A simple and rapid external standard calibration HPLC method for determination of lumefantrine in dried blood spot samples from malaria patients in Botswana.

A simple external calibration liquid chromatography-diode array detector method was developed, validated, and applied for the determination of lumefantrine (Lum) in dried blood spot (DBS) samples collected from malaria patients in Botswana. The samples were validated in accordance with the United States Food and Drug Administration guidelines for bioanalytical methods after sample preparation using solid-liquid extraction. Separation was achieved using an XTerra C18 column (50 × 4.6 mm, 5 µm), and a binary solvent system of acetonitrile and water adjusted to pH 2.3 was used as the mobile phase. The validated method was applied for the determination of Lum in DBS samples collected from malaria patients infected with Plasmodium falciparum in Botswana. The calibration curve was linear between 0.5 and 12 µg/mL with a coefficient of determination (R2 ) of 0.9996. The limit of detection and the lower limit of quantification were 0.5 and 1.4 µg/mL, respectively. The efficiency of extraction measured as percentage recovery ranged between 84.2% and 107.8% at the three quality control (QC) levels, that is, low QC, mid QC, and high QC. In conclusion, data suggest that the method is suitable for the determination of trace Lum in biofluids and can also be used for therapeutic drug monitoring and pharmacokinetic profiling.

Patients' perspectives on optimal doctor-patient interactions during medical consultation: Lessons for medical educators.

Background: Perceptions of patients of the nature and quality of the interaction with their doctors during consultation are potentially an important factor determining patient satisfaction and doctors' success. Failure by medical doctors to understand how patients perceive them or what patients desire from them may hinder the establishment of strong, trust-based doctor-patient relationships. The purpose of this study was to explore the health service users' views in a region of Botswana on what constitutes optimal doctor-patient interaction during consultation and propose recommendations for integration into medical education curricula. Methods: A purposive sample of 12 individuals was selected in a setting where health and well-being are underpinned by principles of interdependence and interpersonal connections (botho/ubuntu philosophy) for semi-structured interviews using the critical incident technique to elicit desired behaviors and actions of medical doctors during consultation. The participants described their good and bad consultation experiences with medical doctors. The data were analyzed using thematic analysis. Results: Three themes, derived from 11 codes, were identified from the data: conversational skills (welcoming and focused), interpersonal skills (humane, respectful, empathetic, unprejudiced, and personal), and professional traits (humble, trustworthy, thorough, and empowering). Discussion: Many expressed desires or expectations correspond with humanistic attributes described in the literature, suggesting their universal value. More importantly, the desires and expectations align with the principles of the Bantu philosophy of botho/ubuntu. Accordingly, educators should intentionally provide learning opportunities for students to promote the development of the desired attributes that enhance an effective doctor-patient relationship but should employ culturally relevant pedagogy.

Assessment of prescribing practices at the primary healthcare facilities in Botswana with an emphasis on antibiotics: Findings and implications.

BACKGROUND AND AIMS: Inappropriate drug prescribing has increased especially in developing countries where systems for monitoring medicine use are not well developed. This increases the rate of antimicrobial resistance. The study aim was to assess the prescribing patterns among urban primary health facilities in Botswana to provide future guidance including developing future quality indicators. METHODS: Retrospective data from patients' records between January and December 2013 in 19 clinics were collected in a cross-sectional study. The WHO/International Network for Rational Use of Drugs indicators were used to assess prescribing patterns in the study clinics. RESULTS: Average number of drugs per prescription was 2.8; 78.6% of the prescribed antibiotics were by International Non-proprietary Name and 96.1% complied with the Botswana Essential Drugs List. Overall rate of antibiotic prescribing was high (42.7%) with 14.7%, 5.9% and 1.3% of prescriptions having two, three and four antibiotics, respectively. Systemic antibiotics (JO1C) accounted for 45.4% of prescribed antibiotics of which amoxicillin accounted for 28.4% and metronidazole 14.4% of all antibiotic prescriptions. There was low use of co-amoxiclav (0.3% of all antibiotic prescriptions). Third generation cephalosporins and macrolides accounted for 9.8% and 6.2% of antibiotic prescriptions respectively, with no prescribing of fluoroquinolones. The majority of indications (87%) for antibiotic prescriptions were according to ICD classification. CONCLUSIONS: While most indications for antibiotic prescriptions were based on signs and symptoms according to ICD, antibiotic prescribing rates were high with some conditions not requiring antibiotics because they are viral infections. There is a need to further improve prescribing practices through induction and training of in-service prescribers. An effective management tool for monitoring antibiotic prescribing practices at Primary Health Care facilities should be designed and implemented, including developing robust quality indicators.

Identification of a Large Pool of Microorganisms with an Array of Porphyrin Based Gas Sensors.

The association between volatile compounds (VCs) and microorganisms, as demonstrated by several studies, may offer the ground for a rapid identification of pathogens. To this regard, chemical sensors are a key enabling technology for the exploitation of this opportunity. In this study, we investigated the performance of an array of porphyrin-coated quartz microbalance gas sensors in the identification of a panel of 12 bacteria and fungi. The porphyrins were metal complexes and the free base of a functionalized tetraphenylporphyrin. Our results show that the sensor array distinguishes the VC patterns produced by microorganisms in vitro. Besides being individually identified, bacteria are also sorted into Gram-positive and Gram-negative.

Advanced immune suppression is associated with increased prevalence of mixed-strain Mycobacterium tuberculosis infections among persons at high risk for drug-resistant tuberculosis in Botswana.

We examined factors associated with mixed-strain Mycobacterium tuberculosis infections among patients at high risk for drug-resistant tuberculosis in Botswana. Thirty-seven (10.0%) of 370 patients with tuberculosis had mixed M. tuberculosis infections, based on 24-locus mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping. In log-binomial regression analysis, age <37 years (adjusted prevalence ratio [PR], 1.92; 95% confidence interval [CI], 1.01-3.57) and prior tuberculosis treatment (adjusted PR, 2.31; 95% CI, 1.09-4.89) were associated with mixed M. tuberculosis infections. Among human immunodeficiency virus-infected patients, prior tuberculosis treatment (adjusted PR, 2.11; 95% CI, 1.04-4.31) and CD4(+) T-cell count of <100 cells/µl (adjusted PR, 10.18; 95% CI, 2.48-41.71) were associated with mixed M. tuberculosis infections. Clinical suspicion of mixed M. tuberculosis infections should be high for patients with advanced immunosuppression and a prior history of tuberculosis treatment.

Clinical outcomes among persons with pulmonary tuberculosis caused by Mycobacterium tuberculosis isolates with phenotypic heterogeneity in results of drug-susceptibility tests.

BACKGROUND: Patients with multidrug-resistant (MDR) tuberculosis may have phenotypic heterogeneity in results of drug-susceptibility tests (DSTs). However, the impact of this on clinical outcomes among patients treated for MDR tuberculosis is unknown. METHODS: Phenotypic DST heterogeneity was defined as presence of at least 1 Mycobacterium tuberculosis isolate susceptible to rifampicin and isoniazid recovered <3 months after MDR tuberculosis treatment initiation from a patient with previous documented tuberculosis due to M. tuberculosis resistant to at least rifampicin and isoniazid. The primary outcome was defined as good (ie, cure or treatment completion) or poor (ie, treatment failure, treatment default, or death). A secondary outcome was time to culture conversion. Cox proportional hazard models were used to determine the association between phenotypic DST heterogeneity and outcomes. RESULTS: Phenotypic DST heterogeneity was identified in 33 of 475 patients (7%) with MDR tuberculosis. Poor outcome occurred in 126 patients (28%). Overall, patients with MDR tuberculosis who had phenotypic DST heterogeneity were at greater risk of poor outcome than those with MDR tuberculosis but no phenotypic DST heterogeneity (adjusted hazard ratio [aHR], 2.1; 95% confidence interval [CI], 1.2-3.6). Among HIV-infected patients with MDR tuberculosis, the adjusted hazard for a poor outcome for those with phenotypic DST heterogeneity was 2.4 (95% CI, 1.3-4.2) times that for those without phenotypic DST heterogeneity, whereas among HIV-negative patients with MDR tuberculosis, the adjusted hazard for those with phenotypic DST heterogeneity was 1.5 (95% CI, .5-4.3) times that for those without phenotypic DST heterogeneity. HIV-infected patients with MDR tuberculosis with phenotypic DST heterogeneity also had a longer time to culture conversion than with HIV-infected patients with MDR tuberculosis without phenotypic DST heterogeneity (aHR, 2.9; 95% CI, 1.4-6.0). CONCLUSIONS: Phenotypic DST heterogeneity among persons with HIV infection who are being treated for MDR tuberculosis is associated with poor outcomes and longer times to culture conversion.

Deteriorating pneumococcal-specific B-cell memory in minimally symptomatic African children with HIV infection.

Invasive pneumococcal disease is a leading cause of human immunodeficiency virus (HIV)-associated mortality in sub-Saharan African children. Defective T-cell-mediated immunity partially explains this high disease burden, but there is an increased risk of invasive pneumococcal disease even in the context of a relatively preserved percentage of CD4 cells. We hypothesized that impaired B-cell immunity to this pathogen further amplifies the immune defect. We report a shift in the B-cell compartment toward an apoptosis-prone phenotype evident early in HIV disease progression. We show that, although healthy HIV-uninfected and minimally symptomatic HIV-infected children have similar numbers of isotype-switched memory B cells, numbers of pneumococcal protein antigen-specific memory B cells were lower in HIV-infected than in HIV-uninfected children. Our data implicate defective naturally acquired B-cell pneumococcal immunity in invasive pneumococcal disease causation in HIV-infected children and highlight the need to study the functionality and duration of immune memory to novel pneumococcal protein vaccine candidates in order to optimize their effectiveness in this population.

Bronchoalveolar CD4+ T cell responses to respiratory antigens are impaired in HIV-infected adults.

RATIONALE: HIV-infected adults are at an increased risk of lower respiratory tract infections. HIV infection impairs systemic acquired immunity, but there is limited information in humans on HIV-related cell-mediated immune defects in the lung. OBJECTIVE: To investigate antigen-specific CD4(+) T cell responses to influenza virus, Streptococcus pneumoniae and Mycobacterium tuberculosis antigens in bronchoalveolar lavage (BAL) and peripheral blood between HIV-infected individuals and HIV-uninfected Malawian adults. METHODS: We obtained BAL fluid and blood from HIV-infected individuals (n=21) and HIV-uninfected adults (n=24). We determined the proportion of T cell subsets including naive, memory and regulatory T cells using flow cytometry, and used intracellular cytokine staining to identify CD4(+) T cells recognising influenza virus-, S pneumoniae- and M tuberculosis-antigens. MAIN RESULTS: CD4(+) T cells in BAL were predominantly of effector memory phenotype compared to blood, irrespective of HIV status (p<0.001). There was immune compartmentalisation with a higher frequency of antigen-specific CD4(+) T cells against influenza virus, S pneumoniae and M tuberculosis retained in BAL compared to blood in HIV-uninfected adults (p<0.001 in each case). Influenza virus- and M tuberculosis-specific CD4(+) T cell responses in BAL were impaired in HIV-infected individuals: proportions of total antigen-specific CD4(+) T cells and of polyfunctional IFN-γ and TNF-α-secreting cells were lower in HIV-infected individuals than in HIV-uninfected adults (p<0.05 in each case). CONCLUSIONS: BAL antigen-specific CD4(+) T cell responses against important viral and bacterial respiratory pathogens are impaired in HIV-infected adults. This might contribute to the susceptibility of HIV-infected adults to lower respiratory tract infections such as pneumonia and tuberculosis.

Comparison of efficiencies of selected sample extraction techniques for the analysis of selected antiretroviral drugs in human plasma using LC-MS.

INTRODUCTION: Sample preparation in bio analytical chemistry poses a challenge because it can be compound dependent. We compared six sample extraction techniques i.e. QuEChERS (Q), liquid extraction (LE), protein precipitation (PPT), Q-PPT, Q-LE and LE-PPT for the extraction of antiretroviral drugs emtricitabine, tenofovir, efavirenz, lopinavir and rotinavir in human blood plasma. METHOD: A multiple reaction monitoring liquid chromatography- tandem mass spectrometry method for the determination of the same antiretroviral drugs developed and validated in this laboratory was used. Comparisons were based on the efficiencies of extraction, the precisions and accuracies. Using United States Food and Drug Administration guidelines, analytical performance characteristics i.e. limits of detection, lower limits of quantification and upper limits of quantification were also compared. RESULTS: The percent mean recoveries ranged between 68.8 and 81.2% for single modes and 52.4-70.5% for mixed mode techniques. The precisions of all the extraction techniques were within the Using United States Food and Drug Administration guidelines acceptable range of <15% at all concentration levels for all analytes. Accuracy ranged between 8.73 and 65.94% for single mode techniques and between 21.73 and 51.59% for mixed mode techniques. DISCUSSION: The mixed modes gave slightly lower recoveries but Q-LE compared well with the single modes at slightly higher spike levels. Limits of detection for all the six sample preparation techniques fell below the clinically relevant therapeutic range of approximately 3-8ppm. Therefore all techniques can be employed for routine therapeutic drug monitoring studies.

Diagnosis of pulmonary tuberculosis and assessment of treatment response through analyses of volatile compound patterns in exhaled breath samples.

OBJECTIVES: We determined the performance of a sensor array (an electronic nose) made of 8 metalloporphyrins coated quartz microbalances sensors for the diagnosis and prognosis of pulmonary tuberculosis (TB) using exhaled breath samples. METHODS: TB cases and healthy controls were prospectively enrolled. Signals from volatile organic compounds (VOCs) in breath samples were measured at days 0, 2, 7, 14, and 30 of TB therapy and correlated with clinical and microbiological measurements. RESULTS: Fifty one pulmonary TB cases and 20 healthy HIV-uninfected controls were enrolled in the study. 31 (61%) of the 51 pulmonary TB cases were coinfected with HIV. At day 0 (before TB treatment initiation) the sensitivity of our device was estimated at 94.1% (95% confidence interval [CI], 83.8-98.8%) and specificity was 90.0% (95% CI, 68.3-98.8%) for distinguishing TB cases from controls. Time-dependent changes in the breath signals were identified as time on TB treatment progressed. Time-dependent signal changes were more pronounced among HIV-uninfected patients. CONCLUSION: The identification of VOCs' signals in breath samples using a sensor array achieved high sensitivity and specificity for the diagnosis of TB and allowed following signal changes during TB treatment.

An Investigation of Liquid Chromatography-Mass Spectral Attributes and Analytical Performance Characteristics of Tenofovir, Emtricitabine and Efavirenz in Human Plasma.

Liquid chromatography (LC) and mass spectral behavior and analytical performance characteristics of efavirenz (EFV), emtricitabine (EMT) and tenofovir (TFV), i.e., individual components of Atripla(®), were probed. This was followed by estimation of their analytical performance characteristics employing LC and a parallel direct infusion sample introduction procedure. Performance characteristics using both types of sample introduction procedures were compared. Using liquid chromatography-mass spectrometry (LC-MS), linearities, i.e., correlation coefficients of the calibration curves of EFV, EMT and TFV, ranged between 0.9300 and 0.9990 in the full scan, selected ion monitoring and mass spectrometry/mass spectrometry (MS-MS) modes. The limits of detection (LODs) ranged between 0.5 and 11.6 µg/L. The lower limits of quantification (LLOQs) and the upper limits of quantification (ULOQs) were in the ranges of 0.9-23.2 and 1.6-38.7 µg/L, respectively. The LODs ranged between 0.8 and 114.7 µg/L. The LLOQs and the ULOQs were in the ranges of 1.6-29.4 and 2.7-49.0 µg/L, respectively. In the case of EMT, sodiated molecular ion at m/z 270 was used to adduce analytical performance characteristics from which lower detection limits were obtained compared with those in the literature where [M+H](+) at m/z 248 was used.

Incomplete recovery of pneumococcal CD4 T cell immunity after initiation of antiretroviral therapy in HIV-infected malawian adults.

HIV-infected African adults are at a considerably increased risk of life-threatening invasive pneumococcal disease (IPD) which persists despite antiretroviral therapy (ART). Defects in naturally acquired pneumococcal-specific T-cell immunity have been identified in HIV-infected adults. We have therefore determined the extent and nature of pneumococcal antigen-specific immune recovery following ART. HIV-infected adults were followed up at 3, 6 and 12 months after initiating ART. Nasopharyngeal swabs were cultured to determine carriage rates. Pneumococcal-specific CD4 T-cell immunity was assessed by IFN-γ ELISpot, proliferation assay, CD154 expression and intracellular cytokine assay. S. pneumoniae colonization was detected in 27% (13/48) of HIV-infected patients prior to ART. The rates remained elevated after 12 months ART, 41% (16/39) (p = 0.17) and significantly higher than in HIV-uninfected individuals (HIVneg 14%(4/29); p = 0.0147). CD4+ T-cell proliferative responses to pneumococcal antigens increased significantly to levels comparable with HIV-negative individuals at 12 months ART (p = 0.0799). However, recovery of the pneumococcal-specific CD154 expression was incomplete (p = 0.0015) as were IFN-γ ELISpot responses (p = 0.0040) and polyfunctional CD4+ T-cell responses (TNF-α, IL-2 and IFN-γ expression) (p = 0.0040) to a pneumolysin-deficient mutant strain. Impaired control of pneumococcal colonisation and incomplete restoration of pneumococcal-specific immunity may explain the persistently higher risk of IPD amongst HIV-infected adults on ART. Whether vaccination and prolonged ART can overcome this immunological defect and reduce the high levels of pneumococcal colonisation requires further evaluation.

Naturally-acquired influenza-specific CD4+ T-cell proliferative responses are impaired in HIV-infected African adults.

BACKGROUND: Seasonal influenza has been associated with greater morbidity and mortality in AIDS patients. Highly-active antiretroviral therapy (HAART) has led to some reduction in influenza-related complications but the nature of naturally-acquired T-cell immunity to influenza virus in an African setting, and how this changes with immune reconstitution following HAART is unknown. We measured influenza-specific CD4(+) T-cell immunity in unimmunized HIV-infected Malawian adults and then investigated immune reconstitution following HAART. METHODS: Peripheral blood mononuclear cells were isolated from HIV-infected and HIV-uninfected Malawian adults. CFSE proliferation and CD154 expression flow cytometry-based assays were used to measure influenza-specific CD4(+) T-cell immunity. RESULTS: We found lower naturally-acquired proliferative influenza-specific CD4(+) T-cell responses in AIDS patients that was also present in asymptomatic HIV-infected adults with relatively high CD4 counts (>350 cells/µl). Influenza-specific CD4(+) T-cell immune reconstitution in HIV-infected patients on HAART for 12 months was poor despite a marked reduction in viral load and an increase in CD4 count. This poor immune reconstitution was characterised by a low influenza-specific proliferative CD4(+) T-cell response and reduced proportions of CD154-expressing influenza-specific CD4(+) T-cells in peripheral blood. CONCLUSION: Our data suggest that asymptomatic HIV-infected adults may also be at risk of influenza-related complications and that HAART alone may not circumvent this risk in AIDS patients. This study highlights the need to identify possible interventions early in HIV infection to reduce the risk of influenza and to intensify influenza surveillance in these susceptible African populations.

Impaired CD4 T cell memory response to Streptococcus pneumoniae precedes CD4 T cell depletion in HIV-infected Malawian adults.

OBJECTIVE: Invasive pneumococcal disease (IPD) is a leading cause of morbidity and mortality in HIV-infected African adults. CD4 T cell depletion may partially explain this high disease burden but those with relatively preserved T cell numbers are still at increased risk of IPD. This study evaluated the extent of pneumococcal-specific T cell memory dysfunction in asymptomatic HIV infection early on in the evolution of the disease. METHODS: Peripheral blood mononuclear cells were isolated from asymptomatic HIV-infected and HIV-uninfected Malawian adults and stained to characterize the underlying degree of CD4 T cell immune activation, senescence and regulation. Pneumococcal-specific T cell proliferation, IFN-γ, IL-17 production and CD154 expression was assessed using flow cytometry and ELISpot. RESULTS: We find that in asymptomatic HIV-infected Malawian adults, there is considerable immune disruption with an increase in activated and senescent CD4+CD38+PD-1+ and CD4+CD25(high)Foxp3+ Treg cells. In the context of high pneumococcal exposure and therefore immune stimulation, show a failure in pneumococcal-specific memory T cell proliferation, skewing of T cell cytokine production with preservation of interleukin-17 but decreased interferon-gamma responses, and failure of activated T cells to express the co-stimulatory molecule CD154. CONCLUSION: Asymptomatic HIV-infected Malawian adults show early signs of pneumococcal- specific immune dysregulation with a shift in the balance of CD4 memory, T helper 17 cells and Treg. Together these data offer a mechanistic understanding of how antigen-specific T cell dysfunction occurs prior to T cell depletion and may explain the early susceptibility to IPD in those with relatively preserved CD4 T cell numbers.

Potential role for mucosally active vaccines against pneumococcal pneumonia.

Pneumococcal pneumonia is a life-threatening disease with high mortality and morbidity among children under 5 years of age, the elderly and immunocompromised individuals worldwide. Protection against pneumococcal pneumonia relies on successful regulation of colonisation in the nasopharynx and a brisk alveolar macrophage-mediated immune response in the lung. Therefore, enhancing pulmonary mucosal immunity (which includes a combination of innate, humoral and cell-mediated immunity) through mucosal vaccination might be the key to prevention of pneumococcal infection. Current challenges include a lack of information in humans on mucosal immunity against pneumococci and a lack of suitable adjuvants for new vaccines. Data from mouse models, however, suggest that mucosally active vaccines will enhance mucosal and systemic immunity for protection against pneumococcal infection.

HIV-1 subtype C in vitro growth and coreceptor utilization.

Human immunodeficiency virus type 1 subtype C (HIV-1C) accounts for about 50% of all HIV infections in the pandemic and is the predominant subtype in the heavily burdened region of southern Africa. HIV-1C possesses unique genetic and phenotypic features that might be associated with biological differences compared to other subtypes. Here, we generated virus isolates from individuals at different stages of HIV-1C infection and investigated the chemokine receptor repertoire that the derived HIV-1C isolates may utilize for entry. Our results show that the R5 phenotype predominates among viruses in Botswana, with a lesser contribution of viruses showing the dualtropic X4R5 phenotype. No viruses of pure X4 phenotype were found, which suggests no discernable evolution of HIV-1C to a monotropic X4 phenotype as the epidemic ages in Botswana. Usage of other coreceptors was rare and apparently insignificant. These results enhance our understanding of HIV-1C biology, with implications for designing and testing therapeutic and prophylactic agents.

Major histocompatibility complex class II (HLA-DRB and -DQB) allele frequencies in Botswana: association with human immunodeficiency virus type 1 infection.

Southern Africa is facing an unprecedented public health crisis due to the high prevalence of human immunodeficiency virus type 1 (HIV-1). Vaccine development and testing efforts, mainly based on elicitation of HIV-specific T cells, are under way. To understand the role of human leukocyte antigen (HLA) class II alleles in HIV pathogenesis and to facilitate HLA-based HIV-1 vaccine design, we analyzed the frequencies of HLA class II alleles within the southern African country of Botswana. Common HLA class II alleles were identified within the Botswana population through the molecular genotyping of DRB and DQB1 loci. The DRB1 allele groups DRB1*01, DRB1*02/15, DRB1*03, DRB1*11, and DRB1*13 were encountered at frequencies above 20%. Within the DQB1 locus, DQB1*06 (47.7%) was the most common allele group, followed by DQB1*03 (39.2%) and DQB1*04 (25.8%). We found that DRB1*01 was more common in HIV-negative than in HIV-positive individuals and that those who expressed DRB1*08 had lower median viral loads. We demonstrate that the frequencies of certain HLA class II alleles in this Botswana population differ substantially from those in North American populations, including African-Americans. Common allele groups within Botswana cover large percentages of other African populations and could be targeted in regional vaccine designs.