How Introducing a Registry With Automated Alerts for Carbapenem-resistant Enterobacteriaceae (CRE) May Help Control CRE Spread in a Region.

BACKGROUND: Regions are considering the use of electronic registries to track patients who carry antibiotic-resistant bacteria, including carbapenem-resistant Enterobacteriaceae (CRE). Implementing such a registry can be challenging and requires time, effort, and resources; therefore, there is a need to better understand the potential impact. METHODS: We developed an agent-based model of all inpatient healthcare facilities (90 acute care hospitals, 9 long-term acute care hospitals, 351 skilled nursing facilities, and 12 ventilator-capable skilled nursing facilities) in the Chicago metropolitan area, surrounding communities, and patient flow using our Regional Healthcare Ecosystem Analyst software platform. Scenarios explored the impact of a registry that tracked patients carrying CRE to help guide infection prevention and control. RESULTS: When all Illinois facilities participated (n = 402), the registry reduced the number of new carriers by 11.7% and CRE prevalence by 7.6% over a 3-year period. When 75% of the largest Illinois facilities participated (n = 304), registry use resulted in a 11.6% relative reduction in new carriers (16.9% and 1.2% in participating and nonparticipating facilities, respectively) and 5.0% relative reduction in prevalence. When 50% participated (n = 201), there were 10.7% and 5.6% relative reductions in incident carriers and prevalence, respectively. When 25% participated (n = 101), there was a 9.1% relative reduction in incident carriers (20.4% and 1.6% in participating and nonparticipating facilities, respectively) and 2.8% relative reduction in prevalence. CONCLUSIONS: Implementing an extensively drug-resistant organism registry reduced CRE spread, even when only 25% of the largest Illinois facilities participated due to patient sharing. Nonparticipating facilities garnered benefits, with reductions in new carriers.

Exploring the potential public health benefits of universal influenza vaccine.

Background: Broadly protective, long-lasting universal influenza vaccines are under development in response to low-moderate seasonal vaccine effectiveness, frequent genetic changes in circulating viruses and extended turnaround for vaccine manufacture. Because a long-lasting vaccine might be less effective than a seasonal vaccine that has been matched to current circulating strains, the public health impact of its introduction should be evaluated.Methods: A modified agent-based model (ABM) examined multi-year effects of a universal vaccine among 18 to 49-year-olds, given in Year 1 only. The proportion of vaccinated 18 to 49-year-olds who received universal vaccine was varied from 0% to 100%. Model parameters were drawn from US databases and the medical literature. Outcomes were 4-year cumulative and annual influenza cases as well as annual cases averted/100,000 population for 3 age groups, 0-17 years, 18-49 years and 50+ years.Results: In Year 1 when universal vaccine was given to 50% or 100% of all vaccinated 18 to 49-year-olds, more influenza cases occurred, compared to no universal vaccine, but fewer cases occurred in Years 2-4 as overall protection increased. Cumulative averted cases over 4 years in 18 to 49-year-olds were 892/100,000 and 1,687/100,000 population for the 50% and 100% universal vaccine for 18 to 49-year-olds scenarios, respectively, with additional benefits to children and older adults through indirect effects.Conclusions: In ABM, the universal vaccine with a conservative VE estimate given once to 18 to 49-year-olds reduced influenza cases among all age groups in Years 2-4 following its introduction. Reduced influenza burden may occur sooner if VE of universal vaccines exceeds that assumed in these models.

The value of tailoring vial sizes to populations and locations.

BACKGROUND: Frequently, a country will procure a single vaccine vial size, but the question remains whether tailoring the use of different size vaccine vial presentations based on populations or location characteristics within a single country could provide additional benefits, such as reducing open vial wastage (OVW) or reducing missed vaccination opportunities. METHODS: Using the Highly Extensible Resource for Modeling Supply Chains (HERMES) software, we built a simulation model of the Zambia routine vaccine supply chain. At baseline, we distributed 10-dose Measles-Rubella (MR) vials to all locations, and then distributed 5-dose and 1-dose MR vials to (1) all locations, (2) rural districts, (3) rural health facilities, (4) outreach sites, and (5) locations with average MR session sizes <5 and <10 children. We ran sensitivity on each scenario using MR vial opening thresholds of 0% and 50%, i.e. a healthcare worker opens an MR vaccine for any number of children (0%) or if at least half will be used (50%). RESULTS: Replacing 10-dose MR with 5-dose MR vials everywhere led to the largest reduction in MR OVW, saving 573,892 doses (103,161 doses with the 50% vial opening threshold) and improving MR availability by 1% (9%). This scenario, however, increased cold chain utilization and led to a 1% decrease in availability of other vaccines. Tailoring 5-dose MR vials to rural health facilities or based on average session size reduced cold transport constraints, increased total vaccine availability (+1%) and reduced total cost per dose administered (-$0.01) compared to baseline. CONCLUSIONS: In Zambia, tailoring 5-dose MR vials to rural health facilities or by average session size results in the highest total vaccine availability compared to all other scenarios (regardless of OVT policy) by reducing open vial wastage without increasing cold chain utilization.

The potential effects of introducing microneedle patch vaccines into routine vaccine supply chains.

BACKGROUND: Microneedle patch (MNP) technology is designed to simplify the process of vaccine administration; however, depending on its characteristics, MNP technology may provide additional benefits beyond the point-of-use, particularly for vaccine supply chains. METHODS: Using the HERMES modeling software, we examined replacing four routine vaccines - Measles-containing vaccine (MCV), Tetanus toxoid (TT), Rotavirus (Rota) and Pentavalent (Penta) - with MNP versions in the routine vaccine supply chains of Benin, Bihar (India), and Mozambique. RESULTS: Replacing MCV with an MNP (5 cm3-per-dose, 2-month thermostability, current single-dose price-per-dose) improved MCV availability by 13%, 1% and 6% in Benin, Bihar and Mozambique, respectively, and total vaccine availability by 1% in Benin and Mozambique, while increasing the total cost per dose administered by $0.07 in Benin, $0.56 in Bihar and $0.11 in Mozambique. Replacing TT with an MNP improved TT and total vaccine availability (3% and <1%) in Mozambique only, when the patch was 5 cm3 and 2-months thermostable but increased total cost per dose administered by $0.14. Replacing Rota with an MNP (at 5-15 cm3-per-dose, 1-2 month thermostable) improved Rota and total vaccine availability, but only improved Rota vaccine availability in Bihar (at 5 cm3, 1-2 months thermostable), while decreasing total vaccine availability by 1%. Finally, replacing Penta with an MNP (at 5 cm3, 2-months thermostable) improved Penta vaccine availability by 1-8% and total availability by <1-9%. CONCLUSIONS: An MNP for MCV, TT, Rota, or Penta would need to have a smaller or equal volume-per-dose than existing vaccine formulations and be able to be stored outside the cold chain for a continuous period of at least two months to provide additional benefits to all three supply chains under modeled conditions.

Epidemiologic and economic impact of pharmacies as vaccination locations during an influenza epidemic.

INTRODUCTION: During an influenza epidemic, where early vaccination is crucial, pharmacies may be a resource to increase vaccine distribution reach and capacity. METHODS: We utilized an agent-based model of the US and a clinical and economics outcomes model to simulate the impact of different influenza epidemics and the impact of utilizing pharmacies in addition to traditional (hospitals, clinic/physician offices, and urgent care centers) locations for vaccination for the year 2017. RESULTS: For an epidemic with a reproductive rate (R0) of 1.30, adding pharmacies with typical business hours averted 11.9 million symptomatic influenza cases, 23,577 to 94,307 deaths, $1.0 billion in direct (vaccine administration and healthcare) costs, $4.2-44.4 billion in productivity losses, and $5.2-45.3 billion in overall costs (varying with mortality rate). Increasing the epidemic severity (R0 of 1.63), averted 16.0 million symptomatic influenza cases, 35,407 to 141,625 deaths, $1.9 billion in direct costs, $6.0-65.5 billion in productivity losses, and $7.8-67.3 billion in overall costs (varying with mortality rate). Extending pharmacy hours averted up to 16.5 million symptomatic influenza cases, 145,278 deaths, $1.9 billion direct costs, $4.1 billion in productivity loss, and $69.5 billion in overall costs. Adding pharmacies resulted in a cost-benefit of $4.1 to $11.5 billion, varying epidemic severity, mortality rate, pharmacy hours, location vaccination rate, and delay in the availability of the vaccine. CONCLUSIONS: Administering vaccines through pharmacies in addition to traditional locations in the event of an epidemic can increase vaccination coverage, mitigating up to 23.7 million symptomatic influenza cases, providing cost-savings up to $2.8 billion to third-party payers and $99.8 billion to society. Pharmacies should be considered as points of dispensing epidemic vaccines in addition to traditional settings as soon as vaccines become available.

The Economic Value of Long-Lasting Insecticidal Nets and Indoor Residual Spraying Implementation in Mozambique.

AbstractMalaria-endemic countries have to decide how much of their limited resources for vector control to allocate toward implementing long-lasting insecticidal nets (LLINs) versus indoor residual spraying (IRS). To help the Mozambique Ministry of Health use an evidence-based approach to determine funding allocation toward various malaria control strategies, the Global Fund convened the Mozambique Modeling Working Group which then used JANUS, a software platform that includes integrated computational economic, operational, and clinical outcome models that can link with different transmission models (in this case, OpenMalaria) to determine the economic value of vector control strategies. Any increase in LLINs (from 80% baseline coverage) or IRS (from 80% baseline coverage) would be cost-effective (incremental cost-effectiveness ratios ≤ $114/disability-adjusted life year averted). However, LLIN coverage increases tend to be more cost-effective than similar IRS coverage increases, except where both pyrethroid resistance is high and LLIN usage is low. In high-transmission northern regions, increasing LLIN coverage would be more cost-effective than increasing IRS coverage. In medium-transmission central regions, changing from LLINs to IRS would be more costly and less effective. In low-transmission southern regions, LLINs were more costly and less effective than IRS, due to low LLIN usage. In regions where LLINs are more cost-effective than IRS, it is worth considering prioritizing LLIN coverage and use. However, IRS may have an important role in insecticide resistance management and epidemic control. Malaria intervention campaigns are not a one-size-fits-all solution, and tailored approaches are necessary to account for the heterogeneity of malaria epidemiology.

Does cost-effectiveness of influenza vaccine choice vary across the U.S.? An agent-based modeling study.

BACKGROUND: In a prior agent-based modeling study, offering a choice of influenza vaccine type was shown to be cost-effective when the simulated population represented the large, Washington DC metropolitan area. This study calculated the public health impact and cost-effectiveness of the same four strategies: No Choice, Pediatric Choice, Adult Choice, or Choice for Both Age Groups in five United States (U.S.) counties selected to represent extremes in population age distribution. METHODS: The choice offered was either inactivated influenza vaccine delivered intramuscularly with a needle (IIV-IM) or an age-appropriate needle-sparing vaccine, specifically, the nasal spray (LAIV) or intradermal (IIV-ID) delivery system. Using agent-based modeling, individuals were simulated as they interacted with others, and influenza was tracked as it spread through each population. Influenza vaccination coverage derived from Centers for Disease Control and Prevention (CDC) data, was increased by 6.5% (range 3.25%-11.25%) to reflect the effects of vaccine choice. RESULTS: Assuming moderate influenza infectivity, the number of averted cases was highest for the Choice for Both Age Groups in all five counties despite differing demographic profiles. In a cost-effectiveness analysis, Choice for Both Age Groups was the dominant strategy. Sensitivity analyses varying influenza infectivity, costs, and degrees of vaccine coverage increase due to choice, supported the base case findings. CONCLUSION: Offering a choice to receive a needle-sparing influenza vaccine has the potential to significantly reduce influenza disease burden and to be cost saving. Consistent findings across diverse populations confirmed these findings.

Potential Consequences of Not Using Live Attenuated Influenza Vaccine.

INTRODUCTION: Decreased live attenuated influenza vaccine (LAIV) effectiveness in the U.S. prompted the Advisory Committee on Immunization Practices in August 2016 to recommend against this vaccine's use. However, overall influenza uptake increases when LAIV is available and, unlike the U.S., LAIV has retained its effectiveness in other countries. These opposing countercurrents create a dilemma. METHODS: To examine the potential consequences of the decision to not recommend LAIV, which may result in decreased influenza vaccination coverage in the U.S. population, a Markov decision analysis model was used to examine influenza vaccination options in U.S. children aged 2-8 years. Data were compiled and analyzed in 2016. RESULTS: Using recently observed low LAIV effectiveness values, fewer influenza cases will occur if LAIV is not used compared with having LAIV as a vaccine option. However, having the option to use LAIV may be favored if LAIV effectiveness returns to prior levels or if the absence of vaccine choice substantially decreases overall vaccine uptake. CONCLUSIONS: Continued surveillance of LAIV effectiveness and influenza vaccine uptake is warranted, given their importance in influenza vaccination policy decisions.

Does Choice of Influenza Vaccine Type Change Disease Burden and Cost-Effectiveness in the United States? An Agent-Based Modeling Study.

Offering a choice of influenza vaccine type may increase vaccine coverage and reduce disease burden, but it is more costly. This study calculated the public health impact and cost-effectiveness of 4 strategies: no choice, pediatric choice, adult choice, or choice for both age groups. Using agent-based modeling, individuals were simulated as they interacted with others, and influenza was tracked as it spread through a population in Washington, DC. Influenza vaccination coverage derived from data from the Centers for Disease Control and Prevention was increased by 6.5% (range, 3.25%-11.25%), reflecting changes due to vaccine choice. With moderate influenza infectivity, the number of cases averaged 1,117,285 for no choice, 1,083,126 for pediatric choice, 1,009,026 for adult choice, and 975,818 for choice for both age groups. Averted cases increased with increased coverage and were highest for the choice-for-both-age-groups strategy; adult choice also reduced cases in children. In cost-effectiveness analysis, choice for both age groups was dominant when choice increased vaccine coverage by ≥3.25%. Offering a choice of influenza vaccines, with reasonable resultant increases in coverage, decreased influenza cases by >100,000 with a favorable cost-effectiveness profile. Clinical trials testing the predictions made based on these simulation results and deliberation of policies and procedures to facilitate choice should be considered.

Cost Effectiveness of Influenza Vaccine Choices in Children Aged 2-8 Years in the U.S.

INTRODUCTION: Prior evidence found live attenuated influenza vaccine (LAIV) more effective than inactivated influenza vaccine (IIV) in children aged 2-8 years, leading CDC in 2014 to prefer LAIV use in this group. However, since 2013, LAIV has not proven superior, leading CDC in 2015 to rescind their LAIV preference statement. Here, the cost effectiveness of preferred LAIV use compared with IIV in children aged 2-8 years is estimated. METHODS: A Markov model estimated vaccination strategy cost effectiveness in terms of cost per quality-adjusted life-year gained. Base case assumptions were equal vaccine uptake; IIV use when LAIV was not indicated (in 11.7% of the cohort); and no indirect vaccination effects. Sensitivity analyses included estimates of indirect effects from both equation- and agent-based models. Analyses were performed in 2014-2015. RESULTS: Using prior effectiveness data in children aged 2-8 years (LAIV=83%, IIV=64%), preferred LAIV use was less costly and more effective than IIV (dominant), with results sensitive only to LAIV and IIV effectiveness variation. Using 2014-2015 U.S. effectiveness data (LAIV=0%, IIV=15%), IIV was dominant. In two-way sensitivity analyses, LAIV use was cost saving over the entire range of IIV effectiveness (0%-81%) when absolute LAIV effectiveness was >7.1% higher than IIV, but never cost saving when absolute LAIV effectiveness was <3.5% higher than IIV. CONCLUSIONS: Results support CDC's decision to no longer prefer LAIV use and provide guidance on effectiveness differences between influenza vaccines that might lead to preferential LAIV recommendation for children aged 2-8 years.

Quantifying influenza virus diversity and transmission in humans.

Influenza A virus is characterized by high genetic diversity. However, most of what is known about influenza evolution has come from consensus sequences sampled at the epidemiological scale that only represent the dominant virus lineage within each infected host. Less is known about the extent of within-host virus diversity and what proportion of this diversity is transmitted between individuals. To characterize virus variants that achieve sustainable transmission in new hosts, we examined within-host virus genetic diversity in household donor-recipient pairs from the first wave of the 2009 H1N1 pandemic when seasonal H3N2 was co-circulating. Although the same variants were found in multiple members of the community, the relative frequencies of variants fluctuated, with patterns of genetic variation more similar within than between households. We estimated the effective population size of influenza A virus across donor-recipient pairs to be approximately 100-200 contributing members, which enabled the transmission of multiple lineages, including antigenic variants.

Identification of mammalian-adapting mutations in the polymerase complex of an avian H5N1 influenza virus.

Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population. The factors that enable avian H5N1 influenza viruses to replicate in humans are not completely understood. Here we use a high-throughput screening approach to identify novel mutations in the polymerase genes of an avian H5N1 virus that confer efficient polymerase activity in mammalian cells. Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus. The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans.

Genome scale evolution of myxoma virus reveals host-pathogen adaptation and rapid geographic spread.

The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.

Comparative analysis of the complete genome sequence of the California MSW strain of myxoma virus reveals potential host adaptations.

Myxomatosis is a rapidly lethal disease of European rabbits that is caused by myxoma virus (MYXV). The introduction of a South American strain of MYXV into the European rabbit population of Australia is the classic case of host-pathogen coevolution following cross-species transmission. The most virulent strains of MYXV for European rabbits are the Californian viruses, found in the Pacific states of the United States and the Baja Peninsula, Mexico. The natural host of Californian MYXV is the brush rabbit, Sylvilagus bachmani. We determined the complete sequence of the MSW strain of Californian MYXV and performed a comparative analysis with other MYXV genomes. The MSW genome is larger than that of the South American Lausanne (type) strain of MYXV due to an expansion of the terminal inverted repeats (TIRs) of the genome, with duplication of the M156R, M154L, M153R, M152R, and M151R genes and part of the M150R gene from the right-hand (RH) end of the genome at the left-hand (LH) TIR. Despite the extreme virulence of MSW, no novel genes were identified; five genes were disrupted by multiple indels or mutations to the ATG start codon, including two genes, M008.1L/R and M152R, with major virulence functions in European rabbits, and a sixth gene, M000.5L/R, was absent. The loss of these gene functions suggests that S. bachmani is a relatively recent host for MYXV and that duplication of virulence genes in the TIRs, gene loss, or sequence variation in other genes can compensate for the loss of M008.1L/R and M152R in infections of European rabbits.

Mammalian adaptation in the PB2 gene of avian H5N1 influenza virus.

The substitution of glutamic acid (E) for lysine (K) at position 627 of the PB2 protein of avian H5N1 viruses has been identified as a virulence and host range determinant for infection of mammals. Here, we report that the E-to-K host-adaptive mutation in the PB2 gene appeared from day 4 and 5 along the respiratory tracts of mice and was complete by day 6 postinoculation. This mutation correlated with efficient replication of the virus in mice.

Sequence analysis of in vivo defective interfering-like RNA of influenza A H1N1 pandemic virus.

Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.

Evolutionary history and attenuation of myxoma virus on two continents.

The attenuation of myxoma virus (MYXV) following its introduction as a biological control into the European rabbit populations of Australia and Europe is the canonical study of the evolution of virulence. However, the evolutionary genetics of this profound change in host-pathogen relationship is unknown. We describe the genome-scale evolution of MYXV covering a range of virulence grades sampled over 49 years from the parallel Australian and European epidemics, including the high-virulence progenitor strains released in the early 1950s. MYXV evolved rapidly over the sampling period, exhibiting one of the highest nucleotide substitution rates ever reported for a double-stranded DNA virus, and indicative of a relatively high mutation rate and/or a continually changing selective environment. Our comparative sequence data reveal that changes in virulence involved multiple genes, likely losses of gene function due to insertion-deletion events, and no mutations common to specific virulence grades. Hence, despite the similarity in selection pressures there are multiple genetic routes to attain either highly virulent or attenuated phenotypes in MYXV, resulting in convergence for phenotype but not genotype.

Presence of oseltamivir-resistant pandemic A/H1N1 minor variants before drug therapy with subsequent selection and transmission.

A small proportion (1%-1.5%) of 2009 pandemic influenza A/H1N1 virus strains (A[H1N1]pdm09) are oseltamivir resistant, almost exclusively because of a H275Y mutation in the neuraminidase protein. However, many individuals infected with resistant strains had not received antivirals. Whether drug-resistant viruses are initially present as minor variants in untreated individuals before they emerge as the dominant strain in a virus population is of great importance for predicting the speed at which resistance will arise. To address this issue, we used ultra-deep sequencing of viral populations from serial nasopharyngeal specimens from an immunocompromised child and from 2 individuals in a household outbreak. We observed that the Y275 mutation was present as a minor variant in infected hosts before the onset of therapy. We also found evidence for the transmission of this drug-resistant variant with drug-susceptible viruses. These observations provide important information on the relative fitness of the Y275 mutation in the absence of oseltamivir treatment.

Brugia malayi gene expression in response to the targeting of the Wolbachia endosymbiont by tetracycline treatment.

BACKGROUND: Brugia malayi, like most human filarial parasite species, harbors an endosymbiotic bacterium of the genus Wolbachia. Elimination of the endosymbiont leads to sterilization of the adult female. Previous biochemical and genetic studies have established that communication with its endobacterium is essential for survival of the worm. METHODOLOGY/PRINCIPAL FINDINGS: We used electron microscopy to examine the effects of antibiotic treatment on Wolbachia cell structure. We have also used microarray and quantitative RT-PCR analyses to examine the regulation of the B. malayi transcripts altered in response to the anti-Wolbachia treatment. Microscopy of worms taken from animals treated with tetracycline for 14 and 21 days (14 d and 21 d) demonstrated substantial morphologic effects on the Wolbachia endobacterium by 14 d and complete degeneration of the endobacterial structures by 21 d. We observed upregulation of transcripts primarily encoding proteins involved in amino acid synthesis and protein translation, and downregulation of transcripts involved in cuticle biosynthesis after both 7 d and 14 d of treatment. In worms exposed to tetracycline in culture, substantial effects on endobacteria morphology were evident by day 3, and extensive death of the endobacteria was observed by day 5. In a detailed examination of the expression kinetics of selected signaling genes carried out on such cultured worms, a bimodal pattern of regulation was observed. The selected genes were upregulated during the early phase of antibiotic treatment and quickly downregulated in the following days. These same genes were upregulated once more at 6 days post-treatment. CONCLUSIONS/SIGNIFICANCE: Upregulation of protein translation and amino acid synthesis may indicate a generalized stress response induced in B. malayi due to a shortage of essential nutrients/factors that are otherwise supplied by Wolbachia. Downregulation of transcripts involved in cuticle biosynthesis perhaps reflects a disruption in the normal embryogenic program. This is confirmed by the expression pattern of transcripts that may be representative of the worms' response to Wolbachia in different tissues; the early peak potentially reflects the effect of bacteria death on the embryogenic program while the second peak may be a manifestation of the adult worm response to the affected bacteria within the hypodermis.