Dental healthcare personnel (DHCP) are subjected to microbe-containing aerosols and splatters in their everyday work. Safer work conditions must be developed to ensure the functioning of the healthcare system. By simulating dental procedures, we aimed to compare the virus-containing aerosol generation of four common dental instruments, and high-volume evacuation (HVE) in their mitigation. Moreover, we combined the detection of infectious viruses with RT-qPCR to form a fuller view of virus-containing aerosol spread in dental procedures. The air–water syringe produced the highest number of aerosols. HVE greatly reduced aerosol concentrations during procedures. The air–water syringe spread infectious virus-containing aerosols throughout the room, while other instruments only did so to close proximity. Additionally, infectious viruses were detected on the face shields of DHCP. Virus genomes were detected throughout the room with all instruments, indicating that more resilient viruses might remain infectious and pose a health hazard. HVE reduced the spread of both infectious viruses and viral genomes, however, it did not fully prevent them. We recommend meticulous use of HVE, a well-fitting mask and face shields in dental procedures. We advise particular caution when operating with the air–water syringe. Due to limited repetitions, this study should be considered a proof-of-concept report. Human saliva contains a multitude of microorganisms, including respiratory viruses such as corona and influenza viruses3. These viruses are known to spread through aerosol and droplet transmission4, causing a potential risk of infection for dental healthcare personnel (DHCP) during dental procedures.Dentists are subjected to saliva and blood-containing aerosols and splatters formed by aerosol-generating instruments on a daily basis7. Aerosols and splatters are not only formed by these instruments but also when patients talk and breathe8. Aerosols can persist in the air for extended periods of time and disperse over long distances, posing in the healthcare setting a potential risk to staff and subsequent patients9. Source control using masks or respirators is not possible during dental procedures, and thus, other methods, such as mouth disinfectants, rubber dams, and four-handed dentistry employing a high-volume evacuator, have been used to reduce infection risk during the COVID-19 pandemic10.Use of dental instruments, such as the ultrasonic scaler, the air–water syringe, and the air turbine handpiece, are considered aerosol-generating procedures10. These safety measures were introduced quickly out of necessity. However, a well-informed consensus on virus-containing aerosol generation during dental procedures and the infection risk posed was lacking. With this study, we aimed to measure the virus-containing aerosol production of different dental instruments to evaluate the risk that their use poses to DHCP and patients at the dental clinic. Moreover, we combined viral RNA detection with infectious viruses to get a fuller picture of virus-containing aerosol spread in dental procedures.With the added threat posed by SARS-CoV-2, routine dental treatments were postponed at the beginning of the pandemic and restrictions were placed on aerosol-generating procedures12.To examine these phenomena, we simulated dental procedures using common dental instruments: an air turbine, a high-speed dental handpiece, an ultrasonic scaler, and an air–water syringe. A dental phantom head with simulated saliva secretion served as the patient. Bacteriophage Phi6 was chosen as a surrogate as it has previously been used to simulate the spread of influenza and SARS viruses11 pfu / ml of Phi6  was used to mitigate the aerosol production. To evaluate its effect, procedures were carried out with HVE (HVE +) and without it (HVE-). At least one HVE + and two HVE- repetitions were measured for each instrument, excluding the air–water syringe  per milliliter, with plaque referring to an empty circular area in the bacterial lawn on a petri dish where bacterial cells are lysed due to viral infection. One plaque is formed by the progeny of the first virus infecting the cell. The total virus concentration, including inactive viruses, was analyzed by measuring genome copy numbers with reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)15.Viruses were collected passively using Luria–Bertani agar plates containing host bacteria, Pseudomonas syringae sp. HB10Y (HB10Y plates), and actively, using pumps, with 5 ml Biosamplers , Button Aerosol samplers (SKC Inc.), and an Andersen 6-stage cascade impactor. Passive collection reflects how aerosols deposit naturally onto surfaces, indicating potential surface contaminants, while active collections represent breathing in contaminated air. HB10Y plates were also used to collect deposing aerosols after the procedures (after plates) in the air turbine handpiece and nebulizer experiments. Additionally, face shield were swabbed after procedures to evaluate possible face contaminations. The face shield was swabbed with a sterile nitrile gloved finger, which was then pressed gently onto an HB10Y plate. The protocol used had been developed by Oksanen et al.Aerosol particle number, size distribution and concentration were measured with an Optical Particle Sizer  model 3330 and the total aerosol number concentration with a Condensation Particle Counter  model 3007. Aerosol levels were monitored between procedures until they returned to baseline.The dental clinic layout is presented in Supplement Fig. Infectious viruses were found on plate 1 in HVE + and HVE- procedures, and in the Andersen impactor only in HVE- procedures   and plate 6.(HVE-) Fig. .Infectious viruses were observed on plate 1 and in four Andersen impactor compartments in HVE- procedures. However, in HVE + procedures they were only detected in one Andersen impactor compartment (1.1–2.1 µm) , however, the RT-qPCR reliability threshold was not crossed in HVE + samples  more infectious viruses were detected on HB10Y plates with the air–water and air sprays compared to any other instruments, without HVE. Moreover, significantly more infectious viruses were also detected with the Andersen impactor in HVE + (p = 0.02–0.03) and HVE- (p < 0.01–0.03) procedures.Significantly (p = 0.05) or air spray (p = 0.04). However, infectious virus count decrease with HVE in the Andersen impactor was significant only when using air spray   and all air–water spray HVE- procedures (p =  < 0.01–0.01)  . Virus genome copy numbers were significantly higher compared to other instruments for all air spray procedures  and OPS  when using the air turbine handpiece. Particle concentration curves from CPC and OPS resembled each other; however, with the high-speed dental handpiece, only particle concentrations in the OPS (i.e. larger particles) were clearly elevated, while measurements with CPC were only slightly elevated. Baseline particle concentrations were 1000–3000 particles/cm3 for CPC and 4–6 particles/cm3 for OPS, varying slightly between experiments  reduced the aerosol generation of all instruments on both ranges.Aerosol concentrations remained near baseline in HVE + procedures, whereas in HVE- procedures elevated concentrations were observed. The highest aerosol concentrations were measured with both CPC  reduced the aerosol concentration on both ranges and the aerosol concentration remained near baseline  reduced the number of infectious viruses detected with the Andersen impactor (Fig. No infectious viruses were detected on HB10Y plates in the HVE + procedure, while in the HVE- procedure, they were detected on all plates. Use of HVE also significantly (Infectious virus plaques were observed on cell culture plates inoculated with face shield swabs after procedures with the ultrasonic scaler and air spray Table .Table 2IHere, by comparing infectious virus and viral genome detections, we provide a new perspective on the spread of viruses in dental procedures. Moreover, this study shows that virus cultivation combined with PCR detection is required to understand the full picture of virus-containing aerosol spread during dental procedures. We demonstrated the importance of HVE in mitigating virus spread and lowering the risk for aerosol exposure of dental healthcare personnel. Air turbine and air–water syringe were found to be associated with a high risk of aerosol generation and virus spread. The use of HVE markedly reduced aerosol concentrations and viral findings. Moreover, we showed that dentists faces are also subjected to infectious virus-containing droplet spills during procedures. Our findings encourage the use of HVE, face shields, and well-fitting masks in dental procedures, not only during pandemics but also in everyday work. However, because this study had only limited repetitions for most instruments, it should be considered a proof-of-concept report, and more studies are needed to bolster these findings.17 and ultrasonic scalers19. The present study provides data on virus-containing aerosol generation of additional common dental instruments and compares them in the same experimental setup. Moreover, RT-qPCR and infectious virus detection have not been combined before to compare the spread of infectious and inactive viruses in dental procedures. Studying these side by side gives a more complete view on the subject.Previous studies examining the spread of virus-containing aerosols in dental procedures have focused on drill-type instrumentsIn air turbine, high-speed dental handpiece, and ultrasonic scaler procedures, infectious viruses were only detected near the source, indicating that while viral aerosol transmission is possible during these procedures the risk seems to be high mainly at close proximity. Some of the viruses found on deposition plates near the source are of larger droplet spill origin, as visible droplets could be seen on these plates and around them. However, as some viruses were detected with the Andersen impactor situated behind the phantom head in HVE- procedures, both origins should be considered in risk management measures.20 and IAV H1N1 aerosols21. This size range, which is commonly observed for virus-containing aerosols4, seems to apply to the other instruments as well, but because the virus numbers were low in those procedures, more studies are needed to confirm this.In the air–water syringe procedures, more infectious viruses were observed compared to other instruments, especially in the Andersen impactor. The air–water syringe procedures were the only ones where infectious viruses were detected on deposition plates other than next to the source, indicating that it produces infectious virus-containing aerosols. The results from the Andersen impactor are particularly interesting, as infectious viruses were mostly detected from particles smaller than 4.7 µm, with the peak being at 2.1–3.3 µm. This size range is similar to that of SARS-CoV-2 aerosols22. This effect of drying on the inactivation of viruses is supported by our results with the air–water syringe, where using the air spray reduced the number of infectious viruses detected with all collection methods, compared with using the air–water spray, without reducing the number of detected genome copies. Regarding virus genome analysis, no virus genome was detected in air turbine handpiece HVE- procedures, even though most of the HVE + procedure’s samples crossed the RT-qPCR reliability threshold. We suspect that the collection plates were not properly rinsed during sample collection.By contrast, viral RNA was detected at all sampling points in all but the high-speed dental handpiece procedures, indicating that aerosols formed during these procedures include viruses, but they are inactivated during aerosolization. This can be due to the extreme physical stress caused by the high rotary speed or the air and water jets of the instruments. The drying of the aerosols also plays a key role in virus inactivation23, it has been found to have similar survivability as SARS or influenza viruses in some respects such as temperature and chemical stress25. However, because different viruses vary in the loss of infectivity, these results highlight the need for proper protective gear for personnel.Regarding Phi6 as a survival model for other enveloped viruses, it is important to mention that while it does not perfectly resemble them3 genome copies / ml15. Still, even with a high virus concentration, some of the samples did not reach the reliability threshold and therefore cannot be reported with certainty. This was especially a problem with the ultrasonic scaler. Nevertheless, as it was the only instrument with which most sampling points did not cross the threshold, we can estimate that it is the least dangerous of the investigated instruments in terms of virus-containing aerosol generation.A higher-than-normal virus concentration had to be used in the mock saliva, as infectious viruses would have been impossible to reliably detect otherwise. The RT-qPCR protocol applied also required this, as it has a high reliability threshold of 2⦁1026, could remain infectious and spread throughout the room, and should therefore be studied in a similar setting. Moreover, bacteria or parts of bacteria27, such as endotoxins, spreading in the room could also pose a health hazard29.Our results show that viral aerosol transmission during dental procedures is possible, and thus, appropriate safety measures should be taken. Moreover, with viral genomes detected at many of the sampling points where no infectious viruses were found, it is possible that more resilient viruses, such as non-enveloped viruses17. This is especially important if a rubber dam or HVE cannot be used.Based on these results, we recommend restrictions on the use of the air–water syringe in worse epidemic situations, as it was found to allow more infectious viruses to spread than any other instrument. We also conclude that the use of a high-speed dental handpiece is safer in terms of aerosol production and virus spread than the air turbine handpiece. Moreover, it can be used as a substitute for the air turbine handpiece during a pandemic or with infectious patients, as has been shown before31. Our results support this finding, as HVE was observed to play a key role in mitigating the spread of virus-containing aerosols. This is further supported by the positive control, where HVE was able to almost fully negate the spread of aerosols created with the nebulizer, even though it produces a much higher concentration of aerosols than any of the dental instruments used in this study. HVE also seems to be able to slightly reduce the number of droplets escaping the mouth, as demonstrated by the reduction of viral plaques on the nearest deposition plate during HVE + procedures. However, differences in the work of the dental staff might also be the cause.HVE has previously been shown to reduce aerosol spread during dental procedures32. In the nebulizer test, HVE remained in an optimal position regarding the nebulizer outlet. However, an optimal positioning cannot always be achieved with patients, which can result in peaks in aerosol concentrations, as seen in some of the HVE + procedures (Fig. 34. Training is also required to ensure its proper use and positioning.Generally, HVE is advised to be positioned as close to the working area as possible, but its effectiveness is also affected by the operator35 found, splatters formed in dental procedures are found all around the DHCP’s clothes and face. Moreover, in the present study, we were able to detect infectious viruses in the face shields of the DHCP, indicating that without protection their faces would be directly subjected to infective viruses. Based on these results, we recommend that all DHCP in the room wear face shields in addition to well-fitting masks during procedures to prevent splatters from reaching the face. Wearing proper face protection is especially important when using high splatter-causing instruments such as the air–water syringe or the air turbine handpiece.As Teichert-Filho et al.36, indicating that viruses found on the shields are most likely of splatter origin. Moreover, the plates directly in front of the mouth almost invariably had viruses on them, indicating that viruses from the mouth spread well via splatters formed during procedures. While it is important to acknowledge that the positioning of DHCPs also plays a key role in avoiding these splatters, a safe position cannot always be taken.Although not all procedures gave virus-positive results from shield swabs, they showed that DHCP’s faces are at risk of being exposed to infectious viruses from patient’s saliva and blood. Previously, HVE has been found to not significantly affect the viral genome copy numbers detected on DHCP’s face shieldsWe demonstrated the importance of high-volume evacuation in mitigating viral aerosol spread during dental procedures. We also discovered that most, but not all, viruses are inactivated in the process of aerosolization with dental instruments. We recommend caution when operating with the air–water syringe, as it was found to preserve virus infectivity in aerosolization better than any other instrument. Using a face shield, instead of routine goggles, in addition to a well-fitting mask, should be considered in all dental procedures to decrease exposure to infectious agents. Replacing the surgical mask with a respirator is reasonable when treating patients with confirmed or probable infection transmissible by aerosols or droplets.Supplementary Information.


Information on the average and incremental costs of implementing alternative strategies for treating young infants 0-59 days old in primary health facilities with signs of possible serious bacterial infection (PSBI) when a referral is not feasible is limited but valuable for policymakers.Direct activity costs were calculated for outpatient treatment of PSBI and pneumonia in two districts of India: Palwal, Haryana and Lucknow, Uttar Pradesh. These included costs of staff time and consumables for initial assessment, classification, and referrals; recommended treatment of fast breathing  and PSBI ; and daily assessments. Indirect operational costs included staff training; staff time cost for general management, supervision, and coordination; referral transport; and communication.The average cost per young infant treated for recommended and acceptable treatment for PSBI was 16 US dollars (US$) (95% CI = US$15.4-16.3) in 2018-19 and US$18.5 in 2022 (adjusted for inflation) when all direct and indirect operational costs were considered. The average cost of recommended treatment for pneumonia was US$10.1 (95% CI = US$9.7-10.6) or US$11.7 in 2022, per treated young infant. The incremental cost 2018-2019 for supplies, medicines, and operations (excluding staff time costs) per infant treated for PSBI was US$6.1 and US$4.3 and for pneumonia was US$3.5 and US$2.2 in Palwal and Lucknow, respectively. Operation and administrative costs were 25% in Palwal and 12% in Lucknow of the total PSBI treatment costs. The average cost per live birth for treating PSBI in each population was US$5 in Palwal and US$3 in Lucknow. Higher operation costs for social mobilisation activities in Palwal led to the empowerment of families and timely care-seeking.Costs of treatment of PSBI with the recommended regimen in an outpatient setting, when a referral is not feasible, are under US$20 per treated child and must be budgeted to reduce deaths from neonatal sepsis. The investment must be made in activities that lead to successful identification, prompt care seeking, timely initiation of treatment and follow-up. The incidence of neonatal sepsis in India is the highest in the world and is the second leading cause of neonatal mortality in India ,2. With Randomised controlled trials conducted in Africa and Asia demonstrated that simplified antibiotic regimens given to young infants with PSBI in outpatient settings when a referral is not feasible were effective -13. To iData about the costs of treatment for the management of neonatal infections, especially in low-resource settings, is quite limited. The information on the cost of making such guidelines operational is limited too. A study from Ethiopia assessed the costs of managing PSBI for young infants at health posts when a referral was not possible and found it cost-effective . A systeRecently, multi-centric implementation research was undertaken at four districts in India to contextualise strategies to address implementation bottlenecks of the national PSBI Management Guideline  and strengthen its on-ground implementation of treatment in outpatient primary health care settings -21. At tThe costing data was collected from two PSBI implementation research sites  ,19, whicThe framework for determining the cost of PSBI management was conceived following national program guidelines -16 and tThese included costs of staff’s additional time, medicine, and essential supplies for strengthening activities that were directed to each mother-child in outpatient settings. The intervention cost framework had three core domains.Six home visits for facility-based birth and seven postnatal visits for home-based births were made by the Accredited Social Health Activist (ASHA) for birth identification, measuring young infant’s weight, promoting optimal care practices such as breastfeeding, keeping the baby warm, promote hygiene, recognise “danger signs” in mothers and newborns and promote appropriate and prompt care seeking. Mothers were given cards to identify danger signs.Once the ASHA identified any danger signs at the community level, young infants were referred / mobilised to a higher-level facility for further assessment, generally a primary health centre (PHC) or community health centre (CHC), but rarely a subcenter. Families usually travelled to health facilities by themselves, however, in some instances, they were accompanied by ASHAs and occasionally used government ambulances for transportation. ASHA also revisited the homes to check the outcome of the referral or treatment.Once sick young infants were presented in health facilities, the IMNCI-trained Auxiliary Nurse Midwife (ANM) or nurse or a Medical Officer (MO) assessed and classified them as per the IMNCI algorithm . Those wAll young infants who had any sign of PSBI were referred to a higher-level facility  for further treatment as per guidelines. Staff time costs for assessment and counselling on referral and care during referral transportation were included.Those who accepted referrals were given a referral form / slips, and a pre-referral dose of injection gentamicin and oral amoxicillin. The costs of both were included under supplies.Young infants 7-59 days old with fast breathing (60 breaths or more per minute) were treated as outpatients in a subcenter, PHC or CHC with oral amoxicillin. Mothers were taught to administer the dosage twice daily at home for seven days.The families, who did not accept the referral, were treated at a primary health facility on an ambulatory basis following national guidelines. Treatment of these sick young infants was based on re-classification into the following three categories: 1) young infants zero-six days old with severe pneumonia (60 breaths or more per minute) were treated with intra-muscular (IM) gentamicin (once daily) and oral amoxicillin (twice daily for seven days); 2) infants with any sign of clinical severe infection were treated with daily IM gentamicin and twice daily oral amoxicillin for seven days; 3) infants with PSBI critical illness were to be treated with a daily dose of IM gentamicin and twice daily IM ampicillin. Under each PSBI subclassification, costs of staff time to provide treatment, costs for medicines and consumable supplies such as syringes, needles, methylated spirit, and a cotton swab were calculated.While Lucknow had follow-up visits on days four and seven after the initiation of outpatient treatment, Haryana reported that mothers were asked to return to the facility on the fourth day of treatment for pneumonia, whereas for PSBI, the follow-up visits happened on all days infants were brought in for gentamicin injection. Staff costs were calculated for time spent on follow-up and the number of visits reported at each site. The follow-up visit was not differentiated by the type of treatment received.The treatments received by sick young infants were classified as recommended, acceptable, partial, and non-compliant, based on the number of days and types of medicines given to them  1) coordination and supervision by senior medical officers such as handholding and confidence building for medical officers and nurses at PHC and CHC 2) for ASHA coordinators, ANM, lady health visitors (LHV) at facility and field level.Recurrent costs of training for an average of one to four days were included for different providers at each site. The training of trainers was considered a startup cost and not included in annual recurrent costs.These include 1) demand generation activities, including communication and community mobilisation activities such as organising school rallies and counselling sessions at Village Health and Nutrition Day (VHND), along with the cost for information, education, and communication (IEC) materials like counselling cards, wall painting, banners, leaflets, danger sign cards for mothers and 2) job aids such as training manual, and treatment displays. Research and most health system costs were not included. Costs required for implementation research such as the development of research instruments (data collection formats), study protocol development, translation of PSBI management guidelines into the local language (Hindi), initial training of the research staff, staff hired to monitor the effectiveness of the antibiotic regimen; costs of baseline surveys, workshops, were not included. Infrastructure and equipment costs for health systems such as power, computing / office, transport, and communication equipment; data management, reporting and logistics; non-consumable items such as thermometers and timers were also not included. Fifty percent of the costs of HBNC kits and weighing scales were included as these were considered important for strengthening correct weighing and identifying danger signs.Data were collected from each site on the types and the total number of staff involved in PSBI activities and their average annual salaries; the number of live births; the number of young infants receiving one-seven postnatal visits; and the number of infants referred, assessed, and treated under different activities. The total number of treated young infants was estimated by adding all those who received recommended or acceptable treatment for PSBI or pneumonia. If an infant was likely to get more than one visit for an activity such as postnatal visits, then the data was compiled for the number of visits for that intervention / activity, otherwise was compiled on the number of infants receiving that intervention.We also collected data on time duration (in minutes) taken by the providers for each activity, using structured interview schedules from medical officers, nurses and ANMs working at sub-centres, PHC and CHC. We collected the data using two approaches: 1) third-party observation – where the researcher directly observed the time taken for each sub-activity, and 2) self-observed / self-reported method – wherein the health providers noted the timing by themselves for activities undertaken . The numData were collected on price and quantities per episode of treatment for medicines and supplies required for fast breathing and any PSBI sub-classifications. The price of medicines and consumables used are presented in Table S3 in the Costs were analysed separately for 1) pre-outpatient activities 2) activities related to outpatient treatment 3) operation and administrative costs.Costs for pre-outpatient activities were estimated using weighted average costs under identification, assessment, referral, and management of sick young infants using IMNCI  250 (US$3.65) when they completed six or seven postnatal home visits and 50% (Indian ₹125) if they made two-five home visits. Based on the live births who got at least six visits, and those who got two-five visits, total costs for postnatal visits were calculated by assuming that 20% of the costs were attributed to PSBI . ReferraThe outpatient treatment costs were calculated per infant treated with “recommended / acceptable” treatment separately for fast breathing and PSBI . Total outpatient recurrent costs were estimated per young infant treated by adding costs of staff time, medicines, and consumables per treated child. Weighted average costs were calculated for PSBI depending on the number of infants treated and total costs under each PSBI sub-classification. While the outpatient costs for each PSBI sub-classification were estimated using infants treated with recommended treatment, the costs per follow-up visit were calculated using all treated young infants with fast breathing and PSBI.Operational costs were estimated per live birth and not per child treated, as these activities benefited all newborns. The costs for administrative staff were calculated based on the full-time equivalent of each staff type  and their salaries. The total costs for the training of the staff were calculated based on per day per trainee costs and the number of staff trained. The per trainee daily costs were estimated using the number of days of training provided, the number of trainees, honorarium for trainers, per diem for trainees, costs of training material and costs for rental of facilities and refreshments. Fifty per cent of the costs reported for IEC and job aids were assigned to PSBI. In addition, in the Palwal district, costs were included for specific activities such as 100% of costs for school rallies and the super village challenge as a special initiative for demand generation, and 10% of the transport per diem paid to ASHAs for attending the supervision meetings during the year.Total costs per infant treated were estimated separately for treatment of any PSBI sub-classification and fast breathing by adding the pre-outpatient costs per infant treated, outpatient costs per infant treated, and operational costs per live birth. The operational cost per live birth was calculated by adding the total recurrent costs for staff time, supplies, operations, and administration and dividing it by the total live births at each site.Local currencies are converted into US$ based on the mid-market exchange rate for July 2018 at 1US$ = Indian ₹68.5 .The study was approved by the institutional ethics committees of each participating institution and the WHO Ethics Review Committee. Written informed consent was obtained from caregivers for data collected.Direct intervention costs for staff time, medicines, and consumables were estimated per young infant treated with recommended / acceptable treatment for pneumonia and PSBI sub-classifications  and administrative costs were 25% in Palwal compared to 12% in Lucknow of the total costs of treating PSBI. The incremental costs, excluding staff costs, were US$6.1 and US$4.3 for PSBI, and US$3.5 and US$2.2 in Palwal and Lucknow, respectively. This implies the share of staff costs in the total (direct + indirect) costs of treating PSBI with recommended treatment was 64% in Palwal and 72% in Lucknow. The larger operation costs in Palwal were due to communication costs for the demand generation activities and transport incentives paid for supervisory visits, and higher staff costs in Lucknow were due to higher salaries and longer time reported per activity. Costs per live birth were US$5 in Palwal compared to US$3 in Lucknow.The weighted average costs across both sites for treating young infants with PSBI in an outpatient setting with recommended / acceptable treatment was US$15.9 (95% CI = US$15.4-16.3) considering all costs, i.e., the cost for identification, postnatal home visits, referrals, treatment, daily assessments, operation, and administration. The corresponding costs for treating pneumonia were US$10.1 (95% CI = US$9.7-10.6). Additionally, in a population of 4271 annual live births in the study area of Palwal and 13895 in Lucknow, investment for management and treatment of PSBI or pneumonia was estimated at an average cost of US$5 per live birth in Palwal and US$3 per live birth in Lucknow. Our results show that recommended direct treatment costs, including pre-outpatient and follow-up assessments for PSBI, were US$13.0 in Palwal and US$13.6 in Lucknow. For pneumonia, the direct costs of treatment with oral amoxicillin were US$7.5 in Palwal and US$7.9 in Lucknow. Low direct costs for pneumonia are primarily due to low staff time costs (mothers are trained to give the antibiotic to the infant) and no consumable costs.While the total costs at the two sites are a bit different per treated infant, with variations in components of direct costs and operational costs, the direct costs of treatment were higher in Lucknow because of higher staff  costs. In Palwal, there were more contractual nurses, who had lower average salaries than regular staff. The pre-outpatient costs per treated infant for the identification of sick infants, and their subsequent management were more than a quarter of the total costs at both sites. The WHO and the Government of India both recommend postnatal home visits to improve maternal and newborn survival ,27,30,31Marginal operational and administrative costs of staff for management, supervision, training, and communications, are an important component of the effective implementation of the program. These formed 24% of the total costs for recommended treatment in Palwal and 12% in Lucknow. The share and absolute costs were higher at the Palwal site, as more was spent on communication, demand generation activities, and the staff implementing these activities. The lower costs per live birth in Lucknow were due to lower operational and administrative costs serving a larger number of live births.Our results showed that staff costs were one of the major components of costs for treatment. These comprised 64% and 72% of the total costs of treatment for PSBI in Palwal and Lucknow, respectively. The incremental costs  were US$6.1 in Palwal and US$4.3 in Lucknow per infant receiving recommended/ acceptable treatment for PSBI. These incremental costs were US$3.5 and US$2.2 for pneumonia in Palwal and Lucknow, respectively.In summary, while pre-outpatient costs for mobilisation may be important, the costs paid in the form of ASHA incentives may not imply better mobilisation. While mobilisation by ASHA worked in the case of Lucknow, Palwal had to implement additional social mobilisation methods to educate mothers to identify danger signs and bring the infant to the primary care facility, which led to higher operating costs for them. This might have important national policy-level implications on strategies to improve coverage while implementing PSBI management guidelines in similar settings.Our results show lower costs for both pneumonia and PSBI, as we did not include the costs of antenatal care and treatment of mild infections for non-PSBI cases, and restricted the assessment costs only for the infants identified with PSBI. Further, as this was the implementation research, a large part of the supplies for pre-outpatient and outpatient treatment, and operational costs were considered health system costs and were not included. Our results show costs to be lower than those found in the AFRINEST study for three African countries , and in The average costs of treating pneumonia across the two sites were less than half (US$10.1) compared to US$26.2 in the AFRINEST study in an outpatient setting . A studyThe WHO recommended outpatient treatment when a referral is not feasible for PSBI was reportedly cost-effective at US$32.3 in the AFRINEST study . In our Our study has strength in terms of estimating the true costs of scaling up the existing program. This implementation research was conducted in a program setting, and both average and incremental costs were estimated based on actual implementation costs incurred for treatment and strengthening of the program. The estimated average costs are marginal in the sense that these did not include the capital and equipment costs, which were considered part of the health system. Further, only additional time for the staff for activities and fractional costs of certain consumables and non-consumables, considered important for strengthening health systems for additional infants treated, were included. Operation costs- communication, demand generation and training were only considered for system strengthening. Scaling up at these costs is only possible if the health system continues to function at least in its current state. We did not estimate societal or household costs. We also were unable to estimate the cost-effectiveness in this study, because we did not have pre-intervention coverage or mortality data, and the scope of the study was to estimate costs for budgeting purposes and provide policymakers guidelines to strengthen the PSBI management in outpatient settings. The cost data have been presented for 2018-19, the year for which the data was collected. However, in 2022, when adjusted for inflation, the average costs per young infant treated for PSBI and pneumonia would be US$18.5 and US$11.7, respectively.Outpatient treatment is not only beneficial in terms of reduced costs but is also less disruptive for the families and has less risk of getting hospital-acquired infections ,36. Outp


Genital injury has a forensic relevance after a sexual assault and it has been discussed and investigated among professionals who work in this field. To analyze the studies published in the last decades, the present review examines different factors that may influence this finding, first clarifying terms of the forensic field, such as the peculiarity of the legal medical examination, and the distinction of the terms “legal” and “anatomical” vagina. Finally, it analyses if it is possible that the existence of these injuries in victims explain the lack of consent in sexual contact, and to clarify the meaning of the absence of injuries. In the usual context of medical practice, the clinicians assess, diagnose, document and treat body injuries according to the medical needs of the patients. When a victim of sexual violence is evaluated in the forensic context, much of the effort is put on the genital examination, giving too much emphasis to the presence of the genital lesions as evidence to take a legal action. For this reason, the forensic importance of genital lesions after a sexual assault has been subject to discussion and research among professionals working in this field.Albeit understanding the psychosocial context and consequences of sexual violence is crucial for a better management of the victims, the aims of the present manuscript is, on the one hand, to refer only to genital injuries in adult females as an evidence of sexual assault. On the other hand, the present review intends to clarify the research in this area, which is full of uncontrollable variables, and therefore, it is not possible to make reliable conclusions considering genital lesion as the only evidence, even when they may be present after a consented intercourse. Before analyzing this current research and delineating the importance of genital lesions, it seems pertinent to clarify some terms and characteristics of this forensic field.The victim of sexual abuse requires to be examined by a competent clinician with a comprehensive knowledge of his/her forensic and therapeutic role. Essential components of a sexual forensic examination  must be It is important consider the clinical context in which the clinical environment in which physical and genital examinations were performed, and the expertise of the clinician. It is clear that clinicians or nurses with different training levels will bring different competencies to the clinical forensic examination, and this will influence their findings. The venue can be a referral unit, center or any variant of this model care . Both examiner and place heterogeneities may explain the different outcomes that a sexual forensic examination can have.The forensic examination can be done with several techniques to watch the genital zone, some of them allow covering the skin and mucosa with different solutions. Some people only use naked-eye inspection; others use the magnification given by a colposcopy, adding or not toluidine dye.The vaginal epithelium is characteristically thin in childhood, but after puberty, it begins to thicken in response to estrogen stimulation with progressive cellular proliferation and growth that results in the formation of intermediate and superficial layers of cells,Prior to continuing, it is important to define a legal frame of sexual violence. Jewkes et alIn many cases, legal reforms have reframed sexual violence as a criminal rather than a moral offense - as it was historically conceptualized in many LAC legal systems. In some settings, discriminatory clauses against the victims have been eliminated, such as allowing victims to be questioned about their previous sexual history, their conduct during the attack or their “honour.” Also, legal reforms have introduced marital rape as a criminal offense, which did not exist before in some countries, such as Mexico. For instance, in Brazil in 1983, Maria da Penha Maia, who was a woman who survived to two murder attempts by her husband. She became a paraplegic as a result of the abuse. She battled for twenty years to bring her case to justice, appealing to international organizations such as the Inter American Commission on Human Rights. The story gained international attention and finally the national domestic violence law in Brazil was signed in 2006 and named “Maria da Penha” in her recognition. The law specifically defines sexual violence as a crime, and includes preventive, punitive and protective legal mechanisms. It is considered one of the most advanced laws in the world addressing violence against women.Despite significant improvements to the laws addressing sexual violence in LAC, sexual violence still remains as a major concern. While in some countries marital rape is not addressed by the legal code and others still consider rape and sexual assault as an offense against “morals” or honor rather than a criminal act against the individual woman,Otherwise, it is quite interesting the difference between medical and legal definitions of vagina in some countries, as the UK, which may guide to confusion and error amongst professionals involved in rape allegation. The medical definition considers the vagina as a muscular tube that has the cervix as its proximal end and the hymen  as the distal end. The British legal definition according to the Sexual Offenses Act 2003  considers the distal end of the vagina as the beginning part of the vulva, therefore, “vagina” includes the vulva (between the labia). For legal purposes, penetration of the vagina does not have to involve penetration of the hymen. By contrast, in other nations there is no difference between both meanings in their domestic law.The wide variations found in the literature are attributable to diverse examination variables such as inconsistent definitions of “findings,” variable time span from sexual intercourse to examination, inclusion criteria of complainants, and divergent statistical methods. These variations demonstrate the difficulty of interpreting the findings in a group of victims, and it is even harder to try to compare them.The prevalence of genital injuries reported after sexual assault ranges between 5 and 87%, according to a meta-analysis conducted by Kennedy.Tears (lacerations), Ecchymosis (bruises), Abrasions, Redness and Swelling. Another classification considers abrasions, bruises and wounds, which can be lacerations or incisions.To improve the accuracy of interpretation of physical findings, some years ago a pattern of genital injuries in female victims was defined, whose acronym is TEARSThe frequency and type of injury also vary according to the region of penetration.Another issue to consider is the timing of sexual assault and healing. The knowledge of injury healing may assist to decide the urgency with which an examination is performed. In some cases, the age of an injury might assist in determining who had access to the victim during the specified timeframe. Police or the court may ask the clinician to consider how old a genital injury is, and this may help to determine whether it was a result of previous consensual intercourse or a later alleged assault. On the other hand, many studies did not stipulate injury rates as genitalia tend to heal quickly.In terms of healing times, the evidence has shown that non-hymenal genital injuries heal at diverse rates depending on the type, location and severity. Nonetheless, there is no statistical difference in the rate of healing between pre- and postpubertal girls. In case of laceration, its depth determined the time required to heal. While superficial vestibular lacerations seemed to heal in 2 days, deep perineal lacerations required up to 20 days.The timing of examination determines the diagnostic rates of genital injuries,From a therapeutic and forensic perspective, a differential diagnosis is crucial. In both settings, the professional could be asked whether the genital findings resulted from an alleged assault or have another explanation. Therefore, awareness of medical conditions that affect the genitals can significantly reduce stress in patients and their surroundings, and lead to an accurate diagnosis. There are several conditions that might be confused with injuries such as allergy, eczema, psoriasis, infections , and normal anatomical variations, amongst others. Consequently, obtaining a full history, when indicated, is a critical element to establish the context in which these findings should be interpreted.Decades ago, there was a wrong view about the “normal” response of women in consensual intercourse. This included vaginal lengthening, increased lubrication and changes in muscular tension, which protected her from genital injury.Another myth that must be tackled is that rapes or sexual assaults are necessarily violent. Modern legal definitions have now replaced the ‘use of force’ with ‘lack of consent’ as the defining feature of rape. In the Sexual Offenses Act 2003, a person consents if he/she agrees by choice, and has the freedom and capacity to make that choice,Specifically about resistance, many women feel paralyzed when are attacked, especially when they fear for their life.The likelihood of injury in the first intercourse of a woman deserves a special mention. During many years, the bleeding from the first hymenal laceration has had sociological/religious significances in many cultures across the world. Nevertheless, hymenal injury is not always present, as 40 to 80% of women do not bleed in the initial coitus,This is the most crucial question, which is not always answered ‘beyond reasonable doubt’. This situation usually frustrates prosecutors of sexual crime as it might explain low conviction rates for rape.case-control OR case AND “control” OR “consensual” AND “nonconsensual” AND genital OR anal OR genitoanal OR vagina OR vulva OR vulvar AND lesion OR injury AND sexual abuse OR sexual violence OR rape OR sexual assault. The search retrieved 49 different articles, 41 of them were excluded after title and abstract reading, and full-text reading was performed for the remaining 8 articles, independently of the language. Finally, 6 articles were included considering the following inclusion criteriaAll case-control studies published until 31st December 2018 in PubMed, CINAHL and EMBASE databases were retrieved using the terms Despite the works of Anderson et alResearchers have been motivated to determine whether factors associated with genital injury can assist in obtaining evidence, to corroborate something that typically occurs between two people without direct witnesses.Based upon the theory that genital injuries might be more likely to occur or be more severe in those cases without consent, the presence, pattern and/or severity of genital injury might be helpful in answering the question about consent. However, this hypothesis is outdated. Although the aforementioned studies done by Astrup et alAstrup et alAccording to Lincoln et al,Taking into consideration the relative irrelevance of common types of genital injury, nongenital examination and documentation of injuries elsewhere on the body may be invaluable. In the meta-analysis published in 2013Penetration with finger(s) and possible pre-existing genital ‘infection’ were found to be significantly associated with the presence of genital injury in the univariate analysis performed by Lincoln et al.Although in the Lincoln et alHilden et alWhen White et alRecently, Zilkens et alWhile Hilden et alBoth the studies by Astrup et alThe percentage of participants without body or genital injury in diverse studies was extremely wide, between 18 and 68%.It is known that police approach to the rape crime is significantly influenced by the presence of injuries. However, the examiner clinician must emphasize in his/her report that “the absence of genital trauma does not preclude the possibility of nonconsensual sexual intercourse.” As health care providers, we have an important obligation in ensuring that police officers fully understand the meaning and the significance of this assertion. Therefore, the same analysis that is discussed about genital injuries should be provided to the police, lawyers, the judiciary and the general public.Many factors influence the presence or absence of genital injuries during a sexual assault, and therefore, they are not strong evidence for this type of crime per se, but the existence of a mutual consent among two – or more – people involved in an intercourse. Although two serious studies demonstrated a significant difference between consented and nonconsented intercourse groups in prevalence of genital injury, they have serious methodological issues that may be affecting these results. Therefore, the presence or absence of genital injury should not be used to render an opinion regarding consent to sexual intercourse.There are many reports in the literature concerning the prevalence of genital injury following alleged sexual assault. Unfortunately, the variety of examination/visualization techniques, participant inclusion criteria, injury definitions, and time to examination seen in these studies make them difficult to assess and get to a convincing conclusion.Regarding the severity or pattern of genital injuries and their association with the consent, some skepticism must be applied to all research that tries to prove this association.Finally, the lack of injuries highlights the importance of ensuring that all those who are involved in these cases, such as medical and legal professionals, police officers, and the public in general, are aware that injury is absolutely not a necessary outcome of sexual violence.In conclusion, it is important to emphasize that the absence of genital lesions does not translate the absence of sexual violence. This is something that we, as healthcare professionals, must be sensitive to and aware of, especially knowing the reality of the legal framework in our LAC region. Therefore, the forensic examination, although relevant, must be accompanied by other social, psychological, medical, etc. evaluations and care by the corresponding professionals to give evidence of the occurrence of a sexual assault as well as to give a comprehensive, holistic management to the victim.


Cannabis sativa) cultivation. We used Illumina small (s)RNA sequencing for virome reconstruction and characterization of antiviral RNA interference (RNAi) in monoecious and dioecious hemp varieties, which exhibited different virus-like symptoms. Through de novo and reference-based sRNA assembly, we identified and reconstructed Cannabis cryptic virus (family Partitiviridae), Cannabis sativa mitovirus 1 (Mitoviridae) and Grapevine line pattern virus (Bromoviridae) as well as a novel virus tentatively classified into Partitiviridae. Members of both Partitiviridae and Bromoviridae were targeted by antiviral RNAi, generating 21 nt and, less abundant, 22 nt sRNAs from both strands of the entire virus genome, suggesting the involvement of Dicer-like (DCL) 4 and DCL2 in viral sRNA biogenesis, respectively. Mitovirus sRNAs represented predominantly the positive-sense strand and had a wider size range, with the 21 nt class being most abundant on both strands. For all viruses, 21 and 22 nt sRNAs had predominantly 5′-terminal uridine or cytosine, suggesting their binding to antiviral Argonaute (AGO) 1 and AGO5, respectively. As no clear association of any virus with symptoms was observed, further studies should clarify if these viruses individually or in combination can cause hemp diseases.Viral infections pose an emerging threat to hemp ( Cannabis sativa L.) is currently undergoing rediscovery in various economical fields, from industrial to medicinal 7], BCTV


Cynopterus bat collected in Yunnan Province, China, in 2011. Full genomes of two cressdnaviruses , and a nearly complete genome for a third  were obtained. Phylogenetic analyses and the characteristics of these viral genomes suggest a high degree of ssDNA virus diversity. These results shed light on cressdnavirus diversity and the probable role of Cynopterus bats as their hosts.Bats serve as reservoirs for many emerging viruses. Cressdnaviruses can infect a wide range of animals, including agricultural species, such as pigs, in which porcine circoviruses cause severe gastroenteritis. New cressdnaviruses have also attracted considerable attention recently, due to their involvement with infectious diseases. However, little is known about their host range and many cressdnaviruses remain poorly characterized. We identified and characterized 11 contigs consisting of previously unknown cressdnaviruses from a rectal swab sample of a  ANSO-CR-SP-2020-02), the Shanghai Municipal Science and Technology Major Project (Grant No. 2019SHZDZX02), G4 funding from Institut Pasteur, Fondation Merieux and Chinese Academy of Sciences to G.W., and the International Affairs Department of the Institut Pasteur of Paris, and the 2021 Project of Guangdong Forestry Bureau to J.C. Y.K. is supported by the CAS-TWAS Fellowship for International Doctoral Students.This project was supported by the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Dedicated to the 130th anniversary of Dmitry Ivanovsky’s discoveryof the virus kingdom as a new form of biological life.The genome of some RNA-containing viruses comprises ambipolar genes that arearranged in stacks (one above the other) encoding proteins in oppositedirections. Ambipolar genes provide a new approach for developing viraldiversity when virions possessing an identical genome may differ in itsexpression scheme (strategy) and have distinct types of progeny virions varyingin the genomic RNA polarity and the composition of proteins expressed bypositive- or negative-sense genes, the so-called ambipolar virions. So far,this pathway of viral genome expression remains hypothetical and hidden fromus, like the dark side of the Moon, and deserves a detailed study. Ivanovskyreported having discovered a new form of biological life, the so-called“contagium vivum fixum” [m fixum” , which wm fixum” . Accordim fixum” .According to the well-known classification by D. Baltimore , which iDifferences in the viral genome structure and variations in the patterns of itsexpression in an infected cell underlie virus diversity, pantropic adaptation of viruses to various organismssuch as bacteria, fungi, plants, fish, and animals, in particular humans, andensure the global spread of viruses on Earth, and possibly in space and otherplanets .The genetic diversity of viruses, which underlies the Baltimore classification,was considered as follows: one unique viral genome develops one genomestrategy; i.e., one genome has one replication scheme and directs the formationof one structural and functional class of virions . This implies a uniform and unified process for the synthesis ofviral particles (virions) within one viral genus (or family) , 8.HowePB1,PB2, PA, HA,NP, NA, M,NS) with negative coding polarity in the genomic RNA in the3’ → 5’ direction, had additional positive coding polarity(in the 5’ → 3’ direction of the genomic molecule).The peculiarity of these ambipolar genes was theirlocalization in genome regions overlapping the corresponding classicalnegative-sense genes; the so-called stacking arrangement [Fig. 1B).The ambipolar genes identified in the genomes oforthomyxo- and coronaviruses were found to be characterized by the presence ofall the functional elements necessary for expression of these geneticframeworks as translational genes [In 2007, we analyzed the negative-sense genome of influenza A viruses(orthomyxovirus family) and found extended open reading frames (ORFs) that,unlike the canonical influenza virus genes , and thal genes  possessial genes , 14, 18.Today, the genome of one virus species (genus) is believed to have one strategythat determines the formation of viral particles of a certain structure and a characteristic range of hosts. The discovery of ambipolarstacking genes in the genomes of RNA viruses suggests the existence ofalternative strategies in the genome of one virus species (genus) whoseexpression pathways may (1) provide the synthesis of several structural andfunctional classes of virions that differ in both their protein composition andthe structural form (polarity) of genomic RNA and/or (2) develop severaldifferent strategies for virus replication and its pathogenesis in an infectedmacroorganism. The presence of several strategies in one viral genome providesa reserve of viral adaptive properties, which may be considered as a pathway(or modification) of genetic bet-hedging .Fig. 2 forthe influenza virus and coronavirus models. Theinfluenza virus comprising genomic (–)-RNA is characterized by thepossibility of both a classical pathway of genome implementation and alternative strategies. Implementation of alternative genomestrategies may lead to the formation of ambipolar virions that may contain bothclassical proteins  and additionalproteins–products of the ambipolar genesNSP1–NSP8 (NSP –Negative Strand Protein) of appropriate genomic RNA segments.Expression of the classic coronavirus strategy also leads to theformation of virions containing the canonical (+)-RNA genome and classicalstructural proteins: N (nucleocapsid protein), S (surface glycoprotein), E(membrane protein), and M  and a number of auxiliarynon-structural regulatory proteins (nsp1-nsp16) that support viral replicationin target cells and suppression of the host’s immune response. However,the products of the main ambipolar genesNGP1–NGP5 , escape the attention of researchers. So far, these proteinsencoded by open ambipolar genes have not been found in infected cells. Apossible reason lies in either the minor level of their synthesis or theirstrictly selective expression only in specialized body cells containing theunique factors necessary for the expression of these viral stacking genes undercertain conditions of the intracellular and/or surrounding extracellularenvironment. At the same time, there are indirect approaches to observeambipolar gene expression in an infected macroorganism. Animals infected withthe influenza A virus were found to develop clones of cytotoxic lymphocytesthat recognize specific peptide domains of the influenza virus ambipolarproteins, in particular the NSP8 protein encoded by the ambipolar NSP8gene of the influenza A virus NS segment [The multiple strategies of the genome in one virus species (genus) and theexpression schemes of its classical and alternative strategies are shown inproteins), which  segment , 25,26.Fig. 2).Thus, ambipolar viral particles may containboth ambipolar proteins and ambipolar genomic RNA replicas, providing analternative pathway for the viral genome strategy. As a result, one uniqueviral genome may be implemented in several, alternative strategies – withor without involvement of ambipolar genes – and viruses may possessseveral possible life pathways, depending on the context of the surroundingcellular processes. This idea is illustratedin Fig. 2. Thismultivariant mechanism of a unique viral genome strategy may be considered as away of bet-hedging by viruses, which promotes the establishment of alternativeways of virus replication and the creation of reserve adaptive potentials forviruses of various families. In this aspect, RNA viruses may be similar to DNAviruses and RNA-containing retroviral (virus-like) transposons that have adual-track lifestyle: as a DNA provirus and a mature virus, respectively, whichdetermines the vertical  and horizontal (mature virions) ways of their existence in the host,depending on the propagation environment and the range of hosts [We may posit that these non-canonical proteins are able to decorate the viralgenome from a new class of viral particles performing unique regulatoryfunctions, and altering the virus behavior in an infected organism; e.g.,switching from productive virus infection to a latent persistent (lowreproductive) viral infection process. Furthermore, there may be an alternativewhen a genome molecule becomes an RNA chain complementary (ambipolar replica)to the canonical virus genome: the coronavirus (–)RNA or influenza virus(+)RNA . So far, these pathwaysof multiple expression strategy of the viral genome remain as hypothetical andenigmatic as the “dark side of the Moon.” Experimental verificationof this crystal-ball reading exercise will enable us to evaluate the possibleexistence of ambipolar classes of stealth virions hidden from the eye ofresearchers. To date, mature protein products encoded by identified ambipolarviral genes in an infected organism have not yet been detected. But this doesnot mean that expression of these viral stacking genes is not implemented innature. Identification of the expression of these genes requires a targetedsearch using original approaches and highly sensitive methods for identifyingproteins in various organs and the specific cells of an infected hostmacroorganism. It is possible that the unraveling of alternative strategies ofviral genomes may be important for understanding virus evolution and thepathogenesis of viral infections, as was the case in covid-2019 when long-termand severe complications of the viral infection could develop due to theformation of ambipolar virions hidden from the attention of researchers andmedical practitioners.The ambipolar genes of viruses are endowed with high evolution stability. Inparticular, in the natural population of highly variable influenza viruses,these genes have been observed in the genome with all the necessary regulatoryelements for more than 100 years, despite a noticeable population variabilityin both canonical and identified ambipolar genes with a characteristic highdN/dS coefficient that indicates pronounced immunological pressure from thehost macroorganism in nature . TheevoFig. 3 usingan arenavirus model . In this regard, the difference in stacking allows us toconsider two major groups of ambipolar viruses. To date, the following divisionseems logical: in the first group of viruses with gene stacking in the viral genome, pathways of ambipolar genome strategiesmay have an alternative  character, while in viruses lacking genestacking , the implementation of theambisense genome strategy should be considered as an obligatory (mandatory)reality for virus replication. Further targeted search for the expressionpathways of alternative genome strategies in one viral species andidentification of a hypothetical class of ambipolar virions will answer thequestion of the existence of this type of viral life diversity and its role inthe evolution of viruses of various genera. This knowledge will come handy inthe development of new vaccines and antiviral drugs and add to ourunderstanding of the molecular basis of viral disease pathogenesis.Obviously, the ambipolar stacking of genes found in RNA viruses provides thevirus with, first, an enhanced information capacity of the genome. Second, itunderlies the linked  evolution of viral genes when mutations inone gene generate changes in a stacking gene and, thus, represent a kind ofgenetic synteny. Third, the protein products of stacked genes may befunctionally linked and have a predetermined structural correspondence to eachother, which remains a hypothetical and requires experimental evidence, 17.The


To (a) describe the demographics of opioid abusers; (b) compare the prevalence rates of selected comorbidities and the medical and drug utilization patterns of opioid abusers with patients from a control group, for the period from 1998 to 2002; and (c) calculate the mean annual per-patient total health care costs  from the perspective of a private payer.International Classification of Diseases, 9th Revision, Clinical Modification) codes for opioid abuse . A control group of nonabusers was selected using a matched sample  in a 3:1 ratio. Per-patient annual health care costs  were measured in 2003 U.S. dollars. Multivariate regression techniques were also used to control for comorbidities and to compare costs with a benchmark of depressed patients.An administrative database of medical and pharmacy claims from 1998 to 2002 of 16 self-insured employer health plans with approximately 2 million lives was used to identify “opioid abusers”—patients with claims associated with ICD-9-CM (P<0.01) times higher, respectively, compared with nonabusers. Opioid abusers also had higher levels of medical and prescription drug utilization. Almost 60% of opioid abusers had prescription drug claims for opioids compared with approximately 20% for nonabusers. Prevalence rates for hospital inpatient visits for opioid abusers were more than 12 times higher compared with nonabusers (P<0.01).740 patients were identified as opioid abusers, a prevalence of 8 in 10,000 persons aged 12 to 64 years continuously enrolled in health care plans for whom 12 months of data were available for calculating costs. Opioid abusers, compared with nonabusers, had significantly higher prevalence rates for a number of specific comorbidities, including nonopioid poisoning, hepatitis , psychiatric illnesses, and pancreatitis, which were approximately 78, 36, 9, and 21 (P < 0.01). Hospital inpatient and physician-outpatient costs accounted for 46%  and 31%  of opioid abusers’ health care costs, compared with 17% ($310) and 50% ($906), respectively, for nonabusers. Mean drug costs for opioid abusers were more than 5 times higher than costs for nonabusers , driven by higher drug utilization (including opioids) for opioid abusers. Even when controlling for comorbidities using a multivariate regression model of a matched control of depressed patients, the average health care costs of opioid abusers were 1.8 times higher than the average health care costs of depressed patients.Mean annual direct health care costs for opioid abusers were more than 8 times higher than for nonabusers ($15,884 versus $1,830, respectively, CONCLUSION: The high costs of opioid abuse were driven primarily by high prevalence rates of costly comorbidites and high utilization rates of medical services and prescription drugs. Opioid abuse can impose substantial costs on society and to payers as well as to the users themselves. Although there has been some research on the overall costs of opioid abuse, few studies have undertaken systematic research of costs from the payer perspective and quantified the payer burden of opioid abuse. In this article, we analyzed the economic burden of opioid abuse (both prescription and non-prescription) from a private payer’s perspective. The analysis focused on average per-patient direct health care costs  measured in 2003 United States dollars.3 Findings from the National Survey on Drug Use and Health showed that nonmedical use of pain relievers among persons aged 12 years and older increased from 11.0 million to 11.7 million.4 Although these results are not directly comparable with those of the NHSDA, they indicate the growing prevalence of opioid abuse in the United States.A number of studies have discussed the growing prevalence of opioid abuse in the United States. The National Household Survey on Drug Abuse (NHSDA) estimated that more than 6 million Americans used prescription pain relievers  for nonmedical purposes in 1999. By 2001, this number had increased to 8.4 million, approximately 4 % of the U.S. population.5 Furthermore, the number of emergency room visits related to illegal drug use or nonmedical use of prescription drugs increased from approximately 250,000 in the latter half of 1997 to more than 325,000 in the latter half of 2001.6 The growing prevalence of drug and opioid abuse has caused an increase in government attention devoted to this problem; in 2001, the National Institute of Drug Abuse launched a public information campaign to educate patients, health care providers, and pharmacists regarding the potential dangers of prescription drug abuse.7From 1999 to 2001, the number of patients admitted for the treatment of prescription opioid abuse increased from approximately 23,000 in 1999 to more than 38,000 in 2001.8 His study focused on 3 major cost categories: health care, criminal justice, and workplace burden costs. The National Institutes of Health reported that, in 1998, the overall cost of drug abuse was $143.4 billion, with 69% in lost productivity costs, 9% in health costs, and 22% in other costs such as criminal justice.9 There have also been a few studies that have focused on the costs associated with illicit drug abuse.10,11Several researchers have attempted to quantify the costs associated with drug abuse, including opioid abuse. Without distinguishing between legal and illegal drug use, Harwood estimated that overall costs of drug abuse in 1998 were $97.7 billion.12 Given the trends in increasing prevalence rates of prescription opioid abuse, its economic burden on society is very likely to escalate in the coming years. Furthermore, since the condition is likely to be underdiagnosed because of the associated stigma, the true burden is probably much greater.Only one study was found that focused on prescription opioid abuse in particular. Birnbaum et al. found that the cost of prescription opioid abuse in the United States in 2001 was $9.2 billion; approximately 30% ($2.8 billion) was for health care, 50% ($4.6 billion) for lost productivity, and 20% ($1.8 billion) for criminal justice.This article focuses on the payer burden of opioid abuse by measuring the per-patient costs incurred by payers arising from all medical and pharmacy payments made for patients diagnosed with opioid abuse .International Classification of Diseases, 9th Revision, Clinical Modification) codes for opioid abuse. The difference in costs between these 2 groups was used to calculate the excess burden of opioid abuse . Costs were measured as payer payments for services provided during a 12-month period for each patient during the years 1998-2002, and were adjusted to 2003 U.S. dollars using the medical consumer price subindex. Descriptive statistics for medical services and drug utilization, prevalence rates of selected comorbidities, and direct health care costs were compared for opioid abusers and nonabusers.A group of patients with a diagnosis of opioid abuse was identified and randomly matched to a group of patients with no diagnoses of opioid abuse. Medical and pharmacy claims and eligibility data were used to compare the demographic characteristics, medical services and drug utilization, prevalence rates of selected comorbidities, and average annual direct health care  costs for patients for both groups. Patient samples were identified using ICD-9-CM  as well as prescription drug claims for employees, spouses, and dependents. Prescription drug utilization was identified using both National Drug Codes (NDCs) for drug claims and Health Care Financing Administration Common Procedure Coding System (HCPCS) codes for medical claims where drugs were administered as part of a medical procedure. The data allowed for disaggregation of costs by place of service . The data could therefore be used to compare the drivers of total payer costs across opioid abusers and nonabusers. The data also allowed comparison of medical and drug utilization and prevalence rates of selected pre-specified comorbidities across opioid abusers and nonabusers. The prevalence rates were compared by calculating relative risk ratios , and statistical differences in prevalence rates were assessed using a chi-squared analysis. Differences in averages between the 2 samples were assessed using The patient research samples were identified as patients between the ages of 12 and 64 years who were continuously enrolled in health care plans for a 12-month period around the index date (defined below). Patients were identified as opioid abusers based on having at least 1 claim with at least 1 of the following 4 ICD-9-CM codes over the period 1998-2002:304.0 opioid type dependence304.7 combinations of opioid abuse with any other305.5 opioid abuse965.0 poisoning by opiates and related narcotics excluding poisoning by heroin (965.01)The index date in this study was defined as the first observed date of diagnosis of opioid abuse. Patients were included if they had claims history for at least 6 months preindex date and 6 months postindex date. The index date was not necessarily the first date of diagnosis of opioid abuse, given the requirement of 6 months of medical claims history prior to the index date. If a patient was diagnosed with opioid abuse in February 1998, then this patient would not be included in the analysis, given that the patient would not have had at least 6 months of preindex date medical claims history (assuming there were no subsequent claims associated with opioid abuse). Patients were required to be continuously enrolled in health care plans during all 12 months of the study . The sample is likely to contain both prescription opioid abusers and nonprescription opioid abusers, since ICD-9-CM codes do not distinguish between the 2 types of opioid abuse.Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria for addiction (opioid dependence).7 In fact DSM-IV has specific criteria for opioid abuse. Definitions of prescription opioid abuse span a spectrum from episodic nonmedical use of a drug  to nonmedical use resulting in harm (Institute of Medicine and Drug Abuse Warning Network) to very specific criteria such as those in DSM-IV.7 In 2001, The American Academy of Pain Medicine issued recommendations for distinguishing between “physical dependence” and “addiction,” where physical dependence referred to a condition characterized by withdrawal symptoms when one stops using opioids. By contrast, addiction “is characterized by behaviors that include one or more of the following: impaired control over drug use, compulsive use, continued use despite harm, and craving.”13It is worth noting that the term “opioid abuse” is a general term, used differently by different people in various contexts. In some cases, it means a less serious form of addiction, i.e., someone appears to be addicted but doesn’t meet the full Given the different uses of the term opioid abuse and the possibility for miscoding with the ICD-9-CM codes, we grouped all such ICD-9-CM codes that related to opioid abuse for the purposes of our cost calculations and analyses and labeled these patients “opioid abusers.” However, recognizing that abuse and dependence may not necessarily be synonymous, we report some sensitivity analyses based on different cohorts defined by ICD-9-CM codes. These are discussed below.A random sample was also drawn from the same overall patient population to serve as a comparison group. The same basic inclusion criteria imposed on the opioid abuse sample were also imposed on the matched sample: aged 12 to 64 years, continuously enrolled in a health care plan during all 12 months of the study period, and having at least 1 medical or prescription drug claim during that year. The random sample excluded patients with opioid abuse diagnoses and was matched with the opioid abuse sample by gender, age, employment status, and census geographic region on a 3:1 matching ratio.An important feature of the analysis is that there was no “wash-out” period before or after claims associated with the ICD-9-CM codes for the opioid abusers in the study. Studies that impose a wash-out period before the first diagnosis and after the end of a “disease episode” are generally focused on capturing the cost of illness associated with the onset of the disease in question and its immediate treatment. In contrast, the goal of the current study was to capture a payer’s typical direct medical cost burden for the average annual cost of illness for all patients diagnosed with opioid abuse who may have been in different stages of their abuse: onset, treatment, management, and/or recovery. In addition, the nature of opioid abuse and the fact that it likely lasts for several years with associated higher prevalence rates of certain comorbidities creates difficulties in identifying the beginning or end of abuse and the costs associated with it. Therefore, by taking a more general cross-sectional approach in calculating the average direct health care costs associated with opioid abuse, patients with varying degrees of severity and in different stages of their illnesses were captured. Thus, we selected patients on the basis of a claim for opioid abuse, conditioned on having their medical claims history for the 6 months preindex and postindex date.Direct health care costs were calculated based on payments made by payers for inpatient, outpatient, physician, and prescription drug claims as well as for all other medical services. Since the focus of this study was on the payer burden of opioid abuse, these payments did not include deductibles or copayments paid by the patients . The dollar amounts for all claims for each patient during the 12 months including the 6-month preindex and 6-month postindex date were summed to arrive at their total direct medical costs. These costs were then averaged over all patients in the research samples to arrive at the average cost per patient for opioid abusers and nonabusers.The resulting sample sizes for the study were 740 opioid abusers and 2,220 nonabusers. Males accounted for 57% of opioid abusers and nonabusers . The aveThe prevalence of opioid abuse among the members in our database increased from 5 per 10,000 in 1998 to 8 per 10,000 in 2002, where the denominator was the total annual number of insured members in each year, and the numerator was the cumulative number of patients with claims associated with ICD-9-CM codes for opioid abuse in the year. This represents a 60% increase.P < 0.01) times, respectively, as prevalent as those for nonabusers. Similarly, an analysis of painful comorbidities found that almost half of all opioid abusers had any chronic pain comorbidity (47.6%), with low-back pain being the most prevalent . Furthermore, opioid abusers averaged 18.7 physician or outpatient visits compared with 7 for nonabusers (P<0.01). Opioid abusers were also 4 times more likely to have had an emergency room visit than nonabusers (P < 0.01). The high rates of use of medical services by opioid abusers, in combination with their comorbidity profile, likely contributed to their higher average health care costs.Opioid abusers had higher utilization rates of various medical services as measured by the number of claims for medical services . For exaP < 0.01). These drugs were identified using NDCs for drug claims and HCPCS codes for medical claims in the cases of drugs administered as medical procedures. About 58% of opioid abusers had claims for short-acting opioids (SAOs), long-acting opioids (LAOs), or both, compared with about 20% of nonabusers. More than 40% of opioid abusers had claims for SAOs, and about 17% had claims for both SAOs and LAOs. As noted above, almost 50% of opioid abusers had a diagnosis of a chronically painful condition . Statistically significant differences from the matched sample of nonabusers are noted in Calculations of the average health care costs for opioid abusers and nonabusers showed that patients who were opioid abusers had health care costs that were more than 8 times higher than those of nonabusers . The totP<0.01, or more than 20 times higher). Physician and outpatient visits accounted for 31% of opioid abusers’ costs compared with 50 % for nonabusers. Although such visits comprised a greater percentage of the costs for nonabusers, the costs of such visits for opioid abusers were more than 5 times higher compared with nonabusers . Average drug costs for opioid abusers were more than 5 times higher compared with those for nonabusers , while such drug costs accounted for 21% of overall costs for nonabusers compared with 13% for opioid abusers.Disaggregated direct health care costs were also calculated (P<0.01). For Group B, the total average per-patient direct health care cost was $16,204 compared with $2,179 for matched controls (P < 0.01). In both instances, the direct health care costs of Groups A and B were approximately 8 times as high as the matched controls, consistent with the findings above for the broader group used for this study. It appears that Group A has costs that are higher than Group B, indicating that those patients diagnosed as opioid “abusers” have higher costs than those diagnosed as opioid “dependent.” This finding should be interpreted with caution given the different uses of the term opioid abuse. Thus, the range of cost estimates for our sample of opioid abusers based on alternative groupings of ICD-9-CM codes is $15,884 to $18,388, and $1,830 to $2,210 for our matched sample.We also conducted sensitivity analyses on direct health care costs based on different groupings of the ICD-9-CM codes. In particular we divided our sample into 2 subgroups: one based on ICD-9-CM codes 304.7 and 305.5, codes for “abuse” , and another based on the ICD-9-CM codes 304.0 and 965.0, codes for “dependence” and “poisoning” . The subgroups are not mutually exclusive since it is possible for a patient to be diagnosed with more than one ICD-9-CM code for opioid abuse. Using the same methodology as above, the total average per-patient direct health care payer cost for Group A was $18,388 compared with $2,210 for matched controls  would allow for a more direct comparison of costs between opioid abusers and a matched control while simultaneously controlling for a variety of factors such as comorbidity profiles and demographics that may drive costs. Furthermore, it would be useful to have an alternative benchmark control group with which to compare the costs of opioid abuse; for this purpose, we have chosen a control group of patients with a diagnosis of depression .15 This regression allows us to compare the costs of an opioid abuser to those of a depressed patient, holding other factors constant, such as age, gender, and the presence of other comorbid conditions. This allows for a more direct and meaningful comparison of the costs associated with opioid abuse.Depression is a mental health illness that is common and is diagnosed on a relatively consistent basis. Similar to opioid abuse, this condition is managed by primary care doctors and specialists and is also a costly condition of interest to payers.We constructed a multivariate regression to account for this, where the left-hand variable was the per-patient costs during the 12-month period encompassing the 6 months preindex and 6 months postindex date. Patients were matched on a 3:1 basis (as before), and overlap between the 2 groups was controlled for . The regression specification is as below:female is a dummy variable taking the value 1 if the patient is female, 0 otherwise; ri is a dummy variable taking the value 1 if the patient is in region i, 0 otherwise; abuser is a dummy variable taking the value 1 if the patient is an opioid abuser, 0 otherwise; abuser x depressed is an interaction term taking the value of 1 if the patient has at least 1 diagnosis for opioid abuse and at least 1 diagnosis for depression, 0 otherwise; and comorbidj are dummy variables taking the value 1 if the patient has a given comorbid condition, 0 otherwise. The variables comorbidkx abuser are interaction terms, taking the value 1 if the patient is an opioid abuser and has a given comorbid condition; ε is the error term.where When we calculated descriptive statistics for our 2 samples, we found that the per-patient costs of opioid abuse were 3.4 times higher than the matched control of depressed patients, $16,722 for opioid abusers compared with $4,875 for depressed patients. Based on the regression results that control for the effects of comorbid conditions, we found that the incremental costs of opioid abuse relative to depressed patients was $3,040, indicating that opioid abusers were 1.8 times as costly as depressed patients  . The coeThe analyses presented here highlight the substantial payer burden associated with patients who are diagnosed as opioid abusers. We are not aware of any other studies that have systematically quantified and compared the direct medical and drug costs of opioid abusers with a cohort of nonabusers. This group of opioid abusers has direct health care costs that are more than 8 times higher than those of nonabusers. These cost measures were the total per-patient direct health care costs and included costs associated with other comorbid conditions. Even when one controls for comorbidities using a multivariate regression analysis, we found that opioid abusers were 1.8 times as costly to the payer as a matched control of depressed patients. In fact, much of the cost of opioid abuse is driven by such things as hospitalizations and emergency room visits. This analysis has shown that opioid abusers have higher prevalence rates of certain comorbidities  and have higher utilization rates of medical services such as hospital inpatient visits. This suggests that these factors may have jointly contributed to the higher level of per-patient costs associated with opioid abusers compared with nonabusers.12Focusing only on the direct health care costs understates the full cost burden imposed by indirect costs associated with disability, absenteeism, and presenteeism (defined as the feeling that one must go to work even if one is too ill to be productive or effective). It is important for payers to consider indirect costs in calculating the true overall burden of opioid abuse. Birnbaum et al. note that lost productivity costs accounted for 50% of the societal cost of prescription opioid abuse in the United States in 2001.Since the diagnosis codes in medical claims do not distinguish between prescription and nonprescription opioid abuse, it was not possible in this study to examine the relationship of nonmedical use of nonprescription  drugs to prescription opioid  and total direct health care costs. Since 58% of opioid abusers had claims for SAOs, LAOs, or both, 42% of opioid abusers (as defined here) may be abusing other prescription drugs or nonprescription (illicit) narcotics. Further research should seek to distinguish between abuse of prescription narcotics and illicit narcotics and measure indirect costs as well as direct costs for prescription and illicit opioids.A more thorough analysis of the comorbidity profile of opioid abuse patients, including an assessment of the temporal pattern of such comorbidities, would be valuable in understanding cost drivers. For example, if the onset of opioid abuse is associated with human immunodeficiency virus (HIV) or hepatitis , then a cost offset may be realized by advocating better treatment of opioid abuse; however, longitudinal examinations of temporal patterns may not be possible in administrative claims databases due to relatively limited duration of membership of opioid abusers.Future research with Medicaid data is also necessary to shed light on the prevalence and costs of opioid abuse among a Medicaid population and possibly to help understand more clearly the causal relationship between opioid abuse and the comorbidities discussed here. The costs of such patients in a Medicaid population could be quite different compared with those reported here from commercial health plan members. It would also be useful to extend the multivariate regression analysis to account for additional factors that may drive costs, including duration of abuse, prior medical history, and the presence of multiple substance abuse treatments.16 we attempted to include all ICD-9-CM codes that related to opioid abuse, recognizing the possibility that those codes may not always capture the phenomenon of opioid abuse.As was noted earlier, the concept of opioid abuse is often interpreted differently by different people. Given the possibility for miscoding with ICD-9-CM codes,Opioid abuse is a costly condition that imposes a large direct cost burden on private payers. Opioid abusers have direct health care costs that are approximately 8 times higher than those of nonabusers. These costs are likely driven by higher prevalence rates of costly comorbidities.