Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Evaluation of the Dengue NS1 Ag Strip® for Detection of Dengue Virus Antigen in Aedes aegypti (Diptera: Culicidae)

Dengue fever is currently one of the most important mosquito-borne diseases that affect humans. With neither vaccines nor treatment available, prevention of the disease relies heavily on surveillance and control of mosquito vectors. In the present study, we have evaluated and showed the potential use of the Dengue NS1 Ag Strip® for the detection of dengue virus (DENV) in Aedes aegypti. Initial results showed that the sensitivity of the test kit in detecting DENV in wild-caught mosquitoes is comparable to that of real-time reverse transcriptase–polymerase chain reaction. The detection of naturally infected Ae. aegypti with the NS1 rapid test kit in our dengue cluster investigation further illustrates its potential use for surveillance of DENV in wild mosquito populations. The kit can easily be used in a simple field station, and minimal training is required. The results can be obtained in less than an hour. Employment of the kit in the field could help guide mosquito control operations in the prioritization of resources in controlling the transmission of DENV. In this study the potential of the kit for field surveillance of infected dengue vectors, which are epidemiologically important, has been demonstrated

Aedes (Stegomyia) albopictus (Skuse) : a potential vector of Zika virus in Singapore

Background: Zika virus (ZIKV) is a little known arbovirus until it caused a major outbreak in the Pacific Island of Yap in 2007. Although the virus has a wide geographic distribution, most of the known vectors are sylvatic Aedes mosquitoes from Africa where the virus was first isolated. Presently, Ae. aegypti is the only known vector to transmit the virus outside the African continent, though Ae. albopictus has long been a suspected vector. Currently, Ae. albopictus has been shown capable of transmitting more than 20 arboviruses and its notoriety as an important vector came to light during the recent chikungunya pandemic. The vulnerability of Singapore to emerging infectious arboviruses has stimulated our interest to determine the competence of local Ae. albopictus to transmit ZIKV. Methodology/Principal Findings: To determine the competence of Ae. albopictus to ZIKV, we orally infected local mosquito strains to a Ugandan strain virus. Fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 80–85%RH. Twelve mosquitoes were then sampled daily from day one to seven and on day 10 and 14 post infection (pi). Zika virus titre in the midgut and salivary glands of each mosquito were determined using tissue culture infectious dose50 assay, while transmissibility of the virus was determined by detecting viral antigen in the mosquito saliva by qRT-PCR. High dissemination and transmission rate of ZIKV were observed. By day 7-pi, all mosquitoes have disseminated infection and 73% of these mosquitoes have ZIKV in their saliva. By day 10-pi, all mosquitoes were potentially infectious. Conclusions/Significance: The study highlighted the potential of Ae. albopictus to transmit ZIKV and the possibility that the virus could be established locally. Nonetheless, the threat of ZIKV can be mitigated by existing dengue and chikungunya control program being implemented in Singapore.Published versio

Lineage Replacement Associated with Fitness Gain in Mammalian Cells and Aedes aegypti: A Catalyst for Dengue Virus Type 2 Transmission

Shifting of virus serotypes and clade replacement events are known to drive dengue epidemics. However, only a few studies have attempted to elucidate the virus attributes that contribute to such epidemics. In 2007, Singapore experienced a dengue outbreak affecting more than 8000 individuals. The outbreak ensued with the shuffling of dominant clades (from clade I to clade II) of Dengue virus 2 (DENV-2) cosmopolitan genotype, at a time when the Aedes premise index was significantly low. Therefore, we hypothesized that clade II had higher epidemic potential and fitness than clade I. To test this hypothesis, we tested the replication and apoptotic qualities of clade I and II isolates in mammalian cells and their ability to infect and disseminate in a field strain of Ae. Aegypti. Our findings indicated that clade II replicated more efficiently in mammalian cells than clade I and possessed higher transmission potential in local vectors. This could collectively improve the epidemic potential of clade II, which dominated during the outbreak in 2007. The findings exemplify complex interactions between the emergence, adaptation and transmission potential of DENV, and testify the epidemiological importance of a deeper understanding of virus and vector dynamics in endemic regions

Infection, dissemination and transmission rates for <i>Ae. albopictus</i> orally fed with ZIKV and held at 29°C at various days post-infection.

<p>MG = midgut; SG = salivary gland.</p

Molecular Epidemiological Investigation of Plasmodium knowlesi in Humans and Macaques in Singapore

Singapore reported its first locally acquired human Plasmodium knowlesi infection in 2007, involving a soldier who had undergone training in a forested area where long-tailed macaques are frequently seen. Comprehensive disease surveillance and monitoring system that was set up after the initial case detected four additional human P. knowlesi cases in 2007 and one in 2008. All involved military personnel who had undergone training in the forested area, and none had traveled out of Singapore 1 month before the onset of symptoms. Screening for malaria parasites on blood obtained from long-tailed macaques revealed that wild monkeys (n = 3) caught from the forested area were infected with P. knowlesi, whereas peri-domestic monkeys (n = 10) caught from a nature reserve park were not infected with any malaria parasites. Phylogenetic analysis of the nonrepeat region of the P. knowlesi csp genes showed that the sequences obtained from the human cases were not distinct from those obtained from wild monkeys. Further, certain genotypes were shared between samples from humans and macaques. Our findings provide evidence that long-tailed macaques are the natural hosts of P. knowlesi in Singapore and the human cases acquired their infection in the same vicinity where these monkeys are found. Further, the risk of acquiring P. knowlesi infection among the general population of Singapore is small as evident from the absence of P. knowlesi in peri-domestic monkeys

Entomological Investigation and Control of a Chikungunya Cluster in Singapore

In August 2008, a team from the National Environmental Agency conducted an entomological investigation of a chikungunya cluster in Singapore, with the primary aim of identifying the vector responsible for the outbreak and to assess the vector control operation. A total of 173 adult mosquitoes were caught using both the sweep-net method and the BG Sentinel Traps in and around the affected workers' quarters. Of these, 120 (69.4%) were Aedes albopictus and the rest were Culex quinquefasciatus. More than 2700 Ae. albopictus larvae were also collected from 33 breeding habitats detected. No Aedes aegypti was found. During the preintervention period, 6 (8.4%) out of 71 adult female Ae. albopictus were found positive for the chikungunya virus (CHIKV). Vector control measures resulted in a 90% reduction of adult Ae. albopictus caught by BG Sentinel Traps. Postintervention surveillance revealed the presence of CHIKV-positive mosquitoes. These findings led to continued intensive vector control operation in the affected area that further reduced vector population and interrupted the transmission of the disease. The E1 gene sequence of the CHIKV was identical to those of CHIKV isolated from human chikungunya cases working in the affected area, and contained the A226V mutation. The incrimination of Ae. albopictus as a major vector involved in the transmission of A226V CHIKV had led to the revision of chikungunya control strategy in Singapore. This study suggests the benefit of a vector control program that includes the evaluation of control measures in conjunction to virological surveillance in vector population