Vision Screening in Children in the Canton of St. Gallen.

BACKGROUND: Pediatric vision screening has been shown to reduce the prevalence of amblyopia. To assess the current local situation in the Canton of St. Gallen, a survey study was performed. METHODS: A standardized questionnaire was sent to 191 general practitioners and school doctors to obtain information on the currently applied vision screening protocols for children. The questionnaire included 17 questions with multiple answers possible. RESULTS: Sixty-seven participants (35%) answered the survey. Overall, 61 (91%) of the repliers confirmed that some sort of vision screening exists. According to 45 (67%) repliers, a mandatory examination exists. As to the question about the person performing the examination, multiple answers were given; 39 (44%) answered that the secretary or the medical practical assistant performs the vision tests, 15 (17%) indicated the school doctor, and 25 (28%) indicated the general practitioner or the pediatrician. Most screening tests are performed in a general practitioner's practice (32; 40%), some are done in the kindergartens (17; 21%), and others are done in a pediatrician's practice (17; 21%). The majority of children are tested at the age of 5 - 6 years (58, 87%). Sixty-one (91%) of the repliers indicated that at least the children's visual acuity is measured. Acuity is assessed with different methods, mainly, with pictures (20; 30%), numbers (23; 34%), and Snellen optotypes (33; 49%). CONCLUSIONS: There are no consistent regulations regarding vision screening in the Canton of St. Gallen. Actually, rather different screening scenarios exist. Tests are performed by a variety of people in different locations with diverse testing procedures. A standardization of the applied screening procedures at cantonal and national levels seems reasonable.

Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.

BACKGROUND: Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT). METHODS: Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System. RESULTS: Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage. CONCLUSIONS: Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.

Population pharmacokinetic analysis of a nevirapine-based HIV-1 prevention of mother-to-child transmission program in Uganda to assess the impact of different dosing regimens for newborns.

Single-dose nevirapine for mothers and newborns at delivery is the simplest prevention strategy for vertical HIV-1 transmission and hence widely used in resource-constrained settings. HIV-1-positive mothers and newborns received single-dose nevirapine in a prevention of mother-to-child HIV-1 transmission (PMTCT) program in Uganda. In a pharmacokinetic investigation, breast milk and plasma samples of mothers and newborns were collected. The nonlinear mixed-effects modeling approach was suitable for analysis (average: 1.8 samples/matrix/individual). For describing the nevirapine pharmacokinetics in mothers and newborns, a 1-compartment model was demonstrated to be sufficient. The plasma-placenta transfer could be quantified, revealing a transfer fraction of 11% to 25% (with a significant influence of time span between maternal nevirapine intake and birth) and a high transfer rate constant from maternal drug administration. Interindividual variability was moderate between mothers and high between newborns. Simulations revealed that newborns born early (<1 hour) after maternal nevirapine intake would benefit from a 3-fold higher nevirapine dosage (6 mg/kg) after birth for analogous protective plasma concentrations over the first 2 weeks. In contrast, postnatal nevirapine dosage seemed to be dispensable for newborns born late (>24 hours) after maternal nevirapine intake. These dosing recommendations should be evaluated in prospective studies, including additional antiretroviral drugs in accordance with current PMTCT guidelines.

Hematological changes in women and infants exposed to an AZT-containing regimen for prevention of mother-to-child-transmission of HIV in Tanzania.

INTRODUCTION: Tanzanian guidelines for prevention of mother-to-child-transmission of HIV (PMTCT) recommend an antiretroviral combination regimen involving zidovudine (AZT) during pregnancy, single-dosed nevirapine at labor onset, AZT plus Lamivudine (3TC) during delivery, and AZT/3TC for 1-4 weeks postpartum. As drug toxicities are a relevant concern, we assessed hematological alterations in AZT-exposed women and their infants. METHODS AND MATERIALS: A cohort of HIV-positive women, either with AZT intake (n = 82, group 1) or without AZT intake (n = 62, group 2) for PMTCT during pregnancy, was established at Kyela District Hospital, Tanzania. The cohort also included the infants of group 1 with an in-utero AZT exposure ≥4 weeks, receiving AZT for 1 week postpartum (n = 41), and infants of group 2 without in-utero AZT exposure, receiving a prolonged 4-week AZT tail (n = 58). Complete blood counts were evaluated during pregnancy, birth, weeks 4-6 and 12. RESULTS: For women of group 1 with antenatal AZT intake, we found a statistically significant decrease in hemoglobin level, red blood cells, white blood cells, granulocytes, as well as an increase in red cell distribution width and platelet count. At delivery, the median red blood cell count was significantly lower and the median platelet count was significantly higher in women of group 1 compared to group 2. At birth, infants from group 1 showed a lower median hemoglobin level and granulocyte count and a higher frequency of anemia and granulocytopenia. At 4-6 weeks postpartum, the mean neutrophil granulocyte count was significantly lower and neutropenia was significantly more frequent in infants of group 2. CONCLUSIONS: AZT exposure during pregnancy as well as after birth resulted in significant hematological alterations for women and their newborns, although these changes were mostly mild and transient in nature. Research involving larger cohorts is needed to further analyze the impact of AZT-containing regimens on maternal and infant health.

Zidovudine exposure in HIV-1 infected Tanzanian women increases mitochondrial DNA levels in placenta and umbilical cords.

BACKGROUND: Zidovudine (AZT) constitutes part of the recommended regimens for prevention and treatment of HIV-1 infection. At the same time, AZT as well as HIV-1 infection itself may induce mitochondrial damage. In this study, we analyzed the impact of prenatal AZT-exposure on mitochondrial alterations in HIV-infected women and their infants. METHODS: Mitochondrial DNA (mtDNA) levels in placentas of HIV-1 infected Tanzanian women with and without prenatal AZT exposure, and in the umbilical cords of their AZT-exposed/unexposed infants were quantified using real-time PCR. Furthermore, we checked for the most common mitochondrial deletion in humans, the 4977 base pair deletion (dmtDNA4977) as a marker for mitochondrial stress. RESULTS: 83 women fulfilled the inclusion criteria. 30 women had been treated with AZT (median duration 56 days; IQR 43-70 days) while 53 women had not taken AZT during pregnancy. Baseline maternal characteristics in the two groups were similar. The median mtDNA levels in placentas and umbilical cords of women (311 copies/cell) and infants (190 copies/cell) exposed to AZT were significantly higher than in AZT-unexposed women (187 copies/cell; p = 0.021) and infants (127 copies/cell; p = 0.037). The dmtDNA4977 was found in placentas of one woman of each group and in 3 umbilical cords of AZT-unexposed infants but not in umbilical cords of AZT-exposed infants. CONCLUSIONS: Antenatal AZT intake did not increase the risk for the common mitochondrial deletion dmtDNA4977. Our data suggests that AZT exposure elevates mtDNA levels in placentas and umbilical cords possibly by positively influencing the course of maternal HIV-1 infection.

Emergence of minor drug-resistant HIV-1 variants after triple antiretroviral prophylaxis for prevention of vertical HIV-1 transmission.

BACKGROUND: WHO-guidelines for prevention of mother-to-child transmission of HIV-1 in resource-limited settings recommend complex maternal antiretroviral prophylaxis comprising antenatal zidovudine (AZT), nevirapine single-dose (NVP-SD) at labor onset and AZT/lamivudine (3TC) during labor and one week postpartum. Data on resistance development selected by this regimen is not available. We therefore analyzed the emergence of minor drug-resistant HIV-1 variants in Tanzanian women following complex prophylaxis. METHOD: 1395 pregnant women were tested for HIV-1 at Kyela District Hospital, Tanzania. 87/202 HIV-positive women started complex prophylaxis. Blood samples were collected before start of prophylaxis, at birth and 1-2, 4-6 and 12-16 weeks postpartum. Allele-specific real-time PCR assays specific for HIV-1 subtypes A, C and D were developed and applied on samples of mothers and their vertically infected infants to quantify key resistance mutations of AZT (K70R/T215Y/T215F), NVP (K103N/Y181C) and 3TC (M184V) at detection limits of <1%. RESULTS: 50/87 HIV-infected women having started complex prophylaxis were eligible for the study. All women took AZT with a median duration of 53 days (IQR 39-64); all women ingested NVP-SD, 86% took 3TC. HIV-1 resistance mutations were detected in 20/50 (40%) women, of which 70% displayed minority species. Variants with AZT-resistance mutations were found in 11/50 (22%), NVP-resistant variants in 9/50 (18%) and 3TC-resistant variants in 4/50 women (8%). Three women harbored resistant HIV-1 against more than one drug. 49/50 infants, including the seven vertically HIV-infected were breastfed, 3/7 infants exhibited drug-resistant virus. CONCLUSION: Complex prophylaxis resulted in lower levels of NVP-selected resistance as compared to NVP-SD, but AZT-resistant HIV-1 emerged in a substantial proportion of women. Starting AZT in pregnancy week 14 instead of 28 as recommended by the current WHO-guidelines may further increase the frequency of AZT-resistance mutations. Given its impact on HIV-transmission rate and drug-resistance development, HAART for all HIV-positive pregnant women should be considered.

Adherence to combination prophylaxis for prevention of mother-to-child-transmission of HIV in Tanzania.

BACKGROUND: Since 2008, Tanzanian guidelines for prevention of mother-to-child-transmission of HIV (PMTCT) recommend combination regimen for mother and infant starting in gestational week 28. Combination prophylaxis is assumed to be more effective and less prone to resistance formation compared to single-drug interventions, but the required continuous collection and intake of drugs might pose a challenge on adherence especially in peripheral resource-limited settings. This study aimed at analyzing adherence to combination prophylaxis under field conditions in a rural health facility in Kyela, Tanzania. METHODS AND FINDINGS: A cohort of 122 pregnant women willing to start combination prophylaxis in Kyela District Hospital was enrolled in an observational study. Risk factors for decline of prophylaxis were determined, and adherence levels before, during and after delivery were calculated. In multivariate analysis, identified risk factors for declining pre-delivery prophylaxis included maternal age below 24 years, no income-generating activity, and enrolment before 24.5 gestational weeks, with odds ratios of 5.8 (P = 0.002), 4.4 (P = 0.015) and 7.8 (P = 0.001), respectively. Women who stated to have disclosed their HIV status were significantly more adherent in the pre-delivery period than women who did not (P = 0.004). In the intra- and postpartum period, rather low drug adherence rates during hospitalization indicated unsatisfactory staff performance. Only ten mother-child pairs were at least 80% adherent during all intervention phases; one single mother-child pair met a 95% adherence threshold. CONCLUSIONS: Achieving adherence to combination prophylaxis has shown to be challenging in this rural study setting. Our findings underline the need for additional supervision for PMTCT staff as well as for clients, especially by encouraging them to seek social support through status disclosure. Prophylaxis uptake might be improved by preponing drug intake to an earlier gestational age. Limited structural conditions of a healthcare setting should be taken into serious account when implementing PMTCT combination prophylaxis.

Emergence and persistence of minor drug-resistant HIV-1 variants in Ugandan women after nevirapine single-dose prophylaxis.

BACKGROUND: Nevirapine (NVP) single-dose is still a widely used antiretroviral prophylaxis for the prevention of vertical HIV-1 transmission in resource-limited settings. However, the main disadvantage of the Non-nucleoside Reverse Transcriptase Inhibitor (NNRTI) NVP is the rapid selection of NVP-resistant virus with negative implications for subsequent NNRTI-based long-term antiretroviral therapy (ART). Here, we analysed the emergence of drug-resistant HIV-1 including minor variants in the early phase after NVP single-dose prophylaxis and the persistence of drug-resistant virus over time. METHODS AND FINDINGS: NVP-resistant HIV-1 harbouring the K103N and/or Y181C resistance mutations in the HIV-1 reverse transcriptase gene was measured from 1 week up to 18 months after NVP single-dose prophylaxis in 29 Ugandan women using allele-specific PCR assays capable of detecting drug-resistant variants representing less than 1% of the whole viral population. In total, drug-resistant HIV-1 was identified in 18/29 (62%) women; rates increased from 18% to 38% and 44% at week 1, 2, 6, respectively, and decreased to 18%, 25%, 13% and 4% at month 3, 6, 12 and 18, respectively. The proportion of NVP-resistant virus of the total viral population was significantly higher in women infected with subtype D (median 40.5%) as compared to subtype A (median 1.3%; p = 0.032, Mann-Whitney U test). 33% of resistant virus was not detectable at week 2 but was for the first time measurable 6-12 weeks after NVP single-dose prophylaxis. Three (10%) women harboured resistant virus in proportions >10% still at month 6. CONCLUSIONS: Current WHO guidelines recommend an additional postnatal intake of AZT and 3TC for one week to avoid NVP resistance formation. Our findings indicate that a 1-week medication might be too short to impede the emergence of NVP resistance in a substantial proportion of women. Furthermore, subsequent NNRTI-based ART should not be started earlier than 12 months after NVP single-dose prophylaxis.

Intraoperative conversion rate to a large, limbal opening in minimally invasive strabismus surgery (MISS).

BACKGROUND: To evaluate which factors predispose to an intraoperative conversion to the usual limbal approach in minimally invasive strabismus surgery (MISS). METHODS: This study included 451 consecutive patients operated on by one surgeon at Kantonsspital St Gallen, Switzerland, with minimally invasive rectus muscle surgery between February 2003 and December 2007. We evaluated the intraoperative conversion rate to the usual limbal approach over time, and performed a retrospective determination of date of surgery, age of patient, motility of the eye, primary or revision surgery, and the type and dose of surgery in 982 consecutive rectus muscle procedures. RESULTS: In 3.6% (35/982) of MISS procedures, an intraoperative conversion to a large, limbal approach was necessary. The overall conversion rate decreased over time, from 8.4% in 2003 to 0.4% in 2007. The multivariate regression analysis showed a significant negative influence between the date of surgery and the conversion rate (p < 0.005). Muscle resections were associated with a higher conversion rate (p < 0.001). The other evaluated factors had no significant influence on an intraoperative enlargement of the conjunctival opening. CONCLUSIONS: This study confirms the reliability of the new MISS technique, and shows a low conversion rate to the usual limbal approach. The conversion rate decreased over time with increasing surgical experience. Muscle resections were associated with a higher conversion rate, while the age of the patient, the motility of the eye, revision surgery and the dose of surgery had no significant influence on an intraoperative conversion.

Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis.

Compound A3 was identified in a high-throughput screen for inhibitors of influenza virus replication. It displays broad-spectrum antiviral activity, and at noncytotoxic concentrations it is shown to inhibit the replication of negative-sense RNA viruses (influenza viruses A and B, Newcastle disease virus, and vesicular stomatitis virus), positive-sense RNA viruses (Sindbis virus, hepatitis C virus, West Nile virus, and dengue virus), DNA viruses (vaccinia virus and human adenovirus), and retroviruses (HIV). In contrast to mammalian cells, inhibition of viral replication by A3 is absent in chicken cells, which suggests species-specific activity of A3. Correspondingly, the antiviral activity of A3 can be linked to a cellular protein, dihydroorotate dehydrogenase (DHODH), which is an enzyme in the de novo pyrimidine biosynthesis pathway. Viral replication of both RNA and DNA viruses can be restored in the presence of excess uracil, which promotes pyrimidine salvage, or excess orotic acid, which is the product of DHODH in the de novo pyrimidine biosynthesis pathway. Based on these findings, it is proposed that A3 acts by depleting pyrimidine pools, which are crucial for efficient virus replication.

Minor drug-resistant HIV type-1 variants in breast milk and plasma of HIV type-1-infected Ugandan women after nevirapine single-dose prophylaxis.

BACKGROUND: Nevirapine single-dose (NVP-SD) reduces mother-to-child transmission of HIV type-1 (HIV-1), but frequently induces resistance mutations in the HIV-1 genome. Little is known about drug-resistant HIV-1 variants in the breast milk of women who have taken NVP-SD. METHODS: Blood and breast milk samples of 39 HIV-1-infected Ugandan women were taken 6-12 weeks after NVP-SD intake. Samples were analysed by population sequencing and allele-specific real-time PCR (AS-PCR) with detection limits for NVP-resistant HIV-1 variants (K103N and Y181C) of < 1% of the total viral population. RESULTS: AS-PCR results for both plasma and breast milk were obtained for 19 women who constituted the final study group (HIV-1 subtype frequencies were A1 n = 11, D n = 5, G n = 2 and C n = 1). A total of 7 (37%) and 10 (53%) women carried NVP-resistant virus in breast milk and plasma, respectively. Overall, 71% (5/7) women with NVP-resistant HIV-1 in breast milk displayed >1 drug-resistant variant. Resistance in breast milk was higher at week 6 (6/13 samples [46%]) compared with week 12 (1/6 samples [17%]). In total, 10 drug-resistant populations harbouring the K103N and/or Y181C mutation were detected in the 19 breast milk samples; 7 (70%) were caused by resistant minorities (< 5% of the total HIV-1 population). In the four women with drug-resistant virus in both plasma and breast milk, the mutation patterns differed between the two compartments. CONCLUSIONS: Minor populations of drug-resistant HIV-1 were frequently found in breast milk of Ugandan women after exposure to NVP-SD. Further studies need to explore the role of minor drug-resistant variants in the postnatal transmission of (resistant) HIV-1.

Strabismus and discrimination in children: are children with strabismus invited to fewer birthday parties?

AIM: To determine the social acceptance of children with strabismus by their peers and to determine the age at which the negative impact of strabismus on psychosocial interactions emerges. METHODS: Photographs of six children were digitally altered in order to create pictures of identical twins except for the position of the eyes (orthotropic, exotropic and exotropic) and the colour of the shirt. One hundred and eighteen children aged 3-12 years were asked to select, for each of the six twin pairs, one of the twins to invite to their birthday party. The grouping of the pictures and the composition of the twin pairs were determined by Latin squares. RESULTS: Children younger than 6 years old did not make any significant distinctions between orthotropic children and children with strabismus. Respondents aged 6 years or older invited children with a squint to their birthday parties significantly less often than orthotropic children. The authors found no impact (p>0.1) of gender, of the colour of the shirt or of the type of strabismus, but did find a highly significant impact of age on the number of invited children with strabismus. CONCLUSIONS: Children aged 6 years or older with a visible squint seem to be less likely to be accepted by their peers. Because this negative attitude towards strabismus appears to emerge at approximately the age of 6 years, corrective surgery for strabismus without prospects for binocular vision should be performed before this age.

The perception of strabismus by children and adults.

BACKGROUND: Visible strabismus has been shown to have adverse psychosocial consequences. It remains controversial if esotropia or exotropia is perceived more negatively. The aim of this study was to determine if esotropia or exotropia and the eye (side) in which strabismus is present are perceived differently. We also asked our adult participants: (1) if they thought visible strabismus should be corrected by surgery, (2) if they thought that strabismus surgery should only be to improve the cosmesis, and (3) if they thought that the surgery should be paid for by health insurance. METHODS: One hundred adults and 61 children rated four photographs of a digitally altered picture of a boy and four of a girl, showing a large-angle esotropia or exotropia either in the left or on the right eye. The adults were additionally asked if a squint should be operated, if they considered strabismus surgery to be a cosmetic procedure, if in their opinion strabismus surgery should be covered by compulsory health insurance, and if children with strabismus are disadvantaged. Comparisons were performed using ANOVA and regression analysis. RESULTS: Adults perceived a squinting right eye as more disturbing than a squinting left eye p < 0.001). The direction of strabismus, the age, gender, and the number of persons with a squint among family and friends of the respondents did not influence the perception of strabismus by adults (p > 0.1 for each). Children also found that a squinting right eye is more disturbing (p < 0.001) than a left one. Additionally, children ranked esotropia worse than exotropia (p < 0.001). Neither age nor gender had an impact on the perception of strabismus by children. Of the adults, 94% would recommend surgery for all forms of strabismus, 18% thought that surgery is only cosmetic, and 94% found that health insurance should cover strabismus surgery for everybody. Problems of squinting children named by the adults included: being made fun of by other children (53%), problems with eyesight (39%), people looking strangely at them (21%), less acceptance by peers (17%), less self confidence (6%), problems judging distances (4%), and that they are perceived as less intelligent (3%). CONCLUSIONS: Adults and children rated a squinting right eye as worse compared to a left one. Children perceived esotropia as more disturbing than exotropia. Neither age, nor gender, nor the fact that the respondents have friends or family members with a squint, had an impact on this ranking. Almost all adults would correct all forms of strabismus, and think that surgery should be covered by compulsory health insurance.

Moderate influence of human APOBEC3F on HIV-1 replication in primary lymphocytes.

Multiple APOBEC3 proteins are expressed in HIV-1 target cells, but their individual contributions to viral suppression when expressed at endogenous levels remain largely unknown. We used an HIV NL4-3 mutant that selectively counteracts APOBEC3G (A3G) but not APOBEC3F (A3F) to dissect the relative contribution of A3F to the inhibition of HIV-1 replication in primary human lymphocytes (peripheral blood mononuclear cells [PBMCs]). This HIV Vif mutant replicated similarly to wild-type virus in PBMCs, suggesting that the effect of A3F on HIV restriction in these cells is limited. The different A3F variants found in PMBC donors displayed either comparable activity or less activity than wild-type A3F. Lastly, the endogenous A3F mRNA and protein expression levels in PBMCs were considerably lower than those of A3G. Our results suggest that A3F neutralization is dispensable for HIV-1 replication in primary human T-lymphocytes.

Effects of moxifloxacin and clinafloxacin on murine limb buds cultured in regular and in magnesium-deficient medium.

Evaluation of the prenatal toxicity of a substance in rats or other animals according to the current guidelines is often hampered by the rapid metabolism of the test compound and/or by maternal toxicity. One example for such a compound is moxifloxacin. In vitro systems offer the possibility to study the direct effects of the test compound on embryonic tissues. The aim of this study was to evaluate the embryotoxic potential of moxifloxacin in vitro using the murine limb bud culture. Clinafloxacin, which was found to be teratogenic when tested in rats, was used for comparison. The effects of various concentrations of moxifloxacin (10, 30, 60 and 100 mg/L) and clinafloxacin (3, 10 and 30 mg/L) on growth and differentiation of 12-day-old murine limb buds were studied in a standard and in a magnesium-deficient medium. After termination of the culture, the respective front limb buds were examined by different methods. Clinafloxacin showed clear-cut effects at a concentration of 30 mg/L in both media. Effects were similarly pronounced as the effects observed with moxifloxacin at a concentration of 100 mg/L. Lower concentrations of moxifloxacin, which are achieved during therapy in humans, did not impair growth and differentiation of limb buds. Using electron microscopy, slight ultrastructural changes could be seen after exposure to 3 mg clinafloxacin/L medium. Ultrastructurally, clinafloxacin caused a concentration-dependent decrease of the extracellular matrix, swelling of cell organelles and at higher concentrations necrotic chondrocytes. These effects were significantly enhanced in a magnesium-deficient medium. In conclusion, the effects of moxifloxacin on murine limb buds in vitro were definitely less pronounced than those of clinafloxacin. Effects on growth and differentiation occurred with moxifloxacin only at concentrations that are higher than plasma concentrations observed during therapy. This result is of special interest, because due to rapid metabolism of moxifloxacin in rats results from a routinely performed segment II type study cannot be used for a risk assessment.

Stimulus parameters for goldmann kinetic perimetry in nonorganic visual loss.

OBJECTIVE: To investigate the influence of the stimulus parameters on perimetry at various distances and draw conclusions for the clinical exploration of nonorganic visual loss. METHODS: Visual field testing using Goldmann kinetic perimetry was performed on 15 healthy volunteers. The I/1e isopter at 33 cm was compared to the I/1e, II/1e and I/2e isopters at 66 cm. The 0/1e isopter at 33 cm was compared to the 0/1e, I/1e and 0/2e isopters at 66 cm. RESULTS: Doubling the examination distance without adjusting the stimulus parameters resulted in significant perimetric visual field constriction. Doubling the stimulus diameter resulted in perimetric visual field expansion by a factor of 2.26 and 3.32 for I/1e and 0/1e, respectively. Increasing stimulus luminance by a factor of 3.17 caused expansion by a factor of 2.15 and 2.32 for I/1e and 0/1e, respectively. CONCLUSIONS: To avoid falsely diagnosing visual field constriction, stimulus parameters need to be adjusted when visual field testing is performed at double distance. Increasing stimulus luminance was more appropriate than augmenting stimulus size.

Detection and quantification of minor human immunodeficiency virus type 1 variants harboring K103N and Y181C resistance mutations in subtype A and D isolates by allele-specific real-time PCR.

Nevirapine (single dose), commonly used to prevent the mother-to-child transmission of human immunodeficiency virus (HIV) in developing countries, frequently induces viral resistance. Even mutations which occur only in a minor population of the HIV quasispecies (<20%) are associated with subsequent treatment failure but cannot be detected by population-based sequencing. We developed sensitive allele-specific real-time PCR (ASPCR) assays for two key resistance mutations of nevirapine. The assays were specifically designed to analyze HIV-1 subtype A and D isolates accounting for the majority of HIV infections in Uganda. Assays were evaluated using DNA standards and clinical samples of Ugandan women having preventively taken single-dose nevirapine. Lower detection limits of drug-resistant HIV type 1 (HIV-1) variants carrying reverse transcriptase mutations were 0.019% (K103N [AAC]), 0.013% (K103N [AAT]), and 0.29% (Y181C [TGT]), respectively. Accuracy and precision were high, with coefficients of variation (the standard ratio divided by the mean) of 0.02 to 0.15 for intra-assay variability and those of 0.07 to 0.15 (K103N) and 0.28 to 0.52 (Y181C) for inter-assay variability. ASPCR assays enabled the additional identification of 12 (20%) minor drug-resistant HIV variants in the 20 clinical Ugandan samples (3 mutation analyses per patient; 60 analyses in total) which were not detectable by population-based sequencing. The individual patient cutoff derived from the clinical baseline sample was more appropriate than the standard-based cutoff from cloned DNA. The latter is a suitable alternative since the presence/absence of drug-resistant HIV-1 strains was concordantly identified in 92% (55/60) of the analyses. These assays are useful to monitor the emergence and persistence of drug-resistant HIV-1 variants in subjects infected with HIV-1 subtypes A and D.

Male involvement in PMTCT services in Mbeya Region, Tanzania.

Throughout all stages of programmes for the prevention of mother-to-child-transmission of HIV (PMTCT), high dropout rates are common. Increased male involvement and couples' joint HIV counselling/testing during antenatal care (ANC) seem crucial for improving PMTCT outcomes. Our study assessed male attitudes regarding partner involvement into ANC/PMTCT services in Mbeya Region, Tanzania, conducting 124 individual interviews and six focus group discussions. Almost all respondents generally supported PMTCT interventions. Mentioned barriers to ANC/PMTCT attendance included lacking information/knowledge, no time, neglected importance, the services representing a female responsibility, or fear of HIV-test results. Only few perceived couple HIV counselling/testing as disadvantageous. Among fathers who had refused previous ANC/PMTCT attendance, most had done so even though they were not perceiving a disadvantage about couple counselling/testing. The contradiction between men's beneficial attitudes towards their involvement and low participation rates suggests that external barriers play a large role in this decision-making process and that partner's needs should be more specifically addressed in ANC/PMTCT services.

Persistence of nevirapine in breast milk and plasma of mothers and their children after single-dose administration.

OBJECTIVES: Nevirapine is widely used in the developing world for the prevention of mother-to-child transmission (PMTCT) of HIV. A single mutation in the HIV genome is sufficient to lead to significant nevirapine resistance. Persistence of low-level drug concentrations in body compartments can foster resistance formation. In this study, concentration-time courses of nevirapine after single-dose administration were analysed over an extended post-partum period. PATIENTS AND METHODS: Breast milk and plasma samples of 62 HIV-positive Ugandan mother-child pairs who had received single-dose nevirapine were collected at delivery and 1, 2 and 6 weeks post-partum. Nevirapine concentrations were quantified by LC/tandem-mass-spectrometry using a quantification limit of 15 ng/mL, and a population pharmacokinetic (PK) analysis was performed. RESULTS: Concentration-time profiles in breast milk, maternal plasma and child plasma showed similar shapes. At week 1, median nevirapine concentrations were 164 ng/mL in maternal plasma, 114 ng/mL in breast milk and 183 ng/mL in child plasma. The population PK model predicted nevirapine concentrations>10 ng/mL (IC50 for nevirapine) for 13 days in breast milk, 14 days in maternal plasma and 18 days in child plasma in 80% of the samples. CONCLUSIONS: Nevirapine concentrations were present for 2-3 weeks in the three compartments. The concentrations are probably sufficiently high to protect most breastfed children from HIV transmission during the first 2 weeks. The long presence of slowly decreasing levels of nevirapine is likely to induce resistance formation. Post-natal addition of antiretrovirals for 1 week only, as recommended in the current PMTCT guidelines, will not suffice to avoid nevirapine resistance formation.

Risk factors for treatment denial and loss to follow-up in an antiretroviral treatment cohort in Kenya.

OBJECTIVES: To evaluate risk factors for treatment denial and loss to follow-up in an antiretroviral treatment (ART) cohort in a rural African setting in western Kenya. METHOD: Sociodemographic and clinical data of patients enrolled in an ART cohort were collected within 18 months of an observational longitudinal study and analysed by logistic and Cox regression models. RESULTS: Of 159 patients with treatment indication 35 (22%) never started ART. Pregnancy [adjusted odds ratio (AOR) 3.60, 95% confidence interval (CI) 1.10-11.8; P = 0.035] and lower level of education (AOR 3.80, 95% CI 1.14-12.7; P = 0.03) were independently associated with treatment denial. The incidence of total loss of patients under therapy was 43.2 per 100 person years (pys) (mortality rate 19.2 per 100 pys plus drop out rate 24 per 100 pys). Older age [adjusted hazard ratio (AHR) 1.06, 95% CI 1.01-1.12; P = 0.04], AIDS before starting treatment (AHR 5.83, 95% CI 1.15-29.5; P = 0.03) and incomplete adherence to treatment (AHR 1.05, 95% CI 1.03-1.07; P < 0.001) were independent risk factors for death. Incomplete adherence also independently predicted drop out because of other reasons (AHR 1.06, 95% CI 1.04-1.09; P < 0.001). CONCLUSION: Pregnancy and lower level of education, higher age, advanced AIDS stage and impaired compliance to ART were identified as risk factors for treatment denial and death, respectively. Adequate counselling strategies for patients with these characteristics could help to improve adherence and outcome of treatment programmes in resource-limited settings.