Optimization of Multiplex-PCR Technique To Determine Azf Deletions in infertility Male Patients

Tung Nguyen Thanh,1 Sang Trieu Tien,2 Phong Nguyen Van,2 Son Dang Thai,3 Thuc Luong Cong,4 Tuan Dinh Le,5 Son Tien Nguyen,5 Tuan Tran Van,1 Hoang Huy Duong,6 Tien Minh Bui,7 Kien Trung Nguyen7 1Military Institute of Clinical Embryology and Histology, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 2Department of Biology and Medical Genetics, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 3Institute of Biological and Food Technology, Hanoi Open University, Hanoi, 100000, Vietnam; 4Cardiovascular Center, Military Hospital 103, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 5Department of Rheumatology and Endocrinology, Military Hospital 103, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 6Department of Neurology, Thai Binh University of Medicine and Pharmacy, Thai Binh, 410000, Vietnam; 7Department of Obstetrics and Gynecology, Thai Binh University of Medicine and Pharmacy, Thai Binh, 410000, VietnamCorrespondence: Sang Trieu Tien, Department of Biology and Medical Genetics, Vietnam Military Medical University, Hanoi, 100000, Vietnam, Email [email protected]: To optimize the multiplex polymerase chain reaction (M-PCR) technique to diagnose microdeletions of azoospermia factors (AZF) on the Y chromosome and initially apply the technique to diagnose male patients with sperm density less than 5× 106 million sperm/mL was assigned to do a test to check for AZF microdeletions on the Y chromosome.Methods: Based on the positive control samples which belong to male subjects who have had 2 healthy children without any assisted reproductive technologies, the M-PCR method was developed to detect simultaneously and accurately AZF microdeletions on 32 male patients with sperm densities below 5× 106 million sperm/mL of semen at the Department of Biology and Medical Genetics – Vietnam Military Medical University.Results: Successful optimization of the M-PCR technique including 7 reactions arranged according to each AZFabc region using 24 STS/gene on the Y chromosome. Initial application to diagnose AZF deletion on 32 azoospermic and oligospermic men reveals that AZFa deletion accounts for 6.25% (2/32); deletion of all 3 regions AZFa,b,c with 18.75% (6/32 cases); The combined deletion rate of AZFb,c is highest, accounting for 56.24% (18/32 patients).Conclusion: Successfully optimized the M-PCR technique in identifying AZF microdeletions using 24 sequence tagged sites (STS)/gene for azoospermic and oligozoospermic men. The M-PCR technique has great potential in the application of AZF deletion diagnosis.Keywords: male infertility, azoospermia factors, AZF, multiplex polymerase chain reaction, M-PCR, sequence tagged sites, ST

Triple therapy with artemether-lumefantrine plus amodiaquine versus artemether-lumefantrine alone for artemisinin-resistant, uncomplicated falciparum malaria: an open-label, randomised, multicentre trial

Background: Late treatment failures after artemisinin-based combination therapies (ACTs) for falciparum malaria have increased in the Greater Mekong subregion in southeast Asia. Addition of amodiaquine to artemether-lumefantrine could provide an efficacious treatment for multidrug-resistant infections. Methods: We conducted an open-label, randomised trial at five hospitals or health centres in three locations (western Cambodia, eastern Cambodia, and Vietnam). Eligible participants were male and female patients aged 2-65 years with uncomplicated Plasmodium falciparum malaria. Patients were randomly allocated (1:1 in blocks of eight to 12) to either artemether-lumefantrine alone (dosed according to WHO guidelines) or artemether-lumefantrine plus amodiaquine (10 mg base per kg/day), both given orally as six doses over 3 days. All received a single dose of primaquine (0·25 mg/kg) 24 h after the start of study treatment to limit transmission of the parasite. Parasites were genotyped, identifying artemisinin resistance. The primary outcome was Kaplan-Meier 42-day PCR-corrected efficacy against recrudescence of the original parasite, assessed by intent-to-treat. Safety was a secondary outcome. This completed trial is registered at ClinicalTrials.gov (NCT03355664). Findings: Between March 18, 2018, and Jan 30, 2020, 310 patients received randomly allocated treatment; 154 received artemether-lumefantrine alone and 156 received artemether-lumefantrine plus amodiaquine. Parasites from 305 of these patients were genotyped. 42-day PCR-corrected treatment efficacy was noted in 151 (97%, 95% CI 92-99) of 156 patients with artemether-lumefantrine plus amodiaquine versus 146 (95%, 89-97) of 154 patients with artemether-lumefantrine alone; hazard ratio (HR) for recrudescence 0·6 (95% CI 0·2-1·9, p=0·38). Of the 13 recrudescences, 12 were in 174 (57%) of 305 infections with pfkelch13 mutations indicating artemisinin resistance, for which 42-day efficacy was noted in 89 (96%) of 93 infections with artemether-lumefantrine plus amodiaquine versus 73 (90%) of 81 infections with artemether-lumefantrine alone; HR for recrudescence 0·44 (95% CI 0·14-1·40, p=0·17). Artemether-lumefantrine plus amodiaquine was generally well tolerated, but the number of mild (grade 1-2) adverse events, mainly gastrointestinal, was greater in this group compared with artemether-lumefantrine alone (vomiting, 12 [8%] with artemether-lumefantrine plus amodiaquine vs three [2%] with artemether-lumefantrine alone, p=0·03; and nausea, 11 [7%] with artemether-lumefantrine plus amodiaquine vs three [2%] with artemether-lumefantrine alone, p=0·05). Early vomiting within 1 h of treatment, requiring retreatment, occurred in no patients of 154 with artemether-lumefantrine alone versus five (3%) of 156 with artemether-lumefantrine plus amodiaquine, p=0·06. Bradycardia (≤54 beats/min) of any grade was noted in 59 (38%) of 154 patients with artemether-lumefantrine alone and 95 (61%) of 156 with artemether-lumefantrine plus amodiaquine, p=0·0001. Interpretation: Artemether-lumefantrine plus amodiaquine provides an alternative to artemether-lumefantrine alone as first-line treatment for multidrug-resistant P falciparum malaria in the Greater Mekong subregion, and could prolong the therapeutic lifetime of artemether-lumefantrine in malaria-endemic populations

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Disparities in Geographic Access to Cardiac Rehabilitation in Los Angeles County

Background Exercise‐based cardiac rehabilitation (CR) is known to reduce morbidity and mortality for patients with cardiac conditions. Sociodemographic disparities in accessing CR persist and could be related to the distance between where patients live and where CR facilities are located. Our objective is to determine the association between sociodemographic characteristics and geographic proximity to CR facilities. Methods and Results We identified actively operating CR facilities across Los Angeles County and used multivariable Poisson regression to examine the association between sociodemographic characteristics of residential proximity to the nearest CR facility. We also calculated the proportion of residents per area lacking geographic proximity to CR facilities across sociodemographic characteristics, from which we calculated prevalence ratios. We found that racial and ethnic minorities, compared with non‐Hispanic White individuals, more frequently live ≥5 miles from a CR facility. The greatest geographic disparity was seen for non‐Hispanic Black individuals, with a 2.73 (95% CI, 2.66–2.79) prevalence ratio of living at least 5 miles from a CR facility. Notably, the municipal region with the largest proportion of census tracts comprising mostly non‐White residents (those identifying as Hispanic or a race other than White), with median annual household income <$60 000, contained no CR facilities despite ranking among the county's highest in population density. Conclusions Racial, ethnic, and socioeconomic characteristics are significantly associated with lack of geographic proximity to a CR facility. Interventions targeting geographic as well as nongeographic factors may be needed to reduce disparities in access to exercise‐based CR programs. Such interventions could increase the potential of CR to benefit patients at high risk for developing adverse cardiovascular outcomes