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Publications (19)

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    Mqondisi Tshabalala · Justen Manasa · Lynn S Zijenah · [...] · David Katzenstein
    [Show abstract] [Hide abstract] ABSTRACT: The rapid scale-up of highly active antiretroviral therapy (HAART) and use of single dose Nevirapine (SD NVP) for prevention of mother-to-child transmission (pMTCT) have raised fears about the emergence of resistance to the first line antiretroviral drug regimens. A cross-sectional study was conducted to determine the prevalence of primary drug resistance (PDR) in a cohort of young (<25 yrs) HAART-naïve HIV pregnant women attending antenatal clinics in Chitungwiza, Zimbabwe. Whole blood was collected in EDTA for CD4 counts, viral load, serological estimation of duration of infection using the BED Calypte assay and genotyping for drug resistance. Four hundred and seventy-one women, mean age 21 years; SD: 2.1 were enrolled into the study between 2006 and 2007. Their median CD4 count was 371cells/µL; IQR: 255-511 cells/µL. Two hundred and thirty-six samples were genotyped for drug resistance. Based on the BED assay, 27% were recently infected (RI) whilst 73% had long-term infection (LTI). Median CD4 count was higher (p<0.05) in RI than in women with LTI. Only 2 women had drug resistance mutations; protease I85V and reverse transcriptase Y181C. Prevalence of PDR in Chitungwiza, 4 years after commencement of the national ART program remained below WHO threshold limit (5%). Frequency of recent infection BED testing is consistent with high HIV acquisition during pregnancy. With the scale-up of long-term ART programs, maintenance of proper prescribing practices, continuous monitoring of patients and reinforcement of adherence may prevent the acquisition and transmission of PDR.
    Full-text available · Article · Jun 2011 · PLoS ONE
  • [Show abstract] [Hide abstract] ABSTRACT: Background: Plasma virus RNA (vRNA) and proviral DNA from archived provirus (pvDNA) may be sequenced to identify drug resistance-associated mutations (RAMS). pvDNA is the more stable analyte and may be stored and shipped at room temperature, while vRNA is more fragile and requires cryopreservation. pvDNA and vRNA genotype and RAMs were compared among 46 viremic subjects. Methods: vRNA and pvDNA were extracted from EDTA anti-coagulated blood and gag-pol amplification and genotyping was performed using in-house RT-PCR and dideoxyterminator sequencing. Antiretroviral drug susceptibility was predicted using the Genotypic Resistance Interpretation Algorithm ( Results: Among 46 subjects median vRNA was 3.54 ± 1.09 log copies/ml, 17 had < 1,000 copies/ml and CD4 counts ranged from 29 to 652 per cu mm. Complete sequences from RT and protease were obtained from all 46 vRNA and 44/46 pvDNA. pvDNA sequence could not be obtained from two subjects with 637 and 359 CD4 cells per cu mm and < 1,000 copies/ml of RNA. Comparing drug resistance interpretation, there were no significant differences in drug susceptibility between vRNA and pvDNA. However, among 17 subjects with RAMs, there were significant differences in the sensitivity of detection of one or more major reverse transcriptase inhibitor (RTI) mutations among 12 subjects; 11/12 (92%) were detected in pRNA and only 5/12 (42%) in pvDNA, (p=0.027 Fisher’s exact test). Notably, RTI mutations were detected in 7/10 samples with vRNA < 1,000 copies/ml and were not detected in the pvDNA. However, in 4 of the 17 samples with RTI drug resistance, specific RAMs were identified only in pvDNA. Conclusions: More accurate drug resistance genotypes were obtained from vRNA compared to pvDNA, particularly in early failure with low levels (< 1,000 copies/ml) of HIV-1 RNA. However, there may be logistic and cost advantages to pvDNA sequencing for population-based and public health surveillance. pvDNA provided subtyping and phylogenetic information and in some cases added new evidence of archival drug resistance.
    Conference Paper · Oct 2009
  • Maya Balamane · Elizabeth Johnston · David Katzenstein · [...] · Nancy Shulman
    [Show abstract] [Hide abstract] ABSTRACT: Background: Zidovudine (AZT) monotherapy and Syncytium Inducing virus (SI) are each associated with more rapid disease progression among nucleoside-treated subjects. To determine the relationship between viral tropism and AZT drug resistance mutations we sequenced pol and env from 78 AZT treated individuals who entered the ACTG175 protocol in 1992. Methods: Virus isolates or plasma samples were available from 78 subjects who entered ACTG175 with a history of AZT treatment for > 200 days. Tropism (SI or NSI) was determined by MT-2 cell co-culture, supplemented with consensus env V3 loop sequence (using PSSM). Drug resistance of population sequences was estimated with HIVSeq ( Tropism, AZT resistance, specific AZT mutations, CD4, viral RNA and AZT treatment duration were examined in SPSS 16.0 using Mann-Whitney and Pearson analysis. Results: At study entry 54 (69%) of the AZT experienced subjects’ viruses were NSI and 24 (31%) SI. The mean duration of AZT was 741.5 (SI) and 808 days (NSI) and mean baseline CD4 cells, 292 and 307 per cu mm were not different (p> 0.2, Kruskal Wallis Chi Square test). However, median HIV RNA was significantly lower among NSI subjects, 4.18 vs 4.71 log copies / ml (p=0.013). Among 75 subjects with > 6 months of AZT, 18 (24%) were susceptible to AZT, 17 (22%) had low level resistance and (40) 53% had intermediate or high level AZT resistance. AZT resistance was significantly associated with higher HIV RNA, a longer duration of AZT treatment and older age (p < 0.03, Mann Whitney U test). However comparing those with SI and NSI virus there were no differences in AZT resistance, or the prevalence of AZT associated drug resistance mutations, or patterns of multiple AZT associated drug resistance. Conclusion: SI viruses were identified in 30% of subjects with > 200 CD4 cells in association with significantly higher HIV-1 RNA. There was no relationship between SI virus and AZT resistance or specific thymidine analog drug resistance mutations among monotherapy recipients. AZT resistance was associated with plasma HIV-1 RNA, the duration of monotherapy and age, but not CD4 cell numbers or envelope coreceptor tropism.
    Conference Paper · Oct 2009
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    [Show abstract] [Hide abstract] ABSTRACT: To investigate the origins and evolutionary history of subtype C HIV-1 in Zimbabwe in a context of regional conflict and migration. HIV-1C pol sequence datasets were generated from four sequential cohorts of antenatal women in Harare, Zimbabwe sampled over 15 years (1991-2006). One hundred and seventy-seven HIV-1C pol sequences were obtained from four successive cohorts in Zimbabwe. Maximum-likelihood methods were used to explore phylogenetic relationships between Zimbabwean HIV-1C sequences and subtype C strains from other regions. A Bayesian coalescent-based framework was used to estimate evolutionary parameters for HIV-1C in Zimbabwe, including origin and demographic growth patterns. Zimbabwe HIV-1C pol demonstrated increasing sequence divergence over the 15-year period. Nearly all Zimbabwe sequences clustered phylogenetically with subtype C strains from neighboring countries. Bayesian evolutionary analysis indicated a most recent common ancestor date of 1973 with three epidemic growth phases: an initial, slow phase (1970s) followed by exponential growth (1980s), and a linearly expanding epidemic to the present. Bayesian trees provided evidence for multiple HIV-1C introductions into Zimbabwe during 1979-1981, corresponding with Zimbabwean national independence following a period of socio-political instability. The Zimbabwean HIV-1C epidemic likely originated from multiple introductions in the late 1970s and grew exponentially during the 1980s, corresponding to changing political boundaries and rapid population influx from neighboring countries. The timing and phylogenetic clustering of the Zimbabwean sequences is consistent with an origin in southern Africa and subsequent expansion. HIV-1 sequence data contain important epidemiological information, which can help focus treatment and prevention strategies in light of more recent political volatility in Zimbabwe.
    Full-text available · Article · Sep 2009 · AIDS (London, England)
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    [Show abstract] [Hide abstract] ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) subtype C is the dominant subtype globally, due largely to the incidence of subtype C infections in sub-Saharan Africa and east Asia. We compared the relative replicative fitness (ex vivo) of the major (M) group of HIV-1 subtypes A, B, C, D, and CRF01_AE and group O isolates. To estimate pathogenic fitness, pairwise competitions were performed between CCR5-tropic (R5) or CXCR4-tropic (X4) virus isolates in peripheral blood mononuclear cells (PBMC). A general fitness order was observed among 33 HIV-1 isolates; subtype B and D HIV-1 isolates were slightly more fit than the subtype A and dramatically more fit than the 12 subtype C isolates. All group M isolates were more fit (ex vivo) than the group O isolates. To estimate ex vivo transmission fitness, a subset of primary HIV-1 isolates were examined in primary human explants from penile, cervical, and rectal tissues. Only R5 isolates and no X4 HIV-1 isolates could replicate in these tissues, whereas the spread to PM1 cells was dependent on active replication and passive virus transfer. In tissue competition experiments, subtype C isolates could compete with and, in some cases, even win over subtype A and D isolates. However, when the migratory cells from infected tissues were mixed with a susceptible cell line, the subtype C isolates were outcompeted by other subtypes, as observed in experiments with PBMC. These findings suggest that subtype C HIV-1 isolates might have equal transmission fitness but reduced pathogenic fitness relative to other group M HIV-1 isolates.
    Full-text available · Article · Apr 2009 · Journal of Virology
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    Seble Kassaye · Elizabeth Johnston · Bryan McColgan · [...] · David Katzenstein
    [Show abstract] [Hide abstract] ABSTRACT: In resource-constrained settings, antiretroviral treatment (ART) is often continued based on clinical and CD4 responses, without virologic monitoring. ART with incomplete viral suppression was assessed in 27 subjects with subtype C HIV-1. Plasma HIV-1 RNA, drug resistance, viral tropism, and evolution in polymerase (pol) and envelope (env) genes were measured. The association between these viral parameters and CD4 cell change over time was analyzed using linear regression models. Increased area under the curve of HIV-1 RNA replication was a predictor of lower CD4 cell gains (P < 0.007), while less drug resistance measured as a genotypic susceptibility score (GSS) (P = 0.065), and lower rates of evolution in pol and env genes (P = 0.08 and 0.097, respectively) measured as genetic distance were modestly associated with increasing CD4 cell counts. Evolution of pol and env were correlated (R2 = 0.48, P = 0.005), however, greater evolution was identified in env vs. pol (P < 0.05). CXCR4-usage (X4) was detected in 14/27 (52%) but no differences in CD4 cell change or plasma viremia were associated with X4-usage. Among subtype C HIV-1 infected patients in Zimbabwe receiving incompletely suppressive ART, higher virus replication and lower CD4 cell gains were associated with drug resistance and evolution of polymerase and envelope.
    Full-text available · Article · Jan 2009 · JAIDS Journal of Acquired Immune Deficiency Syndromes
  • [Show abstract] [Hide abstract] ABSTRACT: Single-dose nevirapine (SD NVP) reduces intrapartum HIV-1 transmission, but nonnucleoside reverse transcription (NNRTI) resistance mutations can emerge. Population sequencing among 32 subtype C HIV-1-infected, SD NVP-exposed Zimbawean women demonstrated NNRTI resistance in 25/32 (78%) women: 23/30 (77%) at 2 weeks, 11/31 (35%) at 8 weeks, and 5/27 (19%) at 24 weeks. A total of 447 unique TA clones (median = 28 per time point), from four women with resistance at 8 weeks but wild-type virus by population sequence at 24 weeks, identified NNRTI mutations in a median of 76% (range: 55-96%) of individual clones at 2 weeks, 48% (range: 33-80%) at 8 weeks, and 5% (range: 0-15%) by 24 weeks. NNRTI mutations in breast milk clones at 2 and weeks from one woman varied significantly from plasma. Population sequencing underestimates the diversity of NNRTI resistance mutations within minority populations following SD NVP in subtype C HIV-1 viral RNA in plasma and breast milk.
    Article · Sep 2007 · AIDS Research and Human Retroviruses
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    Full-text available · Article · Aug 2007 · The Journal of Infectious Diseases
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    [Show abstract] [Hide abstract] ABSTRACT: Single-dose nevirapine reduces intrapartum human immunodeficiency virus 1 type (HIV-1) transmission but may also select for nonnucleoside reverse-transcriptase inhibitor (NNRTI) resistance in breast milk (BM) and plasma. Among 32 Zimbabwean women, median 8-week postpartum plasma and BM HIV-1 RNA levels were 4.57 and 2.13 log10 copies/mL, respectively. BM samples from women with laboratory-diagnosed mastitis (defined as elevated BM Na+ levels) were 5.4-fold more likely to have HIV-1 RNA levels above the median. BM RT sequences were not obtained for 12 women with BM HIV-1 RNA levels below the lower limit of detection of the assay used. In 20 paired BM and plasma samples, 65% of BM and 50% of plasma RT sequences had NNRTI-resistance mutations, with divergent mutation patterns
    Full-text available · Article · Nov 2005 · The Journal of Infectious Diseases
  • [Show abstract] [Hide abstract] ABSTRACT: HIV-1 isolates harboring multiple nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations are more susceptible ("hypersusceptible") to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) than isolates lacking NRTI resistance mutations, but this has only been reported with a single-cycle replication phenotypic assay. In fact, there was a report that a commercial multicycle assay did not readily detect hypersusceptibility. To see whether NNRTI hypersusceptibility can be demonstrated in other types of phenotypic assays, including multicycle assays and enzyme inhibition assays. The susceptibility of HIV-1 clones derived from different patients in multicycle assays was tested in peripheral blood mononuclear cells (PBMCs) and in an established cell line. In addition, the reverse transcriptase (RT) of many of these clones was expressed and their susceptibility tested in an RT inhibition assay. Nevirapine and efavirenz susceptibilities were tested and compared with a control wild-type virus or RT. Hypersusceptibility to nevirapine and efavirenz was detected using each of the methods described above. R values correlating the other methods with single-cycle assay values were between 0.66 and 0.96. In addition to the high correlations, the different methods gave similar numeric results. NNRTI hypersusceptibility is readily seen in multicycle susceptibility assays and in enzyme inhibition assays.
    Article · Jun 2005 · JAIDS Journal of Acquired Immune Deficiency Syndromes
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    Elizabeth Johnston · Kathryn M Dupnik · Matthew J Gonzales · [...] · Robert W Shafer
    [Show abstract] [Hide abstract] ABSTRACT: We have created a panel of recombinant HIV-1 infectious clones containing common patterns of reverse transcriptase (RT) mutations responsible for resistance to each of the currently available nucleoside reverse transcriptase inhibitors (NRTI), and we have submitted the panel to the National Institutes of Health AIDS Research and Reference Reagent Programme. Testing the activity of new antiretroviral compounds against this panel of drug-resistant clones will determine their relative activity against many clinically relevant NRTI-resistant viruses.
    Full-text available · Article · May 2005 · AIDS
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    Elizabeth Johnston · Mark A Winters · Soo-Yon Rhee · [...] · Robert W Shafer
    [Show abstract] [Hide abstract] ABSTRACT: We observed a previously uncharacterized mutation in the protease substrate cleft, L23I, in 31 of 4,303 persons undergoing human immunodeficiency virus type 1 genotypic resistance testing. In combination with V82I, L23I was associated with a sevenfold reduction in nelfinavir susceptibility and a decrease in replication capacity. In combination with other drug resistance mutations, L23I was associated with multidrug resistance and a compensatory increase in replication capacity.
    Full-text available · Article · Jan 2005 · Antimicrobial Agents and Chemotherapy
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    Elizabeth R Johnston · Lynn S Zijenah · Solomon Mutetwa · [...] · David A Katzenstein
    [Show abstract] [Hide abstract] ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) subtype C viruses have been found to almost exclusively use the chemokine receptor CCR5 as a coreceptor for entry, even in patients with advanced AIDS. We have characterized subtype C virus isolates from 28 patients from Harare, Zimbabwe, 20 of whom were receiving antiretroviral treatment. Virus from 10 of the treated patients induced syncytium formation (SI virus) when cultured with MT2 cells. Only non-syncytium-inducing (NSI) virus was cultured from the peripheral blood mononuclear cells of the eight patients who had not received treatment. The majority of these subtype C SI viruses were capable of using both CCR5 and CXCR4 as coreceptors for viral entry, and the consensus V3 loop sequences from the SI viruses displayed a high net charge compared to those of NSI viruses. While those on treatment had reverse transcriptase (RT) and protease mutations, there was no clear association between RT and protease drug resistance mutations and coreceptor tropism. These results suggest that CXCR4-tropic viruses are present within the quasispecies of patients infected with subtype C virus and that antiretroviral treatment may create an environment for the emergence of CXCR4 tropism.
    Full-text available · Article · Aug 2003 · Journal of Virology
  • R Kantor · E Lee · E Johnston · [...] · D Katzenstein
    Conference Paper · Jun 2003
  • Conference Paper · Jun 2003
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    [Show abstract] [Hide abstract] ABSTRACT: HIV-1 drug resistance mutations have been identified and characterized mostly in subtype B HIV-1 infection. The extent to which antiretroviral drugs select for drug resistance mutations in non-subtype B HIV-1 is not known. We obtained HIV-1 reverse transcriptase (RT) and protease sequences from 21 Zimbabwean patients failing antiretroviral drug therapy. We compared these sequences with 56 published RT and protease subtype C sequences from untreated patients, 990 RT and 1140 protease subtype B sequences from treated patients, and 340 RT and 907 protease subtype B sequences from untreated patients and identified four mutation categories of subtype C HIV-1. Seventeen of the 21 patients (81%) had known drug resistance mutations. Mutations at 15 RT and 11 protease positions were more common in subtype C isolates than in subtype B isolates. HIV-1 subtype C-infected individuals receiving antiretroviral therapy develop many of the known subtype B drug resistance mutations. Comparison of subtype C RT and protease sequences with a large database of subtype B sequences identified subtype C-specific polymorphisms and candidate drug resistance mutations.
    Full-text available · Article · Jan 2003 · AIDS Research and Human Retroviruses
  • Hugo Guevara · Elizabeth Johnston · Lynn Zijenah · [...] · David Katzenstein
    [Show abstract] [Hide abstract] ABSTRACT: : Maternal and cord samples from HIV-seropositive women and their infants in Zimbabwe, where subtype C is the predominant strain of HIV, were analyzed to determine the frequency of detection of HIV RNA and DNA. HIV RNA was detected in 90% of maternal and in 38% of cord plasma at levels at least 25% of maternal plasma. Heteroduplex mobility assays and sequencing of virus envelope (C2-V5) demonstrated closely related, but unique, subtype C viruses in maternal and cord RNA, and a significantly greater frequency of cord viremia among women with homogenous, compared with heterogeneous viral envelope RNA. Quantification of RNA, measures of envelope viral diversity, and phylogenetic analysis of maternal and cord plasma RNA provide evidence for the frequent exposure and potential transmission of HIV from mother to infant before birth. (C) 2000 Lippincott Williams & Wilkins, Inc.
    Article · Dec 2000 · JAIDS Journal of Acquired Immune Deficiency Syndromes
  • Hugo Guevara · Elizabeth Johnston · Lynn Zijenah · [...] · David Katzenstein
    Article · Dec 2000 · JAIDS Journal of Acquired Immune Deficiency Syndromes
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    E Johnston · S Mutetwa · L Zijenah · [...] · D Katzenstein
    [Show abstract] [Hide abstract] ABSTRACT: Two fold difference between viral load of treatment groups and non-treatment group but not significant difference (p=0.9) Difference between CD4 counts for Tx groups and nonTx group is significant (p=.01) No significant differences between SI and NSI ARV treatment groups.
    Full-text available · Article ·