An integrated insight into the response of bacterial communities to anthropogenic contaminants in a river: A case study of the Wonderfonteinspruit catchment area, South Africa.

Bacterial communities in human-impacted rivers and streams are exposed to multiple anthropogenic contaminants, which can eventually lead to biodiversity loss and function. The Wonderfonteinspruit catchment area is impacted by operational and abandoned gold mines, farms, and formal and informal settlements. In this study, we used 16S rRNA gene high-throughput sequencing to characterize bacterial communities in the lower Wonderfonteinspruit and their response to various contaminant sources. The results showed that composition and structure of bacterial communities differed significantly (P<0.05) between less (downstream) and more (upstream) polluted sites. The taxonomic and functional gene dissimilarities significantly correlated with each other, while downstream sites had more distinct functional genes. The relative abundance of Proteobacteria, Bacteroidetes and Actinobacteria was higher at upstream sites, while Acidobacteria, Cyanobacteria, Firmicutes and Verrucomicrobia were prominent at downstream sites. In addition, upstream sites were rich in genera pathogenic and/or potentially pathogenic to humans. Multivariate and correlation analyses suggest that bacterial diversity was significantly (P<0.05) impacted by pH and heavy metals (cobalt, arsenic, chromium, nickel and uranium). A significant fraction (~14%) of the compositional variation was explained by a combination of anthropogenic inputs, of which mining (~6%) was the main contributor to bacterial community variation. Network analysis indicated that bacterial communities had non-random inter- and intra-phyla associations and that the main taxa showed both positive and negative linkages to environmental parameters. Our results suggest that species sorting, due to environmental parameters, was the main process that structured bacterial communities. Furthermore, upstream sites had higher relative abundances of genes involved in xenobiotic degradation, suggesting stronger removal of polycyclic aromatic hydrocarbons and other organic compounds. This study provides insights into the influences of anthropogenic land use on bacterial community structure and functions in the lower Wonderfonteinspruit.

Genetic counseling after implementation of statewide cystic fibrosis newborn screening: Two years' experience in one medical center.

PURPOSE: To study the follow-up of genetic counseling performed in families with a newborn detected with one cystic fibrosis (CF) mutation in a statewide newborn screening pilot program. METHODS: Newborns in Massachusetts with an elevated trypsinogen level on newborn screen who are found to have one mutation for CF on a selected mutation assay undergo sweat testing for CF, and their families receive genetic counseling. The genetic counseling focuses on carrier risk for the parents of the newborn and offers carrier testing. We studied the yield of genetic counseling and the resulting genetic testing performed on the families of infants found to be CF carriers who underwent sweat testing in a single institution. RESULTS: Of 102 newborns evaluated with a single CF mutation, 2 (twins) had sweat test results consistent with CF. A total of 101 families were counseled, and 95 were offered DNA-based CF carrier testing. Eighty-two percent of all parents chose to have CF carrier testing, and in five couples, both members were carriers. One of these couples (whose newborn was only a carrier) had an older child who was unexpectedly found to have CF. CONCLUSIONS: Sweat testing of newborns at increased risk for CF in conjunction with genetic counseling for their parents allows identification of infants with CF, finds couples at high risk for having a child with CF, identifies previously undiagnosed siblings with CF, and allows for potential identification of CF carriers in the extended family.

Tandem mass spectrometric analysis for amino, organic, and fatty acid disorders in newborn dried blood spots: a two-year summary from the New England Newborn Screening Program.

BACKGROUND: Tandem mass spectrometry (MS/MS) is rapidly being adopted by newborn screening programs to screen dried blood spots for >20 markers of disease in a single assay. Limited information is available for setting the marker cutoffs and for the resulting positive predictive values. METHODS: We screened >160 000 newborns by MS/MS. The markers were extracted from blood spots into a methanol solution with deuterium-labeled internal standards and then were derivatized before analysis by MS/MS. Multiple reaction monitoring of each sample for the markers of interest was accomplished in approximately 1.9 min. Cutoffs for each marker were set at 6-13 SD above the population mean. RESULTS: We identified 22 babies with amino acid disorders (7 phenylketonuria, 11 hyperphenylalaninemia, 1 maple syrup urine disease, 1 hypermethioninemia, 1 arginosuccinate lyase deficiency, and 1 argininemia) and 20 infants with fatty and organic acid disorders (10 medium-chain acyl-CoA dehydrogenase deficiencies, 5 presumptive short-chain acyl-CoA dehydrogenase deficiencies, 2 propionic acidemias, 1 carnitine palmitoyltransferase II deficiency, 1 methylcrotonyl-CoA carboxylase deficiency, and 1 presumptive very-long chain acyl-CoA dehydrogenase deficiency). Approximately 0.3% of all newborns screened were flagged for either amino acid or acylcarnitine markers; approximately one-half of all the flagged infants were from the 5% of newborns who required neonatal intensive care or had birth weights <1500 g. CONCLUSIONS: In screening for 23 metabolic disorders by MS/MS, an mean positive predictive value of 8% can be achieved when using cutoffs for individual markers determined empirically on newborns.

A trial of shortened zidovudine regimens to prevent mother-to-child transmission of human immunodeficiency virus type 1. Perinatal HIV Prevention Trial (Thailand) Investigators.

BACKGROUND: The optimal duration of zidovudine administration to prevent perinatal transmission of human immunodeficiency virus type 1 (HIV-1) should be determined to facilitate its use in areas where resources are limited. METHODS: We conducted a randomized, double-blind equivalence trial of zidovudine starting in the mother at 28 weeks' gestation, with 6 weeks of treatment in the infant (the long-long regimen), which is similar to protocol 076; zidovudine starting at 35 weeks' gestation, with 3 days of treatment in the infant (the short-short regimen); a long-short regimen; and a short-long regimen. The mothers received zidovudine orally during labor. The infants were fed formula and were tested for HIV DNA at 1, 45, 120, and 180 days. After the first interim analysis, the short-short regimen was stopped. RESULTS: A total of 1437 women were enrolled. At the first interim analysis, the rates of HIV transmission were 4.1 percent for the long-long regimen and 10.5 percent for the short-short regimen (P=0.004). For the entire study period, the transmission rates were 6.5 percent (95 percent confidence interval, 4.1 to 8.9 percent) for the long-long regimen, 4.7 percent (95 percent confidence interval, 2.4 to 7.0 percent) for the long-short regimen, and 8.6 percent (95 percent confidence interval, 5.6 to 11.6 percent) for the short-long regimen. The rate of in utero transmission was significantly higher with the two regimens with shorter maternal treatment (5.1 percent) than with the two with longer maternal treatment (1.6 percent). CONCLUSIONS: The short-short zidovudine regimen is inferior to the long-long regimen and leads to a higher rate of perinatal HIV transmission. The long-short, short-long, and long-long regimens had equivalent efficacy. However, the higher rate of in utero transmission with the short-long regimen suggests that longer treatment of the infant cannot substitute for longer treatment of the mother.

Anonymous testing of newborn infants for HIV antibodies as a basis for estimating prevalence of HIV in childbearing women: the 1991-1994 study in Spain.

During 1991-1994, anonymous screening of newborn infants for maternal antibody to human immunodeficiency virus (HIV) was carried out in three regions of Spain: Valencia, Galicia and Sevilla. The newborn infants whose heel-stick blood eluates were satisfactory for HIV antibody tests were a consecutive series of 104 876, representing 99.3% of all newborn infants undergoing routine metabolic screening and estimated as comprising at least 98% of all births in the three regions. Enzyme immunoassay (EIA) positives were confirmed by immunoblot, yielding 246 confirmations: a rate of 2.3 per 1000. Seropositivity rates ranged from 1.4 per 1000 in Galicia to 2.1 in Sevilla and 3.1 in Valencia, and remained relatively stable in each region during the years of the study. Within socioeconomically defined subgroups of birth hospitals in Valencia and Galicia, all subgroups contained seropositives, even though there was a twofold to fivefold over-representation in the "inner city" public hospitals. To estimate the proportion of HIV-1-seropositive newborn infants who were positive for HIV-1 DNA, polymerase chain reaction (PCR) assays were performed on 165 dried blood spots that had been retained following positive immunoblot assays. Fifteen (9%) were PCR positive, and when this proportion is adjusted for the age-specific sensitivity of the method, it translates into an estimated HIV-1 transmission rate of 24% (range 18-36%). For 94,906 of the 104,876 newborn infants screened, the EIA used could detect antibodies that react with epitopes of HIV-1 and HIV-2. There were 30 newborn infants whose blood eluate was positive by this combined HIV-1/HIV-2 antibody screen and whose secondary screening with monovalent HIV-2 and HIV-1 EIA indicated that the HIV-2 reactivity was above the cut-off whereas the HIV-1 was not. Ranking these 30 results according to absolute HIV-2 reactivity and relative reactivity with respect to HIV-1 indicated that four infants were probable true HIV-2 seropositives and a total of 12 were possible HIV-2 seropositives, a prevalence of the order of 1:10000 to 1:20000 newborn infants. These anonymous population-based serological studies provide "leading-indicator" data to complement traditional AIDS surveillance for epidemiological and planning purposes.

Transmission of HIV-1 in infants born to seropositive mothers: PCR-amplified proviral DNA detected by flow cytometric analysis of immunoreactive beads.

The diagnosis of HIV infection in newborns is established by amplification of proviral DNA using the polymerase chain reaction (PCR). We developed a nonisotopic method for heminested PCR using a biotinylated primer among sets of three oligonucleotides, each selected from the HIV long terminal repeat (LTR) and gag sequences. An internal probe incorporating digoxigenin-dUTP was also synthesized by PCR. The PCR products, hybridized with LTR region or gag region probes, were captured with streptavidin-coated magnetic beads and detected by fluorescein isothiocyanate-labeled antidigoxigenin in flow cytometric analysis. This immunoreactive bead assay (PCR-IRB) detected about three copies of HIV proviral DNA. A panel of 50 coded DNA specimens of infants previously assayed by conventional PCR and with known clinical results revealed that the PCR-IRB findings using LTR, but not gag, were in agreement. A double-blind prospective study of blood samples from 14 mother-infant pairs using the PCR-IRB amplification of LTR gave results similar to the commercial Amplicor HIV-1 PCR test and were consistent with the clinical outcomes. PCR-IRB results were positive for 11 mothers and three infants, one at birth, one at 2 weeks after birth, and one at 8 weeks after birth. PCR-IRB is a simple, reliable, specific, and automatable assay useful in the early diagnosis of perinatal HIV infection in clinical practice and regional screening programs.

A cost effective base-matching assay with low backgrounds.

Base-matching or so-called mini-sequencing is a powerful technique for genotyping and mutation identification. However, its application is often hampered by high background and high cost. We have decreased the background by approximately 5-fold by incorporating an end-blocking step and using only 1/10 of the usual nucleotide concentrations.

Early detection of human immunodeficiency virus on dried blood spot specimens: sensitivity across serial specimens. Women and Infants Transmission Study Group.

OBJECTIVE: To evaluate the use of dried blood spot (DBS) specimens and the early diagnostic value of the polymerase chain reaction (PCR) for detection of the human immunodeficiency virus (HIV) in DBS specimens collected at predefined age intervals from a large cohort of U.S. infants at risk of congenital or perinatal HIV infection. DESIGN: We assayed available DBS specimens (n = 272) obtained during the first 4 months of life from 144 infants (41 infected, 103 uninfected) born to HIV-infected mothers enrolled in the Women and Infants Transmission Study. The DBS PCR results were compared with infant HIV infection status, PCR on liquid blood, and viral culture results. Analyses also included sensitivity and specificity of assay as related to the age of the infant when the specimen was obtained. RESULTS: The DBS specimen PCR results were concordant with results from liquid blood specimens and with results from viral culture. The DBS PCR was highly specific for all age groups. Sensitivity in detecting HIV infection status rapidly increased during the first month of life, from 19% (5/26) by 1 week to 96% (25/26) by 1 month of age. Specimens obtained on the day of birth or the next day were the least likely to have detectable HIV DNA. CONCLUSIONS: The PCR assay of DBS specimens is a reliable tool for the early diagnosis of HIV infection and has important advantage over that of liquid blood DNA PCR and viral culture. These advantages include a lower volume of blood required for testing, increased safety, and ease of storage or transport of specimens. Thus DBS PCR is a useful test for clinical and epidemiologic tracking of infants at risk of HIV infection.

The utility of IgA antibody to human immunodeficiency virus type 1 in early diagnosis of vertically transmitted infection. National Institute of Allergy and Infectious Diseases and National Institute of Child Health and Human Development Women and Infants Transmission Study Group.

OBJECTIVE: To determine the sensitivity and specificity of anti-human immunodeficiency virus (HIV) IgA in identifying infected infants at or before 6 months of age among the offspring of HIV-infected mothers. DESIGN: Prospective comparison of anti-HIV IgA measurement performed in 2 different laboratories by 2 different methods with the criterion standard of blood culture. SETTING: Five centers in the United States and Puerto Rico. PATIENTS: Population-based sample of 156 infants of HIV-infected mothers in the Women and Infants Transmission Study. MAIN OUTCOME MEASURES: Results of anti-HIV IgA test in relation to the infection status of the infants as measured by blood culture. RESULTS: Six-month plasma or serum samples were first tested in the 2 laboratories. The sensitivity and specificity of anti-HIV IgA in detecting infected infants at this age by laboratories 1 and 2 were 69% and 63% and 100% and 99%, respectively. A look-back study of samples obtained at birth, 1, 2, and 4 months was then performed on all infected children and a matched set of uninfected children. The performance of the test at birth was unsatisfactory in both laboratories (sensitivity 44% and 33%, specificity 43% and 60%), whether peripheral or cord blood was examined. At 1, 2, and 4 months, the sensitivity of the test was lower than at 6 months, but specificity was high. A modest correlation of absent anti-HIV IgA antibody and low percentage of CD4 cells in peripheral blood was seen at 6 months of age. CONCLUSIONS: The anti-HIV IgA test has moderate sensitivity and high specificity for the diagnosis of HIV infection at 6 months of age in the offspring of infected mothers.

Application of the polymerase chain reaction for the detection of HIV in specimens from newborn screening programs.

The use of the polymerase chain reaction (PCR) permits detection of HIV proviral DNA in the universally collected "dried blood spot specimens" of newborn screening programs. Detection of HIV proviral DNA among the annual cohorts of seropositive specimens from ongoing anonymous newborn HIV seroprevalence studies can provide a laboratory-based estimate of maternal-infant transmission rates. Preliminary data suggest that maternal-infant transmission rates may be higher in areas where the newborn seroprevalence rates are highest.

Identifying human immunodeficiency virus infection at birth: application of polymerase chain reaction to Guthrie cards.

Guthrie cards containing dried blood spots from 67 children now known to be infected with human immunodeficiency virus (HIV) and 63 children now classified as having had seroreversion were retrieved from the newborn infant archives from 1986 through 1991 to determine whether the polymerase chain reaction (PCR) could predict the infection at birth. The PCR assays operating at a sensitivity capable of detecting 2 to 10 copies of HIV proviral DNA per microgram were able to detect HIV proviral DNA in 52% (35/67) of the infected neonatal blood specimens. Longer storage times did not decrease PCR positivity rates, an advantage over assays for HIV antibody. Children whose clinical progression has been aggressive had high rates of PCR positivity in neonatal specimens, 50% (7/14) in those with low CD4 cell counts during the first year of life, 71% (10/14) in those with Pneumocystis pneumonia or disseminated cytomegalovirus infection by age 1 year, 62% (18/29) in those with onset of acquired immunodeficiency syndrome by 18 months, and 66% (14/21) in those who died of the disease by 36 months of age. No evidence of HIV proviral DNA was found in any of the 63 specimens from children with seroreversion. We conclude that PCR, using routinely available dried blood spots from neonates, has applications in early diagnosis and in epidemiologic projections going beyond current seroprevalence studies.

Polymerase chain reaction in detecting HIV infection among seropositive infants: relation to clinical status and age and to results of other assays.

The polymerase chain reaction (PCR) for human immunodeficiency virus (HIV) was evaluated using coded blood specimens from infants whose clinical status is now known. A micromethod for the efficient isolation of mononuclear cells from small volumes of blood, and definitions of PCR positivity that took into account the number and purity of these mononuclear cells, were established in an attempt to define parameters for quality assurance. Results of HIV culture, p-24 antigen, and HIV-specific IgA obtained on the same specimens were compared to PCR results. PCR had a specificity of 100% among 83 specimens from 50 babies known to be uninfected. Sensitivity among 26 HIV-infected infants older than 3 months was 98% (44 of 45 specimens); the one negative specimen, which had also been culture negative, gave a positive PCR result on the remaining aliquot when tested after decoding. Among infected infants less than 3 months old, which is an age when diagnosis by other assays is most problematic, PCR identified 10 of 10 patients (10 of 11 specimens) including two younger than one month. Viral culture showed the best concordance with PCR; however, in three infants, positive PCR results were observed several months before positive results were observed by viral culture.

Kinetics, distribution, and biotransformation of the chemical HI-6 in the rat, dog, and rhesus monkey.

Disposition of the bis-pyridinium mono-oxime, HI-6, following intramuscular injection in rats (200 mg/kg bw), beagle dogs (10 and 50 mg/kg bw), and rhesus monkeys (50 mg/kg bw) revealed that the oxime was absorbed rapidly and completely from the site of injection, was distributed rapidly in the tissues, and that tissue concentrations decreased below the limits of detection by 4 h after treatment. No overt signs of toxicity were observed in any species at the concentrations given. Tissue analysis for HI-6 and degradation products was conducted by extraction followed by ion-pair, reverse phase, HPLC chromatography. The estimated plasma half-life values were 20, 40-55, and 25-30 min for rats, dogs, and monkeys, respectively. HI-6 and the degradation products were excreted via the urine. A marked species difference in disposition was observed in that HI-6 selectively accumulated in the diaphragmatic muscle of the rat to a level 10- to 20-fold higher than the level in blood plasma, whereas in the dog and monkey, diaphragmatic concentrations were comparable with those in the plasma. Three degradation products of HI-6 were detected in the plasma of the three species. One excreted product formed spontaneously since it was also detected in buffered solutions used for abiotic stability studies. The second product, the picolinic acid analog of HI-6, appeared to be metabolically formed in vivo. A third product remains unidentified.

Disposition and elimination of BIOLF-143, an antiviral agent, in the rabbit.

BIOLF-143, (N-(dimethylamino)methylene-9- [[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine), an experimental, purine-based, acyclic nucleoside was administered by iv or ip injection to adult, male and female, albino New Zealand rabbits in order to determine: (1) the pharmacokinetic disposition, (2) the route and rate of excretion, (3) the biotransformation, and (4) the acute toxicity of the agent. HPLC analysis of blood plasma concentrations of BIOLF-143 was conducted following iv injections of 50 or 100 mg/kg and ip injections of 250 mg/kg. Tissue levels of BIOLF-143 were analyzed at 60 min following an ip injection of 100 mg/kg. Metabolism/excretion studies were conducted over a 48-hr period following ip injections of BIOLF-143 (100 mg/kg). The nucleoside was rapidly distributed in the body, with the dose-dependent, estimated plasma half-life being 21-44 min. The drug molecule was not extensively bound to proteins, being quantitatively recovered from plasma (94.4 +/- 3.2%) and a variety of tissues (85-100%). The bulk of the drug (80-87%) was recovered in the urine within 48 hr of treatment, with no metabolites or unique, unidentifiable peaks being detected in HPLC chromatograms. No drug residue was found in feces. No overt toxicity or untoward signs of latent toxicity were observed in animals receiving acute doses of BIOLF-143 up to 250 mg/kg ip. A potential target organ might be the kidney since high levels of drug residue were detected 60 min post-treatment and this appeared to be the route of elimination from the body.

Characterization of a RNA virus from the parasite Leishmania.

We were interested in screening a series of isolates of the protozoan Leishmania for the presence of viruses. The experimental procedure we used was based on an enzymatic assay originally developed for viral RNA-dependent RNA polymerases. Simultaneously, total promastigote nucleic acid preparations were analyzed for the presence of viral genome and/or transcripts. Two isolates, both classified as L. braziliensis guyanensis, were found to be positive for RNA polymerase activity and to carry a large (6 kilobases) RNA species. The polymerase reaction products hybridized to the 6-kilobase RNA, believed to be the viral genome. In conjunction with electron microscopical observations these results indicate the presence of an RNA virus in these Leishmania isolates.

Acetaminophen metabolism in vivo by pregnant, fetal, and neonatal guinea pigs.

Acetaminophen (50 mg/kg body weight) was administered by iv injection to pregnant guinea pigs (60-65 days of gestation) and by ip injection to cesarean-derived term (67 days of gestation) pups. At suitable time intervals after treatment, the concentrations of drug, glucuronide (GLU), and sulfate (SO4) in blood plasma, urine, and bile were measured by high-performance liquid chromatography (HPLC). At 60-65 days of gestation, guinea pig fetuses formed both GLU and SO4, an approximate ratio of 2:1 being observed with mean concentrations of the order of 43 and 27 micrograms/mL being measured for GLU and SO4, respectively at 180 min post-treatment. At the same time interval, the major detoxification product found in the blood plasma of the pregnant dams was GLU (104 micrograms/mL) with only minute amounts (4.2 micrograms/mL) of SO4 being detected. In cesarean-derived and acetaminophen-treated pups, euthanized at 2 or 4 hr post-treatment, plasma levels of GLU were approximately twofold higher relative to the concentration of SO4 at both time intervals. Significant differences were not observed in either bile or urine at 2 hr post-treatment but by 4 hr after treatment the levels of GLU found in the bile and urine were two- or threefold higher than those of SO4. In contrast to the adult guinea pig where GLU forms some 90% of the urinary excretory product and SO4 accounts for only 7%, the SO4 pathway of detoxification appears to be of significant importance to the fetal and neonatal animal.

Chromosome location of four genes in Leishmania.

Chromosome-sized DNA molecules from Leishmania isolates (L. mexicana amazonensis, L. mexicana mexicana, L. chagasi, L. major, L. donovani, and L. braziliensis) were separated by orthogonal field alternation gel electrophoresis. The chromosome locations of four genes were mapped. The alpha-tubulin and rRNA genes each mapped to a single chromosome size class. The beta-tubulin and the 5'-spliced-leader-sequence genes were found on more than one chromosome size class and showed variation of hybridization profiles across species.