Nanopore sequencing technology for clinical diagnosis of infectious diseases where laboratory capacity is meager: A case report.

In resource-limited settings, patients are often first presented to clinical settings when seriously ill and access to proper clinical microbial diagnostics is often very limited or non-existing. On February 16th, 2022 we were on a field trip to test a completely field-deployable metagenomics sequencing set-up, that includes DNA purification, sequencing, and bioinformatics analyses using bioinformatics tools installed on a laptop for water samples, just outside Moshi, Tanzania. On our way to the test site, we were contacted by the nearby Machame hospital regarding a child seriously ill with diarrhea and not responding to treatment. Within the same day, we conducted an onsite metagenomics examination of a fecal sample from the child, and Campylobacter jejuni was identified as the causative agent. The treatment was subsequently changed, with almost immediate improvement, and the child was discharged on February 21st.

Whole-genome sequencing of SARS-CoV-2 isolates from symptomatic and asymptomatic individuals in Tanzania.

Background: Coronavirus Disease-2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accounts for considerable morbidity and mortality globally. Paucity of SARS-CoV-2 genetic data from Tanzania challenges in-country tracking of the pandemic. We sequenced SARS-CoV-2 isolated in the country to determine circulating strains, mutations and phylogenies and finally enrich international genetic databases especially with sequences from Africa. Methods: This cross-sectional study utilized nasopharyngeal swabs of symptomatic and asymptomatic adults with positive polymerase chain reaction tests for COVID-19 from January to May 2021. Viral genomic libraries were prepared using ARTIC nCoV-2019 sequencing protocol version three. Whole-genome sequencing (WGS) was performed using Oxford Nanopore Technologies MinION device. In silico genomic data analysis was done on ARTIC pipeline version 1.2.1 using ARTIC nCoV-2019 bioinformatics protocol version 1.1.0. Results: Twenty-nine (42%) out of 69 samples qualified for sequencing based on gel electrophoretic band intensity of multiplex PCR amplicons. Out of 29 isolates, 26 were variants of concern [Beta (n = 22); and Delta (n = 4)]. Other variants included Eta (n = 2) and B.1.530 (n = 1). We found combination of mutations (S: D80A, S: D215G, S: K417N, ORF3a: Q57H, E: P71L) in all Beta variants and absent in other lineages. The B.1.530 lineage carried mutations with very low cumulative global prevalence, these were nsp13:M233I, nsp14:S434G, ORF3a:A99S, S: T22I and S: N164H. The B.1.530 lineage clustered phylogenetically with isolates first reported in south-east Kenya, suggesting regional evolution of SARS-CoV-2. Conclusion: We provide evidence of existence of Beta, Delta, Eta variants and a locally evolving lineage (B.1.530) from samples collected in early 2021 in Tanzania. This work provides a model for ongoing WGS surveillance that will be required to inform on emerging and circulating SARS-CoV-2 diversity in Tanzania and East Africa.

Effect of Digital Adherence Tools on Adherence to Antiretroviral Treatment Among Adults Living With HIV in Kilimanjaro, Tanzania: A Randomized Controlled Trial.

BACKGROUND: Lifelong adherence to antiretroviral treatment remains challenging for people living with HIV (PLHIV). The aim of this study was to investigate whether any of 2 digital adherence tools could improve adherence among PLHIV in Kilimanjaro, Tanzania. METHODS: We performed a parallel 3-arm, nonblinded, randomized controlled trial with 1:1:1 allocation. We included adults aged between 18 and 65 years, living in Kilimanjaro region, and who were on antiretroviral treatment for at least 6 months. Their adherence, as judged by the study nurses, had to be suboptimal. In one arm, participants received reminder short message service (SMS) texts, followed by a question SMS. In the second arm, participants received a real-time medication monitoring (RTMM) device (Wisepill) with SMS reminders. In the third arm, participants received standard care only. The primary outcome of mean adherence over 48 weeks was compared between arms using between-group t tests in a modified intention-to-treat analysis. RESULTS: In each arm, we randomized 83 participants: data of 82 participants in the RTMM arm, 80 in the SMS arm, and 81 in the standard care arm were analyzed. The average (over 48 weeks) adherence in the SMS, RTMM, and control arms was 89.6%, 90.6%, and 87.9% for pharmacy refill; 95.9%, 95.0%, and 95.2% for self-report in the past week; and 97.5%, 96.6%, and 96.9% for self-report in the past month, respectively (P values not statistically significant). CONCLUSIONS: Receiving reminder SMS or RTMM combined with feedback about adherence levels and discussion of strategies to overcome barriers to adherence did not improve adherence to treatment and treatment outcome in PLHIV. CLINICAL TRIAL NUMBER: PACTR201712002844286.

Patterns of acquired HIV-1 drug resistance mutations and predictors of virological failure in Moshi, Northern Tanzania.

Emergence of HIV drug resistance poses a serious risk of inactivity to all currently approved antiretroviral drugs. Profiles of HIV drug resistance mutations (HIVDRM) and virological failure (VF) are not extensively studied in Tanzania. This study aimed to determine HIVDRM and predictors of VF in HIV-infected individuals failing first-line HIV drugs in Moshi, Northern Tanzania. A case-control study was conducted at Kilimanjaro Christian Medical Centre, Mawenzi, Pasua and Majengo health facilities with HIV-care and treatment clinics from October, 2017 to August, 2018. Cases and controls were HIV-infected individuals with VF and viral suppression (VS) respectively. HIV-1 reverse transcriptase and protease genes were amplified and sequenced. Stanford University's HIV drug resistance database and REGA subtyping tool 3.0 determined HIVDRM and HIV-1 subtypes respectively. Odds ratios (OR) with 95% confidence interval (95% CI) investigated predictors of VF. P-value < 5% was considered statistically significant. A total of 124 participants were recruited, of whom 63 (50.8%) had VF, 61 (49.2%) had VS and 82 (66.1%) were females. Median [IQR] age and duration on ART were 45 [35-52] years and 72 [48-104] months respectively. Twenty-five out of 26 selected samples from cases were successfully sequenced. Twenty-four samples (96%) had at least one major mutation conferring resistance to HIV drugs, with non-nucleoside analogue reverse transcriptase inhibitor (NNRTI)-resistance associated mutations as the majority (92%). Frequent NNRTI-resistance associated mutations were K103N (n = 11), V106M (n = 5) and G190A (n = 5). Prevalent nucleoside analogue reverse transcriptase inhibitors-resistance associated mutations were M184V (n = 17), K70R (n = 7) and D67N (n = 6). Dual-class resistance was observed in 16 (64%) samples. Thirteen samples (52%) had at least one thymidine analogue-resistance associated mutation (TAM). Three samples (12%) had T69D mutation with at least 1 TAM. Two samples (8%) had at least one mutation associated with protease inhibitor resistance. Age [aOR = 0.94, 95% CI (0.90-0.97), p < 0.001] and occupation [aOR = 0.35, 95% CI (0.12-1.04), p = 0.059] associated with VF. In conclusion, HIV drug resistance is common among people failing antiretroviral therapy. Resistance testing will help to guide switching of HIV drugs.

Ceftriaxone use in a tertiary care hospital in Kilimanjaro, Tanzania: A need for a hospital antibiotic stewardship programme.

Excessive use of antibiotics, especially watch group antibiotics such as ceftriaxone leads to emergence and spread of antimicrobial resistance (AMR). In low and middle-income countries (LMICs), antibiotics are overused but data on consumption is scarcely available. We aimed at determining the extent and predictors of ceftriaxone use in a tertiary care university teaching hospital in Kilimanjaro, Tanzania. A hospital-based cross-sectional study was conducted from August 2013 through August 2015. Patients admitted in the medical, surgical wards and their respective intensive care units, receiving antimicrobials and other medications for various ailments were enrolled. Socio-demographic and clinical data were recorded in a structured questionnaire from patients' files and logistic regression was performed to determine the predictors for ceftriaxone use. Out of the 630 patients included in this study, 322 (51.1%) patients were on ceftriaxone during their time of hospitalization. Twenty-two patients out of 320 (6.9%) had been on ceftriaxone treatment without evidence of infection. Ceftriaxone use for surgical prophylaxis was 44 (40.7%), of which 32 (72.7%) and 9 (20.5%) received ceftriaxone prophylaxis before and after surgery, respectively. Three (6.8%) received ceftriaxone prophylaxis during surgery. Predicting factors for that the health facility administered ceftriaxone were identified as history of any medication use before referral to hospital [OR = 3.4, 95% CI (1.0-11.4), p = 0.047], bacterial infection [OR = 18.0, 95% CI (1.4-225.7, p = 0.025)], surgical ward [OR = 2.9, 95% CI (0.9-9.4), p = 0.078] and medical wards [OR = 5.0, 95% CI (0.9-28.3), p = 0.070]. Overall, a high ceftriaxone use at KCMC hospital was observed. Antimicrobial stewardship programs are highly needed to monitor and regulate hospital antimicrobial consumption, which in turn could help in halting the rising crisis of antimicrobial resistance.

Prescription and non-prescription antibiotic dispensing practices in part I and part II pharmacies in Moshi Municipality, Kilimanjaro Region in Tanzania: A simulated clients approach.

Antibiotic dispensing without a prescription poses a threat to public health as it leads to excessive antibiotic consumption. Inappropriate antibiotic availability to the community has been documented to be amongst drivers of antimicrobial resistance emergence. Community pharmacies are a source of antibiotics in low and middle-income countries (LMICs). We aimed at assessing antibiotic dispensing practices by community pharmacy retailers in Moshi urban, Kilimanjaro, Tanzania and recommend interventions to improve practice. Using a Simulated Client (SC) Method, an observational cross-sectional survey of antibiotic dispensing practices was conducted from 10th June to 10th July 2017. Data analysis was done using Stata 13 (StataCorp, College Station, TX, USA). A total of 82 pharmacies were visited. Part I pharmacies were 26 (31.71%) and 56 (68.29%) were part II. Overall 92.3% (95% CI 77.8-97.6) of retailers dispensed antibiotics without prescriptions. The antibiotics most commonly dispensed without a prescription were ampiclox for cough (3 encounters) and azithromycin for painful urination (3 encounters). An oral third generation cephalosporin (cefixime) was dispensed once for painful urination without prescription by a part I pharmacy retailer. Out of 21, 15(71.43%) prescriptions with incomplete doses were accepted and had antibiotics dispensed. Out of 68, 4(5.9%) retailers gave instructions for medicine use voluntarily. None of the retailers voluntarily explained drug side-effects. In Moshi pharmacies, a high proportion of antibiotics are sold and dispensed without prescriptions. Instructions for medicine use are rarely given and none of the retailers explain side effects. These findings support the need for a legislative enforcement of prescription-only antibiotic dispensing rules and regulations. Initiation of clinician and community antibiotic stewardship and educational programs on proper antibiotic use to both pharmacists and public by the regulatory bodies are highly needed.

Prevalence, determinants and knowledge of antibacterial self-medication: A cross sectional study in North-eastern Tanzania.

Self-medication is very common especially in developing countries and is documented to be associated with many health risks including antibiotic resistance. This study investigated the prevalence, determinants and knowledge of self-medication among residents of Siha District in Tanzania. A cross-sectional study was conducted among 300 residents in a rural District of Kilimanjaro region, North-eastern Tanzania from 1st to 28th April 2017. A semi-structured questionnaire was used to collect information regarding drugs used, knowledge, history and reasons for antibiotic self-medication. Log-binomial regression analysis was done using STATA 13 to examine factors associated with self-medication. A slightly majority of the respondents (58%) admitted to self-medication. Antibiotics most commonly utilized were amoxycillin (43%) and an antiprotozoal drug metronidazole (10%). The most common symptoms that led to self-medication were cough (51.17%), headache/ fever/ malaria (25.57%) and diarrhoea (21.59%). The most common reasons for self-medication were emergency illness (24.00%), health facility charges (20.33%), proximity of pharmacy to home (17.00%) and no reason (16.66%). Almost all reported that self-medication is not better than seeking medical consultation, 98% can result into harmful effects and 96% can result to drug resistance. The level of self-medication in this study is comparable with findings from other studies in developing countries. Pharmacies were commonly used as the first point of medical care. There is therefore a need for educative antibiotic legislative intervention to mitigate the adverse effects of antibiotic self-medication in Siha district in Tanzania.

Applying the diabetes quality improvement project indicators in the Indian Health Service primary care setting.

OBJECTIVE: With publication of the Diabetes Quality Improvement Project (DQIP) measures, the Indian Health Service National Diabetes Program applied the DQIP format to its IHS Diabetes Care and Outcomes Audit for comparison and benchmarks. RESEARCH DESIGN AND METHODS: Since 1986 the IHS Diabetes Care and Outcomes Audit has been conducted by medical record review in >75% of IHS and tribal facilities. Each year systematic random sample of charts is drawn from local diabetes registries. Chart reviews are conducted by, trained professionals according to standard definitions and instructions. Abstracted data are entered into a microcomputer-based epidemiologic software package. Local, regional, and national rates are constructed for each item. During the period 1995-1997, 150 facilities submitted data for compilation, representing participation from all 12 IHS administrative regions. The IHS Diabetes Care and Outcomes Audit collected virtually all of the DQIP measures, with the exception of LDL cholesterol (which was added to the record review in 1998). RESULTS: In 1995, 1996, and 1997, a total of 9,557, 9,985, and 9,626 individuals, respectively, were included in the total IHS audit sample. The reviews for 1995, 1996, and 1997 revealed that of all subjects: 55, 65, and 80%, respectively, had more than one HbA1c test during the year (P < 0.001); 42, 38, and 34%, respectively; had a high-risk HbA1c (>9.5%) (P < 0.001); 83, 81, and 84%, respectively, were tested for macroproteinuria (P < 0.11) and 16, 17, and 23%, respectively were tested for microproteinuria (P < 0.001); total cholesterol was assessed in 80, 81, and 85%, respectively (P < 0.001), and corresponding proportions of those with values <5.17 mmol/l were 48, 50, and 52%, respectively; triglyceride values were measured for 75,75, and 80%, respectively (P < 0.001), and the corresponding median triglyceride levels were 199, 198, and 193 mg/dl, respectively (P < 0.001); the proportion of clients with a blood pressure <140/90 mmHg was 64, 64, and 66%, respectively (P < 0.05); 55, 56, and 55%, respectively, had a dilated eye exam (P < 0.053); and the proportion of clients who had a comprehensive foot exam were 59, 59, and 61%, respectively (P < 0.05). CONCLUSIONS: The DQIP accountability and quality improvement measures could be easily applied to the IHS Diabetes Care and Outcomes Audit, and the process can prove to be practical. However, data alone are not sufficient to effect change. Use of the measures to ensure that the quality of care improves must also be stressed, because measuring alone will not guarantee such improvement.

The prevalence and risk of diabetic retinopathy among Indians of southwest Oklahoma.

The overall prevalence of diabetic retinopathy was 49.3% among 142 diabetic Indians examined at the Indian Health Service at Clinton, Oklahoma. Univariate analysis suggested a positive relationship between duration of diabetes and prevalence of retinopathy. This Indian diabetic population should be examined yearly for diabetic retinopathy.

Brain ascorbate depletion as a response to stress.

Ascorbate-depleted rainbow trout were fed a single dose of L-1-14C-ascorbic acid, then held in metabolism chambers for four consecutive five-day periods. Tissue samples were analyzed for 14C, ascorbate, and ascorbate-2-sulfate. Brain ascorbate showed a long turnover time following a very slow uptake. The loss of brain ascorbate after five days in metabolism chambers was highly significant (p less than 0.001) when compared with similar dosed fish not chambered. A brain ascorbate pool which does not exchange with the body pool is proposed. Possible mechanisms are discussed and a kinetic model is suggested.

Activation of yeast pyruvate carboxylase: interactions between acyl coenzyme A compounds, aspartate, and substrates of the reaction.

Chicken liver pyruvate carboxylase has an absolute requirement for short-chain acyl coenzyme A (CoA), whereas the same enzyme from yeast has less stringent requirements. The yeast enzyme has now been studied in an effort to elucidate the mechanism by which acyl-CoA stimulates pyruvate carboxylase activity. Yeast pyruvate carboxylase has an apparent basal level of activity above which CoA and acyl-CoAs of 2-20 carbons activate; the concentration of acyl-CoA required for half-maximum activation (K0.5) decreases as the chain length of the acyl moiety increases to 16 carbons. Activation of yeast pyruvate carboxylase by acyl-CoA is brought about in part by increasing the affinity of pyruvate carboxylase for two substrates, bicarbonate and pyruvate. The affinity of pyruvate carboxylase for bicarbonate is also increased by potassium ions. The observation of only low levels of activity in the absence of acyl-CoA or potassium ion leads to the conclusion that the basal activity so frequently referred to is probably due to the presence of activating monovalent cations. Pyruvate carboxylase from yeast probably has an absolute requirement for monovalent cations or acyl-CoA with a combination of the two being required for optimum conditions for maximal activity. Stimulation by acyl-CoA and inhibition by aspartate are mutually antagonistic with each affecting the activation or inhibition constant and the degree of cooperativity brought about by the other. The enzyme from liver is unaffected by aspartate.

Graft-versus-host disease from leukocyte transfusions.

Transfusion of blood products containing immunocompetent lymphocytes may cause graft-versus-host disease (GVHD) in immunosuppressed recipients. We describe fatal GVHD which occurred in a 30-year-old renal transplant patient 2 weeks after transfusion of 1.0 x 10(8) lymphocytes/kilogram. In addition to typical cutaneous and gastrointestinal manifestations, she developed irreversible bone marrow failure. Acute GVHD following blood product transfusion from normal donors has a mortality of 88%. Treatment is unsatisfactory, but irradiation of blood products prior to administration is preventive.

Sibling visiting in a neonatal intensive care unit.

The effect of sibling visiting in a neonatal intensive care unit was studied. Sixteen siblings of 13 infants were randomly assigned to a visiting or nonvisiting group. Behavioral patterns were measured by questionnaires administered to the parents and by direct observation and interviews with the children. There were no significant changes in the behavior of the children following the birth of their sibling, and there was no significant difference between the behavior scores of the two groups 1 week after the experimental (or control) intervention. The visiting children did not show signs of fear or anxiety during the visit. These data suggest that sibling visiting to a neonatal intensive care unit is not likely to be harmful and might be beneficial to the siblings and their families.

Protein, amino acid, and caloric intakes of selected pregnant women.

Toxemia of pregnancy is characterized by a combination of at least two of the following clinical symptoms: hypertension, edema, and proteinuria. In three successive trials over three consecutive years, the dietary intakes of a selected number of young pregnant women attending a Maternal and Infant Care Clinic at Tuskegee Institute were evaluated for protein, amino acids, and total calories. Women with toxemia were identified, and women without toxemia served as controls. The toxemic group generally consumed more protein than the controls, but values were statistically significant only in the first trial. However, all essential amino acids were consumed in significantly greater amounts by the toxemic group. Protein and essential amino acids were consumed in adequate amounts (at least two-thirds of the RDA) by both groups but in amounts smaller than the national average. Non-essential amino acids were also consumed in adequate amounts, with the toxemic group consuming larger quantities than the controls. Caloric intakes were adequate for young pregnant women. The relationships of glucosuria and of toxemia to protein and amino acid intake were similar and were opposite to the relationship of anemia to protein and amino acid intake. Meats and grains contributed the greatest quantity of protein and amino acids to the diet in all groups. Data seem to imply that any relationship of protein and amino acids with toxemia of pregnancy is a complex one involving several possibly interrelated nutritional parameters.