Association of 410L, 1016I and 1534C kdr mutations with pyrethroid resistance in Aedes aegypti from Ouagadougou, Burkina Faso, and development of a one-step multiplex PCR method for the simultaneous detection of 1534C and 1016I kdr mutations.

BACKGROUND: Since 2000, Burkina Faso has experienced regular dengue cases and outbreaks, making dengue an increasingly important health concern for the country. Previous studies in Burkina Faso reported that resistance of Aedes aegypti to pyrethroid insecticides was associated with the F1534C and V1016I kdr mutations. The current study reports high resistance of Ae. aegypti populations to pyrethroid insecticides, likely supported by mutations in the voltage-gated sodium channel, here evidenced by genotyping the kdr SNPs V410L, V1016I and F1534C. We also describe a new multiplex PCR-based diagnostic of F1534C and V1016I kdr SNPs. METHODS: Larvae of Ae. aegypti were collected from three health districts of Ouagadougou in 2018. The resistance status of Ae. aegypti to permethrin (15 µg/ml) and deltamethrin (10 µg/ml) was tested using bottles and to malathion (5%) using WHO tube tests. All bioassays used 1-h exposure and mortality recorded 24 h post-exposure. Bioassay results were interpreted according to WHO thresholds for resistance diagnosis. The kdr mutations were screened using AS-PCR and TaqMan methods in exposed and non-exposed Aedes mosquitoes. RESULTS: Females from all health districts were resistant to permethrin and deltamethrin (< 20% mortality) but were fully susceptible to 5% malathion. The F1534C and V1016I kdr mutations were successfully detected using a newly developed multiplex PCR in perfect agreement with TaqMan method. The 1534C/1016I/410L haplotype was correlated with permethrin resistance but not with deltamethrin resistance; however, the test power was limited by a low frequency of dead individuals in deltamethrin exposure. CONCLUSIONS: Resistance to pyrethroid insecticides is associated with kdr mutant haplotypes, while the absence of substantial resistance to malathion suggests that it remains a viable option for dengue vector control in Ouagadougou.

Effectiveness of Rapid Antigen Testing in Forensic Cases of Severe Acute Respiratory Syndrome Coronavirus 2 Infection, Including Delta Variant.

ABSTRACT: The polymerase chain reaction is indispensable for diagnosing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in forensic cases. However, studies regarding the effectiveness of rapid antigen testing (RAT) in forensic cases remain limited. Therefore, we investigated the efficacy of RAT compared with reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for confirming SARS-CoV-2 infection (including the delta variant). Before the external examination or autopsy, we collected samples from the nasopharyngeal mucosa, which were then assessed via RAT (QuickNavi COVID-19 Ag kit, QuickNavi-Flu+COVID-19 Ag kit) and RT-qPCR. Reverse-transcription quantitative polymerase chain reaction results were positive in 73 of 1255 cases, and 21 cases were identified as those of delta variants. Low RT-qPCR threshold cycle value cases and delta variant infections were more likely to result in coronavirus disease-related deaths. The sensitivity of the QuickNavi COVID-19 Ag kit was 76.32%, and that of the QuickNavi-Flu+COVID-19 Ag kit was 77.14%. The specificity of both RATs was 100%. In QuickNavi COVID-19 Ag kit cases, delta variant cases showed lower sensitivity than non-delta variant cases, even for a similar viral load. Thus, RAT in forensic cases is sufficiently useful as a screening test for SARS-CoV-2 infection. However, RAT carries a risk of false negatives, especially for delta variant cases.

A human tissue-based assay identifies a novel carrion blowfly strain for maggot debridement therapy.

Maggot debridement therapy (MDT) is a form of therapeutic wound treatment in which live fly larvae are used intentionally to debride necrotic tissues. MDT has been widely used to treat chronic wounds in humans or animals, such as diabetic foot ulcers. Larvae of a carrion blowfly, Lucilia sericata (green bottle fly), debride wounds by consuming necrotic tissue and removing pathogenic bacteria, promoting effective wound healing. Most medical L. sericata strains were initially collected from natural environments using animal meat as bait and reared on artificial protein-rich media or ground meat. It remains to be examined which strain would be more appropriate for MDT, whereas any method for evaluating the fly's therapeutic potential in humans has not been available. A feeding assay was developed using minced human tissues obtained from surgical waste. To establish L. sericata strains highly eligible for MDT, carrion fly larvae were collected from 45 corpses subjected to forensic autopsy (such as decomposed bodies). Four corpse-derived L. sericata strains were obtained and evaluated using the feeding assay. One strain showed that its feeding activity was 1.4 times higher than the control strain used in conventional MDT. The body length of the adult fly of the corpse-derived strain was longer than the control, which was consistent with the observation that its cell size was enlarged. The human tissue-based assay developed in this study accurately evaluated the ability of fly larvae to debride necrotic wounds. The L. sericata strain newly established from human corpses harboring high feeding activity may offer a clinically significant improvement in MDT.

Determining the effect of water temperature on the T1 and T2 relaxation times of the lung tissue at 9.4 T MRI: A drowning mouse model.

Japanese individuals have a unique culture of soaking in a bathtub, and forensic pathologists have experienced fatal cases due to drowning. However, T1 and T2 relaxation times of a drowning lung are poorly documented. In the present study, we investigated the relationship between drowning water temperature and T1 and T2 relaxation times of drowning lung tissues at 9.4 T MRI (Bruker, BioSpec94/20USR). The mice used as animal drowning models were directly submerged in freshwater. Water temperature was set to 8 °C-10 °C (cold), 20 °C-22 °C (normal), 30 °C, and 45 °C. The regions of interest (ROIs) on the axial section of the third slice were set at the central and peripheral areas of each-the left and the right-lung. T1 relaxation times measured immediately after death differed by the presence or absence of soaking water, except in case of cold water temperature. In the drowning groups, T1 relaxation time showed a linear dependency on water temperature. By contrast, T2 relaxation time was almost constant regardless of the presence of drowning under the same temperature condition; when compared in the lung areas of the same individuals, the times were uniformly reduced in drowning models. To minimize the effects of hypostasis and decomposition, we performed measurements immediately after death and were able to determine the noticeable difference in drowning water temperature. These results may be useful for qualitative assessments of a drowning lung and may serve as a basis when imaging the human body during forensic autopsy cases.

Intensive diagnostic management of coronavirus disease 2019 (COVID-19) in academic settings in Japan: challenge and future.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first emerged in Wuhan, China, and has spread globally to most countries. In Japan, the first COVID-19 patient was identified on January 15, 2020. By June 30, the total number of patients diagnosed with COVID-19 reached 18,000. The impact of molecular detection of pathogens is significant in acute-care settings where rapid and accurate diagnostic measures are critical for decisions in patient treatment and outcomes of infectious diseases. Polymerase chain reaction (PCR)-based methods, such as quantitative PCR (qPCR), are the most established gene amplification tools and have a comprehensive range of clinical applications, including detecting a variety of pathogens, even novel agents causing emerging infections. Because SARS-CoV-2 contains a single-stranded RNA genome, reverse-transcription qPCR (RT-qPCR) has been broadly employed for rapid and sensitive quantitative measurements of viral RNA copy numbers. The RT-qPCR method, however, still requires time-consuming reactions with two different enzymes in addition to isolation of RNA from patient samples, limiting the numbers of testing institutions for diagnosing SARS-CoV-2 infection. Japan is known to have performed a relatively small number of PCR tests as well as confirmed cases among developed nations; as of June 30, 2020, approximately 390,000 people in Japan had undergone PCR tests. Given the devastating impact on medical services and the scale of demand for diagnostic testing of COVID-19, it has been proposed that academic settings such as basic research departments in university/college can be engaged in diagnosing, especially in university hospitals or academic medical centers. In collaboration with established diagnostic laboratories, academic facilities can divert their function to detecting virus from patients with suspected COVID-19, adopting existing specialized expertise in virus handling, molecular work, and data analysis. This in-house testing strategy facilitates the rapid diagnosing of thousands of samples per day and reduces sample turnaround time from 1 week to less than 24 h. This review provides an overview of the general principles, diagnostic value, and limitations of COVID-19 diagnosis platforms in Japan, in particular in-house testing at academic settings.

Seroprevalence and associated factors of Toxoplasma gondii among HIV-infected patients in Tokyo: A cross sectional study.

HIV infection, in particular in patients with developing AIDS, carries a risk of causing toxoplasmosis with encephalitis, which is mostly caused by a form (bradyzoite) of the protozoan parasite Toxoplasma gondii. HIV/AIDS in Japan has been recognized as a serious health issue in recent years. In this study, to elucidate T. gondii seroprevalence in HIV-positive patients in Japan and associated characteristics with Toxoplasma parasite infection, the titer of T. gondii IgG (Tg-IgG) was measured in 399 HIV-positive patients who visited a hospital in Tokyo, Japan, between 2015 and 2017. A questionnaire survey was also conducted to investigate associations between lifestyle and customs. As a result, the overall prevalence of Tg-IgG-positive serum was 8.27% (33 cases of 399). All the cases positive for Tg-IgG were confirmed using the Sabin-Feldman dye test; the titers between each examination correlated robustly (p < 0.001, r = 0.6). A correlation between Toxoplasma infection rate and age was determined (p < 0.001), whereas there was no significant correlation with lifestyle customs such as consuming undercooked meat or owning a cat. An association between Toxoplasma infection and experience of dwelling in the Hokkaido area, the northern part of Japan, was observed (p = 0.001). These results suggested that the proportion of those who were previously exposed to Toxoplasma parasites in the HIV-positive population has been maintained at a similar level as that of the HIV-negative population in Japan, providing clear information about the potential risk of toxoplasmic encephalitis.

Seroprevalence of Toxoplasma gondii in wild sika deer in Japan.

Toxoplasmosis is a food-borne infection that is widespread around the world, causing congenital disorders and opportunistic infections. Ingestion of undercooked meat is one of the risk factors for infection with the causative agent, Toxoplasma gondii. Japanese people occasionally eat rare meat as a traditional cuisine style called "Sashimi". A rapid increase in venison consumption in Japan has occurred mainly due to enhanced population control of wild Japanese deer (Cervus nippon) in recent decades. In particular, Yezo-sika deer (C. n. yesoensis) in Hokkaido (the northernmost and largest prefecture in Japan) is frequently supplied to markets as branded game/bushmeat. To study the possible burden of Toxoplasma gondii among wild Yezo-sika deer, plasma samples of Yezo-sika deer hunted during two seasons, 2010-2012, in Eastern Hokkaido were investigated. A total 80 samples were examined using the Sabin-Feldman dye test, which is highly specific and sensitive for identifying the development and persistence of antibodies after primary Toxoplasma infection, demonstrating that 38 cases (47.5%) were seropositive (cut-off titer <1:16). Antibody prevalence of T. gondii in female deer was higher than in males. Adult deer aged 3 years or over showed higher seroprevalence compared with younger animals. The overall seroprevalence fluctuated significantly according to the season when the deer were hunted. These results indicated widespread infection of T. gondii among Japanese wild Yezo-sika deer, suggesting that both appropriate handling and treatment of bushmeat are required to prevent food-borne toxoplasmosis in Japan.

High frequencies of F1534C and V1016I kdr mutations and association with pyrethroid resistance in Aedes aegypti from Somgandé (Ouagadougou), Burkina Faso.

BACKGROUND: Resistance to pyrethroid insecticides involving kdr mutations is widespread in Aedes aegypti (L.) (Diptera: Culicidae) and potentially could impact control efforts in endemic countries. Dengue cases had been sporadic in Burkina Faso for over a decade prior to the 2016-2017 outbreak that resulted in 15,074 suspected cases and 36 deaths, mainly in Ouagadougou. These outbreaks highlighted the lack of information on numerous aspects of the biology, behaviour and insecticide status of local dengue vector populations that are fundamental to vector control. RESULTS: We investigated the insecticide resistance profiles and the kdr mutations involved in pyrethroid resistance of Ae. aegypti from Somgandé, a district of Ouagadougou. WHO bioassays revealed that the local Ae. aegypti populations were highly resistant to pyrethroids with mortalities of 15% for permethrin and 37% for deltamethrin. Resistance to carbamates was also detected with mortalities of 55% for propoxur and 90% for bendiocarb, but high mortalities (> 97%) to organophosphates (malathion and fenitrothion) indicated susceptibility. Allele-specific PCR and voltage-gated sodium channel gene sequencing showed a very high frequency (97%) of the F1534C kdr allele whilst the V1016I kdr mutation frequency was 46%. Association of dual-locus kdr mutations was detected for permethrin resistance. CONCLUSION: We conclude that in this locality of Burkina Faso, Ae. aegypti is resistant to pyrethroid and carbamate insecticides but remains susceptible to organophosphates, providing useful information for possible future control.

Phylogenetic comparison of avian haemosporidian parasites from resident and migratory birds in northern Japan.

We analyzed blood samples of resident and migratory Japanese birds to evaluate the prevalence and genetic background of avian blood parasites in northern Japan. We used PCR targeting the mitochondrial cytochrome b gene to examine infections of Leucocytozoon, Haemoproteus, and Plasmodium parasites in blood samples from 243 birds of 14 species in three orders (Passeriformes, Columbiformes, and Anseriformes). Sequences were subjected to phylogenetic analysis. The infection rate was 21% in pigeons (Columbiformes) and 17% in Anseriformes. A high infection rate of 93.8% was found in crow species (Passeriformes). Haemoproteus and Plasmodium parasites were detected in only two species. Infected blood samples obtained from seven bird species involved two major clades of Leucocytozoon, which were divided between resident and migratory birds. The parasites, which are genetically distinct from parasites in Japanese resident birds, may have been introduced to Japan by migratory bird species.

Multivariable analysis of host amino acids in plasma and liver during infection of malaria parasite Plasmodium yoelii.

BACKGROUND: Malaria is the most significant human parasitic disease, and yet understanding of the energy metabolism of the principle pathogen, Plasmodium falciparum, remains to be fully elucidated. Amino acids were shown to be essential nutritional requirements since early times and much of the current knowledge of Plasmodium energy metabolism is based on early biochemical work, performed using basic analytical techniques, carried out almost exclusively on human plasma with considerable inter-individual variability. METHODS: In order to further characterize the fate of amino acid metabolism in malaria parasite, multivariate analysis using statistical modelling of amino acid concentrations (aminogram) of plasma and liver were determined in host infected with rodent malaria parasite, Plasmodium yoelii. RESULTS AND CONCLUSION: Comprehensive and statistical aminogram analysis revealed that P. yoelii infection caused drastic change of plasma and liver aminogram, and altered intra- and inter-correlation of amino acid concentration in plasma and liver. These findings of the interactions between amino acids and Plasmodium infection may provide insight to reveal the interaction between nutrients and parasites.

Toxoplasma gondii deoxyribose phosphate aldolase-like protein (TgDPA) interacts with actin depolymerizing factor (TgADF) to enhance the actin filament dynamics in the bradyzoite stage.

The Toxoplasma gondii deoxyribose phosphate aldolase-like (TgDPA) gene is expressed predominantly in bradyzoites. This finding allowed us to infer that TgDPA is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. We conducted yeast two-hybrid screening to identify proteins interacting with TgDPA, and the actin depolymerizing factor (TgADF) gene was obtained. Co-immunoprecipitation and a GST pull-down assay demonstrated that TgDPA interacts with TgADF. To reveal the significance of the protein-protein interaction between TgDPA and TgADF, actin polymerization and disassembly kinetics were examined. Addition of GST-TgDPA to TgADF lowered the extent of actin polymerization and enhanced the filamentous actin disassembly. These results demonstrated that this is the novel protein-protein interaction in T. gondii, and that TgDPA can enhance the activity of TgADF. This phenomenon might play an important role in T. gondii bradyzoites by affecting the actin turnover.