Genome and transcriptome studies of Plasmodium nucleic acids obtained from parasitized whole blood are greatly improved by depletion of human DNA or enrichment of parasite DNA prior to next-generation sequencing and microarray hybridization. The most effective method currently used is a two-step procedure to deplete leukocytes: centrifugation using density gradient media followed by filtration through expensive, commercially available columns. This method is not easily implemented in field studies that collect hundreds of samples and simultaneously process samples for multiple laboratory analyses. Inexpensive syringes, hand-packed with CF11 cellulose powder, were recently shown to improve ex vivo cultivation of Plasmodium vivax obtained from parasitized whole blood. This study was undertaken to determine whether CF11 columns could be adapted to isolate Plasmodium falciparum DNA from parasitized whole blood and achieve current quantity and purity requirements for Illumina sequencing.

Equatorial Guinea (EG) has implemented a successful malaria control program on Bioko Island. A highly effective vaccine would be an ideal complement to this effort and could lead to halting transmission and eliminating malaria. Sanaria® PfSPZ Vaccine (Plasmodium falciparum sporozoite Vaccine) is being developed for this purpose. To begin the process of establishing the efficacy of and implementing a PfSPZ Vaccine mass vaccination program in EG, we decided to conduct a series of clinical trials of PfSPZ Vaccine on Bioko Island. Because no clinical trial had ever been conducted in EG, we first successfully established the ethical, regulatory, quality, and clinical foundation for conducting trials. We now report the safety, tolerability, and immunogenicity results of the first clinical trial in the history of the country. Thirty adult males were randomized in the ratio 2:1 to receive three doses of 2.7 × 105 PfSPZ of PfSPZ Vaccine (N = 20) or normal saline placebo (N = 10) by direct venous inoculation at 8-week intervals. The vaccine was safe and well tolerated. Seventy percent, 65%, and 45% of vaccinees developed antibodies to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) by enzyme-linked immunosorbent assay, PfSPZ by automated immunofluorescence assay, and PfSPZ by inhibition of sporozoite invasion assay, respectively. Antibody responses were significantly lower than responses in U.S. adults who received the same dosage regimen, but not significantly different than responses in young adult Malians. Based on these results, a clinical trial enrolling 135 subjects aged 6 months to 65 years has been initiated in EG; it includes PfSPZ Vaccine and first assessment in Africa of PfSPZ-CVac. ClinicalTrials.gov identifier: NCT02418962.

Semiquantitative dipstick tests are utilized for albuminuria screening.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

Controlled human malaria infection (CHMI) by mosquito bite has been used to assess anti-malaria interventions in > 1,500 volunteers since development of methods for infecting mosquitoes by feeding on Plasmodium falciparum (Pf) gametocyte cultures. Such CHMIs have never been used in Africa. Aseptic, purified, cryopreserved Pf sporozoites, PfSPZ Challenge, were used to infect Dutch volunteers by intradermal injection. We conducted a double-blind, placebo-controlled trial to assess safety and infectivity of PfSPZ Challenge in adult male Tanzanians. Volunteers were injected intradermally with 10,000 (N = 12) or 25,000 (N = 12) PfSPZ or normal saline (N = 6). PfSPZ Challenge was well tolerated and safe. Eleven of 12 and 10 of 11 subjects, who received 10,000 and 25,000 PfSPZ respectively, developed parasitemia. In 10,000 versus 25,000 PfSPZ groups geometric mean days from injection to Pf positivity by thick blood film was 15.4 versus 13.5 (P = 0.023). Alpha-thalassemia heterozygosity had no apparent effect on infectivity. PfSPZ Challenge was safe, well tolerated, and infectious.

Epidemiological data about diabetes mellitus (DM) for sub-Saharan Africa (SSA) are scarce and the utility of glycated hemoglobin (HbA1c) to diagnose DM is uncertain in African populations with a high proportion of anemia.

Disrupted development of oropharyngeal structures as well as cranial nerve and brainstem circuits may lead to feeding and swallowing difficulties in children with 22q11. 2 deletion syndrome (22q11DS). We previously demonstrated aspiration-based dysphagia during early postnatal life in the LgDel mouse model of 22q11DS along with disrupted oropharyngeal morphogenesis and divergent differentiation and function of cranial motor and sensory nerves. We now ask whether feeding and swallowing deficits persist in adult LgDel mice using methods analogous to those used in human patients to evaluate feeding and swallowing dysfunction. Compared to wild-type mice, videofluoroscopic swallow study revealed that LgDel mice have altered feeding and swallowing behaviors, including slower lick rates, longer inter-lick intervals, and longer pharyngeal transit times with liquid consistency. Transoral endoscopic assessment identified minor structural anomalies of the palate and larynx in one-third of the LgDel mice examined. Video surveillance of feeding-related behaviors showed that LgDel mice eat and drink more frequently. Furthermore, LgDel animals engage in another oromotor behavior, grooming, more frequently, implying that divergent craniofacial and cranial nerve structure and function result in altered oromotor coordination. Finally, LgDel mice have significantly increased lung inflammation, a potential sign of aspiration-based dysphagia, consistent with results from our previous studies of early postnatal animals showing aspiration-related lung inflammation. Thus, oromotor dysfunction, feeding, and swallowing difficulties and their consequences persist in the LgDel 22q11DS mouse model. Apparently, postnatal growth and/or neural plasticity does not fully resolve deficits due to anomalous hindbrain, craniofacial, and cranial nerve development that prefigure perinatal dysphagia in 22q11DS. This new recognition of persistent challenges with feeding and swallowing may provide opportunities for improved therapeutic intervention for adolescents and adults with 22q11DS, as well as others with a history of perinatal feeding and swallowing disorders.

Plasmodium vivax is the most prevalent malaria species in Pakistan, with a distribution that coincides with Plasmodium falciparum in many parts of the country. Both species are likely exposed to drug pressure from a number of anti-malarials including chloroquine, sulphadoxine-pyrimethamine (SP), and artemisinin combination therapy, yet little is known regarding the effects of drug pressure on parasite genes associated with drug resistance. The aims of this study were to determine the prevalence of polymorphisms in the SP resistance-associated genes pvdhfr, pvdhps and chloroquine resistance-associated gene pvmdr1 in P. vivax isolates collected from across the country.

The World Health Organization recommends regular therapeutic efficacy studies (TES) to monitor the performance of first and second-line anti-malarials. In 2016, efficacy and safety of artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria were assessed through a TES conducted between April and October 2016 at four sentinel sites of Kibaha, Mkuzi, Mlimba, and Ujiji in Tanzania. The study also assessed molecular markers of artemisinin and lumefantrine (partner drug) resistance.

The recurrent laryngeal nerve (RLN) is responsible for normal vocal-fold (VF) movement, and is at risk for iatrogenic injury during anterior neck surgical procedures in human patients. Injury, resulting in VF paralysis, may contribute to subsequent swallowing, voice, and respiratory dysfunction. Unfortunately, treatment for RLN injury does little to restore physiologic function of the VFs. Thus, we sought to create a mouse model with translational functional outcomes to further investigate RLN regeneration and potential therapeutic interventions. To do so, we performed ventral neck surgery in 21 C57BL/6J male mice, divided into two groups: Unilateral RLN Transection (n = 11) and Sham Injury (n = 10). Mice underwent behavioral assays to determine upper airway function at multiple time points prior to and following surgery. Transoral endoscopy, videofluoroscopy, ultrasonic vocalizations, and whole-body plethysmography were used to assess VF motion, swallow function, vocal function, and respiratory function, respectively. Affected outcome metrics, such as VF motion correlation, intervocalization interval, and peak inspiratory flow were identified to increase the translational potential of this model. Additionally, immunohistochemistry was used to investigate neuronal cell death in the nucleus ambiguus. Results revealed that RLN transection created ipsilateral VF paralysis that did not recover by 13 weeks postsurgery. Furthermore, there was evidence of significant vocal and respiratory dysfunction in the RLN transection group, but not the sham injury group. No significant differences in swallow function or neuronal cell death were found between the two groups. In conclusion, our mouse model of RLN injury provides several novel functional outcome measures to increase the translational potential of findings in preclinical animal studies. We will use this model and behavioral assays to assess various treatment options in future studies.

In sub-Saharan Africa (SSA), epidemiological data for chronic kidney disease (CKD) are scarce. We conducted a prospective cross-sectional study including 952 patients in an outpatient clinic in Tanzania to explore CKD prevalence estimates and the association with cardiovascular and infectious disorders. According to KDIGO, we measured albumin-to-creatinine ratio and calculated eGFR using CKD-EPI formula. Factors associated with CKD were calculated by logistic regression. Venn diagrams were modelled to visualize interaction between associated factors and CKD. Overall, the estimated CKD prevalence was 13.6% (95% CI 11-16%). Ninety-eight patients (11.2%) (95% CI 9-14%) were categorized as moderate, 12 (1.4%) (95% CI 0-4%) as high, and 9 (1%) (95% CI 0-3%) as very high risk according to KDIGO. History of tuberculosis (OR 3.75, 95% CI 1.66-8.18; p = 0.001) and schistosomiasis (OR 2.49, 95% CI 1.13-5.18; p = 0.02) were associated with CKD. A trend was seen for increasing systolic blood pressure (OR 1.02 per 1 mmHg, 95% CI 1.00-1.03; p = 0.01). Increasing BMI (OR 0.92 per 1kg/m2, 95% CI 0.88-0.96; p = <0.001) and haemoglobin (OR 0.82 per 1g/dL, 95% CI 0.72-0.94; p = 0.004) were associated with risk reduction. Diabetes was associated with albuminuria (OR 2.81, 95% CI 1.26-6.00; p = 0.009). In 85% of all CKD cases at least one of the four most common factors (hypertension, diabetes, anaemia, and history of tuberculosis or schistosomiasis) was associated with CKD. A singular associated factor was found in 61%, two in 14%, and ≥3 in 10% of all CKD cases. We observed a high prevalence estimate for CKD and found that both classical cardiovascular and neglected infectious diseases might be associated with CKD in a semi-rural population of SSA. Our finding provides further evidence for the hypothesis that the "double burden" of non-communicable and endemic infectious diseases might affect kidney health in SSA.

The tongue plays a crucial role in the swallowing process, and impairment can lead to dysphagia, particularly in motor neuron diseases (MNDs) resulting in hypoglossal-tongue axis degeneration (e.g., amyotrophic lateral sclerosis and progressive bulbar palsy). This study utilized our previously established inducible rodent model of dysphagia due to targeted degeneration of the hypoglossal-tongue axis. This model was created by injecting cholera toxin B conjugated to saporin (CTB-SAP) into the genioglossus muscle of the tongue base for retrograde transport to the hypoglossal (XII) nucleus via the hypoglossal nerve, which provides the sole motor control of the tongue. Our goal was to investigate the effect of high-repetition/low-resistance tongue exercise on tongue function, strength, and structure in four groups of male rats: (1) control + sham exercise (n = 13); (2) control + exercise (n = 10); (3) CTB-SAP + sham exercise (n = 13); and (4) CTB-SAP + exercise (n = 12). For each group, a custom spout with adjustable lick force requirement for fluid access was placed in the home cage overnight on days 4 and 6 post-tongue injection. For the two sham exercise groups, the lick force requirement was negligible. For the two exercise groups, the lick force requirement was set to ∼40% greater than the maximum voluntary lick force for individual rats. Following exercise exposure, we evaluated the effect on hypoglossal-tongue axis function (via videofluoroscopy), strength (via force-lickometer), and structure [via Magnetic Resonance Imaging (MRI) of the brainstem and tongue in a subset of rats]. Results showed that sham-exercised CTB-SAP rats had significant deficits in lick rate, swallow timing, and lick force. In exercised CTB-SAP rats, lick rate and lick force were preserved; however, swallow timing deficits persisted. MRI revealed corresponding degenerative changes in the hypoglossal-tongue axis that were mitigated by tongue exercise. These collective findings suggest that high-repetition/low-resistance tongue exercise in our model is a safe and effective treatment to prevent/diminish signs of hypoglossal-tongue axis degeneration. The next step is to leverage our rat model to optimize exercise dosing parameters and investigate corresponding treatment mechanisms of action for future translation to MND clinical trials.

Diverse vaccination outcomes and protection levels among different populations pose a serious challenge to the development of an effective malaria vaccine. Co-infections are among many factors associated with immune dysfunction and sub-optimal vaccination outcomes. Chronic, asymptomatic viral infections can contribute to the modulation of vaccine efficacy through various mechanisms. Human Pegivirus-1 (HPgV-1) persists in immune cells thereby potentially modulating immune responses. We investigated whether Pegivirus infection influences vaccine-induced responses and protection in African volunteers undergoing whole P. falciparum sporozoites-based malaria vaccination and controlled human malaria infections (CHMI).

The radiation-attenuated Plasmodium falciparum sporozoites (PfSPZ) Vaccine has demonstrated safety and immunogenicity in 5-month-old to 50-year-old Africans in multiple trials. Except for one, each trial has restricted enrollment to either infants and children or adults < 50 years old. This trial was conducted in Equatorial Guinea and assessed the safety, tolerability, and immunogenicity of three direct venous inoculations of 1.8 × 106 or 2.7 × 106 PfSPZ, of PfSPZ Vaccine, or normal saline administered at 8-week intervals in a randomized, double-blind, placebo-controlled trial stratified by age (6-11 months and 1-5, 6-10, 11-17, 18-35, and 36-61 years). All doses were successfully administered. In all, 192/207 injections (93%) in those aged 6-61 years were rated as causing no or mild pain. There were no significant differences in solicited adverse events (AEs) between vaccinees and controls in any age group (P ≥ 0.17). There were no significant differences between vaccinees and controls with respect to the rates or severity of unsolicited AEs or laboratory abnormalities. Development of antibodies to P. falciparum circumsporozoite protein occurred in 67/69 vaccinees (97%) and 0/15 controls. Median antibody levels were highest in infants and 1-5-year-olds and declined progressively with age. Antibody responses in children were greater than in adults protected against controlled human malaria infection. Robust immunogenicity, combined with a benign AE profile, indicates children are an ideal target for immunization with PfSPZ Vaccine.