Acetyl-CoA Synthetase Mutations S868G and V949I Do Not Confer Resistance to Antimalarial Drugs in vitro in Plasmodium falciparum.

Plasmodium falciparum acetyl-CoA synthetase (PfACAS) protein is an important source of acetyl-CoA. We detected the mutations S868G and V949I in PfACAS by whole-genome sequencing analysis in some recrudescent parasites after antimalarial treatment with artesunate and dihydroartemisinin-piperaquine, suggesting that they may confer drug resistance. Using CRISPR/Cas9 technology, we engineered parasite lines carrying the PfACAS S868G and V949I mutations in two genetic backgrounds and evaluated their susceptibility to antimalarial drugs in vitro. The results demonstrated that PfACAS S868G and V949I mutations alone or in combination were not enough to provide resistance to antimalarial drugs.

An imported malaria case with repeated episodes of neurological syndromes resulting from different Plasmodium species.

BACKGROUND: Imported cerebral malaria (CM) cases in non-endemic areas are often misdiagnosed, which delays treatment. Post-malaria neurological syndrome (PMNS) after recovery from severe malaria can also complicate diagnosis. CASE: We report an imported malaria case from West Africa with two sequential episodes with neurological syndromes within about a month. The first episode was diagnosed as CM with microscopy-positive Plasmodium falciparum infection. The second episode, occurring a month after the recovery from the first CM episode, was consistent with PMNS, since malaria parasites were not detected by microscopy in peripheral blood smears. However, this diagnosis was complicated by the detection of Plasmodium vivax in peripheral blood by PCR, suggesting a potential cause of the second episode by P. vivax. CONCLUSION: This study suggests that PMNS often occurs after severe falciparum malaria. Concurrent P. vivax infection with pathogenic biomass being predominantly extravascular further complicates accurate diagnosis.

Application of a low-cost, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay to detect Plasmodium falciparum imported from Africa.

BACKGROUND: Chinese citizens traveling abroad bring back imported malaria cases to China. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. To complement existing diagnostic methods, we aimed to develop a new loop-mediated isothermal amplification (LAMP) assay to detect and identify Plasmodium falciparum in Chinese travelers returning from Africa. METHODS: We developed a miniaturized LAMP assay to amplify the actin I gene of P. falciparum. Each reaction consumed only 25% of the reagents used in a conventional LAMP assay and the same amount of DNA templates used in nested PCR. We evaluated this LAMP assay's performance and compared it to microscopy and a nested PCR assay using 466 suspected malaria cases imported from Africa. We assessed the sensitivity of the new LAMP assay using cultured P. falciparum, clinical samples, and a plasmid construct, allowing unprecedented precision when quantifying the limit of detection. RESULTS: The new LAMP assay was highly sensitive and detected two more malaria cases than nested PCR. Compared to nested PCR, the sensitivity and specificity of the novel LAMP assay were 100% [95% confidence interval (CI) 98.5-100%] and 99.1% (95% CI 96.7-99.9%), respectively. When evaluated using serial dilutions of the plasmid construct, the detection limit of the new LAMP was as low as 102 copies/µL, 10-fold lower than PCR. The LAMP assay detected 0.01 parasites/µL of blood (equal to 0.04 parasites/µL of DNA) using cultured P. falciparum and 1-7 parasites/µL of blood (4-28 parasites/µL of DNA) in clinical samples, which is as good as or better than previously reported and commercially licensed assays. CONCLUSION: The novel LAMP assay based on the P. falciparum actin I gene was specific, sensitive, and cost-effective, as it consumes 1/4 of the reagents in a typical LAMP reaction.

In vitro susceptibility profile of Plasmodium falciparum clinical isolates from Ghana to antimalarial drugs and polymorphisms in resistance markers.

Drug resistance in Plasmodium falciparum compromises the effectiveness of antimalarial therapy. This study aimed to evaluate the extent of drug resistance in parasites obtained from international travelers returning from Ghana to guide the management of malaria cases. Eighty-two clinical parasite isolates were obtained from patients returning from Ghana in 2016-2018, of which 29 were adapted to continuous in vitro culture. Their geometric mean IC50 values to a panel of 11 antimalarial drugs, assessed using the standard SYBR Green-I drug sensitivity assay, were 2.1, 3.8, 1.0, 2.7, 17.2, 4.6, 8.3, 8.3, 19.6, 55.1, and 11,555 nM for artemether, artesunate, dihydroartemisinin, lumefantrine, mefloquine, piperaquine, naphthoquine, pyronaridine, chloroquine, quinine, and pyrimethamine, respectively. Except for chloroquine and pyrimethamine, the IC50 values for other tested drugs were below the resistance threshold. The mean ring-stage survival assay value was 0.8%, with four isolates exceeding 1%. The mean piperaquine survival assay value was 2.1%, all below 10%. Mutations associated with chloroquine resistance (pfcrt K76T and pfmdr1 N86Y) were scarce, consistent with the discontinuation of chloroquine a decade ago. Instead, the pfmdr1 86N-184F-1246D haplotype was predominant, suggesting selection by the extensive use of artemether-lumefantrine. No mutations in the pfk13 propeller domain were detected. The pfdhfr/pfdhps quadruple mutant IRNGK associated with resistance to sulfadoxine-pyrimethamine reached an 82% prevalence. In addition, five isolates had pfgch1 gene amplification but, intriguingly, increased susceptibilities to pyrimethamine. This study showed that parasites originating from Ghana were susceptible to artemisinins and the partner drugs of artemisinin-based combination therapies. Genotyping drug resistance genes identified the signature of selection by artemether-lumefantrine. Parasites showed substantial levels of resistance to the antifolate drugs. Continuous resistance surveillance is necessary to guide timely changes in drug policy.

First Detection in West Africa of a Mutation That May Contribute to Artemisinin Resistance Plasmodium falciparum.

Background: The spread of drug resistance has seriously impacted the effective treatment of infection with the malaria parasite, Plasmodium falciparum. Continuous monitoring of molecular marker polymorphisms associated with drug resistance in parasites is essential for malaria control and elimination efforts. Our study describes mutations observed in the resistance genes Pfkelch13, Pfcrt, and Pfmdr1 in imported malaria and identifies additional potential drug resistance-associated molecular markers. Methods: Chinese patients infected in Africa with P. falciparum were treated with intravenous (IV) injections of artesunate 240-360 mg for 3-5 days while hospitalized and treated with oral dihydroartemisinin-piperaquine (DHP) for 3 days after hospital discharge. Blood samples were collected and PCR sequencing performed on genes Pfkelch13, Pfcrt, and Pfmdr1 from all isolates. Results: We analyzed a total of 225 patients from Guangxi, China with P. falciparum malaria acquired in Africa between 2016 and 2018. All patients were cured completely after treatment. The F446I mutation of the Pfkelch13 gene was detected for the first time from samples of West African P. falciparum, with a frequency of 1.0%. Five haplotypes of Pfcrt that encode residues 72-76 were found, with the wild-type CVMNK sequence predominating (80.8% of samples), suggesting that the parasites might be chloroquine sensitive. For Pfmdr1, N86Y (13.1%) and Y184F (58.8%) were the most prevalent, suggesting that artemether-lumefantrine may not, in general, be a suitable treatment for the group. Conclusions: For the first time, this study detected the F446I mutation of the Pfkelch13 gene from Africa parasites that lacked clinical evidence of resistance. This study provides the latest data for molecular marker surveillance related to antimalarial drug resistance genes Pfkelch13, Pfcrt, and Pfmdr1 imported from Africa, in Guangxi, China from Chinese migrate workers. Clinical Trial Registration: ChiCTROPC17013106.

Unraveling the Complexity of Imported Malaria Infections by Amplicon Deep Sequencing.

Imported malaria and recurrent infections are becoming an emerging issue in many malaria non-endemic countries. This study aimed to determine the molecular patterns of the imported malaria infections and recurrence. Blood samples were collected from patients with imported malaria infections during 2016-2018 in Guangxi Zhuang Autonomous Region, China. Next-generation amplicon deep-sequencing approaches were used to assess parasite genetic diversity, multiplexity of infection, relapse, recrudescence, and antimalarial drug resistance. A total of 44 imported malaria cases were examined during the study, of which 35 (79.5%) had recurrent malaria infections within 1 year. The majority (91.4%) had one recurrent malaria episode, whereas two patients had two recurrences and one patient had three recurrences. A total of 19 recurrence patterns (the species responsible for primary and successive clinical episodes) were found in patients returning from malaria epidemic countries. Four parasite species were detected with a higher than usual proportion (46.2%) of non-falciparum infections or mixed-species infections. An increasing trend of recurrence infections and reduced drug treatment efficacy were observed among the cases of imported malaria. The high recurrence rate and complex patterns of imported malaria from Africa to non-endemic countries have the potential to initiate local transmission, thereby undermining efforts to eliminate locally acquired malaria. Our findings highlight the power of amplicon deep-sequencing applications in molecular epidemiological studies of the imported malaria recurrences.

Characterization of the complete mitochondrial genome of Antheraea pernyi strain Luhong.

The present study for the first time describes the complete mitochondrial (mt) genome of Antheraea pernyi Guérin-Méneville 1855 strain Luhong, a genetic lethal mutant exhibiting especially red skin color. The mt genome is 15,563 bp in length that is the smallest among the sequenced A. pernyi inbred strains. This genome displays an identical genomic component and gene order to other six known A. pernyi mt genomes. The mt genome-based phylogenetic analysis clustered Luhong with four strains exhibiting yellow skin color, consistent with the traditional view that all of them belonged to the yellow blood lineage.

Increasing proportions of relapsing parasite species among imported malaria in China's Guangxi Province from Western and Central Africa.

BACKGROUND: Travel-related malaria in non-endemic areas returning from endemic areas presents important challenges to diagnosis and treatment. Imported malaria to newly malaria-free countries poses further threats of malaria re-introduction and potential resurgence. For those traveling to places with high Plasmodium falciparum prevalence, prophylaxis against this parasite is recommended, whereas causal prophylaxis against relapsing malaria is often overlooked. METHODS: We analyzed a cluster of imported malaria among febrile patients in Shanglin County, Guangxi Province, China, who had recent travel histories to Western and Central Africa. Malaria was diagnosed by microscopy and subsequently confirmed by species- and subspecies-specific PCR. Plasmodium vivax was genotyped using a barcode consisting of 42 single nucleotide polymorphisms. RESULTS: Investigations of 344 PCR-confirmed malaria cases revealed that in addition to Plasmodium falciparum being the major parasite species, the relapsing parasites Plasmodium ovale and P. vivax accounted for ~40% of these imported cases. Of the 114 P. ovale infections, 65.8% and 34.2% were P. ovale curtisi and P. ovale wallikeri, respectively, with the two subspecies having a ~2:1 ratio in both Western and Central Africa. Phylogenetic analysis of 14 P. vivax isolates using a genetic barcode demonstrated that 11 formed a distinct clade from P. vivax populations from Eastern Africa. CONCLUSION: This study provides support for active P. vivax transmission in areas with the predominant Duffy-negative blood group. With relapsing malaria making a substantial proportion of the imported malaria, causal prophylaxis should be advocated to travelers with a travel destination to Western and Central Africa.

Loop-mediated isothermal amplification (LAMP) assays targeting 18S ribosomal RNA genes for identifying P. vivax and P. ovale species and mitochondrial DNA for detecting the genus Plasmodium.

BACKGROUND: Loop-mediated isothermal amplification (LAMP) has been widely used to diagnose various infectious diseases. Malaria is a globally distributed infectious disease attributed to parasites in the genus Plasmodium. It is known that persons infected with Plasmodium vivax and P. ovale are prone to clinical relapse of symptomatic blood-stage infections. LAMP has not previously been specifically evaluated for its diagnostic performance in detecting P. ovale in an epidemiological study, and no commercial LAMP or rapid diagnostic test (RDT) kits are available for specifically diagnosing infections with P. ovale. METHODS: An assay was designed to target a portion of mitochondrial DNA (mtDNA) among Plasmodium spp., the five human Plasmodium species and two other assays were designed to target the nuclear 18S ribosomal DNA gene (18S rDNA) of either P. vivax or P. ovale for differentiating the two species. The sensitivity of the assays was compared to that of nested PCR using defined concentrations of plasmids containing the target sequences and using limiting dilutions prepared from clinical isolates derived from Chinese workers who had become infected in Africa or near the Chinese border with Myanmar. RESULTS: The results showed that 102 copies of the mitochondrial target or 102 and 103 copies of 18S rDNA could be detected from Plasmodium spp., P. vivax and P. ovale, respectively. In 279 clinical samples, the malaria Pan mtDNA LAMP test performed well when compared with a nested PCR assay (95% confidence interval [CI] sensitivity 98.48-100%; specificity 90.75-100%). When diagnosing clinical cases of infection with P. vivax, the 18S rDNA assay demonstrated an even great sensitivity (95.85-100%) and specificity (98.1-100%). The same was true for clinical infections with P. ovale (sensitivity 90.76-99.96%; specificity 98.34-100%). Using plasmid-positive controls, the limits of detection of Malaria Pan, 18S rDNA P. vivax and 18S rDNA P. ovale LAMP were 100-, 100- and tenfold lower than those of PCR, respectively. CONCLUSION: The novel LAMP assays can greatly aid the rapid, reliable and highly sensitive diagnosis of infections of Plasmodium spp. transmitted among people, including P. vivax and P. ovale, cases of which are most prone to clinical relapse.

Integration of sample preparation with RNA-Amplification in a hand-held device for airborne virus detection.

Aerosol transmission is one of the three major transmission routes of respiratory viruses. However, the dynamics and significance of the aerosol transmission route are not well understood, partially due to the lack of rapid and efficient tools for on-the-spot detection of airborne viruses. We report a hand-held device that integrates a 3D-printed sample preparation unit with a laminated paper-based RNA amplification unit. The sample preparation unit features an innovative reagent delivery scheme based on a ball-based valve capable of storing and delivering reagents through the rotation of the unit without manual pipetting, while the paper-based unit enables RNA enrichment and reverse transcription loop-mediated isothermal amplification (RT-LAMP). We have determined the detection limit of the integrated sample-preparation/amplification device (SPAD) at 1 TCID50 H1N1 influenza viruses in 140 µL aqueous sample. Further, we integrated SPAD with a previously reported viable virus aerosol sampler (VIVAS), a water-vapor-based condensational growth system capable of collecting aerosolized virus particles (Pan et al., 2016) [1]. Using the combined VIVAS-SPAD platform, we have demonstrated the collection/detection of lab-generated, airborne H1N1 influenza viruses in 65 min, suggesting that the platform has a potential for detecting and monitoring airborne virus transmission during outbreaks. The effective sampling and rapid detection of airborne viruses by the sample-to-answer platform will also help us better understand the dynamics and significance of aerosol transmission of infectious disease.

[Effect of Huoxue Huayu decoction on Wnt/ß-catenin signal pathway expression in rats with severe traumatic brain injury].

OBJECTIVE: To observe the dynamic of neurological severity scores (NSS) and the expressions of Wnt/ß-catenin signaling pathway, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in rats with severe traumatic brain injury (sTBI), and to explore the effect of Huoxue Huayu decoction. METHODS: A total of 126 Sprague-Dawley (SD) rats were randomly divided into seven groups by random number table with 18 rats in each group, namely control group (normal saline 2 kg/L), model group (normal saline 2 kg/L), brain protolysate group (BP group, 5.6 g/kg), Taohong Siwu decoction group (TH group, 10.2 g/kg), Xuefu Zhuyu decoction group (XF group, 15.6 g/kg), Tongqiao Huoxue decoction group (TQ group, 9.6 g/kg) and Buyang Huanwu decoction group (BY group, 28.7 g/kg). The sTBI rat model was reproduced by modified Feeney free fall method, and the rats in the control group were not treated with trauma. The rats in each group were intragastrical administered with corresponding drugs at 6 hours after injury, and the NSS scores were evaluated on the 1st, 3rd and 7th days after injury. After the hippocampus was harvested, the mRNA expressions of Wnt3a and ß-catenin were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the positive expressions of BDNF and NGF were detected by immunohistochemistry. RESULTS: Compared with the control group, the rats in the model group showed obvious symptoms of craniocerebral injury at 1 day after injury, which was manifested as significantly increased NSS score, up-regulated mRNA expressions of Wnt3a and ß-catenin, and increased positive expressions of BDNF and NGF, which indicated that the sTBI rat model was successfully prepared and presented a certain self-repair ability with the extension of time. Compared with the model group, NSS scores in the XF group, TQ group and BY group significantly decreased at 1 day after injury (6.6±1.5, 6.1±2.0, 5.7±2.4 vs. 9.4±1.5, all P < 0.05); however, the NSS scores in the BP group and TH group decreased significantly at 7 days after injury, and the NSS scores in the TQ group and BY group decreased more significantly than those in other drug groups. Compared with the model group, mRNA expressions of Wnt3a and ß-catenin in the hippocampus of the BP group increased significantly at 1 day and 3 days after injury, respectively, and continued to increase with the extension of time. The mRNA expression levels of Wnt3a and ß-catenin in the four groups of Huoxue Huayu decoction fluctuated to varying degrees from 1 day to 3 days after injury, but they were significantly higher than those in the model group at 7 days after injury, and the increase was more significant in the BY group [Wnt3a mRNA (2-ΔΔCt): 154.7±4.1 vs. 17.4±1.0, ß-catenin mRNA (2-ΔΔCt): 17.05±0.45 vs. 2.74±0.13, both P < 0.05], and the second was the TQ group [Wnt3a mRNA (2-ΔΔCt): 126.6±2.8 vs. 17.4±1.0, ß-catenin mRNA (2-ΔΔCt): 8.70±1.19 vs. 2.74±0.13, both P < 0.05]. Compared with the model group, the positive expressions of BDNF and NGF in the BP group increased significantly at 1 day after injury, but decreased after 3 days after peak. The positive expressions of BDNF and NGF in the four Huoxue Huayu decoction groups fluctuated to varying degrees from 1 day to 3 days after injury, but they were significantly higher than those in the model group at 7 days after injury, among which, the positive expressions of BDNF and NGF in the TQ group and BY group were significantly higher than those in the model group at 1 day after injury [BDNF positive cells (cells/MP): 56.4±6.2, 61.6±7.0 vs. 37.4±2.0, NGF positive cells (cells/MP): 58.4±5.0, 62.4±4.4 vs. 53.4±3.6, all P < 0.05], the increase amplitude at 7 days after injury was more significant than those in the other groups. CONCLUSIONS: Taohong Siwu decoction, Xuefu Zhuyu decoction, Tongqiao Huoxue decoction and Buyang Huanwu decoction have curative effect on the nerve regeneration and repair of rats with sTBI at acute stage, but the intensity of the effect is different. Buyang Huanwu decoction and Tongqiao Huoxue decoction have a fast and better effect.

Multiple relapses of Plasmodium vivax malaria acquired from West Africa and association with poor metabolizer CYP2D6 variant: a case report.

BACKGROUND: Plasmodium vivax transmission in West Africa, dominant for the Duffy-negative blood group, has been increasingly recognized from both local residents as well as international travelers who contracted P. vivax malaria there. However, the relapsing pattern and sensitivity to antimalarial treatment of P. vivax strains originated from this region are largely unknown. There is evidence that the efficacy of primaquine for radical cure of relapsing malaria depends on host factors such as the hepatic enzyme cytochrome P450 (CYP) 2D6. CASE PRESENTATION: A 49-year-old Chinese man was admitted to the Shanglin County Hospital in Guangxi Province, China, on December 19, 2016, 39 days after he returned from Ghana, where he stayed for one and a half years. He was diagnosed by microscopy as having uncomplicated P. vivax malaria. Treatment included 3 days of intravenous artesunate (420 mg total), and 3 days of chloroquine (1550 mg total), and 8 days of primaquine (180 mg total). Although parasites and symptoms were cleared rapidly and he was malaria-negative for almost two months, he suffered four relapses with relapse intervals ranging from 58 to 232 days. The last relapse occurred at 491 days from his first vivax attack. For the first three relapses, he was treated similarly with chloroquine and primaquine, sometimes supplemented with additional artemisinin combination therapies (ACTs). For the last relapse, he was treated with intravenous artesunate, 3 days of an ACT, and 7 days of azithromycin, and had remained healthy for 330 days. Molecular studies confirmed P. vivax infections for all the episodes. Although this patient was diagnosed to have normal glucose-6-phosphate dehydrogenase (G6PD) activity, his CYP2D6 genotype corresponded to a *2A/*36 allele variant suggesting of an impaired primaquine metabolizer phenotype. CONCLUSIONS: This clinical case suggests that P. vivax malaria originating from West Africa may produce multiple relapses extending beyond one year. The failures of primaquine as an anti-relapse therapy may be attributed to the patient's impaired metabolizer phenotype of the CYP2D6. This highlights the importance of knowing the host G6PD and CYP2D6 activities for effective radical cure of relapsing malaria by primaquine.

Determination of the distribution of infectious viruses in aerosol particles using water-based condensational growth technology and a bacteriophage MS2 model.

Inhalation of aerosols containing pathogenic viruses can result in morbidity, in some cases leading to mortality. The objective of this study was to develop a model for assessing how infectious viruses might distribute in airborne particles using bacteriophage MS2 as a surrogate for human viruses. Particle deposition in the respiratory system is size-dependent, and small virus-containing particles can be inhaled deeply into the lower lungs, potentially leading to more severe respiratory disease manifestations. Laboratory-generated virus-containing particles were size-selected by a differential mobility analyzer and then collected by the newly introduced Super-Efficient Sampler for Influenza Virus. The number of infectious and total viruses per particle as a function of particle size varied with the spraying medium: it approximated a cubic exponential value scaling for deionized (DI) water, a quartic exponential value for artificial saliva (AS), and between quadratic and cubic exponential value for beef extract solution (BES). The survivability of MS2 did not change significantly with particle size for DI water and BES, while that for AS was maximum at 120 nm. Viruses could be homogeneously distributed or aggregated inside or on the surface of the particles, depending on the composition of the spraying medium.

Performance of silver, zinc, and iron nanoparticles-doped cotton filters against airborne E. coli to minimize bioaerosol exposure.

To overcome limitations of existing air-cleaning filters in capturing and deactivating aerosolized microorganisms, this study was embarked to evaluate novel Ag, Zn, and Fe nanoparticle-doped cotton filters (AgCt, ZnCt, FeCt), as biocidal filters for bioaerosol attenuation. To evaluate the biocidal activity of the nanocomposite filters, the survival of lab-generated E. coli after collection on each filter material was compared to collection on an undoped cotton control filter and in a BioSampler. Relative humidity (RH) affected the survival of bacteria on the filters, and the optimal RH was found to be 50 ± 5%. The physical removal efficiency (PRE) determined by an optical particle counter was 99.9 ± 0.7% for ZnCt, 97.4 ± 1.2% for AgCt, and 97.3 ± 0.6% for FeCt, where the control showed only 77.4 ± 6.3% for particles > 500 nm. The doped filters showed 100% viable removal efficiency (VRE). Importantly, the VRE of the nanocomposite filters after four cycles remained nearly 99% and was greater than the cotton control filter at 76.6 ± 3.2%. Adding to its benefits, the AgCt filters had a lower pressure drop than the FeCt and ZnCt filters and the cotton control. The permeability for the cotton control filter was 3.38 × 10-11 m2 while that for the AgCt filter was slightly higher (3.64 × 10-11 m2) than the other filters as well. Overall, these results suggest that nanocomposite-doped filter media, particularly AgCt, can provide effective protection against airborne pathogens with a lower pressure drop, elevated collection efficiency, and better disinfection capability as compared to untreated cotton filters, which are all important features for practical biocidal applications. Graphical abstract.

Collection of Viable Aerosolized Influenza Virus and Other Respiratory Viruses in a Student Health Care Center through Water-Based Condensation Growth.

The dynamics and significance of aerosol transmission of respiratory viruses are still controversial, for the major reasons that virus aerosols are inefficiently collected by commonly used air samplers and that the collected viruses are inactivated by the collection method. Without knowledge of virus viability, infection risk analyses lack accuracy. This pilot study was performed to (i) determine whether infectious (viable) respiratory viruses in aerosols could be collected from air in a real world environment by the viable virus aerosol sampler (VIVAS), (ii) compare and contrast the efficacy of the standard bioaerosol sampler, the BioSampler, with that of the VIVAS for the collection of airborne viruses in a real world environment, and (iii) gain insights for the use of the VIVAS for respiratory virus sampling. The VIVAS operates via a water vapor condensation process to enlarge aerosolized virus particles to facilitate their capture. A variety of viable human respiratory viruses, including influenza A H1N1 and H3N2 viruses and influenza B viruses, were collected by the VIVAS located at least 2 m from seated patients, during a late-onset 2016 influenza virus outbreak. Whereas the BioSampler when operated following our optimized parameters also collected virus aerosols, it was nevertheless overall less successful based on a lower frequency of virus isolation in most cases. This side-by-side comparison highlights some limitations of past studies based on impingement-based sampling, which may have generated false-negative results due to either poor collection efficiency and/or virus inactivation due to the collection process. IMPORTANCE The significance of virus aerosols in the natural transmission of respiratory diseases has been a contentious issue, primarily because it is difficult to collect or sample virus aerosols using currently available air sampling devices. We tested a new air sampler based on water vapor condensation for efficient sampling of viable airborne respiratory viruses in a student health care center as a model of a real world environment. The new sampler outperformed the industry standard device (the SKC BioSampler) in the collection of natural virus aerosols and in maintaining virus viability. These results using the VIVAS indicate that respiratory virus aerosols are more prevalent and potentially pose a greater inhalation biohazard than previously thought. The VIVAS thus appears to be a useful apparatus for microbiology air quality tests related to the detection of viable airborne viruses.

Drifted Influenza A and B Viruses Collected by a Water-Based Condensation Growth Air Sampler in a Student Health Care Center during an Influenza Outbreak.

A viable virus aerosol sampler (VIVAS) effectively collected viable influenza A and B viruses from air inside a student health care center during an influenza outbreak. The viruses had "drifted" genes, showcasing the usefulness of the VIVAS for air sampling and noninvasive surveillance of viruses in circulation.

Use of RNA amplification and electrophoresis for studying virus aerosol collection efficiency and their comparison with plaque assays.

The spread of virus-induced infectious diseases through airborne routes of transmission is a global concern for economic and medical reasons. To study virus transmission, it is essential to have an effective aerosol collector such as the growth tube collector (GTC) system that utilizes water-based condensation for collecting virus-containing aerosols. In this work, we characterized the GTC system using bacteriophage MS2 as a surrogate for a small RNA virus. We investigated using RNA extraction and reverse transcription- polymerase chain reaction (RT-PCR) to study the total virus collection efficiency of the GTC system. Plaque assays were also used to enumerate viable viruses collected by the GTC system compared to that by a commercially available apparatus, the SKC® Biosampler. The plaque assay counts were used to enumerate viable viruses whereas RT-PCR provides a total virus count, including those viruses inactivated during collection. The effects of relative humidity (RH) and other conditions on collection efficiency were also investigated. Our results suggest that the GTC has a collection efficiency for viable viruses between 0.24 and 1.8% and a total virus collection efficiency between 18.3 and 79.0%, which is 1-2 orders of magnitude higher than that of the SKC® Biosampler. Moreover, higher RH significantly increases both the viable and total collection efficiency of the GTC, while its effect on the collection efficiency of the SKC® Biosampler is not significant.