Laboratory-Enhanced Dengue Sentinel Surveillance in Colombo District,Sri Lanka: 2012-2014

Introduction

Dengue has emerged as a significant public health problem in Sri Lanka. Historically surveillance was passive, with mandatory dengue notifications based on clinical diagnosis with only limited laboratory confirmation. To obtain more accurate data on the disease burden of dengue, we set up a laboratory-based enhanced sentinel surveillance system in Colombo District. Here we describe the study design and report our findings of enhanced surveillance in the years 2012–2014.

Methods

Three outpatient clinics and three government hospitals in Colombo District that covered most of the Colombo metropolitan area were selected for the sentinel surveillance system. Up to 60 patients per week presenting with an undifferentiated fever were enrolled. Acute blood samples from each patient were tested by dengue specific PCR, NS1 ELISA and IgM ELISA. A sub-set of samples was sent to Duke-NUS Singapore for quality assurance, virus isolation and serotyping. Trained medical research assistants used a standardized case report form to record clinical and epidemiological data. Clinical diagnoses by the clinicians-in-charge were recorded for hospitalized cases.

Results

Of 3,127 febrile cases, 43.6% were PCR and/or NS1 positive for dengue. A high proportion of lab confirmed dengue was observed from inpatients (IPD) (53.9%) compared to outpatient (clinics in hospitals and general practice) (7.6%). Dengue hemorrhagic fever (DHF) was diagnosed in 11% of patients at the time of first contact, and the median day of illness at time of presentation to the sentinel sites was 4. Dengue serotype 1 was responsible for 85% of the cases and serotype 4 for 15%. The sensitivity and specificity of the clinicians’ presumptive diagnosis of dengue was 84% and 34%, respectively.

Conclusion

DENV-1, and to a lesser degree DENV-4, infection were responsible for a high proportion of febrile illnesses in Colombo in the years 2012 to 2014. Clinicians’ diagnoses were associated with high sensitivity, but laboratory confirmation is required to enhance specificity.     

A mutation in aspergillus nidulans that blocks the transition from interphase to prophase

In order to develop a method for obtaining mitotic synchrony in aspergillus nidulans, we have characterized previously isolated heat-sensitive nim mutations that block the nuclear division cycle in interphase at restrictive temperature. After 3.5 h at restrictive temperature the mitotic index of a strain carrying one of these mutations, nimA5, was 0, but when this strain was subsequently shifted from restrictive to permissive temperature the mitotic index increased rapidly, reaching a maximum of 78 percent after 7.5 min. When this strain was examined electron-microscopically, mitotic spindles were absent at restrictive temperature. From these data we conclude that at restrictive temperature nimA5 blocks the nuclear division cycle at a point immediately preceding the initiation of chromosomal condensation and mitotic microtubule assembly, and upon shifting to permissive control over the initiation of microtubule assembly and chromosomal condensation in vivo through a simple temperature shift and, consequently, nimA5 should be a powerful tool for studying these processes. Electron-microscopic examination of spindles of material synchronized in this manner reveals that spindle formation, although very rapid, is gradual in the sense that spindle microtubule numbers increase as spindle formation proceeds.     

DEVELOPMENTAL CORRELATES OF GENOME SIZE IN PLETHODONTID SALAMANDERS AND THEIR IMPLICATIONS FOR GENOME EVOLUTION

We present an analysis of the evolutionary relationship between genome size (C-value, mass of DNA per haploid nucleus) and developmental rate using observations of limb regeneration in salamanders of the family Plethodontidae. Rates of growth and differentiation of regenerating limbs are reported for 27 plethodontid species whose C-values range from 14 to 76 picograms. A phylogenetic analysis employing Felsenstein's method of independent contrasts indicates that rate of differentiation is inversely proportional to genome size, although we have not identified any statistically significant association between genome size and the growth rate of regenerating tissue. Our results are consistent with an interpretation that genome size may place a limit on the maximum rate of regeneration attainable in plethodontid salamanders. The implications of our findings for the “junk DNA,” “nucleotypic DNA,” “selfish DNA,” and “skeletal DNA” hypotheses of genome evolution are discussed.     

The biological significance of variation in satellite DNA and heterochromatin in newts of the genus Triturus: an evolutionary perspective

The functional and evolutionary significance of highly repetitive, simple sequence (satellite) DNA is analysed by examining available information on the patterns of variation of heterochromatin and cloned satellites among newts (family Salamandridae), and particularly species of the European genus Triturus. This information is used to develop a model linking evolutionary changes in satellite DNAs and chromosome structure. In this model, satellites accumulate initially in large tandem blocks around centromeres of some or all of the chromosomes, mainly by repeated chromosomal exchanges in these regions. Centromeric blocks later become broken up and dispersed by small, random chromosome rearrangements in these regions. They are dispersed first to pericentric locations and then gradually more distally into the chromosome arms and telomeres. Dispersal of a particular satellite is accompanied by changes in sequence structure (for example, base substitutions, deletions, etc.) and a corresponding decrease in its detectability at either the molecular or cytological level. On the basis of this model, observed satellites in newt species may be classified as 'old', 'young', or of 'intermediate' phylogenetic age. The functions and effects of satellite DNA and heterochromatin at the cellular and organismal levels are also discussed. It is suggested that satellite DNA may have an impact on cell proliferation through the effect of late-replicating satellite-rich heterochromatin on the duration of S-phase of the cell cycle. It is argued that even small alterations in cell cycle time due to changes in heterochromatin amount may have magnified effects on organismal growth that may be of adaptive significance.     

Global gene expression analysis of bovine somatic cell nuclear transfer blastocysts and cotyledons

Low developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos is a universal problem. Abnormal placentation has been commonly reported in SCNT pregnancies from a number of species. The present study employed Affymetrix bovine expression microarrays to examine global gene expression patterns of SCNT and in vivo produced (AI) blastocysts as well as cotyledons from day‐70 SCNT and AI pregnancies. SCNT and AI embryos and cotyledons were analyzed for differential expression. Also in an attempt to establish a link between abnormal gene expression patterns in early embryos and cotyledons, differentially expressed genes were compared between the two studies. Microarray analysis yielded a list of 28 genes differentially expressed between SCNT and AI blastocysts and 19 differentially expressed cotyledon genes. None of the differentially expressed genes were common to both groups, although major histocompatibility complex I (MHCI) was significant in the embryo data and approached significance in the cotyledon data. This is the first study to report global gene expression patterns in bovine AI and SCNT cotyledons. The embryonic gene expression data reported here adds to a growing body of data that indicates the common occurrence of aberrant gene expression in early SCNT embryos. Mol. Reprod. Dev. 76: 471–482, 2009. © 2008 Wiley‐Liss, Inc.     

Colcemid‐treatment of heifer oocytes enhances nuclear transfer embryonic development,establishment of pregnancy and development to term

Four experiments were designed to examine the effects of colcemid, a microtubule assembly inhibitor, on the development of bovine nuclear transfer (NT) embryos in vitro and in vivo. Recipient oocytes matured at different times were exposed to colcemid. Approximately 80–93% of the exposed oocytes, with or without the first polar body (PB1), developed obvious membrane projections. In Experiment 1, oocytes matured for either 14–15 or 16–17 hr, treated with colcemid and used as recipient cytoplasm for NT resulted in over 40% blastocyst development. In Experiment 2, oocytes matured for 16–17 hr were treated with either 0.2 or 0.4 µg/ml colcemid for 2–3 or 5–6 hr, respectively. The percentages of blastocyst development (39–42%) were not statistically different among the different colcemid treatment groups, but were both higher (P < 0.05) than the control group (30%). Colcemid concentrations and length of colcemid treatment of oocytes did not affect their ability to support NT embryo development to the blastocyst and hatched blastocyst stages. Results from Experiment 3 indicate that semi‐defined medium increases morula and blastocyst development of NT embryos derived fromcolcemid‐treated oocytes under 5% CO₂ in air atmosphere. In addition, cell numbers of blastocysts in colcemid‐treated groups were numerically higher than the control groups. After embryo transfer, higher (P < 0.05) pregnant rates were obtained from the colcemid‐treated group than the nontreated group. Five of 40 recipients (12.5%) which received embryos from colcemid‐treated oocytes delivered healthy calves, significantly higher than those recipients (3.3%) that received embryos derived from nontreated oocytes. Mol. Reprod. Dev. 76: 620–628, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of Novel Methane-, Ethane-, and Propane-Oxidizing Bacteria at Marine Hydrocarbon Seeps by Stable Isotope Probing

Marine hydrocarbon seeps supply oil and gas to microorganisms in sediments and overlying water. We used stable isotope probing (SIP) to identify aerobic bacteria oxidizing gaseous hydrocarbons in surface sediment from the Coal Oil Point seep field located offshore of Santa Barbara, California. After incubating sediment with ¹³C-labeled methane, ethane, or propane, we confirmed the incorporation of ¹³C into fatty acids and DNA. Terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of the 16S rRNA and particulate methane monooxygenase (pmoA) genes in ¹³C-DNA revealed groups of microbes not previously thought to contribute to methane, ethane, or propane oxidation. First, ¹³C methane was primarily assimilated by Gammaproteobacteria species from the family Methylococcaceae, Gammaproteobacteria related to Methylophaga, and Betaproteobacteria from the family Methylophilaceae. Species of the latter two genera have not been previously shown to oxidize methane and may have been cross-feeding on methanol, but species of both genera were heavily labeled after just 3 days. pmoA sequences were affiliated with species of Methylococcaceae, but most were not closely related to cultured methanotrophs. Second, ¹³C ethane was consumed by members of a novel group of Methylococcaceae. Growth with ethane as the major carbon source has not previously been observed in members of the Methylococcaceae; a highly divergent pmoA-like gene detected in the ¹³C-labeled DNA may encode an ethane monooxygenase. Third, ¹³C propane was consumed by members of a group of unclassified Gammaproteobacteria species not previously linked to propane oxidation. This study identifies several bacterial lineages as participants in the oxidation of gaseous hydrocarbons in marine seeps and supports the idea of an alternate function for some pmoA-like genes.Hydrocarbon seeps are widespread along continental margins and emit large amounts of oil and gas into the surrounding environment. This gas is primarily composed of methane, a powerful greenhouse gas, and marine hydrocarbon seeps are estimated to contribute 20 Tg year⁻¹ methane to the atmosphere, representing about 5% of the total atmospheric flux (21, 39). Seeps of thermogenic gas also release an estimated 0.45 Tg year⁻¹ ethane and 0.09 Tg year⁻¹ propane to the atmosphere (20). Each of these three fluxes would be substantially larger if not for microbial oxidation in the sediments and water column (68). Methane, ethane, and propane are subject to anaerobic oxidation in anoxic sediments and water columns (44, 53, 68) or to aerobic oxidation in oxic and suboxic water columns and oxygenated surface sediment (10, 47, 53, 80). We focus here on aerobic oxidation.The majority of known aerobic methane-oxidizing bacteria are members of either Gammaproteobacteria (type I) or Alphaproteobacteria (type II) (29), though several strains of highly acidophilic methanotrophic Verrucomicrobia have also been recently isolated (63). Most methanotrophs are capable of growth only on methane or other one-carbon compounds (17, 29), using a methane monooxygenase (MMO) enzyme to oxidize methane to methanol. There are two known forms of this enzyme: soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO). sMMO is a soluble, di-iron-containing monooxygenase found only in certain methanotrophs and typically expressed only under low-copper conditions (57). In contrast, pMMO is a membrane-bound enzyme believed to contain copper and iron (26). It is found in all known methanotrophs, with the exception of species of the genus Methylocella (16). pmoA, the gene encoding the α subunit of pMMO, is often used to identify methanotrophic bacteria (54). Very few methanotrophs from marine environments have been cultured (22, 49, 72, 74), but several previous studies of marine methanotrophs (35, 62, 77, 82, 85) have been performed with culture-independent methods and have almost exclusively detected type I methanotrophs. Many of the pmoA sequences from methane seep sites are quite different from those of cultured organisms, suggesting that these environments may contain many novel methanotrophs (77, 82, 85).Even less is known about the organisms that oxidize ethane or propane in marine environments. The number of such isolates, which primarily represent high G+C Gram-positive bacteria (Nocardia, Pseudonocardia, Gordonia, Mycobacterium, and Rhodococcus) or Pseudomonas species, is limited (70). Nearly all of these strains were isolated from soil and selected for their ability to grow on propane or n-butane as the sole carbon source. Most propane-oxidizing strains can oxidize butane, as well as a range of longer chain n-alkanes, but differ in the ability to oxidize ethane. These strains show little, if any, ability to oxidize methane, and none have been shown to grow with methane as the sole carbon source (13, 27, 38, 45, 65). As with methane metabolism, the first step in aerobic ethane and propane metabolism is the oxidation of the alkane to an alcohol (70). Several different enzymes are known to catalyze this step. Thauera butanivorans uses a soluble di-iron butane monooxygenase related to sMMO to oxidize C₂ through C₉ n-alkanes (18, 73). Gordonia sp. strain TY-5, Mycobacterium sp. strain TY-6, and Pseudonocardia sp. strain TY-7 contain soluble di-iron propane monooxygenases that are capable of both terminal and subterminal propane oxidation and differ in their substrate ranges (45, 46). Nocardioides sp. strain CF8 is believed to possess a copper-containing monooxygenase similar to pMMO and ammonia monooxygenase (27, 28). An alkane hydroxylase typically used to oxidize longer-chain n-alkanes has also shown some ability to oxidize propane and butane but not ethane (38). The variety of enzymes and their substrate ranges make it difficult to identify ethane or propane oxidizers with a single functional gene.In order to identify the organisms responsible for methane, ethane, and propane oxidation at hydrocarbon seeps, we used stable isotope probing (SIP). SIP allows the identification of organisms actively consuming a ¹³C-labeled substrate of interest, based on the incorporation of ¹³C into biomass, including DNA and lipids (67). We collected sediment from the Coal Oil Point seep field and incubated sediment-seawater slurries with ¹³C methane, ethane, or propane. Samples were removed at three time points, chosen to ensure sufficient ¹³C incorporation into DNA while minimizing the spread of ¹³C through the community as a result of cross-feeding on metabolic byproducts. ¹³C-DNA was separated from ¹²C-DNA by CsCl density gradient ultracentrifugation, and we used the fractionated DNA for terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis. We also measured ¹³C incorporation into fatty acids in order to confirm significant ¹³C enrichment in membrane lipids, to determine the carbon labeling pattern for each substrate and lipid, and to further characterize the composition of the microbial community.