The rising use of titanium dental implants has increased the prevalence of peri-implant disease that shortens their useful life. A growing view of peri-implant disease suggests that plaque accumulation and microbiome dysbiogenesis trigger a host immune inflammatory response that destroys soft and hard tissues supporting the implant. The incidence of peri-implant disease is difficult to estimate, but with over 3 million implants placed in the USA alone, and the market growing by 500,000 implants/year, such extensive use demands additional interceptive approaches. We report a water-based, nonsur-gical approach to address peri-implant disease using a bifunctional peptide film, which can be applied during initial implant placement and later reapplied to existing implants to reduce bacterial growth. Bifunctional peptides are based upon a titanium binding peptide (TiBP) optimally linked by a spacer peptide to an antimicrobial peptide (AMP). We show herein that dental implant surfaces covered with a bifunctional peptide film kill bacteria. Further, using a simple protocol for cleaning implant surfaces fouled by bacteria, the surface can be effectively recoated with TiBP-AMP to regain an antimicrobial state. Fouling, cleansing, and rebinding was confirmed for up to four cycles with minimal loss of binding efficacy. After fouling, rebinding with a water-based peptide film extends control over the oral microbiome composition, providing a novel nonsurgical treatment for dental implants. Porphyromonas gin-givalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Prevotella intermedia.1 The relative abundance of commensal bacterial species to periodontal pathogens shifts with dysbiosis and too often induces the host to mount an inflammatory response leading to peri-implant diseases. This starts with peri-implant mucositis and can culminate with peri-implantitis, resulting in loss of soft and hard tissue anchoring the implant in the jaw, thus threatening and/or reducing the implant’s useful life.There is a continuing need to improve patient treatment based on the therapeutic advantages offered by titanium dental implants. However, a complex biofilm rapidly forms on the surface of any dental implant once placed in the oral cavity. Within several weeks, the biofilm consists of typical subgingival bacterial species, including keystone periodontal pathogens such as 3 Recent consensus on peri-implant disease points to plaque accumulation and microbial dysbiogenesis as the major factors triggering the host immune inflammatory response.6 Millions of implants are placed yearly around the world, with 3 million placed in the USA alone and rising at 500,000/annum.7 Reduced service life and eventual implant failure will therefore have a growing adverse impact on public health, with an increase in healthcare cost. On the other hand, effective treatment for peri-implant disease would make the benefits of implants available to a wider group of high-risk patients.9The expanded use of implants in dental treatment coupled with the rising prevalence of peri-implant disease threatens to shorten implant life and lead to implant failure in increasing numbers of patients.12 and a higher percentage fail in patients with chronic illness, advanced age, and/or poor periodontal health.15 A Cochrane metaanalysis report estimated the incidence of peri-implantitis as 1.6% after 3 years and 5.5% at 10 years.17 In contrast, Derks and colleagues placed the incidence of peri- implantitis at ~ 14.5% after 9 years of service,18 while others identified clinically significant, nonlinear bone loss as early as after 3 years of function in more than ~ 80% of patients.18 With implant use growing, increasing implant loss has a profound financial healthcare cost, with the potential for loss of public confidence in the dental profession.Despite a high success rate, within 5 years of placement, a small percentage (4–8%) of implants fail,21 Current state-of-the-art treatments for implants are similar to those for periodontal disease and include mechanical debridement and/or medicinal means intended to retard the biofilm. For implant surfaces, these include abrasive cup polishing, abrasive air blasting, titanium brushes with cleansing agents such as sodium hypochlorite or povidone-iodine, chlorohexidine rinses, antibiotics, and antimicrobials. Each treatment can be used alone or in concert by dental professionals. Successful treatment for peri-implant disease must also recognize the need to maintain biocompatibility, osteogenic competency, and cell viability at the implant surface to obtain favorable host cell responses.22 Thus, a commonly held goal remains to control the oral microbiota in order to arrest or slow hard and soft tissue destruction by the host inflammatory response.There is currently no definitive means for controlling or eliminating the bacterial biofilm on implants.30 However, the majority of these have required passive absorption or chemical coupling to the surface.33 Absorption leads to leaching and poor performance. Covalent coupling has also met with limited success, because coupling agents  creates hostile environments to bioactive molecules, leading to loss of bioactivity34 and/or incorrect display of the bioactivity to the cellular environment. Moreover, these hostile coupling environments can be used only during manufacturing and not at recall appointments, long after placement, when the longevity of the implant is threatened. Ideally, both strong affinity to the implant surface and maintenance of the antimicrobial state are required.There have been numerous attempts to boost implant surface performance using biologically active signals..36Peri-implantitis has become a growing concern in the oral health community due to the increasing popularity of dental implants to restore form and function. Because, at present, none of the nonsur-gical treatments result in a superior outcome, there is a lack of consensus with respect to predictable treatments for peri-implantitis, making treatment choices all but unmanageable for clinicians and patients. With the number of dental implants continuing to rise, there is an urgent need to identify a strategy that can further slow or even prevent peri-implantitis. Successful approaches will require novel and rational engineering design that can leverage the multifaceted characteristics of biomolecules, as well as providing structural and functional properties at the material-tissue interfaceUsing a combination of experimental and computational bioengineering approaches, we engineered a bifunctional peptide to provide a biocompatible, water-based, easy-to-apply, durable peptide film that exhibits antimicrobial activity. The bifunctional peptide can be repeatedly applied at recall appointments for continued treatment of peri-im-plantitis to extend implant longevity. Reapplication offers a safe, inexpensive, water-based bifunctional peptide film to treat existing implants at recall appointments, to arrest disease progression as a viable peri-implantitis treatment strategy compared with current state-of-the-art treatments.23 To remove the Fmoc protecting group, Wang resins preloaded with the first amino acid protected by a Fmoc group were treated with 20% piperidine in dimethylformamide (DMF). Fmoc deprotection was monitored by ultraviolet (UV) absorbance, and the deprotected resin was then washed with DMF to remove piperidine. Modified amino acids with protected side chains and Fmoc were solubilized in DMF at concentration of 0.2 M and added in sevenfold excess. In a separate measuring vessel, amino acids in solution were activated with 0.4 M O-benzotriazole-N,N,N’,N’-te-tramethyluronium hexafluorophosphate (HBTU) in DMF and 1 M 4-methylmorpholine (NMM). The activated amino acid was then added to the reaction vessel and mechanically mixed under nitrogen gas for 45 min to couple the amino acid to the resin. Double coupling was used for each amino acid in the sequences. After amino acid addition, the resin was washed with DMF and the protocol applied again for each subsequent amino acid.Peptides were produced with an AAPPTec Focus XC automated solid-phase peptide synthesizer using standard fluorenylmethyloxycarbonyl (Fmoc) chemistries and Wang resins.2O/phenol/ethanedithiol (87.5:5:5:2.5)] and precipitated using cold ether. Crude peptides were purified using reversed-phase (RP) high-performance liquid chromatography (HPLC), then lyophilized and stored at – 20°C.Following synthesis, the resins with synthesized peptides were dried with reagent-grade ethanol to remove residual DMF. The peptides were cleaved from the resin, and the side-chains were deprotected using Reagent K  in 70% 0.85% NaCl solution was used to stain bacteria on the disc surface. The discs were then transferred to a glass microscope slide and inverted for fluorescence microscopy. Images of five unique locations on the discs were collected at 10 × magnification.Peptide-functionalized discs were evaluated for antibacterial efficacy against Discs were cleaned to remove bacteria, peptide, and salts using a method that is common to implant retreatment visits. An electric toothbrush with a round head slightly larger than the diameter of the disc was secured in a laboratory stand. A Petri dish filled with peri-plast wax was used as a base for insertion of 30-gauge needles in a triangular configuration surrounding the disc to prevent motion of the disc when the electric toothbrush was applied with 100 g force at 10 cm from the secured pivot point. A 1:10 solution of sodium hypochlorite was pipetted into the Petri dish, and the disc was brushed for 2 min, then removed and washed thoroughly with water. The disc was then air-dried in a sterile laminar flow hood in preparation for peptide rebinding. The rebinding protocol was identical to the initial binding protocol.2O, then dried at room temperature. A solution of 50-nm gold nanoparticles  was incubated at room temperature on the peptide-functionalized disc for 20 min. The discs were washed once with DI H2O and dried at room temperature. Next, a solution of 8 μM green fluorescent protein-gold binding peptide fusion (GFPuv-AuBP) was incubated on the surface for 20 min, washed, dried, and imaged using a fluorescence microscope (Zeiss AxioPlus). The same procedure was applied for four repetitions of fouling/cleansing. All images were analyzed using ImageJ software (version 1.52a).A solution of 33 iM TiBP-AuBP in phosphate buffered saline was pipetted onto a titanium disc and incubated at room temperature for 1 h. Discs were washed once with DI HThree-dimensional surface measurements of the discs were visualized using a white-light profilometer . Pictures were taken at 5 ×, 10 ×, and 50 × magnification. Nonoverlapping pictures were taken of the disc. To obtain images from the same disc coordinates, a grid was etched onto a glass slide that repeatedly oriented the disc in the same orientation and position. The profilometer was then manipulated to ensure that the same region of the disc was imaged for consistency and reproducibility.4 PEP- FOLD 3.5 generates 3D structural conformations of linear peptides of between 5 and 50 amino acids and provides PDB models for the five best structures. PEP-FOLD 3.5 generates peptide structures by assigning one of 27 structural alphabet (SA) terms to fragments of four amino acids overlapping by three. The SA generalizes the secondary structure by assigning geometric descriptors emitted by the hidden Markov model described by Maupetit et al.5 Three-dimensional models are then generated from the fragments using a course-grained representation and refined by 30,000 Monte Carlo steps. The chimeric peptide sequences were input into the PEP-FOLD 3.5 online service, and 200 simulations were run assuming aqueous conditions and neutral pH. Once generated, the models were clustered and sorted using the Optimized Potential for Efficient Structure Prediction (sOPEP).7 Nonbiased modeling was applied.The de novo 3D structural modeling approach available through the online service PEP-FOLD 3.5 was implemented to generate Protein Data Bank (PDB) models for the five best models of each bifunctional peptide amino acid sequence.8 The structures were colored according to the peptide domains composing the bifunctional peptides. The first functional domain was colored blue, the spacer was colored black, and the second functional domain was colored red. The structures were oriented so that the first functional domain was located at the bottom. The surface was added to the ribbon structure, and transparency of 80% was applied.PDB files containing the secondary structure models generated by PEP-FOLD 3.5 were visualized and further analyzed using UCSF Chimera.S. mutans bacteria grown in presence of serial dilutions of TiBP-AMP are shown in S. mutans bacterial growth was found to be 64 μM. The lowest concentration evaluated, viz. 8 μM, showed no bacterial inhibition, while in contrast, 256 μM resulted in no growth of S. mutans.The bacterial growth curves of 40 that does not adversely affect clinical attachment. Hypochlorous acid (NaOCl) and a rotary electric toothbrush  was used to identify peptides via the GFP fluorescence signal that were bound to the AuNPs on TiBP-AuBP film attached to the titanium disc surface in a “sandwich” technique. Minimal to no fluorescence was observed for bare titanium discs and for discs function-alized with TiBP-AuBP + AuNP, and no signal for discs functionalized with GFPuv-AuBP fusion protein only. The fluorescence of the discs was measured using ImageJ, and the results for TiBP-AuBP + AuNP + GFPuv-AuBP are depicted as percentage surface coverage in Secondary structures were generated for the three bifunctional peptides studied herein. The secondary structures for TiBP-AMP, Control-AMP, and TiBP-AuBP are depicted in Surface topography characterization is summarized in 41 Similarly, we have previously established the importance of engineering the design of the spacer to optimize the function of the antimicrobial peptide domain in the bifunctional peptide construct.41 The spacer serves as a link between the two functional domains and is designed to preserve the secondary structure of each individual domain, a parameter that is tightly linked to antimicrobial function. Here, a novel AMP domain obtained from literature was linked to the TiBP through a “spacer5” to create a bifunctional peptide, as previously studied.41 The antimicrobial peptide (AMP domain) and the bifunctional peptide was selected through a rational design process based on previously determined antimicrobial “rules”.23 The minimum inhibitory concentration for the TiBP-spacer5-AMP bifunctional peptide was established at 64 iM for an ATCC line of S. mutans  through an engineered spacer . The tit. mutans . The bifTiBP-AMP is designed to incorporate a high-affinity titanium implant anchoring peptide with an antimicrobial peptide linked through a spacer designed to preserve the function of each peptide. The data demonstrate that the selected spacer design preserves not only the antimicrobial activity but also the robust anchoring activity to the implant surface through the TiBP domain, as seen during repeated retreatment of cleansed bacteria-fouled surfaces as would occur for implants already placed in the jaws , Table I40 demonstrate that the described bifunctional peptide can be successfully reapplied and anchored to the surface of a previously fouled and cleaned implant surface.TiBP-AMP can be repeatedly applied to a bacte-ria-fouled implant surface to deliver antimicrobial properties to address the etiopathogenesis of peri-implant disease. In contrast, the Control-AMP (without TiBP) showed minimal absorption to the surface of bacteria-fouled and cleansed titanium discs , Table IS. mutans bacteria. The TiBP-AMP film successfully demonstrated an antibacterial function with 19.3% dead bacteria, compared with 0.2% for the Control- AMP. Additionally, the TiBP-AMP peptide film had antibiofouling properties, with 21% total surface coverage by bacteria (living and dead), compared with 53.5%  P-value of 0.011. Furthermore, the data demonstrate that the amount of peptide bound generally increased slightly with each successive fouling cycle, with the exception of cycle 2. The demonstrated preservation of the TiBP anchored to the surface strengthens the impact of this technology as a nonsurgical, water-based retreatment option for peri-implant disease. Multiple retreatment procedures can be performed, extending the lifetime of the implant.The novel method applied to reveal peptide anchored on the implant surface is demonstrated in The secondary structures generated demonstrate preservation of the a-helix character of the antimicrobial domain, which has previously been linked to antimicrobial function. The engineered spacer is critical to preserving the function of the antimicrobial and binding domains.46 and sterilization following a widely used protocol similar to that used by implant manufacturers to create “active osteoin-tegration” surfaces46 Biocompatibility is an essential feature for all implant surfaces, and preserving the surface properties is a mandatory design requirement. The average, RMS, and range of the roughness did not change significantly after fouling and cleaning for retreatment, or after rebinding of the bifunctional peptide film (Titanium implant discs were prepared by grit blasting with 180–220 micron titanium dioxide particleside film . These r47A water-based, nonsurgical approach to address peri-implant disease using application of a bifunctional peptide film during initial implant placement of new implants or as retreatment for existing implants was demonstrated. The functions of the TiBP and AMP domains linked by an engineered spacer in a bifunctional peptide were confirmed by identification of the minimal inhibitory concentration in solution, as well as by surface antimicrobial activity. Moreover, this simple cleansing protocol likely preserved the biocompatibility of the titanium implant while reexposing the titanium surface after bacterial fouling to permit sufficient rebinding of the bifunctional peptide. The rebound bifunctional TiBP-AMP peptide film exhibited antibacterial and antibiofouling activity over four fouling/cleaning cycles. The modularity of this approach enables it to be combined with our next generation peptide engineering methodologies to improve both the titanium binding and antimicrobial properties of the peptide film, which could lead to better coverage, with higher rebinding affinity and antimicrobial effectiveness, respectively. This technology represents a paradigm shift in prevention of dental implant failure while adding to the range of bioactive molecules that can be anchored to implant surfaces to improve their function, such as osteointegration.Supplemental


Similar results were obtained with CES-D. There was a negative association of BDI-II scores and BSID-III cognitive and language scores at 20–24  and 36 months . CES-D associations with BSID-III cognitive and language scores showed similar trends. BSID-III motor scores were associated with depression scores at 36 months for both BDI-II and CES-D . In conclusion, the group education was associated with reduced maternal depression scores. Moreover, the depression scores were inversely associated with child cognitive and language development outcomes.Optimal nutrition improves child development, and impaired development is associated with maternal depression symptoms, in particular in low resource settings. In this follow-up of an open cluster-randomized education trial, we examined its effects among mothers in rural Uganda on their depression symptoms and the association of these symptoms to child development. The education comprised complementary feeding, stimulation, and hygiene. We assessed 77 intervention mothers and 78 controls using Beck Depression Inventory-II (BDI-II) and Center for Epidemiologic Studies Depression Scale (CES-D) scores. Child development was assessed with Bayley Scales of Infant and Toddler Development-III (BSID-III) composite scores for cognitive, language and motor development. Compared to controls, the intervention reduced depression symptoms’ scores with mean (95% CI) differences: −8.26 (−11.49 to −1.13,  Early childhood is characterized by rapid cognitive and social development changes that require optimal nutrition . IntroduInadequate infant and young child feeding practices increase the risk of morbidity and mortality, especially in low resource settings. Moreover, childhood morbidity has been associated with maternal depression symptoms in such settings . The preAmong mothers with depression symptoms, meta-analysis and observational studies have reported more symptoms, such as negative maternal behaviors, negative maternal affect, and hostile/coercive behaviors as well as disengagement with their infants than among non-depressed mothers . InitiatNotably, recently a Lancet Series indicated an increasing worldwide burden of mental health disorders including maternal depression symptoms . The LanIn a cluster-randomized controlled trial initiated in 2013, we performed a 6-months’ intervention comprising nutrition, stimulation and hygiene education among impoverished mothers of children aged 6–8 months in rural districts of Uganda . The intWe have now examined the possible effects of this intervention on maternal depression symptoms and their associations to child development, among a sub-sample of these mother/child pairs at the child-ages of 12–16, 20–24, and 36 months. In our parental trial, the intervention mothers formed groups that frequently met to practice and share the childcare practices introduced as part of the education intervention. These groups were formed based on the group dynamics theory . AccordiIn this follow-up study, mothers in the intervention group would identify with each other and have shared values, practices and skills on the appropriate complementary feeding taught in the intervention. Moreover, the complementary feeding skills would be learnt through observation, imitation, and positive reinforcement from mother group members. In this follow-up study, we examined if this education intervention (i) would be associated with reduced depression symptoms among the mothers, and (ii) if there were any association between maternal depression symptoms and child development outcomes.This is a follow-up study of a two-armed, open cluster-randomized education intervention regarding nutrition including focus on complementary feeding, stimulation and hygiene among impoverished mothers of children aged 6–8 months conducted in the Kisoro and Kabale districts of South-Western Uganda . In the The study was approved by The AIDS Support Organisation Research Ethics Committee (no. TASOREC/06/15-UG-REC-009) and by the Uganda National Council for Science and Technology (no. UNCST HS 1809) as well as by the Norwegian Regional Committee for Medical and Health Research Ethics (no. 2013/1833). The parental trial was registered with clinicaltrials.gov (ID no. NCT02098031). We report the data according to the CONSORT guidelines and with intention-to-treat analysis.n = 263) or the control (n = 248) group.For the parental trial we used proportionate sampling, 10 sub-counties  were obtained . We used a three-stage procedure to identify eligible households. First, by simple random sampling, three sub-counties in Kabale were allocated to the intervention group and the other three to the control group. Similarly, two sub-counties were allocated to the intervention and the other two to the control group in Kisoro. Second, all the villages in each participating sub-county (intervention or control) were listed alphabetically and assigned numbers in an ascending order. By use of computer-generated random numbers, villages to whose assigned number matched with the random numbers were selected. The intervention villages did not share common geographical boundaries with control villages to minimize contamination of the intervention contents between the two study groups. Third, by complete enumeration, all consenting households with children aged 6–8 months within a participating village were recruited to the study. If a household had more than one eligible child, the youngest was selected, and in the case of twins, we randomly selected one for evaluation. We finally enrolled 511 mother-child pairs in the parental study and they were randomized to the intervention . A score of 10 or above is considered to be indicative of probable depression [https://www.outcometracker.org/library/CES-D.pdf). A total score of 16 or higher is considered to indicate depression in the general population [The primary outcome of this follow-up study was maternal depression symptoms using Beck Depression Inventory II (BDI-II) scores and Center for Epidemiologic Studies Depression Scale (CES-D) scores. The BDI-II is a self-reported tool for assessing symptoms of depression. It asks mothers to report on a 4-point scale from 0 to 3 with 21 questions, giving a possible range of 0–63  score, which is the most comprehensive development measure for children up to 3.5 years and has been adapted and used in Uganda . Inter-oIn a similar low-resource-setting, Van der Heijden et al. reported a mean difference of about 1.5 SD in BDI-II scores between intervention and control groups . Thus, tp < 0.05. According to the skewness test for normality (in the software programme), our data residuals showed a normal distribution. In addition to this, we generated histogram plots, which showed a bell-shaped curve pointing to a normal distribution. We used a mixed effect linear regression to compare the intervention with the control group and estimated ICC. The sub-county (cluster), village, mother, and mothers within villages were the random intercepts, while the time points and group affiliation (intervention or control) were the random slope and fixed variables in the model. Exchangeable variance-covariance structure was used for the random part at the mother level and the models were fitted via the restricted maximum likelihood method. We fitted data by the maximum likelihood method and used a log likelihood-ratio test to determine the overall effect of the intervention for the entire study period. Differences between the two study groups are given as mean (SD or 95% CI).Maternal depression symptoms and child development outcomes were analyzed using Stata/SE  and SPSS version 22.0 . Significance level was set at For the secondary outcomes  we used pooled data from the two study groups (intervention and control) to examine associations between maternal depression symptoms using the BDI-II and CES-D scores and the development outcomes by the BSID-III scores using mixed effects linear regression models to handle three data points per mother. In the association analyses, time, maternal depression symptoms’ scores, and interaction were treated as the fixed variables, the sub-county as the cluster and reml as a fitting method. Additionally, these analyses were adjusted for group affiliation.The flow chart in There were no significant differences in the characteristics between the parental cohort (data obtained at baseline) and the follow-up cohort .On the question about maternal sadness asked at baseline, the number (proportion) of mothers in the control group who responded as being sad (score 1) was 43 (55%), while the corresponding value for the intervention group was 40 (52%) . Notably, the reported depression symptoms assessed by the BDI-II scores were significantly associated with all BSID-III child development outcomes at both 20–24 and 36 months, except the motor child development outcome at 20–24 months . At the 36 months assessment, all BSID-III child development outcomes were significantly associated with the maternal depression symptoms.In their recent Cochrane report, Arikbo and colleagues found evidence for effects of education interventions on complementary feeding, but no effects on child growth were found and neither developmental outcomes nor maternal depression were evaluated . MoreoveIn the parental trial, the 6-months education intervention led to significant improvements in development outcomes when the children reached 20–24 months, but without affecting growth . The folIn the current follow-up study, we found that the education intervention significantly reduced maternal depressive symptoms among intervention mothers compared with the controls. These findings from our randomized trial are consistent with those of interventions focusing on maternal childcare practices. For example, similar findings were reported by Singla et al., whose manualized, parenting intervention in rural Uganda resulted in mothers in the intervention group reporting significantly fewer depressive symptoms than mothers in the control group . FurtherHow our maternal education intervention led to a decrease in the maternal depression symptoms is unknown. In the parental trial we found that the child diet diversity score improved among children in the intervention group , and at Our study findings indicated an inverse relationship between maternal depressive symptoms and child cognitive, language and motor development outcomes. These findings agree with those of observational studies that identified persistent declines in the rate of child development upon exposure to late onset of maternal depressive symptoms ,31. A meThere is vast evidence on the negative effect of maternal depression symptoms on children’s cognitive, behavioral, and socio-emotional development ,36. Our Our results also showed an inverse relationship between maternal depression symptoms and motor development. In support of these findings, previous research reported that maternal depression during early childhood increased the risk of delayed motor (fine and motor) development at three years ,39.Collectively, previous research and our current study highlight the importance of early intervention to address maternal depression in early childhood. Specifically, interventions should possibly be directed at educating the mothers regarding the importance of nutrition, hygiene and sanitation as these measures are likely to improve the health of their children and thereby reduce the many burdens mothers in low-resource settings face every day.This study has several strengths. For example, we adopted a multidisciplinary approach combining aspects of child care practices of nutrition, child stimulation, hygiene, and two independent and validated psychological research instruments for assessing maternal depression symptoms (BDI-II and CES-D) and one well established tool for assessing child development outcomes (BSID-III). Notably, the children were studied for several years with little loss to follow-up and in a randomized controlled trial based in a rural community setting. The study also has some limitations. The use of self-reported maternal depression symptoms could have compromised reporting of as well as mistakes in presenting the symptoms. Notably, the BDI-II and the CES-D tools only provide scoring of maternal depressive symptoms and not a diagnosis of depression per se, although higher scores are considered to be indicating of a depressive disorder as categorized in the ICD-10 and DSM-IV classifications ,41. AddiOur randomized group education trial focusing on complementary feeding, hygiene, and stimulation education among mothers of 6–8 months old children significantly reduced maternal depression symptoms at child-ages of 20–24 and 36 months. We also found inverse associations between maternal depression symptoms and child cognitive and language development outcomes. The reported positive effects from this intervention would call for further studies of similar interventions in other low-income rural settings before consideration of a larger scale-up in the sub-Saharan region and elsewhere.


Oropharyngeal squamous cell carcinoma (OPSCC) is an increasing health problem in the developing and developed world. In recent years, there have been major changes in the treatment paradigms for OPSCC. This is because of a number of reasons: the understanding and discovery of a new viral etiology (the human papillomavirus [HPV]), changes in practice patterns owing to advances in radiotherapy, and then an organ preservation strategy with the increased use of chemotherapy. Next came the development of new surgical technologies and the emergence of a new treatment modality, immunotherapy. In this article, we discuss the evolution of OPSCC treatments, starting with the traditional tobacco era. Treatment paradigms then evolved during the organ preservation era, the HPV era, and the minimally invasive surgery era. We are currently in the immunotherapy era, with a number of new drugs becoming available both on trial and by approval for use in the clinical setting for head and neck cancer patients. We discuss a number of trials and the reasons behind attempts at both treatment escalation and treatment de-escalation. Within these subsites of the oropharynx, the tonsil and tongue base are the most common sites to be affected by carcinoma. These sites differ from the other subsites because they have a high density of lymphoid cells and show a strong association with human papillomavirus (HPV)-related squamous carcinoma. The strong predilection of HPV for the oropharynx is due to the microanatomy of the reticulated epithelium (epithelium with an immune system component) of the base of the tongue and tonsils2. HPV infects the reticulated epithelium lining the deep tonsillar crypts. The deep crypts of tonsils are immune-privileged sites, which means they can tolerate the introduction of antigens without eliciting an inflammatory immune response. This results in inhibition of the effector function of the HPV-specific T cells and thereby facilitates immune evasion at the time of initial HPV infection3.Oropharyngeal squamous cell carcinoma (OPSCC) refers to squamous cancers that develop in the posterior third of the tongue, tonsils, soft palate, and posterior pharyngeal wallIn this article, we will discuss the evolution in understanding of OPSCC and describe how this has impacted on treatment options and research opportunities in this field.4. There was also a high incidence of comorbidity in these patients from associated tobacco exposure conditions. Treatment was typically with surgery, as most tumors were locally advanced at presentation. Open approaches, such as mandibulotomy, were used to access the oropharynx. Long-term support of airway and feeding was often required in the form of a gastrostomy feeding tube and a tracheostomy. Radiotherapy was used post-operatively to optimize loco-regional control. With developments in the delivery of radiation and the recognition that primary non-surgical management was feasible, a move away from traditional open resection was seen. Two multi-center randomized controlled trials in 2004 also showed a survival advantage with the addition of post-operative chemoradiotherapy in high-risk patients6. Radiation-based approaches were adopted more commonly with surgery reserved for salvage. A report of the outcomes and complications comparing surgical and radiotherapy-based approaches in 2002 showed that while both approaches had similar survival and local control rates, surgery was associated with an increased rate of complications7.Traditionally, oropharyngeal carcinoma was closely related to tobacco and alcohol exposure. The incidence of carcinoma in the oropharynx was similar to that in the other sites of the upper aero-digestive tract that all shared common exposure to the carcinogens in tobaccoet al. showed a survival benefit in the use of concomitant chemoradiotherapy (chemotherapy administered during radiation) over induction chemotherapy or radiation alone8. These results were extrapolated to other head and neck sites and provided the initial support for organ preservation strategies. The first major OPSCC-specific trial was authored by the GORTEC group. They randomized 266 patients with OPSCC to concomitant chemoradiotherapy or radiotherapy alone9. An overall survival at 3 years was reported as 51% in the chemoradiotherapy group versus 31% for the radiotherapy-alone group9. At 5 years, a 6.6% overall survival benefit with the addition of chemotherapy was reported10. This has subsequently become a standard therapy for loco-regional advanced OPSCC and was supported by a robust meta-analysis11.Landmark trials fueled the enthusiasm for organ preservation approaches in head and neck cancers and built upon increasing radiation treatment experience and expertise. In 1994, the Veterans Affair study reported outcomes for locally advanced laryngeal carcinoma treated with induction chemotherapy followed by radiotherapy (in responders) compared with total laryngectomy. The results of this study confirmed that a non-surgical organ preservation approach could be adopted, which achieved similar oncological outcomes but avoided laryngectomy in over 60% of cases. A further study in locally advanced laryngeal carcinoma by Forastiere12 and to the pharyngeal constrictors in an attempt to minimize swallowing dysfunction13.Further interest in radiation treatments for OPSCC was stimulated by advancing technologies in the delivery of radiotherapy. Intensity-modulated radiotherapy treatment (IMRT) was introduced in the early 2000s. This was a new radiotherapy technique aimed at reducing the comorbidity of treatment by allowing the oncologist to manipulate radiation dose in a way that would increase accuracy and reduce the dose to bystander structures. Specific potential advantages of this targeted method of delivering radiation include avoidance of high-dose exposure to the parotid glands to minimize xerostomia14. This coincided with changes in the incidence and demographics of OPSCC15. The incidence of tonsillar carcinomas in the United States increased by 1.9% and 2.7% per year from 1973 to 1995 in Caucasian and African-American men, respectively, while all other oral sites remained constant. The impact and relevance of these discoveries were crystallized by Kian Anget al. in 201016. They reported a retrospective analysis of the association between tumor HPV status and survival among patients with stage III or IV OPSCC. In 323 patients, HPV was detected in 206 tumors (63.8%). The HPV-positive tumors had a better 3-year overall survival  and, after adjustment for age, race, tumor and nodal stage, tobacco exposure, and treatment assignment, had a 58% reduction in the risk of death . The effect of HPV status on outcome has now been shown in many series throughout the world. A meta-analysis of studies worldwide reporting on 5,681 patients showed the prevalence of HPV tumors was 22% and this was associated with an improved survival, with HPV-positive tumors having a 68% reduced risk of death compared to HPV-negative tumors 17.The next major development was the discovery of a viral etiological agent in the carcinogenesis of OPSCC. An association of HPV with carcinomas of the tonsil and base of the tongue emerged in the early 1990s, with the identification of HPV DNA in tumor cell nuclei and viral oncogene transcription in tonsillar carcinomas14, but the rate does vary by country. A UK series has shown up to 70% of oropharyngeal tumors are HPV related19. The HPV16 subtype is the most common and is found in 90% of tumors. The other high-risk subtypes 31, 33, and 18 have also been identified14. In an East-Denmark study between 2000 and 2010, Garnæset al. found 58% of tonsillar OPSCC were HPV-related tumors and 51% in the base of the tongue were also HPV related21. However, high-risk HPV DNA showing evidence of transcription is infrequently seen at other sites in the head and neck and at the subsites of the soft palate and posterior pharynx18. A multicenter cross-sectional retrospective study in the UK, however, has confirmed an increase in OPSCC cases but also showed that the percentage of tumors that were HPV related remained static22.The proportion of OPSCC with detectable HPV DNA on testing approached 50% in a number of series23. An analysis of Surveillance, Epidemiology, and End Results (SEER) data from 1973 to 2004 with patients deemed likely to be HPV positive based on if the tumor’s location was in the tonsil or the base of the tongue showed patients were younger 23. A series of 193 patients, using DNA PCR to test for HPV, showed that HPV-positive tumors were more likely to affect patients younger than 55 years old24. Age was recognized as an important distinction between HPV-positive and -negative tumors in a study from Sweden25. Patients with HPV-related cancers were younger (mean age of 59 years [range 42–78] versus 66 years [range 45–89]).This difference in anatomical location has allowed for the investigation of population-level data23. However, when likely HPV-positive tumors are examined, they were seen to have a higher incidence in white men and an increasing incidence in men in all other races23. There was also a correlation between the educated middle class and HPV-positive cancers24.Additionally, USA SEER data indicated that men have a higher incidence of OPSCC than do women and that blacks are more commonly affected26. A 25,500-patient multicenter study found that tobacco is a major risk factor for head and neck and oropharynx carcinoma27. Patients with HPV-positive tumors have been shown to be non-smokers more often28, and the overall tobacco use is lower compared to that in HPV-negative patients. However, smoking is still a risk factor but has less of an impact in HPV-related cancers. Patients with HPV-positive tumors have about 30% non-smokers in their group compared with less than 5% in the HPV-negative groups29. This relationship has been seen in a number of studies30.Other differences between HPV-positive and -negative tumors have also been seen. Tobacco has been identified as a head and neck cancer risk factor; it also acts synergistically with alcoholThe majority of studies that supported the use of chemoradiotherapy in patients with OPSCC were performed in the 1990s and early 2000s. At this time, the link between HPV and OPSCC was being uncovered. Unfortunately, it has not been possible to retrospectively analyze the patients in these studies to understand what proportion of these tumors were HPV related.31 tumors with larger cervical nodal disease32. The nodal disease is often also cystic. However, despite presenting with advanced nodal disease, which is traditionally thought to be associated with poor outcome, they are associated with an improved prognosis. This means HPV status could have acted as a confounder in studies that have not stratified for HPV status. The recurrence and failure rates are lower in the doubly positive HPV/p16 tumors compared to the HPV/p16-negative tumors33. Other important differences in risk factors for HPV-related carcinoma compared to non-HPV-related OPSCC are the male preponderance and sexual exposure34. Recognition of the major differences in risk factors, demographics, clinical behavior, and response to treatment of HPV-positive compared to HPV-negative tumors35 may allow more tailored treatment.Other differences are also seen in the clinical presentation of HPV-related OPSCC. They tend to have smaller primary T stage36. In an analysis of 43,983 patients between 1998 and 2009, the number of patients receiving chemoradiation increased from 22% of all patients in 1998 to 61% in 2009. This has been accompanied by a concurrent decline in the percentage of patients receiving surgery, from 41% in 1998 to 31% in 2009. This coincides with the increasing incidence of OPSCC and increased HPV-related disease.In a study using the National Cancer Database (NCDB), a North American governmental database of cancers and treatments, the trends in the treatment modalities used in OPSCC were examined37 analyzing the survival and toxicity outcomes with primary IMRT, 186 patients received IMRT with 90% of loco-regionally advanced disease receiving concurrent chemotherapy or cetuximab. The estimated 3-year overall survival (OS), disease-free survival (DFS), and disease-specific survival (DSS) rates were 77.2% (70.5–83.9), 72.3% (65.4–79.2), and 80.2% (74.1–86.3), respectively. Estimated 3-year OS, DFS, and DSS rates for HPV-positive patients were 90.9% (85.2–96.6), 87.9% (81.4–94.4), and 91.8% (86.3–97.3), respectively.In recent studies using modern IMRT techniques, outcomes of chemoradiation for OPSCC have been excellent. In a European studyDespite the excellent oncological results achieved with this approach, the changing demographic of patients with OPSCC and the improvement in survival means new challenges are being seen. The complications and sequelae of treatment are now more pertinent because patients are younger, more likely to be cured, and more likely to live longer.et al.37 reported that three (1.6%) patients died during or within 30 days of radiation completion, 74 (40%) were admitted at least once during their radiotherapy, and 76% needed a feeding tube either as a supplement to their oral intake or to meet their complete nutritional requirements. However, long-term toxicities are likely to be more important in understanding which treatment is best for patients, as all treatment approaches are associated with acute toxicity.Bird38. However, access can be challenging, particularly to the posterior tongue base. Since the US Food and Drug Administration’s (FDA) approval of trans-oral robotic surgery (TORS), robotic surgery has offered a minimally invasive approach for head and neck tumors. The da Vinci robot  began its development in urology and cardiac surgery. In 2005, Melder and McLeod reported its first use in head and neck surgery when they performed a resection of a vallecular cyst39. In 2006, Weinsteinet al. at the University of Pennsylvania reported the robot’s first application to head and neck malignancy40; their group conducted most of its early research and coined the term TORS. Subsequently, in 2009, the FDA approved its use in the head and neck, and TORS is now utilized worldwide.Our understanding of OPSCC etiology and its relationship with HPV infection has evolved at a time when non-surgical organ preservation approaches in head and neck oncology have replaced more traditional open surgeries with post-operative radiotherapy. However, over a similar time frame, significant progress has been made in surgical technology. Advances in optics and instrumentation have made a number of options available for the removal of head and neck cancers via a trans-oral endoscopic route. Trans-oral laser techniques have long been used in the larynx, and similar approaches have been adapted to use in the oropharynx41. The advantages of TORS are the ability to operate without line-of-sight restrictions that limit other trans-oral endoscopic or microscopic approaches42. The approach offers a consistent approach in which the pharyngeal muscle constrictors are removed to provide a deep margin. It allows tumors that would normally demand a pharyngotomy or mandibulotomy to be resected. Additionally, it involves instruments with six degrees of freedom, motion scaling, instrument stabilization, and tremor reduction43. Precise three-dimensional visualization is aided by binocular and magnified endoscopic vision. Trans-oral microsurgery offers a different philosophy which was initially popularized by Steiner44, with some centers reproducing excellent results with the technique45. A bespoke tumor resection is performed in which the deep margin is assessed using the microscope and tissue characteristics during resection. The tumor may be carefully divided to allow removal and resection in different sections. Careful histopathological analysis is required to understand where the true margin of the tumor is.Trans-oral surgery has emerged as an approach that offers an alternative to open surgery and primary non-surgical treatments and is being used in the staging/diagnosis of unknown primary patients47. TORS has also shown promising functional outcomes with appropriate adjuvant therapy48. A multi-institutional study of TORS has recently reported a 3-year survival rate of 92.5% and a 3-year recurrence rate of 88.8%49.Trans-oral laser microsurgery (TLM) and TORS have demonstrated excellent oncological results for many indications and subsites, mostly in single institutional studies and oropharyngeal disease50. However, there are currently 16 trials exploring the use of these drugs in the primary setting with curative intent.The most recent development in the treatment of head and neck cancer is immunotherapy. Significant progress has been made in the application of immune checkpoint inhibitors such as nivolumab, pembrolizumab, durvalumab, atezolizumab, and avelumab. The checkpoint inhibitors pembrolizumab and nivolumab are FDA approved in the recurrent and metastatic setting and have an established paradigm for use. The majority of current studies are assessing drugs in the end-stage setting and also using a combination of treatments51.A majority of these drugs act on the PD1 (programmed cell death protein)/PDL1 axis. This is applicable to head and neck and OPSCC because the oropharynx is known to be an immune-privileged site. The reticulated epithelium is known to express the immune checkpoint ligand PDL1, and the resulting reduction in cytotoxic T cell response has been linked to persistent HPV infection at these sites35, the recommended treatment options are still the same, unless the patient is on a trial. Treatment is decided using the Tumor, Node, Metastasis (TNM) stage, the patient’s preferences, the patient’s co-morbidities, and the physician’s experience26. For loco-regionally advanced oropharyngeal cancer, dual modality treatment with either trans-oral surgery and post-operative radiotherapy with or without post-operative chemotherapy or concurrent chemoradiotherapy is usually offered. In cancers of the tonsil and base of the tongue, chemoradiotherapy is used more frequently53. Early stage disease can be treated with single modality treatment, such as surgery or radiotherapy alone.Despite major differences in the risk factors, demographics, clinical behavior, response to treatment, and molecular patterns of HPV-positive compared to HPV-negative tumors54 and have provided impetus and enthusiasm for trans-oral resection as a primary treatment for these tumors55. There are no randomized trials comparing trans-oral approaches versus IMRT, and the majority of the literature is limited to uncontrolled reports56. Comparable oncologic outcomes with TORS compared to IMRT have been reported in these studies and functional outcomesmay be superior. However, current follow-up is relatively short, and the TORS studies include patients with earlier-stage OPSCC on average compared to comparable IMRT studies.The long-term toxicities following chemoradiotherapy have been questioned57. Trans-oral laser surgery (TLS) was first popularized by Steiner in Germany44. There has been increasing experience with TLS, but its use for oropharyngeal tumors has been limited to a few high-volume centers in the USA58 and European units in the UK, France, and Germany57.The use of surgery in HPV-positive oropharyngeal cancer and the application of minimally invasive techniques to avoid or reduce required doses of adjuvant treatment have become important areas of studyDe-escalation based on surgical resection and the neck stage is being assessed in HPV-positive tumors In the Sinai Robotic Surgery Trial (NCT02072148). Another study, conducted at the University of Pennsylvania, is employing robotic surgery to de-escalate adjuvant treatment. Reduced treatment to the primary tumor bed in fully resected tumors (NCT02225496) is being investigated. The Eastern Cooperative Oncology Group (ECOG) 3311 study is a large, randomized, multicenter study comparing normal-dose post-operative radiation with low-dose treatment (NCT01898494) following minimally invasive surgery; patients are stratified post-operatively, with low-risk patients being observed, high-risk patients receiving chemoradiotherapy, and intermediate-risk patients being randomized to normal or low standard-dose radiotherapy.The PATHOS trial (Post-operative Adjuvant Treatment for HPV-positive Tumors) is a UK-based multicenter randomized trial. It is comparing post-operative treatment for HPV-positive disease. With pathological information from the surgical resection, patients will be classified as low, intermediate, or high risk. Patients who are low risk will be observed without adjuvant treatment. Patients who are intermediate and high risk will be randomized to an adjuvant treatment. The intermediate-risk group will be randomized to either high-dose or low-dose radiation and the high-risk group to standard radiotherapy either with or without chemotherapy (NCT02215265).The ADEPT trial  examined patients with fully excised HPV-positive tumors and randomized patients to either radiotherapy or radiotherapy plus cisplatin (NCT01687413). It is now closed to accrual. The use of post-operative docetaxel with hyper-fractioned IMRT is being investigated in another trial following minimally invasive surgery (NCT01932697). In the Canadian ORATOR trial, a phase II randomized trial, patients with early stage OPSCC were randomized to receive radiotherapy or trans-oral robotic surgery with neck dissection (NCT01590355). In this “best of” randomized study, patient-reported swallowing function over the first year will be investigated following allocation to either IMRT or TORS in patients with early stage OPSCC (NCT02984410).There are a number of other trials that are using modification of the standard radiotherapy or chemotherapy techniques. In HPV-positive oropharyngeal tumors, cisplatin alternatives are being trialed, as they are thought to have less toxicity. With concurrent radiation, epidermal growth factor receptor (EGFR) therapies are being investigated. Cetuximab is the most common agent. It is a monoclonal antibody that targets the EGFR extracellular ligand-binding domain on the cell surface.The Radiation Therapy Oncology Group (RTOG) are conducting a randomized trial of cisplatin versus cetuximab with radiation (NCT01302834). De-ESCALaTE (Determination of Cetuximab Versus Cisplatin Early and Late Toxicity Events) is a UK trial comparing either cisplatin or cetuximab with radiation and using toxicity as a primary outcome (NCT01874171). Also, the Australian Trans-Tasman Radiation Oncology Group (TROG) have a trial comparing cetuximab to cisplatin with radiotherapy (NCT01855451). An additional study is investigating cetuximab with pre- and post-treatment biopsies, which will then be compared to a historical series of cisplatin-treated patients (NCT01663259).Reducing or modulating the radiation dose following induction chemotherapy is another strategy. In a RTOG trial, depending on how patients respond to induction chemotherapy with paclitaxel, cisplatin, and cetuximab, cetuximab combined with either low- or standard-dose IMRT will be administered (NCT01084083). The Quarterback Trial (NCT01706939) compares a decreased radiation dose with weekly carboplatin to a standard radiation regimen with weekly carboplatin following induction chemotherapy in patients with a good response. Also, the OPTIMA trial is investigating de-intensifying treatment based on response to induction chemotherapy. Using nab-paclitaxel and carboplatin as an induction regimen, the investigators will administer a response-based therapy. Chemotherapy with high- or low-dose radiation or just radiation alone is being investigated in stage III or IV HPV-related OPSCC (NCT02258659). Paclitaxel and carboplatin used as an induction therapy with concomitant paclitaxel in tumors of HPV-positive patients is another regimen under investigation (NCT02048020).In HPV-positive patients, reducing the radiation dose is another approach. Reduction of the dose of IMRT to 54–60 Gy while simultaneously administering weekly intravenous cisplatin will precede surgical resection of any clinically apparent residual tumor or neck disease (NCT01530997).A randomized trial of HPV-positive oropharynx tumors using a reduced dose of IMRT treatment with randomization to radiotherapy alone or concomitant cisplatin presents an additional approach (NCT02254278). Treatment de-intensification is being investigated in another study alongside cisplatin chemotherapy with a reduced radiation dose, with the experimental arm’s dose decreasing from 70 Gy to 63 Gy (NCT01088802).There are other new agents and immune therapies currently being trialed. Ribavirin is being evaluated as part of a phase I trial. It is a drug that is used in the treatment of hepatitis C, and it is being used with afatinib (a tyrosine kinase inhibitor) as an induction chemotherapy agent with weekly carboplatin/paclitaxel dose (NCT01721525).59.Immune therapy and vaccines are thought to offer an additional modality of treatment because foreign viral antigens could be amenable to targeted therapy. The local presence of HPV16-specific T cell immunity found in HPV16-induced SCC suggests a basis for this60, and it is now being investigated in the head and neck.The use of T cells as an autologous transfusion is a process termed adoptive immunotherapy. Other highly immunogenic tumors such as melanoma have responded to this type of therapyListeria monocytogenes is the base for a vaccine that has been modified to express HPV16 targets . This trial was closed early.Antitumor vaccines are a different immune-modulating therapy aiming to stimulate a host’s immune system in the treatment of cancer. In a phase I trial, recombinantIn contrast to efforts to de-escalate treatment of HPV-positive low-risk tumors, HPV-negative tumors and some HPV-positive tumors in patients with a smoking and alcohol history are deemed high risk. In these patients, there may be benefit in escalating treatment to improve outcomes. In the COMPARE trial (UKCRN Study ID: 18621), additional treatments in conjunction with standard chemoradiotherapy are being investigated compared to standard chemoradiotherapy. Patients are randomized to either the standard or receive surgery followed by chemoradiotherapy, more chemotherapy as induction , a higher dose of radiotherapy as part of their chemoradiotherapy, or durvalumab with chemoradiotherapy.53. The detection of HPV E6/E7 mRNA transcripts correlates with cellular genotoxic damage and gene expression changes that are the hallmarks of cancer. However, the detection of mRNA in the clinical setting is difficult and expensive61. Another approach is using p16 as a surrogate for HPV infection and could utilize the cheaper and more available immunohistochemistry stains. However, when a number of these different assays were tested, only the RT-PCR assay for HPV16 E6*I mRNA developed specifically for formalin-fixed paraffin-embedded (FFPE) material was able to accurately classify samples62. Using the different biomarkers for prediction of outcome, the doubly positive p16/HPV DNA test had the best predictive ability, with p16-positive/HPV-negative tumors having a prognosis closer to more typical non-HPV-related tumors33. Therefore, it has been proposed that the combination of p16 immunohistochemistry and the detection of HPV DNA by PCR is required63. Other testing methods for HPV infection involve the use of saliva samples from swish and spit specimens64. This may provide a potential screening test for OPSCC or may replace the need for formal biopsy65.There has been controversy regarding the different techniques and biomarkers used to determine whether a tumor is related to HPV infection. Sustained and persistent high-risk HPV E6/E7 viral oncogene expression is essential for a HPV-driven malignant tumorAnother recent change has been the introduction of an updated staging system for OPSCC. The eighth edition of the Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) TNM system was introduced into clinical practice in January 2018. However, the current UK policy is to not use the TNM eighth edition for treatment allocation. The data used to revise the system are largely retrospective and therefore treatment de-intensification should currently only occur in the context of an appropriate clinical trial.66. In the T staging category, the Tis and T4b stages have been removed from HPV-related tumors. There has been the introduction of separate clinical and pathological N staging systems. The clinical N staging has been simplified, with the N1 stage now including all patients with ipsilateral nodes less than 6 cm. This will downstage patients who previously had T2b neck disease in the seventh edition. N2 disease includes patients with contralateral or bilateral disease, and N3 is for patients with a neck mass of more than 6 cm. In the pathological staging system, N1 disease is fewer than four positive nodes, N2 is more than four positive nodes, and N3 has been removed.OPSCC has been divided into p16-positive, high-risk HPV-associated tumors and p16-negative tumorsOPSCC is an evolving field. For many years, surgery and radiation therapy were the only options for treatment in the majority of cases. While this is still true, a number of new and exciting options are becoming available. Advances in radiotherapy techniques and the introduction of IMRT allow more accurate dose delivery to the tumor while sparing surrounding structures to minimize side effects. Improvements in our understanding of the biology of HPV-related disease have led to attempts to de-intensify treatment regimens to further reduce the morbidity of therapy in low-risk patients. Surgical techniques have incorporated state-of-the-art technology, such as new robotic and laser systems. These advances offer the potential to reduce the long-term morbidity associated with chemoradiation and may also play a role in the intensification of treatment for the highest-risk HPV-negative patients. There is also a whole new modality, immune therapy, with new drugs receiving FDA approval in the setting of recurrent/metastatic head and neck cancers. It seems likely that, over the next decade, we will see a move away from standard chemoradiotherapy techniques to a more individualized approach based on the patient and tumor factors in an attempt to optimize oncological outcome while minimizing morbidity for a growing number of patients with OPSCC. Other changes have emerged as our understanding of HPV-related tumors has improved. This is particularly shown with the introduction of the new TNM eighth edition staging system.


The recent development of autonomous oceanographic observation platforms provides an opportunity to bridge this gap. Here, we use satellite-derived surface chlorophyll-a (Chl-a) observations, in conjunction with a Biogeochemical-Argo dataset, to assess the capability of remote sensing to estimate phytoplankton phenology metrics in the northern Red Sea – a typical tropical marine ecosystem. We find that phenology metrics derived from both contemporary platforms match with a high degree of precision (within the same 5-day period). The remotely-sensed surface signatures reflect the overall water column dynamics and successfully capture Chl-a variability related to convective mixing. Our findings offer important insights into the capability of remote sensing for monitoring food availability in tropical marine ecosystems, and support the use of satellite-derived phenology as an ecosystem indicator for marine management strategies in regions with limited data availability.The timing of phytoplankton growth (phenology) in tropical oceans is a crucial factor influencing the survival rates of higher trophic levels, food web structure and the functioning of coral reef ecosystems. Phytoplankton phenology is thus categorised as an ‘ecosystem indicator’, which can be utilised to assess ecosystem health in response to environmental and climatic perturbations. Ocean-colour remote sensing is currently the only technique providing global, long-term, synoptic estimates of phenology. However, due to limited available  This translates to economic support, services and well-being for maritime nations via fisheries and tourism5. Phenology characterises the timing of phytoplankton growth periods and is an integral component controlling the structure of marine food webs and marine ecosystem functioning7. Alterations to phytoplankton phenology may influence the survival of higher trophic levels due to variations in the timing of food availability10. Thus, monitoring phenology at seasonal and interannual timescales is necessary for the establishment of management strategies in tropical oceans and associated coral reef ecosystems. Phenology metrics, including the timing of phytoplankton growth initiation, maximum amplitude, termination and duration, are referred to as ‘ecological indicators’, representing objective and quantitative measurements that can be utilised to evaluate the condition of marine ecosystems and their response to environmental change14.In tropical oceans, phytoplankton constitute a direct food source for coral reef fauna and pelagic larvae15. However, remotely-sensed Chl-a observations are representative of the surface oceanic layer , rather than being indicative of the complete vertical phytoplankton distribution within the water column. In particular, stratified tropical ecosystems are characterised by the presence of Subsurface Chl-a Maxima (SCM) that cannot be detected by satellites. To date, attempts to validate satellite-based estimates of phytoplankton phenology with in situ measurements remain sparse, primarily due to the lack of continuous, spatially extensive observations16. The aforesaid limitations of satellite-derived datasets may discourage researchers from utilising remotely-sensed information in ecosystem management schemes. Oceanographic multi-platforms could bridge this gap and provide the necessary information needed to assess the potential of satellite remote sensing in retrieving phenology indices, and also, enable a more holistic quantification of phenology over the whole water column.Ocean-colour remote sensing is currently the only method providing continuous, long-term (~20 years), synoptic time series of phytoplankton abundance (indexed by chlorophyll-a [Chl-a] concentration), from which phytoplankton phenology metrics can be computedAdopting an innovative approach, we synergistically utilise satellite-derived Chl-a observations with data from an autonomous Biogeochemical-Argo float (BGC-Argo float) to evaluate (1) the capability of remote-sensing data to estimate phytoplankton phenology metrics in a typical tropical marine ecosystem – the northern Red Sea; and (2) extend the phenological analysis to the part of the upper water column that is not seen by satellites. We corroborate surface signatures detected by satellites by investigating the physical mechanisms that control vertical phytoplankton dynamics.Sat-Surf] and Chl[Argo-Surf] respectively, whilst Chl[Argo-Int] refers to the time series of integrated BGC-Argo Chl-a over the euphotic depth . First, it is worth noting that Chl[Sat-Surf] exhibits a significant correlation with Chl[Argo-Surf] and Chl[Argo-Int] , highlighting the strong coherence between the BGC-Argo and satellite datasets. Phenology metrics derived from the two datasets match remarkably well , in contrast to Chl[Argo-Int] which is ~2–3 weeks longer.To evaluate the capability of satellite-derived Chl-a observations for the computation of phytoplankton phenology, we directly compare phenology metrics computed using satellite (OC-CCI) and BGC-Argo Chl-a datasets in the Red Sea Fig. . We refeell Fig. . Figure To elucidate how satellite-derived surface Chl-a seasonality relates to the vertical dynamics of the water column, we present seasonal time series of the three Chl-a datasets, alongside bi-monthly averages of BGC-Argo Chl-a concentration and density profiles Fig. .Figure 2Sat-Surf] exhibits similar patterns of variability to the BGC-Argo time series (Chl[Argo-Surf] and Chl[Argo-Int]). Three distinct phases of the Chl-a seasonal succession are observed. First, a period of low, but increasing, Chl-a concentrations occurs in autumn (October–November), coinciding with the observed timing of phytoplankton growth initiation for the Chl[Argo-Surf] and Chl[Sat-Surf] time series , and the presence of a small pycnocline, coinciding with an overall decrease in surface density , temperature, and the Brunt–Väisälä Frequency , the deepening of the MLD to depths of 100–250 metres, and the presence of oxygenated waters  is consistent with estimates attained using an 24. In this respect, biological dynamics in the northern Red Sea follow a regime that is analogous to what is typically observed in other tropical marine ecosystems, where the re-distribution of nutrients from the deeper layers is the dominant factor controlling phytoplankton growth25. The enhancement of Chl-a captured by satellite sensors in early November  exhibit a ‘modified SCM’ shape in early winter, when Chl-a concentrations are higher in the surface layer and peak just below the base of the mixed layer  is reflected by an enhancement in both surface and integrated BGC-Argo Chl-a  within the mixed layer, is well-represented by the Chl[Sat-Surf] time series  in mid February . Satellite-derived surface phenology successfully captures the commencement and culmination of convection-driven vertical mixing, which is the predominant mechanism affecting nutrient availability in the northern Red Sea. Although previous studies have demonstrated the importance of float-based measurements for the validation of satellite ocean-colour products in subtropical waters36, to our knowledge, this study comprises the first float-based assessment highlighting the capability of satellite sensors for retrieving phytoplankton phenology in a tropical marine ecosystem.Using a biogeochemical dataset, acquired by an autonomous BGC-Argo float deployed in a relatively unexplored tropical ecosystem – the Red Sea – we demonstrate that remotely sensed surface phenology matches very closely with phenology metrics derived from an in situ sampling efforts are sparse or not possible, satellite-derived phytoplankton phenology is likely to become a fundamental factor influencing the effective design and implementation of future ecosystem management strategies.The timing of food availability in tropical marine ecosystems may be altered under future scenarios of climate warming, potentially having far-reaching impacts on higher trophic levels, reef-dwelling organisms, and coastal fisheries that are an invaluable economic resource in tropical regions. With the consideration that there are presently two decades of satellite data available, the ability to now retrieve representative estimates of surface phenology synoptically from space, at interannual timescales, is paramount for monitoring how tropical ecosystems and their associated coral reef habitats respond to global climate change. Additionally, in data-limited regions where http://argo.jcommops.org) equipped with biogeochemical and bio-optical sensors was deployed in the northern Red Sea in September 2015 at 33.73°N and 27.66°E. The float acquired vertical profiles of biogeochemical and optical parameters during its vertical ascent from a maximum parking depth of 1000 m. It was programmed to surface at noon, over time periods varying from 1, 2, 5 or 10 days, depending on the mission’s specifications. Based on the float’s sampling track, a total of 139 vertical profiles were analysed between September 30th 2015 and September 27th 2016, spanning a latitudinal range of approximately 4° . Briefly, vertical profiles of Chl-a were adjusted for non-zero deep values, and corrected for non-photochemical quenching using an empirical method for shallow-mixing waters according to Xing et al.42. The uncertainties regarding this method (XB18) are provided in their Table 2. Following Roesler et al.43, the community-established calibration bias of 2 for the WET Labs ECO fluorescence sensor was applied to in situ fluorometric Chl-a measurements. After processing, we corroborated Chl-a data by comparing the first vertical profile acquired during the float’s deployment, with a nearby profile of Chl-a concentration obtained by a CTD cast taken on the day before the BGC-Argo deployment . The CTD Chl-a measurements were obtained using a similar type of WET Labs ECO Puck Triplet sensor, which was calibrated using High Performance Liquid Chromatography (HPLC) measurements acquired from multiple cruises conducted across the Red Sea44. Visual comparison of the two profiles revealed that they were highly similar with regards to their range of Chl-a concentrations, shape and magnitude, providing us with confidence that Chl-a concentrations measured by the BGC-Argo float are representative of the region (figure not shown). DO measurements were corrected by applying a factor of 1.06 to each profile based on the comparison between the surface percent oxygen saturation values and those from the World Ocean Atlas climatology45.Measurements of pressure, temperature and salinity were obtained via a Seabird standard conductivity-temperature-depth profiler . Dissolved Oxygen (DO) concentrations were determined using an Aanderaa Optode sensor (model 4330). Vertical profiles of Chl-a fluorescence were acquired using a WET Labs ECO Puck Triplet sensor, whilst radiometric measurements, including Photosynthetically Available Radiation (PAR), were acquired by an OC4 Satlantic radiometer. Following standard procedures for Argo data managementArgo-Surf]) were computed by averaging Chl-a data over the first optical depth. The first optical depth was computed as the euphotic depth , divided by 4.647. Integrated Chl-a values (Chl[Argo-Int]) were calculated by integrating Chl-a between the surface and the euphotic depth of each profile. This depth was chosen to represent phytoplankton biomass situated within the epipelagic, photic zone. The Mixed Layer Depth (MLD) was computed using the threshold method with a density gradient criterion of 0.03 kg m−3, compared to the density at 10 m48. To evaluate the level of stratification within the water column , Moderate Resolution Imaging Spectroradiometer (MODIS), Medium Resolution Imaging Spectrometer (MERIS) and Visible Infrared Imaging Radiometer Suite (VIIRS) satellite sensors. Level 3, daily, mapped Chl-a data were acquired at a spatial resolution of 4 km from http://www.esa-oceancolour-cci.org. Brewin et al.53 and Racault et al.28 have shown that both standard ocean-colour algorithms and the OC-CCI algorithm perform relatively well in the Red Sea, supporting the use of satellite-derived Chl-a datasets. Previous studies in the Red Sea have also demonstrated that the OC-CCI product is characterised by significantly higher data availability in comparison to single-sensor-based missions28. Thus, we believe that the OC-CCI dataset is an optimum choice for phenological analysis. We refer the reader to the OC-CCI Product User Guide at http://www.esa-oceancolour-cci.org/?q=webfm_send/318 for a more extensive overview of data processing, sensor merging and uncertainty quantification.Version 3.1 of the European Space Agency’s Ocean Colour Climate Change Initiative (ESA OC-CCI)Sat-Surf]) were temporally matched to BGC-Argo data based on the BGC-Argo sampling date. For the phenology analysis, 5-day composites were calculated to reduce the number of missing data and increase the matchups available to compare phenological metrics estimated using Bio-Argo and satellite datasets. Spatial matchups were acquired by locating the closest 4 km pixel (nearest latitude and longitude) to the BGC-Argo sampling location, and computing the average of 3 pixels longitudinally, centred on that 4 km pixel .Satellite Chl-a data  Chl-a time-series as an input, otherwise the phenology metrics cannot be calculated. Hence, to further improve the coverage of Chl-a satellite data, we applied a linear interpolation method that fills gaps in the time series. The interpolation method used is based on the MATLAB subroutine inpaint_nans, which interpolates missing data using a linear least squares approach57.Following the approach published in 59. Using this threshold, Chl-a anomalies were computed by subtracting the threshold criterion from the time series. The cumulative sums of anomalies were then calculated. Increasing (decreasing) trends in the cumulative sums of anomalies represent periods when Chl-a concentrations are above (below) the threshold criterion. The gradient of the cumulative sums of anomalies was then used to identify the timing of the transition between increasing and decreasing trends28. The initiation of the main phytoplankton growth period corresponded to the 5-day period when Chl-a concentrations first rose above the threshold criterion (i.e. when the gradient of the time series first changed sign). The termination of the main phytoplankton growth period was computed as the time when the gradient first changed sign following the occurrence of the maximum Chl-a concentration in the time series (the growth peak). The total duration of the main phytoplankton growth period was calculated as the number of 5-day periods between the timings of initiation and termination.Next, we defined the threshold criterion as the median of the time series plus 5%60. However, such features are usually observed in the southern-central Red Sea, as opposed to the northernmost region, which tends to be more convection-dependant 62. Overall, our results infer that the BGC-Argo float generally captured the seasonal convective mixing that characterises the region.Although not accounted for in the analysis of this study, we note that the transportation of water masses via eddies and surface currents in the northern Red Sea may influence Chl-a concentrations via horizontal advective processes that act to redistribute material towards or away from the BGC-Argo profiling sitebp700) averaged over the first optical depth and the first euphotic depth, generally increases in conjunction with integrated and surface Chl-a concentrations (figure not shown). Thus, although photoacclimation likely exerts some influence on Chl-a concentrations under conditions of reduced light availability, we acknowledge that there is an overall increase in the concentration of particulate matter within the water column during the main phytoplankton growth period, indicating that there is a net increase in Chl-a associated with new production.We also note that variations in Chl-a concentration are not always associated with changes in biomass, but may result from fluctuations in the concentration of intracellular pigments as a result of photoacclimation processes. As the concentration of Chl-a is not a perfect proxy of phytoplankton biomass, we acknowledge that photoacclimation to low light levels, particularly during MLD deepening events, could impact our analysis. We have investigated the potential impact of photoacclimation using measurements of particulate backscattering acquired by the BGC-Argo float. Particulate backscattering at 700 nm (bFinally, the choice of depth used for computing integrated Chl-a concentrations could potentially influence our analysis, particularly if we consider the fact that the SCM may occasionally reach depths that extend below the euphotic zone. In order to capture Chl-a variability in the deeper layers and verify that the winter increase in Chl-a concentrations observed at the surface is in fact new production associated with enhanced nutrient supply from convective mixing, we produced a time series of Chl-a concentrations integrated between 100 and 200 metres (Supplementary Fig. Supplementary Material


Most malaria-endemic countries use artemisinin-based combination therapy (ACT) as their first-line treatment. ACTs are known to be highly effective on asexual stages of the malaria parasite. Malaria transmission and the spread of resistant parasites depend on the infectivity of gametocytes. The effect of the current ACT regimens on gametocyte infectivity is unclear.Anopheles gambiae following ACT treatment in the field.This study aimed to determine the infectivity of gametocytes to During a randomised controlled trial in Bougoula-Hameau, Mali, conducted from July 2005 to July 2007, volunteers with uncomplicated malaria were randomised to receive artemether-lumefantrine, artesunate-amodiaquine, or artesunate-sulfadoxine/pyrimethamine. Volunteers were followed for 28 days, and gametocyte carriage was assessed. Direct skin feeding assays were performed on gametocyte carriers before and after ACT administration.Anopheles gambiae sensu lato (s.l.) (p < 10−4), whereas artesunate-sulfadoxine/pyrimethamine decreased gametocyte infectivity in this setting (p = 0.03).Following artemether-lumefantrine treatment, gametocyte carriage decreased steadily from Day 0 to Day 21 post-treatment initiation. In contrast, for the artesunate-amodiaquine and artesunate-sulfadoxine/pyrimethamine arms, gametocyte carriage increased on Day 3 and remained constant until Day 7 before decreasing afterward. Mosquito feeding assays showed that artemether-lumefantrine and artesunate-amodiaquine significantly increased gametocyte infectivity to Anopheles vector. Frequent assessment of the effect of antimalarials on gametocytogenesis and gametocyte infectivity may be required for the full assessment of treatment efficacy, the potential for spread of drug resistance and malaria transmission in the field.Different ACT regimens could lead to gametocyte populations with different capacity to infect the  Gametocyte development and viability are essential for the perpetuation of Plasmodium life cycle by enabling both transmission from the human host to the mosquito vector3 and the spread of resistant parasites.Malaria is still a major public health problem in numerous parts of the world. Malaria still affects 216 000 million individuals each year with 445 000 deaths worldwide.Plasmodium gametocyte development within the human host is a tedious process involving the differentiation from asexual to sexual forms to accommodate metabolic requirements, environmental changes and sexual reproduction.5Plasmodium gametocytes are conventionally classified into five distinct stages (stages I–V) but only the immature stage I gametocytes and the mature stage V gametocytes are detectable in the peripheral blood of a malaria-infected patient.7 The other stages  are sequestered in the bone marrow and possibly other internal organs.9 Gametocytes do not cause any symptoms in the infected human host, but the presence of competent circulating gametocytes and their duration in the bloodstream, which varies from 3 to 4 weeks,10 are directly responsible for malaria parasite transmission to the Anopheles vector.11 However, gametocytogenesis and gametocyte transmission to the mosquito vector constitute a population bottleneck in the Plasmodium life cycle as only a minute number of parasites enter the mosquito bloodmeal and gut.12 Monitoring the density and infectiousness of circulating gametocytes is necessary for a better assessment of malaria transmission in endemic areas.15 Several clinical and in vitro studies reveal that most antimalarial drugs currently in use can promote or impair gametocytogenesis and to some extent affect sexual reproduction within the mosquito vector.16 The 8-aminoquinoline primaquine is presently the only clinically used antimalarial drug displaying potent activity against all Plasmodium species and gametocyte stages,16 but its side effects on glucose-6-phosphate dehydrogenase-deficient individuals hinder its use in large-scale elimination strategies.18 The 4-aminoquinoline chloroquine was shown to increase the production of fully competent gametocytes both in vitro and in vivo.20 In contrast atovaquone, artemisinin and the antifolates (sulfadoxine and pyrimethamine) have been shown to impair gametocyte development and infectivity.24 The gametocyte developmental stages affected by antimalarial drugs is poorly understood. The antimalarial treatment represents a stress factor that triggers differentiation of the asexual form into the gametocytes.26 This process may be more prevalent with drug-resistant parasites compared to sensitive ones.27 Hence, the selective pressure exerted by the antimalarial drugs on the parasite may contribute to the spread of resistant parasites through the development and transmission of drug-resistant gametocytes.28 Therefore it becomes essential to assess the emergence of resistant strains and the impact of treatment on gametocytogenesis and gametocyte infectivity.Gametocyte development within the human host is influenced by various factors, including host and parasite genetic factors, immune response, mosquitoes’ gut microbiota and the exposure to antimalarial drugs.29 with the expected benefit to reduce gametocyte carriage.30 Indeed, the fast killing action of artemisinin and derivatives on asexual parasites results in the decrease of circulating stage V gametocytes.30 Artesunate has been reported to reduce post-treatment transmission of gametocyte to Anopheles mosquitoes but does not abolish gametocyte infectivity entirely.31Artemisinin-based combination therapies (ACT) are recommended in most malaria-endemic countriesin vivo assessment of the impact of artemether-lumefantrine, artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine on circulating gametocytes density and their infectivity.With the increased interest in malaria elimination, understanding the impact of ACT regimens and other antimalarial drugs with different pharmacodynamic properties on gametocyte development and transmission becomes a key issue. Here, we report an The protocol  was reviewed and approved by the ethical committee of the Faculty of Medicine, Pharmacy, and Dentistry, University of Bamako (No 05-20 dated 22 June 2005). Each participant  gave fully informed written consent before enrolment.Plasmodium falciparum is hyperendemic with seasonal peaks in this village. Parasitemia prevalence rates range from 40% to 50% during the dry season (January–April) and 70% to 85% during the rainy season (May–December).32 Approximately 10% – 20% of the local population are gametocyte positive with an average gametocyte density of 23 gametocytes/µl.33 The main malaria vectors in the Sikasso region are Anopheles gambiae and Anopheles funestus with a sporozoite rate of 6.4% at the end of the rainy season and an entomological inoculation rate (EIR) of 0.032 infected bites per person per night.34This study was conducted in Bougoula-Hameau, a peri-urban village of approximately 7000 people located near the city of Sikasso in southern Mali. ®, Novartis, Basel, Switzerland), artesunate + amodiaquine , and artesunate  + sulfadoxine-pyrimethamine .35 Briefly, patients aged 6 months and above were enrolled in the clinical study if they satisfied the following inclusion criteria: weighed ≥ 35 kg, resided in the study village, were able to receive oral treatment, had an axillary temperature ≥ 37.5°C, and had Plasmodium sp. infection with a parasite density between 2,000 and 200,000 asexual forms per microlitre of blood. As described previously by Sagara et al.,35 780 volunteers were enrolled and 260 were randomly assigned to one of the three treatment arms and drug efficacy was assessed according to the World Health Organization 2003 protocols.36 Briefly, blood samples were collected on Days 1, 2, 3, 7, 14, 21, 28 and any day of recurrent illness. Smears were made, Giemsa-stained and read on site for asexual parasites and gametocytes quantification. Parasite count was performed against 300 leucocytes and gametocyte count was performed against 1000 leucocytes.This is a sub-study of a previously published randomised controlled clinical trial, which was conducted from July 2005 to July 2007 to compare the efficacy of three ACT regimens: artemether + lumefantrine  reared in the insectaries were used. Up to 60 F1 female mosquitoes were starved for about 12 h and held in 2 small screened-cups containing 30 mosquitoes each. They were then allowed to blood-feed for 5–10 min on the leg of a 6–18 years old consenting volunteer.Wild female After feeding, unfed mosquitoes were removed the same day, and only fed mosquitoes were kept in the insectaries as described above. 8 days post feeding, a group of at least 15 mosquitoes per carrier were dissected, their midguts pulled out in 0.5% mercurochrome for oocyst detection and quantification. Oocyst numbers, abdomen stages, date and other observations were recorded on data sheets. Oocysts were counted under light microscopes 10 times for each mosquito.For the analysis of the direct skin feeding experiment, feeding experiments for Days 3–28 were lumped together in each treatment arm. To calculate the oocyst prevalence, for each treatment arm, the number of positive mosquitoes was divided by the total number of mosquitoes dissected for that treatment arm). The oocyst positivity for each ACT regimen was compared to that of the control group using the chi-square test. All data were analysed and reported using Stata software version 14.0 .p = 0.005) and from 7.0% to 9.8% in the artesunate-sulfadoxine-pyrimethamine arm (p = 0.02). Overall, the prevalence of gametocyte carriage in the artemether-lumefantrine arm steadily decreased from Day 0 to Day 28, while for the artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine arms the prevalence of gametocyte carriage increased from Day 0 to Day 7 before declining    (The oocyst positivity rate in the control group was 11.7% (82/698) . For vol < 10−4) . Likewis < 10−4) . In cont = 0.03) .Plasmodium falciparum gametocyte carriage, density and infectivity to Anopheles gambiae s.l. following artemether-lumefantrine, artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine treatment. We measured P. falciparum gametocyte density of consenting volunteers suffering from acute uncomplicated malaria and gametocyte infectivity to Anopheles mosquitoes before and after ACT administration. From the direct skin feeding experiments of this study, we show that post-artemether-lumefantrine and post-artesunate-amodiaquine treatment gametocytes are more infectious to Anopheles gambiae s.l. than post-artesunate-sulfadoxine-pyrimethamine treatment gametocytes or non-drug treated controls. Since artemisinin derivatives, including artesunate and artemether, are rapidly metabolised in vivo into dihydroartemisinin, the observed differences in infectivity could be dependent on the partner drugs . However, one cannot rule out the effect of differences in artemisinin derivative pharmacokinetics and pharmacodynamics between regimens. Co-exposure to artemisinin derivative and the partner drugs albeit for a short period could impact gametocyte biology differently from any of the individual drugs. In addition, drug administration timing differs between artemether-lumefantrine and artesunate-amodiaquine or artesunate-sulfadoxine-pyrimethamine. While artemether-lumefantrine was given twice daily for 3 days, artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine were administered once daily for 3 days. A number of studies have investigated the effect of antimalarial drugs on gametocyte density and infectivity.31 Beavogui et al. revealed that sulfadoxine-pyrimethamine considerably increased gametocyte carriage in the population but the infectivity of these circulating gametocytes was very low.22 Further investigation by Kone et al. showed that the low infectivity following sulfadoxine-pyrimethamine treatment was due to pyrimethamine, which is known to prevent male gametocyte exflagellation.23 In addition, 4-aminoquinoline chloroquine, which is chemically related to amodiaquine, has been shown to induce the production of fully infective gametocytes both in vitro and in vivo.16 These studies support the observed decrease of infectivity with artesunate-sulfadoxine-pyrimethamine treatment and the increase of infectivity with artesunate-amodiaquine seen in this study. Although gametocyte infectivity was different between the tested ACT regimens, the small number of carriers tested may be a limitation of this study. Additional infectivity studies with higher numbers of gametocyte carriers need to be conducted to further investigate these findings. In addition, the mechanisms involved in antimalarial drugs and gametocyte biology ought to be thoroughly studied.The goal of this study was to determine the impact of different ACT regimens on 35 Baseline gametocyte carriage was significantly higher in the artemether-lumefantrine arm compared to both artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine. That observation may have occurred by chance as the study was a randomised controlled trial; therefore this could not be attributed to a selection bias.35 There was no difference in gametocyte density between the treatment arms at baseline. Gametocyte carriage significantly decreased in all treatment arms between Day 0 and Day 28. This result confirms numerous previous findings.30 However, while gametocyte prevalence in the artemether-lumefantrine arm decreased steadily from Day 0 to Day 28, there was a significant increase in the artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine arms from baseline to Day 3 with a plateau until Day 7. This rise after ACT administration corroborates previous work39 and could be a consequence of the stress induced by the treatment.41 These data suggest that artemether-lumefantrine may affect gametocytogenesis differently from artesunate-amodiaquine or artesunate-sulfadoxine-pyrimethamine.43 Conversely, the dosing schedule of artemether-lumefantrine, which is taken every 8 h instead of once daily as for artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine, could also play a role in the above observations.To investigate gametocyte dynamics in the peripheral blood, gametocytes carriers were included in this study.30 However, the few that manage to differentiate into gametocytes appear to be well fit for infecting the mosquitoes, hence their increased infectivity. Conversely, sulfadoxine-pyrimethamine sharply increases gametocytes but those gametocytes appear to be less fit for infecting the mosquitoes.22, 23 Pyrimethamine was also shown to hamper sporogonic development of P. falciparum in the Anopheles mosquitoes.44 These could explain the decrease in infectivity of post artesunate-sulfadoxine-pyrimethamine gametocytes.We show that artesunate-amodiaquine and artemether-lumefantrine decreased gametocyte carriage in the treated population while both ACTs increased gametocyte infectivity to the mosquitoes. The effect of these ACTs in reducing gametocyte carriage can be a direct result of their active and fast killing action on asexual forms, leaving them no or little chance to differentiate into gametocytes.47 and gametocyte quantification after purification38 could have yielded higher proportions of gametocyte carriers and gametocyte density.Gametocytes were detected and quantified by light microscopy, which is an important limitation of this study. Thus, gametocyte carriage and gametocyte density are likely to be underestimated with our approach. The use of molecular toolsAnopheles gut microbiota is known to influence the sporogonic cycle15 and some drugs, such as antibiotics, have been shown to change the composition of mosquitoes’ gut resident microbes.48We also cannot exclude the possible modulation of the mosquitoes’ gut microbiota by the ACTs used in this study. The in vivo, and the infectivity of post-treatment gametocytes. Understanding the potential impact of antimalarial drugs on the spread of resistant strains and malaria transmission will require a fine assessment of their effects on gametocyte biology and the mechanisms involved.Antimalarial drugs influence gametocytogenesis and their impact on gametocyte density and viability are likely to differ from one combination therapy regimen to another. Evaluation of transmission potential in malaria-endemic areas requires more studies assessing the influence of current antimalarial treatment on gametocyte development and clearance