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Objective: To study maternal and neonatal effects of combination nucleoside analog therapy administered to human immunodeficiency virus (HIV)-infected pregnant women for maternal indications.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing—a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies.Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases.Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.

Seven Northern Inuit Dogs (NID) were diagnosed by pedigree analysis with an autosomal recessive inherited oculoskeletal dysplasia (OSD). Short-limbed dwarfism, angular limb deformities and a variable combination of macroglobus, cataracts, lens coloboma, microphakia and vitreopathy were present in all seven dogs, while retinal detachment was diagnosed in five dogs. Autosomal recessive OSD caused by COL9A3 and COL9A2 mutations have previously been identified in the Labrador Retriever (dwarfism with retinal dysplasia 1-drd1) and Samoyed dog (dwarfism with retinal dysplasia 2-drd2) respectively; both of those mutations were excluded in all affected NID. Nine candidate genes were screened in whole genome sequence data; only one variant was identified that was homozygous in two affected NID but absent in controls. This variant was a nonsense single nucleotide polymorphism in COL9A3 predicted to result in a premature termination codon and a truncated protein product. This variant was genotyped in a total of 1,232 dogs. All seven affected NID were homozygous for the variant allele (T/T), while 31/116 OSD-unaffected NID were heterozygous for the variant (C/T) and 85/116 were homozygous for the wildtype allele (C/C); indicating a significant association with OSD (p = 1.41x10-11). A subset of 56 NID unrelated at the parent level were analysed to determine an allele frequency of 0.08, estimating carrier and affected rates to be 15% and 0.6% respectively in NID. All 1,109 non-NID were C/C, suggesting the variant is rare or absent in other breeds. Expression of retinal mRNA was similar between an OSD-affected NID and OSD-unaffected non-NID. In conclusion, a nonsense variant in COL9A3 is strongly associated with OSD in NID, and appears to be widespread in this breed.

Sensory neuropathy in the Border Collie is a severe neurological disorder caused by the degeneration of sensory and, to a lesser extent, motor nerve cells with clinical signs starting between 2 and 7 months of age. Using a genome-wide association study approach with three cases and 170 breed matched controls, a suggestive locus for sensory neuropathy was identified that was followed up using a genome sequencing approach. An inversion disrupting the candidate gene FAM134B was identified. Genotyping of additional cases and controls and RNAseq analysis provided strong evidence that the inversion is causal. Evidence of cryptic splicing resulting in novel exon transcription for FAM134B was identified by RNAseq experiments. This investigation demonstrates the identification of a novel sensory neuropathy associated mutation, by mapping using a minimal set of cases and subsequent genome sequencing. Through mutation screening, it should be possible to reduce the frequency of or completely eliminate this debilitating condition from the Border Collie breed population.

Abstract Background Canine progressive retinal atrophies are a group of hereditary retinal degenerations in dogs characterised by depletion of photoreceptor cells in the retina, which ultimately leads to blindness. PRA in the Lhasa Apso (LA) dog has not previously been clinically characterised or described in the literature, but owners in the UK are advised to have their dog examined through the British Veterinary Association/ Kennel Club/ International Sheep Dog Society (BVA/KC/ISDS) eye scheme annually, and similar schemes that are in operation in other countries. After the exclusion of 25 previously reported canine retinal mutations in LA PRA-affected dogs, we sought to identify the genetic cause of PRA in this breed. Results Analysis of whole-exome sequencing data of three PRA-affected LA and three LA without signs of PRA did not identify any exonic or splice site variants, suggesting the causal variant was non-exonic. We subsequently undertook a genome-wide association study (GWAS), which identified a 1.3 Mb disease-associated region on canine chromosome 33, followed by whole-genome sequencing analysis that revealed a long interspersed element-1 (LINE-1) insertion upstream of the IMPG2 gene. IMPG2 has previously been implicated in human retinal disease; however, until now no canine PRAs have been associated with this gene. The identification of this PRA-associated variant has enabled the development of a DNA test for this form of PRA in the breed, here termed PRA4 to distinguish it from other forms of PRA described in other breeds. This test has been used to determine the genotypes of over 900 LA dogs. A large cohort of genotyped dogs was used to estimate the allele frequency as between 0.07–0.1 in the UK LA population. Conclusions Through the use of GWAS and subsequent sequencing of a PRA case, we have identified a LINE-1 insertion in the retinal candidate gene IMPG2 that is associated with a form of PRA in the LA dog. Validation of this variant in 447 dogs of 123 breeds determined it was private to LA dogs. We envisage that, over time, the developed DNA test will offer breeders the opportunity to avoid producing dogs affected with this form of PRA.

Background: Congenital syphilis (CS) is associated with significant perinatal morbidity and mortality. The study objectives were to compare risk factors among women with syphilis infection whose pregnancies did and did not result in CS cases and to evaluate other geographic and socioeconomic characteristics of county of residence as a measure of healthcare inequity., Methods: This study linked maternal and congenital syphilis data from the Georgia Department of Public Health (DPH), 2008-2015. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline was followed. Demographic, behavioral, and case characteristics were compared among women with syphilis infection who did and did not have an infant with CS. Chi-square, Fisher's exact, and multivariate regression analyses were performed using STATA 14.2 (College Station, TX)., Results: Of 505 women with syphilis infection, 23% had an infant with CS, while 77% did not. After adjusting for race/ethnicity, factors associated with CS outcome were age greater than 35 years (adjusted odds ratio (aOR) 3.88; 95% confidence interval (CI) 1.01-14.89), hospital/emergency department diagnosis of syphilis (aOR 3.43; 95% CI 1.54-7.62), and high-risk behaviors such as exchanging sex for money or drugs (aOR 3.25; 95% CI 1.18-8.98). There were no associations between characteristics of county of residence and CS outcome., Conclusions: This study highlights risk factors that may be associated with CS incidence and the adverse pregnancy outcomes associated with CS. Further work is needed to study improved data collection systems, contributing factors related to CS as well as prevention measures in the United States., Competing Interests: None of our authors has any conflict of interest related to this publication. Outside of this work, Dr. Kachikis reported being a coinvestigator on studies funded by Pfizer and Merck outside the submitted work. Dr. Kachikis reported serving as an unpaid research consultant for Pfizer and GlaxoSmithKline on maternal immunization-related projects outside the submitted work., (Copyright © 2023 Alisa Kachikis et al.)

Background: The presence of hydrogen peroxide (H2O2)-producing lactobacilli in the vagina is associated with decreased rates of preterm birth and HIV acquisition. We hypothesize that this is due to immunomodulatory effects of these species., Methods: Concentrations of interleukin (IL)-1β, IL-6, IL-8, secretory leukocyte protease inhibitor, and human β-defensin 2 were quantified from vaginal swabs from 4 groups of women: women with and without bacterial vaginosis (BV) by Nugent score, further stratified by detection of H2O2-producing lactobacilli by semiquantitative culture. Ten quantitative polymerase chain reaction assays characterized the presence and quantity of select Lactobacillus and BV-associated species in each group. Levels of immune markers and bacteria were compared between the 4 groups using analysis of variance, Kruskal-Wallis, Mann-Whitney U, or χ tests., Results: Swabs from 110 women from 4 groups were included: 26 had a normal Nugent score (BV-), and no H2O2-producing lactobacilli detected (H2O2-); 47 were BV-, H2O2+; 27 BV+, H2O2-; and 10 BV+, H2O2+. The groups were similar in age, marital status, and reproductive history, but not ethnicity: the BV-, H2O2- group had more white participants (P = 0.02). In women with and without BV, IL-1β was lower in the H2O2+ groups. Human β-defensin 2 was lowest in BV+ H2O2- women and highest in BV-, H2O2-. Secretory leukocyte protease inhibitor was lower in women with BV and did not differ by the presence of H2O2-producing lactobacilli. In regression analysis, higher quantities of Lactobacillus crispatus were associated with lower quantities of IL-1β. Detection and quantity of BV-associated species by quantitative polymerase chain reaction was significantly different between women with and without BV, but not between women with and without H2O2-producing lactobacilli within those groups., Conclusions: The presence of H2O2-producing lactobacilli is associated with lower levels of some vaginal proinflammatory cytokines, even in women with BV.

Microbial infection of the amniotic fluid is a significant cause of fetal injury, preterm birth, and newborn infections. Group B Streptococcus (GBS) is an important human bacterial pathogen associated with preterm birth, fetal injury, and neonatal mortality. Although GBS has been isolated from amniotic fluid of women in preterm labor, mechanisms of in utero infection remain unknown. Previous studies indicated that GBS are unable to invade human amniotic epithelial cells (hAECs), which represent the last barrier to the amniotic cavity and fetus. We show that GBS invades hAECs and strains lacking the hemolysin repressor CovR/S accelerate amniotic barrier failure and penetrate chorioamniotic membranes in a hemolysin-dependent manner. Clinical GBS isolates obtained from women in preterm labor are hyperhemolytic and some are associated with covR/S mutations. We demonstrate for the first time that hemolytic and cytolytic activity of GBS is due to the ornithine rhamnolipid pigment and not due to a pore-forming protein toxin. Our studies emphasize the importance of the hemolytic GBS pigment in ascending infection and fetal injury.

The effect of the levonorgestrel-releasing intrauterine device (LNG-IUD) on genital HIV-1 RNA shedding and inflammation among 25 HIV-infected women was evaluated. Blood, endocervical, and cervicovaginal lavage samples were collected from HIV-infected women not taking antiretrovirals before LNG-IUD insertion and 1 month, 3 month, and 6 months thereafter. HIV-1 RNA was quantitated by real-time reverse transcriptase-polymerase chain reaction. Inflammatory markers were measured by enzyme immunoassay. Genital HIV-1 RNA shedding and inflammatory markers did not differ between LNG-IUD placement and month 6, with the exception of interleukin 1β that increased (0.42 log10; 95% confidence interval: 0.10 to 0.75). The LNG-IUD did not increase genital HIV-1 RNA shedding after 6 months of use.

Cervical shedding of HIV-1 DNA may influence HIV-1 sexual transmission. HIV-1 DNA was detected in 250 (80%) of 316 and 207 (79%) of 259 cervical cytobrush specimens from 56 US and 80 Kenyan women, respectively. Plasma HIV-1 RNA concentration was associated with increased HIV-1 DNA shedding among US and Kenyan women. Kenyan women had higher cervicovaginal concentrations of proinflammatory interleukins (IL)-1β, IL-6, IL-8, and anti-inflammatory secretory leukocyte protease inhibitor compared with US women (all P < 0.01). HIV-1 DNA shedding was associated with increased concentrations of IL-1β and IL-6 and lower secretory leukocyte protease inhibitor among US women but not Kenyan women.

Background: The presence of hydrogen peroxide (H(2)O(2)) producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H(2)O(2)-producing Lactobacillus detected by culture with quantitative PCR (qPCR) detection of Lactobacillus species commonly assumed to be H(2)O(2)-producers., Methods: Samples were collected as part of a prospective cohort study of HIV-1 seropositive US women. Cervicovaginal lavage specimens were tested for L. crispatus and L. jensenii using 16S rRNA gene qPCR assays. Vaginal swabs were cultured for Lactobacillus and tested for H(2)O(2)-production. We calculated a kappa statistic to assess concordance between culture and qPCR., Results: Culture and qPCR results were available for 376 visits from 57 women. Lactobacilli were detected by culture at 308 (82%) visits, of which 233 of 308 (76%) produced H(2)O(2). L. crispatus and/or L. jensenii were detected at 215 (57%) visits. Concordance between detection of L. crispatus and/or L. jensenii by qPCR and H(2)O(2)-producing Lactobacillus by culture was 75% (kappa = 0.45)., Conclusions: Among HIV-1 seropositive women, there was a moderate level of concordance between H(2)O(2)-producing Lactobacillus detected by culture and the presence of L. crispatus and/or L. jensenii by qPCR. However, one-quarter of samples with growth of H(2)O(2)-producing lactobacilli did not have L. crispatus or L. jensenii detected by qPCR. This discordance may be due to the presence of other H(2)O(2)-producing Lactobacillus species.

Objective: Compare the risk of HIV drug resistance in women stopping suppressive nelfinavir (NFV)-based or Nevirapine (NVP)-based antiretroviral therapy (ART) after pregnancy., Methods: Specimens collected after stopping ART were tested for drug resistance by an oligonucleotide ligation assay and consensus sequencing. When postpartum drug resistance was detected, specimens obtained at study entry and during ART were evaluated., Results: Sixteen of 38 women with ART-induced suppression of viral replication suspended ART postpartum. Resistance mutations were detected in 75% who stopped NFV-ART and in 50% who stopped NVP-ART. M184V, associated with Lamivudine resistance, was more frequent among those randomized to NFV-ART compared with NVP-ART (6 of 8 versus 1 of 8; P = 0.04), and nonnucleoside reverse transcriptase inhibitor resistance was detected in 4 of 8 stopping NVP-ART., Conclusions: HIV drug resistance was frequently observed among women who stopped suppressive NVP-ART or NFV-ART postpartum. This suggests that NFV-ART may have suboptimal potency, that staggering discontinuation of NVP-ART may be warranted, and/or ART adherence may be lax in women who choose to stop ART postpartum.

To assess the volume of genital fluid collected for measuring the HIV-1 RNA level in cervicovaginal fluid, phosphate-buffered saline containing 10 mM LiCl was used. Thirty-eight women provided 275 cervicovaginal specimens. The estimated median volume of cervicovaginal fluid was 0.51 ml (interquartile range, 0.33, 0.69).

Objective: To compare genital HSV shedding among HIV-positive and HIV-negative women., Methods: Women with and without known HIV infection who delivered at the University of Washington Medical Center between 1989-1996 had HSV serologies done as part of clinical care. Genital swabs from HSV-2-seropositive women were evaluated by real-time quantitative HSV DNA PCR., Results: HSV-2 seroprevalence was 71% and 30% among 75 HIV-positive and 3051 HIV-negative women, respectively, (P < .001). HSV was detected at delivery in the genital tract of 30.8% of HIV-seropositive versus 9.5% of HIV-negative women (RR = 3.2, 95% CI 1.6 to 6.5, P = .001). The number of virion copies shed per mL was similar (log 3.54 for HIV positive versus 3.90 for HIV negative, P = .99)., Conclusions: Our study demonstrated that HIV-, HSV-2-coinfected women are more likely to shed HSV at delivery.

Background: HIV-infected women have a high prevalence of human papillomavirus (HPV) infection and are more likely to be infected with HPV genotypes that are considered high-risk and have the potential for progressing to cervical cancer. The currently available HPV vaccines protect against specific HPV genotypes that may not be the most important causes of dysplasia and potentially of cervical cancer in HIV-1-infected women. African women have been underrepresented in the studies of global prevalence of HPV genotypes., Methods: We compared the HPV genotype distribution in HIV-1-infected women from Seattle, Washington, USA and Nairobi, Kenya. The reverse line blot assay and DNA sequencing on cervicovaginal lavage (CVL) specimens were carried out., Results: The most commonly detected HPV types among the women from Seattle were HPV 56, 66, MM8, and 81; in contrast HPV 53, 33, and 58 were the most common HPV genotypes detected in the CVL specimens from the women in the Nairobi cohort. The HPV types associated with low-grade squamous intraepithelial lesions (LSIL) were HPV 53 and HPV 56. HPV types 58, 52, and 16 were associated with high-grade squamous intraepithelial lesions (HSIL)., Conclusions: A better understanding of HPV genotype distribution in the most affected regions of the world is essential to planning effective vaccine strategies if we are unable to demonstrate cross-protection between HPV genotypes included in the present vaccines and those prevalent in the different populations., (Copyright © 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Mycoplasma genitalium is associated with cervicitis and pelvic inflammatory disease in nonpregnant women. We investigated associations between cervical M genitalium, demographic and behavioral risk factors for sexually transmitted infection and preterm birth among low-income Peruvian women., Methods: This case-control study, conducted at the Instituto Nacional Materno Perinatal, Lima, Peru, included 661 cases with a spontaneous preterm birth at <37 weeks and 667 controls who delivered at >or=37 weeks. Within 48 hours after delivery, subjects underwent interviews, medical record review, and collection of cervicovaginal specimens for M. genitalium, Chlamydia trachomatis, and Neisseria gonorrhoeae by nucleic acid amplification testing, and Trichomonas vaginalis by culture. Odds ratios and 95% confidence intervals were calculated for associations between M. genitalium, other genital infections and risk factors, and preterm birth. Multivariable logistic regression was used to adjust for potential confounders., Results: Cervical M. genitalium was detected in 3% of subjects and was significantly associated with C. trachomatis infection (P < 0.001) and preterm birth (4% vs. 2%; adjusted odds ratio: 2.5, 95% confidence interval: 1.2-5.0, P = 0.014), and marginally associated with T. vaginalis (P = 0.05). M. genitalium detection was also associated with younger maternal age (P = 0.003) but not with other risk factors for preterm birth. The association between cervical M. genitalium detection and preterm birth remained significant after adjustment for maternal age and coinfection with C. trachomatis or T. vaginalis., Conclusions: Cervical M. genitalium detection was independently associated with younger maternal age and preterm birth, suggesting that this organism may be an infectious correlate of spontaneous preterm birth.

We compared vaginal proinflammatory cytokine and secretory leukocyte protease inhibitor (SLPI) concentrations among pregnant and nonpregnant women according to bacterial vaginosis (BV) status. One-hundred and twenty-two women at 12-20 weeks' gestation and 133 nonpregnant controls had vaginal concentrations of interleukin (IL)-1β, IL-6, IL-8, and SLPI measured by enzyme immunoassay. Multivariable linear regression was used to evaluate factors independently associated with vaginal cytokine and SLPI response. Pregnancy and BV were both independently associated with increased vaginal concentrations of IL-1β and IL-8; pregnant women had increased concentrations of SLPI, while women with BV had decreased SLPI concentrations.

Background: Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons., Methodology/principal Findings: Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by >/=2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage., Conclusions/significance: In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood.

Understanding the dynamics and spread of human immunodeficiency virus type 1 (HIV-1) within the body, including within the female genital tract with its central role in heterosexual and peripartum transmission, has important implications for treatment and vaccine development. To study HIV-1 populations within tissues, we compared viruses from across the cervix to those in peripheral blood mononuclear cells (PBMC) during effective and failing antiretroviral therapy (ART) and in patients not receiving ART. Single-genome sequences of the C2-V5 region of HIV-1 env were derived from PBMC and three cervical biopsies per subject. Maximum-likelihood phylogenies were evaluated for differences in genetic diversity and compartmentalization within and between cervical biopsies and PBMC. All subjects had one or more clades with genetically identical HIV-1 env sequences derived from single-genome sequencing. These sequences were from noncontiguous cervical biopsies or from the cervix and circulating PBMC in seven of eight subjects. Compartmentalization of virus between genital tract and blood was observed by statistical methods and tree topologies in six of eight subjects, and potential genital lineages were observed in two of eight subjects. The detection of monotypic sequences across the cervix and blood, especially during effective ART, suggests that cells with provirus undergo clonal expansion. Compartmentalization of viruses within the cervix appears in part due to viruses homing to and/or expanding within the cervix and is rarely due to unique viruses evolving within the genital tract. Further studies are warranted to investigate mechanisms producing monotypic viruses across tissues and, importantly, to determine whether the proliferation of cells with provirus sustain HIV-1 persistence in spite of effective ART.

Background: Gingivitis has been linked to adverse pregnancy outcome (APO). Bacterial vaginosis (BV) has been associated with APO. We assessed if bacterial counts in BV is associated with gingivitis suggesting a systemic infectious susceptibilty., Methods: Vaginal samples were collected from 180 women (mean age 29.4 years, SD +/- 6.8, range: 18 to 46), and at least six months after delivery, and assessed by semi-quantitative DNA-DNA checkerboard hybridization assay (74 bacterial species). BV was defined by Gram stain (Nugent criteria). Gingivitis was defined as bleeding on probing at >or= 20% of tooth sites., Results: A Nugent score of 0-3 (normal vaginal microflora) was found in 83 women (46.1%), and a score of > 7 (BV) in 49 women (27.2%). Gingivitis was diagnosed in 114 women (63.3%). Women with a diagnosis of BV were more likely to have gingivitis (p = 0.01). Independent of gingival conditions, vaginal bacterial counts were higher (p < 0.001) for 38/74 species in BV+ in comparison to BV- women. Counts of four lactobacilli species were higher in BV- women (p < 0.001). Independent of BV diagnosis, women with gingivitis had higher counts of Prevotella bivia (p < 0.001), and Prevotella disiens (p < 0.001). P. bivia, P. disiens, M. curtisii and M. mulieris (all at the p < 0.01 level) were found at higher levels in the BV+/G+ group than in the BV+/G- group. The sum of bacterial load (74 species) was higher in the BV+/G+ group than in the BV+/G- group (p < 0.05). The highest odds ratio for the presence of bacteria in vaginal samples (> 1.0 x 104 cells) and a diagnosis of gingivitis was 3.9 for P. bivia (95% CI 1.5-5.7, p < 0.001) and 3.6 for P. disiens (95%CI: 1.8-7.5, p < 0.001), and a diagnosis of BV for P. bivia (odds ratio: 5.3, 95%CI: 2.6 to 10.4, p < 0.001) and P. disiens (odds ratio: 4.4, 95% CI: 2.2 to 8.8, p < 0.001)., Conclusion: Higher vaginal bacterial counts can be found in women with BV and gingivitis in comparison to women with BV but not gingivitis. P. bivia and P. disiens may be of specific significance in a relationship between vaginal and gingival infections.

Unlabelled: The presence of semen in vaginal fluid, as identified by an acid phosphatase spot test, does not influence vaginal proinflammatory cytokine concentrations., Objective: Determine whether semen, as detected by acid phosphatase, influences vaginal cytokines or secretory leukocyte protease inhibitor concentrations., Methods: 138 pregnant women had vaginal fluid collected for Gram stain, acid phosphatase detection by colorimetric assay, and interleukin 1-Beta, interleukin-6, interleukin-8, and secretory leukocyte protease inhibitor measurement by enzyme immunoassay. Results for women with and without acid phosphatase were compared by Mann-Whitney test., Results: Of 138 subjects, 28 (20%) had acid phosphatase detected; of these, only 19 (68%) reported recent intercourse and 3 (11%) had sperm seen on Gram stain. There were no significant differences in proinflammatory cytokine concentrations; however, secretory leukocyte protease inhibitor concentrations were significantly higher among women with acid phosphatase., Conclusions: Proinflammatory cytokine measurement does not appear to be affected by the presence of semen, but secretory leukocyte protease inhibitor is significantly higher when semen is present. Detection of semen by acid phosphatase was associated with higher vaginal SLPI concentrations, however, the presence of semen did not appear to influence vaginal proinflammatory cytokine concentrations.

Objective: To assess toxicities associated with highly active antiretroviral therapy (HAART) among HIV-1-infected pregnant women treated with nevirapine-based regimens according to Mozambican national guidelines., Study Design: Prospective cohort study., Methods: HIV-1-infected antiretroviral-naive pregnant women with CD4 counts < or =350 cells/microL were initiated on nevirapine, lamivudine, and stavudine or zidovudine and followed monthly. Severe hepatotoxicity was defined as aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels > or =5-fold the upper limit of normal. Analyses were stratified by baseline CD4 count (<250 vs. 250-350 cells/microL)., Results: Among 146 pregnant women, 75 (52%) began nevirapine, lamivudine, and zidovudine and 71 (48%) began nevirapine, lamivudine, and stavudine. Overall, 79 (54%) women had CD4 counts <250 cells/microL, 7 (5%) had grade II hepatotoxicity, and 4 (3%) had severe (grade III or IV) hepatotoxicity. All 4 women with severe hepatotoxicity had baseline CD4 counts > or =250 cells/microL (P = 0.02). Rates of skin toxicity, anemia, and peripheral neuropathy did not differ by CD4 cell count group. Overall, 12 (8%) women changed or discontinued HAART as a result of drug toxicity., Conclusions: Severe hepatotoxicity from nevirapine-containing HAART in this cohort of pregnant women was more common at higher CD4 counts (6% vs. 0% among women with CD4 counts > or =250 cells/microL and CD4 counts <250 cells/microL, respectively), suggesting that laboratory monitoring is necessary when administering nevirapine-containing regimens to pregnant women with CD4 counts > or =250 cells/microL.

Objectives: To determine the effects of vaginal, cervical, and endometrial infections on shedding of HIV-1 RNA in the female genital tract., Design: Cross-sectional., Methods: Antiretroviral-naive women from Nairobi, Kenya with CD4 cell counts >or= 350 cells/mul had plasma and endocervical wick samples collected for HIV quantification by real-time RNA reverse transcriptase-polymerase chain reaction. Vaginal and cervical Gram stains and endometrial biopsies were obtained. Vaginal Gram stain was used to diagnose bacterial vaginosis and to quantify Lactobacillus levels., Results: Twenty-six of 50 (52%) women had detectable endocervical HIV-1 RNA with a median endocervical viral load of 1760 copies/ml (range: undetectable to 1 1,030,000 copies/ml). Women with decreased Lactobacillus had 15.8-fold [95% confidence interval (CI), 2.0-123] greater endocervical HIV-1 RNA than women with normal Lactobacillus levels. Women with plasma cell (PC) endometritis [>or= 1 PC/high-power field (hpf)] had a 15.8-fold (95% CI, 2.0-120) higher endocervical HIV RNA level than women without PC endometritis. Both these associations remained after controlling for plasma viral load. Cervicitis (>or= 30 polymorphonuclear leukocytes/hpf), however, was not associated with endocervical HIV-1 RNA shedding (P = 0.81)., Conclusions: In HIV-1-infected, antiretroviral-naive women without symptoms of pelvic inflammatory disease infection, abnormal vaginal flora and inflammatory cells in the endometrium affected HIV-1 shedding from the lower genital tract. These data suggest that both the upper and lower genital tracts contribute to female HIV-1 genital shedding.

Placental efflux transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) protect the developing fetus from exposure to potentially toxic xenobiotics. However, little is known about the expression of these transporters in human placentae of different gestational ages. Therefore, we quantified the expression of P-gp and BCRP in human placentae of different gestational ages. We also measured the expression of various nuclear regulatory factors such as the pregnane xenobiotic factor to determine whether their expression also changes with gestational age. Syncitial microvillous plasma membranes were isolated from human placentae of various gestational ages (60-90 days, 90-120 days, and full-term C-section placentae). P-gp and BCRP expression (protein) in these preparations were measured by Western blot analysis followed by an ELISA. Expression (mRNA) of P-gp, BCRP, and nuclear regulatory factors in the placentae were quantified by quantitative real-time PCR. P-gp expression (relative to that of alkaline phosphatase) was significantly (P < 0.05) higher (44.8-fold as protein; 6.5-fold as mRNA) in early gestational age human placentae (60-90 days) vs. term placentae. In contrast, BCRP (protein and mRNA) and nuclear regulatory factors (mRNA) expression in placental tissue did not change significantly with gestational age. However, placental expression of P-gp and human chorionic gonadotropin-beta (hCG-beta) transcripts was highly correlated (r = 0.73; P < 0.0001; Spearman rank correlation). Expression of P-gp, but not BCRP, decreases dramatically with gestational age in human placentae. This decrease in P-gp expression is not caused by a change in expression of nuclear receptor transcripts but appears to be related to hCG-beta expression. The placental P-gp expression appears to be upregulated in early pregnancy to protect the fetus from xenobiotic toxicity at a time when it is most vulnerable to such toxicity.

This study evaluates the safety, tolerance, and pharmacokinetics of stavudine (d4T) in human immunodeficiency virus (HIV)-infected zidovudine (ZDV)-intolerant/refusing pregnant women and of single-dose d4T in their infants. Women received d4T and lamivudine (3TC) from enrollment until labor. During labor, women received oral 3TC and either intravenous or oral d4T. Infants received ZDV and 3TC for 6 weeks and a single dose of oral d4T at weeks 1 and 6. Mean maternal antenatal d4T pharmacokinetics (terminal plasma half-life [T1/2], 83.5+/-16.8 min; area under the plasma-concentration time curve [AUC0-infinity), 81.6+/-22.0 microg.min/mL; n=6) were not significantly different from those during labor (T(1/2), 87.3+/-24.7 min; AUC0-infinity, 88.1+/-16.6 microg.min/mL; n=6). Umbilical-cord and maternal plasma concentrations were not significantly different from one another. The oral clearance of d4T in infants was significantly greater at week 6 versus week 1 (6.8+/-1.0 vs. 5.6+/-1.2 mL/min/kg). There were no toxicities, in women or infants, that required discontinuation or modification of the study drug. No infants had positive HIV viral diagnostic tests. d4T with or without 3TC is a potential alternative to ZDV for HIV-infected pregnant women.

Objective: To compare the safety of nelfinavir and nevirapine-based antiretroviral treatment in HIV-1-infected pregnant women., Methods: In Pediatric AIDS Clinical Trials Group Protocol 1022, 38 antiretroviral-naive pregnant women at 10-30 weeks' gestation were randomized to nelfinavir or nevirapine with zidovudine plus lamivudine. The study was suspended because of greater than expected toxicity and changes in nevirapine prescribing information. The incidence of treatment-limiting hepatic or cutaneous toxicity was compared between groups for all subjects and for the subset with CD4 cell counts greater than 250 cells/microL at study entry., Results: Toxicity was seen in 1 (5%) of 21 subjects randomized to nelfinavir and 5 (29%) of 17 subjects randomized to nevirapine (P = 0.07). Within the nevirapine group, 1 subject developed fulminant hepatic failure and died, and another developed Stevens-Johnson syndrome. The one adverse event associated with nelfinavir occurred in a subject with a CD4 cell count less than 250 cells/microL. All 5 events among subjects with a CD4 cell count greater than 250 cells/microL were associated with nevirapine (P = 0.04)., Conclusions: Continuous nevirapine may be associated with increased toxicity among HIV-1-infected pregnant women with CD4 cell counts greater than 250 cells/microL, as has been observed in non-pregnant women.

The ubiquitin-proteasome proteolytic pathway is pivotal in most biological processes. Despite a great level of information available for the eukaryotic 26S proteasome-the protease responsible for the degradation of ubiquitylated proteins-several structural and functional questions remain unanswered. To gain more insight into the assembly and function of the metazoan 26S proteasome, a two-hybrid-based protein interaction map was generated using 30 Caenorhabditis elegans proteasome subunits. The results recapitulate interactions reported for other organisms and reveal new potential interactions both within the 19S regulatory complex and between the 19S and 20S subcomplexes. Moreover, novel potential proteasome interactors were identified, including an E3 ubiquitin ligase, transcription factors, chaperone proteins and other proteins not yet functionally annotated. By providing a wealth of novel biological hypotheses, this interaction map constitutes a framework for further analysis of the ubiquitin-proteasome pathway in a multicellular organism amenable to both classical genetics and functional genomics.

The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.