High Compliance with Newborn Community-to-Facility Referral in Eastern Uganda:.An Opportunity to Improve Newborn Survival

Background

Seventy-five percent of newborn deaths happen in the first-week of life, with the highest risk of death in the first 24-hours after birth.WHO and UNICEF recommend home-visits for babies in the first-week of life to assess for danger-signs and counsel caretakers for immediate referral of sick newborns. We assessed timely compliance with newborn referrals made by community-health workers (CHWs), and its determinants in Iganga and Mayuge Districts in rural eastern Uganda.

Methods

A historical cohort study design was used to retrospectively follow up newborns referred to health facilities between September 2009 and August 2011. Timely compliance was defined as caretakers of newborns complying with CHWs’ referral advice within 24-hours.

Results

A total of 724 newborns were referred by CHWs of whom 700 were successfully traced. Of the 700 newborns, 373 (53%) were referred for immunization and postnatal-care, and 327 (47%) because of a danger-sign. Overall, 439 (63%) complied, and of the 327 sick newborns, 243 (74%) caretakers complied with the referrals. Predictors of referral compliance were; the newborn being sick at the time of referral- Adjusted Odds Ratio (AOR) = 2.3, and 95% Confidence-Interval (CI) of [1.6 - 3.5]), the CHW making a reminder visit to the referred newborn shortly after referral (AOR =1.7; 95% CI: [1.2 -2.7]); and age of mother (25-29) and (30-34) years, (AOR =0.4; 95% CI: [0.2 - 0.8]) and (AOR = 0.4; 95% CI: [0.2 - 0.8]) respectively.

Conclusion

Caretakers’ newborn referral compliance was high in this setting. The newborn being sick, being born to a younger mother and a reminder visit by the CHW to a referred newborn were predictors of newborn referral compliance. Integration of CHWs into maternal and newborn care programs has the potential to increase care seeking for newborns, which may contribute to reduction of newborn mortality.     

Direct Detection and Differentiation of Pathogenic Leptospira Species Using a Multi-Gene Targeted Real Time PCR Approach

Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of Leptospira. Rapid and reliable laboratory tests for the direct detection of leptospiral infections in animals are in high demand not only to improve diagnosis but also for understanding the epidemiology of the disease. In this work we describe a novel and simple TaqMan-based multi-gene targeted real-time PCR approach able to detect and differentiate Leptospira interrogans, L. kirschneri, L. borgpeteresenii and L. noguchii, which constitute the veterinary most relevant pathogenic species of Leptospira. The method uses sets of species-specific probes, and respective flanking primers, designed from ompL1 and secY gene sequences. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal β-actin and the leptospiral lipL32 genes was implemented. The analytical sensitivity of all primer and probe sets was estimated to be <10 genome equivalents (GE) in the reaction mixture. Application of the amplification reactions on genomic DNA from a variety of pathogenic and non-pathogenic Leptospira strains and other non-related bacteria revealed a 100% analytical specificity. Additionally, pathogenic leptospires were successfully detected in five out of 29 tissue samples from animals (Mus spp., Rattus spp., Dolichotis patagonum and Sus domesticus). Two samples were infected with L. borgpetersenii, two with L. interrogans and one with L. kirschneri. The possibility to detect and identify these pathogenic agents to the species level in domestic and wildlife animals reinforces the diagnostic information and will enhance our understanding of the epidemiology of leptopirosis.     

Site fidelity and seasonal habitat preferences of largemouth bass (Micropterus salmoides) in a temperate regulated reservoir

Hydrobiologia - A wide range of fish species navigate and return to previously selected habitats across time scales ranging from days to years. While this ability may be challenged in...     

Complement Regulatory Molecules on Human Myelin and Glial Cells: Differential Expression Affects the Deposition of Activated Complement Proteins

Abstract: The expression of decay-accelerating factor CD55, membrane cofactor protein CD46, and CD59 was studied on Schwann cells cultured from human sural nerve and myelin membranes prepared from human cauda equina and spinal cord. These proteins are regulatory membrane molecules of the complement system. CD55 and CD46 are inhibitors of C3 and C5 convertases and CD59 inhibits C8 and C9 incorporation into C5b-9 complex and C9-C9 polymerization. The presence of these proteins was assessed by using antibodies to each of the proteins by fluorescent microscopy, fluorescence-activated cell sorter analysis, and also sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Schwann cells in culture expressed CD55, CD46, and CD59. It is interesting that only CD59 was detected on myelin from both central and peripheral nerve tissue. The ability of these proteins to limit C3 peptide deposition and C9 polymerization in myelin was studied by western blot analysis. C3b deposition was readily detected on antibody-sensitized myelin incubated with normal human serum used as a source of complement but not with EDTA-treated or heat-inactivated serum. C3b deposition was not affected by anti-CD55 antibody. On the other hand, poly-C9 formation in myelin, which was maximum when 50% normal human serum was used, was increased four- to fivefold when myelin was preincubated with anti-CD59. Our data suggest that complement activation on myelin is down-regulated at the step of the assembly of terminal complement complexes, including C5b-9, due to the presence of CD59.     

Drug susceptibility testing of clinical isolates of streptococci and enterococci by the Phoenix automated microbiology system

Background  

Drug resistance is an emerging problem among streptococcal and enterococcal species. Automated diagnostic systems for species identification and antimicrobial susceptibility testing (AST) have become recently available. We evaluated drug susceptibility of clinical isolates of streptococci and enterococci using the recent Phoenix system (BD, Sparks, MD). Diagnostic tools included the new SMIC/ID-2 panel for streptococci, and the PMIC/ID-14 for enterococci. Two-hundred and fifty isolates have been investigated: β-hemolytic streptococci (n = 65), Streptococcus pneumoniae (n = 50), viridans group streptococci (n = 32), Enterococcus faecium (n = 40), Enterococcus faecalis (n = 43), other catalase-negative cocci (n = 20). When needed, species ID was determined using molecular methods. Test bacterial strains were chosen among those carrying clinically-relevant resistance determinants (penicillin, macrolides, fluoroquinolones, glycopeptides). AST results of the Phoenix system were compared to minimal inhibitory concentration (MIC) values measured by the Etest method (AB Biodisk, Solna, Sweden).     

Whole Genome Sequencing Investigation of a Tuberculosis Outbreak in Port-au-Prince,Haiti Caused by a Strain with a “Low-Level” rpoB Mutation L511P – Insights into a Mechanism of Resistance Escalation

The World Health Organization recommends diagnosing Multidrug-Resistant Tuberculosis (MDR-TB) in high burden countries by detection of mutations in Rifampin (RIF) Resistance Determining Region of Mycobacterium tuberculosis rpoB gene with rapid molecular tests GeneXpert MTB/RIF and Hain MTBDRplus. Such mutations are found in >95% of Mycobacterium tuberculosis strains resistant to RIF by conventional culture-based drug susceptibility testing (DST). However routine diagnostic screening with molecular tests uncovered specific “low level” rpoB mutations conferring resistance to RIF below the critical concentration of 1 μg/ml in some phenotypically susceptible strains. Cases with discrepant phenotypic (susceptible) and genotypic (resistant) results for resistance to RIF account for at least 10% of resistant diagnoses by molecular tests and urgently require new guidelines to inform therapeutic decision making. Eight strains with a “low level” rpoB mutation L511P were isolated by GHESKIO laboratory between 2008 and 2012 from 6 HIV-negative and 2 HIV-positive patients during routine molecular testing. Five isolates with a single L511P mutation and two isolates with double mutation L511P&M515T had MICs for RIF between 0.125 and 0.5 μg/ml and tested susceptible in culture-based DST. The eighth isolate carried a double mutation L511P&D516C and was phenotypically resistant to RIF. All eight strains shared the same spoligotype SIT 53 commonly found in Haiti but classic epidemiological investigation failed to uncover direct contacts between the patients. Whole Genome Sequencing (WGS) revealed that L511P cluster isolates resulted from a clonal expansion of an ancestral strain resistant to Isoniazid and to a very low level of RIF. Under the selective pressure of RIF-based therapy the strain acquired mutation in the M306 codon of embB followed by secondary mutations in rpoB and escalation of resistance level. This scenario highlights the importance of subcritical resistance to RIF for both clinical management of patients and public health and provides support for introducing rpoB mutations as proxy for MICs into laboratory diagnosis of RIF resistance. This study illustrates that WGS is a promising multi-purpose genotyping tool for high-burden settings as it provides both “gold standard” sequencing results for prediction of drug susceptibility and a high-resolution data for epidemiological investigation in a single assay.     

Exposure to Bovine Leukemia Virus Is Associated with Breast Cancer: A Case-Control Study

Background

Age, reproductive history, hormones, genetics, and lifestyle are known risk factors for breast cancer, but the agents that initiate cellular changes from normal to malignant are not understood. We previously detected bovine leukemia virus (BLV), a common oncogenic virus of cattle, in the breast epithelium of humans. The objective of this study was to determine whether the presence of BLV DNA in human mammary epithelium is associated with breast cancer.

Methods

This was a case-control study of archival formalin fixed paraffin embedded breast tissues from 239 donors, received 2002–2008 from the Cooperative Human Tissue Network. Case definition as breast cancer versus normal (women with no history of breast cancer) was established through medical records and examination of tissues by an anatomical pathologist. Breast exposure to BLV was determined by in situ-PCR detection of a biomarker, BLV DNA, localized within mammary epithelium.

Results

The frequency of BLV DNA in mammary epithelium from women with breast cancer (59%) was significantly higher than in normal controls (29%) (multiply- adjusted odds ratio = 3.07, confidence interval = 1.66–5.69, p = .0004, attributable risk = 37%). In women with premalignant breast changes the frequency of BLV DNA was intermediate (38%) between that of women with breast cancer and normal controls (p for trend < .001).

Conclusions

Among the specimens in this study, the presence of amplified BLV DNA was significantly associated with breast cancer. The odds ratio magnitude was comparable to those of well-established breast cancer risk factors related to reproductive history, hormones, and lifestyle and was exceeded only by risk factors related to genetics (familial breast cancer), high dose ionizing radiation, and age. These findings have the potential for primary and secondary prevention of breast cancer.     

Genotyping of Mycoplasma bovis isolates using multiple-locus variable-number tandem-repeat analysis

Mycoplasma bovis has been considered an important cause of mastitis, pneumonia, and arthritis in bovines with a highly detrimental economic impact in the livestock industry. Previous epidemiological studies, using different typing techniques showed that the isolates were usually heterogeneous and results were difficult to compare between different laboratories. Reliable and comparable molecular typing techniques using geographically and temporal distinct isolates have never been used. The objective of this study was to use multiple-locus variable-number tandem-repeat analysis (MLVA) for the differentiation of M. bovis isolates. This typing scheme was developed using the sequenced genome of the M. bovis PG45 type strain. Nine tandem-repeat sequences were selected and the genetic diversity of 37 isolates of wide geographic and temporal origins was analyzed. The results were compared to those obtained with random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) for the same isolates. Cluster concordance between techniques was evaluated using Adjusted Rand and Wallace coefficients, and different cutoff levels of similarity were tested. Acceptable values of ≥0.5 for all techniques for the Wallace coefficient were only observed at the most relaxed cutoff level, i.e., 52% for MLVA, 29% for PFGE and 18% for RAPD. The Simpson's index of diversity was 0.91 for MLVA, 0.99 for RAPD analysis and 0.99 for PFGE. This MLVA assay is compatible with simple PCR and agarose gel-based electrophoresis steps as well as with high-throughput automated methods. The molecular typing scheme presented in this study provides a fast, reliable, and cost-effective typing method for surveillance of M. bovis epidemiology.     

Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation

Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.