The effect of antenatal isoniazid preventive therapy on birth outcomes in Western Kenya.

BACKGROUND: Pregnant women living with HIV (WLHIV) are at high risk for TB. There are limited data to inform whether TB preventive therapy is safe in pregnancy.METHODS: We completed a retrospective study of antenatal and birth records of mother-infant dyads at two health care facilities in Kisumu, Kenya. Among pregnant WLHIV, we assessed the relationship of antenatal isoniazid preventive therapy (IPT) with birth outcomes (preterm birth, low birth weight [LBW], congenital anomalies, and perinatal death).RESULTS: Of 576 mother-infant pairs, most women were on antiretroviral therapy (574, 99.7%) with viral suppression (518, 89.9%) and one-quarter had IPT exposure during pregnancy (152, 26.4%). The prevalence of preterm birth was lower among women with antenatal IPT exposure (21% vs. 30%; P = 0.03). LBW, congenital anomaly and perinatal death were not associated with antenatal IPT; however, we observed a trend toward fewer composite poor birth outcomes among women taking antenatal IPT (26% vs 33%; P = 0.08). Controlling for maternal age and viral load, IPT use during pregnancy was associated with lower odds of preterm birth (aOR 0.62, 95% CI 0.40-0.98; P = 0.04).CONCLUSION: In a programmatic setting in Western Kenya, IPT use was not associated with adverse birth outcomes.

16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria.

AIMS: Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer. METHODS AND RESULTS: Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture. CONCLUSIONS: Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare. SIGNIFICANCE AND IMPACT OF THE STUDY: We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.

Integrating tuberculosis screening in Kenyan Prevention of Mother-To-Child Transmission programs.

BACKGROUND: Tuberculosis (TB) screening in Prevention of Mother-To-Child Transmission (PMTCT) programs is important to improve TB detection, prevention and treatment. METHODS: As part of a national PMTCT program evaluation, mother-infant pairs attending 6-week and 9-month immunization visits were enrolled at 141 maternal and child health clinics throughout Kenya. Clinics were selected using population-proportion-to-size sampling with oversampling in a high human immunodeficiency virus (HIV) prevalence region. The World Health Organization (WHO) TB symptom screen was administered to HIV-infected mothers, and associations with infant cofactors were determined. RESULTS: Among 498 HIV-infected mothers, 165 (33%) had a positive TB symptom screen. Positive maternal TB symptom screen was associated with prior TB (P = 0.04). Women with a positive TB symptom screen were more likely to have an infant with HIV infection (P = 0.02) and non-specific TB symptoms, including cough (P = 0.003), fever (P = 0.05), and difficulty breathing (P = 0.01). TB exposure was reported by 11% of the women, and 15% of the TB-exposed women received isoniazid preventive therapy. CONCLUSIONS: Postpartum HIV-infected mothers frequently had a positive TB symptom screen. Mothers with a positive TB symptom screen were more likely to have infants with HIV or non-specific TB symptoms. Integration of maternal TB screening and prevention into PMTCT programs may improve maternal and infant outcomes.

Incidence and correlates of tuberculosis IGRA conversion among HIV-infected postpartum women.

SETTING: Prevention of maternal-to-child transmission program at a tertiary care hospital in Nairobi, Kenya. The risk of acquiring Mycobacterium tuberculosis infection among peripartum human immunodeficiency virus (HIV) infected women is poorly defined. OBJECTIVE: To determine the incidence of and co-factors for interferon-gamma release assay (IGRA) conversion among postpartum HIV-infected women using T-SPOT.TB. DESIGN: We used data and cryopreserved peripheral blood mononuclear cells from a historical cohort of HIV-infected women enrolled at 32 weeks' gestation and followed for 1 year postpartum between 1999 and 2005. RESULTS: Of 89 women initially IGRA-negative during pregnancy, 11 (12.4%) became positive, 53 (59.5%) remained negative and 25 (28.1%) were indeterminate at 1 year postpartum. Mean interferon-gamma (IFN-γ) response among converters increased from ~1 to >50 spot-forming cells/well (P = 0.015). IGRA conversion was significantly associated with partner HIV infection, flush toilets, maternal illness and cough during follow-up, but not maternal CD4 count or HIV viral load. CONCLUSION: The high rates of IGRA conversion seen among HIV-infected postpartum women in our study are similar to those of other groups at high risk for M. tuberculosis infection. This has important implications for M. tuberculosis infection screening strategies and provision of preventive therapy for the health of women and their infants.

Tuberculosis interferon-gamma responses in the breast milk of human immunodeficiency virus infected mothers.

Tuberculosis (TB) cellular immune responses were examined in the breast milk of human immunodeficiency virus infected mothers using the T-SPOT. TB interferon-gamma release assay (IGRA). Positive TB interferon-gamma (IFN-γ) responses were detected in 6 of 8 (75%) valid breast milk assays. Among 7 mothers with paired breast milk and blood assays, TB IFN-γ responses were higher in breast milk than in blood (P = 0.02). The magnitude of TB IFN-γ responses in maternal breast milk and blood were correlated. Elucidating the influence of TB immune responses in breast milk on infant TB susceptibility and immunity may inform future maternal TB vaccine strategies.

Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy.

In HIV-1-infected individuals on currently recommended antiretroviral therapy (ART), viremia is reduced to <50 copies of HIV-1 RNA per milliliter, but low-level residual viremia appears to persist over the lifetimes of most infected individuals. There is controversy over whether the residual viremia results from ongoing cycles of viral replication. To address this question, we conducted 2 prospective studies to assess the effect of ART intensification with an additional potent drug on residual viremia in 9 HIV-1-infected individuals on successful ART. By using an HIV-1 RNA assay with single-copy sensitivity, we found that levels of viremia were not reduced by ART intensification with any of 3 different antiretroviral drugs (efavirenz, lopinavir/ritonavir, or atazanavir/ritonavir). The lack of response was not associated with the presence of drug-resistant virus or suboptimal drug concentrations. Our results suggest that residual viremia is not the product of ongoing, complete cycles of viral replication, but rather of virus output from stable reservoirs of infection.

Suppression of murine cytomegalovirus (MCMV) replication with a DNA vaccine encoding MCMV M84 (a homolog of human cytomegalovirus pp65).

The cytotoxic T-lymphocyte (CTL) response against the murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) 89-kDa phosphoprotein pp89 plays a major role in protecting BALB/c mice against the lethal effects of the viral infection. CTL populations specific to MCMV early-phase and structural antigens are also generated during infection, but the identities of these antigens and their relative contributions to overall immunity against MCMV are not known. We previously demonstrated that DNA vaccination with a pp89-expressing plasmid effectively generated a CTL response and conferred protection against infection (J. C. Gonzalez Armas, C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 70:7921-7928, 1996). In this report, we have sought (i) to identify other viral antigens that contribute to immunity against MCMV and (ii) to determine whether the protective response is haplotype specific. DNA immunization was used to test the protective efficacies of plasmids encoding MCMV homologs of human cytomegalovirus (HCMV) tegument (M32, M48, M56, M82, M83, M69, and M99), capsid (M85 and M86), and nonstructural antigens (IE1-pp89 and M84). BALB/c (H-2(d)) and C3H/HeN (H-2(k)) mice were immunized by intradermal injection of either single plasmids or cocktails of up to four expression plasmids and then challenged with sublethal doses of virulent MCMV administered intraperitoneally. In this way, we identified a new viral gene product, M84, that conferred protection against viral replication in the spleens of BALB/c mice. M84 is expressed early in the infection and encodes a nonstructural protein that shares significant amino acid homology with the HCMV UL83-pp65 tegument protein, a major target of protective CTLs in humans. Specificity of the immune response to the M84 protein was confirmed by showing that immunization with pp89 DNA, but not M84 DNA, protected mice against subsequent infection with an MCMV deletion mutant lacking the M84 gene. The other MCMV genes tested did not generate a protective response even when mice were immunized with vaccinia viruses expressing the viral proteins. However, the M84 plasmid was protective when injected in combination with nonprotective plasmids, and coimmunization of BALB/c mice with pp89 and M84 provided a synergistic level of protection in the spleen. Viral titers in the salivary glands were also reduced, but not to the same extent as observed in the spleen, and the decrease was seen only when the BALB/c mice were immunized with pp89 plus M84 or with pp89 alone. The experiments with the C3H/HeN mice showed that the immunity conferred by DNA vaccination was haplotype dependent. In this strain of mice, only pp89 elicited a protective response as measured by a reduction in spleen titer. These results suggest that DNA immunization with the appropriate combination of CMV genes may provide a strategy for improving vaccine efficacy.

In vivo replication, latency, and immunogenicity of murine cytomegalovirus mutants with deletions in the M83 and M84 genes, the putative homologs of human cytomegalovirus pp65 (UL83).

We previously identified two open reading frames (ORFs) of murine cytomegalovirus (MCMV), M83 and M84, which are putative homologs of the human cytomegalovirus (HCMV) UL83 tegument phosphoprotein pp65 (L. D. Cranmer, C. L. Clark, C. S. Morello, H. E. Farrell, W. D. Rawlinson, and D. H. Spector, J. Virol. 70:7929-7939, 1996). In this report, we show that unlike the M83 gene product, the M84 protein is expressed at early times in the infection and cannot be detected in the virion. To elucidate the functional differences between the two pp65 homologs in acute and latent MCMV infections, we constructed two MCMV K181 mutants in which either the M83 or M84 ORF was deleted. The resultant viruses, designated DeltaM83 and DeltaM84, respectively, were found to replicate in NIH 3T3 cells with kinetics identical to those of the parent strain. Western blot analysis demonstrated that except for the absence of M83 or M84 protein expression in the respective mutants, no global perturbations of protein expression were detected. When DeltaM83 and DeltaM84 were inoculated intraperitoneally (i.p.) into BALB/c mice, both viruses showed similar attenuated growth in the spleen, liver, and kidney. However, only DeltaM83 was severely growth restricted in the salivary glands, a phenotype that was abolished upon restoration of the M83 ORF. DeltaM83's growth was similarly restricted in the salivary glands of the resistant C3H/HeN or highly sensitive 129/J strain, as well as in the lungs of all three strains following intranasal inoculation. Using a nested-PCR assay, we found that both DeltaM83 and DeltaM84 established latency in BALB/c mice, with slightly decreased levels of DeltaM83 and DeltaM84 genomic DNAs, relative to K181, observed in the salivary glands and lungs. Immunization of BALB/c mice with 10(5) PFU of K181, DeltaM83, or DeltaM84 i.p. provided similar levels of protection against lethal challenge. Although immunization with 200 PFU of DeltaM83 also provided complete protection, this dose allowed both the immunizing and challenge viruses to establish latency in the spleen. Our results show that the two MCMV pp65 homologs differ in their expression kinetics, virion association, and influence on viral tropism and/or dissemination.

DNA immunization confers protection against murine cytomegalovirus infection.

The murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) encodes an 89-kDa phosphoprotein (pp89) which plays a key role in protecting BALB/c mice against the lethal effects of the MCMV infection. In this report, we have addressed the question of whether "naked DNA" vaccination with a eukaryotic expression vector (pcDNA-89) that contains the MCMV IE1 gene driven by a strong enhancer/promoter can confer protection. BALB/c mice were immunized intradermally with pcDNA-89 or with the plasmid backbone pcDNAI/Amp (pcDNA) and then challenged 2 weeks later with either a lethal or a sublethal intraperitoneal dose of the K181 strain of MCMV. Variable results were obtained for the individual experiments in which mice received a lethal challenge. In four separate trials, an average of 63% of the mice immunized with pcDNA-89 survived, compared with 18% of the mice immunized with pcDNA. However, in two other trials there was no specific protection. The results of experiments in which mice were injected with a sublethal dose of MCMV were more consistent, and significant decreases in viral titer in the spleen and salivary glands of pcDNA-89-immunized mice were observed, relative to controls. At the time of peak viral replication, titers in the spleens of immunized mice were reduced 18- to >63-fold, while those in the salivary gland were reduced approximately 24- to 48-fold. Although DNA immunization elicited only a low level of seroconversion in these mice, by 7 weeks postimmunization the mice had generated a cytotoxic T-lymphocyte response against pp89. These results suggest that DNA vaccination with selected CMV genes may provide a safe and efficient means of immunizing against CMV disease.

Identification, analysis, and evolutionary relationships of the putative murine cytomegalovirus homologs of the human cytomegalovirus UL82 (pp71) and UL83 (pp65) matrix phosphoproteins.

We have identified three open reading frames (ORFs) in murine cytomegalovirus (MCMV), designated M82, M83, and M84, which likely encode homologs of the human cytomegalovirus (HCMV) UL82 and UL83 matrix phosphoproteins. These ORFs, in the HindIII C fragment of MCMV, are colinear with the UL82, UL83, and UL84 ORFs of HCMV. M82 encodes a 598-amino-acid (aa) protein with homology to UL82, M83 encodes an 809-aa protein with homology to UL82 and UL83, and M84 encodes a 587-aa protein with homology to UL83 and UL84. Analysis of transcription by Northern (RNA) blotting indicated that the M82 and M83 ORFs are transcribed as 2.2- and 5-kb mRNAs, respectively, at 24 to 48 h postinfection (p.i.), while M84 is transcribed as a 6.9-kb mRNA only at 8 h p.i. All transcripts appear to terminate at the same position 3' of the M82 ORF. Of the products of the three ORFs, only M83 is strongly recognized by hyperimmune mouse serum. The M83 protein is a virion-associated phosphoprotein with an apparent molecular mass of 125 kDa. In MCMV-infected cells, it is detectable by Western blotting (immunoblotting) only at 48 h p.i. in the absence of phosphonoacetic acid, consistent with late gene expression. The M83 ORF is also expressed at high levels in cells infected by a recombinant vaccinia virus and yields a protein which is serologically cross-reactive and comigrates with the authentic MCMV protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Cloning, characterization, and expression of the murine cytomegalovirus homologue of the human cytomegalovirus 28-kDa matrix phosphoprotein (UL99).

We have identified, characterized, and expressed in bacteria and recombinant vaccinia viruses a protein which likely represents the murine cytomegalovirus (MCMV) homologue of the human cytomegalovirus (HCMV) 28-kDa matrix phosphoprotein, the product of the HCMV UL99 open reading frame (ORF). This protein, referred to as the MCMV UL99, is encoded by a 336-nucleotide ORF within the HindIII G fragment of MCMV strain Smith (K181). Using a DNA probe that corresponded to the amino terminus of the ORF, we detected a transcript of 4.8 kb at 8 hr and additional transcripts of 0.88, 2.4, and 5.7 kb at 24-48 hr postinfection (p.i.) of NIH 3T3 cells with MCMV. The smallest transcript is unspliced, initiates 235 nucleotides upstream from the start of the ORF, and utilizes a polyadenylation site located 62 nucleotides downstream from the end of the ORF. The ORF encodes a protein of 112 amino acids, with a predicted mass of 11.8 kDa. Comparison of the derived amino acid sequence with that of the HCMV UL99 gene product reveals 34.8% identity in an overlap of 66 amino acids. Within the amino acid sequence are at least two potential protein kinase C and one potential casein kinase II target motifs for phosphorylation. The ORF was cloned into the pGEX-KG prokaryotic expression vector and bacterially expressed protein was used to generate a specific rabbit antiserum against the protein. Western blotting of MCMV-infected NIH 3T3 cells showed that the ORF was expressed as a doublet of 16.3 and 15.2 kDa at 48 hr p.i. only in the absence of phosphonoacetic acid, thus demonstrating that this protein is a member of the true late gene class. The immunoreactive protein in MCMV-infected cells comigrated with that produced in cells infected with recombinant vaccinia virus containing the ORF. The protein appears to be part of the MCMV virion, is phosphorylated in vivo, and generates a strong humoral immune response following MCMV infection of BALB/c mice.

Gonyauline: a novel endogenous substance shortening the period of the circadian clock of a unicellular alga.

The circadian clock in the unicellular alga Gonyaulax polyedra is accelerated by a substance in extracts from the cells themselves. The extracts have been fractionated using the circadian rhythm of bioluminescence as bioassay. The active substance, termed gonyauline, has been isolated and characterized as a novel low molecular weight cyclopropanecarboxylic acid (S-methyl-cis-2-(methylthio) cyclopropanecarboxylic acid). Synthetic gonyauline has a similar shortening effect on the period of the circadian clock.