IntroductionPancytopenia is a common presentation in the pediatric population. It is a manifestation of various diseases, and its etiology can be explained on the basis of bone marrow examination. The study aims to determine the etiological factors leading to pancytopenia via bone marrow examination in pediatric patients presenting in our hospital.Materials and methodsThis retrospective cross-sectional study was conducted in the Department of Pathology at a public sector tertiary care hospital. Data were recorded by convenience sampling from the patients’ database from January 2015 to April 2018. Patients aged 2 months to 15 years who had pancytopenia on peripheral blood smear and were admitted for bone marrow examination were included in the study. Patients who were beyond these age limits, diagnosed cases of aplastic anemia and leukemia, and those with a recent history of blood transfusion were excluded from the study. The analysis was done via the Statistical Package for Social Sciences (SPSS) v.23.0 , and descriptive statistics were applied.ResultsOf 115 cases, 58 (50.4%) were males and 57 (49.6%) were females. Megaloblastic anemia was present in 32 (27.8%) patients, and it was the most common cause of pancytopenia. Non-malignant disorders were seen in 95 cases (82.6%) and malignant disorders were seen in 20 (17.4%) cases.ConclusionMegaloblastic anemia is the most common cause, and acute lymphoblastic leukemia is the most common malignant cause of pancytopenia in children. There was no significant gender predilection among causes of pancytopenia. Pancytopenia is a condition in which there is a decrease of all the three cellular elements of blood, namely erythrocytes, leukocytes, and platelets -5. It isPancytopenia is not a disease in itself but a laboratory finding of the constitution of diseases , 3, 7. I9/L, and platelet count is less than 100*109/L [Pancytopenia results in a hypoplastic bone marrow . It is dBut, in order to find out the etiological factor causing pancytopenia, bone marrow examination is done , 4. It cThe aim of this study was to determine the spectrum of pancytopenia with its frequency and etiology on the basis of bone marrow examination in children presenting to our hospitals. This has been an area of great interest in South-East Asia, and many articles on a similar topic have been published recently. Studying about the etiology of pancytopenia will equip us with better knowledge on how to tackle this pathology.9/L, and platelet count < 100*109/L. Patients aged 2 months to 15 years who had pancytopenia on a peripheral blood smear and were admitted for bone marrow examination were included in the study. Patients who were beyond these age limits, diagnosed cases of aplastic anemia and leukemia, and those with a recent history of blood transfusion were excluded from the study. The analysis was done using the Statistical Package for Social Sciences (SPSS) v. 23.0 . Simple descriptive statistics were applied for finding out frequency distribution. Chi-square test was applied for finding out the association between gender and causes of pancytopenia, and p-value of less than 0.05 was considered significant. The study was a retrospective descriptive cross-sectional study. It was conducted in the Department of Pathology at a public sector tertiary care hospital. The ethical approval was sought from the Institutional Research Forum of Rawalpindi Medical University. The data were collected by convenience sampling from the pathology department of the hospital, in accordance with the Declaration of Helsinki. The patients' identities were kept anonymous and only the serial number, gender, and etiology for pancytopenia were noted. Patients’ database was explored for patients with pancytopenia during the period of January 2015 to April 2018. Pancytopenia was defined as Hb < 10 gm%, ANC < 1.5*10Of the 115 cases, 58 (50.4%) were males and 57 (49.6%) were females with a male to female ratio of 1.01:1. Megaloblastic anemia was the most common presentation of pancytopenia with 32 (27.8%) cases, followed by acute lymphoblastic leukemia with 16 (13.9%) cases. Non-malignant disorders were seen in 95 (82.6%) cases and malignant disorders were seen in 20 (17.4%) cases. Malignant disorders included acute lymphoblastic leukemia in 16 (13.9%) cases and acute myeloid leukemia in 4 (3.5%) cases. Non-malignant disorders included nutritional disorders in 43 (37.4%) cases and non-nutritional disorders in 52 (45.2%) cases. The further division is described in more detail in Table Pancytopenia is not an uncommon presentation in pediatric wards. A thorough history taking, physical examination, and the right laboratory investigations can lead to proper diagnosis and hence, to the relevant management of the etiology .In the present study, males and females were in similar numbers with a male to female ratio of 1.01:1, but other studies reported different numbers. In the studies conducted by Makheja et al., Memon et al. in Karachi; Jan et al., Gul et al. in Peshawar; Tufail et al. in Faisalabad; Dubey et al. in India; and Basak et al. in Bangladesh, male to female ratios were 1.38:1, 1.6:1, 1.84:1, 1.8:1, 0.76:1, 0.88:1, and 1.7:1, respectively -8.Non-malignant disorders were predominantly high in our study with 95/115 (82.6%) cases as compared to malignant disorders with 20/115 (17.4%) cases. Similar results were obtained in a study by Memon et al. with non-malignant disorders at 190/230 (82.59%) cases and malignant disorders at 40/230 (17.4%) cases [Our study showed that megaloblastic anemia (27.8%) is the most common nutrition-related presentation of pancytopenia. This is consistent with studies done by Makheja et al. and Dubey et al. with megaloblastic anemia as the most common presentation at 41.9% and 47%, respectively , 6. VariFurther results of our study showed that acute lymphoblastic leukemia 13.9%) is the most common malignant presentation of pancytopenia. The study by Memon et al. also reported similar results with acute lymphoblastic leukemia at 8.69% .9% is th, 9. ThisDespite these important findings, the limitations of the study are also noted. This is a small center cross-sectional study done by convenience sampling; therefore, the results may not apply to other regions. Further studies are needed to complement these findings and study the trends in other parts of the world.Pancytopenia is related to nutritional deficiencies in our area. Megaloblastic anemia is the most common presentation of pancytopenia in children. The most common malignant presentation is acute lymphoblastic leukemia. There was no significant gender predilection among causes of pancytopenia.


In the Caribbean region, a notable difference in HPV-prevalence and genotypes distribution between the islands is observed. Recently we found in Curaçao a low incidence of HPV16 and 18 in cervical cancer compared to the standard world population. We aimed to determine HPV-prevalence, HPV-genotype distribution and associated risk-factors in women from Curaçao.5000 women aged 25–65 years were randomly selected from the national Population Register. HPV was detected by means of GP5+/6+PCR EIA and GP 5+/6+amplimers from HPV-positive samples were genotyped with a reverse hybridisation assay. We also collected personal data and data on risk-factors.1075 women were enrolled in the study. Overall HPV-prevalence was 19.7%. Most frequent genotypes were HPV16 (2.3%), 35 (2.1%) and 52 (1.8%). Twenty-seven women detected with abnormal cytology (i.e.≥ASC-US) were referred for biopsy. In women with normal cytology (n = 1048), HPV-prevalence was 17.9% and the most common high-risk HPV (hrHPV)-types were HPV35 (2.0%), 18 (1.8%), 16 (1.5%) and 52 (1.5%). The highest HPV-prevalence (32.8%) was found in the age-group: 25–34 (n = 247). HPV positive women started sex at a younger age (p = 0.032).HPV-prevalence in the overall population is high and HPV16 was the most common genotype followed by 35 and 18. In women with normal cytology HPV35 is the most common genotype followed by HPV18, 52 and 16. The high HPV-prevalence (32.8%) in women of 25–34 years argue for introduction of cervical cancer prevention strategies. HPV-type distribution found in Curaçao should be taken into account when considering the choice for prophylactic vaccination. In the Caribbean region the cervical cancer incidence is high, mainly because of the lack of a structured prevention strategy., 2 AlthoThe Caribbean population is known for its unique diverse ethnic distribution consisting of Afro descendants mixed with other ethnicities settled in the region. PublisheThree HPV prophylactic vaccines are currently registered: a bivalent vaccine against HPV16 and 18, a quadrivalent vaccine against HPV16 and 18 with an additional coverage of low risk HPV (lrHPV) 6 and 11, and a nonavalent vaccine which, in addition to the 4 types in the quadrivalent vaccine, covers high risk HPV (hrHPV) types 31, 33, 45,52 and 58.[When considering cervical cancer prevention strategies i.e. population based screening and prophylactic vaccination, knowledge of the HPV genotype prevalence in the female population and its associated risk factors are important for health policy makers. Here we present data about the prevalence of HPV and HPV genotypes and associated risk factors in a randomly selected age stratified female population aged 25–65 years from Curaçao.From the national Population Register of Curaçao, Fundashon Prevenshon, the prevention centre in Curaçao, we obtained a database containing age and ID-number of all women aged 25–65 years and registered as inhabitant of the island. From each of the age strata 25–34, 35–44, 45–54 and 55–65, 1,250 women were randomly selected. Each selected woman received an invitation letter per mail to participate in the study. If a woman did not respond, a second invitation letter was sent after 2 weeks.In this study, we excluded all women who had a history of hysterectomy, were pregnant or less than 3 months postpartum, or had cervical (pre)cancer in the last 2 years or on-going treatment with chemo- or radiation therapy.The Institutional Review Board of the medical ethics committee of Fundashon Prevenshon Curaçao approved the study registered with the code FP0003/15. The participants received, in systematic order and prior to their participation, detailed information about the study, the objectives and their right to interrupt the study. Documents with extensive information about the study, HPV and cancer were made available in four languages: Papiamentu (native language spoken on Curaçao), Dutch, English and Spanish. All participants signed an informed consent if they agreed to participate in the study, and before they proceed with the sample collection.On arrival at the prevention centre, the participants received a questionnaire in which they were asked about their age, ethnicity, and habits such as smoking, drugs- and alcohol use. Other questions of the questionnaire concerned allergies, co-morbidities, gynaecological and obstetrical background, anti-conceptive methods, sexual habits, lifetime—and current sexual partners, and sexual transmitted diseases (STDs).  and stored at room temperature.Secondly, a sample for HPV detection was collected, with the same type of brush used for cyto-collection. The sample for HPV detection was put in PreservCyt® at room temperature and at the end of each day transported to the Analytical Diagnostic Centre (ADC) Laboratory of Curaçao where the HPV samples were stored at -20°C.Smears were red by 2 cytotechnicians and scored according to the CISOE-A classification as used in The Netherlands and Curaçao. This classification can be easily translated into the Bethesda Classification. In case Biopsy specimens for histological evaluation were read by one cytotechnician and one pathologist and classified as no lesion, CIN1, CIN2, CIN3 and carcinoma according to international criteria. Labo Bio-medical Products, Rijswijk, The Netherlands) according to the manufacturer’s instructions. This kit detects amplified DNA from HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. A 10μL aliquot of GP5+/6+ amplimer from HPV positive samples by EIA was tested with the Genotyping kit HPV GP, version 2 . This kit enables genotyping of 12 hrHPV genotypes (16/ 18/ 31/ 33/ 35/ 39/ 45/ 51/ 52/ 56/ 58/ 59), 6 possible hrHPV types (26/ 53/ 73/ 82/ 66/ 68) and 5 lrHPV types (6/ 11/ 30/ 67/ 70).[The MagnaPure 96 instrument was used for DNA isolation. Ten μL of extracted DNA was used as input for the broad spectrum GP5+/6+-PCR in a total volume of 50μL. Detection of hrHPV was done on 5μL GP5+/6+ amplimer with the Enzyme immune assay  for normally distributed continuous data. HPV type prevalence was assessed for all women and stratified by age. The association between HPV prevalence and sexual risk factors obtained via the questionnaire are tested with the chi-square test or Fisher’s exact test. In case of significant differences in risk factors between the age groups, logistic regression models are used to correct the associations between HPV prevalence and sexual risk-factors for age. A forward selection procedure  was used to build a multivariable logistic regression model to identify all independent risk-factors associated with HPV prevalence. In a subgroup analysis, we excluded all participants who had Pap results ≥BMD (or ≥ASC-US). The significance level α was set at 0.05. SPSS version 22  was used for the statistical analyses.Of the 5000 invited women, 1695 women responded to participate (33.9%). Among the invited women, 310 responded but cancelled the appointment and 119 women responded but withdrew due to no interest (n = 38) or unknown reason (n = 81). Another 191 women were excluded because of hysterectomy (n = 165) and pregnancy (n = 26), leaving 1075 women for the analysis. The majority of our participants were Afro-descendants (87.2%). The sociodemographic and clinical characteristics of this population are described in The majority of the participants were sexually active (80.7%). Mean age at first sexual contact was 19.0 (SD 3.6) year. Most participants responded currently to have one sex-partner (73.4%) while a small proportion (1.6%) responded to have more than one sex-partner, and 24.0% had no sex-partner in the past twelve months. For the lifetime number of sexual partners, the majority (53.6%) had between 2 to 5 partners. Four (0.37%) participants answered they never had sex before. Most of the participants practice oral sex (62.6%) while only few  practice anal sex. Seven hundred and sixty seven participants (71.3%) used (or have used) oral contraceptive and 219 (20.4%) participants currently have or have been using an IUD. One hundred and thirteen (10.3%) participants mentioned history of STD.One hundred and nineteen (11.1%) participants smoked or had smoked cigarettes. Information about the specific period exposed to cigarettes was not available. Of all participants 0.67% used any kind of recreational drugs. Twenty seven participants (2.6%) were detected with abnormal cytology ≥BMD(or ≥ASC-US).  EighteenHPV prevalence in the total study population was 19.7%. Multiple infections with different HPV types were detected in 42 (19.8%) women, yielding 271 infections in total, from which 70.5% were hrHPV-types and HPV 16 (2.3%) was the most common hrHPV genotype. .HPV prevalence differed between the age groups (p<0.001): the youngest age group had the highest prevalence (32.8%). The HPV prevalence of women in the other age groups were comparable . HPV 16 (4.9%) was most common high risk genotype found in women aged 25–34 years, followed by HPV 45 (4.5%) and 52 (4.5%). In women aged 35–44 years and 45–54 years, HPV 66 (2.1% both) was the most common high risk genotype followed by HPV 16 (1.8%) and 18 (1.8%) in the age group of 35–44 years and HPV 16 (1.7%) and 31 (1.7%) in the age group of 45–54 years. In women in the age of 55–65 years, we found more of HPV 35 (2.3%), followed by HPV 52 (2.0%) and 16 (1.3%) .No significant difference between the age groups is observed with regard to the proportional attribution of HPV 16  .In the group with abnormal cytology, 3 were HPV negative while the 3 most common genotypes in referred participants with abnormal cytology were HPV 16 (n = 9), 45 (n = 5) and 58 (n = 4).p = 0.37).  was 20.6% and did not differ significantly from HPV prevalence of women migrated from other Caribbean islands  and from Latin America  (= 0.37). A subgroup analysis of women with normal cytology (n = 1.048) showed 188 HPV positive participants (17.9%). In contrast with the overall population the attribution of the different HPV genotypes changed in ranking order to: HPV 35 (2.0%), 18(1.8%), 16 and 52, each (1.5%) as the most common types. HPV prevalence was found to be significantly associated with young age and age at first sexual intercourse. The mean age of first sexual contact of HPV-positive women was 18.5 (SD 3.4) year and of HPV-negative women was 19.1 (SD 3.7) year (p = 0.032). However, after adjusting for age, this risk factors was not significantly associated with HPV prevalence.  In the fHPV prevalence in the total study population of Curaçao was high 19.7%) and is comparable with the prevalence on some other islands in the Caribbean, i.e. Haiti (19.0%) and Guadeloupe (25.1%) .7% and i Fig 2)  Fig 2) HThe most frequent HPV types in the total population of Curaçao were HPV 16 (2.3%), 35 (2.1%), 18 (1.8%) and 52 (1.8%). This type distribution is largely comparable with a study from Jamaica  and one from Haiti (HPV 16 and 35) ,12. HoweHPV genotype prevalence data are difficult to compare with other Caribbean data because of differences in HPV genotyping assays used. Moreover, interpretation is hampered because women in these studies were derived from different populations i.e. screening populations, gynaecologic outpatient populations or a mixture of these, and consequently include varying proportions of women with abnormal cytology. This has a large influence on HPV positivity and genotype distribution in these populations, because of different preferential risks for CIN 2/3 lesions and cancer of different HPV types. To encompass this problem, we also studied HPV prevalence and HPV genotype distribution in women with normal cytology. In women with normal cytology from Curaçao the most commonly detected HPV genotypes were: HPV 35 (1.9%), 18 (1.8%), 16 and 52 (each 1.5%).  Only frord to 1st place, whereas HPV 35 which was the most prevalent in normal cytology was rare in cervical cancer specimens. HPV45, which is not commonly seen in women with normal cytology from Curaçao appears in the three most common HPV types associated with cancer. HPV 45 is often associated with adenocarcinoma of the cervix which is localised higher in the endocervical canal and this may explain why infection with HPV 45 is not easily detected in exfoliated cells of women with normal cytology and cervical precursor lesions. [Interestingly in Curaçao, the HPV genotype distribution in the population with normal cytology differs from the HPV genotype distribution in women with cervical cancer: HPV 16 shifts in ranking from 3lesions.  An alterIn this survey, only young age, and early age at sexual debut were found to be statistically associated with a high HPV prevalence as has been described by others.,18In our questionnaire only 10.5% of women reported having a history of any type of STD. Probably, a higher number of participants had been infected with an STD in the past, but were unaware that they had contracted an STD because they were not familiar with the recognition of symptoms associated STD’s. Also, 4 participants referred they never had sexual contact while 2 of them were HPV positive. A strong point of our study is that to the best of our knowledge, this is the first attempt in the Caribbean area to describe HPV prevalence and HPV-genotype distribution in relation to sexual behaviour and other risk factors in a randomly selected population of women of all age groups (25–65 years). Moreover, the HPV prevalence and HPV genotype distribution were studied in both women from the total population and women with normal cytology. Another strong point is that one person at one centre collected all samples. In the laboratory, one technician was responsible for the different handling procedures, and evaluated all Pap-smears whereas another technician performed all HPV assays, unaware of the results of the cytology report. All the work was realized over a relatively short period of 3–4 months. A limitation of our study is that our studied population was based on participants 25 years and above while HPV prevalence in the population <25 years is probably higher than in the studied population.In conclusion, we showed in a randomly selected population of women (age 25–65) from Curaçao that HPV prevalence is high, and very high in women of 25–34 years. These HPV prevalence data ask for the introduction of prevention strategies. HPV 16 is the most common HPV type detected in the overall population eligible for cervical screening whereas HPV 35 is the most commonly detected HPV type in women with normal cytology. Young age and early age of sexual debut were the only risk factors associated with a high HPV prevalence.Together with our earlier published HPV genotyping findings in cervical cancer these HPV genotype data in the population of Curaçao should be taken into account in the choice of a prophylactic HPV vaccine when a vaccination programme is implemented in Curaçao.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S1 Table* Data not available BD borderline dyskariosis, MD Mild dyskariosis are comparable to ASCUS ASCUS–H/LSIL.(DOCX)Click here for additional data file.S2 Table28 participants presented 2 HPV infections, 6 presented 3- and 2 presented 6 infections. * The percentage is calculated based on all participating women (n = 1075). ** The percentage is calculated based on 1.048 women excluding all participants with ≥ BMD (ASC-US) or more.(DOCX)Click here for additional data file.


Anaemia during pregnancy is commonly reported in humans and is considered a significant health issue as it is associated with adverse neonatal outcomes including low birth weight, preterm birth and possible perinatal mortality. Pregnant sheep are a common model for research investigating the causes and consequences of preterm birth, but the incidence and significance of anaemia during pregnancy is not known. However, intraoperative anaemia in pregnant sheep has been reported previously. In addition, it has been demonstrated that the cause of intraoperative anaemia is not pregnancy alone as pregnant sheep in a farm environment did not develop anaemia. The aim of this study was to document the red blood cell and protein content of maternal blood before, during and after anaesthesia and surgery to elucidate the cause and duration of intraoperative anaemia in pregnant sheep in a research context. The concentration of red blood cells and protein decreased during anaesthesia and were attributed to the use of anaesthetic drugs and fluids as opposed to the pregnancy status of the sheep.p = 0.003) with an initial decrease of 12.5% from the baseline (0.36 (0.36–0.4) to 0.315 (0.29–0.34), p = 0.044), a further intraoperative decrease of 41.7% from the baseline (0.21 (0.195–0.245), p = 0.002) and an increase the day afterwards (0.3 (0.285–0.35), p > 0.99 compared with baseline). The haemoglobin concentration also changed (p < 0.0001) (baseline: 114 (111.8–123); pre-op: 97 (77.25–104.5), 14.9% decrease, p = 0.022; intra-op: 70 (61.5–83.25), 38.5% decrease, p = 0.0009; post-op: 101.5 (96.25–114) g/L, p > 0.99). Likewise the change in total protein during the study was significant (p = 0.0003) and decreased from the baseline [70 (67.25–70.75) g/L] prior to anaesthesia (61 (58.25–64.5) g/L, 12.9% decrease, p = 0.0437) and further during anaesthesia (55.5 (53.75–63.25) g/L, 20.7% decrease, p = 0.0021) with an increase  on the first post-op day. In conclusion, intraoperative anaemia and hypoproteinaemia occurred in this study. These alterations are attributed to a combination of the side effects of acepromazine and haemodilution.The aim of this study was to document the packed cell volume (PCV), haemoglobin concentration and total protein concentration of maternal blood before, during and after anaesthesia. Six singleton Merino-cross pregnant ewes at 116-117 days of gestation were premedicated with intramuscular acepromazine (0.02 mg/kg) and buprenorphine (0.01 mg/kg), and anaesthesia was induced with intravenous midazolam and ketamine. Anaesthesia was maintained with isoflurane in 100% oxygen. Serial blood samples were collected the day before anaesthesia (baseline), immediately prior to induction of anaesthesia (pre-op), at the end of the procedure (intra-op) and the following day (post-op). There was a significant change in the PCV during the study ( Anaemia during pregnancy is commonly reported in humans and is considered a significant health issue as it is associated with adverse neonatal outcomes including low birth weight, preterm birth and possible perinatal mortality . ApproxiThe aim of this study was to document the packed cell volume (PCV), haemoglobin concentration and total protein concentration of maternal blood before, during and after anaesthesia and surgery to investigate the cause and duration of intraoperative anaemia in pregnant sheep in a research context.The study was approved by the Animal Ethics Committee of the University of Western Australia (RA/3/100/1575) in accordance with the Code of Practice for the Care and use of Animals for Scientific Purposes . The eweSix singleton Merino-cross pregnant ewes at 116–117 days of gestation (term ~150 days) were enrolled in the study. The sheep were sourced from a commercial farm in the south-west of Western Australia and introduced to the Large Animal Facility 2 to 3 weeks prior to surgical intervention. The indoor housing was in shared raised floor pens and then in single pens in the perioperative period. Sheep were fed pellets, oaten chaff and lupins with a mineral mix calculated on bodyweight and pregnancy status. Rooms were controlled for temperature (20.5–21.5 °C) and maintained on a 12:12 hour light:dark cycle.2: 35–45 mmHg) using an Aestiva 5 anaesthetic machine with a Smartvent ventilator . Volume controlled ventilation was utilised with a set tidal volume of 8 to 12 mL/kg, a respiratory rate between 8 and 12 breaths per minute and positive end-expiratory pressure of 5 cmH2O. Intravenous fluid therapy with 0.9% sodium chloride (NaCl) at 10 mL/kg/h was administered throughout anaesthesia. The animals were positioned in dorsal recumbency. Physiological monitoring with a multiparameter monitor included noninvasive blood pressure (oscillometric method) from a cuff around a dorsal pedal artery, oxyhaemoglobin saturation  and side stream capnography (sample rate 150 ± 20 mL/min) . The ventilator variables were also recorded. The ventral abdomen and the neck of the ewe were clipped and prepared for aseptic surgery. A ventral midline laparotomy and hysterotomy were performed for placement of a central venous line in the foetus percutaneously. A maternal central venous catheter was also placed in a maternal jugular vein percutaneously.The sheep were anaesthetised as previously described . Food anSerial blood samples were collected on four occasions: the day before anaesthesia from the jugular vein or cephalic vein (baseline); immediately prior to induction of anaesthesia from the catheter placed in the cephalic vein (pre-op); at the end of the procedure from the maternal jugular catheter (intra-op); and the following day from the maternal jugular catheter (post-op). Blood was collected into EDTA anticoagulant and the analyses were performed by a commercial veterinary pathology laboratory , to determine the PCV, haemoglobin concentration  and total protein concentration .For analgesia the ewes received buprenorphine prior to anaesthesia, ketamine during induction of anaesthesia, a line block for the ventral abdominal wound with ropivacaine  and a transdermal fentanyl patch as previously described . The ewep-value < 0.05 was considered significant and the data are presented as mean (± SD) or median (25–75 percentile).Data were tested for normality and compared with analysis of variance (Freidman test) and Dunn’s multiple comparison test . A The ewes weighed 56.7 (± 5.7) kg and anaesthesia time was 77.5 (± 2) minutes. Anaesthesia, surgery and recovery were uneventful in the six ewes. By pooling all the intraoperative data the mean arterial blood pressure was 50 (± 7.6) mmHg, the heart rate was 82 (± 17) beats per minutes, the oxyhaemoglobin saturation was 93 (± 3) %, the expired carbon dioxide was 41 (± 4) mmHg and the peak inspiratory pressure during mechanical ventilation was 17.2 (± 1.8) cmH2O.p = 0.003) with an initial decrease of 12.5% from the baseline (0.36 (0.36–0.4) to 0.315 (0.29–0.34), p = 0.044), a further intraoperative decrease of 41.7% from the baseline (0.21 (0.195–0.245), p = 0.002) and an increase the day afterwards (0.3 (0.285–0.35), p > 0.99 compared with baseline) (p < 0.0001) (baseline: 114 (111.8–123); pre-op: 97 (77.25–104.5), 14.9% decrease, p = 0.022; intra-op: 70 (61.5–83.25), 38.5% decrease, p = 0.0009; post-op: 101.5 (96.25-114) g/L, p > 0.99) (p = 0.0003) and decreased from the baseline (70 (67.25–70.75) g/L) prior to anaesthesia (61 (58.25-64.5) g/L, 12.9% decrease, p = 0.0437) and further during anaesthesia (55.5 (53.75–63.25) g/L, 20.7% decrease, p = 0.0021) with an increase (63 (61.25–67) g/L, p > 0.99) on the first post-op day (There was a significant change in the PCV during the study (aseline) . The pat > 0.99) . Likewist-op day .Given the reported incidence of intraoperative anaemia in pregnant sheep the aim of this study was to determine the cause and duration of intraoperative anaemia in pregnant sheep in a research context. Intraoperative anaemia and hypoproteinaemia occurred in this study and partially resolved by the first postoperative day. While the values for each of the three measured parameters did not return to the baseline on the first postoperative day, there were no statistically significant differences between these post-op values and the baseline values.From the baseline values there was a decrease in both the PCV and haemoglobin concentration to a level consistent with anaemia . The resThe mechanism of anaemia following the administration of acepromazine has been demonstrated in various species including sheep to be splenic relaxation and consequent increase of red cells held there ,10,11. TThe decrease in the PCV and haemoglobin concentration during anaesthesia and surgery (from pre-op to intra-op) is not likely to be an effect from the acepromazine alone. Normovolaemic haemodilution is a result of progressive exchange of the blood with crystalloids or colloids and the physiological responses to haemodilutional anaemia are similar between species . In our The manifestations of intraoperative anaemia in pregnant sheep were not apparent in this or other studies in our facility, as there were no overt signs of postoperative organ injury and mortality. This tolerance of intraoperative anaemia is attributed to its relatively short duration being associated with anaesthesia, surgery and the capacity of compensatory mechanisms to maintain oxygen delivery to tissues . NeverthThe parallel alterations in haemoglobin concentration in this study are directly related to the changes in the PCV. Given that the haemoglobin protein is contained with red cells, the fewer circulating red cells the lower the circulating haemoglobin concentration. Erroneous causes of low haemoglobin concentration include dilution of red blood cells due to vasodilation and intravenous fluid therapy , but, inThe dose of acepromazine used in this study may be considered as low at 0.02 mg/kg by intramuscular injection. Despite this dose the side effects of acepromazine were apparent as previously discussed. However, the noninvasive blood pressure measurement technique is unlikely to be accurate as the sheep were in dorsal recumbency and the position of the cuff was higher than the right atrium, the site of the cuff was not clipped of wool and other authors have demonstrated that other oscillometric devices underestimate the mean arterial blood pressure . DespiteThe changes in total protein concentration were similar to those for the PCV and haemoglobin concentration. This similarity suggests the cause of intraoperative hypoproteinaemia was also haemodilution. Decreasing total protein concentrations are reported in dogs during anaesthesia and laparotomy attributed to anaesthetic drug administration, dilutional effects of intravenous fluid therapy and loss of plasma protein into the abdominal cavity . In anotThere are a number of limitations to this study, which must be considered when interpreting the results. The study is small and it was an opportunistic study so sample collection occurred at times that did not interfere with the primary objectives of the study proper. Likewise, a control group of nonpregnant sheep could not be included. To understand the side effects of acepromazine invasive blood pressure measurement would have been a better way to evaluate blood pressure. Finally additional samples after the first postoperative day would have provided more information about recovery from the decreased PCV, haemoglobin concentration and TP concentrations.In conclusion, intraoperative anaemia and hypoproteinaemia occurred in the sheep in this descriptive study. The extent of these alterations was statistically significant and is attributed to a combination of the side effects of acepromazine and haemodilution.


The present study aims to investigate the etiology, clinical profile and resistance pattern of the isolated pathogens in Nepalese adults with acute gastroenteritis. This cross-sectional study was conducted at Sukraraj Tropical and Infectious Disease Hospital, from April 2016 to Sep 2017. Subjects’ ages 14 or above, presenting with gastroenteritis with positive stool culture were enrolled for analysis.Vibrio cholerae spp and Shigella spp were detected in 36.6% and 23.28% respectively. The most common resistance among Vibrio cholerae was to nitrofurantoin (92.8%), cotrimoxazole (92.8%) and nalidixic acid (92.8%). Among 17 isolates of Shigella spp, the most frequent drug resistant was observed in ampicillin (64.7%), nalidixic acid (58.8%), ceftriaxone (47%). Chloramphenicol (94.1%), tetracycline (88.2%), and cotrimoxazole (82.3%) were found to be the most sensitive towards this pathogen. High rate of diarrhea due to bacterial infection, especially Shigella spp and Vibrio spp and their high rate of drug resistance emphasize an urgent need of designing a surveillance system for antimicrobial resistance in Nepalese setting.Of total 153 patients, 47.72% subjects confirmed the presence of bacterial infection.  Shigella spp. play an important role in causing inflammatory diarrhea and dysentery, and can be considered a global health problem [Shigella spp and Vibrio cholerae have become progressively resistant to the most widely-used and inexpensive antimicrobials [Diarrhoeal diseases remain a major cause of morbidity and mortality in all age groups in impoverished areas of South East Asia –5. A wid problem –11, caus problem , 9, 12.  problem . Over thcrobials , 12, 13.crobials –16. Conscrobials –19. TherThis was a cross-sectional observational study, conducted over 6 month period from April 2016 to Sep 2017.Setting: The present study, conducted at Sukraraj Tropical and Infectious Disease Hospital (STIDH), established in 1933, is the only Infectious & Tropical Disease Hospital in Kathmandu, Nepal. This is the national referral hospital with a capacity of 100 beds. It receives patients from all over the country and referred patients from hospitals in Kathmandu Valley.Subjects ages 14 or above, presenting with acute gastroenteritis were sampled during 6 month study period. Demographic including age and sex, clinical features, including body temperature, stool type, abdominal pain, and vomiting and laboratory report were assessed at study entry.Shigella and Vibrio. Culture negative specimens on primary solid media were sub-cultured from the enrichment broth to primary solid media to improve recovery of the isolates. All inoculated media were incubated at 37 °C for 18–24 h. After overnight incubation, non-lactose fermenters were further identified by biochemical tests using appropriate media namely: triple sugar Iron agar for carbohydrate fermentation test, urea agar for the urea utilization test, sulphide Indole motility test for indole production, Simmon citrate agar for citrate utilization, motility agar for motility test, lysine agar for lysine utilization test . The plates were incubated at 37  °C for a minimum of 24 h. Plates exhibiting no growth were incubated for another 24 h. All isolated colonies growing in the MAC agar were processed further for identification.Faecal samples were inoculated on MacConkey agar (Hi-media) plates. The feces samples were cultured on MacConkey agar, xylose lysine Deosxycholate agar; Thiosulphate citrate bile salt sucrose agar and Selenite F enrichment broth  were used for isolation of Identification of the isolates was done by the standard microbiological techniques which involved morphological appearance of the colonies, Grams staining reactions, catalase test, oxidase test and other biochemical properties.P. aeruginosa ATCC 27853, E. coli ATCC 25922, S. aureus ATCC 25923.The antimicrobial susceptibility of all identified isolates were done with various antibiotics using Mueller–Hinton agar (MHA) by modified Kirby–Bauer disk diffusion technique following the criteria of Clinical and Laboratory Standards Institute (CLSI) . DiffereDescriptive data were generated. Continuous data were presented in Mean ± SD, and frequency table were generated for categorical variables. Chi square test and Fisher exact test were performed for comparison of categorical variables. A P value < 0.05 was considered to be statistically significant. All data were entered and managed in SPSS version 20.The study complies with the standards of the Declaration of Helsinki and has been approved by research committee of Sukraraj Tropical and Infectious Disease Hospital (STIDH). Anonymous forms were used to collect the data to assure confidentiality.Demographic characteristics as well as clinical profiles of the study subjects are shown in Table Shigella spp and Vibrio spp were 11.1%, and 36.6% respectively. Of total 17 Shigella isolates, Shigella flexneri were identified as 41.17% and Shigella sonnei as 58.8%.Among the 153 stool samples collected, the estimated prevalence of Vibrio cholerae spp were determined (Table Vibrio cholerae was to nitrofurantoin 52 (92.8%), cotrimoxazole 52 (92.8%) and nalidixic acid 52 (92.8%), ciprofloxacin 48 (85.7%), ampicillin 26 (46.4%), chloramphenicol 23 (41%). However, 53 (94.6%) strain of Vibrio spp were sensitive to tetracycline.The drug susceptibility patterns of 56 isolates of ed Table . The higShigella spp, the resistant rate was high for ampicillin 11 (64.7%), nalidixic acid 10 (58.8%), ceftriaxone 8 (47%) and ciprofloxacin 7 (41.17%).Among 17 isolates of However, more than 90% strain of these isolates were sensitive to chloramphenicol 16/17 (94.1%), and more than 80% strain of these isolates were sensitive to tetracycline 15/17 (88.2%) and cotrimoxazole 14/17 82.3%) . Parasite was identified notably in higher number of population (26.1%). In comparison, studies from sub-Saharan country demonstrated 47 (60.2%) and 31 (39.7%) parasitic and bacterial infection respectively .Vibrio spp and Shigella spp 36.6% and 11.1% respectively.In the present study, bacterial infection remained the major etiology of the disease, with estimated prevalence of Shigella spp across the nations, ranges between 5 and 16.8% [of Shigella spp in under 5 years of age in Nepal were found to be 52.2% [Available literatures suggest varying prevalence of nd 16.8% –25. The be 52.2% . This diShigella sonnei 10 (58.8%) remained the most common species followed by Shigella flexneri 7 (41.17%), which is in consistent with the results of the study in pediatric population from Teharan n Farahani et al., where 60.8% reported to be Shigella sonnei and 39.2% S. flexeneri [Shigella sonnei, followed by S. flexneri (27.8%). However, in pediatric population in Nepal a low prevalence of Shigella sonnei (25%) and S. flexneri 20.83% were found [Shigella flexneri (7.5%) and S. sonnei (5.2%) reported in Karaj, Teharan [In the present study, lexeneri . A simillexeneri  in pediare found . The pre Teharan  were shoVibrio cholerae was found to be 21.1%. In comparison, our study showed high prevalent of Vibrio spp (36.6%). In previous studies, bacterial infection occurred more frequently during rainy season [Shigella and Vibrio spp infection in this study were predominantly appeared to be higher during rainy season (June and July). In Nepal, the rainy season includes the warmest month and the period of heavy rain and floods. Heavy rain and floods are responsible for poor sanitation and increase human contact with waste water, in turn infection. However, more data in subsequent years are needed to elucidate the seasonality of this pathogen in this region.In a previous study by Karki Ravindra , Vibrio y season , 13, 21.Available literatures suggest that abdominal pain (72.6%), weakness (41.9%), and vomiting (36.5%) are the most common symptoms in diarrhoeic patients . In our Shigella spp had more than one drug resistant, and among 17 isolates of Shigella spp, the most frequent drug resistant was to ampicillin 11 (64.7%), nalidixic acid 10 (58.8%), ceftriaxone 8 (47%), ciprofloxacin 7 (41.17%).In previous study , the mosVibrio cholerae was detected in 56 (36.6%) of the population. Among these isolates, nitrofurantoin 52 (92.8%), cotrimoxazole 52 (92.8%) and nalidixic acid 52 (92.8%), were found to be highly resistant which shows a consistency to the study revealed by Karki Rabindra [V. cholerae spp towards ciprofloxacin and ampicillin are on increase, and a proper surveillance measure is necessary so as to prevent this emergence of bacterial resistance. In previous study, tetracycline 53 (94.6%) appeared the most sensitive to this isolate which compiles the results of previous study by Karki [In our study, Rabindra , where FRabindra , ciproflby Karki . In our Shigella  spp and Vibrio cholerae in Nepalese adult population, which appeared as an important aetiological agent of acute diarrhea with high rate of drug resistance. Thus, the results of this study emphasize an urgent need of designing a surveillance system for antimicrobial resistance in Nepal and the introduction of integrated guidelines for the appropriate use of antibiotics.Our study confirmed a high rate of This study was single centered hospital based study. So, the isolates resulted in this study may not be the representative of the entire country.


Background: Insecticides resistance inAnopheles mosquitoes limits Long-Lasting Insecticidal Nets (LLIN) used for malaria control in Africa, especially Benin. This study aimed to evaluate the bio-efficacy of current LLINs in an area whereAn. funestus s.l. andAn. gambiae have developed multi-resistance to insecticides, and to assess in experimental huts the performance of a mixed combination of pyrethroids and piperonyl butoxide (PBO) treated nets on these resistant mosquitoes.Methods: The study was conducted at Kpomè, Southern Benin. The bio-efficacy of LLINs againstAn. funestus and An. gambiae was assessed using the World Health Organization (WHO) cone and tunnel tests. A released/recapture experiment following WHO procedures was conducted to compare the efficacy of conventional LLINs treated with pyrethroids only and LLINs with combinations of pyrethroids and PBO. Prior to huts trials, we confirmed the level of insecticide and PBO residues in tested nets using high performance liquid chromatography (HPLC).Results: Conventional LLINs (Type 2 and Type 4) have the lowest effect against local multi-resistantAn. funestus s.s. and An. coluzzii populations from Kpomè. Conversely, when LLINs containing mixtures of pyrethroids and PBO (Type 1 and Type 3) were introduced in trial huts, we recorded a greater effect against the two mosquito populations (P < 0.0001). Tunnel test withAn. funestus s.s. revealed mortalities of over 80% with this new generation of LLINs (Type 1 and Type 3),while conventional LLINs produced 65.53 ± 8.33% mortalities for Type 2 and 71.25 ±7.92% mortalities for Type 4. Similarly, mortalities ranging from 77 to 87% were recorded with the local populations ofAn. coluzzii.Conclusion: This study suggests the reduced efficacy of conventional LLINs  currently distributed in Benin communities whereAnopheles populations have developed multi-insecticide resistance. The new generation nets (pyrethroids+PBO) proved to be more effective on multi-resistant populations of mosquitoes. Malaria vector control tools have been encouraging worldwide, resulting in a decreased morbidity and mortality as of 2016 compared to the 20002. Unfortunately, as this disease has reduced globally, it has been a different case in Africa, where malaria is still a serious challenge2. Long lasting insecticide-treated nets (LLINs) are major components of malaria control tools, and they have helped to combat malaria disease when in good conditions and properly used2. LLINs are effective, simple to use, easy to deliver to rural communities, and cost-effective when used in highly endemic malaria areas3.Malaria is responsible for about 438,000 deaths with an estimated 214 million disease cases annually5. In October 2014, there was a country-wide distribution campaign of mosquito nets to ensure universal coverage, with the free distribution of 6,077,272 LLINs to 2,199,522 households surveyed6. After this exercise, LLINs utilization by children under five rose from 70% in 2012, to 73% in 20146. However, the emergence and spread of resistant malaria vectors to insecticidal components used for treating these nets have threatened the earlier progress made with this malaria vector control tool9. Resistance to insecticides ofone of the main malaria vectors,An. funestus against control tools has since become a serious challenge facing the quest for malaria elimination in Africa. Reported cases of resistance are available in countries such as Cameroon10, Uganda11, Mozambique12, Malawi14, Ghana15, Nigeria16 and Benin18. There are also multiple mechanisms that are driving observed resistance in this mosquito population, although over-expression of detoxification genes remains the main driving force of insecticide resistance in thisAnopheles species19. Another observation is that resistance mechanisms are known to differ from one mosquito population to another suggesting the contribution of geographical differences in resistance profiling20. This revelation is of serious concern because it is becoming a significant threat to existing malaria control tools. Recently, a study by Agossaet al.9 in the northern part of Benin, showed that the efficacy of existing malaria vector control tools treated with pyrethroid have decreased in wildAn. gambiae s.l. populations. Considering the fact thatAn. funestus andAn. gambiae have developed resistance to almost all classes of insecticides across Benin22, it might follow a similar trend as the above study. Indeed, there is a serious quest for alternative insecticides since pyrethroids are becoming less effective with recorded reports of resistance in malaria vectors23. Pyrethroids are very safe, acceptable and suitable for LLINs, but degrade very fast, especially when exposed to sunlight, which can be avoided if nets are well preserved24. A different insecticide resistance management approach combining a chemical synergist, piperonyl butoxide (PBO), with pyrethroids on net fibres could be a promising way to fight insecticide resistance. PBO, a synergist capable of inhibiting the action of oxidase enzymes, has potential to combat the growing problem of oxidase based pyrethroid resistance in mosquito vectors species. Two types of long lasting nets treated with permethrin+PBO and deltamethrin+ PBO are the new generation of LLINs for improved resistance management26. These new generation nets have shown their efficacy on some resistant populations ofAnopheles in experimental hut trials27. In hut trials, the new generation of LLINs increases mortality and inhibits blood feeding against pyrethroid-resistantAn. gambiae in some Africa regions28. In Nigeria, the efficacy of LLINs treated with deltamethrin +PBO was highly effective on resistantAn. gambiae compared with standard treated nets with no PBO29. Also, in Southern Africa (Mozambique), this combination proved to be more effective against resistantAn. funestus andAn. gambiae20.In Benin, malaria control is hugely dependent on LLINs and indoor residual spraying (IRS)An. funestus from South to the North of Benin21, it is important to assess the efficacy of currently used LLINs  and also conduct in experimental huts a comparative assessment of the performance of the new generation of treated LLINs (Pyrethroids+PBO) against conventional LLINs currently used by communities in areas of Benin where the main malaria vectorsAn. gambiae andAn. funestus have developed multiple resistance to insecticides.Due to the widespread of insecticide resistance in most populations ofAn. funestus and other mosquito species18. Previous studies carried out in Kpomè showed thatAn. funestus s.s. is mainly predominant during the dry season and transitional periods, and exhibited high resistance to permethrin and deltamethrin with mortalities rates  susceptibility tests) of 13% and 46.5% respectively17. P450s are the main family of detoxification enzymes involved in observed pyrethroid resistance in theAn. funestus population in this locality17.An. gambiae populations from this same locality have also developed multi-resistance to several insecticides families30. This set of available environmental and entomological data has prompted building of seven experimental huts at Kpomè for trials to identify best LLINs types for improved control of insecticide resistant populations of malaria vectors.The assessment was conducted in the rural locality of Kpomè  located in South Benin, approximately 81 km from Cotonou. The study area has a sub-tropical climate, receives 1,100 mm of mean rainfall annually and has a mean monthly temperature between 27 and 31°C. The rainfall pattern in this area is similar to other southern localities of Benin, with two rainy seasons and two dry seasons. The constant presence of water bodies in this locality favors the development ofAn. funestus resting inside houses were collected using electric aspirators between 06h00 and 10h00 in June 2017 (Consent from head of household was obtained prior to collection). These mosquitoes were identified morphologically using Gillies and De Meillon31 and Gillies and Coetzee32 key asAn. funestus were kept in small cups and immediately transported to the laboratory (Relative humidity of 70–80% and a temperature of 25–30 °C) until fully gravid . Eggs were obtained from F0generation  using the forced egg laying method33 and were allowed to hatch to obtain F1 generation to be used for different experiments.Early morning collections of blood-fed, semi-gravid and fully gravid females of resistantAn. gambiae breeding sites were surveyed and their larvae collected using dipping methods34 and reared in the insectary to adult stage for different experiments.Still in the same locality,An. funestus and 100An. gambiae) of mosquitoes to be used for various assays were subjected to molecular speciation prior to assays. DNA ofAn. funestus s.l. used for forced-laying was extracted using the Livak method35. Species-specific targets were amplified for DNA using method described by Koekemoeret al.36, before distinct separations on a 1.5% agarose gel electrophoresis. ForAn. gambiae, we used also the protocol of Livak35, for DNA extraction followed by an amplification based on the protocol described by Fanelloet al.37 then after, amplified products were migrated on agarose gel for describing banding patterns.A subset  were therefore tested with 2 insecticides: 0.75% permethrin (type I pyrethroid) and 0.05% deltamethrin (type II pyrethroid) by using WHO susceptibility tests38. Approximately 100 mosquitoes (4 replicates of 25 mosquitoes) were used per test. The knockdown rate of mosquito exposed to the insecticides was recorded each 5 min, during 1 h exposure-period. A 10 % of sugar solution was made available to survivors. This test was made under observation at 25°C and 80% relative humidity laboratory condition. Mortality was recorded 24h after exposure to each insecticide. According to WHO criteria38, vectors were considered as being susceptible to a given insecticide if mortality rate was ≥ 98 %, resistant if mortality was <90 % or possibly resistant if mortality was between 90 and 98 %.Prior to assays, a subset ofAn. funestus population has been shown to express P450s genes more than in previous studies40, 2–5 days old F1 mosquitoes were pre-exposed to 4% PBO paper for 1 h and immediately exposed to 0.75% permethrin and 0.05% deltamethrin for 1 h. Two controls were used during this experiment. The first control was the mosquitoes exposed to untreated papers without PBO, and the second comprised of mosquitoes exposed to paper treated with PBO only. Mortalities were recorded 24h post exposure and were later compared to the un-synergized group in order to evaluate the potential role of cytochromes P450 genes in the observed resistance.According to the level of observed resistance against permethrin and deltamethrin, and because of pyrethroids resistant,Five types of LLINs were used for the phase I (Cone and tunnel tests) and phase II  evaluations.The Type 1 LLINs made of monofilament polyethylene (100 mesh size) fabric treated with deltamethrin at 4 g/kg±25% and piperonyl butoxide (PBO) at 25g/kg±25%, side panels made of multifilament polyester fabric with a strengthened border treated with deltamethrin at 2.1 g/kg±25%.The Type 2 LLINs was made of multifilament polyester fabric (100 mesh size), treated with a deltamethrin only (no PBO added) at 1.4 g/kg±25%.The Type 3 LLINs was treated with 20 g/kg of permethrin and 10 g/kg of PBO in the whole polyethylene net fibres (150 mesh size).The Type 4 LLINs made of polyethylene fibers treated with permethrin only (no PBO) at 20 g/kg, incorporated during fibers extrusion (150 mesh size).The Type 5 was an untreated net, a multifilament polyester (100 mesh size) fabric with neither insecticide nor PBO treatments.All nets used had sizes of 160 cm wide, 180 cm long and 150 cm high. All types of nets treated and control nets were procured by the Liverpool School of Tropical Medicine (LSTM), UK, properly wrapped and shipped to us at IITA for our various trials.Anopheles Research, 2010). 2 to 5 days old individuals from the 3 colonies ofAnopheles mosquitoes  were exposed to nets for 3 min, after which they were transferred into recovery paper cups and provided with cotton wool soaked in a 10 % honey solution. A minimum of 50mosquitoes was tested for each net. At least three pieces per net was used for this test. Mosquito knock-down rate (kd) was recorded at 1h post-exposure period and the mortality rate was determined 24 h post-exposure. The mortality rate was corrected using Abbott’s formula if needed. These tests were conducted at a room temperature and relative humidity of 25–30°C and 70–80% respectively.Four cones were fixed in contact to 25 × 25 cm pieces of nets taken from the sides and top panels of LLINs  insert, fitted onto a frame that slots across the tunnel. The LLINs fragments used had been pierced (1-cm diameter holes) to allow mosquitoes to pass through it into the tunnel to the compartment C2 where a guinea pig was placed for mosquitoes feeding. Each guinea pig was used only once for this study. Guinea pigs were sourced from local markets where they are sold for food consumption. At 8.00 AM of the following morning, mosquitoes were collected from both compartments and transferred into plastic cups. The mortality and feeding status (blood-fed or unfed) of each mosquitoes collected from the tunnel were recorded. Blood-feeding rate and penetration rate across the tunnel were also assessed.Tunnel test was carried out with the same samples of LLINs used for cone test. Adults41. The 3.5 × 2 × 2 m huts were made from concrete bricks, with a corrugated iron top and a ceiling of thick polyethylene sheeting lined, and each was built on a concrete base surrounded by a water-filled moat to exclude ants. Mosquito access was through 4 window slits, constructed from pieces of iron fixed at an angle to create a funnel with a 1-cm gap, present on 3 sides of the huts. Mosquitoes had to fly upward to enter through the gaps and downwards to exit; this precluded or limited exodus through the aperture and enabled us to account for most entering mosquitoes. A veranda trap made of concrete bricks and mesh screening (2 m long × 1.5 m wide × 1.5 m high) projected from the back wall of each hut. Movement of mosquitoes between a room and the veranda was unimpeded.Experimental huts newly built in Kpomè are specially designed to test the efficacy of different vector control products against freely entering mosquitoes under natural but controlled conditions. This facility was used for our release and recaptures tests. Huts were typical of the West African model as recommended by WHOAn. funestus and An. gambiae. The control mosquito population used was onlyAn. gambiae Kisumu as we had neither laboratory/field susceptibleAn. funestus, nor field susceptibleAn. gambiae.The described 5 types of mosquito nets were assessed against pyrethroid resistantThe following five comparison arms were tested in separate huts:1- LLINs Type 1 (deltamethrin + PBO)2-LLINs Type 2 (deltamethrin only)3-LLINs Type 3 (permethrin + PBO)4-LLNIs Type 4 (permethrin only)5-Type 5 Untreated Polyester net (control)Prior to introducing nets into huts, we conducted preliminary experiments which showed the huts to be evenly attractive to mosquitoes. Briefly, assessment for freely-entered mosquitoes in the hut was conducted during 2 weeks and the attractiveness effects of each hut were evaluated. Adult male volunteers slept under the untreated net in the huts from 20:00 hours to 05:00 hours each night after cleaning the hut to remove any spiders and ants. To minimize biases in individual attractiveness, sleepers were rotated between huts on successive nights throughout the 2 weeks.An. gambiae Kisumu strain of 2 to 5 days old were put into each cone for 30 min. After this exposure time, they were removed from the cone and put into plastic cups covered with untreated mosquito net and given access to 10% honey solution and mortalities recorded after 24h.Still prior to assessments, an initial series of bioassays was conducted to determine the mortality of susceptible mosquitoes exposed to various surfaces in the huts to know the lethal effect of the huts. Bioassays were performed with WHO cones tests attached to the surfaces with masking tape. In each hut, surfaces tested included doors, walls, screening-mesh of veranda, ceiling and floor. Ten females ofAn. funestus and An. gambiae both from Kpomè and a susceptibleAn. gambiae Kisumu. These 3 populations of mosquitoes were released different days into huts where the 5 described types of nets were erected. The experiment was conducted as described by WHO protocols41. The main trial was conducted in August 2017. The treatments were allocated randomly to five experimental huts in study site. Each net was deliberately holed with six 4cm×4cm holes to simulate a worn net. Before experimental hut evaluation, adult volunteers had been recruited among the inhabitants of the villages where experimental huts were implemented and informed consent to participate in the study was given beforehand and, chemoprophylaxis was provided during the trial. Female mosquitoes aged 5 days were released in each hut at 20:00 h in the night and monitored till morning. Early in the morning, released mosquitoes were recaptured from the hut, veranda and inside the nets and were scored as dead or alive and as fed or unfed. Live mosquitoes were kept in small cups containing sugar solution for 24 hours to assess the delayed mortality. Entomological effects of treatments were compared in-between nets and with the untreated net (control Net Type 5). Target entomological parameters monitored included: induced exiting, blood-feeding inhibition and mortality.A release/recapture experiment was conducted in experimental huts with resistant populations of(i) insecticide-induced exiting, i.e. the proportion of mosquitoes found in hut verandahs relative to control huts; (ii) blood-feeding inhibition, i.e. the proportional reduction in blood-feeding relative to untreated nets; and (iii) mortality, the proportion of mosquitoes killed (immediate plus delayed).Prior to the trial, chemical analysis were conducted on pieces of nets (pieces from holes made on nets) from the five Net Types erected in each hut. This experiment was to confirming the presence, or absence, and the concentration of pyrethroids and PBO in each net to be used in this trial. For the LLINs Type 1, the side panels and top panel were tested separately. Chemical analysis was conducted using high performance liquid chromatography (HPLC) machine . Deltamethrin, permethrin and PBO was extracted using acetonitrile as solvent and the mixture was sonicated for 15min. Afterwards, the solution without the net was transferred into a new flask and filtered through a 0.45µm PTFE syringe filter into an HPLC vial for analysis. For HPLC analysis, standard solutions of each insecticide  purchased from Sigma Aldrich were prepared from stock solution in acetonitrile. Standard curve of each insecticide were drawn. The HPLC system condition was as follow: mobile phase: Acetonitrile /H2O (90:10), C18 Column, flow rate: 1ml/min, injection volume: 50µl and UV detector wavelength: 226nm.The quantities of insecticides were calculated based on the peak area and expressed in g/kg of net.MedCalc easy-to-use online statistical software, version 18.2.142, while the Fisher’s exact test was used to test for significant difference of mortality rates. Significance between treatments was set at 5% level. The proportion of mosquitoes that exited early (induced exophily), the proportion that was killed within the hut  and the proportion that successfully blood-fed (blood feeding rate) were compared and analyzed using the logistic regression with treatments as fixed effects and huts, sleepers as random effects (STATA 9 Software).Data from bioassays were compared between each net usingAn. funestus and 100 samples ofAn. gambiae from Kpomè analyzed for molecular speciation wereAn. funestus s.s. andAn. coluzzii respectively  and deltamethrin (60.11±7.19%). No mortality was recorded when subsets of these mosquitoes species were exposed to papers with no insecticides (Control).ctively . Low morAn. funestus s.s. rose from 14.84% to 96.51% (permethrin) and from 44.15% to 100% (deltamethrin). However withAn. coluzzii population, mortalities reached 95% with deltamethrin + PBO whereas, the combination permethrin + PBO lifted up the mortality from 19.27% to 69.67%.When permethrin and deltamethrin were combined with PBO , mortaliHPLC analysis conducted on net fiber showed that deltamethrin concentration in the side of the Net Type 1(1.570±0.024 g/kg) and in the roof (3.762±0.019g/kg) were all within the standard dose (2.1g/kg±25% on sides and 4g/kg ±25% in the roof) .A mean dAn. funestus s.s.,An. coluzzii andKisumu strain respectively aged 2-5 days were exposed to the five sides of nets following WHO standard cones protocol. No mortality was recorded with mosquitoes exposed to untreated Net Type 5 after 24 hrs.An. gambiae Kisumu showed full susceptibility to all treated net. The Net Type 2 had the lowest lethal effect on both resistantAn. funestus s.s. (56.67±6.38%) andAn. coluzzii (34.67±4.11%) from Kpomè .A total of 275, 461 and 462 females ofm Kpomè . HoweverAn. funestus s.s. were exposed to Net Type 4. Recorded mortality rate with Net Type 4 was 23.3 ± 3.59 %. When PBO was added into the net fibers for Net Type 3 (PBO + permethrin), a significant increase of the mortality rate was recorded .In contrast, whenAn. coluzzii mosquitoes from Kpomè were introduced into the various cones, mortalities slightly rose from 6% to 25.83% with Net Type 3.A similar trend was observed whenAn. gambiae Kisumu,An. funestus s.s. and An. coluzzii) were separately released in tunnels with the untreated Net Type 5, we globally recorded over 45% penetration rates for all 3 mosquito species. When the untreated net was replaced by Net Type 2 and Net Type 1, the penetration rate decreased respectively from 85.66% to 3.4% and 0% forAn. gambiae Kisumu and with side sections of Net Type 1 (1.79%) when compare to the untreated Net Type 5 (66.85%). NoAn. funestus s.s. was able to pass through the top section of the Net Type 1. When the pyrethroid resistantAn. coluzzii population was released in tunnels containing fragments of treated Net Type 2 (deltamethrin only), side and top sections of Net Type 1, we recorded respectively penetration rates of 9.66±2.84%, 7.84±0.86% and 3.66 ±1.22% in the second compartment (C2) of tunnels. With fragments of Net Type 4 (bigger mesh size and treated with permethrin only), we recorded a similar penetration rate as with the control untreated Net Type 5 in resistant population ofAn. funestus s.s. However remarkable decreased penetration of this mosquito species through the Net Type 3 (PBO+permethrin) was observed. With Net Type 4 containing only permethrin and Net Type 3 containing permethrin+PBO, we recorded limited entry ofAn. coluzzii into compartment C2; 16.07±4.44% and 55.52±1.24% penetration rates respectively.When the three mosquito populations ( Kisumu. For theAn. funestus s.s. population (76.54±3.46%), the resistantAn. coluzzii (33.87±10.96%) andKisumu strains (91.92±0.81%) released in tunnel containing untreated Net Type 5  was able to blood feed in the presence of Net Type 4 containing permethrin and having large mesh sizes. Generally, all treated nets provided more blood feeding inhibition withAn. funestus s.s. compare toAn. coluzzii. Indeed, blood feeding was inhibited at respectively 91.98%, 97.66% and 100% with Net Type 2, Net Type 1(Side) and Net Type 1 (Top) inAn. funestus s.s. InAn. coluzzii population, Net Type 2, Net Type 1 (Side) and Net Type 1 (Top) provided respectively 71.48%, 80.30% and 100% blood feeding inhibition rates. Against resistantAn. funestus s.s., blood feeding inhibition rates with Net Type 4 (41.75%) was significantly lower than Net Type 4 (100%). Respectively 9.71% and 63.92% blood feeding inhibition rates were obtained with Net Type 4 and Type 3 inAn. coluzzii.A high blood feeding rate was observed in the insecticide resistant Type 5 . HoweverAn. gambiae Kisumu strain. However, the mortality rate recorded in resistantAn. coluzzii population with Net Type 2 increased when we exposed this mosquito species to Net Type 1. The same observation was noted with Net Type 2 and Type 1 (side and top sections) against resistantAn. funestus s.s. where mortality rate rose from 65.53% to 92.47% and to 98% respectively. Less than 80% mortality was recorded in presence of Net Type 4 againstAn. funestus s.s., while 96% of mortality rate with Net Type 3. High lethal action was provided by Net Type 3 (77.88%) againstAn. coluzzii  followed by hut N°2 (8.41). The mean number of mosquito per night was almost similar in huts N° 6, 5, 4 and 3. The hut N°1 showed a relatively low attractiveness . HoweverAnopheles gambiae Kisumu strain. The mortality rate for all exposed mosquitoes was very low as only one mosquito died out of the total of 73 exposed in hut N°4 and N°7 . A similar trend was observed with the pyrethroids resistantAn. funestus s.s. population from Kpomè with exophily rates ranging from 30 to 40% with treated nets compare to 9.46% with untreated Net Type 5 (An. funestus s.s. was not significant in between huts containing treated nets (induced exophily rate ranging from 23 to 34%)  . As for  to 37% .Blood feeding. NoAnopheles Kisumu bite was recorded when volunteer sleepers spent nights under all treated nets (100% blood feeding inhibition). In contrary, when these nets were replaced with untreated nets (Net Type 5), 71% biting rates were recorded withAnopheles Kisumu, 52% with pyrethroids resistantAn. funestus s.s. and 42% with resistantAn. coluzzii.An. funestus s.s. populations in presence of Net Type 4 (44% blood feeding inhibition) and Net Type 2 (58% blood feeding inhibition) compared to untreated nets. Generally, higher blood-feeding inhibition rates were provided by Net Type 1 containing deltamethrin + PBO (92% blood feeding inhibition) than Type 2 containing deltamethrin only (P<0.0001). Similarly, blood feeding inhibition rates in huts with Net Type 3 containing permethrin + PBO (100% blood feeding inhibition) was higher than those with Net Type 4 containing permethrin alone  than Type 2 which contains deltamethrin only (58% blood feeding inhibition). Respectively, 44% and 57% blood feeding inhibition rates were recorded in huts with Net Type 4 and Net Type 3 . In huts containing Net Type 2, 59.38% mortality rate was recorded with resistantAn. funestus s.s. However, Net Type 1 showed a high lethal effect of 92.5% against this resistant mosquito population. Respectively, 33.03% and 90.28% mortality rates were recorded in the huts containing Net Type 4 and Type 3  compared to Net Type 1 (73.65%). Mortalities rose from 10.7% to 71.8%, when resistantAn. coluzzii were released in huts containing respectively Net Type 4 and Type 3. Consequently, high overall killing effect was provided by Net Type 3 against thisAnopheles specie compared to Net type 4 ( Type 3 . Consequus s.s. . Same th type 4 . A combiAn. funestus s.s. from Benin to pyrethroid treated nets (current LLINs) and to combined PBO + pyrethroid nets for improved control of resistant populations of malaria vectors.This study aimed to assess the response of resistantAn. gambiae Kisumu) to treated nets showed that pyrethroid and pyrethroid + PBO treated nets remained effective for controlling susceptibleAnopheles mosquitoes. It was also observed that the bio-efficacy of nets treated with deltamethrin only (Type 2) was significantly lower when we compared the recorded mortality rates from the cone test in the resistant populations ofAn. funestus s.s. andAn. coluzzii and the susceptible strainKisumu. These observations further confirm the high pyrethroid resistance observed in both malaria vectors in Kpomè like in others localities of Southern Benin21. A more recent study conducted across a South-North transect of Benin, revealed that more than 50% ofAn. gambiae mosquitoes are unaffected by lethal effects of the current form of Net Type 243. However, in the Ivory Coast, this net was effective againstAn. gambiae s.s.44. When resistant mosquitoes were exposed to the combined deltamethrin-PBO (Net Type 1), the mortality rose from 56.67 to 95.77% and from 34.67 to 69.54% for respectivelyAn. funestus s.s. andAn. coluzzii. This finding showed the important involvement of P450s genes in observed pyrethroids resistance in this study and confirms also the results of synergist bioassays test performed with these same resistant mosquitoes as almost all individuals were dead when they were exposed to PBO and immediately after to deltamethrin.Results obtained from the response of susceptible mosquitoes (An. funestus s.s. in the presence of current permethrin treated Net Type 4 was observed compared to the combined Net Type 3 (permethrin + PBO). The loss of bio-efficacy of this current Net Type 4 was also demonstrated in Malawi, Mozambique and Democratic Republic of Congo, where recorded mortality rates ofAn. funestus to Net Type 4 in this study were respectively 3%, 20% and 34%20. The study conducted in Benin in 2013 demonstrated the efficacy of combined permethrin-PBO net (Olyset plus) against resistantAn. gambiae25. Surprisingly, only 25.83% ofAn. coluzzii was affected by lethal effect of this net in this study. It could probably due to the presence of other mechanisms involved in multi-resistance ofAn. coluzzii from Kpomè likekdr mutations30. This result supports the relatively low mortality (69.67%) obtained from the synergist test when we pre-exposedAn. coluzzii to PBO before to permethrin. Therefore, a combined permethrin - PBO net does not provide a solution to pyrethroid resistance withAn. coluzzii from Kpomè, Southern Benin.Similarly, significantly lower mortality of46. Indeed, reduced repellent effect of Net Type 2 against wild resistantAnopheles mosquitoes compared to high repellent effect againstKisumu strain could be as a result of their resistance nature. However, crossing of mosquitoes through Net Type 1was highly inhibited for each resistant population, even for susceptible strain, penetrating the compartment C2 of the tunnel containing Net Type 1. Nevertheless, deltamethrin alone used for the treated net (Type 2) continues to have moderate performance against resistantAnopheles mosquitoes in terms of reducing human and malaria vector contact and also blood feeding rate.Tunnel test performed on the all net Types used in this study confirmed the reduced bio-efficacy of only pyrethroids treated nets, showing a decrease in their effectiveness in areas of high resistance. This observation could be related to the resistance selection pressure generated by the use and misuse of the same class of insecticides for malaria vector control in public health and for pest control in agricultureet al.,27, working withAn. gambiae VKPER a strain originate from Kou valley in Burkina-Faso even after 20 washing times, same elevated crossing rates were still recorded. We made identical observations forAn. coluzzii in this study, with mosquitoes being no more able to pass through the net section of Net Type 3 comparing to the Net Type 4.Crossing rates with Net Type 1 were earlier described by N’GuessanAn. funestus s.s. penetration rate recorded in presence of Net Type 4 (66.67%) was significantly higher than that of Net Type 3 (5.57%) . These observations suggest the ability of resistant mosquitoes to withstand the excito-repellency effect of LLINs and penetrate impregnated bed nets thus, feed on humans raising concerns about the duration of LLIN effective life. This high passage rate recorded with Net Type 4 could be due to the decreasing repellent effect of permethrin, which was previously pointed out withAn. gambiae9.Similarly,An. funestus s.s. (71.25%) andAn. coluzzii (45.91%)] is in line with what was reported by Darrietet al.,47 showing that bed nets treated with pyrethroids (permethrin and deltamethrin) remained effective even in areas whereAn. gambiae s.s. are resistant to these insecticides. This paradox is due to the behavioral changes in the resistant mosquitoes; they were less repelled by the insecticide, remained on the only pyrethroid-treated material for longer periods, and thus received a higher dose of insecticide leading to the death of the mosquito48.But the highest mortality recorded with this Net Type 4 [Anopheles populations were significantly high in presence of Net Type 3 than Type 4 . With Net Type 3 in which PBO is applied, only about 4% mosquito was able to survive the exposure. Interestingly, the same observations were found for Net Type 2 and Type 1 against both resistant mosquito populations. This test gave a slightly synergistic action of PBO on the roof of Net Type 1 with a mortality of 98% forAn. funestus s.s. compared to the side net .However, the mortality rates in both resistantAn. funestus s.s. This is similar to previous studies with permethrin resistantAn. gambiae s.l. which showed that 100% blood feeding inhibition with unwashed PermaNet 3.0, a deltamethrin-PBO combination net27. Blood feeding inhibition rates of resistantAnopheles mosquitoes with Net Type 4 were significantly lower than Net Type 3, suggesting the decreased potency of this standard Net Type 4 in area where pyrethroids resistance is already spread50 but overall, the effectiveness of LLINs treated only with insecticides seems to be significantly lower compared to that of nets treated with insecticides and PBO.Concerning the control untreated Net Type 5, blood feeding was inhibited more by both Net Type 2 and Type 1 against resistantAn. gambiae Kisumu. This result indicates that the current pyrethroid only treated nets continue to exert strong repulsive action on theAnopheles susceptible strain. There was no significant difference between the low induced exophily rates inAn. funestus s.s. andAn. coluzzii in the hut containing Net Type 2 and Type 1 . This observation could be due to the naturally high endophilic behavior of thisAnopheles species51. Contrary, there was a significant difference between induced exiting rates of Net Type 4 and Type 3 against resistantAn. coluzzii.Experimental huts evaluations conducted in this study showed that all treated nets induced significant exophily rate ranged from 50 to 73% relatively compared to untreated net at 30% against susceptibleAn. funestus s.s. andAn. coluzzii survived in the hut with Net Type 2. When resistantAnopheles were released in the hut containing a combined deltamethrin-PBO net Type 1, less than 15% survived. However, almost all susceptible mosquito dead to the exposure with this net. Low lethal effect of Net Type 4 was observed with the resistant strain ofAnopheles compared to Net Type 3. This result correlates with those reported by Malimaet al.52 where the recorded mortality ofAn. funestus was 71.6% against nets treated with permethrin only. Survival rates of these mosquitoes in the huts suggest that the protective nature of currently used net Type 4 in Benin is compromised, as previously reported53. These semi-field controlled experiments confirmed the results from laboratory phase I evaluations and displays faith in combined pyrethroids-PBO nets, despite the multiple resistance mechanisms present in these mosquito species56. However, it is necessary to further investigate the impact of these multiple mechanisms on the efficacy of nets treated with pyrethroids only againstAn. funestus s.s.Respectively more than 40% and 60% of57. The fact that these new generation nets (Type 1 and Type 3) were able to inhibit blood feeding more than current nets (Type 2 and Type 4), could suggest their capability to confer high personal protection against resistant mosquito biting.A combination of the synergist PBO to pyrethroids made treated nets more efficient as PBO acted both as a metabolic enzyme inhibitor and as an adjuvant through its effect on enhanced cuticular penetration of deltamethrinAn. gambiae58. This research has demonstrated that the efficacy of a combined pyrethroids-PBO nets on resistant malaria vector populations could be a promising strategy against pyrethroid resistance populations ofAnopheles as previously highlighted60. This study further confirms a role of oxidases in pyrethroids resistance ofAn. funestus and the need to develop nets combining pyrethroid and synergist against pyrethroid resistant malaria vectors61. Nevertheless, several other studies have been conducted on the insecticide resistance management ofAnopheles especiallyAn. gambiae, using non-pyrethroid insecticides alone or in mixture of pyrethroids67. These studies also provided relatively good pointers for management of resistant mosquitoes but the problems with non-pyrethroid ingredients are human toxicity and their irritant effect68. A more recent study conducted by Malimaet al.69 in an area whereAn. funestus is resistant to pyrethroid, showed that even when non-pyrethroid insecticide-treated nets with durable wall lining (ITWL) are used, this cannot guarantee up to 50% protection against resistantAn. funestus.Studies conducted in Benin and other African countries showed a loss efficacy of pyrethroids treated net againstAn. funestus s.s. andAn. coluzzii in Kpomè is high, and is likely to limit the impact of currently used LLINs. This study showed that the use of new generation bed nets could provide additional protection and reduce malaria burden in endemic environments. This study is of importance to Malaria control programs for improved control of pyrethroids resistant malaria vectors in Benin.Pyrethroid resistance in the major malaria vectorsApproval was obtained from the ethics review boards of the International Institute of Tropical Agriculture (IITA) ref.PJ/CC5339. All volunteers recruited to sleep in the experimental huts gave written and verbal consents. Chemoprophylaxis was provided to volunteer prior to the hut studies.http://doi.org/10.17605/OSF.IO/3YRMS.All data generated and analyzed during this study will be included in the published article. Raw data are available from Open Science Framework. Dataset 1: Experimental huts trial of the efficacy of pyrethroids/piperonyl butoxide (PBO) nets treatments for controlling multi-resistant populations of Anopheles funestus s.s. in Kpomè, Southern Benin.The data is available under a CC0 1.0 Universal License General remarks: The paper presents key results of an operational research topic with high significance for preventing malaria transmission in the context of multiple insecticide resistance among vector populations. The study is well designed and methods used are appropriate and linked with expected outcomes of the study. Though, I noticed some limitations that have not been clearly sound for me (or confusing) while reading the whole manuscript.In Abstract: Comment 1: Key results presented do not meet with the objectives to achieve in the subsection “background”: i) “bioefficacy of current LLINs” and ii) “performance of PBO LLINs in experimental hut”. Key results on the second objective are missing. Suggestion 1: provide key outcomes from experimental huts .In Method section: Comment 2: the sequence of subsections as presented is confusing. The subsection “study design” in page 5 appears as heading for experiments in huts with a description of the five types of LLINs to evaluate. The description of such types of LLINS was made in relation with experimental huts and seems like a redundancy of the subsection “characteristics of LLINs …..” in page 4. But no confirmation if these 5 types of LLINS were different on not to those tested with cones or tunnels. The way you have calculate some key variables in tunnels , and in huts  is not clear. Suggestion 2: Confirm if types of LLINs (1 to 5) described in sections "chemical analysis of …" are also those used for all the bioassays including phase I (cone and tunnel tests) and phase II . - If so , the study design may be moved after the “study area” and adjusted accordingly prior the description of “mosquito collection”, “species identification”, “susceptibility tests with insecticide paper and with PBO synergist”, “cone tests”, “tunnel tests”, “experimental huts” and “data analysis”. Under “experimental hut” subsection may consider subparts like blank assessment of hut effects and release/recapture experiments. - If no (the 5 types of LLINs differ between tests), clearly indicate as “experimental hut design” and keep at this position. - Provide formula (available in WHOPES guidelines 2013) for the estimation of key parameters of LLIN bioefficacy in tunnels  and experimental huts .In Result section: Comment 3: All key results related to methods used have been presented. However, redundant data and information should be limited or avoided to shorter the paper, for example: Suggestion 3: - Table 1 (distribution of members of ….) in page 6 is not necessary , since data have been considered in the text at the subsection “molecular speciation …”; - Figure 1 in page 6 does not add value since same data are considered in figure 2 in page 7. Consider only figure 2 and insert the specific data on Kisumu strain from figure 1 in the main text. - Table 4 and 5 can be combined in one table of blank assessment  of attractiveness and lethal effect of experimental huts  - Check the last sentence of the third paragraph in page 8 (column on the right)…” Against resistant An. funestus s.s., blood feeding inhibition rates (removed “s”) with net type 4 (41.75%) was significantly lower than net Type 4 (against? Should be type 3) (100%). Respectively... - Titles of figures 2 (page 7) and 3 (page 8) may be more comprehensive: Figure 2. “Mortality rates of  Anopheles to pyrethroids and when combined …” Figure 3: “Mortality rates reported with cone tests on different types of nets using pyrethroids  resistant …” - Provide statistical evidences of the conclusion at the end of subsection “blank assessment of hut attractiveness” ..In Conclusion section: Comment 4: The conclusions of this paper are adequately supported by the results. Probably add elsewhere the justification and pertinence for undergoing phase II in experimental huts when LIN efficacy does not meet the WHO criteria using standard methods i.e. cone test  or tunnel tests . For example: I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Anopheles funestus s.s. The study is highly interesting as deals with development of strategies of malaria control in the context of high resistance to pyrethroid observed in main vectors. The manuscript is well written and presented, however there are very few minor concerns I would like the authors to take into consideration so that their work will be more efficient.This manuscript describe results of experimental experiences performed to assess the efficacy of of pyrethroids/piperonyl butoxide (PBO) net treatments for controlling multi-resistant populations ofComments: An. coluzzi as all the results of the manuscript are also presenting data from this speciesThe title could be modified to also take into accountnd paragraph of the introduction, in the 5th sentence : “resistance to insecticide ofone of ………”  replace “ofone” by “of on”2Methods in Anopheles Research, 2010)” by a corresponding number to be uniform in all the manuscript.In the section “Methods”, on the part “WHO cone tests with the nets” authors must keep the same references format. Indeed they should replace “(Release and recapture experiment) the sentence “Target entomological parameters monitored included: included exiting, blood-feeding inhibition and mortality” could be change by “target entomological parameters monitored inclided :(i) insecticide-induced exiting, i.e. the proportion of mosquitoes found in hut verandahs relative to control huts; (ii) blood-feeding inhibition, i.e. the proportional reduction in blood-feeding relative to untreated nets; and (iii) mortality, the proportion of mosquitoes killed (immediate plus delayed)”.In the text (I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Resolution of warts was partial in 3.9 and 24.1% and absent in 9.6 and 13.2% of children and adults respectively. Side effects were present in 41.2% of children and 46.3% of adults (p = .7). Most common side effects were blistering, pain, and burning sensation. No serious adverse events occurred. On a 10‐point scale, median patient satisfaction score was 9.0 (interquartile range 7.8–10.0) and 8.0 (interquartile range 5.1–9.7) for children and adults respectively (p < .01). CPS1 solution is a safe and promising treatment modality with a high clearance and high patient satisfaction rate for the management of cutaneous warts, particularly in children.Patients often request treatment of their burdensome cutaneous warts. However, a safe and effective treatment for cutaneous warts is lacking. This study evaluates treatment outcome, side effects, and patient satisfaction after topical application of cantharidin 1% podophyllin 2% salicylic acid 30% (CPS1) solution in a large series of children and adults with cutaneous warts. Fifty‐two children and 83 adults with warts, treated with CPS1 solution between October 2012 and October 2014, were included. Complete clearance of warts occurred in 86.5% of children and 62.7% of adults treated with CPS1 solution ( Therefore, this research was not subject to the Medical Research Involving Human Subjects Act. Patients who received treatment with topical application of CPS1 solution for cutaneous warts between October 2012 and October 2014 were eligible for inclusion. Exclusion criteria were plane or anogenital warts, seborrheic keratoses, immune compromised patients, pregnant, or lactating women.2.2Treatments were performed by trained health care professionals at our outpatient clinic, under the supervision of a physician. Self‐application of CPS1 solution was not allowed due to its toxicity and potential misuse, as it can be fatal if ingested .Continuous variables with a non‐normal distribution were summarized as median with a corresponding interquartile range (IQR). Categorical variables were summarized as frequencies and percentages. Statistical analysis of continuous variables was performed using the Pearson chi‐square test and for statistical analysis of two independent groups on a continuous variable, the Mann–Whitney 33.1A total of 147 patients were treated with topical application of CPS1 solution for cutaneous warts between October 2012 and October 2014. Five patients declined to participate in the survey and six patients were lost to follow‐up. One patient was excluded as the survey was completed anonymously. A total of 52 children (age < 18 years) with the help of their parents and 83 adults (age ≥ 18 years) completed the survey and were included in the analyses.p = .05). The majority of children had common warts. Warts in children were twice as likely to have been present for <12 months at first presentation compared to adults . Children more frequently had multiple reasons for desiring treatment in comparison to adults . Children experienced social and cosmetic inconveniences of the warts twice as often as adults. Only 3.8% of children and 4.8% of adults had not received previous treatment for their warts. There were no significant differences between children and adults in terms of number of treatments prior, during, or after therapy with CPS1 solution , female (53.3%), and had more than one wart (69.2%). Adults more often had plantar warts in comparison to children  of children and 62.7%  of adults treated with CPS1 solution. Resolution of warts was partial in 3.9%  and 24.1%  and ineffective in 9.6%  and 13.2%  of children and adults respectively (p < .01). Children required significantly fewer treatments with CPS1 solution compared to adults: 65.4% of children received 1–3 treatments with CPS1 solution as opposed to 37.3% of adults (p < .01). A majority of the adults (62.7%) required ≥4 treatments with CPS1 solution for their warts. Children were completely cleared from their warts at a median of 3.0 months (IQR 1.6–6.0) after onset of treatment with CPS1 solution, while for adults complete clearance occurred at a median of 5.0 months . Of the warts that initially cleared with CPS1 solution, recurrence of the warts occurred in 10.6% of children and 19.4% of adults (p = .3) (Table Clearance of warts was complete in 86.5% p = .01). The median patient satisfaction score was higher for children than adults, namely 9.0 (IQR 7.8–10.0) versus 8.0 (IQR 5.1–9.7), respectively, . The most common side effects were blistering, pain, and burning sensation. No serious adverse events occurred  showed no significant difference in terms of treatment outcomes.4p < .01) with only mild side effects and a high degree of patient satisfaction  of cutaneous warts treated with CPS1 solution in children and adults, respectively.This study reports the largest series of patients with warts treated with CPS1 solution to date. This study demonstrated a high complete clearance rate  randomized trial is indicated to further assess this promising safe and effective treatment for cutaneous warts.The authors report no conflict of interest.SUPPLEMENTARY TABLE: Patient characteristicsClick here for additional data file.