Choice of DNA extraction method affects stool microbiome recovery and subsequent phenotypic association analyses.

The lack of standardization in the methods of DNA extraction from fecal samples represents the major source of experimental variation in the microbiome research field. In this study, we aimed to compare the metagenomic profiles and microbiome-phenotype associations obtained by applying two commercially available DNA extraction kits: the AllPrep DNA/RNA Mini Kit (APK) and the QIAamp Fast DNA Stool Mini Kit (FSK). Using metagenomic sequencing data available from 745 paired fecal samples from two independent population cohorts, Lifelines-DEEP (LLD, n = 292) and the 500 Functional Genomics project (500FG, n = 453), we confirmed significant differences in DNA yield and the recovered microbial communities between protocols, with the APK method resulting in a higher DNA concentration and microbial diversity. Further, we observed a massive difference in bacterial relative abundances at species-level between the APK and the FSK protocols, with > 75% of species differentially abundant between protocols in both cohorts. Specifically, comparison with a standard mock community revealed that the APK method provided higher accuracy in the recovery of microbial relative abundances, with the absence of a bead-beating step in the FSK protocol causing an underrepresentation of gram-positive bacteria. This heterogeneity in the recovered microbial composition led to remarkable differences in the association with anthropometric and lifestyle phenotypes. The results of this study further reinforce that the choice of DNA extraction method impacts the metagenomic profile of human gut microbiota and highlight the importance of harmonizing protocols in microbiome studies.

Impact of occupational pesticide exposure on the human gut microbiome.

The rising use of pesticides in modern agriculture has led to a shift in disease burden in which exposure to these chemicals plays an increasingly important role. The human gut microbiome, which is partially responsible for the biotransformation of xenobiotics, is also known to promote biotransformation of environmental pollutants. Understanding the effects of occupational pesticide exposure on the gut microbiome can thus provide valuable insights into the mechanisms underlying the impact of pesticide exposure on health. Here we investigate the impact of occupational pesticide exposure on human gut microbiome composition in 7198 participants from the Dutch Microbiome Project of the Lifelines Study. We used job-exposure matrices in combination with occupational codes to retrieve categorical and cumulative estimates of occupational exposures to general pesticides, herbicides, insecticides and fungicides. Approximately 4% of our cohort was occupationally exposed to at least one class of pesticides, with predominant exposure to multiple pesticide classes. Most participants reported long-term employment, suggesting a cumulative profile of exposure. We demonstrate that contact with insecticides, fungicides and a general "all pesticides" class was consistently associated with changes in the gut microbiome, showing significant associations with decreased alpha diversity and a differing beta diversity. We also report changes in the abundance of 39 different bacterial taxa upon exposure to the different pesticide classes included in this study. Together, the extent of statistically relevant associations between gut microbial changes and pesticide exposure in our findings highlights the impact of these compounds on the human gut microbiome.

Modified Mach-Zehnder interferometer for spatial coherence measurement.

Spatial coherence of light sources is usually obtained by using the classical Young's interferometer. Although the original experiment was improved upon in successive works, some drawbacks still remain. For example, several pairs of points must be used to obtain the complex coherence degree (normalized first-order correlation function) of the source. In this work, a modified Mach-Zehnder interferometer which includes a pair of lenses and is able to measure the spatial coherence degree is presented. With this modified Mach-Zehnder interferometer, it is possible to measure the full 4D spatial coherence function by displacing the incoming beam laterally. To test it, we have measured only a 2D projection (zero shear) of the 4D spatial coherence, which is enough to characterize some types of sources. The setup has no movable parts, making it robust and portable. To test it, the two-dimensional spatial coherence of a high-speed laser with two cavities was measured for different pulse energy values. We observe from the experimental measurements that the complex degree of coherence changes with the selected output energy. Both laser cavities seem to have similar complex coherence degrees for the maximum energy, although it is not symmetrical. Thus, this analysis will allow us to determine the best configuration of the double-cavity laser for interferometric applications. Furthermore, the proposed approach can be applied to any other light sources.

Effect of host genetics on the gut microbiome in 7,738 participants of the Dutch Microbiome Project.

Host genetics are known to influence the gut microbiome, yet their role remains poorly understood. To robustly characterize these effects, we performed a genome-wide association study of 207 taxa and 205 pathways representing microbial composition and function in 7,738 participants of the Dutch Microbiome Project. Two robust, study-wide significant (P < 1.89 × 10-10) signals near the LCT and ABO genes were found to be associated with multiple microbial taxa and pathways and were replicated in two independent cohorts. The LCT locus associations seemed modulated by lactose intake, whereas those at ABO could be explained by participant secretor status determined by their FUT2 genotype. Twenty-two other loci showed suggestive evidence (P < 5 × 10-8) of association with microbial taxa and pathways. At a more lenient threshold, the number of loci we identified strongly correlated with trait heritability, suggesting that much larger sample sizes are needed to elucidate the remaining effects of host genetics on the gut microbiome.

Environmental risk factors of type 2 diabetes-an exposome approach.

Type 2 diabetes is one of the major chronic diseases accounting for a substantial proportion of disease burden in Western countries. The majority of the burden of type 2 diabetes is attributed to environmental risks and modifiable risk factors such as lifestyle. The environment we live in, and changes to it, can thus contribute substantially to the prevention of type 2 diabetes at a population level. The 'exposome' represents the (measurable) totality of environmental, i.e. nongenetic, drivers of health and disease. The external exposome comprises aspects of the built environment, the social environment, the physico-chemical environment and the lifestyle/food environment. The internal exposome comprises measurements at the epigenetic, transcript, proteome, microbiome or metabolome level to study either the exposures directly, the imprints these exposures leave in the biological system, the potential of the body to combat environmental insults and/or the biology itself. In this review, we describe the evidence for environmental risk factors of type 2 diabetes, focusing on both the general external exposome and imprints of this on the internal exposome. Studies provided established associations of air pollution, residential noise and area-level socioeconomic deprivation with an increased risk of type 2 diabetes, while neighbourhood walkability and green space are consistently associated with a reduced risk of type 2 diabetes. There is little or inconsistent evidence on the contribution of the food environment, other aspects of the social environment and outdoor temperature. These environmental factors are thought to affect type 2 diabetes risk mainly through mechanisms incorporating lifestyle factors such as physical activity or diet, the microbiome, inflammation or chronic stress. To further assess causality of these associations, future studies should focus on investigating the longitudinal effects of our environment (and changes to it) in relation to type 2 diabetes risk and whether these associations are explained by these proposed mechanisms.

Fast optical source for quantum key distribution based on semiconductor optical amplifiers.

A novel integrated optical source capable of emitting faint pulses with different polarization states and with different intensity levels at 100 MHz has been developed. The source relies on a single laser diode followed by four semiconductor optical amplifiers and thin film polarizers, connected through a fiber network. The use of a single laser ensures high level of indistinguishability in time and spectrum of the pulses for the four different polarizations and three different levels of intensity. The applicability of the source is demonstrated in the lab through a free space quantum key distribution experiment which makes use of the decoy state BB84 protocol. We achieved a lower bound secure key rate of the order of 3.64 Mbps and a quantum bit error ratio as low as 1.14×10⁻² while the lower bound secure key rate became 187 bps for an equivalent attenuation of 35 dB. To our knowledge, this is the fastest polarization encoded QKD system which has been reported so far. The performance, reduced size, low power consumption and the fact that the components used can be space qualified make the source particularly suitable for secure satellite communication.

Bone marrow changes in anorexia nervosa are correlated with the amount of weight loss and not with other clinical findings.

The clinical history and biochemical and hematologic variables for 44 consecutive patients diagnosed with anorexia nervosa were recorded. Bone marrow aspirates and biopsy specimens were analyzed by standard morphologic procedures, and bone marrow adipocytes were studied morphometrically. The bone marrow of the 44 patients was classified as normal (5 cases [11%]), hypoplastic or aplastic (17 [39%]), with partial or focal gelatinous degeneration (13 [30%]), or with complete gelatinous degeneration of the bone marrow (GDBM; 9 [20%]). These patterns correlated with amount of weight loss (P = .005) but not other clinical findings. WBC counts were lower in patients with GDBM (P = .0189), but this and other peripheral blood variables did not always reflect the severity of bone marrow damage. Hypoplastic or aplastic bone marrow showed an increase in bone marrow fat fraction due to an increase in adipocyte diameters, while in GDBM, fat fraction and adipocyte diameters decreased. Morphologic changes in bone marrow and stereologic alterations in bone marrow adipocytes may be observed in anorexia nervosa. The extent of damage is related to the amount of weight loss, not to other factors. Peripheral blood cell counts may not reflect the extent of damage. In some patients, this process may be reversible with reestablishment of adequate nutritional intake.

[Pneumonia in patients with chronic lymphocytic leukemia. Study of 30 episodes]. / Neumonías en pacientes con leucemia linfática crónica. Estudio de 30 episodios.

BACKGROUND: To analyse the etiology, diagnostic methods and response to therapy in 30 episodes of pneumonia diagnosed in 17 patients with chronic lymphocytic leukemia (CLL) between 1995 and 2000. PATIENTS AND METHOD: In each episode of pneumonia the following data were analysed: age, gender, treatment of CLL, antiinfectious prophylaxis, granulocytopenia, CD4/CD8 lymphocytes ratio, hipogammaglobulinemia, origin of pneumonia (nosocomial or community-acquired), localisation, respiratory insufficiency, need for mechanical ventilation, antimicrobial therapy and response. Diagnostic methods included blood and sputum cultures, fiberoptic bronchoscopy and search for antigens in urine (Legionella pneumophila serogroup 1, galactomannan, and Streptococcus pneumoniae). RESULTS: Median age of the series was 60 yr. (range 50-86) and 12 patients were male. Chlorambucil and prednisone were used in 13 cases and fludarabine in 8. Granulocytopenia was present in 14 episodes, hypogammaglobulinemia was seen in 22 and CD4/CD8 ratio was lower than 1 in 8 out of 14 evaluable cases. Etiology of pneumonia was established in 16 episodes (53%). Fiberoptic bronchoscopy was the most useful technique (83% of positive diagnoses) followed by blood cultures (38%). Two patients were diagnosed of aspergillosis at autopsy. Pneumococcus was the most frequent agent (5 cases) followed by Pseudomonas aeruginosa (4), Pneumocystis carinii (2) and Aspergillus fumigatus (2). One out of the two patients with P. carinii pneumonia had received fludarabin and the remaining was treated with prednisone for long time. Ten patients (30%) had died: P. aeruginosa (3 cases), P. carinii (2), A. fumigatus (2), Mycobacterium xenopi (1), and unknown microorganism (2). CONCLUSIONS: In this series of CLL patients the frequency of etiologic diagnosis of pneumonias was good. Pneumococcus was the most frequent microorganism. Pneumonias caused by opportunistic microorganisms were associated to the treatment with fludarabin or prednisone and were associated to a high mortality rate.

Influence of highly active anti-retroviral therapy on response to treatment and survival in patients with acquired immunodeficiency syndrome-related non-Hodgkin's lymphoma treated with cyclophosphamide, hydroxydoxorubicin, vincristine and prednisone.

Combined highly active anti-retroviral therapy (HAART) with protease and reverse transcriptase inhibitors has modified the natural history of opportunistic infections and neoplasms in human immunodeficiency virus (HIV)-infected patients. We analysed the influence of HAART on the response to treatment and survival in a series of 58 patients with acquired immune deficiency syndrome (AIDS)-related non-Hodgkin's lymphoma (NHL) treated with CHOP (cyclophosphamide, hydroxydoxorubicin, vincristine and prednisone). Two groups of patients were included: (i) forty-one patients diagnosed with NHL between 1988 and 1996 who were not treated with HAART; (ii) seventeen patients diagnosed since 1996, who were receiving or commenced HAART when NHL was diagnosed. The response rate to CHOP was higher in group 2 (13 out of 17 cases; 75%) than in group 1 (14 out of 41 cases; 34%) (P = 0.003). The 2-year probability of event-free survival (EFS) [95% confidence interval (CI)] for group 1 was 0.5 (0.24-0.74), whereas for group 2 it was 0.85 (0.61-0.90) (P = 0.024). The lymphoma-free survival (LFS) was also significantly different for both groups (2-year LFS probability 0.53 vs. 1.0, P = 0.04). The median (95% CI) overall survival (OS) for group 1 was 7 months (range, 3-10.8 months), whereas it was not reached in group 2 (P = 0.0015). In the multivariate analysis for remission attainment, the only variables with a higher probability to achieve complete remission (CR) were HAART (P = 0.01) and International Prognostic Index score 1 (P = 0.02). The only statistically significant variable in the multivariate analysis for EFS was HAART (P = 0.049) and the variables with prognostic value for OS in the multivariate analysis were B symptoms (P = 0.01) and HAART (P = 0.003). Patients with AIDS-related NHL treated with CHOP and HAART had a higher CR rate than those treated only with CHOP. In this study, HAART was an independent prognostic factor for CR, OS and EFS in patients with AIDS-related NHL.

Usefulness and reproducibility of cytomorphologic evaluations to differentiate myeloma from monoclonal gammopathies of unknown significance.

We attempted to differentiate monoclonal gammopathies of unknown significance (MGUS) and multiple myeloma (MM) on morphologic grounds and to determine interobserver reproducibility of the differentiation. Cytologists blindly evaluated bone marrow smears from 154 patients with bone marrow plasmacytosis for the proportion of plasma cells with predefined cellular atypias. The single morphologic characteristic that most strongly differentiated MM from MGUS was the presence of nucleoli. The percentage of plasma cells, cytoplasmic contour irregularities, and anisocytosis also predicted a diagnosis of myeloma in multivariate analysis. Six cytologists independently evaluated 68 consecutive cases to determine sensitivity and specificity of these cytomorphologic features. The interobserver coefficient of variation for the plasma cell count was 33%. On consideration of the diagnosis, 36 of 41 MGUS cases and all 24 cases of myeloma were classified correctly. The use of a predesigned score system did not present such a bias, although it did not improve overall efficiency. The plasma cell count is the most predictive characteristic of myeloma from a cytologic viewpoint, but the interobserver variability is high. Interobserver variability is also high in the assessment of morphologic atypia, and atypical traits are not uncommon in plasma cells in MGUS.

[Hodgkin's disease in patients infected by the human immunodeficiency virus. Study of fifteen cases]. / Enfermedad de Hodgkin en pacientes con infección por el virus de la inmunodeficiencia humana. Estudio de 15 casos.

BACKGROUND: In spite of not being considered as an AIDS defining illness, Hodgkin's disease (HD) has specific clinical and biological features in HIV-infected patients. PATIENTS AND METHODS: Study of clinicopathologic and analytic characteristics, Epstein-Barr virus (EBV) detection (polymerase chain reaction), and prognosis in 15 patients with HD and HIV infection. RESULTS: Thirteen patients had B symptoms, 10 extranodal involvement and 12 advanced HD. The most frequent histologic subtypes were mixed cellularity (6) and lymphocyte depletion (6). The mean (SD) of CD4 lymphocytes was 0.10 (0.08) x 10(9)/l. The presence of EBV in lymph node biopsy was demonstrated in 3 out of 4 patients investigated. Complete remission (CR) was achieved in 7 out of 14 treated cases (50%), the median overall survival was 26 months and the 2 year event-free survival probability was 60%. CONCLUSIONS: In HIV-infected patients, HD presents in advanced stages, unfavourable histologic subtypes, frequent extranodal involvement and B symptoms. The prognosis is poor, mainly because of a low CR rate.

Immune hemolytic anemia induced by 6-mercaptopurine.

BACKGROUND: Myelosuppression is the main hematotoxic effect of 6-mercaptopurine (6-MP), which is an antimetabolite chemotherapy drug. Immune hemolytic anemia associated with this drug has not been previously reported. CASE REPORT: A 67-year-old man with chronic myelomonocytic leukemia presented with anemia 2 weeks after 6-MP therapy had been initiated. Additional tests provided laboratory evidence of hemolysis. When treatment was stopped, the patient's condition and laboratory results showed a progressive improvement. RESULTS: The direct antiglobulin test was positive for IgG. The eluate and the serum were not reactive with panel red cells but reacted with 6-MP-treated red cells, while the normal serum pool was unreactive. The direct antiglobulin test was no longer positive by 20 days after the cessation of 6-MP therapy. CONCLUSION: This drug, 6-MP, should be added to the list of drugs that have been reported to cause immune hemolytic anemia by means of the so-called hapten mechanism.

[Changes in the long arm of chromosome 3 (3q) in malignant myeloid hemopathies. A study of 10 cases]. / Alteraciones del brazo largo del cromosoma 3 (3q) en hemopatías malignas mieloides. Estudio de 10 casos.

BACKGROUND: Abnormalities of the long arm of chromosome 3 (3q) involving bands 3q21 and/or 3q26 occur in 2-6% of myeloid malignancies. Trilineage myelodysplasia, especially in the megakaryocytic line, is a characteristic feature. Additional abnormalities of chromosomes 5 and 7 are usually present. The response to treatment and prognosis are poor. PATIENTS AND METHODS: The main clinical, cytologic (bone marrow aspirate and biopsy) and cytogenetic characteristics as well as the response to the treatment in 10 patients with 3q abnormalities diagnosed in a single hospital in a period of 8 years are referred. RESULTS: Eight patients had acute non-lymphoblastic leukemia, being de novo in five of them. The median value of hemoglobin was 87 g/l (range: 51-148), white blood cells count 5.8 x 10(9)/l (1.2-47.1) and platelet count 34 x 10(9)/l (5-182). The morphological findings in the study of the bone marrow were: dyserythropoietic features (7 patients), dysgranulopoietic abnormalities (5 patients) and small-sized megakaryocytes with hypolobulated nuclei (8 patients). Fibrosis was observed in the 4 cases in which a bone marrow biopsy was performed. In addition to the 3q alteration, abnormalities of chromosomes 7 (4 patients), 5q (2 patients) and +8 (2 patients) were present. Four patients received intensive chemotherapy and in two of them a complete remission was achieved, but relapse occurred at 3 and 5 months, respectively. All patients have died, the median survival being 7 months. CONCLUSIONS: 3q abnormalities define a subtype of myeloid malignancies with characteristic clinical and morphological features. The response to therapy and survival are poor.

[The usefulness of the bone marrow examination in the etiological diagnosis of prolonged fever in patients with HIV infection]. / Utilidad del examen de la médula ósea en el diagnóstico etiológico de la fiebre prolongada en los pacientes con infección por el virus de la inmunodeficiencia humana.

BACKGROUND: To analyze the usefulness of bone marrow (BM) study in the diagnosis of fever of unknown origin (FUO) in patients infected by the human immunodeficiency virus (HIV) in a single center during a period of 42 months. PATIENTS AND METHODS: 182 episodes of FUO in 162 patients p3th HIV infection were studied. Age, sex, risk factor for HIV infection, hemoglobin level, counts of leucocytes, neutrophils, lymphocytes, CD4 positive lymphocytes, platelets and levels of hepatic enzymes, albumin and beta 2-microglobulin were studied. BM aspirate was performed in all episodes for cytologic and microbiologic examination, and BM biopsy was done in 43. Analysis of factors related with the probability of diagnosis by BM examination was carried out. RESULTS: The median age was 33 years (range, 22-70), and 123 were males. Drug abuse was the most frequent risk factor for HIV infection (63%). One hundred thirty patients had previous AIDS diagnosis before they were evaluated for unexplained fever. A specific diagnosis was achieved in 161 episodes (88%) and the most frequent diagnoses were Mycobacterium spp. (55%) and Leishmania spp. (14%) infections. Fifty-four episodes (30%) were diagnosed by BM examination, and in 36 (20%) BM study was the only diagnostic tool. Examination of the BM aspirate yielded the diagnosis in 40 out of the 178 episodes (13%), whereas BM biopsy was a diagnostic tool in 8 (19%); in 9 additional cases (21%) granulomas were observed. Microbiologic study of BM smears for mycobacterial infections was positive in 28 of the 143 episodes (19%), and the culture for Leishmania was positive in 2 out of the 42 cases. The presence of thrombocytopenia (< 75 x 10(9)/l) and elevated serum levels of aspartate-aminotransferase (AST) (> 100 U/l) were the factors associated with a high probability to obtain the diagnosis through BM study. CONCLUSIONS: In patients infected by the HIV and unexplained fever, BM examination is an useful procedure for the diagnosis, particularly in areas where infections by Mycobacterium spp. and Leishmania are prevalent. So that, in our setting, systematic use of this procedure is justified for diagnosis of FUO in those patients.

[Pneumonias in patients with malignant hemopathies. Their etiology, response to treatment and prognostic factors in 69 patients (88 episodes)]. / Neumonías en los pacientes con hemopatías malignas. Etiología, respuesta al tratamiento y factores pronósticos en 69 pacientes (88 episodios).

BACKGROUND: To analyze the etiology, treatment response and prognostic factors in 88 episodes of pneumonia in patients with hematological malignancies diagnosed in one center during a period of 30 months. PATIENTS AND METHODS: 88 episodes of pneumonia in 69 adult patients were studied. Age, sex, hematological malignancy and its treatment, existence of neutropenia and hypogammaglobulinemia, administration of immunosuppressive agents, type (hospital or community-acquired) and localization of pneumonia, presence of respiratory failure and treatment response were reported. For etiologic diagnosis of pneumonias, cultures of blood, sputum, pleural fluid and bronchoalveolar lavage (BAL), as well as Legionella pneumophila, Aspergillus fumigatus and Streptococcus pneumoniae antigen detection in urine were performed. Univariate and multivariate analyses of prognostic factors for pneumonia evolution were carried out. RESULTS: The median age was 63 years. Acute leukemias (AL) were the predominant hematological malignancies. Microbiologic documentation was obtained in 40 (45%) of pneumonias. Fiberoptic bronchoscopy with BAL (71%) was the diagnostic procedure with highest yield followed by blood cultures (25%). Streptococcus pneumoniae (13) was the most frequent isolated pathogen, followed by Legionella pneumophila (6) and Pseudomonas aeruginosa (6). A significantly higher prevalence of Streptococcus pneumoniae was observed in community-acquired pneumonia. The overall mortality rate was 20%. Respiratory failure (p = 0.0009), existence of neutropenia (p = 0.0023), age equal or higher than 60 years (p = 0.012) and prolonged administration of immunosuppressive agents (p = 0.015) were the prognostic factors associated with unfavourable evolution of pneumonias in the multivariate analysis. CONCLUSIONS: The etiologic diagnosis of pneumonia in patients with hematological malignancies was only achieved in a half of cases. In our series, the high prevalence of Legionella pneumophila can be attributed to the special epidemiologic characteristics of our hospital. Prognostic factors of pneumonia are related to individual factors as well as to the hematological status of patients.