyme found in some bacteria and mammalians uses N5-methyltetrahydrofolate but first transfers the CH3 group to Cbl as the CH3 donor to produce methylcobalamin during methionine production. Methylcobalamin receives the CH3 groups from N5-methyltetrahydrofolate and this is the only reaction that uses N5-methyltetrahydrofolate in mammalians. The reaction that converts the N5,N10-methylene form into the N5-methyltetrahydrofolate form  is irreversible. The metabolic folates therefore become trapped in the N5-methyl form if the coenzyme Cbl is not available for methylcobalamin synthesis. Megaloblastic anemia develops for the same reasons in folate deficiency where all forms of tetrahydrofolate are decreased.34A form of methionin synthase uses 21 The problem in the development of erythroid precursors is the result of the direct consumption of N5,N10-methylenetetrahydrofolate. Thymidylate synthase is used as a C1-donor for the synthesis of thymidine nucleotides in the biosynthesis of the folate cofactor N5,N10-methylenetetrahydrofolate during DNA biosynthesis. A methylene group (–CH2–) bound to tetrahydrofolate is used by the thymidylate synthase enzyme to convert the uracil-type pyrimidine base in RNA (deoxyuridine monophosphate) to a thymine-type base in DNA (deoxythymidine monophosphate). Cbl deficiency that causes pseudofolate and folate deficiency decreases deoxythymidine triphosphate synthesis  and DNA with high uracil content forms after misinsertion of uracil into DNA during replication.36Erythroid precursors have increased RNA content but normal DNA content. The amount of RNA per unit of erythroid precursor DNA is increased and the cell is therefore larger than a normal cell at the same level of maturation. The nuclear chromatin of these cells appears looser than normal on the peripheral smear. This gives the megaloblasts their characteristic appearance. The maturation of the nucleus and cytoplasm is asynchronous and the nucleus is less mature than the cytoplasm.36 Treatment makes the coenzyme Cbl available for methylcobalamin synthesis and the metabolic folates are no longer trapped in their N5-methyl form. They are converted into tetrahydrofolate, restarting the production of N5,N10-methylenetetrahydrofolate. Thymidylate synthase uses the folate cofactor N5,N10-methylenetetrahydrofolate for the resynthesis of thymidine nucleotides (DNA synthesis). Cell division starts to increase again and this may cause the megaloblastic changes in the bone marrow to recover.Decreased Cbl (pseudofolate)/folate leads to decreased purine and pyrimidine biosynthesis and therefore decreased DNA biosynthesis and cell division. This is reflected in the decreased number of erythrocytes, causing anemia in the most direct manner.37 One of these other factors may be the inadequate synthesis of the succinyl-CoA required for heme synthesis.However, the disturbed de novo synthesis of thymidine nucleotides may partially explain the megaloblastic anemia. Observations in animal models and nonanemic patients with Cbl deficiency have shown that thymidylate synthase deficiency by itself is not enough to cause anemia. There may be other contributing factors.21 Although our patient number is inadequate  was less than half of the level at diagnosis (89.5 ± 37.4 before treatment). This decrease in the serum ferritin level may indicate that the iron required for heme synthesis is not utilized adequately.A “positive therapeutic test” criteria for Cbl deficiency diagnosis is a 50% decrease in SI and LDH levels.39Cbl is primarily required for hematopoiesis and nervous system development. Cbl deficiency in adults can be seen as megaloblastic anemia, polyneuropathy, subacute combined neurodegeneration of the spinal cord, dementia, or depression. Although a clinical disorder due to Cbl deficiency develops slowly over months or years in the mature (adult) nervous system, significant clinical problems can develop within a couple of weeks in infants with rapid cerebral growth and development. The most commonly seen symptoms are growth retardation, hypotonia, hyperirritability or lethargy, neuromotor delay or retardation, epilepsy, and abnormal movements.41 The level of methylmalonic acid increases at CSF in the deficiency of Cbl. Because of this reason, the level of glycine is expected to increase in the CNS.The cerebrospinal fluid (CSF) methylmalonic acid concentration had increased (the mean level was 600 times that of controls) in a disproportionate manner to the serum values in 3 patients with neuropsychiatric syndrome. However, the potential benefit of CSF metabolite levels in the diagnosis of Cbl deficiency has not been determined.2Urinary concentrations of methylmalonic acid and homocysteine are typically high in Cbl deficiency. Hyperglycinuria is occasionally present. There are 2 reports on infants with Cbl deficiency showing abnormal movements after the start of the treatment. Methylmalonic acid and homocysteine were reported to disappear quickly after the start of treatment in 2 infants where abnormal movements were present but the hyperglycinuria that was present at the beginning of the abnormal movements continued.43 This indicates the possible presence of other catabolism/elimination routes outside the CNS other than the cleavage enzyme complex. The increase of glycine primarily in the nervous system due to Cbl deficiency, even if at the cell and tissue level and not in the blood, can explain the abnormal clinical findings stemming from the nervous system.The fact that glycine does not always increase in the blood and urine such as methylmalonic acid and homocysteine can be related to the cleavage enzyme complex or other metabolic pathways that can catabolize/eliminate glycine easily. Although glycine cannot pass the blood–brain barrier easily, the glycine increase in the CSF in nonketotic hyperglycinemia is almost 2 to 4 times the increase in the serum.A receptors. Strychnine antagonizes the effect of glycine not at the glycine receptor level but at the level of the chloride channel. Some glycine-binding sites on the glutamate N-methyl-d-aspartate (NMDA) receptors that are chloride channel locked cannot be blocked with strychnine. These are called glycineB receptors.46Glycine is an important excitatory neurotransmitter released from the inhibitory interneurons in the spinal cord and brain stem, and is an agonist of the glycine receptors. A large part of the interneurons in the spinal cord gray matter consists of glycinergic neurons whereas a smaller part is Gamma Amino Butyric Acid (GABA)ergic neurons. Glycine is effective on 2 different receptors in the CNS. The glycine receptor consists of α, β, and other subunits; it has a chloride channel in the middle such as the GABA receptor. The receptors clamped to the chloride channel are called glycineA) are mainly found in the spinal cord, brainstem, and retina.45 Stimulation of the glycine inhibitor receptor causes lethargy and respiratory depression.48 The most common neurological findings in infants with severe Cbl deficiency have been reported as hypotonia, growth retardation, and the loss of acquired neuromotor skills.49 All our patients had hypotonia and our study was consistent with the literature in this regard. Although we failed to show glycine elevation in our study, the presence of hypotonia before the treatment, its improvement with treatment, and the lack of any sequel indicates the presence of a functional inhibitory neurotransmitter such as glycine. The reason for the neurological findings could be the inhibitory effect of the increasing glycine level on glycinergic neurons (stimulating glycineA receptors), although at the cellular level, before treatment and the disappearance of the inhibitory effect due to the decreased glycine level after the treatment. In addition, a large part of the interneurons in the spinal cord gray matter consists of glycinergic neurons that are especially responsible for the glycinergic inhibitory neurotransmission (with α3β glycine receptors) in the dorsal horn of the spinal cord.45 The subacute combined neurodegeneration in the posterolateral spinal cord of adult patients with Cbl deficiency indicates that the glycinergic neurons that we believe glycine causes hypotonia through (stimulating glycineA receptors) and the neurons where subacute combined neurodegeneration are observed could be similar neurons.Inhibitor glycine receptors (glycine+/Cl− dependent and is expressed in the glia. GlyT2 transporter is expressed in the neurons and recovers the released glycine from the synaptic gap. Glycine concentration in inhibitory neurons is higher than the glycine concentration in other parts of the CNS (2–3 times difference). Glycinergic inhibitory neurotransmission ends with recovery of GlyT2 into neuronal terminals and GlyT1 into glia. GlyT1 also prevents the saturation of the glycine-binding site over the NMDA receptors.42 The stimulation of NMDA receptors with glycine leads to hyperirritability and an inability to perform voluntary movements.48 The excessive stimulation of the NMDA receptor usually also contributes to the neurological damage related to nonketotic hyperglycinemia.50 Abnormal or severe involuntary movements accompanied by tremor and myoclonus, twitching, and chorea affecting particularly the tongue, face, pharynx, and legs can be seen in about half of the infants with Cbl deficiency. These movements may improve before or a few days after the start of the treatment. The duration of the movements change between 10 and 30 days.52 All our patients had neuromotor growth retardation and most of them had lost their acquired neuromotor skills. We believe that the presence of neuromotor development delay and acquired neuromotor ability loss before the treatment and their improvement with treatment, generally without any sequelae, indicates a role for a functional inhibitory neurotransmitter such as glycine. The stimulation of glycineB receptors by glycine may be responsible for the abnormal movements before treatment as the increased glycine at the cellular level leads to an inability of GlyT1 to saturate the glycine-binding area through the NMDA receptor. Normal functioning of glycinergic neurons is necessary to provide a balance between the excitatory and inhibitory activity in the integrative centers in the spinal cord that regulate skeletal muscle contractions. We believe that the reason of the abnormal movements or convulsions is the temporary imbalance brought by the rearrangement of glycineA/glycineB receptors and GlyT1/GlyT2 together with the decrease of glycine at the cellular level. Glycine is the coagonist of glutamate in glutamatergic NMDA receptors in the brain and may therefore have both excitatory and inhibitory functions and be the cause of abnormal movements or convulsions.Glycine is also the coagonist of glutamate in glutamatergic NMDA receptors in the brain  and therefore has both excitatory and inhibitory functions. Glycine transporters (GlyT) eliminate glycine from the synaptic cleft of the glycinergic inhibitory neurons in synapses containing glycine receptors in the CNS and this process maintains the low glycine extracellular concentration necessary to saturate the glycine site over the glutaminergic NMDA receptors. The human GlyT1 transporter is NaN5,N10-methylenetetrahydrofolate) used for thymidylate synthesis can alternatively also be channeled into the “methylation cycle.” This ensures adequate S-adenosylmethionine for the cell at all times. S-adenosylmethionine is a methionin-activated form that acts as a CH3 donor for many methyltransferase enzymes. These enzymes add a CH3 group to a wide range of substrates such as lipids, hormones, proteins, and DNA. One of these important methylations concerns myelin basic protein. This protein constitutes a third of the myelin cover of nerves, providing insulation. The methylation cycle is interrupted in Cbl deficiency and causes nerve demyelinization.35The folate form  can be a reason of growth retardation. In addition, the cause of the neurological abnormalities can be increased glycine in tissues due to the heme synthesis (succinyl-CoA) deficiency.Despite the limitations of the inadequate number of patients and the inability to measure methylmalonic acid, homocysteine, and glycine levels in our study, we think that it is important to systematically summarize the reasons of growth retardation, neuromotor skill loss, and hyperglycinemia even at the hypothesis level in light of essential information and believe that our study will shed light on future studies.In conclusion, nutritional Cbl deficiency may be observed in various clinical forms from asymptomatic disease to severe disease according to the Cbl level. Severe Cbl deficiency is an important nutritional disorder and the complications can be prevented with early treatment. When evaluating the pathogenesis of the disorder, it should be noted that nutritional Cbl deficiency is a succinyl-CoA synthesis deficiency.


For the past 30 years, many communities and countries in sub-Saharan Africa have suffered from the ravages of the HIV epidemic. During this time the mortality rates in HIV-infected people have been 10–20 times higher than in HIV-uninfected people , 5.This supplement puts together evidence from different sources in order to estimate the impact of ART on adult HIV mortality. The papers in this supplement use different measures to estimate the effect of ART, and the stories from the different papers provide a consistent picture of the reduction in HIV-related mortality with the advent of ART in sub-Saharan Africa. However, in developed countries the mortality in HIV-positive people on ART is similar to the mortality in the general population , but basThe AIDS impact module within the Spectrum software is a modeling tool for estimating many facets of the HIV epidemic in the population, usually using HIV prevalence and program data available at the national level . We can The paper by Kanjala et al. documents the changes in age-specific mortality rates (ASMR) from 1994 to 2010 in a cohort in Tanzania . This stFour papers in this supplement reported the use of InterVA-4 to interpret the cause of death from VA data. Byass et al. validated the InterVA-4 model against pre-mortem serological data from six ALPHA sites . This shTwo papers used Spectrum model estimates and showed good agreement with empirical data in Kenya and Tanzania , 13. Oti3, but new guidelines in 2013 recommend initiation of ART in all those with CD4 counts less than 500 cells per mm3 , and 2.5 times higher than the general population. The excess HIV mortality in those with CD4 counts between 350 and 499 cells per mm3 would be prevented with the implementation of the WHO guidelines for people living with HIV in developing countries.Most countries have now adopted the 2010 WHO guidelines to initiate ART for those with HIV infection and CD4 counts under 350 cells per mm per mm3 . Masiirapulation . MortaliThe final paper in the supplement, by Levira et al., looks at a different impact that ART may have on mortality . Many peThe message from the papers in this supplement across the first decade of ART roll-out in sub-Saharan Africa shows a consistent reduction of overall mortality rates in the population of around 30%, with the proportion of deaths attributable to HIV falling by 30–50%. In this period, excess mortality among HIV-positive adults has halved, but the mortality rates in HIV positives are still up to 10 times higher than among HIV negatives . In the Jim ToddEmma SlaymakerBasia Zaba London School of Hygiene and Tropical Medicine, Keppel Street WC1E 7HT, London, UKMary Mahy Epidemiology Section, UNAIDS, Geneva Umeå Centre for Global Health Research Department of PublicPeter Byass Health and Clinical Medicine, Umeå University Umeå, Sweden


SRY gene by RT-PCR. Two hundred and twenty six pregnancies have been tested so far in these 5 years. Neither false positives nor false negatives diagnoses have been registered, thus giving a diagnostic accuracy of 100%.Since the discovery of the presence of fetal DNA in maternal blood, non-invasive fetal sex determination has been the test most widely translated into clinical practice. To date there is no agreement between the different laboratories performing such tests in relation to which is the best protocol. As a consequence there are almost as many protocols as laboratories offering the service, using different methodologies and thus obtaining different diagnostic accuracies. By the end of 2007, after a validation study performed in 316 maternal samples collected between the 5th and 12th week of gestation, the fetal sex determination was incorporated into clinical practice in our Service. The test is performed in the first trimester of pregnancy, and it is offered as part of the genetic counseling process for couples at risk of X-linked disorders. As a general rule and in order to avoid misdiagnosis, two samples at different gestational ages are tested per patient. The analysis is performed by the study of the  Lo’s discovery in 1997 of the presence of cell-free fetal DNA circulating (ccffDNA) in maternal blood by means of the detection of Y chromosome sequences in male bearing pregnancies was a milestone not only for the discovery itself, but also because it opened the door to the first non-invasive diagnosis that has been incorporated into clinical practice ,3,4. Fetal sexing is nowadays performed by many laboratories worldwide and it is one of the crucial steps in the management of pregnancies at risk of an X-linked disorder, since it may mean avoiding invasive prenatal diagnosis (PD) . In factSRY gene by RT-PCR. Two hundred and twenty six pregnancies have been tested so far in these 5 years. Neither false positives nor false negatives diagnoses have been registered, thus giving a diagnostic accuracy of 100%.By the end of 2007, after a validation study performed in 316 maternal samples collected between the 5th and 12th weeks of gestation, the non-invasive fetal sex determination was incorporated into clinical practice in our Service [From September 2007 to December 2012, 226 pregnant women have been tested, comprising a total of 30 different X-linked disorders ; Table 1Twenty milliliters of maternal peripheral blood are collected in tubes with ethylene diamine tetra-acetic acid (EDTA) anticoagulant. Processing is preferably carried out by female personnel to avoid contamination with male genetic material. Specimens are centrifuged at 1,600 ×g for 10 min at room temperature in the original collection tube. Plasma is collected, divided in 1 mL aliquots and centrifuged at 13,000 ×g for 10 min. The supernatant is removed from each aliquot and placed in new collection tubes and stored at −20 °C. An aliquot of 1 mL of plasma sample is used for DNA extraction.Samples are thawed at room temperature and DNA is automatically extracted using the QIACube with QIAamp DNA blood Mini Kit  according to the protocol previously described . DNA is SRY gene). In addition, the glyceraldehyde phosphate dehydrogenase gene (GAPDH gene) is used as internal control for the DNA extraction process and polymerase chain reactions (PCR). Both targets are analysed independently in singleplex reactions to prevent outcompetition of the SRY amplification. Each sample is tested three times for the SRY gene and twice for the GAPDH gene. In each replicate, 8 μL of DNA extracted from the plasma sample is used. A sample of male genomic DNA and a female genomic DNA is included in the PCR as controls. For these samples, only 1 μL of genomic DNA is 115 used for the PCR amplification. All reactions are carried out in a final volume of 25 μL. Reactions for the SRY amplification contain 300 nM of the forward primer (SRY-F: 5'-GCGACCCATGAACGCATT-3'), 900 nM of the reverse primer (SRY-R: 5'-GCCATCTTGCGCCTCTGA-3'), 200 nM of the Taqman probe (SRY-P: 6-FAM CGTGTGGTCTCGCG-MGB) and 12.5 μL of Taqman Universal PCR Master Mix . Reactions for the GAPDH amplification contain 300 nM of each primer (forward primer GAPDH-F: 5'-CCCCACACACATGCACTTACC-3' and reverse primer GAPDH-R: 5'-CCTAGTCCCAGGGCTTTGATT-3') and 200 nM of the Taqman probe (GAPDH-R: VIC-AAAGAGCTAGGAAGGACAGGCAACTTGGC- TAMRA) and 12.5 μL of Taqman Universal PCR Master Mix (Applied Biosystems). Thermal cycling was initiated with 2 min incubation at 50 °C, followed by a first denaturation phase at 95 °C for 10 min and then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All probes and primers are supplied by Applied Biosystems. Amplification data are collected and analysed with an ABI Prism 7000 Sequence Detection System (Applied Biosystems).Fetal sex assessment is based on detecting the presence of the sex-determining region of the Y chromosome , only one replicate of the -SRY gene is allowed among replicates of both samples.for negative results , only one positive result for 139 the SRY replicated are allowed.In pregnancies tested once no discrepancies between the three Because some of the centers that were sending samples for the first time were not aware of the timing for sampling, in eight pregnancies the first sample was collected before the 7th week of gestation. In these cases a third sample was required to confirm the diagnosis. SRY and GAPDH genes were 35.91 (SD 1.20) and 30.31 (SD 1.81) respectively. A total of 111 males and 115 females were diagnosed. All female pregnancies (50%) avoided conventional prenatal diagnosis.The median gestational age for the first sample was 8 weeks (ranging from 7–9) and for the second sample was 10 weeks (ranging from 9–11). As described above, some samples were collected under exceptional circumstances (before the 7th week and after the 10th) and so do not fulfill these criteria. Only 4 out of the 226 pregnancies tested no confirmation after the test could be done due to a spontaneous miscarriage. All 4 were only tested once, resulting in 3 females and 1 male. The remaining 222 pregnancies were correctly diagnosed. One hundred and eighty one pregnancies were tested twice, 33 only tested once and 8 three times. The mean Ct for the Confirmation methods: Two different confirmation methods were used, conventional prenatal diagnosis and ultrasound scan.SRY gene in two samples per pregnant women, with a time lapse of 2 weeks between them, allows us to have a 100% accuracy in the diagnosis. This level of certainty has been pursued by many groups using different methodologies, diagnostic algorithms, scoring criteria [It is widely accepted that non-invasive fetal sex determination based on the identification of ccffDNA in maternal blood provides a safe alternative to conventional invasive genetic studies of the fetus. Here we show how a simple protocol that implies the study of the criteria ,16,17,18criteria . HoweverDYS14 and SRY polymerase chain reaction is the analysis most commonly used [DYS14 is firstly tested due to its highest sensitivity and those cases that tested positive are afterwards tested for SRY for confirmation to avoid false positives. However, evidence of positive results in female bearing pregnancies are reported and for this reason some groups have introduced another marker to the algorithm [et al. [et al. [AMELX/Y and SRY genes. The study compares the sensitivity and specificity obtained testing only for SRY, AMELX/Y and the combination of both. By combining the analysis of both genes they get the highest sensitivity and specificity but a false negative rate of 1.2%. Multiplex QF-PCR not only has the problem of competition between primers but also that it is less sensitive than RT-PCR. A small study performed in our laboratory in the plasma samples from the 1st trimester of pregnancy (data not shown) has demonstrated that the detection of the SRY gene can be done earlier in gestation by RT-PCR than with QF-PCR. In the absence of replicates for the SRY gene, and to make sure that the absence of Y chromosome is because the fetus is a female, other groups have designed new tests to prove the presence of fetal DNA in the maternal blood. The majority of them focus on the differences in methylation between fetal and maternal sequences [et al. [et al. [SRY gene and differences in methylation. They documented that an incomplete digestion of the RASSF1A sequences was observed in plasma samples and that the protocol has to be modified to test maternal plasma samples. Though both assays have identical reaction conditions and show good results, they still find unreportable levels of hypermethylated RASSF1A together with positive SRY replicates.A diagnostic algorithm combining the results of nly used ,11,18. Glgorithm . However [et al.  have recequences ,16 but oSRY gene  and that has shown to be 100% accurate. Recently, new reports looking for certainty in the confirmation of the presence of fetal DNA in the maternal blood are describing new diagnostic algorithms that imply many different tests. However, the test is getting less simple and more expensive. From a clinical laboratory perspective, incorporation of this study in the daily routine should be effortless. Besides, for this specific analysis, it is important to avoid too much handling to avoid the chance of contamination. Collection of maternal blood samples based on sonographic gestational age and study of two independent samples reduces the risk of misdiagnosis. This is especially important to consider for those samples collected at early gestational ages (before the 9th week). In fact, there were 8 pregnancies in which we had to ask for a third sample for an accurate result. These cases were coming from other centers and sonographic confirmation of gestational age could not be done beforehand.Here, we have reported a simple diagnostic flow for fetal sex assessment that uses an automated system for DNA extraction and only tests for the In conclusion, fetal sex assessment in maternal blood is at present an essential tool in the management of pregnancies at risk of X-linked disorders. This test has widely shown to be accurate from the 7th week of gestation. Although there is not a general consensus about the best algorithm to be followed, time consuming and expensive diagnostic approaches should be avoided in a clinical setting.


Early infant male circumcision (EIMC) conducted by nurse-midwives using the AccuCirc device proved safe, feasible, and acceptable to parents in Zimbabwe. The AccuCirc device has the potential to facilitate widespread scale-up of safe EIMC in sub-Saharan Africa. Early infant male circumcision (EIMC) conducted by nurse-midwives using the AccuCirc device proved safe, feasible, and acceptable to parents in Zimbabwe. The AccuCirc device has the potential to facilitate widespread scale-up of safe EIMC in sub-Saharan Africa. For prevention of HIV, early infant male circumcision (EIMC) needs to be scaled up in countries with high HIV prevalence. Routine EIMC will maintain the HIV prevention gains anticipated from current adult male circumcision initiatives. We present here the results of a field study of EIMC conducted in Zimbabwe.The study was observational and based on the World Health Organization (WHO) framework for clinical evaluation of male circumcision devices. We recruited parents of newborn male infants between August 2013 and July 2014 from 2 clinics. Nurse-midwives used the AccuCirc device to circumcise eligible infants. We followed participants for 14 days after EIMC. Outcome measures were EIMC safety, acceptability, and feasibility.We enrolled 500 male infants in the field study (uptake 11%). The infants were circumcised between 6 and 60 days postpartum. The procedure took a median of 17 minutes (interquartile range of 5 to 18 minutes). Mothers’ knowledge of male circumcision was extensive. Of the 498 mothers who completed the study questionnaire, 91% knew that male circumcision decreases the risk of HIV acquisition, and 83% correctly stated that this prevention is partial. Asked about their community’s perception of EIMC, 40% felt that EIMC will likely be viewed positively in their community; 13% said negatively; and 47% said the perception could be both ways. We observed 7 moderate or severe adverse events . All resolved without lasting effects. Nearly all mothers (99%) reported great satisfaction with the outcome, would recommend EIMC to other parents, and would circumcise their next sons.This first field study in sub-Saharan Africa of the AccuCirc device for EIMC demonstrated that EIMC conducted by nurse-midwives with this device is safe, feasible, and acceptable to parents. As the WHO “Framework for Clinical Evaluation of Devices for Male Circumcision”,In August and September 2013, doctors trained 4 study nurse-midwives over the course of 5 days to use the AccuCirc device. The trainers had previously performed circumcisions for the Zimbabwe EIMC comparative trial, described in detail elsewhere.Mothers and infants were enrolled between August 2013 and July 2014 at Edith Opperman and Mabvuku, 2 polyclinics in Harare, with 400 and 250 deliveries per month, respectively.We collected data on eligibility criteria from all mothers and infants. Maternal eligibility criteria were: (1) ability to attend follow-up appointments at the study clinic through 2 weeks postpartum, (2) readiness to provide a home address, and (3) written informed consent. Only infants ages 6 to 60 days were eligible. Although WHO guidelinesBefore enrollment, study staff performed a physical examination to exclude infants with abnormalities precluding circumcision. We referred to a specialist all 10 infants who were ineligible due to genital abnormalities. The 4 trained nurse-midwives (2 at each study clinic) performed all the circumcisions, assisted by 2 registered general nurses. To minimize bleeding, all infants received vitamin K (1 mg) an hour before the procedure. Additionally, all infants had approximately 1 gram of EMLA Cream  applied to the outer foreskin and shaft of the penis about 45 minutes before the procedure. Administration of vitamin K and the anesthetic is part of standard operating procedures for conducting EIMCs and not specific to the AccuCirc device.We assessed achievement of anesthetic effect by holding the foreskin using artery forceps. If there was no pain response from the infant, subsequent steps would commence. Otherwise, providers would wait until the EMLA Cream had achieved anesthetic effect . When the EMLA Cream had achieved anesthetic effect, the surgical area was cleaned with povidone-iodine. Physiologic adhesions between the foreskin and the glans were released by manual technique using the flexible foreskin probe that comes with the AccuCirc kit. The nurse-midwife then marked the circumcision site (around the corona) with a surgical pen that is also part of each AccuCirc kit. The pen mark helps to minimize excessive or insufficient skin removal.www.accucirc.com) and as recommended by the study previously conducted in Botswana.,,In addition to the flexible foreskin probe, the AccuCirc device consists of a shielding ring plus a single-action clamp  that contains a circular blade. It is available in 2 sizes: 1.1 cm and 1.3 cm (penile diameter).Follow-up appointments at the clinic took place at days 2, 7, and 14 after circumcision. At these visits, nurse-midwives asked parents about complications. The EIMC providers also conducted physical examinations of the infants, including inspection of the circumcision site. Seven days after the procedure we asked parents their views on the acceptability of circumcision.Outcome measures for the field study were (1) EIMC safety, (2) acceptability, and (3) feasibility. Safety was indicated by the number of moderate and severe adverse events (AEs). EIMC-related AEs were categorized as bleeding, infection, inadequate or excessive skin removal, or penile injury  . We reco,We asked parents to rate their satisfaction with and acceptability of the procedure on a numerical scale ranging from 0 (dissatisfaction) to 10 (very satisfied). Satisfaction was defined as a score between 6 and 10. We also asked parents to rate on a scale of 0 to 10 the likelihood that they would recommend EIMC to friends or relatives. A score of 0 was classified as “would not recommend” and 10 as “would definitely recommend.” Feasibility was defined as spending the least amount of effort on a procedure and gauged by the time that the nurse-midwives needed to perform EIMC using AccuCirc (timed using a stopwatch plus video recording of each procedure), and the ability of nurse-midwives to safely perform the procedure. We considered15 to 30 minutes a “feasible procedure time,” based on the typical time spent in previous studies of the AccuCirc device.We chose a sample size of 500 subjects, as specified by the WHO “Framework for Clinical Evaluation of Devices for Male Circumcision.”We used Stata 13  to perform statistical analyses. We summarized characteristics of parents and infants, including socio‐demographic characteristics, father’s reported circumcision status, and circumcision and HIV knowledge and attitudes. To evaluate the safety of the procedure, we calculated the proportion of procedures associated with AEs and the associated 95% CI. We calculated similar proportions and 95% CIs for the parental acceptability outcomes. To assess feasibility, we calculated the mean time for the procedure and an associated 95% CI.The Medical Research Council of Zimbabwe and the ethics committees of University College London and the London School of Hygiene and Tropical Medicine approved the study. We obtained written informed consent from the infant’s mother and verbal consent from the infant’s father (if available) before enrollment. After the procedure, mothers received a 20-liter plastic bucket, 2 bars of washing soap, a 100-ml bottle of Vaseline (for wound care), and 3 disposable diapers . At each scheduled clinic visit, mothers also received the equivalent of US$5 for bus fare.To enroll 500 infants in the field study, we approached 4,617 parents of newborn male infants. Thus, the uptake rate was 11% . Some 4,107 parents (89%) declined to participate. Reasons for refusal included fear of harm and sociocultural considerations.The median age of male infants at the time of circumcision was 22 days  . Eighty-Approximately 40% of fathers were reportedly circumcised. Among mothers whose partner was not circumcised, 80% would like their partner to be circumcised.Completing the procedure took a median of 17 minutes . Mild post-circumcision bleeding occurred in 49 male infants (10%). For 46 of these infants (94%), simple pressure stopped this bleeding. Three cases of bleeding could not be stopped by simple pressure. We classified these as moderate or severe procedure-related AEs; we detail these cases below.Completing the procedure took a median of 17 minutes.We observed 7 AEs  . These iModerate or severe adverse events occurred in 7 of 500 circumcisions.The 1 case of moderate bleeding required clamping of the bleeding vessel with forceps and an acetylated glucosamine dressing at the time of the procedure. The 2 cases of severe bleeding required hospitalization. In 1 case, the infant bled immediately post-circumcision. The bleeding was controlled with pressure but started again at home, and he was referred to a hospital. Three sutures were required to stem the bleeding. The other case of severe bleeding was in an infant who had a family history of hemophilia (unreported and undetected at the time of the procedure). The infant was given a blood transfusion (200 ml). He was hospitalized for 3 days.One male infant left the study 2 days after the procedure without any formal follow-up visits. This infant developed an acute respiratory infection after the procedure was performed and died 36 hours post-EIMC. The respiratory infection was due to an outbreak of severe bronchiolitis and was deemed unrelated to the EIMC procedure.All mothers who answered the question on satisfaction with EIMC (n = 498) reported being satisfied with the outcome (scores  =  6 to 10), with 491 (99%) reporting that they were very satisfied (scores  =  8 or greater). Nearly all mothers  said they would recommend EIMC to other parents and would circumcise their next newborn son . Among fathers who answered the question on satisfaction with EIMC, all 112 gave a score of 6 or greater. Nearly all the fathers would recommend the procedure to a friend  and would have a future son circumcised  . The mosWe have implemented the first field study in sub-Saharan Africa of EIMC conducted by nurse-midwives using the AccuCirc device. We found that the procedure is safe, feasible, and acceptable to parents who choose it for their infant sons.,,,,,,,Overall, the rate of AEs was low and similar to those reported in the 2 previous AccuCirc studies conducted in BotswanaSimilar AE rates for physicians and nurse-midwives suggest that it is safe and feasible to roll out EIMC using non-physician providers.Nonetheless, the fact that we encountered 2 cases of severe bleeding (including a case of unreported hemophilia) that required hospitalization for monitoring and management has important implications for EIMC planning and subsequent roll-out:Although EIMC can be offered at the lowest-level health facilities, there should be easily accessible backup services to deal with any life-threatening complications .Intensive screening and history-taking before the procedure are required in order to exclude any infants with a family history of bleeding disorder.Screening for other non-circumcision-related health issues is necessary to ensure that infants who are circumcised are clinically well.The infant mortality rate is high in sub-Saharan Africa. Recent Zimbabwe Demographic and Health Survey findings suggest that infant and neonatal mortality rates are 57 and 31 deaths per 1,000 live births, respectively.,,As in the Zimbabwe comparative trial,Actual uptake of the procedure—11%—was lower than previously suggested by hypothetical acceptability studies.,,,Sustained uptake and acceptability will depend greatly on perceptions of the safety and aesthetic aspects of EIMC.,Most women who sought EIMC for their sons were knowledgeable about male circumcision and its HIV-preventive benefits. Our findings corroborate those from previous studies on the hypothetical acceptability of VMMC and EIMC; these studies found that this knowledge is likely to be associated with uptake.-A few women  felt that EIMC will likely be viewed negatively in their community. Hypothetical acceptability studies have suggested that fear of their son’s future social exclusion, including ostracism, derision, and rejection, may discourage parents from opting for EIMC.Current initiatives that portray VMMC as a lifestyle choice likely are playing a critical role in changing community norms.-EIMC may provide a rare opportunity to reach fathers via health care facilities. In this study, for example, about one-third of fathers accompanied their sons to the EIMC clinic. Otherwise, men are known to be hard to reach via health services.It may be possible to use EIMC as a platform to strengthen other maternal and child health programs and to promote other health interventions to men.The sample size of this field study was guided by the WHO “Framework for Clinical Evaluation of Devices for Male Circumcision,”We circumcised 500 male infants in a field study of the AccuCirc device for EIMC in Zimbabwe. We found that EIMC can be safely and acceptably offered by nurse-midwives using the AccuCirc device. The AccuCirc device has the potential to facilitate widespread scale-up of safe EIMC in sub-Saharan Africa.


Ovarian cancer has the lowest survival rate of all gynaecologic cancers and is characterised by a lack of early symptoms and frequent late stage diagnosis. There is a paucity of robust molecular markers that are independent of and complementary to clinical parameters such as disease stage and tumour grade.OvMark algorithm combines data from multiple microarray platforms (including probesets targeting miRNAs) and correlates them with clinical parameters  and outcomes , overall survival). In total, OvMark combines 14 datasets from 7 different array platforms measuring the expression of ~17,000 genes and 341 miRNAs across 2,129 ovarian cancer samples.We have developed a user-friendly, web-based system to evaluate the association of genes/miRNAs with outcome in ovarian cancer. The −6). Surprisingly, those genes found to have the greatest correlation with outcome have not been heavily studied in ovarian cancer, or in some cases in any cancer. For instance, the three genes with the greatest association with survival are SNAI3, VWA3A and DNAH12.To demonstrate the utility of the system we confirmed the prognostic ability of 14 genes and 2 miRNAs known to play a role in ovarian cancer. Of these genes, CXCL12 was the most significant predictor of DFS . The impact of this tool will be in the preliminary assessment of putative biomarkers in ovarian cancer, particularly for research groups with limited bioinformatics facilities.The online version of this article (doi:10.1186/1476-4598-13-241) contains supplementary material, which is available to authorized users. Ovarian cancer is the most lethal gynecological malignancy. Due to its lack of early symptoms, this disease is usually diagnosed at an advanced stage when the cancer has already spread to secondary sites . While iOvMark) which allows the association of gene(s) of interest with patient outcomes in ovarian cancer. This algorithm integrates gene expression data from 2,129 patients in 14 DNA microarray studies and corresponding clinical  and treatment (chemotherapy) data. Among all ovarian cancers in included in OvMark, the vast majority are grade 2/3 (high-grade) serous cancers, which compose more than 80% of ovarian cancers that occur in women. The other ovarian cancers in OvMark represent less common epithelial ovarian cancer subtypes that are biologically distinct from high-grade serous ovarian cancers, mainly grade 1 (low-grade) serous cancers and low and high-grade endometrioid ovarian cancers. The user can stratify samples based on subtype and these clinical data for investigation of prognostic markers in the context of disease-free survival (DFS) and overall survival (OS). A similar approach has been “previously used” in integrating gene expression and detailed clinical data including survival information in breast cancer [For several years now, efforts to identify novel prognostic and predictive factors have focused on molecular markers, with a large number having been identified and investigated . Howevert cancer , 6.OvMark to allow their evaluation as putative prognostic markers in ovarian cancer from gene expression data.The association of small non-coding RNAs known as microRNAs (miRNAs) with ovarian cancer has been well established . Over thOvMark was able to confirm the robustness of these prognostic marker across a large and diverse patient dataset.We confirm the utility of this approach following comparisons of outputs with a panel of 14 previously identified markers of prognosis in ovarian cancer. In addition, we also conducted an unbiased analysis of all genes present in the dataset to assess their prognostic potential and found that the list of the most significant genes is dominated by novel markers of prognosis in ovarian cancer. The feasibility of using miRNA host gene expression as a surrogate for miRNA levels was confirmed using the known miRNA prognostic markers, let-7f-2  and miR-OvMark is a powerful tool for examining putative gene/miRNA prognostic biomarkers in ovarian cancer. The value of this tool will be in the preliminary assessment of biomarkers in ovarian cancer, particularly for research groups with limited bioinformatics facilities.Our novel user-friendly algorithm http://www.ncbi.nlm.nih.gov/geo/) in the form of raw data files, where possible. Only ovarian cancer datasets with survival information and at least 50 patients were included. In total, 2,129 samples across 14 datasets incorporating 7 different array platforms were utilised to develop the OvMark system. Table http://www.bioconductor.org). For the dual-channel platforms, data were loess normalised [http://www.biomart.org/) and the Bioconductor annotation libraries. Probes that mapped to multiple genes were filtered out. If there were multiple probes for the same gene, the probe values were averaged for that gene. This resulted in expression data for a total of 20,017 Entrez gene IDs across 2,129 samples. These 20,017 Entrez gene IDs corresponding to approximately 17,000 genes.Gene expression data sets were downloaded from the Gene Expression Omnibus . U133A and U133plus2 microarrays have 22,277 probe sets in common. Using this information, it is possible to infer the expression of 341 miRNAs across 1,358 samples based on a previously published mapping of Affymetrix probesets to miRBase . As withOvMark survival analysis dichotomises the expression of the gene of interest based on a median, high (within the 75% quartile) or low expression (within 25% quartile) cutoff. For example, if median expression is chosen, the expression of the gene of interest in a particular dataset is calculated. Those samples where the expression of the gene is greater than the median expression of that gene for that dataset are placed in the high expression group and those with less than median expression are placed in the low group. To account for study-to-study variation this phase is conducted separately for each of the 14 datasets. Once stratification is complete the individual datasets are combined and a global pooled survival analysis is performed to determine if the gene is associated with either OS and DFS. It is important to treat each dataset separately when determining if a sample belongs to the high or low expression groups, as the expression of the gene of interest will vary greatly across the different experiments/platforms. This approach is robust enough to detect the expression changes at low levels. A gene that goes from not detected to low expression, will “change quartiles”. In this presence or absence scenario, those samples where the gene is absent would be in the lower quartile, the “low expression cohort” and those samples where the gene is present would be in the higher quartile, the “high expression cohort” even though the absolute expression of the gene is very low. This is because though the interquartile range is low, it is still sufficient to distinguish between the two groups.The first stage in http://cran.r-project.org/). For further details see Madden et al. [Survival curves are based on Kaplan-Meier estimates and the log-rank p-value is shown for difference in survival. Cox regression analysis is used to calculate hazard ratios. The R package survival is used to calculate and plot the Kaplan-Meier survival curve. All calculations are carried out in the R statistical environment  is available on a publically accessible a web server at http://glados.ucd.ie/OvMark/index.html and will be updated on a regular basis. The software uses Common Gateway interface (CGI) to link the web server with the R/PERL based algorithm. All calculations are carried out in real-time. All data/scripts are available upon request from the authors.The interface  stage and histology subtype. The software also allows for the median expression and the upper or lower quartiles of the expression of the gene of interest to be used to determine high and low groups within each of the 14 individual datasets.OvMark was run using 14 previously identified prognostic markers, LPR [OvMark database using the median expression option to dichotomise the data and DFS and OS as the survival endpoints.ers, LPR , PRL [34ers, LPR , MIF [36ers, LPR , BRCA1 [ers, LPR , MLH1 [3ers, LPR , ApoA1 [ers, LPR , SNAI2 [ers, LPR , CXCL12 ers, LPR  and IFNGers, LPR . Each ofOvMark database. The database was dichotomised using median gene expression and overall survival was chosen as the survival endpoint. No other software parameters were used. The resultant p-values are adjusted for multiple testing using the Benjamini-Hochberg method [All ~20,000 Entrez gene IDs corresponding to approximately 17,000 genes were queried in the g method . The sigOvMark system we identified the 10 genes most closely associated with survival (i.e. highest and lowest hazard ratios from Cox regression). To further demonstrate their potential as biomarkers for ovarian cancer prognosis the concordance index (C-index) was calculated for each of these genes, using the bioconductor pack survcomp [To illustrate the utility of the survcomp . The C-isurvcomp , 47. BriOvMark. Many other miRNAs are significantly associated with survival depending on the combination of clinical parameters chosen.In order to test the robustness of our gene-centred survival meta-analysis we used a panel of 14 known markers of prognosis in ovarian cancer. In addition we chose to screen all human genes for their prognostic potential in ovarian cancer. As there is currently no large-scale robust signature for miRNAs in ovarian cancer, we tested our approach on known individual miRNAs which have previously been shown to be prognostic markers, namely let-7f-2  and mir-OvMark using median expression to dichotomise the data and DFS and OS as the survival end points. All patients in the OvMark database were chosen for this analysis without sub-selection based on any clinical parameters. This information is summarised in Table −5, n = 996). Combining the markers can improve HRs in comparison with single markers alone. For example, the Kaplan-Meier DFS plot for BRCA2 and PRL in combination  is shown in Figure −4 n = 996). Patients with high-level expression of both BRCA2 and PRL in their ovarian cancers have a worse prognosis than those with high expression of BRCA2 or PRL alone in their ovarian cancers (with a HR of 1.43 versus a HR of 1.38 or 1.25 respectively). When greater than median expression of CDKN1B is combined with greater than median expression of BRCA2 and PRL .Each of the 14 ovarian cancer gene expression markers identified above were analysed separately within OvMark database. Our results for miR-16-1 and let-7f-2 are shown in Figure −3, n = 1241).The miRNA MiR-16-1 has previously been associated with poor prognosis in ovarian cancer  and let-OvMark database for known ovarian cancer biomarkers are consistent with previously published data, thus validating the potential utility of this database for the study of the clinical and outcome implications of the expression of other genes and miRNAs in ovarian cancer.In summary, outcome predictions by the OvMark. We then queried all human genes in OvMark so as to identify which genes had the greatest association with OS of ovarian cancer patients. The results for the top ten genes are summarised in Table −5) and Figure Although all of the prognostic markers above chosen for testing have been well studied in ovarian cancer (in some cases with mixed results), only BRCA2, SNAI2 and CXCL12 were a particularly convincing marker of outcome in OvMark provides a user-friendly tool for examining putative prognostic biomarkers in ovarian cancer. It builds on our previous work in breast cancer where we successfully combined multiple datasets to perform cross-dataset survival analysis [OvMark system allows users to assess prognostic markers across multiple microarray platforms by utilising study by study dicotimisiation to reduce batch effects. We are therefore not reliant on complex dataset transformations. Also, as the database is gene-centred, rather than probe-centred, we are not limited to the gene coverage of a particular platform. In summary, OvMark allows the analysis of ~20,000 unique Entrez gene IDs in 2,129 ovarian cancer samples. It is our intension to increase this number as more ovarian cancer datasets become available.analysis , 6. The analysis , the OvMSurprisingly, those genes found to have the greatest correlation with outcome across the dataset have not been studied in ovarian cancer, or in some cases in any cancer. For example, of the three genes with the greatest association with overall survival SNAI3, VWA3A and DNAH12, only SANI3 has been previously associated with carcinogenesis where it is involved in epithelial to mesenchymal transition .After confirming the robustness of our algorithm using genes already identified as biomarkers in ovarian cancer we proceeded to examine its potential for inferring the prognostic ability of miRNAs from the gene expression data. The attraction of miRNA biology to cancer researchers arises from the potential of miRNAs to alter an entire pathway or indeed, pathways. miRNAs have been heavily studied in ovarian cancer; however, their role as prognostic markers are not well characterised. There are only a few large-scale studies which incorporate miRNA profiling integrated with detailed clinical data , 57. DesOvMark. This issue cannot be fully resolved until such time as there is a better understanding of which miRNAs are co-expressed with their host genes (using the TCGA data we estimate this number to be ~60%) and, further, if those that are not significantly co-expressed do so in a disease/tissue-specific manner. It also needs to be determined whether the miRNAs themselves are subject to some level of post-transcriptional regulation.Again, our algorithm gene centres the data, and allows us to examine miRNAs as prognostic markers in ovarian cancer. We were able to confirm the results of other studies , 13, 48,OvMark has some limitations. It can not overcome the inherent problems associated with transcriptomic analysis of ovarian cancer, in that often samples are taken at a late stage. The identification of biomarkers through the OvMark system should only be considered as part of the discovery phase. In order to confirm the utility of genes identified by OvMark further validation is required to assess biomarker candidates in an independent replication cohort. The OvMark system will allow researchers to easily asses the prognostic performance of their targets of interest within a large scale dataset and reduce false discovery rates when prioritising putative biomarkers for subsequent validation in their laboratories.Our new algorithm In this study, we have developed a simple user-friendly tool for examining putative gene/miRNA prognostic markers in ovarian cancer. The value of this tool is both in the simplicity of its design and the robustness of its approach. It is designed with non-bioinformatic research groups in mind and will be of great value in the preliminary assessment of putative biomarkers in ovarian cancer.miRNAs significantly correlated with their host gene expression data.(XLSX 14 KB)Additional file 1:Forest plot illustrating the concordance index (CI) for each of the 10 genes most closely associated with patient outcome. The statistical significance of each CI was demonstrated by shuffling the survival information 10,000 times and calculating an empirical p-value. (TIFF 37 KB)Additional file 2: